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WORLD ORGANISATION FOR ANIMAL HEALTH
MANUAL OF DIAGNOSTIC TESTS AND VACCINES

FOR TERRESTRIAL ANIMALS
(mammals, birds and bees)
Sixth Edition
Volume 1
2008
This Terrestrial Manual has been edited by the
OIE Biological Standards Commission and adopted by
the International Committee of the OIE
Reference to commercial kits does not mean their endorsement by the OIE. All commercial kits should
be validated; tests on the OIE register have already met this condition (the register can be consulted at:
www.oie.int).
OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals

Sixth Edition, 2008
Manual of Recommended Diagnostic Techniques and Requirements for Biological Products:

Volume I, 1989; Volume II, 1990; Volume III, 1991.
Manual of Standards for Diagnostic Tests and Vaccines:
Second Edition, 1992
Third Edition, 1996
Fourth Edition, 2000
Fifth Edition, 2004
ISBN 978-92-9044-718-4
© Copyright
OFFICE INTERNATIONAL DES EPIZOOTIES, 2008
12, rue de Prony, 75017 Paris, FRANCE
Telephone: 33-(0)1 44 15 18 88
Fax: 33-(0)1 42 67 09 87
Electronic mail: [email protected]
http://www.oie.int
All OIE (World Organisation for Animal Health) publications are protected by international copyright law. Extracts
may be copied, reproduced, translated, adapted or published in journals, documents, books, electronic media
and any other medium destined for the public, for information, educational or commercial purposes, provided
prior written permission has been granted by the OIE.
The designations and denominations employed and the presentation of the material in this publication do not
imply the expression of any opinion whatsoever on the part of the OIE concerning the legal status of any country,
territory, city or area or of its authorities, or concerning the delimitation of its frontiers and boundaries.
FOREWORD
The Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Terrestrial Manual) aims to
facilitate international trade in animals and animal products and to contribute to the improvement of
animal health services world-wide. The principal target readership is laboratories carrying out
veterinary diagnostic tests and surveillance, plus vaccine manufacturers and regulatory authorities
in Member Countries. The objective is to provide internationally agreed diagnostic laboratory
methods and requirements for the production and control of vaccines and other biological
products.
This ambitious task has required the cooperation of highly renowned animal health specialists from
many countries. The OIE, the World Organisation for Animal Health, is clearly the most appropriate
organisation to undertake this task on a global level. The main activities of the organisation, which
was established in 1924 and in 2008 comprised 172 Member Countries and Territories, are as
follows:
1. To ensure transparency in the global animal disease and zoonosis situation.
2. To collect, analyse and disseminate scientific veterinary information on animal disease
control methods.
3. To provide expertise and encourage international solidarity in the control of animal diseases.
4. Within its mandate under the WTO (World Trade Organization) Agreement on Sanitary and
Phytosanitary Measures (SPS Agreement), to safeguard world trade by publishing health
standards for international trade in animals and animal products.
5. To improve the legal framework and resources of national Veterinary Services.
6. To provide a better guarantee of the safety of food of animal origin and to promote animal
welfare through a science-based approach.
The Terrestrial Manual, covering infectious and parasitic diseases of mammals, birds and bees,
was first published in 1989. Each successive edition has extended and updated the information
provided. This sixth edition includes new chapters on Guidelines for international standards for
vaccine banks, Turkey rhinopneumonitis, Small hive beetle infestation (Aethina tumida) and
camelpox, and Mycoplasma synoviae has been added to the chapter on Avian mycoplasmosis
(previously the chapter focused on Mycoplasma gallisepticum. As a companion volume to the
Terrestrial Animal Health Code, the Terrestrial Manual sets laboratory standards for all OIE listed
diseases as well as several other diseases of global importance. In particular it specifies (in blue
font) those “Prescribed Tests” that are recommended for use in health screening for international
trade or movement of animals. The Terrestrial Manual has become widely adopted as a key
reference book for veterinary laboratories around the world. Aquatic animal diseases are included
in a separate Aquatic Manual.
The task of commissioning chapters and compiling the Terrestrial Manual was assigned to the OIE
Biological Standards Commission by the International Committee of the OIE (General Assembly of
national Delegates of Member Countries and Territories). Manuscripts were requested from
specialists in each of the diseases or the other topics covered. After initial scrutiny by the
Consultant Technical Editor, the chapters were sent to scientific reviewers and to experts at OIE
Reference Laboratories. They were also circulated to all OIE Member Countries for review and
comment. The Biological Standards Commission and the Consultant Technical Editor took all the
resulting comments into consideration, often referring back to the authors for further help, before
finalising the chapters. The final text has the approval of the International Committee of the OIE.
A procedure for the official recognition of commercialised diagnostic tests, under the authority of
the International Committee, was finalised in September 2004. Data are submitted using a
validation template that was developed by the Biological Standards Commission. Submissions are
evaluated by appointed experts, who advise the Biological Standards Commission before the final
opinion of the OIE International Committee is sought. All information on the submission of
applications can be found on the OIE Web site.
OIE Terrestrial Manual 2008 iii

How to use this Terrestrial Manual
The Terrestrial Manual continues to expand and to extend its range of topics covered. It is our
sincere hope that it will grow in usefulness to veterinary diagnosticians and vaccine manufacturers
in all the OIE Member Countries. A new paper edition of the Terrestrial Manual is published every
4 years. It is important to note that annual updates to the Terrestrial Manual will be published on
the OIE website once approved by the International Committee, so readers are advised to check
there for the latest information. The Terrestrial Manual is published in English, French and
Spanish.
Doctor Bernard Vallat Professor Steven Edwards

Director General, OIE President, OIE Biological Standards Commission
January 2008
iv OIE Terrestrial Manual 2008

ACKNOWLEDGEMENTS
I am most grateful to the many people whose combined efforts have gone into the preparation of
this Terrestrial Manual. In particular, I would like to express my thanks to:
Dr Bernard Vallat, Director General of the OIE from 2001 to the present, who gave his
encouragement and support to the project of preparing the new edition of this Terrestrial
Manual,
The Members of the OIE Standards Commission, Prof. Steven Edwards, Dr Beverly Schmitt,
Dr Anatoly Golovko, Dr Mehdi El Harrak and Dr Santanu K. Bandhopadhyay who were
responsible for commissioning chapters and, with the Consultant Technical Editor, for editing
all the contributions so as to finalise this edition of the Terrestrial Manual,
The contributors listed on pages xxii to xxxv who contributed their invaluable time and
expertise to write the chapters,
The expert advisers to the Biological Standards Commission’s meeting, Dr Adama Diallo and
Dr Peter Wright, the OIE Reference Laboratory experts and other reviewers who also gave
their time and expertise to scrutinising the chapters,
Those OIE Member Countries that submitted comments on the draft chapters that were
circulated to them. These were essential in making the Terrestrial Manual internationally
acceptable,
Ms Sara Linnane who, as Scientific Editor, organised this complex project and made major
contributions to the quality of the text,
Dr James E. Pearson, Consultant Technical Editor of the Terrestrial Manual, who contributed
hugely to editing and harmonising the contents, but also in collating and incorporating
Member Country comments,
Members of both the OIE Scientific and Technical Department and the Publications
Department, for their assistance.
Dr Barry O’Neill
President of the OIE International Committee
January 2008
OIE Terrestrial Manual 2008 v

CONTENTS
VOLUME 1
Introduction (How to use this Terrestrial Manual)..................................................... ix
List of tests for International trade............................................................................ xi
Common abbreviations used in this Terrestrial Manual............................................ xv
Glossary of terms...................................................................................................... xvii
Contributors.............................................................................................................. xxii
PART 1 GENERAL INFORMATION
SECTION 1.1. INTRODUCTORY CHAPTERS

Chapter 1.1.1. Collection and shipment of diagnostic specimens.................................................... 3
Chapter 1.1.2. Biosafety and biosecurity in the veterinary microbiology laboratory 15
and animal facilities..................................................................................................
Chapter 1.1.3. Quality management in veterinary testing laboratories............................................. 27
Chapter 1.1.4. Principles of validation of diagnostic assays for infectious diseases........................ 34
Chapter 1.1.5. Validation and quality control of polymerase chain reaction methods 46
used for the diagnosis of infectious diseases...........................................................
Chapter 1.1.6. Laboratory methodologies for bacterial antimicrobial susceptibility testing………. 56
Chapter 1.1.7. Biotechnology in the diagnosis of infectious diseases and vaccine development.... 66
Chapter 1.1.8. Principles of veterinary vaccine production............................................................... 90
Chapter 1.1.9. Tests for sterility and freedom from contamination of biological materials............... 105
Chapter 1.1.10. Guidelines for international standards for vaccine banks…………..………………… 115
Chapter 1.1.11. The role of official bodies in the international regulation of veterinary biologicals.... 120
PART 2 OIE LISTED DISEASES AND OTHER DISEASES OF IMPORTANCE TO

INTERNATIONAL TRADE
SECTION 2.1. MULTIPLE SPECIES

Chapter 2.1.1. Anthrax...................................................................................................................... 135
Chapter 2.1.2. Aujeszky’s disease.................................................................................................... 145
Chapter 2.1.3. Bluetongue................................................................................................................ 158
Chapter 2.1.4. Echinococcosis/hydatidosis...................................................................................... 175
Chapter 2.1.5. Foot and mouth disease........................................................................................... 190
Chapter 2.1.6. Heartwater................................................................................................................ 217
Chapter 2.1.7. Japanese encephalitis.............................................................................................. 231
Chapter 2.1.8. Leishmaniosis........................................................................................................... 240
Chapter 2.1.9. Leptospirosis............................................................................................................. 251
Chapter 2.1.10. New World screwworm (Cochliomyia hominivorax) and 265
Old World screwworm (Chrysomya bezziana).........................................................
Chapter 2.1.11. Paratuberculosis (Johne’s disease).......................................................................... 276
Chapter 2.1.12. Q fever...................................................................................................................... 292
Chapter 2.1.13. Rabies....................................................................................................................... 304
Chapter 2.1.14. Rift Valley fever......................................................................................................... 323
Chapter 2.1.15. Rinderpest................................................................................................................. 334
Chapter 2.1.16. Trichinellosis............................................................................................................. 344
Chapter 2.1.17. Trypanosoma evansi infections (including surra)….................................................. 352
OIE Terrestrial Manual 2008 vii

Contents
Chapter 2.1.18. Tularemia.................................................................................................................. 361

Chapter 2.1.19. Vesicular stomatitis................................................................................................... 367
Chapter 2.1.20. West Nile fever......................................................................................................... 377
SECTION 2.2. APIDAE

Introductory note on bee diseases............................................................................ 387
Chapter 2.2.1. Acarapisosis of honey bees...................................................................................... 388
Chapter 2.2.2. American foulbrood of honey bees........................................................................... 395
Chapter 2.2.3. European foulbrood of honey bees........................................................................... 405
Chapter 2.2.4. Nosemosis of honey bees......................................................................................... 410
Chapter 2.2.5. Small hive beetle infestation (Aethina tumida).......................................................... 415
Chapter 2.2.6. Tropilaelaps infestation of honey bees (Tropilaelaps spp.)...................................... 419
Chapter 2.2.7. Varroosis of honey bees........................................................................................... 424
SECTION 2.2. AVES

Chapter 2.3.1. Avian chlamydiosis................................................................................................... 431
Chapter 2.3.2. Avian infectious bronchitis........................................................................................ 443
Chapter 2.3.3. Avian infectious laryngotracheitis.............................................................................. 456
Chapter 2.3.4. Avian influenza.......................................................................................................... 465
Chapter 2.3.5. Avian mycoplasmosis (Mycoplasma gallisepticum, M. synoviae)............................. 482
Chapter 2.3.6. Avian tuberculosis..................................................................................................... 497
Chapter 2.3.7. Duck virus enteritis.................................................................................................... 507
Chapter 2.3.8. Duck virus hepatitis................................................................................................... 515
Chapter 2.3.9. Fowl cholera……………………................................................................................. 524
Chapter 2.3.10. Fowl pox.................................................................................................................... 531
Chapter 2.3.11. Fowl typhoid and Pullorum disease.......................................................................... 538
Chapter 2.3.12. Infectious bursal disease (Gumboro disease).......................................................... 549
Chapter 2.3.13. Marek’s disease........................................................................................................ 566
Chapter 2.3.14. Newcastle disease.................................................................................................... 576
Chapter 2.3.15. Turkey rhinopneumonitis (avian metapneumovirus).………………………….………. 590
viii OIE Terrestrial Manual 2008

INTRODUCTION
(How to use this Terrestrial Manual)
• Arrangement of the Terrestrial Manual
Part 1, the beginning of this Terrestrial Manual, contains eleven introductory chapters that deal with
a variety of general subjects of interest to veterinary laboratory diagnosticians. These chapters are
intended to give a brief introduction to their subjects. They are to be regarded as background
information rather than standards.
The main part of the Terrestrial Manual (Part 2) covers standards for diagnostic tests and vaccines
for the diseases listed in the OIE Terrestrial Animal Health Code. The diseases are in alphabetical
order, subdivided by animal host species. OIE listed diseases are transmissible diseases that have
the potential for very serious and rapid spread, irrespective of national borders. They have
particularly serious socio-economic or public health consequences and are of major importance in
the international trade of animals and animal products.
Four of the diseases in Section 2.9 are included in some individual species sections, but these
chapters cover several species and thus give a broader description. Some additional diseases that
may also be of importance to trade but that do not have a chapter in the Terrestrial Code are also
included in Section 2.9.
The contributors of all the chapters are listed on pages xxii–xxxv, but the final responsibility for the
content of the Terrestrial Manual lies with the International Committee of the OIE.
There is an alphabetical index of the diseases at the end of Volume 2.
• Format of chapters
Each disease chapter includes a summary intended to provide information for veterinary officials
and other readers who need a general overview of the tests and vaccines available for the disease.
This is followed by a text giving greater detail for laboratory workers. In each disease chapter,
Part A gives a general introduction to the disease, Part B deals with laboratory diagnosis of the
disease, and Part C (where appropriate) with the requirements for vaccines or in vivo diagnostic
biologicals. The information concerning production and control of vaccines or diagnostics is given
as an example; it is not always necessary to follow these when there are scientifically justifiable
reasons for using alternative approaches. Bibliographic references that provide further information
are listed at the end of each chapter.
• Explanation of the tests described and of the table on pages xi–xiv
The table on pages xi–xiv lists diagnostic tests in two categories: ‘prescribed’ and ‘alternative’.
Prescribed tests are those that are required by the Terrestrial Animal Health Code for the testing of
animals before they are moved internationally. In the Terrestrial Manual these tests are printed in
blue. At present it is not possible to have prescribed tests for every listed disease. ‘Alternative
tests’ are those that are suitable for the diagnosis of disease within a local setting, and can also be
used in the import/export of animals after bilateral agreement. There are often other tests
described in the chapters, which may also be of some practical value in local situations or which
may still be under development.
OIE Terrestrial Manual 2008 ix

Introduction
• List of OIE Reference Laboratories
A list of OIE Reference Laboratories is given in Part 3 of this Terrestrial Manual. These laboratories
have been designated by the OIE as centres of excellence with expertise in their particular field.
They are able to provide advice to other laboratories on methodology. In some cases standard
strains of micro-organisms or reference reagents (e.g. antisera, antigens) can also be obtained
from the reference laboratories.
The list of OIE Reference Laboratories will be updated by the International Committee of the OIE
each year. The revised list is available on the OIE Web site.
*
* *
x OIE Terrestrial Manual 2008

LIST OF TESTS FOR INTERNATIONAL TRADE
The table below lists diagnostic tests in two categories: ‘prescribed’ and ‘alternative’. Prescribed
tests are required by the OIE Terrestrial Animal Health Code for the international movement of
animals and animal products and are considered optimal for determining the health status of
animals. In the Terrestrial Manual these tests are printed in blue. At present it is not possible to have
prescribed tests for every listed disease. Alternative tests are those that are suitable for the
diagnosis of disease within a local setting, and can also be used in the import/export of animals after
bilateral agreement. There are often other tests described in the chapters that may also be of some
practical value in local situations or that may still be under development.
Chapter No. Disease name Prescribed tests Alternative tests
2.1.1. Anthrax – –
2.1.2. Aujeszky’s disease ELISA, VN –
2.1.3. Bluetongue Agent id., AGID, VN
ELISA, PCR
2.1.4. Echinococcosis/Hydatidosis – –
2.1.5. Foot and mouth disease ELISA *, VN CF
2.1.6. Heartwater – ELISA, IFA

2.1.7. Japanese encephalitis – –
2.1.8. Leishmaniosis – Agent id.
2.1.9. Leptospirosis – MAT
2.1.10. New World screwworm (Cochliomyia – Agent id.
hominivorax) and Old World screwworm
(Chrysomya bezziana)
2.1.11. Paratuberculosis (Johne’s disease) – DTH, ELISA

2.1.12. Q fever – CF
2.1.13. Rabies ELISA, VN –
2.1.14. Rift Valley fever VN ELISA, HI
2.1.15. Rinderpest ELISA VN
2.1.16. Trichinellosis Agent id. ELISA

2.1.17. Trypanosoma evansi infections (including – –
surra)
2.1.18. Tularemia – Agent id.
2.1.19. Vesicular stomatitis CF, ELISA, VN –
2.1.20. West Nile fever – –
2.2.1. Acarapisosis of honey bees – –
2.2.2. American foulbrood of honey bees – –
* Please refer to Terrestrial Manual chapters to verify which method is prescribed.
OIE Terrestrial Manual 2008 xi

List of tests for international trade
2.2.3. European foulbrood of honey bees – –

2.2.4. Nosemosis of bees – –
2.2.5. Small hive beetle infestation – –
(Aethina tumida)
2.2.6. Tropilaelaps infestation of honey bees – –
(Tropilaelaps spp.)
2.2.7 Varroosis of honey bees – –
2.3.1. Avian chlamydiosis – –
2.3.2. Avian infectious bronchitis – ELISA, HI, VN
2.3.3. Avian infectious laryngotracheitis – AGID, ELISA, VN
2.3.4. Avian influenza Virus isolation with AGID, HI
pathogenicity
testing
2.3.5. Avian mycoplasmosis – Agg., HI
(Mycoplasma gallisepticum, M. synoviae)
2.3.6. Avian tuberculosis – Agent id.,
Tuberculin test
2.3.7. Duck virus enteritis – –
2.3.8. Duck virus hepatitis – –
2.3.9. Fowl cholera – –
2.3.10. Fowl pox – –
2.3.11. Fowl typhoid and Pullorum disease – Agent id., Agg.
2.3.12. Infectious bursal disease – AGID, ELISA
(Gumboro disease)
2.3.13. Marek’s disease – AGID
2.3.14. Newcastle disease – HI
2.3.15. Turkey rhinotracheitis (avian – –
metapneumovirus)
2.4.1. Bovine anaplasmosis – CAT, CF
2.4.2. Bovine babesiosis – CF, ELISA, IFA
2.4.3. Bovine brucellosis BBAT, CF, –
ELISA, FPA
2.4.4. Bovine cysticercosis – Agent id.
2.4.5. Bovine genital campylobacteriosis Agent id. –
2.4.6. Bovine spongiform encephalopathy – –
2.4.7. Bovine tuberculosis Tuberculin test Gamma interferon test
2.4.8. Bovine viral diarrhoea Agent id. –
2.4.9. Contagious bovine pleuropneumonia CF, ELISA –
2.4.10. Dermatophilosis – –
2.4.11. Enzootic bovine leukosis AGID, ELISA PCR
2.4.12. Haemorrhagic septicaemia – Agent id.
2.4.13. Infectious bovine rhinotracheitis/ Agent id. (semen –
infectious pustular vulvovaginitis only), ELISA, PCR,
VN
2.4.14. Lumpy skin disease – VN
xii OIE Terrestrial Manual 2008

2.4.15. Malignant catarrhal fever – IFA, PCR, VN

2.4.16. Theileriosis Agent id., IFA –
2.4.17. Trichomonosis Agent id. Mucus agg.
2.4.18. Trypanosomosis (Tsetse-transmitted) – IFA
2.5.1. African horse sickness CF, ELISA Agent id. (real-time
PCR), VN
2.5.2. Contagious equine metritis Agent id. –
2.5.3. Dourine CF ELISA, IFA
2.5.4. Epizootic lymphangitis – –
2.5.5. Equine encephalomyelitis – CF, HI, PRN
(Eastern and Western)
2.5.6. Equine infectious anaemia AGID ELISA
2.5.7. Equine influenza – HI
2.5.8. Equine piroplasmosis ELISA, IFA CF
2.5.9. Equine rhinopneumonitis – VN
2.5.10. Equine viral arteritis Agent id. (semen –

only), VN
2.5.11. Glanders CF, Mallein test –
2.5.12. Horse mange – Agent id.

2.5.13. Horse pox – –
2.5.14. Venezuelan equine encephalomyelitis – CF, HI, PRN
2.6.1. Myxomatosis – AGID, CF, IFA
2.6.2. Rabbit haemorrhagic disease – HI
2.7.1. Border disease Agent id. –
2.7.2. Caprine and ovine brucellosis BBAT, CF Brucellin test, FPA
(excluding Brucella ovis)
2.7.3/4. Caprine arthritis/encephalitis & Maedi-visna AGID, ELISA –
2.7.5. Contagious agalactia – –
2.7.6. Contagious caprine pleuropneumonia CF –
2.7.7. Enzootic abortion of ewes – CF
(ovine chlamydiosis)
2.7.8. Nairobi sheep disease – –
2.7.9. Ovine epididymitis (Brucella ovis) CF ELISA
2.7.10. Ovine pulmonary adenocarcinoma – –
(adenomatosis)
2.7.11. Peste des petits ruminants VN ELISA
2.7.12. Salmonellosis (S. abortusovis) – –
2.7.13. Scrapie – –
2.7.14. Sheep pox and goat pox – VN
2.8.1. African swine fever ELISA IFA
2.8.2. Atrophic rhinitis of swine – –
2.8.3. Classical swine fever (hog cholera) ELISA, FAVN, –
NPLA
OIE Terrestrial Manual 2008 xiii

2.8.4. Nipah virus encephalitis – –

2.8.5. Porcine brucellosis ELISA BBAT, FPA
2.8.6. Porcine cysticercosis – –
2.8.7. Porcine reproductive and – ELISA, IFA, IPMA
respiratory syndrome
2.8.8. Swine influenza – –
2.8.9. Swine vesicular disease VN ELISA
2.8.10. Teschovirus encephalomyelitis (previously – VN
enterovirus encephalomyelitis or
Teschen/Talfan disease)
2.8.11. Transmissible gastroenteritis – VN, ELISA
2.9.1. Bunyaviral diseases of animals (excluding – –
Rift Valley fever)
2.9.2. Camelpox – –
2.9.3. Campylobacter jejuni and C. coli – –
2.9.4. Cryptosporidiosis – –
2.9.5. Cysticercosis – Agent id.
2.9.6. Hendra and Nipah virus diseases – –
2.9.8. Listeria monocytogenes – –
2.9.8. Mange – Agent id.
2.9.9. Salmonellosis – Agent id.

2.9.10. Toxoplasmosis – –
2.9.11. Verocytotoxigenic Escherichia coli – –
2.9.12. Zoonoses transmissible from non-human – –
primates
Note: The tests prescribed by the Terrestrial Animal Health Code for the purposes of international trade are printed
in blue in this Terrestrial Manual.
Abbreviations
Agent id. Agent identification HI Haemagglutination inhibition

Agg. Agglutination test IFA Indirect fluorescent antibody
AGID Agar gel immunodiffusion IPMA Immunoperoxidase monolayer assay
BBAT Buffered Brucella antigen test MAT Microscopic agglutination test
CAT Card agglutination test NPLA Neutralising peroxidase-linked assay
CF Complement fixation PCR Polymerase chain reaction
DTH Delayed-type hypersensitivity PRN Plaque reduction neutralisation
ELISA Enzyme-linked immunosorbent assay VN Virus neutralisation
FAVN Fluorescent antibody virus neutralisation – No test designated yet
FPA Fluorescence polarisation assay
xiv OIE Terrestrial Manual 2008

COMMON ABBREVIATIONS USED
IN THIS TERRESTRIAL MANUAL
ABTS 2,2’-azino-di-(3-ethyl-benzthiazoline)-6- FBS Fetal bovine serum

sulphonic acid
AGID Agar gel immunodiffusion FITC Fluorescein isothiocyanate
ATCC 1 American type culture collection FLK Fetal lamb kidney (cells)
BBAT Buffered Brucella antigen test FPA Fluorescence polarisation assay
BFK Bovine fetal kidney (cells) G Relative centrifugal force
BGPS Beef extract-glucose-peptone-serum GIT Growth inhibition test
(medium)
BHK Baby hamster kidney (cell line) HA Haemagglutination
BLP Buffered lactose peptone HAD Haemadsorption
BPAT Buffered plate antigen test HBSS Hanks’ balanced salt solution
BSA Bovine serum albumin HEP High-egg-passage (virus)
BSF Bovine serum factors HEPES N-2-hydroxyethylpiperazine, N-2-
ethanesulphonic acid (buffer)
CAM Chorioallantoic membrane HI Haemagglutination inhibition
CEF Chicken embryo fibroblast HRPO Horseradish peroxidase
CF Complement fixation (test) IB Immunoblot test
CFU Colony-forming unit ICFTU International complement fixation test unit
CIEP Counter immunoelectrophoresis ICPI Intracerebral pathogenicity index
CK Calf kidney (cells) ID50 Median infectious dose
CNS Central nervous system IFA Indirect fluorescent antibody (test)
CPE Cytopathic effect IHA Indirect haemagglutination
CPLM Cysteine-peptone-liver infusion maltose IPMA Immunoperoxidase monolayer assay
(medium)
CSY Casein-sucrose-yeast (agar) IU International units
DEPC Diethyl pyrocarbonate IVPI Intravenous pathogenicity index
DEAE Diethylaminoethyl LA Latex agglutination
DEPC Diethylpyrocarbonate LD Lethal dose
DMEM Dulbecco’s modified Eagle’s medium LEP Low egg passage (virus)
DMSO Dimethyl sulphide LPS Lipopolysaccharide
DTH Delayed-type hypersensitivity MAb Monoclonal antibody
EDTA Ethylene diamine tetra-acetic acid MAT Microscopic agglutination test
EGTA Ethylene glycol tetra-acetic acid MCS Master cell stock
EID Egg-infective dose MDBK Madin-Darby bovine kidney (cell line)
ELISA Enzyme-linked immunosorbent assay MDT Mean death time
EMTM Evans’ modified Tobie’s medium MEM Minimal essential medium
EYL Earle’s yeast lactalbumin (balanced salt MHC Major histocompatibility complex
solution)
FAT Fluorescent antibody test MLV Modified live virus (vaccine)
FAVN Fluorescent antibody virus neutralisation m.o.i. multiplicity of infection
1 American Type Culture Collection, P.O. Box 1549, Manassas, Virginia 20108, United States of America.
OIE Terrestrial Manual 2008 xv

Common abbreviations used in this Terrestrial Manual
MSV Master seed virus RBC Red blood cell

NI Neutralisation index RFLP Restriction fragment length polymorphism
OGP 1-octyl-beta-D-glucopyranoside (buffer) RK Rabbit kidney
OPD Orthophenyldiamine (chromogen) RPM Revolutions per minute
OPG Oxalase-phenol-glycerin (preservative RSA Rapid serum agglutination
solution)
ORF Open reading frame RT-PCR Reverse-transcription polymerase chain
reaction
PAGE Polyacrylamide gel electrophoresis SAT Serum agglutination test
PAP Peroxidase–antiperoxidase (staining SDS Sodium dodecyl sulphate
procedure)
PAS Periodic acid-Schiff (reaction) SPF Specific pathogen free
PBS Phosphate buffered saline SPG Sucrose phosphate glutamic acid
PCR Polymerase chain reaction SRBC Sheep red blood cells
PD Protective dose TCID50 Median tissue culture infective dose
PFGE Pulsed field gel electrophoresis TMB Tetramethyl benzidine
PFU Plaque-forming unit TSI Triple sugar iron (medium)
PHA Passive haemagglutination (test) VB Veronal buffer
PPD Purified protein derivative VBS Veronal buffered saline
PPLO Pleuropneumonia-like organisms Vero African green monkey kidney (cells)
PRN Plaque reduction neutralisation VN Virus neutralisation
PSG Phosphate buffered saline glucose
*
* *
xvi OIE Terrestrial Manual 2008

GLOSSARY OF TERMS
The definitions given below have been selected and restricted to those that are likely to be useful
to users of this OIE Terrestrial Manual.
• Absorbance/optical density
Absorbance and optical density are terms used to indicate the strength of reaction. A spectrophotometer is used
to measure the amount of light of a specific wave length that a sample absorbs and the absorbance is
proportional to the amount of a particular analyte present.
• Accuracy
Nearness of a test value to the expected value for a reference standard reagent of known activity or titre.
• Assay
Synonymous with test or test method, e.g. enzyme immunoassay, complement fixation test or polymerase chain
reaction tests.
• Batch
All vaccine or other reagent, such as antigen or antisera, derived from the same homogeneous bulk and
identified by a unique code number.
• Cell line
A stably transformed line of cells that has a high capacity for multiplication in vitro.
• Centrifugation
Throughout the text, the rate of centrifugation has been expressed as the Relative Centrifugal Force, denoted by
‘g’. The formula is:
(RPM × 0.10472)2 × Radius (cm) = g
980
where RPM is the rotor speed in revolutions per minute, and where Radius (cm) is the radius of the rotor arm, to
the bottom of the tube, in centimetres.
It may be necessary to calculate the RPM required to achieve a given value of g, with a particular rotor. The
formula is:
RPM = « g × 980 /Radius (cm)
0.10472
• Cross-reaction
See ’False-positive reaction’.
• Cut-off/threshold
Test result value selected for distinguishing between negative and positive results; may include indeterminate or
suspicious zone.
OIE Terrestrial Manual 2008 xvii

Glossary of terms
• Dilutions
Where dilutions are given for making up liquid reagents, they are expressed as, for example, 1 in 4 or 1/4,
meaning one part added to three parts, i.e. a 25% solution of A in B.
• v/v – This is volume to volume (two liquids).
• w/v – This is weight to volume (solid added to a liquid).
• Dilutions used in virus neutralisation tests

There are two different conventions used in expressing the dilution used in virus neutralisation (VN) tests. In
Europe, it is customary to express the dilution before the addition of the antigen, but in the United States of
America and elsewhere, it is usual to express dilutions after the addition of antigen.
These alternative conventions are expressed in the Terrestrial Manual as ‘initial dilution’ or ‘final dilution’,
respectively.
• Efficacy
Specific ability of the biological product to produce the result for which it is offered when used under the
conditions recommended by the manufacturer.
• Equivalency testing
Determination of certain assay performance characteristics of new and/or different test methods by means of an
interlaboratory comparison to a standard test method; implied in this definition is that participating laboratories
are using their own test methods, reagents and controls and that results are expressed qualitatively.
• False-negative reaction
Negative reactivity in an assay of a test sample obtained from an animal exposed to or infected with the organism
in question, may be due to lack of analytical sensitivity, restricted analytical specificity or analyte degradation,
decreases diagnostic sensitivity.
• False-positive reaction
Positive reactivity in an assay that is not attributable to exposure to or infection with the organism in question,
maybe due to immunological cross-reactivity, cross-contamination of the test sample or non-specific reactions,
decreases diagnostic specificity.
• Final product (lot)

All sealed final containers that have been filled from the same homogenous batch of vaccine in one working
session, freeze-dried together in one continuous operation (if applicable), sealed in one working session, and
identified by a unique code number.
• Harmonisation
The result of an agreement between laboratories to calibrate similar test methods, adjust diagnostic thresholds
and express test data in such a manner as to allow uniform interpretation of results between laboratories.
• Incidence
Estimate of the rate of new infections in a susceptible population over a defined period of time; not to be
confused with prevalence.
• In-house checks
All quality assurance activities within a laboratory directly related to the monitoring, validation, and maintenance
of assay performance and technical proficiency.
• In-process control
Test procedures carried out during manufacture of a biological product to ensure that the product will comply with
the agreed quality standards.
xviii OIE Terrestrial Manual 2008

Glossary of terms
• Inter-laboratory comparison (ring test)

Any evaluation of assay performance and/or laboratory competence in the testing of defined samples by two or
more laboratories; one laboratory may act as the reference in defining test sample attributes.
• Master cell (line, seed, stock)

Collection of aliquots of cells of defined passage level, for use in the preparation or testing of a biological
product, distributed into containers in a single operation, processed together and stored in such a manner as to
ensure uniformity and stability and to prevent contamination.
• Master seed (agent, strain)

Collection of aliquots of an organism at a specific passage level, from which all other seed passages are derived,
which are obtained from a single bulk, distributed into containers in a single operation and processed together
and stored in such a manner as to ensure uniformity and stability and to prevent contamination.
• Performance characteristic
An attribute of a test method that may include analytical sensitivity and specificity, accuracy and precision,
diagnostic sensitivity and specificity and/or repeatability and reproducibility.
• Potency
Relative strength of a biological product as determined by appropriate test methods. (Initially the potency is
measured using an efficacy test in animals. Later this may be correlated with tests of antigen content, or antibody
response, for routine batch potency tests.)
• Precision
The degree of dispersion of results for a repeatedly tested sample expressed by statistical methods such as
standard deviation or confidence limits.
• Predictive value (negative)

The probability that an animal is free from exposure or infection given that it tests negative; predictive values are
a function of the DSe (diagnostic sensitivity) and DSp (diagnostic specificity) of the diagnostic assay and the
prevalence of infection.
• Predictive value (positive)

The probability that an animal has been exposed or infected given that it tests positive; predictive values are a
function of the DSe and DSp of the diagnostic assay and the prevalence of infection.
• Prevalence
Estimate of the proportion of infected animals in a population at one given point in time; not to be confused with
incidence.
• Primary cells
A pool of original cells derived from normal tissue up to and including the tenth subculture.
• Production seed
An organism at a specified passage level that is used without further propagation for initiating preparation of a
production bulk.
• Proficiency testing
One measure of laboratory competence derived by means of an interlaboratory comparison; implied in this
definition is that participating laboratories are using the same test methods, reagents and controls and that
results are expressed qualitatively.
OIE Terrestrial Manual 2008 xix

Glossary of terms
• Purity
Quality of a biological product prepared to a final form and:
a) Relatively free from any extraneous microorganisms and extraneous material (organic or inorganic) as
determined by test methods appropriate to the product; and
b) Free from extraneous microorganisms or material which could adversely affect the safety, potency or
efficacy of the product.
• Reference animal
Any animal for which the infection status can be defined in unequivocal terms; may include diseased, infected,
vaccinated, immunised or naïve animals.
• Reference Laboratory
Laboratory of recognised scientific and diagnostic expertise for a particular animal disease and/or testing
methodology; includes capability for characterising and assigning values to reference reagents and samples.
• Repeatability
Level of agreement between replicates of a sample both within and between runs of the same test method in a
given laboratory.
• Reproducibility
Ability of a test method to provide consistent results when applied to aliquots of the same sample tested by the
same method in different laboratories.
• Room temperature
The term ‘room temperature’ is intended to imply the temperature of a comfortable working environment. Precise
limits for this cannot be set, but guiding figures are 18–25°C. Where a test specifies room temperature, this
should be achieved, with air conditioning if necessary; otherwise the test parameters may be affected.
• Safety
Freedom from properties causing undue local or systemic reactions when used as recommended or suggested
by the manufacturer and without known hazard to in-contact animals, humans and the environment.
• Sample
Material that is derived from a specimen and used for testing purposes.
• Sensitivity (analytical)
Synonymous with ‘Limit of Detection’, smallest detectable amount of analyte that can be measured with a defined
certainty; analyte may include antibodies, antigens, nucleic acids or live organisms.
• Sensitivity (diagnostic)
Proportion of known infected reference animals that test positive in the assay; infected animals that test negative
are considered to have false-negative results.
• Sensitivity (relative)
Proportion of reference animals defined as positive by one or a combination of test methods that also test
positive in the assay being compared.
• Specific pathogen free (SPF)

Animals that have been shown by the use of appropriate tests to be free from specified pathogenic
microorganisms, and also refers to eggs derived from SPF birds.
xx OIE Terrestrial Manual 2008

Glossary of terms
• Specificity (analytical)
Degree to which the assay distinguishes between the target analyte and other components in the sample matrix;
the higher the analytical specificity, the lower the level of false-positives.
• Specificity (diagnostic)
Proportion of known uninfected reference animals that test negative in the assay; uninfected reference animals
that test positive are considered to have false-positive results.
• Specificity (relative)
Proportion of reference animals defined as negative by one or a combination of test methods that also test
negative in the assay being compared.
• Specimen
Material submitted for testing.
• Standard Reagents
• International Standard Reagents

Standard reagents by which all other reagents and assays are calibrated; prepared and distributed by an
International Reference Laboratory.
• National Standard Reagents
Standard reagents calibrated by comparison with International Standard Reagents; prepared and
distributed by a National Reference Laboratory.
• Working Standards (reagents)
Standard reagents calibrated by comparison with the National Standard Reagent, or, in the absence of a
National Standard Reagent, calibrated against a well-characterised in-house standard reagent; included in
routine diagnostic tests as a control and/or for normalisation of test results.
• Sterility
Freedom from viable contaminating microorganisms, as demonstrated by approved and appropriate tests.
• Test method
Specified technical procedure for detection of an analyte (synonymous with assay).
• Tests
• Prescribed
Test methods that are required by the OIE Terrestrial Animal Health Code for the international movement of
animals and animal products and that are considered optimal for determining the health status of animals.
• Alternative
Test methods considered in this Terrestrial Manual to be suitable for the diagnosis of disease in a local
situation, and that can also be used for import/ export by bilateral agreement.
• Screening
Tests of high diagnostic sensitivity suitable for large-scale application.
• Confirmatory
Test methods of high diagnostic specificity that are used to confirm results, usually positive results, derived
from other test methods
• Working seed
Organism at a passage level between master seed and production seed.
OIE Terrestrial Manual 2008 xxi

CONTRIBUTORS
C O NT RIBUT O RS A ND P RO F E S S IO NA L A DDRE S S A T T IME O F W RIT ING
The chapters in the Terrestrial Manual are prepared by invited contributors. In accordance with OIE
standard procedure, all chapters are circulated to OIE Member Countries and to other experts in
the disease for comment. The OIE Biological Standards Commission and the Consultant Editor
then modify the text to take account of comments received. Once this review process is complete
and the text is finalised, the Terrestrial Manual is presented to the OIE International Committee
during its annual General Session for adoption before it is printed. The Terrestrial Manual is thus
deemed to be an OIE Standard Text that has come into being by international agreement. For this
reason, the names of the contributors are not shown on individual chapters but are listed below.
The Biological Standards Commission greatly appreciates the work of the following contributors:
1.1.1. Collection and shipment of diagnostic Dr J.E. Pearson

specimens 4016 Phoenix St., Ames, Iowa 50014, USA.
1.1.2. Biosafety and biosecurity in the veterinary Dr B. Schmitt

microbiology laboratory and animal facilities National Veterinary Services Laboratories,
Diagnostic Virology Laboratory, P.O. Box 844,
Ames, IA 50010, USA.
Dr P. Le Blanc Smith
Biocontainment Microbiologist, CSIRO Livestock
Industries, Australian Animal Health Laboratory
(AAHL), Private Bag 24, Geelong, Victoria 3220,
Australia.
1.1.3. Quality management in veterinary testing Dr A. Wiegers

laboratories USDA, APHIS, Veterinary Services, Center for
Veterinary Biologics, 510 South 17th. Street,
Suite 104, Ames, Iowa 50010, USA.
1.1.4. Principles of validation of diagnostic assays for Dr R.H. Jacobson

infectious diseases 27801 Skyridge Drive, Eugene, Oregon 97405,
USA.
Dr P. Wright
Aquatic Animal Health, Fisheries and Oceans
Canada, 343 University Avenue, Moncton,
New Brunswick, E1C 9B6, Canada.
1.1.5. Validation and quality control of polymerase Dr S. Belak & Dr P. Thorén

chain reaction methods for the diagnosis of Department of Virology, National Veterinary
infectious diseases Institute, Ulls väg 2B, SE-751 89 Uppsala,
Sweden.
1.1.6. Laboratory methodologies for bacterial Dr D. White

antimicrobial susceptibility testing Division of Animal and Food Microbiology,
National Antimicrobial Resistance Monitoring
System (NARMS), US Food and Drug
Administration, Center for Veterinary Medicine,
Office of Research, 8401 Muirkirk Road, Laurel,
MD 20708, USA.
xxii OIE Terrestrial Manual 2008

Contributors
1.1.7. Biotechnology in the diagnosis of infectious Dr D. Knowles & Dr H. Li

diseases and vaccine development Animal Disease Research Unit, ARS, USDA,
Washington State University, Pullman,
Washington 99164-6630, USA.
Prof. P.-P. Pastoret

World Organisation for Animal Health (OIE),
12 rue de Prony, 75017 Paris, France.
1.1.8. Principles of veterinary vaccine production Dr B. Rippke

Center for Veterinary Biologics, USDA, Animal
and Plant Health Inspection Service, Veterinary
Services, Suite 104, 510 South 17th Street,
Dr D.J.K. Mackay
European Medicines Agency, Veterinary
Medicines and Inspections, 7 Westferry Circus,
Canary Wharf, London E14 4HB, UK.
Dr M. Lombard
International Association for Biologicals (IABs),
22 Rue Crillon, 69006 Lyon, France.
1.1.9. Tests for sterility and freedom from Dr L. Elsken

contamination of biological materials USDA, APHIS, Center for Veterinary Biologics,
Suite 104, 510 South 17th Street, Ames, Iowa
50010, USA.
1.1.10. Guidelines for international standards for OIE Ad hoc Group on Antigen and Vaccine Banks
vaccine banks for Foot and Mouth Disease
1.1.11. The role of international bodies in the Dr Ph. Vannier

regulation of veterinary biologicals AFSSA Ploufragan, Laboratoire d’études et de
recherches avicoles et porcines, Zoopôle des
Côtes d’Armor-Les Croix, BP 53, 22440
Ploufragan, France.
Dr R. Hill
Center for Veterinary Biologics, USDA, APHIS,
Veterinary Services, P.O. Box 844, Ames
Iowa 50010, USA.
Dr O. Itoh
National Veterinary Assay Laboratory, JMAFF,
1-15-1 Tokura, Kokubunji, Tokyo 185-8511,
Japan.
Dr P. Dehaumont
AFSSA Fougères, Agence nationale du
médicament vétérinaire, B.P. 203, 35302
Fougères Cedex, France.
2.1.1. Anthrax Dr P.R. Coker

Pathogen Research & Consulting, LLC,
Shreveport, Louisiana 71104, USA.
OIE Terrestrial Manual 2008 xxiii

Contributors
2.1.2. Aujeszky’s disease Prof. B. Toma (retired) & Prof. N. Haddad

École nationale vétérinaire d’Alfort, 7 avenue du
Général de Gaulle, 94704 Maisons-Alfort Cedex,
France.
Dr Ph. Vannier
AFSSA Ploufragan, Laboratoire d’études et de
recherches avicoles et porcines, Zoopôle des
Côtes d’Armor-Les Croix, BP 53, 22440
Ploufragan, France.
2.1.3. Bluetongue Dr P. Daniels

Australian Animal Health Laboratory, CSIRO
Livestock Industries, 5 Portarlington Road,
Geelong, Victoria 3220, Australia.
2.1.4. Echinococcosis/Hydatidosis Dr M. Kamiya

Laboratory of Environmental Zoology, Department
of Biosphere and Environmental Sciences,
Faculty of Environmental Systems, Rakuno
Gakuen University, Midori-machi 582,Ebetsu 069-
8501, Hokkaido, Japan.
2.1.5. Foot and mouth disease Dr R.P. Kitching

National Centre for Foreign Animal Disease,
1015 Arlington Street, Winnipeg, Manitoba
R3E 3M4, Canada.
Dr P.V. Barnett & Dr D. Paton

Institute for Animal Health, Pirbright Laboratory,
Ash Road, Pirbright, Surrey GU24 0NF, UK.
Dr D. Mackay
European Medicines Agency, Veterinary
Medicines and Inspections, 7 Westferry Circus,
Canary Wharf, London E14 4HB, UK.
2.1.6. Heartwater Dr D. Martinez

CIRAD-EMVT, Campus International de
Baillarguet - TA30/G, 34398 Montpellier Cedex 5,
France.
Dr N. Vachiéry
CIRAD-EMVT, Domaine de Duclos, Prise d'Eau,
97170 Petit-Bourg, Guadeloupe.
Prof. F. Jongejan
Department of Parasitology & Tropical Veterinary
Medicine, Faculty of Veterinary Medicine, Utrecht
University, P.O. Box 80.165, 3508 TD Utrecht,
The Netherlands
AND
Department of Veterinary Tropical Diseases,
Faculty of Veterinary Science, University of
Pretoria, Onderstepoort 0110, South Africa.
2.1.7. Japanese encephalitis Dr T. Kondo

Epizootic Research Center, Equine Research
Institute, Japan Racing Association, 1400-4
Shiba, Shimotsuke, Tochigi 329-0412, Japan.
xxiv OIE Terrestrial Manual 2008

Contributors
2.1.8. Leishmaniosis Dr L. Gradoni & Dr M. Gramiccia

Dipartimento di Malattie Infettive, Parassitarie ed
Immunomediate, Istituto Superiore di Sanità,
Viale Regina Elena 299, I-00161 Rome, Italy.
2.1.9. Leptospirosis Prof. C.A. Bolin

Diagnostic Center for Population & Animal Health,
College of Veterinary Medicine, Michigan State
University, 4125 Beaumont Rd, Lansing, Michigan
48910, USA.
2.1.10. New World screwworm (Cochliomyia Dr M.J.R. Hall

hominivorax) and Old World screwworm Department of Entomology, The Natural History
(Chrysomya bezziana) Museum, Cromwell Road, London SW7 5BD, UK.
2.1.11. Paratuberculosis (Johne’s disease) Dr J. Gwozdz

Department of Primary Industries, Victoria,
475 Mickleham Road, Attwood, VIC 3049,
Australia.
2.1.12. Q fever Dr E. Rousset, Dr V. Duquesne, Dr P. Russo &

M.F. Aubert
AFSSA Sophia Antipolis, Laboratoire d’Études et
de Recherches sur les Petits Ruminants et les
Abeilles (LERPRA), Les Templiers, 105 route des
Chappes, BP 111, 06902 Sophia Antipolis Cedex,
France.
2.1.13. Rabies Dr F. Cliquet & Dr J. Barrat

AFSSA Site de Nancy, Technopôle Agricole et
Vétérinaire, B.P. 40009, 54220 Malzeville,
France.
2.1.14. Rift Valley fever Dr G.H. Gerdes

Onderstepoort Veterinary Institute, Private Bag
X05, Onderstepoort 0110, South Africa.
2.1.15. Rinderpest Dr W.P. Taylor

16 Mill Road, Angmering, Littlehampton, West
Sussex BN16 4HT, UK.
Dr P. Roeder
Hollyhedge Cottage, Spats Lane, Headley Down,
Bordon, Hampshire GU35 8SY, UK.
2.1.16. Trichinellosis Dr A. Gajadhar & Dr L. Forbes

Canadian Food Inspection Agency, Centre for
Foodborne & Animal Parasitology, 116 Veterinary
Road, Saskatoon, Saskatchewan S7N 2R3
Canada.
2.1.17. Trypanosoma evansi infections Dr A.G. Luckins

(including surra) Centre for Tropical Veterinary Medicine, Easter
Bush Veterinary Centre, Roslin, Midlothian,
Scotland EH25 9RG, UK.
2.1.18. Tularemia Dr T. Mörner

Department of Pathology and Wildlife Diseases,
Swedish National Veterinary Institute, Sweden.
2.1.19. Vesicular stomatitis Dr S.L. Swenson

USDA, APHIS, National Veterinary Services
Laboratories, P.O. Box 844, Ames, Iowa 50010,
USA.
OIE Terrestrial Manual 2008 xxv

Contributors
2.1.20. West Nile fever Dr E.N. Ostlund

United States of America.
2.2.1. Acarapisosis of honey bees Dr W. Ritter

CVUA-Freiberg, FB: Bienen (bee team), Am
Moosweiher 2, D79018 Freiberg, Germany.
2.2.2. American foulbrood of honey bees Dr D.C. de Graaf

2.2.3. European foulbrood of honey bees Laboratory of Zoophysiology, University of Ghent,
K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
2.2.4. Nosemosis of honey bees Dr W. Ritter

CVUA-Freiberg, Animal Health, Am Moosweiher
2, D79018 Freiberg, Germany.
2.2.5. Small hive beetle infestation (Aethina tumida) Dr W. Ritter

CVUA-Freiberg, Animal Health, Am Moosweiher
2, D79018 Freiberg, Germany.
Dr P. Neumann
Swiss Bee Research Centre, Agroscope
Liebefeld-Posieux, Research Station ALP,
Schwarzenburgstrasse 161, CH-3003 Bern,
Switzerland.
Dr J.D. Ellis
Department of Entomology, The University of
Georgia, Athens, GA 30602, USA.
2.2.6. Tropilaelaps infestation of honey bees Dr W. Ritter

(Tropilaelaps spp.) CVUA-Freiberg, FB: Bienen (bee team), Am
2.2.7. Varroosis of honey bees Moosweiher 2, D79018 Freiberg, Germany.
2.3.1. Avian chlamydiosis Dr A.A. Andersen (retired)

Formerly USDA, ARS, National Animal Disease
Center, P.O. Box 70, Ames, Iowa 50010, USA.
2.3.2. Avian infectious bronchitis Dr J. Gelb

Dept of Animal and Food Sciences and the Avian
Biosciences Center, University of Delaware,
531 South College Avenue, Newark, Delaware
19716-2150, USA.
2.3.3. Avian infectious laryngotracheitis Dr R.C. Jones

Department of Veterinary Pathology, University of
Liverpool, Jordan Building, Veterinary Field
Station, ‘Leahurst’, Neston, South Wirral
CH64 7TE, UK.
2.3.4. Avian influenza Dr D.J. Alexander

VLA Weybridge, New Haw, Addlestone,
Surrey KT15 3NB, UK.
xxvi OIE Terrestrial Manual 2008

Contributors
2.3.5. Avian mycoplasmosis Dr S.H. Kleven

(Mycoplasma gallisepticum, M. synoviae) University of Georgia, Poultry Diagnostic and
Research Center, 953 College Stantion Road,
Athens, Georgia 30602-4875, USA.
Dr J.M. Bradbury
University of Liverpool, Department of Veterinary
Pathology, Veterinary Teaching Hospital,
Leahurst, Neston H64 7TE, UK.
2.3.6. Avian tuberculosis Dr D.V. Cousins

Australian Reference Laboratory for Bovine
Tuberculosis, Western Australia Dept of
Agriculture and Food, Locked Bag N° 4, Bentley
Delivery Centre, Bentley WA 6983, Australia.
2.3.7. Duck virus enteritis Dr P.R. Woolcock

2.3.8. Duck virus hepatitis California Animal Health and Food Safety,
University of California, Davis, 2789 South
Orange Avenue, Fresno, California 93725, USA.
2.3.9. Fowl cholera Dr R. Kunkle,

National Animal Disease Center, P.O. Box 70,
Ames, Iowa 50010, USA.
Dr M.A. Wilson
National Animal Disease Center, 1800 N. Dayton
Avenue, Ames, Iowa 50010, USA.
2.3.10. Fowl pox Dr D.N. Tripathy

University of Illinois at Urbana-Champaign,
College of Veterinary Medicine, Department of
Veterinary Pathbiology, 2001 South Lincoln
Avenue, Urbana, Illinois 61802, USA.
2.3.11. Fowl typhoid and Pullorum disease Dr R. Davies

VLA Weybridge, New Haw, Addlestone, Surrey
KT15 3NB, UK.
2.3.12. Infectious bursal disease (Gumboro disease) Dr N. Eterradossi

AFSSA-site de Ploufragan/Brest, Laboratoire
d’Etudes et de Recherches Avicoles, Porcines et
Piscicoles (LERAPP), Unité de virologie,
immunologie et parasitologie aviaires et cunicoles
(VIPAC), BP 53, 22440 Ploufragan, France.
2.3.13. Marek’s disease Dr V.K. Nair

Institute for Animal Health, Compton Laboratory,
Compton, Berkshire RG20 7NN, UK.
2.3.14. Newcastle disease Dr D.J. Alexander

2.3.15. Turkey rhinopneumonitis (avian Dr J. Pedersen

metapneumovirus) National Veterinary Services Laboratories,
Diagnostic Virology Laboratory, P.O. Box 844,
Dr R. Gough
OIE Terrestrial Manual 2008 xxvii

Contributors
2.4.1. Bovine anaplasmosis Prof. T.F. McElwain

Animal Health Research Center, College of
Veterinary Medicine, 155N Bustad Hall, P.O. Box
647034, Pullman, WA 99164-7034, USA.
2.4.2. Bovine babesiosis Dr R.E. Bock

Queensland Department of Primary Industries,
Animal and Plant Health Service, Tick Fever
Research Centre, 280 Grindle Road, Wacol,
Queensland 4076, Australia.
Dr W.K. Jorgensen & Mr J.B. Molloy

Queensland Department of Primary Industries,
Delivery, Emerging Technologies, 665 Fairfield
Rd, Yeerongpilly, Queensland 4105, Australia.
2.4.3. Bovine brucellosis Dr K. Nielsen

Canadian Food Inspection Agency, Animal
Diseases Research Institute, P.O. Box 11300,
Station H, Nepean, Ontario K2H 8P9, Canada.
Dr D.R. Ewalt
Pathobiology Laboratory, National Veterinary
Services Laboratories, 1800 Dayton Road, Ames,
Iowa 50010, USA.
2.4.4. Bovine cysticercosis (see chapter 2.9.5) Dr S. Lloyd

Department of Clinical Veterinary Medicine,
University of Cambridge, Madingley Road,
Cambridge CB3 0ES, UK.
2.4.5. Bovine genital campylobacteriosis Prof. J.A. Wagenaar

Utrecht University, Faculty of Veterinary Medicine,
P.O. Box 80.165, 3508 TD Utrecht, The
Netherlands.
Dr M.A.P. Van Bergen

Central Veterinary Institute of Wageningen UR,
P.O. Box 65, 8200 AB Lelystad, The Netherlands.
2.4.6. Bovine spongiform encephalopathy Dr D. Matthews, Dr M.M. Simmons, Mr M. Stack &

Prof. G.A.H. Wells
2.4.7. Bovine tuberculosis Dr D.V. Cousins

Australian Reference Laboratory for Bovine
Tuberculosis, Western Australia Dept of
Agriculture and Food, Locked Bag N° 4, Bentley
Delivery Centre, Bentley WA 6983, Australia.
2.4.8. Bovine viral diarrhoea Dr T. Drew

KT15 3NB, UK.
2.4.9. Contagious bovine pleuropneumonia Dr F. Thiaucourt

CIRAD-EMVT, Campus international de
Baillarguet, Montferriez-sur-Lez, B.P. 5035,
34032 Montpellier Cedex 1, France.
2.4.10. Dermatophilosis Dr D. Martinez

CIRAD, Campus International de Baillarguet –
TA-A15 / G, 34398 Montpellier Cedex 5, France.
xxviii OIE Terrestrial Manual 2008

Contributors
2.4.11. Enzootic bovine leukosis Dr D. Beier (retired)

Bundesforschungsanstalt für Viruskrankheiten der
Tiere, Institute für epidemiologische Diagnostik,
Seestrasse 55, 16868 Wusterhausen/Dosse
Germany.
Dr T.W. Vahlenkamp
Friedrich-Loeffler-Institut, Südufer 10,
17493 Greifswald-Insel, Germany.
2.4.12. Haemorrhagic septicaemia Dr S.K. Srivastava, Dr A.A. Kumar,

Dr P. Chaudhuri & Dr M.P. Yadav
Indian Veterinary Research Institute, Izatnagar
243122 U.P., India
2.4.13. Infectious bovine rhinotracheitis/ Dr J.A. Kramps

infectious pustular vulvovaginitis Central Institute for Animal Disease Control
Lelystad (CIDC-Lelystad), P.O. Box 2004, 8203
AA Lelystad, The Netherlands.
2.4.14. Lumpy skin disease Dr R.P. Kitching

R3E 3M4, Canada.
Dr V. Carn
Brewishay, Main Street, Barton St David, Dorset
DT9 6QD, UK.
2.4.15. Malignant catarrhal fever Dr H.W. Reid

Moredun Research Institute, International
Research Centre, Pentlands Science Park, Bush
Loan, Penicuik EH26 0PZ, Scotland, UK.
2.4.16. Theileriosis Prof. E. Pipano

Koret School of Veterinary Medicine, The Hebrew
University of Jerusalem, P.O. Box 12 Rehovot,
Israel.
Dr S. Morzaria
FAO Regional Office for Asia and the Pacific,
39 Phra Athit Road, Bangkok 10200, Thailand.
Dr P. Spooner
International Livestock Research Institute,
Naivasha Road, Nairobi 00100, Kenya.
2.4.17. Trichomonosis Dr A.A. Gajadhar

Centre for Food-borne and Animal Parasitology,
Canadian Food Inspection Agency,
116 Veterinary Road, Saskatoon,
Saskatchewan S7N 2R3, Canada.
Dr S. Parker
Large Animal Clinical Sciences, Western College
of Veterinary Medicine,52 Campus Drive,
Saskatoon, Saskatchewan S7N 5B4, Canada.
Prof. M. Taylor
Veterinary Surveillance, Central Science
Laboratory, Sand Hutton, York YO41 1LZ, UK.
OIE Terrestrial Manual 2008 xxix

Contributors
2.4.18. Trypanosomosis (Tsetse-transmitted) Dr M. Desquesnes

Cirad-Bios, UMR177-Trypanosomes, Campus
international de Baillarguet, TA A-17 / G, 34398
Montpellier Cedex 5, France.
2.5.1. African horse sickness Prof. J.M. Sánchez-Vizcaíno

Catedrático del Área de Sanidad Animal,
Universidad Complutense, Facultad de
Veterinaria, Avda. Puerta de Hierro s/n, 28040
Madrid, Spain.
2.5.2. Contagious equine metritis Dr P. Heath (retired)

VLA Bury St Edmunds, Rougham Hill, Bury St
Edmunds, UK
Dr P.J. Timoney
Maxwell H. Gluck Equine Research Center, Dept
of Veterinary Science, University of Kentucky,
108 Gluck Equine Research Center, Lexington,
Kentucky 40546-0099, USA
2.5.3. Dourine Dr J.B. Katz

USA.
2.5.4. Epizootic lymphangitis Dr J. Picard

Department of Veterinary Tropical Diseases,
Faculty of Veterinary Science, University of
Pretoria, Private Bag X04, Onderstepoort 0110,
South Africa.
2.5.5. Equine encephalomyelitis Dr E.N. Ostlund

(Eastern and Western) USDA, APHIS, National Veterinary Services
2.5.6. Equine infectious anaemia Laboratories, P.O. Box 844, Ames, Iowa 50010,
USA.
2.5.7. Equine influenza Dr J. Daly (formerly)

Animal Health Trust, Lanwades Park, Kentford,
Newmarket, Suffolk CB8 7UU, UK.
Dr J.A. Mumford
Cambridge Infectious Diseases Consortium,
Department of Veterinary Medicine, Madingley
Road, Cambridge CB3 0ES, UK.
2.5.8. Equine piroplasmosis Dr T. de Waal

University College Dublin, School of Agriculture,
Food Science and Veterinary Medicine,
Veterinary Sciences Centre, Belfield, Dublin 4,
Ireland.
2.5.9. Equine rhinopneumonitis Dr G.P. Allen

Department of Veterinary Science,
College of Agriculture, University of Kentucky,
108 M.H. Gluck Equine Research Center,
Lexington, Kentucky 40546-0099, USA.
2.5.10. Equine viral arteritis Dr P.J. Timoney

University of Kentucky, Department of Veterinary
Science, 108 Gluck Equine Research Center,
Lexington, Kentucky 40546-0099, USA.
xxx OIE Terrestrial Manual 2008

Contributors
2.5.11. Glanders Dr H. Neubauer

Friedrich-Loeffler Institut, Institut für Bakterielle
Infektionen und Zoonosen, Naumburger Strasse
96a, 07743 Jena, Germany.
2.5.13. Horse mange (see chapter 2.9.8) Dr J.L. Schlater & Dr J.W. Mertins
Parasitology and Clinical Pathology Section,
Services Laboratories, USDA, APHIS, VS, P.O.
Box 844, Ames, Iowa 50010, USA.
2.5.14. Venezuelan equine encephalomyelitis Dr E.N. Ostlund

USA.
2.6.1. Myxomatosis Prof. S. Bertagnoli

École Nationale Vétérinaire, 23 Chemin de
Capelles, BP 87614, 31076 Toulouse Cedex 03,
France.
2.6.2. Rabbit haemorrhagic disease Dr A. Lavazza & Dr L. Capucci

Istituto Zooprofilattico Sperimentale della
Lombardia e dell’Emilia Romagna, Via Bianchi
7/9, 25124 Brescia, Italy.
2.7.1. Border disease Dr P.F. Nettleton & Dr K. Willoughby

Research Centre, Pentlands Science Park, Bush
Loan, Penicuik EH26 0PZ, Scotland, UK.
2.7.2. Caprine and ovine brucellosis (excluding Dr B. Garin-Bastuji

Brucella ovis) EU Community/OIE & FAO Reference Laboratory
for Brucellosis, Unité Zoonoses Bactériennes,
AFSSA, 94706 Maisons-Alfort Cedex, France.
Dr J.M. Blasco
Centro de Investigación y Tecnología
Agroalimentaria de Aragón (CITAA), Apartado
727, 50080 Zaragoza, Spain.
2.7.3/4. Caprine arthritis/encephalitis & Maedi-visna Dr D. Knowles & Dr L.M. Herrmann

USDA- ARS, Animal Disease Research Unit,
3003 ADBF, Washington State University,
Pullman, Washington 99164-6630, USA.
2.7.5. Contagious agalactia Dr R. Nicholas

Dr G.R. Loria
Istituto Zooprofilattico Sperimentale della Sicilia,
Palermo, Italy.
2.7.6. Contagious caprine pleuropneumonia Dr F. Thiaucourt

CIRAD-EMVT, Campus international de
Baillarguet, Montferriez-sur-Lez, B.P. 5035,
34032 Montpellier Cedex 1, France.
2.7.7. Enzootic abortion of ewes (ovine chlamydiosis) Dr D. Longbottom

Research Centre, Pentlands Science Park
Bush Loan, Penicuik EH26 0PZ, UK.
OIE Terrestrial Manual 2008 xxxi

Contributors
2.7.8. Nairobi sheep disease (see chapter 2.9.1) Dr G.H. Gerdes

Onderstepoort Veterinary Institute, Private Bag
2.7.9. Ovine epididymitis (Brucella ovis) Dr B. Garin-Bastuji

EU Community/OIE & FAO Reference Laboratory
for Brucellosis, Unité Zoonoses Bactériennes,
AFSSA, 94706 Maisons-Alfort Cedex, France.
Dr J.M. Blasco
Centro de Investigación y Tecnología
Agroalimentaria de Aragón (CITAA), Apartado
727, 50080 Zaragoza, Spain.
2.7.10. Ovine pulmonary adenocarcinoma Dr M.J. Sharp

(adenomatosis) VLA, Lasswade Laboratory, Pentlands Science
Park, Bush Loan, Penicuik EH26 0PZ, Scotland,
UK.
2.7.11. Peste des petits ruminants Dr A. Diallo

Animal Health Service, Food and Agriculture
Organization of the United Nations Viale delle
Terme di Caracalla, 00153 Rome, Italy.
2.7.12. Salmonellosis (S. abortusovis) Dr R. Davies

(see chapter 2.9.9) VLA Weybridge, New Haw, Addlestone, Surrey
KT15 3NB, UK.
2.7.13. Scrapie Dr D. Matthews, Dr M.M. Simmons, Mr M. Stack&

Prof. G.A.H. Wells
KT15 3NB, UK.
2.7.14. Sheep pox and goat pox Dr R.P. Kitching

R3E 3M4, Canada.
Dr V. Carn
Brewishay, Main Street, Barton St David, Dorset
DT9 6QD, UK.
2.8.1. African swine fever Dr C.A.L. Oura

Dr M. Arias
Centro de Investigación en Sanidad Animal
(CISA-INIA), Valdeolmos, 28130 Madrid, Spain.
2.8.2. Atrophic rhinitis of swine Dr K.B. Register

USDA, ARS, National Animal Disease Center,
2300 Dayton Avenue, Ames, Iowa 50010, USA.
2.8.3. Classical swine fever (hog cholera) Dr T. Drew

KT15 3NB, UK.
xxxii OIE Terrestrial Manual 2008

Contributors
2.8.4. Nipah virus encephalitis (see chapter 2.9.6) Dr P. Daniels

Dr M. Narasiman
Veterinary Research Institute, 59, Jalan Sultan
Azlan Shah, 31400 Ipoh, Perak, Malaysia.
2.8.5. Porcine brucellosis Dr S. Olsen,

USDA, ARS, National Animal Disease Center,
2300 Dayton Avenue, Ames, Iowa 50010, USA.
2.8.6. Porcine cysticercosis (see chapter 2.9.5) Dr S. Lloyd

2.8.7. Porcine reproductive and respiratory syndrome Dr L.R. Ludemann

USDA, APHIS, Center for Veterinary Biologics,
Laboratory, P.O. Box 844, Ames, Iowa 50010,
USA.
Dr K.M. Lager
Virus and Prion Diseases of Livestock Research
Unit, National Animal Disease Center, USDA-
ARS, Ames, Iowa 50010, USA.
2.8.8. Swine influenza Dr S.L. Swenson

National Veterinary Services Laboratories, P.O.
Dr P.L. Foley
Center for Veterinary Biologics, 510 S. 17th St.,
Suite 104, Ames, IA 50010 USA
Dr C.W. Olsen
Department of Pathobiological Sciences, School
of Veterinary Medicine, University of Wisconsin-
Madison,2015 Linden Drive, Madison, WI 53706,
USA
2.8.9. Swine vesicular disease Dr R.P. Kitching

R3E 3M4, Canada.
Dr D. Paton
Dr A.I. Donaldson,
290 London Road, Burpham, Guildford, Surrey
GU4 7LB, UK.
2.8.10. Teschovirus encephalomyelitis (previously Mr N. Knowles

enterovirus encephalomyelitis or Institute for Animal Health, Pirbright Laboratory,
Teschen/Talfan disease) Ash Road, Pirbright, Woking, Surrey GU24 0NF,
UK.
OIE Terrestrial Manual 2008 xxxiii

Contributors
2.8.11. Transmissible gastroenteritis Dr L.J. Saif

The Ohio State Universtiy, Ohio Agricultural
Research and Development Center, Food Animal
Health Research Program, 1680 Madison
Avenue, Wooster, Ohio 44691-4096, USA.
2.9.1. Bunyaviral diseases of animals (excluding Dr G.H. Gerdes

Rift Valley fever) Onderstepoort Veterinary Institute, Private Bag
2.9.2. Camelpox Dr H. Elliott

International Animal Health Division , DEFRA,
1A Page Street, London SW1P 4PQ, UK.
Dr E. Tuppurainen
2.9.3. Campylobacter jejuni and C. coli Prof. J.A. Wagenaar

Utrecht University, Faculty of Veterinary Medicine,
P.O. Box 80.165, 3508 TD Utrecht, The
Netherlands.
Dr W.F. Jacobs-Reitsma
RIKILT Institute of Food Safety, Wageningen-UR
P.O. Box 230, 6700 AE Wageningen, The
Netherlands.
2.9.4. Cryptosporidiosis Prof. H. Smith

Scottish Parasite Diagnostic Laboratory, Stobhill
General Hospital, Glasgow G21 3UW, UK.
2.9.5. Cysticercosis Dr S. Lloyd

2.9.6. Hendra and Nipah virus diseases Dr P. Daniels

Dr M. Narasiman
Veterinary Research Institute, 59, Jalan Sultan
Azlan Shah, 31400 Ipoh, Perak, Malaysia.
2.9.7. Listeria monocytogenes Dr J. Lopez

Canadian Food Inspection Agency,
R3E 3M4, Canada.
2.9.8. Mange Dr J.L. Schlater & Dr J.W. Mertins

Parasitology and Clinical Pathology Section,
Services Laboratories, USDA, APHIS, VS, P.O.
2.9.9. Salmonellosis Dr R. Davies

KT15 3NB, UK.
xxxiv OIE Terrestrial Manual 2008

Contributors
2.9.10. Toxoplasmosis Dr D. Buxton & Dr S.W. Maley

Moredun Research Institute, Pentlands Science
Park, Bush Loan, by Edinburgh EH26 0PZ,
Scotland, UK.
2.9.11. Verocytotoxigenic Escherichia coli Dr F.A. Clifton-Hadley

KT15 3NB, UK.
2.9.12. Zoonoses transmissible from non-human FELASA Working Group on Non-Human Primate
primates Health: H. Weber (Convenor), E. Berge, J. Finch,
P. Heidt, F.-J. Kaup, G. Perretta, B. .Verschuere &
S. Wolfensohn
FELASA, BCM Box 2989, London WC1N 3XX,
UK.
OIE Terrestrial Manual 2008 xxxv

PART 1
GENERAL INFORMATION
OIE Terrestrial Manual 2008 1

SECTION 1.1.
INTRODUCTORY CHAPTERS
CHAPTER 1.1.1.
COLLECTION AND SHIPMENT

OF DIAGNOSTIC SPECIMENS
INTRODUCTION
The starting point for the laboratory investigation of an animal disease is the taking of samples. This
first introductory chapter considers some of the general principles involved in sample collection,
submission and storage. Each of the disease chapters of this Terrestrial Manual provides specific
information on sample collection for that particular disease. Samples may be taken from animals or
the environment for a variety of purposes, such as disease diagnosis, disease surveillance, health
certification or monitoring the response to treatment or vaccination. The samples collected should
be appropriate for the intended purpose, and adequate in number and amount to provide
statistically valid results. Diagnostic laboratories require the submission of appropriate samples that
arrive at the laboratory in good condition. For disease diagnosis, the tissues sampled should be
representative of the condition being investigated and the lesions observed. Samples should be
taken with care, to avoid undue stress or injury to the animal or danger to the operator. Where
appropriate, samples should be collected aseptically, and care should be taken to avoid cross-
contamination between samples.
The samples should be carefully packaged, labelled, and transmitted to the laboratory by the
fastest practicable method, with the appropriate temperature control. There are specific
requirements for the packaging and shipping of infectious substances, including diagnostic
specimens that must be followed. If material is sent to a laboratory in another country, this
laboratory should be consulted in advance to ensure that it is willing to receive the material and to
obtain the appropriate import licence. All samples should be accompanied by a letter or submission
form, which includes the name and address of the submitter, the origin of the material, the relevant
history, animal identification and corresponding specimens, and the tests requested.
A. COLLECTION OF SAMPLES
Before taking samples, careful consideration should be given to the purpose for which they are required. This will
determine the type and number of samples needed to provide valid results. When samples are taken from live
animals, care should be taken to avoid injury or distress to the animal or danger to the operator and attendants. It
may be necessary to use mechanical restraint, tranquillisation or anaesthesia. Whenever handling biological
material, from either live or dead animals, the risk of zoonotic disease should be kept in mind and precautions
taken to avoid human infection (see also Chapter 1.1.2 Biosafety and biosecurity in the veterinary microbiology
laboratory and animal facilities). Post-mortem examinations should be carried out under as aseptic conditions as
is practicable. Care should be taken to avoid environmental contamination, or risk of spread of disease through
insects or fomites. Arrangements should be made for appropriate safe disposal of animals and tissues.
Considerable skill and care are required to decide on the correct samples to be sent to the laboratory. The
samples collected should be representative of the condition being investigated and the lesions observed. Also the
stage of the disease and lesion development should be considered, as well as the type of test(s) that will be
performed. Frequently, a combination of blood samples for serology and tissues from dead or culled animals for

Chapter 1.1.1. — Collection and shipment of diagnostic specimens
microbiological culture and pathological examination will be required. Recommendations for transport are
described later in this chapter.
The disease chapters in this Terrestrial Manual provide guidance on samples that should be collected so that
information will not be repeated here. In addition, procedures for sample collection and submission have been
prepared by national and international authorities (3, 5, 9, 11, 12). These publications provide detailed
recommendations of specific samples that should be collected from different species and for a wide variety of
suspected diseases. They also provide information on post-mortem procedures, lists of appropriate media, and
instructions on submission of samples. The laboratory that is going to perform the assay(s) should be contacted if
there are specific questions concerning the type of sample that should be collected.
1. Sample collection from live animals
a) Blood
Blood samples may be taken for haematology or for culture and/or direct examination for bacteria, viruses,
or protozoa, in which case it is usual to use anticoagulants, such as ethylene diamine tetra-acetic acid
(EDTA) or heparin. They may also be taken for serology, which requires a clotted sample. Blood plasma is
also used for some procedures. A blood sample is taken, as cleanly as possible, by venipuncture. In most
large mammals, the jugular vein or a caudal vein is selected, but brachial veins and mammary veins are also
used. Vena cava veins are also used in pigs. In birds, a wing vein (brachial vein) is usually selected. For
techniques for sampling small laboratory animals, see refs 1 and 6. Blood may be taken by syringe and
needle or by needle and vacuum tube (not easy in delicate veins but convenient in strong veins). Small
quantities of blood are conveniently obtained by pricking with a triangular, solid-pointed needle. Ideally the
skin at the site of venipuncture should first be shaved (plucked) and swabbed with 70% alcohol and allowed
to dry.
For samples that are collected with anticoagulant, thorough mixing, using gentle agitation only, is necessary
as soon as the sample has been taken. It may also be necessary to make a smear of fresh blood on a
microscope slide; both thick and thin smears may be prepared. For polymerase chain reactions, EDTA is the
preferred anticoagulant. For serum samples, the blood should be left to stand at ambient temperature (but
protected from excessive heat or cold) for 1–2 hours until the clot begins to contract. The clot can then be
ringed round with a sterile rod and the bottles placed in a refrigerator at 4°C. After several hours, or
overnight, the sample can be centrifuged at about 1000 g for 10–15 minutes and the serum can be decanted
or removed with a pipette. In order to establish the significance of antibody titres, paired serum samples will
often need to be collected 7–14 days apart. An alternative method for collecting and transporting blood that
is to be used for serology is to place a drop of blood on to filter paper, the blood is dried at room temperature
and the sample can then be shipped unrefrigerated. Contact the laboratory to enquire if this method of
collection is validated for the required tests.
b) Faeces
At least 10 g of freshly voided faeces should be selected. Faeces for parasitology should fill the container
and be sent to arrive at the laboratory within 24 hours. If transport times are likely to be longer than
24 hours, the sample should be sent on ice or refrigerated to prevent the hatching of parasite eggs. Screw
top containers or sterile plastic bags should be used for shipment; avoid tubes with rubber stoppers as gas
generated can result in blowing the stopper off the tube, ruining the integrity of the sample and
contaminating other samples in the package. An alternative and sometimes preferable method is to take
swabs from the rectum (or cloaca), taking care to swab the mucosal surface. The swabs should be visibly
coated with faecal material; however, samples collected with a swab are inadequate for parasitology. Care
should be taken when collecting swabs from small, delicate animals or birds to avoid injury to the animal;
small swabs are commercially available that should be used. Swabs should be transported in appropriate
transport medium. Faeces are best stored and transported at 4°C.
c) Skin
In diseases producing vesicular lesions, collect, if possible, 2 g of affected epithelial tissue as aseptically as
possible and place it in 5 ml phosphate buffered glycerine or Tris-buffered tryptose broth virus transport
medium at pH 7.6. Additionally, the vesicular fluid should be sampled where unruptured vesicles are
present; if possible, vesicular fluid should be aspirated with a syringe and placed in a separate sterile tube.
Plucked hair or wool samples are useful for surface-feeding mites, lice and fungal infections. Deep skin
scrapings, using the edge of a scalpel blade, are useful for burrowing mites and, in birds, feather tips can be
taken for detection of viral antigen where Marek’s disease is suspected.
d) Genital tract and semen

Samples may be taken by vaginal or preputial washing, or by the use of suitable swabs. The cervix or
urethra may be sampled by swabbing. Samples of semen are best obtained using an artificial vagina or by
4 OIE Terrestrial Manual 2008

extrusion of the penis and artificial stimulation. The sperm-rich fraction should be present in the sample and
contamination by antiseptic washing solutions should be avoided. Specific transport media and conditions
are often required.
e) Eye
A sample from the conjunctiva can be taken by holding the palpebra apart and gently swabbing the surface.
The swab is then put into transport medium. Scrapings may also be taken on to a microscope slide. The
handles of metal-handled swabs are useful for this, to ensure that sufficient cells are removed for
microscopic examination. Mucopurulent nasal and lacrimal discharges are rarely useful.
f) Nasal discharge (saliva, tears)

Samples may be taken with dacron, cotton or gauze swabs, preferably on wire handles as wood is inflexible
and may snap. It may be helpful if the swab is first moistened with transport medium. The swab should be
allowed to remain in contact with the secretions for up to 1 minute, then placed in transport medium and sent
to the laboratory without delay at 4°C. Long protected nasopharyngeal swabs should be used to collect
samples for some suspected viral infections.
g) Milk
Milk samples should be taken after cleansing and drying the tip of the teat, the use of antiseptics should be
avoided. The initial stream of milk should be discarded and a tube filled with the next stream(s), a sample of
bulk tank milk can be used for some tests. Milk for serological tests should not have been frozen, heated or
subjected to violent shaking. If there is going to be a delay in submitting them to the laboratory,
preservatives can be added to milk samples that are being collected for serological testing. If necessary,
milk for bacterial examination can be frozen.
2. Sample collection at post-mortem
Samples of tissue from a variety of organs can be taken at post-mortem. Detailed procedures for conducting a
post-mortem examination and collecting samples are described in most pathology text books; a guide to necropsy
procedures has been published (10). Post-mortem techniques are also included in some of the national guidelines
(3, 5, 9). A summary of these procedures will be provided here.
Animal health personnel should be trained in the correct procedures for post-mortem examination of the species
of animals with which they work. The equipment required will depend on the size and species of animal, but a
knife, saw and cleaver will be required, and also scalpel, forceps and scissors, including scissors with a rounded
tip on one blade, for opening intestines. A plentiful supply of containers and tubes of transport media appropriate
to the nature of the sample required should be available, along with labels and report forms. Containers should be
fully labelled with the date, tissue and animal identification. Special media may be required for transport of
samples from the field. The operator should wear protective clothing: overalls, washable apron, rubber gloves and
rubber boots. Additionally, if potential zoonotic diseases are being investigated, the post-mortem examination
should be conducted in a biological safety cabinet; if this is not possible, an efficient face mask and eye protection
should be worn. If rabies or transmissible spongiform encephalopathies (TSEs) are suspected, it is usual to
detach the animal’s head.
Tissues may be collected for microbiological culture, parasitology, biochemistry, histopathology and/or immuno-
histochemistry, and for detection of proteins or genome nucleic acids. In addition buccal, oropharyngeal or rectal
(cloacal) swabs may be collected. The person conducting the post-mortem examination should have sufficient
knowledge of anatomy and pathology to select the most promising organs and lesions for sampling. Each piece of
tissue should be placed in a fully labelled separate plastic bag or sterile screw-capped jar. Swabs should always
be submitted in appropriate transport media. Sterile instruments should be used for collecting specimens for
microbiological culture and care should be taken not to contaminate tissues with intestinal contents. Disinfectants
should not be used on or near tissues to be sampled for bacterial culture or virus isolation.
The tissues may be sent to the laboratory dry or in bacterial or virus transport medium, depending on the type of
specimen and the examinations required; swabs should be sent in transport medium. After collection, the samples
for microbiological examination should be refrigerated until shipped. If shipment cannot be made within 48 hours,
the samples should be frozen; however, prolonged storage at –20°C may be detrimental to virus isolation. For
histopathology, blocks of tissue not more than 0.5 cm thick and 1–2 cm long are cut and placed in neutral buffered
4–10% formalin, which should be at least ten times the volume of the tissue sample. For certain suspected
diseases, larger portions of brain are required; the brain is sectioned using a sagittal cut, half is submitted fresh,
on ice, and the other half is submitted in 10% buffered formalin. For scrapie, bovine spongiform encephalopathy
and other TSEs, details of sample collection are provided in the individual disease chapters in this Terrestrial
Manual. Store and pack formalin-fixed tissues separately from fresh tissues, blood and smears. Care should be
taken to insure that formalin-fixed tissues are not frozen. Once fixed, tissues can be removed from formalin and,

as long as they are kept moist and protected (e.g. by wrapping in formalin-soaked paper towels, then sealed in
screw-capped jars), they can be forwarded to the laboratory without formalin.
3. Environmental and feed sampling
Samples may be taken to monitor hygiene or as part of a disease enquiry. Environmental samples are commonly
taken from litter or bedding and voided faeces or urine. Swabs may be taken from the surface of ventilation ducts,
feed troughs and drains. This kind of sampling is particularly important in hatcheries, artificial insemination centres
and slaughterhouses in which specialised equipment is maintained. Samples may also be taken from animal feed,
in troughs or bulk containers. Water may be sampled in troughs, drinkers, header tanks or from the natural or
artificial supply.
4. Honey bees
Adult bees, either dead or moribund, may be collected in the vicinity of the colonies. Live bees should be killed by
freezing. Brood samples are taken by removing a piece of brood comb that shows abnormalities. This should be
wrapped in paper and placed in a box for transport to the laboratory. Hive debris may be collected for
examination, preferably on a sticky board to trap mobile parasites.
B. SAMPLE SIZE
When investigating a case of clinical disease, the specimens collected should be representative of the condition
being investigated and the lesions observed. When developing a programme of surveillance and monitoring for
animal health in the absence of clinically evident disease, some general statistical sampling methods should be
used. These sampling methods are needed to perform the scientifically based surveys specified in the OIE
Terrestrial Animal Health Code (14). It is possible to calculate how many animals should be sampled from a
herd/flock of a certain size, to achieve a 95% probability of detecting infection or previous exposure assumed to
be present in a certain percentage of the animals. The following formulae can give approximate numbers, but a
specific sampling programme for the planned surveillance programme should be based on complete formulas
available in the references (2, 4) or by the use of a program (FreeCalc) available off the internet:
http://www.ausvet.com.au/content.php?page=res_software#freecalc. All calculation examples provided in the
following paragraphs can be calculated using FreeCalc. This software also includes “a “pooled prevalence
calculator”, which describes the calculation of prevalence using pooled samples.
The following formula could be used to calculate the sample size n to detect at least one infection with a test that
has a 100% sensitivity and specificity; where α is the significance level and 1–α is the level of confidence, p is the
prevalence in the population. If disease were present in 5% of a herd of 500 animals, it would be necessary to
collect specimens from 56 animals to be 95% confident of finding at least one positive, assuming that both the
sensitivity and specificity of the test were 100%. In order to make a prediction of disease prevalence, it is critical
that the sample be selected from the population by a formal random sampling procedure. As most diagnostic tests
do not have specificity and sensitivity of 100%, the number of specimens collected must be adjusted to the
sensitivity and specificity of the test that will be used (see also Chapter 1.1.4 Principles of validation of diagnostic
assays for infectious diseases).
ln (α)
n=
–p)
In the above example α = 0.05, 1– α = 95%, p = 0.05 and n = 59
If the sensitivity (Se) is less than 100%, the above formula should be modified as follows:
ln (α)
n=
ln (1–p.Se)
In the above example with α = 0.05, p = 0.05, specificity (Sp) = 1 and Se = 0.95, a minimum of n = 62 animals
instead of 59 would need to be sampled to have a probability of at least 0.95 of finding a positive animal. The
increase in the sample size from 59 to 62 is due to the decrease in the sensitivity of the test from 1 to 0.95. The
graph below gives the minimum sample size required for finding at least one positive for several sensitivity and
prevalence combinations at α = 0.05 and Sp = 1.

If the test is known to have a specificity of less than 1, the positive results should be confirmed by a test with a
higher specificity. If the prevalence is very low and the test used has a specificity of less than 1, it is very likely
that a positive test result is a false positive.
Fig. 1. Minimum sample size required to be 95% confident of finding infection

at various sensitivity and prevalence combinations.
C. INFORMATION TO BE SENT WITH SAMPLES

It is essential that individual samples be clearly identified using appropriate methods. Marking instruments should
be able to withstand the condition of use, i.e. being wet or frozen (use indelible marking pen). Pencil has a
tendency to rub off containers and labels attached to plastic will fall off when stored at –70°C. Information and
case history should always accompany the samples to the laboratory, and should be placed in a plastic envelope
on the outside of the shipping container. As outlined in the following section on transport of samples, this
information must also be inside the shipping container. The following are suggested items that should be
addressed. It would be advisable to contact the receiving laboratory to determine if it has a submission form that it
would like to have submitted with the samples or if it needs other information.
i) Name and address of owner/occupier and geolocation (latitude and longitude, if available) where disease
occurred, with telephone and fax numbers.
ii) Name, postal and e-mail address, telephone and fax numbers of the sender.
iii) Diseases suspected and tests requested.
iv) The species, breed, sex, age and identity of the animals sampled.
v) Date samples were collected and submitted.
vi) List of samples submitted with transport media used.
vii) A complete history would be beneficial for the laboratory and should be included if possible. Some of the
components of the history are:
a) A list and description of the animals examined and the findings of the post-mortem examination.
b) The length of time sick animals have been on the farm; if they are recent arrivals, from where did they
originate.

c) The date of the first cases and of subsequent cases or losses, with any appropriate previous
submission reference numbers.
d) A description of the spread of infection in the herd or flock.
e) The number of animals on the farm, the number of animals dead, the number showing clinical signs,
and their age, sex and breed.
f) The clinical signs and their duration including the temperature of sick animals, condition of mouth, eyes
and feet, and milk or egg production data.
g) The type and standard of husbandry, including the type of feed available, possible contact with poison
or poisonous plants.
h) History of foreign travel by owner or of introduction of animals from other countries or regions.
i) Any medication given to the animals, and when given.
j) Any vaccines given, and when given.
k) Other observations about the disease, husbandry practices and other disease conditions present.
D. PACKAGING AND TRANSPORT OF SAMPLES
1. Approval to ship specimens
The laboratory that is going to receive the samples should be contacted to ensure that it has the capability to do
the testing requested and to see if there are any special packaging or shipping requirements. It is essential to
contact the receiving laboratory when material is sent to another country. A special import licence will usually be
required for shipment of any biological material to other countries and must be obtained in advance. This licence
should be placed in an envelope on the outside of the parcel.
Shipments must be made in accordance with the dangerous goods rules for the particular mode of transport. For
air transport it is the International Civil Aviation Organization (ICAO) technical instructions for the safe transport of
dangerous goods by air. These are reflected in the International Air Transport Association (IATA) Dangerous
Goods Regulations which is the interpretation of ICAO instructions applied to shipments by air (7). These
regulations have been described in a United Nations World Health Organization publication (13). The shipper is
responsible for checking the variations guidelines to insure that restrictions are met.
2. Transportation of specimens
The specimens should be forwarded to the laboratory by the fastest method available. If they can reach the
laboratory within 48 hours, samples should be sent refrigerated. If dry ice is used, the additional packaging
requirements must be met. Infectious substances, which can include diagnostic specimens, are not permitted to
be shipped as checked luggage or as carry on luggage and must be shipped as cargo.
3. Packaging
The shipper should ensure that the specimens are packaged so they arrive at the laboratory in good condition
and there is no leakage during shipment. The Dangerous Goods Regulations (DGR) have explicit requirements
for packaging and shipment of diagnostic specimens, by all commercial means of air transport (7, 13). In some
countries, there are similar requirements for ground shipments and the postal service, but these requirements
should be reviewed before shipping. These requirements for air transport are covered in detail in the IATA
publication, which are updated every year. The shipper is expected to know and follow the procedures outlined in
the current DGR. The following is a summary of the regulations at the time that this revision of the Terrestrial
Manual was published and it should only be used as a guide for shipping. Shippers must also always check the
latest version of the DGR prior to shipping diagnostic specimens. In addition, three of the national guidelines
provide explicit directions for packaging and shipping diagnostic specimens and are based on IATA requirements
(3, 5, 9).
The DGR outline the procedures for the shipment of infectious substances, which can include diagnostic
specimens. Infectious substances are defined in the DGR as substances that are known or are reasonably
expected to contain pathogens. Pathogens are defined as micro-organisms (including bacteria, viruses,
rickettsiae, parasites, fungi) or recombinant micro-organisms (hybrid or mutant) that are known or reasonably
expected to cause disease in humans or animals.

The IATA (7, 13) lists the following exemption from the Dangerous Goods Regulations:
3.6.2.2.3.1 Substances which do not contain infectious substances or substances which are unlikely to
cause disease in humans or animals are not subject to these Regulations unless they meet the criteria for
inclusion in another class.
3.6.2.2.3.2 Substances containing microorganisms which are non-pathogenic to humans or animals are not
subject to these Regulations unless they meet the criteria for inclusion in another class.
3.6.2.2.3.3 Substances in a form that any present pathogens have been neutralised or inactivated such that
they no longer pose a health risk are not subject to these Regulations unless they meet the criteria for
inclusion in another class.
3.6.2.2.3.4 Environmental samples (including food and water samples), which are not considered to pose a
significant risk of infection, are not subject to these Regulations unless they meet the criteria for inclusion in
another class.
3.6.2.2.3.5 Dried blood spots, collected by applying a drop of blood on to absorbent material, or faecal occult
blood screening tests and blood or blood components that have been collected for the purposes of
transfusion or for the preparation of blood products to be used for transfusion or transplantation and any
tissues or organs intended for use in transplantation.
3.6.2.2.3.6 Patient specimens for which there is minimal likelihood that pathogens are present are not
subject to these Regulations if the specimen is transported in a packaging which will prevent any leakage
and which is marked with the words “Exempt human specimen” or “Exempt animal specimen”, as
appropriate. The packaging should meet the following conditions:
(a) The packaging should consist of three components:

(1) a leak-proof primary receptacle(s);
(2) a leak-proof secondary packaging; and
(3) an outer packaging of adequate strength for its capacity, mass and intended use, and with at least
one surface having minimum dimensions of 100 mm × 100 mm;
(b) For liquids, absorbent material in sufficient quantity to absorb the entire contents must be placed
between the primary receptacle(s) and the secondary packaging so that, during transport, any release
or leak of a liquid substance will not reach the outer packaging and will not compromise the integrity of
the cushioning material;
(c) When multiple fragile primary receptacles are placed in a single secondary packaging, they should be
either individually wrapped or separated to prevent contact between them.
“Note: In determining whether a patient specimen has a minimal likelihood that pathogens are present, an
element of professional judgment is required to determine if a substance is exempt under this paragraph. That
judgment should be based on the known medical history, symptoms and individual circumstances of the source,
human or animal, and endemic local conditions. Examples of specimens which may be transported under this
paragraph include the blood or urine tests to monitor cholesterol levels, blood glucose levels, hormone levels, or
prostate specific antibodies (PSA); tests required to monitor organ function such as heart, liver or kidney function
for humans or animals with non-infectious diseases, or therapeutic drug monitoring; tests conducted for insurance
or employment purposes and are intended to determine the presence of drugs or alcohol; pregnancy test;
biopsies to detect cancer; and antibody detection in humans or animals. ”
There are also exceptions for some biological products and the shipper of these products is referred to the IATA
Regulations for these requirements as not all biological products are exempt. The following is the DGR definition
of Biological Products (7, 13):
“Biological products are those products derived from living organisms which are manufactured and
distributed in accordance with the requirements of appropriate national authorities, which may have special
licensing requirements, and are used either for prevention, treatment, or diagnosis of disease in humans or
animals, or for development, experimental or investigational purposes related thereto. They include, but are
not limited to, finished or unfinished products such as vaccines.”
The DGR state that infectious substances (including diagnostic specimens likely to contain animal or human
pathogens) are designated as Category A and B and assigned to UN 2814, UN 2900 or UN 3373.

Category A is defined as an: “Infectious substance, which is transported in a form that when exposure to it
occurs, is capable of causing permanent disability, life threatening or fatal disease in otherwise healthy
humans or animals, indicative examples of substances that meet these criteria are given in Table 1 and 2”.
Infectious substances meeting this definition that affect humans, including zoonotic agents, are designated
UN 2814 and given the shipping name of “Infectious substance, affecting humans” those affecting
animals only are designated UN 2900 and given the shipping name of “Infectious substance, affecting
animals”.
Infectious substances shipped for diagnostic purposes that do not meet the criteria for assignment to UN 2814 or
UN 2900 are assigned to Category B and must be assigned to UN 3373 and designated as “DIAGNOSTIC
SPECIMENS or CLINICAL SPECIMENS or BIOLOGICAL SUBSTANCES CATEGORY B”.
The IATA DGR contains an indicative list of pathogens that must be assigned to UN 2814 or UN 2900 (Tables 1
and 2). The pathogens on these lists cannot be assigned to UN 3373 (7, 13).
Table 1. Infectious substances affecting humans that must be designated UN 2814
Bacillus anthracis (cultures only) Japanese encephalitis virus (cultures only)
Brucella abortus (cultures only) Junin virus
Brucella melitensis (cultures only) Kyasanur Forest disease virus
Brucella suis (cultures only) Lassa virus
Burkholderia mallei – Pseudomonas mallei – Machupo virus

Glanders (cultures only)
Burkholderia pseudomallei – Marburg virus

Pseudomonas pseudomallei (cultures only)
Chlamydia psittaci – avian strains (cultures only) Mycobacterium tuberculosis (cultures only)
Clostridium botulinum (cultures only) Monkeypox virus
Coccidioides immitis (cultures only) Nipah virus
Coxiella burnetii (cultures only) Omsk hemorrhagic fever virus
Crimean-Congo hemorrhagic fever virus Poliovirus (cultures only)
Dengue virus (cultures only) Rabies virus (cultures only)
Eastern equine encephalitis virus (cultures only) Rickettsia prowazekii (cultures only)
Escherichia coli, verotoxigenic (cultures only) Rickettsia rickettsii (cultures only)
Ebola virus Rift Valley fever virus (cultures only)
Flexal virus Russian spring-summer encephalitis virus (cultures only)
Francisella tularensis (cultures only) Sabia virus
Guanarito virus Shigella dysenteriae type 1 (cultures only)
Hantaan virus Tick-borne encephalitis virus (cultures only)
Hantavirus causing haemorrhagic fever with renal syndrome Variola virus
Hendra virus Venezuelan equine encephalitis virus (cultures only)
Hepatitis B virus (cultures only) West Nile virus (cultures only)
Herpes B virus (cultures only) Yellow fever virus (cultures only)
Human immunodeficiency virus (cultures only) Yersinia pestis (cultures only)
Highly pathogenic avian influenza virus (cultures only)

Table 2. Indicative examples of animal pathogens forbidden as diagnostic specimens that must
be shipped as infectious substances affecting animals (UN 2900)
African swine fever virus (cultures only) Peste des petits ruminants virus (cultures only)
Avian paramyxovirus Type 1 – Velogenic Newcastle disease Rinderpest virus (cultures only)
virus (cultures only)
Classical swine fever virus (cultures only) Sheep-pox virus (cultures only)
Foot and mouth disease virus (cultures only) Goatpox virus (cultures only)
Lumpy skin disease virus (cultures only) Swine vesicular disease virus (cultures only)
Mycoplasma mycoides – Contagious bovine pleuropneumonia Vesicular stomatitis virus (cultures only)
(cultures only)
New or emerging pathogens must also be assigned to UN 2814 or UN 2900.
The following is the IATA definition of amplification in culture:
“Cultures are the result of a process by which pathogens are intentionally propagated. This definition does not
include patient specimens.”
“Patient specimens are those collected directly from humans or animals, including, but not limited to, excreta,
secreta, blood and its components, tissue and tissue fluid swabs, and body parts being transported for purposes
such as research, diagnosis, investigational activities, disease treatment and prevention.”
Note: Cultures of organisms that do not fit into the definition of Category A infectious substance can be
transported as Biological Substances, Category B.
The following flow chart summarises the classification of DIAGNOSTIC SPECIMENS or CLINICAL SPECIMENS
or BIOLOGICAL SUBSTANCES CATEGORY B.
Is the substance likely to contain micro-organisms

No
Yes
Is the substance unlikely to cause Exempt from dangerous goods

human or animal disease regulations – send in leak proof packaging
Yes
No
Is the infectious substance capable of causing permanent

disability, life threatening or fatal disease to humans or
animals (indicative examples are given in Tables 1 and 2)?
No
Yes
Is the substance being transported for

diagnostic purposes?
Yes
Consign as a diagnostic specimen (UN 3373) Consign as an infectious substance

(UN 2814 or UN 2900)
Live animals must not be used to transport infectious substances.
Animal carcasses affected by pathogens of category A or which would be assigned to Category A in cultures only,
must be assigned to UN 2814 or UN 2900 as appropriate. Other animal carcasses affected by pathogens
included in Category B must be transported in accordance with provisions determined by the Competent
Authority.

The packaging of infectious substances and specimens from suspected serious animal diseases, UN 2814 or UN
2900, are outlined in packing instruction 620; a Shippers Declaration of Dangerous Goods must be completed
and submitted with these samples. There is also a requirement that the shipper receive training on the IATA-
approved shipping procedures for UN 2814 and UN 2900 shipments. Due to the complexity of these guidelines
the shipper is referred to the regulations for further information on all UN 2814 or 2900 shipments (7, 13).
The other group, UN 3373, covers ‘Diagnostic Specimens or Clinical Specimens or Biological Substances
Category B’. This category has a lower risk and packages containing these specimens should be labelled as
‘Diagnostic Specimens or Clinical Specimens or Biological Substances Category B’; a Declaration of Dangerous
Goods is not needed. IATA packing instruction 650 provides the guidelines for packaging infectious substances
assigned to UN 3373 and the following is a summary of these packing instructions. However, the complete
procedure, as outlined in the most recent IATA Dangerous Good Regulations, must be followed (7, 13).
i) Infectious substances assigned to UN 3373 ‘Diagnostic Specimens’ must be packed in good quality
packaging, which must be strong enough to withstand the shocks and loadings normally encountered during
transport. Packaging must be constructed and closed so as to prevent any loss of contents, which might be
caused under normal conditions of transport.
ii) The packaging must consist of three components:

a primary receptacle;
a secondary packaging; and
a rigid outer packaging.
iii) For liquid substances:

the primary receptacle(s) must be leak-proof and must not contain more than 1 litre; the secondary
packaging must also be leak-proof;
adequate adsorbent material must be packed around the primary receptacle(s) to absorb all the fluid in
the primary receptacle(s);
if multiple primary receptacles are used they should be individually wrapped or separated so as to
prevent contact;
the primary receptacle or the secondary packaging must be capable of withstanding without leakage an
internal pressure of 95 kPa in the range of –40°C to 55°C (–40°F to 130°F);
the outer packaging must not contain more than 4 litres. This quantity excludes ice, dry ice, or liquid
nitrogen when used to keep specimens cold.
iv) For solid substances:

the primary receptacle(s) must be sift-proof and must not exceed the outer packaging weight limit; the
secondary packaging must be sift-proof;
adequate adsorbent material must be packed around the primary receptacle(s) to absorb all the fluid in
the primary receptacle(s);
except for packages containing body parts, organs or whole bodies, the outer packaging must not
contain more than 4 kg. This quantity excludes ice, dry ice or liquid nitrogen when used to keep
specimens cold;
if there is any doubt as to whether or not residual liquid may be present in the primary receptacle
during transport then packaging suitable for liquids, including absorbent materials, must be used.
v) An itemised list of contents must be enclosed between the secondary packaging and the outer packaging.
vi) If shipped at ambient temperatures or higher, the primary receptacle must have a positive means of ensuring
that it is leak proof, such as a leak proof seal, heat seal or skirted stopper. If screw caps are used they
should be sealed with parafilm or tape.
vii) Prefrozen packs or dry ice can be packed around the secondary receptacle. If dry ice is used, there must be
an internal support to secure the secondary receptacle in the original position after the dry ice has been
dissipated. The outer packaging must permit the release of carbon dioxide. There are additional
requirements if liquid nitrogen is used and these are described in the DGR.
viii) Packages containing diagnostic or clinical specimens are not required to have the net quantity marked on
the outside of the package. However, where dry ice is used as a refrigerant, the net quantity of dry ice must
be shown.
ix) The primary and secondary receptacles must be put into a shipping container with adequate cushioning
material.

x) The packaging must be able to withstand a 1.2 metre drop test. (There are additional strength requirements
for packaging used for UN 2900 and UN 2814 specimens.)
xi) At least one surface of the outer packaging must have a minimum dimension of 100 mm × 100 mm.
xii) For transport, the label 3373 must be displayed on the external surface of the outer packaging on a
background of a contrasting colour and must be clearly visible and legible. The mark must be in the form of a
square set an angle of 45° (diamond-shaped) with each side having a length of at least 50 mm, the width of
the line must be at least 2 mm, and the letters and numbers must be at least 6 mm high. The proper
shipping name “Diagnostic specimen”, “Clinical specimen” or “Biological substance category B” in letters at
least 6 mm high must be marked on the outer package adjacent to the diamond-shaped mark.
4. Shipping forms
All shipping forms, including the import licence and submission form must be put in an envelope attached to the
outside of the shipping container. The forms and labels must be completed as outlined in the DGR and also put
on the outside of the container.
E. PRESERVATION OF SAMPLES FOR PROLONGED STORAGE

Establishing a collection of samples for future studies can be very useful. This can include cultures for comparison
with future isolates, tissue or serum samples that can be used for the validation of new tests and a collection of
fixed tissues, or paraffin blocks, for future histological examination. Possibly the most useful collection is the
storage of serum samples. These samples may be useful if a retrospective investigation is carried out to compare
the present disease status with that of earlier times.
Serum banks
Serum samples can provide information about the animals from which the sera were taken. The samples can be
tested for a variety of constituents, such as immunoglobulins, trace elements, toxins, hormones and enzymes. If a
sufficient number of serum samples have been collected at random from a population, comparisons can be made
on the affect of sex, age, breed and geographical location. Results from this comparison can identify high risk
groups, vaccination priorities can be established, and patterns and rates of disease determined (8).
A serum bank is a catalogued collection of sera that are stored so as to preserve their immunological and other
biochemical properties. Both the catalogue and the storage conditions are essential for a successful serum bank.
Each individual sample should be fully documented and identified. The database should contain all relevant
information about the origin of the sample and test results obtained. Additional data that may be of interest, such
as weather conditions and the animal’s productivity may also be included. Accurate records are essential and
must be obtained when the blood samples are collected. The first essential is the complete identification of the
animal. The amount of detail recorded should be appropriate to the abilities of the operator, accuracy being more
important than quantity of information. Although pooling of sera reduces documentation and storage space, it
should be avoided as it greatly reduces the usefulness of the material. Care should be taken to collect the blood
as aseptically as possible and sterility should be maintained during separation of the serum and all other
manipulations. The serum bank catalogue should be well organised and maintained on a computer database with
appropriate backup. A suggested methodology has been described in detail (8).
Sera may be stored for periodic use or kept in long-term storage for historical purposes and these two functions
should be separated. Storage conditions should minimise loss of immunological and other biochemical properties
of the sera. There are three methods: deep freezing, dry storage on paper disks at ambient temperature and
lyophilisation (freeze-drying). For long-term storage of sera by deep freezing, a core temperature below –60°C
should be maintained. The lower the temperature the better, but lower temperatures are more expensive to
maintain. Liquid phase N2 is at –196°C, vapour phase N2 is at –100°C and an ultra-low deep freezer will maintain
–90°C. Some serum banks have been maintained at –20°C, but the serum may deteriorate and not be suitable for
detection of some properties, especially if stored for long periods at this temperature. Deep-freezers should have
a system to provide a warning if the temperature rises due to mechanical break down or power failure. A stand-by
generator is essential together with alternative cold storage space in case the contents of a freezer must be
transferred. Paper disk storage is a simple and inexpensive method, but it allows only a small quantity of serum to
be stored and the eluted serum is only suitable for a limited number of tests. The disks should be kept in a cool,
dry atmosphere. They can probably provide satisfactory results for up to about 5 years. Lyophilisation is generally
regarded as the best method for long-term storage of sera. If freeze-drying conditions are optimised the loss of
serum characteristics are minimised. Lyophilisation requires expensive equipment and is a time-consuming
process. Lyophilised vials should be stored at 4°C.

REFERENCES
1. ANON (1993). Removal of blood from laboratory mammals and birds. First Report of the
BVA/FRAME/RSPCA/UFAW/Joint Working Group on Refinement. Laboratory Animals, 27, 1–22.
2. CAMERON A.R. & BALDOCK F.C. (1998). A new probability formula for surveys to substantiate freedom from
disease. Prev. Vet. Med., 34, 1–17.
3. CANADIAN FOOD INSPECTION AGENCY, LABORATORY DIRECTORATE: ANIMAL HEALTH (2002). Manual of Common
Procedures. Section: Specimen Collection and Submission; Specimen Packaging; Specimen Transportation.
Canadian Food Inspection Agency, Ottawa, Canada, 61 pp (http://www.inspection.gc.ca/english/animal/
heasan/disemala/cpm-mpc/indexe.shtml).
4. CANNON R.M. & ROE R.T. (1982). Livestock Disease Surveys – A Field Manual for Veterinarians. Department
of Primacy Industry, Water and Environment, Australia, 15 pp.
5. COOK R., BARTON M., GLEESON L. & MAIN C. (1996). AUSVETPLAN Management Manual: Laboratory
Preparedness. Animal Health Australia, Canberra, 56 pp. http://www.aahc.com.au/ausvetplan/index.htm.
6. HEM A., SMITH A.J. & SOLBERG P. (1998). Saphenous vein puncture for blood sampling of the mouse, rat,
hamster, gerbil, guinea pig, ferret and mink. Laboratory animals, 32 (4), 364–368.
7. INTERNATIONAL AIR TRANSPORT ASSOCIATION (2006). Dangerous Goods Regulations, 44th Edition.
International Air Transport Association, 800 Place Victoria, P.O. Box 113, Montreal, Quebec H4Z 1M1,
Canada, 824 pp.
8. MOORHOUSE P.D. & HUGH-JONES M.E. (1981). Serum banks. Vet. Bull., 51, 277–290.
9. NATIONAL VETERINARY SERVICES LABORATORIES (2006). Procedures for Collection and Submission of
Specimens. National Veterinary Services Laboratories, Ames, Iowa, USA.
http://www.aphis.usda.gov/vs/nvsl/html/shipping.html
10. STRAFUSS A.C. (1988). Necropsy: Procedures and Basic Diagnostic Methods for Practicing Veterinarians.
Charles C. Thomas, Springfield, IL, USA, 244 pp.
11. VETERINARY LABORATORIES AGENCY (2003). Submission of Samples to the Veterinary Laboratories Agency.
Veterinary Laboratories Agency, New Haw, Addleston, Surrey, United Kingdom, 27 pp.
www.vla.gov.uk/servtovet/documents/Submissions.pdf
12. VETERINARY SERVICES (OF THE UNITED STATES DEPARTMENT OF AGRICULTURE) (2005). Regulations for
Classifying Infectious Substances and Diagnostic Specimens, USDA Veterinary Services Notice NO. 06-02
13. WORLD HEALTH ORGANIZATION (2005). Guidance on regulations for the transport of infectious substances.
http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2005_22/en/
14. WORLD ORGANISATION FOR ANIMAL HEALTH (OIE: OFFICE INTERNATIONAL DES EPIZOOTIES) (2006). Terrestrial
Animal Health Code. OIE, Paris, France, www.oie.int.
*
* *

CHAPTER 1.1.2.
BIOSAFETY AND BIOSECURITY IN THE

VETERINARY MICROBIOLOGY LABORATORY AND
ANIMAL FACILITIES
INTRODUCTION
Laboratory work of the type described in this Terrestrial Manual should be carried out with a
minimum of risk to the health of the staff (biosafety) and the environment (biocontainment). This
requires careful consideration of the risks involved in a particular procedure, followed by
appropriate measures to minimise the risk of human disease and of possible release into the
environment. This is a complex subject that can only be considered in outline in an introductory
chapter. This chapter is concerned almost exclusively with risks from infectious agents, but
physical and chemical injuries in microbiology laboratories must also be prevented. Risks from
infection are reduced by good laboratory techniques and secure facilities, which aid in the
containment of pathogens. It is important to understand that containment of pathogens can be
used for two purposes. One is to prevent disease in humans in the laboratory; the other is to
prevent the release of the pathogen into the environment and causing disease in animals or
humans. Often the same methods of containment are used for both preventing laboratory-acquired
infection in humans and for preventing escape of pathogens that could cause an outbreak of
animal diseases. Although the methods, techniques and facilities required may be the same, the
list of pathogens and categorisation into levels of risk will differ depending on whether it is human
or animal diseases control that is the primary objective.
Existing national and international reference laboratories have considerable experience in the
operation of safe working practices and provision of appropriate facilities. When new laboratories
are being established, it would be prudent to seek advice from the relevant regulatory authorities
and the competent authorities at established institutes. It is important to comply with legislative
requirements.
A. ASSESSMENT OF RISK FROM PATHOGENS

It is necessary first to assess the risk from a pathogen, so that it can be assigned to a Risk Group. A further risk
assessment can be conducted, based on the proposed work, to determine the appropriate containment level. To
assess the risk to humans and animals from a particular pathogen it is necessary to know whether infection with
that organism can cause clinical disease and/or mortality in humans and animals, and whether it could then
spread to cause disease in the general human and/or animal population. There are additional requirements
related to the containment of animal pathogens and the prevention of the spread of infection to animals. To
assess these risks it is necessary to know the epidemiological background of the organism and also such
attributes of the organism as infectivity for humans and animals, stability in the environment, ability to infect by
different routes of exposure, and susceptibility to specific treatments or prophylaxis (1, 2, 5, 6). It is relatively
easy to obtain this information when working with a known pathogen, but the problem is more complex in a
diagnostic laboratory receiving clinical material that may be infected with a variety of unknown pathogens, some
of which could be extremely hazardous to human health or pose a significant threat to animal populations. Some
of the considerations to take into account when evaluating risk are:
1. Known occurrence of human and animal infection with the organism or related organisms with similar
characteristics, any history of laboratory-acquired infection, infective dose and disease severity; production
of toxins or allergens.
2. The volume of culture to be handled and the concentration of the organism likely to be present. (Procedures
such as antigen or vaccine production that require large quantities of organisms usually carry a higher risk
than attempted isolation procedures.)

Chapter 1.1.2. — Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities
3. The origin of the sample, for example samples from wildlife species may contain human or animal
pathogens not normally encountered.
4. The history of the isolate being handled. Pathogens on primary isolation or of low passage level are often
more dangerous than pathogens of high passage level. In some cases, pathogenicity may be enhanced by
passage or subculture using different media.
5. The possibility of aerosol formation should be especially taken into consideration when handling fluid
samples or, for example, during grinding, homogenisation and centrifugation.
6. The threat that the organism may pose to food-producing or companion animals or to wildlife, irrespective of
the threat to laboratory personnel. Additional precautions for handling and storage are required for animal
disease agents from foreign countries.
7. The physical state of the employees. For example, in the case of pregnancy, immunodeficiency or allergy,
special precautions may be required. Sometimes certain individuals have to be excluded from particular
types of work that would be especially hazardous to them.
8. A higher level of risk may arise when agents such as Brucella or Mycobacterium are inoculated into
animals. To evaluate the impact of animal inoculation, a risk assessment should be conducted and the
following factors should be considered:
i) Host species versus inoculated species;
ii) Strain/treatment and concentration of the inoculum;
iii) Route of inoculation;
iv) Animal housing;
v) Types of sampling during the experiment.
9. Some pathogens need to be transmitted by specific vectors or require intermediate hosts to complete their
life cycles before they can infect animals and cause disease. In countries where such vectors or
intermediate hosts do not occur, or where climatic or environmental factors mitigate against their survival,
the pathogen poses a lower risk to animal health than in countries where such vectors or intermediate hosts
occur naturally or could survive.
B. GROUPING OF MICROORGANISMS BY HUMAN AND ANIMAL HEALTH RISK
The considerations outlined above have been used by several national authorities to designate microorganisms
into four Risk Groups (2, 4) representing increasing risks to human health. Such categorisation of pathogens
makes no allowance for people who are particularly susceptible, for example due to pre-existing disease, a
compromised immune system or pregnancy. The four Groups may be summarised thus:
• Group 1 – Organisms that are unlikely to cause human or animal disease and are disease-producing
organisms in animals that are enzootic but not subject to official control.
• Group 2 – Organisms that may cause human or animal disease but are unlikely to be spread in the
community or animal population and for which effective prophylaxis and treatment are available; examples
of Group 2 animal pathogens:
i) They do not depend on vectors or intermediate hosts for transmission.
ii) There is very limited or no transmission between different animal species.
iii) Geographical spread if released from the laboratory is limited.
iv) Direct animal to animal transmission is relatively limited.
v) Mode of transmission is primarily through ingestion, inoculation or mucus membrane route.
vi) The need to confine diseased or infected nondiseased animals is minimal.
vii) The disease is of limited economic and/or clinical significance.
viii) Short-term survival in the environment and effective treatment or prevention is available.
ix) May be either exotic or enzootic but are subject to official control and have a low risk of spread from
the laboratory.

• Group 3 – Organisms that can cause severe human or animal disease and may spread in the community
and/or animal population but for which there is usually effective prophylaxis and treatment; examples of
Group 3 animal pathogens:
i) They may depend on vectors or intermediate hosts for transmission.
ii) Transmission between different animal species may readily occur.
iii) Geographical spread if released from the laboratory is moderate.
iv) Direct animal to animal transmission occurs relatively easily.
v) The statutory confinement of diseased, infected and in-contact animals is necessary.
vi) The disease is of severe economic and/or clinical significance.
vii) Prophylactic and/or therapeutic treatments are not readily available or of limited benefit.
viii) Mode of transmission may be through the airborne route or direct contact.
ix) Are either exotic or enzootic but are subject to official control and that have a moderate risk of spread
from the laboratory.
• Group 4 – Organisms that cause severe human or animal disease, may represent a high risk of spread in
the community or animal population and for which there is usually no effective prophylaxis or treatment.
i) They may depend on vectors or intermediate hosts for transmission.
ii) Transmission between different animal species may occur very readily.
iii) Geographical spread if released from the laboratory is widespread.
iv) Direct animal to animal transmission occurs very easily.
v) Can be transmitted through casual contact or indirectly.
vi) The statutory confinement of diseased, infected and in-contact animals is necessary.
vii) The statutory control of animal movements over a wide area is necessary.
viii) The disease is of extremely severe economic and/or clinical significance.
ix) No satisfactory prophylactic and/or therapeutic treatments are available.
x) Have a high risk of spread from the laboratory into the environment and the national animal
population.
Infectious organisms that might be encountered in laboratory work have been assigned to Risk Groups 1–4 by
authorities in several countries (2, 4). Some examples of pathogens that may cause disease in humans, and also
may be found in a veterinary laboratory, are listed in Table 1 Also, some very serious Group 4 agents, including
Hendra and Nipah, have been isolated from diagnostic specimens in veterinary laboratories.
Table 1. Examples of some of the microorganisms in Risk Groups 2 and 3 that

are capable of causing human disease and that may be present in a veterinary laboratory
Group 2
Viruses: Influenza viruses types A, B, C other than notifiable avian influenza (NAI); Newcastle disease virus; Orf
(parapox virus)
Bacteria: Alcaligenes spp.; Arizona spp.; Campylobacter spp.; Chlamydophila psittaci (nonavian); Clostridium tetani;
Clostridium botulinum; Corynebacterium spp.; Erysipelothrix rhusiopathiae; Escherichia coli; Haemophilus spp.;
Leptospira spp.; Listeria monocytogenes; Moraxella spp.; Mycobacterium avium; Pasteurella spp.; Proteus spp.;
Pseudomonas spp.; Salmonella spp.; Staphylococcus spp.; Yersinia enterocolitica; Yersinia pseudotuberculosis
Fungi: Aspergillus fumigatus; Microsporum spp.; Trichophyton spp.
Group 3
Viruses: Rabies virus; Equine encephalomyelitis virus (Eastern, Western and Venezuelan); Japanese B encephalitis
virus; Louping ill virus
Bacteria: Bacillus anthracis; Burkholderia mallei (Pseudomonas mallei); Brucella spp.; Chlamydia psittaci (avian
strains only); Coxiella burnetti; Mycobacterium bovis

C. REQUIREMENTS FOR WORK WITH INFECTIOUS AGENTS
1. Known pathogens
Having decided the risk level of certain work it is then possible to decide the appropriate ‘containment level’ that
is needed to minimise the risk of human disease, and the risk of spread of disease to animals and the
environment. The containment level is defined by a combination of the physical facilities and working practices
employed. Organisms of the four Risk Groups indicated above may be placed into containment levels
appropriate for safe working, see below. Laboratories usually appoint a Biological Safety Officer, responsible for
ensuring that microorganisms are handled at the appropriate containment level. They should have sufficient
expertise and be of sufficient seniority to oversee and advise on all safety matters. In large organisations with a
network of laboratories, it is appropriate to appoint a central Safety Officer to advise on and coordinate safety
matters of a corporate nature, which are implemented by local laboratory Safety Officers at each site. The
working methods for a particular procedure or work station should be written out and readily available. Staff must
be fully trained and fully aware of any health risks associated with their work and in procedures for reporting
incidents or accidents. Staff should also be given a medical card indicating pathogens to which they might be
exposed. In some cases, staff can be specially vaccinated to give additional protection, e.g. when working with
the rabies virus; this should also be recorded on the medical card. Such information is useful for a medical
practitioner in the event of illness occurring. Regular medical examinations of employees are recommended and,
as appropriate, monitoring tests of employees working with the organisms that cause certain serious human
diseases, such as brucellosis and tuberculosis.
Much information is available on containment of pathogens, and sophisticated apparatus and buildings may be
constructed for containment of the more hazardous organisms as required by the guidelines, standards and
regulations of each country. The requirements depend on the containment required, from the most basic to the
highest level.
Essential requirements for all laboratory work. The essential requirements for any work with infectious
agents, however innocuous they may seem, are as follows:
1. The laboratory should be easy to clean, with surfaces that are impervious to water and resistant to
chemicals. There shall be a wash-hand basin and emergency shower, including an eye bath, in each
laboratory suite as appropriate for the chemicals and other hazards present. Procedures shall be
established for frequent cleaning and disinfection during and at the end of the work period;
2. Personnel access to the work area should be restricted; appropriate security measures such as controlled
electronic access may be necessary with higher risk agents.
3. Personal protective equipment such as long-sleeved lab coats or gowns, closed-toe footwear, disposable
gloves, masks, safety glasses, face shields, and oro-nasal respirators, as appropriate, shall be worn in the
laboratory and removed when leaving the laboratory
4. The laboratory door should be closed when work is in progress and ventilation should be provided by
extracting air from the room. (Where biosafety cabinets are used, care shall be taken to balance ventilation
systems.);
5. Food (including chewing gum, candy, throat lozenges and cough drops) and/or drink shall not be stored or
consumed in laboratories;
6. Smoking and/or application of cosmetics shall not take place in the laboratory;
7. Pipetting shall not be done by mouth;
8. Care shall be taken to minimise the production of aerosols;
9. Emergency response plans should be developed to deal with the biohazard of spills. Some of the items
addressed in the plans should include having effective disinfectant available for cleaning spills, removal of
and decontamination of contaminated protective clothing, washing of hands, and cleaning and disinfection
of bench tops;
10. Used laboratory glassware and other contaminated material shall be stored safely.. Materials for disposal
shall be transported without spillage in strong containers. Waste material should be autoclaved, incinerated
or otherwise decontaminated before disposal. Reusable material shall be decontaminated by appropriate
means;
11. No infectious material shall be discarded down laboratory sinks or any other drain;
12. Any accidents or incidents shall be recorded and reported to the Safety Officer.

Containment level for Group 2 pathogens, in addition to the points given above, a Class I, II or III
microbiological safety cabinet should be used when there is potential for generating aerosols or when handling
large quantities of culture or where there is a real need to protect the biological product (see Section D).
Appropriate signs are required at all entry doors to indicate the hazard present and the name and telephone
number of the person(s) responsible. Emergency protocols should be posted within the laboratory to advise
personnel of procedures to follow in case of a pathogen spill or the need to evacuate the laboratory in the event
of a fire or other emergency.
Containment level for Group 3 pathogens, it is advisable that the laboratory be in an isolated location; access
should be limited to appropriately trained level 3 staff. Emergency protocols should be posted within the
laboratory to advise personnel of procedures to follow in case of a pathogen spill or the need to evacuate the
laboratory in the event of a fire. OIE containment level for Group 3 pathogens surpasses biosafety level-3 (BSL-
3) guidelines as outlined by the United States Department of Health and Human Services (DHHS) (16) and the
United States Department of Agriculture (USDA) (15).
In addition to the previous requirements, the laboratory shall be under negative pressure and the pressure
differentials should be monitored; a procedure should be developed to provide an alarm if there is a problem and
personnel to respond to the alarm. A ventilation system is required that removes air from the laboratory through a
high efficiency particulate air (HEPA) filter. HEPA filters shall be verified regularly (usually annually); this would
include HEPA filters in biosafety cabinets and on room and equipment exhausts. The laboratory should be
sealable for fumigation and contain an airlock entry. There is a requirement to treat effluent depending on the
pathogen. Biological safety cabinets of Class I, II or III shall be used whenever the process to be undertaken is
likely to generate an aerosol (17). It may be necessary for staff to shower on exit from the laboratory and they
must wear dedicated laboratory clothing that is left in the laboratory before leaving the building.
Note. Because of the link between bovine spongiform encephalopathy (BSE) and new variant Creutzfeldt-Jakob
disease in humans, BSE and related agents are now categorised with the human transmissible spongiform
encephalopathies in Risk Group 3. Consequently, veterinarians and laboratory workers conducting necropsies on
BSE-suspect animals or handling tissues derived from such animals must conduct the work under appropriately
strict containment conditions, sometimes with derogations allowed by the nature of the work and the results of
local risk assessment. It is important that appropriate protective clothing be worn and that a strict code of practice
be followed to prevent exposure to the agent. Laboratories conducting work on BSE must comply with national
biocontainment and biosafety regulations (3).
Containment level for Group 4 pathogens, the most stringent precautions are required, including access to the
building through air locks, and the building being maintained under negative air pressure. Inlet air to the
laboratory shall be filtered through a single HEPA filter and extracted air through double HEPA filters in series. All
work with infective materials shall be conducted in a Class III cabinet or in a Class II cabinet in conjunction with
the use of one-piece positive-pressure suits. All sewage from the laboratory, laboratory effluent and autoclave
drain effluent shall be treated by appropriate means to ensure that all infectious material is destroyed before
entering the sewerage outside the laboratory. Staff shall shower and change their clothing before leaving the
building. Other precautions as described for Group 3 would also apply. The use of one-piece positive-pressure
suits is now an internationally accepted way of providing additional protection at level 4.
OIE guidelines for the containment level for Group 4 pathogens are generally equal to the USDA’s biosafety
level 3 Ag guidelines (15). The primary difference between OIE level 4 and BSL-3 Ag is that the BSL-3 Ag
guidelines specify that the laboratory will be airtight and shall pass a pressure decay test to confirm that it does
not surpass the prescribed maximum leak rate.
2. Diagnostic specimens
Veterinary diagnostic centres readily receive specimens that are submitted because they are suspect for a
variety of diseases. The infectious nature of the specimens is usually unknown, but they have the potential to
contain biological agents that may cause disease in animals and humans. Practices and procedures need to be
in place that will minimise the risk of occupational exposure of employees to such pathogens. Unless suspected
of containing a pathogen requiring a higher containment level, it is advisable that initial processing of all unknown
specimens should be carried out as though the material contained a Group 2 pathogen. The most important
aspects are to prevent percutaneous, mucous membrane exposure, particularly inhalation and ingestion.
Biological safety cabinets should be used for all manipulations that may generate aerosols. Class I or II are
appropriate depending on the need for protection of the samples from contamination. Additionally, there should
be no mouth pipetting, personal protective clothing shall be worn with, in some cases, eye and respiratory
protection, depending on the anticipated level of exposure. Although initial diagnostic procedures may be carried
out at level 2, once a Group 3 or 4 organism has been isolated (or suspected) further work must be carried out at
the higher containment level.

D. MICROBIOLOGICAL SAFETY CABINETS

These are used at the different containment levels, as described in Section C above. They are of three types:
Class I: An open-fronted cabinet designed specifically to provide operator and environmental protection and not
to give protection to the work being handled.
Class II: An open-fronted safety cabinet, sometimes referred to as a laminar flow recirculating cabinet. They are
designed to give operator, product and environment protection.
Class III: These cabinets are closed, with glove ports at the front, and provide the highest degree of containment
by complete separation of work and worker. Some cabinets have a removable glove port and are known as Class
III/I cabinets, i.e. they can be used in either mode.
Descriptions of safety cabinets and safe working practices have been published (8, 10, 17).
E. STORAGE OF PATHOGENS
Storage of live pathogens requires appropriate containment and security to avoid risks due to breakage or
unauthorised use of material. Storage facilities should be appropriately labelled to indicate the nature of the
pathogens (e.g. their Group) and the contact information for the person(s) responsible for them. A complete
inventory of the pathogens in storage should be kept up to date and available. Special care must be taken when
opening glass vials of freeze-dried pathogens, as these can sometimes shatter. Care must be taken when
working with liquid nitrogen or rooms where asphyxiating gasses may be produced.
Many of the considerations given above relate not only to human safety but also to prevention of the spread of
infection to animals. In a veterinary laboratory an important responsibility is to minimise any risk of escape of
pathogens to animals, either wild or domestic, in the outside community. Close communication must be
maintained with the veterinary authorities. There may be national requirements for special licences to work with
certain microorganisms.
F. PHYSICAL AND CHEMICAL HAZARDS

Laboratory work involves many manipulations that are potentially dangerous, such as handling glassware and
work with needles or other sharp instruments. There shall be appropriate procedures and equipment for the safe
and proper disposal of needles and other ‘sharps’.
Laboratory staff should be protected from the risk of receiving a burn from hot solids or liquids. Autoclaves shall
be fitted with safety devices to prevent accidental opening of doors when under pressure, and be regularly
serviced and tested. Heat-protective gloves, apron and face shields with brow and chin guards shall be provided.
Extreme cold can also be a risk, for example when working with liquid nitrogen; splashes on exposed skin can be
very damaging. Gloves should be worn that provide insulation from cold and that are also waterproof, to prevent
penetration of the liquid nitrogen. Face shields with brow and chin guards and boots should also be worn when
working with liquid nitrogen. Nitrogen evaporating from liquid nitrogen storage in poorly ventilated rooms can lead
to depletion of oxygen with fatal consequences.
Irradiation is a serious health risk that may be present due to the use of X-ray machines, or use of gamma-
emitters or other sources. Equipment shall be regularly serviced and tested. All use of radioactive material must
be meticulously recorded. All staff must wear a personal radiation-monitoring device and have annual health
checks. Local and national regulations must be followed (10).
A wide range of chemicals are used in veterinary laboratories, many of which may be toxic or mutagenic, and
some may be carcinogenic. It should be remembered that it is the dose that makes the poison. Vapours are
especially hazardous, and some chemicals can be absorbed by penetration of intact skin. Steam sterilisation
may make toxic chemicals volatile and endanger personnel who unload the autoclave/pressure steam steriliser.
Procedures sufficient to protect pregnant laboratory workers should be followed at all times. A list of hazardous
chemicals shall be maintained, and a file record kept of chemicals to which individual staff members could be
exposed. This is now a legal requirement in some countries. Chemicals shall be correctly stored in appropriate
containers and at the correct temperature. Those that are flammable shall be kept in a fireproof chemical store. A
record must be maintained of the purchase and use of hazardous chemicals: how much, when used, by whom
and for what purpose. Disposal of some chemicals is subject to official regulation.
Further information on physical and chemical safety precautions can be found in the literature (11, 12).

G. LABORATORY ANIMAL FACILITIES

Work with pathogens in laboratory animals poses special risks. Animal rooms have to be constructed to
appropriate standards and containment levels, just as laboratories. Containment in animal houses is very
important because of the large amount of infectious agents that they may generate. Similar considerations also
apply regarding the training of staff, protective clothing and the recording of working procedures. Special care
must be taken to avoid injury to staff, e.g. through animals biting and kicking or self inoculation accidents. Any
such incidents must be recorded and wounds appropriately treated. There shall be provision for autoclaving
steam sterilisation, incineration or rendering of carcasses and for the thorough cleansing and disinfection of
animal rooms. The animal rooms should not only provide a suitable environment for the animals themselves but
should be constructed and ventilated in such a way as to ensure comfort for the attending personnel. This is a
large subject that can only be referred to briefly here (4, 7). Also, an excellent book on health and safety in
laboratory animal facilities is available (18).
H. EMERGENCY PROVISIONS
First-aid equipment should be readily available, but stored in a location that is unlikely to be contaminated by
work conducted in the laboratory (for example, in the air-lock or ante-room). This equipment shall be appropriate
to the work and properly maintained. It shall be kept ready to hand for immediate emergency use by trained first
aid personnel. Bandages and dressings should be available. Some staff shall receive training in safety and first
aid from recognised authorities and shall possess a valid certificate as evidence of competence. Personnel
working in Containment Level 4 facilities shall have advanced first aid competence. Their names and locations
should be known to everyone and posted on notice boards. All staff should be aware of the importance of safety.
There must be suitable procedures and equipment for dealing with spillages and decontamination. A record must
be kept of all incidents and in some countries there may be a legal obligation to report incidents to the enforcing
authority.
There must be written procedures for dealing with emergency failure of all safety and containment systems, for
example in biosafety cabinets or biocontainment rooms, which can lead to loss of containment.
Many laboratories have a staff safety committee to increase safety awareness and to discuss safety issues with
management. Personnel are responsible for their own safety and those around them. Managers are equally
responsible for safety in their area of command and should not allow consideration of speed or cost of work to
come before the safety of personnel or containment of animal disease agents.
There must be an emergency procedure for obtaining medical assistance if required, and for hospitalisation in
appropriate infectious disease facilities when needed. Fire alarms shall be fitted, and tested regularly. The
institute or laboratory must designate a warden to control and communicate in emergency situations and conduct
periodic drills to make staff aware of what to do and where to assemble in the event of an emergency. The
warden is responsible for checking that everyone is in a safe location. Procedures for natural disasters, such as
hurricanes and earthquakes, should be in place where they present a risk. All these procedures should be written
down and periodically reviewed.
I. TRANSPORT OF INFECTIOUS MATERIAL

Great care must be taken when preparing and packing diagnostic specimens, infectious materials and pathogens
for transport, to ensure that there is no breakage of containers or leakage of contents that could put at risk
personnel in the transport system or animals that may come in contact with contamination. Applicable local,
national and international regulations for the transportation of dangerous goods (diagnostic or clinical sample and
infectious materials) and importation of animal pathogens must be followed. These are summarised in Chapter
1.1.1 Collection and shipment of diagnostic specimens.
When categorising animal pathogens into specific Groups, the following criteria should be taken into account:
a) Group 1 animal pathogens

Disease-producing organisms that are enzootic but not subject to official control.
b) Group 2 animal pathogens

Disease-producing organisms that are either exotic or enzootic but subject to official control and that have a
low risk of spread from the laboratory.

c) Group 3 animal pathogens

moderate risk of spread from the laboratory.
d) Group 4 animal pathogens

high risk of spread from the laboratory into the environment and the national animal population.
J. CONTAINMENT GROUPS
1. The principal purpose of containment is to prevent the escape of the pathogen from the laboratory into the
national animal population. Some animal pathogens can infect humans. In these instances the risk to
human health may demand additional containment than would otherwise be considered necessary from
purely animal health considerations. The risk of human to animal transmission of disease must also be
considered and controlled. In addition, other animals being used for experimental work on the pathogen
should be held in the appropriate containment level.
2. The level of physical containment and biosafety procedures and practices should be not less than the
Group into which the pathogen has been placed and the detailed requirements should be appropriate to the
type of organism (i.e. bacterium, virus, fungus or parasite). The lowest containment level will be required for
pathogens in Group 1 and the highest level for those in Group 4. Guidance on the containment
requirements for Groups 2, 3 and 4 is provided in Section K.
3. Arthropods may be pathogens or vectors for pathogens. If they are a vector for a pathogen being used in
the laboratory, the appropriate containment level for the pathogen will be necessary in addition to the
containment facilities for the arthropod.
K. GUIDANCE ON THE LABORATORY/ANIMAL FACILITY REQUIREMENTS FOR

THE DIFFERENT CONTAINMENT GROUPS
Containment Group
Requirements of the laboratory/animal facility 2 3 4
A) Laboratory/animal facility setting and structure
1. It is advisable that the laboratory/animal facility be in an Yes Yes

isolated location
2. Not next to known fire hazard Yes Yes Yes
3. Workplace separated from other activities Yes Yes Yes
4. Personnel access limited Yes Yes Yes
5. Protected against entry/exit of rodents and insects Yes Yes Yes
6. Liquid effluent must be sterilised and monitored Yes Yes
7. Liquid effluent from steam sterilisers shall be sterilised Yes

and monitored
8. Isolated by airlock. Continuous internal airflow Yes Yes
9. The laboratory/animal facility shall be under negative Yes Yes

pressure and the pressure differentials should be
monitored
10. Input air to be filtered using HEPA or equivalent such as Single on extract Single for input,
gas tight damper; exhaust air to be single HEPA filtration double for extract
for laboratories and double HEPA filtration for animal
facilities.

Containment Group
A) Laboratory/animal facility setting and structure (cont.)
11. HEPA filters shall be verified regularly (usually annually) Yes Yes
12. Mechanical air supply system with fail-safe system and Yes Yes
an alarm provided if there is a problem
13. Laboratory/animal facility sealable to permit fumigation Yes Yes
14. Incinerator, pressure steam steriliser or renderer for Available Yes Yes on site
disposal of carcasses and waste
15. The laboratory/animal facility should be easy to clean, Yes Yes Yes
with surfaces that are impervious to water and resistant
to chemicals. There shall be a wash-hand basin and
emergency shower, including an eye bath, in each
laboratory suite as appropriate for the chemicals and
other hazards present. Procedures shall be established
for frequent cleaning and disinfection during and at the
end of the work period
B) Additional Laboratory facility requirements
16. Class I or II biological safety cabinet available Yes Yes Yes

17. Class III biological safety cabinet available Yes Yes
18 HEPA filters shall be verified regularly (usually annually) Yes Yes Yes
19. Direct access to autoclave/pressure steam steriliser Yes Yes with Yes with
double doors double doors
20. Specified pathogens stored in laboratory Yes Yes Yes
21. Double-ended dunk tank required Preferable Yes
22. Personal protective clothing and equipment not worn Yes Yes Yes
outside laboratory
23. Full body shower and change of clothing required before It may be necessary Yes
exiting laboratory for staff to shower on
exit from the
laboratory and they
must wear dedicated
laboratory clothing
that is left in the
laboratory before
leaving the building
24. Safety Officer responsible for containment Yes Yes Yes
25. Staff receive special training and demonstrate Yes Yes Yes
competence in the requirements needed
C) Laboratory discipline
26. Warning notices for containment area to indicate the Yes Yes Yes
hazard present and the name and telephone number of
the person(s) responsible
27. Emergency protocols should be posted within the Yes Yes Yes
laboratory to advise personnel of procedures to follow in
case of a pathogen spill or the need to evacuate the
laboratory in the event of a fire or other emergency
28. Laboratory must be lockable Yes Yes Yes
29. Authorised entry of personnel Yes Yes Yes
30. Protective clothing, including gloves, masks, eye shields, Yes Yes Yes
and oro-nasal respirators, as appropriate, shall be worn in
the laboratory and removed when leaving the laboratory

Containment Group
C) Laboratory discipline (cont.)
31. The laboratory door should be closed when work is in Yes Yes Yes
progress and ventilation should be provided by extracting
air from the room. (Where biosafety cabinets are used,
care shall be taken to balance ventilation systems.)
32. Food and/or drink shall not be stored or consumed in Yes Yes Yes
laboratories
33. Smoking and/or application of cosmetics shall not take Yes Yes Yes
place in the laboratory
34. Pipetting shall not be done by mouth Yes Yes Yes
35. Care shall be taken to minimise the production of Yes Yes Yes
aerosols
36. No infectious material shall be discarded down laboratory Yes Yes Yes
sinks or any other drain
37. Used laboratory glassware and other materials shall be Yes Yes Yes
stored safely before disinfection. Materials for disposal
shall be transported without spillage in strong containers.
Waste material should be autoclaved, incinerated or
otherwise made safe before disposal. Reusable material
shall be decontaminated by appropriate means
38. Any accidents or incidents shall be recorded and reported Yes Yes Yes
to the Safety Officer
39. On entering all clothing removed and clean clothes put on Yes Yes
40. On exiting all laboratory clothes removed, individual shall Yes
wash and transfer to clean side
41. Individual shall shower prior to transfer to clean side Yes
D) Handling of specimens
42. Packaging requirements to be advised prior to Yes Yes Yes

submission
43. Incoming packages opened by trained staff in Yes Yes Yes
appropriately contained reception area
44. Movement of pathogens from an approved laboratory to Yes Yes Yes
another requires a licence
45. Standard Operating Procedures covering all areas must Yes Yes Yes
be available
1. Additional requirements for work in animal facilities
1. The animal facility should be easy to clean, with surfaces that are impervious to water and resistant to
chemicals used in the area.
2. Personnel access to the work area should be restricted; appropriate security measures such as controlled
electronic access may be necessary with higher risk agents.
3. Personal protective equipment such as coveralls, boots, disposable gloves, masks, safety glasses, face
shields, and oro-nasal respirators, as appropriate, shall be worn in the animal facility and removed when
leaving the animal facility.
4. The animal facility door should be closed when work is in progress and ventilation should be provided by
extracting air from the room. (Where biosafety cabinets are used, care shall be taken to balance ventilation
systems.)

5. Food (including chewing gum, candy, throat lozenges and cough drops) and/or drink shall not be stored or
consumed in animal facilities.
6. Smoking and/or application of cosmetics shall not take place in the animal facility.
10. Used laboratory glassware and other materials shall be stored safely before disinfection. Materials for
disposal shall be transported without spillage in strong containers. Waste material should be autoclaved,
incinerated or otherwise made safe before disposal. Reusable material shall be decontaminated by
appropriate means.
11. No infectious material shall be discarded down animal facility drains without appropriate waste treatment in
place.
12. Any accidents or incidents shall be recorded and reported to the Safety Officer.
L. CONCLUSION
High standards of laboratory safety and containment that will ensure healthy working conditions for laboratory
staff and protection of the environment must be of the greatest priority. They can only be achieved by careful
study of the principles involved followed by practical application to premises, facilities, operating procedures and
hygiene. Training of all laboratory personnel must be a high priority and no personnel should be allowed to work
until appropriate training and competence has been demonstrated and documented. There is a large published
literature on all aspects of the subject, and further reading is recommended (6, 9, 13, 14, 19).
REFERENCES
1. ACHA P.N. & SZYFRES B. (2001). Zoonoses and Communicable Diseases Common to Man and Animals,
Third Edition. Pan American Health Organisation, Scientific Publication. PAHO, Washington DC, USA.
2. ADVISORY COMMITTEE ON DANGEROUS PATHOGENS (1995). Categorisation of Biological Agents According to

Hazard and Categories of Containment, Fourth Edition. Health and Safety Executive Books, P.O. Box 1999,
Sudbury, Suffolk, CO10 6FS, UK. ISBN 0-7176-1038-1.
3. ADVISORY COMMITTEE ON DANGEROUS PATHOGENS (1998). Transmissible Spongiform Encephalopathy Agents:

Safe Working and the Prevention of Infection. Spongiform Encephalopathy Advisory Committee. Her
Majesty’s Stationery Office, London, UK.
4. BARBEITO M.S., ABRAHAM G., BEST M., CAIRNS P., LANGEVIN P., STERRITT W.G., BARR D., MEULEPAS W.,
SANCHEZ-VIZCAINO J.M., SARAZA M., REQUENA E., COLLADO M., MANI P., BREEZE R., BRUNNER H., MEBUS C.A.,
MORGAN R.L., RUSK S., SIEGFRIED L.M. & THOMPSON L.H. (1995). Recommended biocontainment features for
research and diagnostic facilities where animal pathogens are used. Rev. sci. tech. Off. int. Epiz., 14, 873–
887.
5. BELL J.C., PALMER S.R. & PAYNE J.M. (1988). The Zoonoses. Edward Arnold, London, UK.
6. BERAN G.W. & STEELE J.H. (eds) (1994). Handbook of Zoonoses, Second Edition. Section A. (Bacterial,
Rickettsial, Chlamydial and Mycotic Zoonoses) and Section B (Viral Zoonoses), CRC Press, USA.
7. CANADIAN FOOD INSPECTION AGENCY (1996). Containment Standards for Veterinary Facilities, Best M., ed.
Canadian Food Inspection Agency, Ottawa, Ontario, Canada.
8. COLLINS C.H. (1990). Health hazards in microbiology. In: Handbook of Laboratory Health and Safety
Measures, Pal S.B., ed. Kluwer Academic Publications, Dordrecht, The Netherlands, 159–188.
9. COMITE EUROPEAN DE NORMALISATION (2000). EN12469:2000; Performance Criteria for Microbiological Safety
Cabinets. Comité European de Normalisation, Rue de Strassart 36, B-1040 Brussels, Belgium.
10. INTERNATIONAL ATOMIC ENERGY AGENCY (IAEA) (1994). International Basic Safety Standards for Protection
against Ionizing Radiation and for the Safety of Radiation Sources, Interim Edition. Safety Series No. 115-I.
IAEA, Vienna, Austria.
11. OFFICE OF BIOSAFETY, LABORATORY CENTRE FOR DISEASE CONTROL, HEALTH AND W ELFARE CANADA (1996).
Laboratory Biosafety Guidelines, Second Edition. Ministry of Supply and Services Canada, Cat. No. MR 21-
2/1996.

12. RAYBURN S.R. (1990). The Foundations of Laboratory Safety (a Guide for the Biomedical Laboratory).
Springer-Verlag, New York, USA.
13. RICHMOND Y. (ed) (1996–2002). Anthology of Biosafety: I–V. American Biological Safety Association.
Mundelein, Illinois, USA.
14. SEWELL D.L. (1995). Laboratory associated infections and biosafety. Clin. Microbiol. Rev., 8, 389–405.
15. UNITED STATES DEPARTMENT OF AGRICULTURE (2002). ARS Facilities Design Standards, Section 9 Biohazard
Containment Design. http://www.afm.ars.usda.gov/ppweb/242-01m.htm#H304
16. US DEPARTMENT OF HEALTH AND HUMAN SERVICES, CENTRES FOR DISEASE CONTROL AND PREVENTION AND
NATIONAL INSTITUTES OF HEALTH (2007). Biosafety in Microbiological and Biomedical Laboratories, Fifth
Edition., Washington DC, USA. http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm
17. U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, PUBLIC HEALTH SERVICE, CENTERS FOR DISEASE CONTROL
AND PREVENTION, AND NATIONAL INSTITUTES OF HEALTH (2000). Primary Containment for Biohazards: Selection,
Installation and Use of Biological Safety Cabinets, Second Edition. United States Government Printing
Office, Washington DC, USA.
18. W OOD M. & SMITH M.W. (EDS) (1999). Health and Safety in Laboratory Animal Facilities. Royal Society of
Medicine Press, London, UK, pp. 249.
19. W ORLD HEALTH ORGANIZATION (2004). Laboratory Biosafety Manual, Third Edition. WHO, Geneva,
Switzerland.
*
* *

CHAPTER 1.1.3.
QUALITY MANAGEMENT IN VETERINARY

TESTING LABORATORIES
SUMMARY
Valid laboratory results are essential for diagnosis, surveillance, and trade. Such results are
achieved by the use of good management practices, valid test and calibration methods, proper
technique, quality control, and quality assurance, all working together within a quality management
system. These subjects comprise one complex area of critical importance in the conduct of testing
and in the interpretation of test results. This subject may be called laboratory quality management,
and includes managerial, operational, and technical elements. A quality management programme
enables the laboratory to demonstrate that it operates a viable quality system and is able to
generate technically valid results. Additionally the quality management programme should enable
the laboratory to show that it meets the needs of its clients or customers. The need for the mutual
recognition of test results for international trade and the acceptance of international standards such
as the ISO/IEC 1 International Standard 17025 (7) for laboratory accreditation also affect the need
and requirements for laboratory quality management programmes. The OIE has published a
detailed standard on this subject (10). This chapter is not intended to reiterate the requirements of
these ISO or OIE documents. Rather, it outlines the important issues and considerations a
laboratory should address in the design and maintenance of its quality management programme.
KEY CONSIDERATIONS FOR THE DESIGN AND MAINTENANCE OF A

LABORATORY QUALITY MANAGEMENT PROGRAMME
In order to ensure that the quality management programme is appropriate and effective, the design must be
carefully thought out. The major categories of consideration and the key issues and activities within each of these
categories are outlined in the following seven sections of this chapter.
1. The work, responsibilities, and goals of the laboratory
Many factors affect the necessary elements and requirements of a quality management programme. These
factors include:
i) The type of testing done;

ii) The use of the test results;
iii) The impact of a questionable or erroneous result;
iv) The tolerance level of risk and liability;
v) Customer needs (e.g. sensitivity and specificity of the test method, costs, turnaround time);
vi) The role of the laboratory in legal work or in regulatory programmes;
vii) The role of the laboratory in assisting with, confirming, and/or overseeing the work of other laboratories; and
viii) The business goals of the laboratory, including the need for any third party recognition and/or accreditation.
1 International Organization for Standardization/International Electrochemical Commission.

Chapter 1.1.3. — Quality management in veterinary testing laboratories
2. Standards, guides, and references

It is recommended that the laboratory choose reputable and accepted standards and guides to assist in
designing the quality management programme. The OIE standard on this subject is a useful guideline (10). For
laboratories seeking accreditation, the use of ISO/IEC 17025 (7) and/or the OIE standard (10) will be essential.
Further information on standards may be obtained from the national standards body of each country, from the
International Laboratory Accreditation Cooperation (ILAC), and from accreditation bodies (e.g. the National
Association of Testing Authorities [NATA], Australia and the American Association for Laboratory Accreditation
[A2LA], United States of America. Technical and international organisations such as the AOAC International
(formerly the Association of Official Analytical Chemists) and the ISO publish useful references, guides, and/or
standards that supplement the general requirements of ISO/IEC 17025. ISO International Standard 9001 (8), a
general standard for quality management systems and one of the many standards in the group commonly termed
the ‘ISO 9000 series’, is not usable for accreditation, as conformity with its requirements does not necessarily
ensure or imply technical competence (see Section 3. below). While a laboratory may implement a quality
management system meeting the requirements of ISO 9001, registration or certification is used to indicate
conformity with this standard, not accreditation, as ISO 9001 is not a competence standard: see Section 3,
below.
3. Accreditation
If the laboratory has determined that it needs formal recognition of its quality management programme, then third
party verification of its conformity with the selected standard(s) will be necessary. ILAC has published specific
requirements and guides for laboratories and accreditation bodies. Under the ILAC system, ISO/IEC 17025 is to
be used for accreditation. Definitions regarding laboratory accreditation may be found in ISO/IEC International
Standard 17000 (5). Accreditation is tied to competence and this is significant as it means much more than
having and following documented procedures. Having competence also means that the laboratory:
i) Has technically valid and validated test methods, procedures, and specifications that are documented in
accordance with the requirements of the selected standard(s) and/or guidelines;
ii) Has adequate qualified and appropriately trained personnel who understand the science behind the
procedures;
iii) Has correct and adequate equipment;
iv) Has adequate facilities and environmental control;
v) Has procedures and specifications that ensure accurate and reliable results;
vi) Can foresee technical needs and problems and implement continual improvements;
vii) Can cope with and prevent technical problems that may arise;
viii) Can accurately estimate and control the uncertainty in testing; and
ix) Can demonstrate proficiency to conduct the test methods used.
x) Has demonstrated competence to generate technically valid results.
4. Selection of an accreditation body
In order for accreditation to facilitate the acceptance of the laboratory’s test results for trade, the accreditation
must be recognised by the international community. Therefore, the accreditation body should be recognised as
competent to accredit laboratories. Programmes for the recognition of accreditation bodies are, in the ILAC
scheme, based on the requirements of ISO/IEC International Standard 17011 (6). One may obtain information on
recognised accreditation bodies from the organisations that recognise them, such as the Asia-Pacific Laboratory
Accreditation Cooperation (APLAC), the Interamerican Accreditation Cooperation (IAAC), and the European Co-
operation for Accreditation (EA).
5. Determination of the scope of the quality management programme and/or of the

laboratory’s accreditation
The quality management programme should ideally cover all areas of activity affecting all testing that is done at
the laboratory. However, for the purpose of accreditation, the laboratory should determine the scope of testing to
be included in the accreditation. Factors that might affect the laboratory’s choice of scope of accreditation
include:
i) The availability and cost of necessary personnel, facilities and equipment;

ii) The cost of environmental monitoring against the possibility of cross contamination;

iii) The deadline for accreditation;

iv) The impact of the test results;
v) The number of tests done;
vi) Whether the testing done is routine or non-routine;
vii) Whether any part of testing is subcontracted out;
viii) The quality assurance necessary for materials, reagents and media;
ix) The availability of reference standards (e.g. standardised reagents, internal quality control samples,
reference cultures);
x) The availability of proficiency testing;
xi) The availability, from reputable sources, of standard and/or fully validated test methods;
xii) The evaluation and validation of test methods to be done,
xiii) The technical complexity of the method (s); and
xiv) The cost of maintaining staff competence to do the testing.
Accreditation bodies also accredit the providers and operators of proficiency testing programmes, and may
require the use of an accredited provider, where available and feasible, in order to issue the laboratory a
certificate of accreditation. Accreditation against ISO/IEC Guide 43-1 (assessment against ILAC G13:08/2007) is
recommended (3, 4).
6. Test methods
ISO/IEC 17025 requires the use of appropriate test methods and has requirements for selection, development,
and validation. The OIE document (10) also provides requirements for selection and validation.
This Terrestrial Manual provides recommendations on the selection of test methods for trade and diagnostic
purposes in the chapters on specific diseases. In addition, a list of tests for international trade is provided. As
stated in the introduction to this list, the prescribed tests that are listed are those that are required by the OIE
Terrestrial Animal Health Code. These tests are considered to be adequately validated to give reliable results to
qualify animals for international movement. Also listed are alternative tests that are suitable for the diagnosis of
disease within a local setting, but that may have had limited validation. These tests are generally serological
tests.
In the veterinary profession, other standard methods 2 or fully validated methods 3, while preferable to use, may
not be available. Many veterinary laboratories develop or modify methods, and most of these laboratories have
test programmes that use non-standard methods, or a combination of standard and non-standard methods. In
veterinary laboratories, even with the use of standard methods, some in-house evaluation, optimisation, and/or
validation generally must be done to ensure valid results.
Customers and laboratory staff must have a clear understanding of the performance characteristics of the test,
and customers should be informed if the method is non-standard. Many veterinary testing laboratories will
therefore need to demonstrate competence in the development, adaptation, and validation of test methods.
This Terrestrial Manual provides more detailed and specific guidance on test selection, optimisation,
standardisation, and validation in Chapter 1.1.4 Principles of validation of diagnostic assays for infectious
diseases. The following items discuss test method issues that are of most interest to those involved in the quality
management of the laboratory.
a) Selection of the test method

Valid results begin with the selection of a test method that meets the needs of the laboratory’s customers in
addressing the diagnostic issues at hand. Considerations for the selection of a test method include:
i) International acceptance;
ii) Scientific acceptance;
2 Standard Methods: Methods published in international, regional, or national standards.

3 Validated Methods: Methods having undergone a full collaborative study and that are published or issued by an
authoritative technical body such as the AOAC International.

iii) Method is the current technology or a recent version;

iv) Performance characteristics (e.g. analytical and diagnostic sensitivity and specificity, repeatability,
reproducibility, isolation rate, lower limit of detection, precision, trueness, and uncertainty);
v) Behaviour in species and population of interest;
vi) Resources and time available for development, adaptation, and/or evaluation;
vii) Performance time and turnaround time;
viii) Type of sample (e.g. serum, tissue) and its expected quality or state on arrival at the laboratory;
ix) Analyte (e.g. antibody, antigen);
x) Resources and technology of the laboratory;
xi) Nature of the intended use (e.g. export, import, surveillance, screening, confirmatory, individual animal
use, herd use);
xii) Customer expectations;
xiii) Safety factors;
xiv) Number of tests to be done;
xv) Cost of test, per sample;
xvi) Existence of reference standards, including reference materials; and
xvi) Availability of proficiency testing schemes.
b) Optimisation and standardisation of the test method

Once the method has been selected, it must be set up at the laboratory. Whether the method was
developed in-house or imported from an outside source, generally some additional optimisation is
necessary. Optimisation is a series of experiments and subsequent data analysis. Optimisation establishes
critical specifications and performance standards for the test process and for use in monitoring the correct
performance of the test. Optimisation should ensure that a method is brought under statistical control.
Optimisation should also determine:
i) Critical specifications for equipment and instruments;

ii) Critical specifications for reagents (e.g. chemicals, biologicals);
iii) Critical specifications for reference standards, reference materials, and internal controls;
iv) Robustness (if applicable);
v) Critical control points and acceptable ranges, attributes or behaviour at critical control points, using
statistically acceptable procedures;
vi) The quality control activities necessary to monitor critical control points;
vii) The type, number, range, frequency, and/or arrangement of test run controls needed;
viii) The requirements for control behaviour for the non-subjective acceptance or rejection of test results;
ix) The elements of a fixed, documented test method for use by laboratory staff; and
x) The level of technical competence required of those who carry out and/or interpret the test.
c) Validation of the test method

Validation further evaluates the test for its fitness for a given use. Validation establishes performance
characteristics for the test method, such as sensitivity, specificity, and isolation rate; and diagnostic
parameters such as positive/negative cut-off, and titre of interest or significance. Validation should be done
using an optimised, documented, and fixed procedure. Depending on logistical and risk factors, validation
may involve any number of activities and amount of data, with subsequent data analysis using appropriate
statistics. Test validation is covered in Chapter 1.1.4 Principles of validation of diagnostic assays for
infectious diseases, and Chapter 1.1.5 Validation and quality control of polymerase chain reaction methods
used for the diagnosis of infectious diseases.
Validation activities might include:
i) Field and/or epidemiological studies;

ii) Comparison with other methods, preferably standard methods;

iii) Comparison with reference standards (if available);

iv) Collaborative studies with other laboratories using the same documented method, and including the
exchange of samples, preferably of undisclosed composition or titre. It is preferable that these be
issued by a qualified conducting laboratory that organises the study and evaluates the results provided
by the participants;
v) Reproduction of data from an accepted standard method, or from a reputable publication;
vi) Experimental infection studies; and
vii) Analysis of internal quality control data.
Validation is always a balance between costs, risks, and technical possibilities. Experienced accreditation
bodies know that there are many cases in which quantities such as accuracy and precision can only be
given in a simplified way.
It is also important to develop criteria and procedures for the correlation of test results for diagnosis of
disease status or regulatory action, including retesting, screening methods, and confirmatory testing.
d) Uncertainty
Laboratories should be able to estimate the uncertainty of the test methods as performed in the laboratory.
This includes methods used by the laboratory to calibrate equipment (7).
The determination of measurement uncertainty (MU) is not new to some areas of measurement sciences.
However, the application of the principles of MU to laboratories for the life sciences is new. Most of the work
to date regarding MU is for areas of testing other than the life sciences, and much of the work has been
theoretical. However, as accreditation becomes more important, applications are being developed for the
other areas. It is important to note that MU does not imply doubt about the validity of a test result or other
measurement, nor is it equivalent to error, as it may be applied to all test results derived from a particular
procedure. It may be viewed as a quantitative expression of reliability, and is commonly expressed as a
number after a +/– sign (i.e. the true value lies within the stated range, as MU is expressed as a range).
Standard deviation and confidence interval are examples of the expression of MU. An example of the use of
standard deviation to express uncertainty is the allowed limits on the test run controls for an enzyme-linked
immunosorbent assay, commonly expressed as +/– n SD.
Although the determination and expression of MU has not been standardised for veterinary testing
laboratories (or medical, food, or environmental), some sound guidance exists.
MU must be estimated in the laboratory for each method included in the scope of accreditation. This can be
estimated by a series of tests on control samples. MU can also be estimated using published characteristics
(9), but the laboratory must first demonstrate acceptable performance with the method. Government
agencies may also set goals for MU values for official methods (e.g. Health Canada). Reputable technical
organisations and accreditation bodies (e.g. AOAC International, ISO, NATA, A2LA, SCC, UKAS,
Eurachem, and the Co-Operation on International Traceability in Analytical Chemistry [CITAC]) teach
courses and/or provide guidance on MU for laboratories seeking accreditation. Codex Alimentarius, which
specifies standards for food testing, has taken the approach that it is not necessary for a laboratory to take
a further estimate of MU if the laboratory complies with Codex principles regarding quality: i.e. that the
laboratory is accredited to ISO/IEC 17025, and therefore uses validated methods (e.g. knows applicable
parameters such as sensitivity and specificity, as well as the confidence interval around such parameters),
participates in proficiency testing programmes and collaborative studies, and uses appropriate internal
quality control procedures.
The requirement for “use of appropriate internal quality control procedures” implies that the laboratory must
use quality control procedures that cover all major sources of uncertainty. There is no requirement to cover
each component separately. Components can be estimated with experiments in the laboratory (Type A
estimates), or from other sources (reference materials, calibration certificates, etc.) (Type B estimates). A
traditional control sample procedure, run many times by all analysts and over all shifts, usually covers all the
major sources of uncertainty except perhaps sample preparation. The variation of the control samples can
be used as an estimate of those combined sources of uncertainty.
ISO/IEC 17025 requires the laboratory to identify all major sources of uncertainty, and to obtain reliable
estimates of MU. Laboratories may wish to establish acceptable specifications, criteria, and/or ranges at
critical control points for each component. Where appropriate, laboratories can implement appropriate
quality control at the critical points associated with each source, or seek to reduce the size of a component.
Sources of uncertainty include sampling, storage conditions, sample effects, extraction and recovery,
reagent quality, reference material purity, volumetric manipulations, environmental conditions,
contamination, equipment effects, analyst or operator bias, biological variability, and other unknown or
random effects. The laboratory would also be expected to account for any known systematic error (see also

Section 6.b. steps i–vii). Systematic errors (bias) must either be corrected by changes in the method,
adjusted mathematically, or have the bias noted in the report statement. If an adjustment is made to the
procedure, there may or may not be a need to reassess uncertainty. If there is an adjustment made to
correct for bias, then a new source of uncertainty is introduced (the uncertainty of the correction). This must
be added to the MU estimate.
There are three principal ways to estimate MU:
1. The components approach (or ‘bottom-up’ approach), where all the sources of uncertainty are
identified, reasonable estimates are made for each component, a mathematical model is developed
that links the components, and the variations are combined using rules for the propagation of error (1).
2. The control sample approach (or ‘top-down’ approach), where measurements on a stable control
material are used to estimate the combined variation of many components. Variation from additional
sources needs to be added.
3. The method characteristics approach, where performance data from a valid collaborative study are
used as combined uncertainties (other sources may need to be added). Laboratories must meet
defined criteria for bias and repeatability for the MU estimates to be valid. These should be larger than
would be obtained by competent laboratories using their own control samples or components model.
Additional information on the analysis of uncertainty may be found in the Eurachem Guide to Quantifying
Uncertainty in Measurement (2).
e) Implementation and use of the test method

Analysts should be able to demonstrate proficiency in using the test method prior to producing reported
results, and on an ongoing basis.
The laboratory should ensure, using appropriate and documented project management, records keeping,
data management, and archiving procedures, that it can recreate at need all events relating to test
selection, development, optimisation, standardisation, validation, implementation, and use. This includes
quality control and quality assurance activities.
7. Strategic planning
Continual improvement is essential. It is recommended that the laboratory be knowledgeable of and stay current
with the standards and methods used to demonstrate laboratory competence and to establish and maintain
technical validity. The methods by which this may be accomplished include:
i) Attendance at conferences;
ii) Participation in local and international organisations;
iii) Participation in writing national and international standards (e.g. participation on ILAC and ISO committees);
iv) Consulting publications;
v) Visits to other laboratories;
vi) Conducting research;
vii) Participation in cooperative programmes (e.g. Inter-American Institute for Cooperation in Agriculture);
viii) Exchange of procedures, methods, reagents, samples, personnel, and ideas;
ix) Wherever possible, accreditation and maintenance thereof by a third party that is itself recognised as
competent to issue the accreditation;
x) Preplanned, continual professional development and technical training;
xi) Management reviews;
xii) Analysis of customer feedback; and
xiii) Preventive action implementation
REFERENCES
1. AMERICAN NATIONAL STANDARDS INSTITUTE (1997). ANSI/NCSL Z540-2-1997, US Guide to the Expression of
Uncertainty in Measurement, First Edition. American National Standards Institute, 1819 L Street, NW,
Washington, DC 20036, USA.

2. EURACHEM (2000). Guide to Quantifying Uncertainty in Analytical Measurement, Second Edition. Eurachem
Secretariat, as Secretary, Mr Nick Boley, LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY,
United Kingdom.
3. ILAC G13:08/2007 (2007). Guidelines for the Requirements for the Competence of Providers of Proficiency
Testing Schemes. International Laboratory Accreditation Cooperation (ILAC). Secretariat, NATA, 7 Leeds
Street, Rhodes, MSW 2138, Australia.
4. ISO/IEC GUIDE 43-1 (1997). Proficiency testing by interlaboratory comparisons – Part 1: Development and
operation of proficiency testing schemes. International Organisation for Standardisation (ISO), ISO Central
Secretariat, 1 rue de Varembé, Case Postale 56, CH-1211, Geneva 20, Switzerland.
5. ISO/IEC INTERNATIONAL STANDARD 17000 (2004). Conformity assessment – Vocabulary and general
principles. International Organisation for Standardisation (ISO), ISO Central Secretariat, 1 rue de Varembé,
Case Postale 56, CH - 1211, Geneva 20, Switzerland.
6. ISO/IEC INTERNATIONAL STANDARD 17011 (2004) 4. Conformity assessment -- General requirements for
accreditation bodies accrediting conformity assessment bodies. International Organisation for
Standardisation (ISO), ISO Central Secretariat, 1 rue de Varembé, Case Postale 56, CH - 1211, Geneva 20,
Switzerland.
7. ISO/IEC INTERNATIONAL STANDARD 17025 (2005). General requirements for the competence of testing and
calibration laboratories. International Organisation for Standardisation (ISO), ISO Central Secretariat, 1 rue
de Varembé, Case Postale 56, CH - 1211, Geneva 20, Switzerland.
8. ISO INTERNATIONAL STANDARD 9001 (2000). Quality management systems – Requirements. International
Organization for Standardization (ISO), ISO Central Secretariat, 1 rue de Varembé, Case Postale 56, CH -
1211, Geneva 20, Switzerland.
9. ISO/TS 21748 (2004). Guidance for the use of repeatability, reproducibility and trueness estimates in
measurement uncertainty estimation. International Organisation for Standardisation (ISO), ISO Central
Secretariat, 1 rue de Varembé, Case Postale 56, CH - 1211, Geneva 20, Switzerland.
10. W ORLD ORGANISATION FOR ANIMAL HEALTH (2008). Standard for Management and Technical Requirements for
Laboratories Conducting Tests for Infectious Animal Diseases. In: OIE Quality Standard and Guidelines for
Veterinary Laboratories: Infectious Diseases, Second Edition. World Organisation for Animal Health (OIE:
Office International des Epizooties), 12 rue de Prony, 75017 Paris, France, 1–25.
*
* *
4 ISO/IEC International Standard 17011 replaces ISO/IEC Guide 58 (1993). Calibration and Testing Laboratory
Accreditation Systems – General Requirements for Operation and Recognition.

CHAPTER 1.1.4.
PRINCIPLES OF VALIDATION OF DIAGNOSTIC

ASSAYS FOR INFECTIOUS DISEASES
INTRODUCTION
Validation is the evaluation of a process to determine its fitness for a particular use and includes
assay optimisation and demonstration of performance characteristics. An assay validated for an
infectious disease yields test results that identify the presence of a particular analyte (e.g.
components of an infectious agent or antibody induced by it) and allows predictions to be made
about the status of the test subjects. Assays applied to individuals or populations have many
purposes, such as aiding in: documenting freedom from disease in a country or region, preventing
spread of disease through trade, eradicating an infection from a region or country, confirming
diagnosis of clinical cases, estimating infection prevalence to facilitate risk analysis, identifying
infected animals toward implementation of control measures, and classifying animals for herd
health or immune status post-vaccination. A single assay may be validated for one or several
intended purposes by optimising its performance characteristics for each purpose (e.g. setting
diagnostic sensitivity [DSe] high [such as 99.99%] with associated lower diagnostic specificity
[DSp] for a screening assay, or conversely, setting DSp high with associated lower DSe for a
confirmatory assay).
The principles of validation discussed in this chapter will focus primarily on methods to detect
antibody in sera using an ELISA as an example. However, these same principles are applicable to
validation of tests for other analytes in sera or tissues. Chapter 1.1.5 Validation and quality control
of polymerase chain reaction methods used for the diagnosis of infectious diseases extends the
principles outlined here to a direct method of infectious agent detection, the molecular diagnostic
assays.
By considering the variables that affect an assay’s performance, the criteria that must be
addressed in assay validation become clearer. The variables can be grouped into three categories:
(a) the sample – host/organism interactions affecting the analyte composition and concentration in
the serum sample; (b) the assay system – physical, chemical, biological and technician-related
factors affecting the capacity of the assay to detect a specific analyte in the sample; and (c) the
test result – the capacity of a test result, derived from the assay system, to predict accurately the
status of the individual or population relative to the analyte in question.
Factors that affect the concentration and composition of analyte in the serum sample are mainly
attributable to the host and are either inherent (e.g. age, sex, breed, nutritional status, pregnancy,
immunological responsiveness) or acquired (e.g. passively acquired antibody, active immunity
elicited by vaccination or infection). Nonhost factors, such as contamination or deterioration of the
sample, may also affect the analyte in the sample.
Factors that interfere with the analytical accuracy of the assay system include instrumentation,
technician error, reagent choice (both chemical and biological) and calibration, accuracy and
acceptance limits of controls, reaction vessels, water quality, pH and ionicity of buffers and
diluents, incubation temperatures and durations, and error introduced by detection of closely
related analytes, such as antibody to cross-reactive organisms, rheumatoid factor, or heterophile
antibody.

Chapter 1.1.4. — Principles of validation of diagnostic assays for infectious diseases
Measures that influence the capacity of the test result to predict accurately the infection or analyte
status of the host 1 are DSe, DSp, and prevalence of the disease in the population targeted by the
assay. DSe and DSp are derived from test results on samples obtained from selected reference
animals. The methods used to select the reference animals are critical to the accuracy of the
estimates (5). The degree to which the reference animals represent all of the host and
environmental variables in the population targeted by the assay has a major impact on the
accuracy of test-result interpretation. For example, experienced diagnosticians are aware that an
assay, validated by using samples from northern European cattle, may not give valid results for the
distinctive populations of cattle in Africa.
The capacity of a positive or negative test result to predict accurately the infection and/or exposure
status of the animal or population of animals is the most important consideration of assay
validation. This capacity is not only dependent on a highly precise and accurate assay
(incorporating well-characterised and standardised reagents) and carefully derived estimates of
DSe and DSp, but is heavily influenced by prevalence of the infection in the targeted population or
the likelihood that an animal is infected based on clinical criteria. Without a current estimate of the
disease prevalence in that population or likelihood of infection in an individual animal, the
interpretation of a positive or negative test result may be compromised.
Many factors obviously must be addressed before an assay can be considered to be ‘validated’ (5,
16). However, there is no consensus whether the concept of assay validation is a time-limited
process during which only those factors intrinsic to the assay are optimised and standardised, or
whether the concept includes an ongoing validation of assay performance for as long as the assay
is used. Accordingly, the term ‘validated assay’ elicits various interpretations among laboratory
diagnosticians and veterinary clinicians. Therefore, a working definition of assay validation is
offered as a context for the guidelines outlined below. Ideally, all diagnostic assays would be fully
validated for one or more purposes, but in practice there are sometimes limitations to the
completeness of validation.
DEFINITIONS OF ASSAY VALIDATION
A validated assay consistently provides test results that identify animals as positive or negative for an analyte or
process (e.g. antibody, antigen, or induration at skin test site) and, by inference, accurately predicts the infection
and/or exposure status of animals with a predetermined degree of statistical certainty 2. Implicit in this definition is
the requirement that the test method was properly developed, optimised, and standardised to achieve
performance characteristics that are consistent with the purpose for which the assay is intended.
This chapter will focus on the principles underlying development and maintenance of a validated assay. Previous
iterations of this chapter (12) were condensed renditions of a review article (9). At that time, the goal was to
provide general principles of assay validation. In this update, the content is reorganised into the parts of assay
validation consistent with the format of the OIE Validation Template, and emphasises the absolute necessity of
pre-determining the specific purpose(s) for which the assay is intended. In addition to the validation process per
se, guidance is offered on scientifically sound methods for development, maintenance, and extension of
validation criteria for a given assay.
It must be emphasised that an assay, when applied to target populations, will minimise misclassifications of
animals as false positive or false negative only to the extent that validity is assured for all elements of the assay
validation process (see Section B. Assay Validation – Part I). This assumes that the assay is fit for the purpose
for which it is intended (e.g. a confirmatory assay will likely yield many false-negative results if used as a
1 In this chapter, the terms ‘positive’ and ‘negative’ have been reserved for test results and never refer to infection or
antibody/antigen status of the host. Whenever reference is made to ‘infection’ or ‘analyte’, any method of exposure to an
infectious agent that could be detected directly (e.g. antigen) or indirectly (e.g. antibody) by an assay, should be inferred.
2 In this definition, the DSe and DSp are performance characteristics of the assay for a given target population. They
determine – together with the disease prevalence in the population – the probability that a given test result reflects the
true status of the animal. An assay can be recognised as validated if reliable estimates of DSe and DSp for a given target
population are available. This does not imply any minimum threshold values for these parameters. In practical
applications, low values of DSe and DSp or diagnostic problems due to low disease prevalence are compensated by the
sampling design or by combining multiple diagnostic assays into parallel or serial testing regimens. The selection of
assays, the sampling process, the combination of multiple assays into a testing regimen and the interpretation rule for the
results define the diagnostic process.

screening assay). It also assumes that a well conceived, designed, and documented test method and proper
standardised reagents, in combination with well-trained technicians, will give a stable assay within the laboratory.
Furthermore, it assumes a thorough use of the most rigorous experimental design and epidemiological and
statistical tools. These are required to reduce bias, random error, and false assumptions about the reference
population of animals upon which the assay performance estimates are made (5). Finally, it assumes that when
placed in practice, the assay is conducted within the context of a rigorous quality assurance programme.
A. ESSENTIAL PREREQUESITES BEFORE VALIDATION OF AN ASSAY

1. Selection of an assay fit for its intended purpose
The OIE Standard for Management and Technical Requirements for Laboratories Conducting Tests for Infectious
Diseases (14) 3. This Standard states that test methods and related procedures must be appropriate for specific
diagnostic applications in order for the test results to be of any relevance. In other words, the assay must be ‘fit
for purpose’.
As outlined in the background information in Certification of diagnostic assays on the OIE website (www.oie.int),
the first step is selection of an assay type that likely can be validated for a particular use. The intended
purpose(s) of an assay have been broadly defined:
1) Demonstrate freedom from infection in a defined population (country/zone/compartment/herd) (prevalence

apparently zero):
1a) ‘Free’ with and/or without vaccination,
1b) Historical freedom,
1c) Re-establishment of freedom after outbreaks.
2) Certify freedom from infection or agent in individual animals or products for trade/movement purposes.
3) Eradication of infection from defined populations.
4) Confirmatory diagnosis of suspect or clinical cases (includes confirmation of positive screening test).
5) Estimate prevalence of infection or exposure to facilitate risk analysis (surveys, herd health status, disease
control measures).
6) Determine immune status of individual animals or populations (post-vaccination).
The OIE Standard further states that in order for a test method to be considered appropriate, it must be properly
validated and that this validation must respect the principles outlined in the validation chapters of the this
Terrestrial Manual.
While this chapter deals with validation and fitness for purpose from a scientific perspective, it should also be
noted that other factors might impact the relevance of an assay with respect to fitness for purpose. These factors
include not only the diagnostic suitability of the assay, but also its acceptability by scientific and regulatory
communities, acceptability to the client, and feasibility given available laboratory resources. An inability to meet
operational requirements of an assay also may make it unfit for its intended purpose. Such requirements may
include running costs, equipment availability, level of technical sophistication and interpretation skills, kit/reagent
availability, shelf life, transport requirements, safety, biosecurity, sample throughput, test turn-around times,
aspects of quality control and quality assurance.
2. Initial assay development considerations

An indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibody will be used in this chapter to
illustrate the principles of assay validation. It is a type of assay that can be difficult to validate because of signal
amplification of both specific and nonspecific components (2). This methodology serves to highlight the problems
that need to be addressed in any assay validation process. The same basic principles are used in validation of
other complex or simple assay formats. However, each unique type of assay, such as an antigen detection
ELISA, may have different sample collection and storage requirements. This chapter assumes that the assay
developer will have a high level of relevant scientific expertise to achieve proper preparation and use of protocols
and reagents, leading to a validated assay that is publishable in peer reviewed journals. Chapter 1.1.5 Validation
and quality control of polymerase chain reaction methods used for the diagnosis of infectious diseases describes
the principles for validating gene-amplification techniques.
3 This is a specific interpretation of the more generally stated requirements of the ISO/IEC 17025:2005 international quality
standard for testing laboratories (8).

Selection of appropriate samples, calibrated instrumentation and a relevant methodology to achieve the intended
purpose are critical elements in assay validation. Continuity in experiments is assured when reagents and
samples are chosen, properly prepared, aliquoted, and stored for use in each experiment. This reduces to a
minimum the number of variables and guards against failure when the validation process commences. This
approach reduces the variability and provides data needed to establish appropriate controls for ensuring each
run of the assay is valid.
a) Feasibility studies — selection of samples

Samples are needed for experiments to determine if the proposed assay is feasible. For this preliminary
step, it is useful to select four or five sera (in our example) that range from high to low levels of antibodies
against the infectious agent in question. In addition, a sample containing no antibody is required. These
samples ideally should represent known infected and uninfected animals from the population that eventually
will become the target of the assay once it is validated. The samples should have given expected results in
one or more serological assay(s) other than the one being validated. The samples are preferably derived
from individual animals, but they may represent pools of samples from several animals. These samples can
be used in experiments to determine if the assay is able to distinguish between varying quantities of analyte
(antibody in our example), and for optimising the reagent concentrations and perfecting the protocol.
A good practice is to prepare a large volume (e.g. 10 ml or more if possible) of each sample and divide it
into 0.1 ml aliquots for storage at or below –20°C. One aliquot of each sample is thawed, used for
experiments, and ideally then discarded. If it is impractical to discard the aliquot, it may be held at +4°C
between experiments for up to about 2 weeks; however, there is a possibility of sample deterioration under
these circumstances. Then, another aliquot is thawed for further experimentation. This method provides the
same source of serum with the same number of freeze–thaw cycles for all experiments (repeated freezing
and thawing of serum can denature antibodies so should be avoided). Also, variation between experiments
is reduced when the same source of serum is used for all experiments rather than switching among various
sera between experiments. This approach has the added advantage of generating a data trail for the
repeatedly run samples.
Repeated runs using these samples also can provide preliminary repeatability assessments both within and
between runs of the assay. When compared with international standards to establish their activity
(concentration or titre), one or more of these samples may also serve as secondary standards; such
standards provide assurance that runs of the assay are producing accurate data (16).
Finally, these pools of sera may be used as controls in future routine runs of the assay once all steps of the
validation process have been completed.
It is highly desirable to include OIE International Standard Sera or other international standard sera (if they
are available) at an early stage in assay development. This may facilitate harmonisation between the assay
under development and a standard test method in which international standard sera are normally used (15).
b) Selection of method to achieve normalised results

Normalisation adjusts raw test results of all samples relative to values of controls included in each run of the
assay (not to be confused with transformation of data to achieve a ‘normal’ [Gaussian] distribution). The
method of normalisation and expression of data should be determined, preferably no later than at the end of
the feasibility studies. Comparisons of results from day to day and between laboratories are most accurate
when normalised data are used. For example, in ELISA systems, raw optical density (absorbance) values
are absolute measurements that are influenced by ambient temperatures, test parameters, and photometric
instrumentation. To account for this variability, results are expressed as a function of the reactivity of one or
more serum control samples that are included in each run of the assay. Data normalisation is accomplished
in the indirect ELISA by expressing absorbance values in one of several ways (16). A simple and useful
method is to express all absorbance values as a percentage of a single high-positive serum control that is
included in each plate (Sample/Positive or S/P ratio). (This control must yield a result that is in the linear
range of measurement.) This method is adequate for most applications. More rigour can be brought to the
normalisation procedure by calculating results from a standard curve generated by several serum controls.
It requires a more sophisticated algorithm, such as linear regression or log-logit analysis. This approach is
more precise because it does not rely on only one high-positive control sample for data normalisation, but
rather uses several serum controls, adjusted to expected values, to plot a standard curve from which the
sample value is extrapolated. This method also allows for exclusion of a control value that may fall outside
expected confidence limits.
For assays that are end-pointed by sample titration, such as serum (viral) neutralisation, each run of the
assay is accepted or rejected based on whether control values fall within predetermined limits. Because
sample values usually are not adjusted to a control value, the data are not normalised by the strict definition
of the term.

Whatever method is used for normalisation of the data, it is essential to include additional controls for any
reagent that may introduce variability and thus undermine attempts to achieve a validated assay. The
normalised values for those controls need to fall within predetermined limits (e.g. within an appropriate
multiple of the standard deviation of the mean of many runs of each control). The chosen limits should
reflect a reasonable and tolerable assay run rejection rate and an acceptable risk that some test samples
may be misclassified.
B. ASSAY VALIDATION — PART 1
1. Optimisation and standardisation of reagents
Using several well-defined sera, such in-house standards as outlined in Section A.2.a of this chapter, or
reference standards from outside sources, the optimal concentrations/dilutions of the antigen adsorbed to the
plate, serum, enzyme–antibody conjugate, and substrate solution are determined through ‘checkerboard’
titrations of each reagent against all other reagents, following confirmation of the best choice of reaction vessels
(usually evaluation of two or three types of microtitre plates, each with its different binding characteristics, to
minimise background activity while achieving the maximum spread in activity between negative and high-positive
samples). Additional experiments determine the optimal temporal, chemical, and physical variables in the
protocol, including incubation temperatures and durations; the type, pH, and molarity of diluent, washing and
blocking buffers; and equipment used in each step of the assay (for instance pipettes and washers that give the
best reproducibility).
The choice of reagents and their characterisation must be carefully addressed, or the assay’s performance
characteristics likely will be compromised. For example, increased assay specificity can be accomplished
through recombinant expression of antigens or by use of monoclonal antibodies in antigen capture or antibody
competition assays. Alternatively, the method of reagent production can also lead to reduced specificity and
increased variability. For example, if a viral antigen used in the assay is derived from a viral culture system that is
also used to produce viral vaccines commonly used in the species targeted by the assay, nonspecific cross
reactivity may occur. Absorption of cross reactive antigens that are in both the vaccine and the antigen used in
the assay is necessary, or a cell culture control needs to be tested on each serum sample in routine runs of the
assay to identify and account for the extent of such cross reactivity. Obviously, anticipation of the negative or
positive impacts of reagent choice on the assay under development/validation is a major consideration, and
careful experimentation is necessary to establish an optimal assay.
When a reagent such as a serum control sample is nearing depletion, it is essential to prepare and repeatedly
test a replacement before such a control is depleted. The prospective control sample is included in 10–20 runs of
the assay before depletion of the original control to establish its proportional relationship to the nearly depleted
control. If the depleted sample was a positive control in ELISAs where the normalised value is expressed as a
per cent of that positive control, the proportional difference in ELISA activity between the original and
replacement sera must be factored into the normalisation algorithm to retain the same cut-off, and thus the same
DSe and DSp in the assay. When other reagents, such as antigen for capture of antibody, must be replaced,
they should be produced using the same criteria as for the original reagents, and tested in at least five runs of
the assay using a panel of sera that has been designed for this purpose. Reagent lots (serials) need to be
evaluated for consistency so variability is minimised in the assay as new lots are required. Whenever possible, it
is important to change only one reagent at a time to avoid the compound problem of evaluating more than one
variable at a time. Variability is minimised when reagents are well-characterised using methods other than that of
the target assay.
a) Linear operating range of the assay

The range of values that constitute the linear operating range of an assay is best determined by a dilution
series in which a high positive serum is serially diluted in a negative serum. Each dilution is then run at the
optimal working dilution in buffer, and the results plotted in the form of a ‘response-curve’. This curve,
sometimes referred to as a ‘dose–response curve’ as in pharmacological applications, establishes the linear
range of assay values that are valid for use in the assay.
b) Calibration against reference reagents

i) International standards
Serum standards and other reagents, available from OIE, WHO, FAO, or other international
organisations, can be used to harmonise the assay with expected results gained from reference
reagents of known activity.

ii) In-house standards

The in-house serum controls (used for normalisation of data) and additional secondary serum
standards, such as low positive, high positive, and negative sera (used for repeatability estimates in
subsequent routine runs of the assay) can be fitted to the response curve to achieve expected values
for such sera.
2. Repeatability
Preliminary evidence of repeatability (agreement between replicates within and between runs of the assay) is
necessary to warrant further development of the assay. This is accomplished by evaluating results from a
minimum of three in-house samples representing activity within the linear range of the assay. Quadruplicates of
these samples are tested in at least four runs of the assay to determine within-run (intraplate) variation. Between-
run variation is determined by using the same samples in a minimum of 20 runs (total), by two or more operators,
preferably on separate days. All runs must be independent of each other.
For reporting purposes, ELISA raw absorbance values are usually used to calculate repeatability during this part
of validation because it is uncertain whether the results of the high-positive control serum, which could be used
for calculating normalised values, are reproducible in early runs of the assay format. Also, expected values for
the controls have not yet been established. Coefficients of variation (CV: standard deviation of replicates ÷ mean
of replicates), generally less than 20% for raw absorbance values for most samples (low-titred samples may have
larger CVs), indicates adequate repeatability at this stage of assay development. However, if evidence of
excessive variation (>30%) is apparent for most samples within and/or between runs of the assay, more
preliminary studies should be done to determine whether stabilisation of the assay is possible, or whether the
test format should be abandoned. This is important because an assay that is inherently variable has a high
probability of not withstanding the rigours of day-to-day testing on samples from the targeted population of
animals.
Additional evidence of repeatability is obtained from the many additional runs of the assay that are required later
in the validation process to fully validate the assay. This is accomplished by running replicates of each control,
standard, and test sample when experiments are conducted to establish other validation parameters (see
Section C. Assay Validation – Part 2, below). Such data will lend confidence to repeatability estimates because
they will be based on within-run and between-run assay performance using reagents prepared daily, including
different lots of reagents that could affect repeatability.
3. Determination of analytical specificity and sensitivity
Analytical specificity of the assay is the degree to which the assay does not cross-react with other analytes and
analytical sensitivity is the smallest detectable amount of the analyte in question, i.e., the lowest detection limit of
the assay.
Analytical specificity is assessed by use of a panel of samples derived from animals that have been exposed to
genetically related organisms that may stimulate cross-reactive antibodies, or sera from animals with similar
clinical presentations. This ‘near neighbour analysis’ is useful in determining the probability of false-positive
reactions in the assay. It is also appropriate to document a group specificity criterion that includes detection of
the analyte of interest in sera from animals that have experienced infections/exposure to an entire group or
serotype of organisms of interest. It is also important to evaluate the analytical specificity of the assay using
samples from animals that have been vaccinated. If the assay targets antibody elicited by a virus, vaccination
against that virus may produce antibody that interferes with the assay’s inferences about infection. Also, if the
viral antigen used in the assay is derived from a whole-cell viral culture preparation, containing antigenic
reagents (carrier proteins, etc.) in addition to the virus, a vaccinated animal may test falsely positive due to
detection of nonviral antibodies.
Analytical sensitivity of an assay can be assessed by quantifying the least amount of analyte that is detectable in
the sample. This can be done by limiting dilutions of a standard of known concentration of the analyte. However,
such an objective absolute measure is often impossible to achieve due to lack of samples or standards of known
concentration or activity. Another approach is to use end-point dilution analysis of samples from known positive
animals, to define the penultimate dilution of sample in which the analyte is no longer detectable, or at least, is
indistinguishable from the activity of negative sera. When the results for the assay under development are
compared with other assay(s) run on the same samples, a relative measure of analytical sensitivity can be
estimated.
In addition to analyte standards or samples for which titers have been determined by other assays, it is possible
to create samples by spiking a negative sample matrix with known amounts of the analyte in question. In this
case, however, spiked samples may be intrinsically different from samples obtained from clinical cases, thus
leading to inferences that may not be accurate.

If the intended purpose of the assay is for screening of animals for antibody activity, analytical sensitivity needs
to be high to achieve the greatest probability possible for detecting infected animals. If very high analytical
sensitivity is not achievable, the assay may not be fit as a screening assay. Alternatively, if confirmation of
another independent diagnostic procedure is the purpose for which the assay is intended, analytical specificity is
required that minimises the amount of cross-reactivity. If neither of these objectives is obtainable, the reagents
need to be recalibrated, replaced, or the assay should be abandoned.
C. ASSAY VALIDATION — PART 2
1. Determining assay performance characteristics after establishment of a standard assay

method and reagent criteria
Estimates of DSe and DSp are the primary performance indicators established during validation of an
assay. These must be established after the assay and reagents are optimised and standardised; alteration
of protocols or reagents may require reestablishment of performance characteristics. They are the basis for
calculation of other parameters from which inferences are made about test results. Therefore, it is
imperative that estimates of DSe and DSp are as accurate as possible. Ideally, they are derived from
testing a series of samples from reference animals of known history and infection status relative to the
disease/infection in question and relevant to the country or region in which the test is to be used, but that is
not always possible. A sampling design must be chosen that will allow estimation of diagnostic performance
characteristics. However this is a difficult process complicated by logistical and financial limitations. It is also
limited by the fact that reference populations and gold standards may be lacking. The following are
examples of reference populations and methodologies that may aid in determining performance
characteristics of the test being validated.
a) Reference animal populations

i) Infected or exposed and uninfected or nonexposed reference animals
Selection of reference animals to evaluate performance characteristics requires that the variables
attributable to the target population are represented in the infected/exposed and uninfected/unexposed
reference animal populations. The variables include but are not limited to species, age, sex, breed,
nutritional status, pregnancy, stage of infection, immunological status including vaccination history,
and historical, epidemiological, and/or clinical data including herd disease history should be noted and
considered.
ii) Reference animal status determined by other assays

In serology, the ‘standard of comparison’ is the results of a method or combination of methods with
which the new assay is compared. Although the term ‘gold standard’ is commonly used to describe
any standard of comparison, it should be limited to methods that unequivocally classify animals as
infected/exposed or uninfected. Some isolation methods themselves have problems of repeatability
and sensitivity. Gold standard methods include unequivocal isolation of the agent or pathognomonic
histopathological criteria.
Because a true gold standard may be lacking or is impossible to achieve, relative standards of
comparison are often necessary; the most common of these include results from other serological
assays. Calculations of DSe and DSp are most reliable when the gold standard of comparison is
available. When only relative standards of comparison are available, estimates of DSe and DSp for
the new assay may be compromised because the error in the estimates of DSe and DSp for the
relative standard is carried over into those estimates for the new assay. Indeed, when using imperfect
reference tests without efforts to control for any biases, the DSe and DSp performance estimates of
the new test will be flawed and thus unacceptable.
iii) Experimentally infected or vaccinated reference animals

Sera obtained sequentially from experimentally infected or vaccinated animals have been used to
‘validate’ a new assay. Such repeated observations, pre- and post-seroconversion, from the same
animals are not acceptable for establishing estimates of DSe and DSp because the statistical
requirement of independent observations is violated. Thus, time-point sampling of individual
experimental animals is necessary. Also, exposure to organisms under experimental conditions, or
vaccination may elicit antibody responses that are not quantitatively and qualitatively typical of natural
infection in the target population (9). The strain of organism, dose, and route of administration to
experimental animals are examples of variables that may introduce error when extrapolating DSe and
DSp estimates to the target population. For these reasons, validation of an assay should not be based
solely on experimental animals.

iv) Reference animals – Status unknown

When it is not possible to assemble sera from animals of known infection status, it is possible to
estimate DSe and DSp by non-gold standard methods or latent class models (3, 7). Because these
statistical models are complex, an expert should be consulted to provide assistance on proper ways to
conduct and describe the sampling from the target population(s), the characteristics of other tests
included in the analysis, the appropriate choice of model and the estimation methods based on peer-
reviewed literature.
2. Threshold determination
To achieve performance estimates of DSe and DSp of the new assay, the test results first must be reduced to
categorical (positive or negative) status. This is accomplished by insertion of a cut-off point (threshold or decision
limit) on the continuous scale of test results. Although many methods have been described for this purpose,
three examples will illustrate different approaches, together with their advantages and disadvantages. The first is
a cut-off based on the frequency distributions (9) of test results from uninfected and infected reference animals.
This cut-off can be established empirically by visual inspection of the frequency distributions, by receiver-
operator characteristics (ROC) analysis (6, 17), or by selection that favours either DSe or DSp, depending on the
intended use for a given assay (11). A second approach is establishing a cut-off based only on uninfected
reference animals, for example the 99th percentile in a frequency distribution of assay values for uninfected
reference animals; this provides an estimate of DSp but not DSe. The third method provides an ‘intrinsic cut-off’
based on test results from sera drawn randomly from within the target population with no prior knowledge of the
animals’ infection status (4).
If considerable overlap occurs in the distributions of test values from known infected and uninfected animals, it is
difficult to select a cut-off that will accurately classify these animals according to their infection status. Rather
than a single cut-off, two cut-offs can be selected that define a high DSe (e.g. inclusion of 99% of the values from
infected animals), and a high DSp (e.g. 99% of the values from uninfected animals). The values that fall between
these percentiles would then be classified as suspicious or equivocal, and would require testing by a
confirmatory assay or retesting for detection of seroconversion.
The selection of the cut-off will typically reflect the intended purpose of the assay. For example, a screening
assay designed for high DSe versus a confirmatory assay designed for high DSp will require different cut-offs in
the same assay system. Although the intended purpose will dictate the cut-off, a ROC analysis is still desirable,
as it will show the potential performance of the assay in other epidemiological settings.
3. Assay performance estimates
a) Number of reference animals required

The number and source of reference samples coupled with the methodologies used to derive DSe and DSp
estimates are of paramount importance if the assay is ever to be properly validated for use in the population
of animals targeted by the assay. It is possible to calculate the number of reference samples, from animals
of known infection/exposure status, required for determinations of DSe and DSp that will have statistically
defined limits. Formulae and tables for determining the number of samples required are provided elsewhere
(5, 9). Table 1, page 474 of reference 9 reveals how the number of samples tested affects the confidence
levels in the calculated estimates of DSe and DSp for the assay. For example, an estimated DSe or DSp of
92% with a confidence level of 75% in that estimate requires 161 analyte-positive (known infected) animals
from the population targeted by the assay (with an allowable error of ± 2%). However, to increase
confidence in the estimate to a 95% level requires that 542 samples/animals be tested. The number of
samples theoretically required to achieve confidence levels ranging from 75% to 99% can be found in this
reference table for assays that are anticipated to have DSe or DSp ranging from 80% to 99%.
b) DSe and DSp estimates based on reference animals with defined infection status
The selection of a cut-off allows classification of test results into positive or negative categories.
Calculations of DSe and DSp are aided by associating the positive/negative categorical data with the known
infection status for each animal using a two-way (2 × 2) table (Table 1). After the cut-off is established,
results of tests on standard sera can be classified as true positive (TP) or true negative (TN) if they are in
agreement with those of the gold standard (or other standard of comparison). Alternatively, they are
classified as false positive (FP) or false negative (FN) if they disagree with the standard. Diagnostic
sensitivity is calculated as TP/ (TP + FN) whereas diagnostic specificity is TN/ (TN + FP); the results of both
calculations are usually expressed as percentages (Table 1).

Table 1. Calculations of DSe and DSp aided by a 2 × 2 table that associates infection status with
test results from 2000 reference animals
Reference animals of known

infection status
Infected (n = 600) Uninfected (n = 1400)
Positive 570 46
Test TP FP
Result FN TN
Negative 30 1354
Diagnostic sensitivity Diagnostic specificity
TP 570 TN 1354
= = 95.0% = = 96.7%
TP + FN 600 TN + FP 1400
c) DSe and DSp estimates based on animals with infection status not defined
DSe and DSp can be estimated when infection or analyte status of the animals are not defined; however,
these latent class statistical models are complex. Expert advice should be sought not only in the design of
the evaluation study but the interpretation of the estimates of DSe and DSp as well. It has been
recommended to the OIE that an expert group be formed to address the application of latent class models
and to draft guidelines for models as they apply to the validation and certification assays by the OIE.
4. Comparison and harmonisation of assays
New assays usually are developed to improve on existing techniques. In order to demonstrate that a new assay
is an improvement over an existing technique, there must be some form of comparison that demonstrates the
improvement. The comparison may be related to analytical and/or diagnostic performance characteristics. It may
also be related to operational characteristics such as cost, ruggedness, turn-around-times, throughput, etc. If the
new assay is to be incorporated into a diagnostic regimen involving other test methods, the rationale for its use,
interpretation of data and decision-making should be stated.
When an international standard method (15) is available for detection of an analyte, it is possible to harmonise
the performance of that method with the one under development. This process requires use of the same serum
controls and/or standards in both assays. If OIE Standard Sera or other international standard sera are available,
preferably at least three (negative, low positive, and high positive), they should be included in the assay-
comparison study. This could lead to a new assay that is indexed to an international standard method and
international standard sera (15). Harmonisation of the two assays may then be realised.
It is critical that all samples, test reagents, and the protocol or instructions for running the assay be properly
controlled. If the reagents will not be supplied from a common source, the laboratories should produce and
characterise the reagents independently. This will allow determination of the adequacy of the protocol for reagent
production and characterisation. This provides data needed to determine whether it is necessary to establish a
single shared source of well-characterised reagents. Part of the evaluation is the determination that the protocol
or instructions are complete, clear and precise. If verbal instructions are required, the developer should consider
revision of the protocol to ensure they are comprehensive. If it is determined that the protocol or instructions were
interpreted in a different manner, then they should be rewritten and the reproducibility may need to be re-
established using the revised protocol or instructions.
D. ASSAY VALIDATION — PART 3
1. Establishing reproducibility and augmenting repeatability estimates of the assay
An assay intended for distribution to many laboratories (such as a commercial kit) must be evaluated for
reproducibility, which is defined as the ability of a test method to provide consistent results when applied to
aliquots of the same samples tested at different laboratories. This is accomplished by testing a panel of sera in a
minimum of three laboratories using the identical test method and serum panels.

A test panel consisting of a minimum of 20 samples is assembled for this purpose. Ideally, these will be
individual samples from animals within the target population, representing the range of assay activity anticipated
in that population. If such samples are not available, dilution of a high positive with a negative serum to achieve
the range of activity is acceptable but not optimal. Replicates of about 20% of the samples are desirable as a
check on repeatability within each participating laboratory. Each sample is aliquoted, rendering a series of
identical panels for distribution to other laboratories. The sample identity is encoded for blind testing, and each
panel is handled, transported to participating laboratories, and stored identically.
The descriptive statistics for test panel data accumulated from the laboratories includes between-laboratory
mean, standard deviation, and range of results for each sample as well as controls. Evaluation of precision and
accuracy at each laboratory is facilitated by Youden plots. The data will help to inform the legitimacy of the upper
and lower control limits of the assay as established by the developer.
In addition, when the panel of samples is tested in each laboratory, it is advisable to run each sample in duplicate
or triplicate. This provides a basis for an expanded analysis of repeatability within each laboratory using the
assay. Also, when the assay is placed into routine use, repeatability is also monitored by inclusion of at least
duplicates for the controls and preferably for each sample as well.
E. ASSAY VALIDATION — PART 4
1. Programme implementation
Ultimate proof of the usefulness of an assay is its successful application(s). These would include international,
regional or national programs. As new and improved assays are developed and come on-line, they will ultimately
replace existing assays if they prove a better fitness for purpose. However, this will only happen if they are
actually put into routine use and their usefulness documented over time. In the natural progression of diagnostic
and/or technological improvement, some new assays will become the new standard of comparison. As such, they
may progressively achieve national, regional and international recognition. As a recognised standard, these
assays will also be used to develop reference reagents for quality control, proficiency and harmonisation
purposes. These reference reagents may also become international standards, as well. The last level of
validation in the OIE Registry involves documentation related to actual application and levels of recognition for
the assay in question. This is intended to provide potential users with an informed and unbiased source of
information.
2. Monitoring validity of assay performance
a) Interpretation of test results — factors affecting assay validity

An assay’s test results are useful only if the inferences made from them are accurate. A common error is to
assume that an assay with 99% DSe and 99% DSp will generate one false-positive and one false-negative
result for approximately every 100 tests on animals from the target population. Such an assay may be
precise and accurate, but produce test results that do not accurately predict infection status. For example, if
the prevalence of disease in a population targeted by the assay is only 1 per 1000 animals, and the false-
positive test rate is 1 per 100 animals (99% DSp), for every 1000 tests on that population, ten will be false
positive and one will be true positive. Hence, only approximately 9% of positive test results will accurately
predict the infection status of the animal; the positive test results will misclassify the animal 91% of the time.
This illustrates that the capacity of a positive or negative test result to predict infection status is dependent
on the prevalence of the infection in the target population (10). Of course, the prevalence will probably have
been determined by use of a serological test with its own inherent misclassification of results.
An estimate of prevalence in the target population is necessary for calculation of the predictive values of
positive (PV+) or negative (PV–) test results. When test values are reported without providing estimates of
the assay’s DSp and DSe, it is not possible to make informed predictions of infection status from test results
(9). It is, therefore, highly desirable to provide an interpretation statement with test results accompanied by
a small table indicating PV+ and PV– for a range of expected prevalences of infection in the target
population. Without provision of such information, test results from the assay may have failed to accurately
classify the infection status of animals, and thus do not reflect a fully validated assay.
b) Maintenance of validation criteria

A validated assay needs constant monitoring and maintenance to retain that designation. Once the assay is
put into routine use, internal quality control is accomplished by consistently monitoring the assay for
assessment of precision and accuracy (1).

Reproducibility between laboratories should be assessed at least twice each year. It is highly desirable to
become part of a consortium of laboratories that are interested in evaluating their output. In the near future,
good laboratory practice, including implementation of a total quality assurance programme, will become
essential for laboratories seeking to meet national and international certification requirements (see Chapter
1.1.3 Quality management in veterinary testing laboratories).
Proficiency testing is a form of external quality control for an assay. It is usually administered by a reference
laboratory that distributes panels of samples, receives the results from the laboratories, analyses the data,
and reports the results back to the laboratories. If results from an assay at a given laboratory remain within
acceptable limits and show evidence of accuracy and reproducibility, the laboratory may be certified by
government agencies or reference laboratories as an official laboratory for that assay (13). Panels of sera
for proficiency testing should contain a full representation of an analyte’s concentration in animals of the
target population. If the panels only have high-positive and low-positive sera (with none near the assay’s
cut-off), the exercise will only give evidence of reproducibility at the extremes of analyte concentration, and
will not clarify whether routine test results on the target population properly classify infection status of
animals.
c) Enhancement and extension of validation criteria

Because of the extraordinary set of variables that impact on the performance of serodiagnostic assays, it is
highly desirable to expand the number of standard sera from animals of known infection status because of
the principle that confidence in the estimates of DSe and DSp is enhanced with increasing sample size.
Furthermore, when the assay is to be applied in a completely different geographical region, it is essential to
re-validate the assay for its new intended use by subjecting it to sera from populations of animals that
reside under local conditions. The same is true for establishing DSe and DSp for subpopulations (e.g. age
groups, vaccinated/nonvaccinated, etc.).
REFERENCES
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Chemistry, Bishop M.L., Duben-Engelkirk J.L. & Fody E.P., eds. Lippincott, Philadelphia, USA, 63–101.
2. CROWTHER J.R. (1995). ELISA theory and practice. In: Methods in Molecular Biology. Humana Press,
Totowa, NJ, USA, 1–256.
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the sero-epidemiological study of Trypanosoma evansi infections in a canine population in Brazil: a new
approach towards unbiased estimation of prevalence. Acta Trop., 56, 97–109.
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Prev. Med., 45, 3–22.
6. GREINER M., PFEIFFER D. & SMITH R.D. (2000). Principles and practical application of the receiver operating
characteristic (ROC) analysis for diagnostic tests. Vet. Prev. Med., 45, 23–41.
7. HUI S.L. & W ALTER S.D. (1980). Estimating the error rates of diagnostic tests. Biometrics, 36, 167–171.
8. INTERNATIONAL ORGANIZATION FOR STANDARDIZATION/INTERNATIONAL ELECTROTECHNICAL COMMISSION (ISO/IEC)

(2005). ISO/IEC 17025:2005, General requirements for the competence of testing and calibration
laboratories.
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Off. int. Epiz., 17, 469–486.
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J. Am. Vet. Med. Assoc., 199, 1343–1347.
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infectious diseases. In: OIE Manual of Standards for Diagnostic Tests and Vaccines, Third Edition. OIE,
Paris, France, 8–15.

13. W ORLD ORGANISATION FOR ANIMAL HEALTH (OIE) (2002). OIE Guide 3: Laboratory Proficiency Testing. In: OIE
Quality Standard and Guidelines for Veterinary Laboratories: Infectious Diseases. OIE, Paris, France, 53–
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Requirements for Laboratories Conducting Tests for Infectious Diseases. In: OIE Quality Standard and
Guidelines for Veterinary Laboratories: Infectious Diseases. OIE, Paris, France, 1–31.
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antibody detection. Rev. sci. tech. Off. int. Epiz., 17, 527–533.
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of enzyme-linked immunosorbent assay techniques for the detection of antibody in infectious disease
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*
* *

CHAPTER 1.1.5.
VALIDATION AND QUALITY CONTROL OF

POLYMERASE CHAIN REACTION METHODS USED
FOR THE DIAGNOSIS OF INFECTIOUS DISEASES
INTRODUCTION
The diagnosis of infectious diseases is performed by direct and/or indirect detection of infectious
agents. By direct methods, the particles of the agents and/or their components, such as nucleic
acids, structural or non-structural proteins, enzymes, etc., are detected. The indirect methods
demonstrate the antibodies induced by the infections.
The most common direct detection methods are isolation or in-vitro cultivation, electron
microscopy, immunofluorescence, immunohistochemistry, antigen enzyme-linked immunosorbent
assay (antigen-ELISA), nucleic-acid hybridisation (NAH), macro- and microarrays and the various
techniques of nucleic acid amplification, such as the polymerase chain reaction (PCR) or the
isothermal amplification methods, such as nucleic acid sequence based amplification (NASBA),
Invader or loop-mediated isothermal amplification (LAMP). As NAH, macro- and microarrays and
the various amplification assays have nucleic acid molecules as targets, they are also termed
methods of molecular diagnosis.
The most common indirect methods of infectious agent detection are serological assays, such as
virus neutralisation, antibody-ELISA, haemagglutination inhibition tests, followed by the recently
appearing novel methods, such as biosensors, bioluminometry, fluorescence polarisation,
chemoluminescence, etc. In general, diagnostic laboratories simultaneously apply both the direct
and the indirect methods, in order to provide more certainty in a diagnosis.
The experiences of the last two decades indicate that the PCR techniques will eventually
supersede many of the classical direct methods of infectious agent detection. It is clear that the
PCR is replacing virus isolation or bacteria cultivation for the detection of agents that are difficult or
impossible to culture. There are several reasons for this trend, including that virus isolation
requires: i) the presence of replicating organisms (viruses or bacteria); ii) expensive cell culture
and maintenance facilities; iii) as long as several weeks to complete the diagnosis in some
instances; and iv) special expertise, which is missing or diminishing today in many laboratories.
Although PCR assays were initially expensive and cumbersome to use, they have now become
relatively inexpensive, safe and user-friendly tools in diagnostic laboratories (2–4, 6, 7, 11–13).
The sensitivity and specificity of PCR is generally greater than isolation or antigen capture ELISA
procedures. The introduction of various real-time PCR methods, nucleic acid extraction robots, and
automated work stations has resulted today in a large arsenal of high throughput, robust, very
rapid and reliable assays for molecular diagnosis. In this chapter the diagnostic applicability of the
various PCR methods is summarised with special regard to international harmonisation and
validation.
A. PCR METHODS USED IN ROUTINE MOLECULAR DIAGNOSTICS

1. The principles of the PCR
Polymerase chain reaction (PCR) implies that there is an enzyme-based amplification reaction in the assay. The
term ‘chain reaction’ refers to several cycles of copying a specified stretch of DNA, in this case from the genome
of an infectious agent. The region to be amplified is defined by two (or more) short nucleotide sequences, termed
primer sites that flank the target sequence. Primers, short oligonucleotides that are complementary to the primer
sites, bind to the DNA strand to be copied. Using a polymerase, which is not denatured during heat cycling, it is

Chapter 1.1.5. — Validation and quality control of PCR methods for the diagnosis of infectious disease
possible to copy the target sequence by joining free nucleotides to the primers. By repeating the heat-cycling
regime 20–40 times, the amount of copied target DNA increases exponentially, producing enough for further
operations, such as detection, cloning or sequencing. The diagnostic sensitivity of the PCR is very high because
several million copies of the selected target are produced. The specificity may also be very high, as determined
by the specific nucleotide sequences of the selected target, as well as primer design. The primers can be
designed to detect very specific nucleotide sequences in the genomes of the selected target infectious agents, or
can be designed to be complementary to more conserved regions, thus enabling detection of members within a
family or genus of infectious agent. A more detailed overview of molecular techniques has been published (17).
a) DNA amplification
If the genome of the infectious agent is DNA, the amplification is performed directly, with or without previous
purification of the target DNA. In many cases, use of DNA extracted and purified from the material to be
tested (e.g. blood) will result in increased analytical and diagnostic sensitivity.
b) RNA amplification (reverse-transcription PCR)

The genomes of many infectious agents contain ribonucleic acid (RNA) that cannot be amplified directly by
the PCR. For PCR amplification, a single-stranded DNA target is necessary, and this is not available in the
case of RNA viruses. This problem can be solved by the addition of a step before the PCR is begun. Using
reverse transcriptase, it is possible to transcribe the RNA into complementary DNA (cDNA), which can be
used in a PCR assay (the procedure is termed reverse transcriptase PCR). Traditionally, the reverse
transcription reaction is performed in a separate reaction vessel and the cDNA produced is then transferred
to a new tube for the PCR. However, heat-stable DNA polymerases with reverse transcriptase activity and
specific buffers in which RT and DNA polymerases are active are now readily available. Both allow a
reverse-transcription PCR amplification to take place in the same tube in direct sequence without any
further handling and with less chance of carry-over contamination. In most cases, it will be necessary to
extract and purify RNA prior to reverse transcription.
c) PCR amplicon detection

The PCR product, or amplicon, can be detected using a variety of procedures. The most common include
nonspecific detection of the PCR product based on amplicon size using electrophoresis in agarose gel and
staining of the DNA with a nonspecific, intercalating dye, such as ethidium bromide (it is now possible to
replace the latter with non-carcinogenic dyes, e.g. GelRed), or specific recognition of the amplified target
sequence using Southern blot transfer of the DNA followed by hybridisation with oligonucleotide probes
complementary to the target sequence. Hybridisation probes can be enzyme, chemiluminescent, or
radionucleotide-labelled to allow visual detection of the specific target sequence.
Some examples of PCR methods currently used are given below.
2. Conventional PCR
‘Conventional PCR’ (or simply PCR) uses one pair of oligonucleotide primers to amplify a small part of the genome
of the infectious agent. Analytical sensitivity is typically high with a minimum number of 100 to 1000 copies of the
target DNA detectable. Analytical specificity can be high, dependent on target selection, primer design, and assay
optimisation. Both analytical sensitivity and specificity can be further improved by applying nested PCR (see point 3
below). Detection methods, such as Southern blotting followed by hybridisation probes, can further improve
sensitivity and specificity, but are time-consuming, require laboratory handling of amplified DNA, and the
interpretation of results can be technically subjective. Based on complexity and expense, these detection methods
are not generally considered suitable procedures for common practice in diagnostic laboratories today.
3. Nested PCR
Nested PCR assays use two sets of amplification cycles with four primers, termed external and internal primers.
In general, nested PCR assays provide higher analytical sensitivity and specificity compared with conventional
PCR assays. However, there is a substantial increased risk of cross contamination as products from the first
round of amplification are often used as the starting template in the second round, resulting in the transfer of
material between different PCR tubes. The nested PCR has been largely replaced by real-time PCR protocols,
which are equally sensitive but have much less risk of contamination. The limit of detection with the nested PCR
is typically <10 genomic copies of the target DNA, and analytical specificity is also enhanced because in the
nested PCR, four oligonucleotide primers have to bind specifically to the selected targets in order to yield a
positive reaction (4).
4. Real-time PCR
Real-time PCR differs from standard PCR; here the amplified PCR products are detected directly during the
amplification cycles, using hybridisation probes, which enhance assay specificity. Various real-time methods,

such as TaqMan, Scorpion primers, fluorescence resonance energy transfer (FRET), Primer-Probe Energy
Transfer (PriProET), SybrGreen, Light-Upon-eXtension (LUX) and the Molecular Beacon assays have become
popular tools for detection of infectious agents. Real-time PCR has been used for the detection of bacteria,
viruses or parasites from a range of animal species (2–4, 14, 17). These assays have several advantages over
the ‘classical’ conventional or nested PCR methods. In general, only one primer pair is used, providing sensitivity
often close or equal to traditional nested PCR but with a much lower risk of contamination. Fluorescence,
indicating the presence of the amplified product, is measured through the lid or side of the reaction vessel, thus
there is no need for post-PCR handling of the amplified DNA. These procedures are considerably less time-
consuming compared with traditional post-amplification PCR product detection in agarose gels followed by
ethidium bromide or equivalent DNA detection stain and again, the risk of contamination is reduced. The use of a
96-well microtitre plate format, without the need for nested PCR, allows the procedure to be automated and
suitable for large-scale testing (10, 17). Diagnosis can be further automated by using robots for DNA/RNA
extractions and pipetting. Compared with classical amplification methods, a further advantage of the real-time
PCR is that it is possible to perform quantitative assays (6, 7). Using real time PCR, the diagnostic time can be
shortened from hours to minutes. Real-time PCR can also be used for reverse-transcription PCR using one-step
protocols, thus enabling the RT-step and PCR to take place in the same tube during the same PCR protocol (17).
5. Multiplex PCR
PCR using multiple primers directed at different targets in a single assay are referred to as multiplex PCR
assays. In multiplex PCR, various infectious agents can be detected and differentiated in a single reaction vessel
at the same time. The different PCR targets amplified in a standard PCR assay are identified based on PCR
product size. The use of ‘classical’ nested PCR methods for the construction of a multiplex assay is complicated
by the need for targets of different sizes, as well as primers that may ‘compete’ with each other in the same
reaction mix, both of which can negatively impact PCR efficiency. In contrast, the concept of real-time PCR
(single primer pairs) provides excellent possibilities for the construction of highly sensitive multiplex systems (4,
9) based on more uniform target size, uniform amplification conditions, and differential detection of targets using
specific hybridisation probes labelled with different fluorophores. It should also be noted that common primers
can be used to amplify specific regions of the genome of a group of pathogens and fluorogenic (TaqMan) probes
can then be employed to discriminate between members of the group. This is not strictly multiplex PCR although
it may mistakenly be described as such.
6. Further methods of molecular diagnosis

Although this chapter focuses on the PCR-based molecular diagnosis, it should be briefly mentioned that besides
PCR, there are many other novel methods being introduced for the molecular detection of pathogens, for example
the various isothermal amplification methods (such as nucleic acid sequence based amplification [NASBA], Invader
or loop-mediated isothermal amplification [LAMP] technologies), various macro- and microarrays using padlock
probes, rolling cycle amplification and other molecular approaches. There are also other approaches being
assessed to detect and analyse PCR products, such as MALDI and Luminex. The advantage of such approaches, in
combination with the multiplex PCR, is that typing of different strains or types by of an organism becomes possible. It
is evident that the arsenal of molecular diagnosis is further strengthened by these methods.
B. PRINCIPLES OF ASSAY VALIDATION FOR NUCLEIC ACID DETECTION TESTS

When performing analyses of clinical material it is important to produce data of good quality. For this, some key
criteria have to be fulfilled. The establishment of quality assurance (QA) and quality control (QC) systems is
required, i.e. a set of quality protocols, including the use of control samples that ensure that the system is
working properly and confirms data reproducibility and quality. QA and QC systems, together with trained and
competent personnel, have already been established in many laboratories world-wide. Assay validation is
another essential factor for assuring that test results reflect the true status of the samples (8).
To predict the diagnostic performance of a diagnostic assay, it is necessary to use a validation methodology to
document the expected performance of the assay in question. Validation is the evaluation of a diagnostic assay
for the purpose of determining how fit the assay is for a particular use. The general principles of assay validation
can be found in Chapter 1.1.4 Principles of validation of diagnostic assays for infectious disease. This chapter
extends these validation principles to molecular diagnostic assays. For explanations of terms and definitions
please consult Chapter 1.1.4.
C. ASSAY VALIDATION — INTRODUCTION

1. Selection of an assay fit for its intended purpose
The fitness of PCR assays for various purposes is broad. Wherever there is a need for direct detection of an
infectious agent, it is generally possible to use PCR. During the first years of PCR diagnostic development, many

laboratories had problems with contamination and performance; thus PCR had a poor reputation as a technique
suitable for diagnostic use. Achievements in recent years have reversed that view. New technology (i.e. real-time
PCR) has made the technique less prone to producing false positive results caused by contamination and is
easier to use. Furthermore, automating the extraction and pipetting procedures using robots has substantially
lowered the costs, enhanced repeatability and reduced the required work-load. During the ‘early years’ many in-
house assays were developed, and harmonisation and validation were poor or non-existent. The OIE, National
Laboratories and the European Community Reference Laboratories (ECRLs) have an important role to play in
driving the validation and harmonisation work forward. It is fair to say that PCR, as it is performed today, is safe
(substantially lower risk of false-positive results), usually validated in some form and fit for its intended purposes.
Some specific examples of the importance of PCR are given below, definitions of intended purpose(s) can be
found in Chapter 1.1.4.
• To diagnose infection when antibody levels are so low that previous exposure cannot be confirmed by an
antibody test (e.g. enzyme-linked immunosorbent assay [ELISA] repeatedly in the ‘gray zone’ during the
bovine leukaemia eradication programmes).
• To discriminate between infection and maternal immunity in young animals (e.g. young calves in eradication
programmes).
• To detect viral or bacterial nucleic acid when the diagnostic specimen is not suitable for virus isolation due
to toxicity (e.g. semen, exam of mummified fetus).
• In the final stage of eradication programmes, when thorough investigation of single cases is necessary (e.g.
herpesvirus latency and single reactor animals during the Aujeszky’s disease eradication programmes).
• To discriminate vaccine strains from field viruses (DIVA [differentiating infected from vaccinated animals]
approaches).
• To determine phylogenetic relationship of viruses and use this information for molecular epizootiology.
• To enable fast and safe first diagnosis in outbreak situations (e.g. the 2006 outbreaks of highly pathogenic
avian influenza).
• To determine the viral load (e.g. in porcine circovirus type 2 infections).
• Rapid monitoring of vaccinated animals that appear to have clinical signs.
• Detection of drug resistant mutants of pathogens, etc.
• To demonstrate freedom of infection in live animals or animal products. However, it has to be noted that
some infected animals may have no detectable nucleic acid in the tissues being examined.
2. Initial assay development considerations
a) Precautions and controls

Considering the uncertainty about the safety and reliability of the PCR in routine diagnosis, special
precautions should be applied in any laboratory using PCR for detecting infectious agents so as to avoid
false-positive or false-negative results. These, together with internal controls (e.g. mimics) assure the safe
evaluation of the results, free from false-positive results caused by contamination. True internal controls
using house-keeping genes also complete with the target PCR but only for reagents that are in vast excess
such as the polymerase and nucleotides. Minimal competition implies that the level of the true target is
known, which cannot be the case for field samples. Armoured RNAs allow the mimic to be added in the
extraction process, which is a step towards knowing that the extraction has worked. A true internal control
provides more confidence that the extraction has been performed correctly. The down side of using
housekeeping genes as internal controls is that they can be present in greater amounts than target
pathogens.
b) Precautions taken to avoid false-positive results

False-positive results (negative samples showing a positive reaction), may arise from either laboratory-
related issues, such as cross-contamination, or assay-related factors, such as inefficient optimisation or
assay performance. Product carry-over from positive samples or, more commonly, from cross-
contamination by PCR products from earlier experiments is a possible source of error, and various practices
and tools have been applied to prevent false-positive PCR results. Samples and reagents should be

handled in separate laminar air-flow hoods, which are regularly decontaminated using UV light (the use of
UV-light demands very careful maintenance to be effective) and bleach. Constructing and using special
tube-holders and openers can also help to prevent false-positives (2). In addition, good laboratory practices
should be applied, i.e. to perform the basic steps (DNA extraction, mix and primer preparation, sample
preparation, agarose gel electrophoresis of amplification products, etc.) in separated laboratory areas or
rooms (Figure 1; refs 1, 4, 17). Different sets of pipettes should be used for each of the steps. The use of
positive displacement and filtered tips is advisable. It is also, if possible, advisable to have different persons
perform the different steps, who are restricted to the respective laboratory areas. Precautions should be
taken to prevent the introduction of amplified material from potentially contaminated laboratories into ‘clean’
laboratory areas by movement restrictions on samples, papers, equipment, persons or any other potential
method of contamination. Movement in the opposite direction should only occur after surface
decontamination of equipment and tubes etc. and changing of laboratory coats and gloves. If the sample is
expected to have a high amount of agent or target nucleic acid, it is preferable to dilute it prior to introducing
it into ‘clean’ laboratory areas.
Figure 1. Recommended laboratory set-up for diagnostic real-time PCR. Samples to be analysed are transferred
into the ‘Preparation laboratory’ for extraction of nucleic acid. PCR master mix is prepared in the ‘Clean
laboratory’ and transferred to the ‘Preparation laboratory’ for dispensing into PCR plates and adding template.
Ready-made reaction tubes/plates are subsequently transferred into the ‘Apparatus room’ for the PCR run. The
‘Clean laboratory’ is used only for preparing PCR master mix; no DNA or PCR products are allowed in the
room. It is advisable to have an air lock entryway into the ‘Clean laboratory’ for changing into a lab coat and
shoes that are only used herein. The ‘Preparation laboratory’ is used for processing samples and setting up
PCR reactions (with master mix prepared in the ‘Clean laboratory’). No PCR products are allowed in this room
and nothing from the ‘Preparation laboratory’ goes back to the ‘Clean laboratory’. The ‘Apparatus room’
contains the PCR machines and nothing from this room goes back to the ‘Clean laboratory’ or the
‘Preparation laboratory’. If the laboratory has a controllable air system, the clean lab should be positive and
the other two negative. If nested PCR is going to be performed, two more rooms are recommended.
A ‘Second PCR laboratory’ for setting up the second PCR reaction (which will involve handling
PCR products, thus making it impossible to do this in the “Preparation laboratory’) and an ‘Electrophoreses
laboratory’ for analysing PCR products in agarose gels.
It is also very important to include negative controls, i.e. samples that are as similar to the test samples as
possible but without having the target. In laboratories experiencing problems with cross-contamination, at
least one negative control per five diagnostic samples has been recommended. Both positive and negative
control samples should routinely be interspersed with diagnostic samples to assess PCR assay
performance.
c) Precautions taken to avoid false-negative results

PCR has proven to be a very effective method of detecting nucleic acids, such as viral genomes in clinical
specimens. However, an infected animal in the later phases of infection may no longer have viral nucleic
acid in the tissues being examined. Consequently, in such cases the negative PCR results should be
considered as one part of a complex diagnostic examination.

False-negative results (samples containing the agent of interest but tested as negative) occur mostly due to
inhibitory effects and/or pipetting errors; however, issues attributable to sample handling can also yield
false-negative results. Therefore, internal controls can be used as indicators of PCR assay efficiency. PCR
internal controls may include foreign DNA added to the sample or ubiquitous DNA naturally occurring in the
sample. Foreign DNA added to the sample, may include DNA or RNA mimics. DNA mimics, manufactured
oligonucleotides, have the same primer-binding sequences as the PCR target, but flank a heterologous
DNA fragment of a different size. The identical primer-binding nucleotide sequences allow co-amplification
of the target and the mimic in the same tube with minimal competition. The size differences provide easy
discrimination by Southern blot analysis. Armored RNA®, an identical concept to DNA mimics, uses a
control RNA fragment packaged in bacteriophage coat proteins to protect or stabilise the RNA for control or
standardisation of RT-PCR assays (further details on internal controls, see above).
With real-time PCR assays, it is also possible to use internal controls, a naturally occurring housekeeping
gene, a selected fragment of the host animal’s genome such as beta-actin, GAPDH, or ribosomal RNA. By
multiplexing such an intrinsic control with a specifically coloured reporter fluorophore, it is possible to check
the sample quality and confirm PCR efficiency, as the target agent and intrinsic DNA are simultaneously
detected (14).
Internal controls (for example ‘mimics’) increase the reliability of diagnostic PCR (1, 4). Caution must be
used when designing and validating internal controls. Extensive testing is necessary to ensure that PCR
amplification of the added internal control does not compete with the diagnostic PCR and thus lower the
analytical sensitivity. Internal controls are used in concentrations slightly higher than the detection limit of
the diagnostic PCR to ensure the test’s performance. It should also be remembered that internal controls
have a disadvantage, similar to spiked samples, in not being representative of target nucleic acid and can
lead to false-negative results.
d) Preparation of standards
Reference laboratories should provide standard samples representative of a given infectious agent. Such
samples can be cultivated infectious agents or clinical specimens, etc., which are distributed in such a
manner that the infectious agent is well preserved. Thus the samples are distributed frozen, in organic
solvents (e.g. Trizol) or other suitable ways. The samples can also be sent as nucleic acids (frozen, freeze-
dried or in ethanol). For specific details, see the individual disease chapters. Reference laboratories should
also provide the appropriate mimics.
The availability of standard samples is crucial for a successful assay validation. Unfortunately this is often
the most problematic issue to solve when planning a validation project. It is not sufficient to use only
cultivated agents or spiked samples; true samples from the field may have very different characteristics
than these laboratory-generated samples. It might prove very difficult to obtain samples from the field that
are truly negative or positive, especially as PCR generally has a higher analytical sensitivity than most ‘Gold
Standard’ methods, thus making it difficult to determine the status of the intended validation samples using
already established assays. As mentioned previously, reference laboratories might be one potential source
for such standard samples. Alternatively, Bayesian methods offer probabilistic approaches to validate
diagnostic tests in the absence of a gold standard (5), but are not further discussed in this chapter.
D. ASSAY VALIDATION — PART 1
1. Optimisation and standardisation of reagents and determination of critical control

parameters
Sample collection, preparation and transport (see Chapter 1.1.1 Collection and shipment of diagnostic
specimens) and nucleic acid extraction methods (see Chapter 1.1.7 Biotechnology in the diagnosis of infectious
diseases and vaccine development) are all critical parameters in test performance and should be optimised for
disease diagnosis. Suitable methods vary depending on sample and organism type. In general, blood serum,
body tissues and swab samples are suitable samples for easy extraction of target nucleic acids, while faeces,
autolysed material and semen samples are more difficult to handle. Extraction of RNA targets differs from
extraction of DNA targets, and RNA is more prone to degradation. Both commercial (robotic, spin columns,
magnet-based extractions, etc.) and standard chemistry-based methods are used for DNA or RNA extraction. It
is crucial to determine the most reproducible and efficient extraction method before further validation of the assay
is performed. If the method of extraction is changed, equivalency data should be generated or the entire
validation procedure should be repeated.
All equipment used during the process must be properly maintained. Apparatus (heating blocks, refrigerators,
freezers, thermocyclers, pipettes, etc.) that require calibration must be calibrated according to the laboratory’s
quality assurance protocols. It is also important to properly validate the equipment and protocols used. One good

example is the recent implementation of robotic extraction methods for routine diagnostic processing. It is not
sufficient to compare the characteristics of this technique with that of the previously used extraction method. The
robot and the protocol must be validated to confirm that there is no danger of cross-contamination, e.g. by
running a set of mixed positive and negative samples.
When developing ‘classical’ or real-time PCR assays, all parameters, protocols and reagents need to be
optimised. A standardised assay is a method that consistently gives the same result for a given sample when
repeated several times and when performed by different analysts in different laboratories.
During the optimisation of the PCR assay, it is also possible to estimate the capacity of the method to remain
unaffected by small changes in the main parameters. To achieve an optimised PCR assay, it is essential to
evaluate critical parameters in the assay. Examples of such parameters include: incubation times and
temperatures, concentrations of buffers, primers, MgCl2, etc., pH, amounts of other components added (e.g.
dNTP, bovine serum albumin, etc.). The characterisation of critical control parameters is crucial for identifying
critical points that must be properly controlled in the assay. Intentional variations of parameters can lead to a
preliminary expression of assay robustness.
2. Repeatability
Agreement between replicates within and between runs of the assay should be accessed at this stage. This gives
important information about the assay before further validation is carried out. If excessive variability is
encountered, it should be corrected before continuing the validation process.
Repeatability of a PCR assay requires that each replicate be treated as an independent sample. According to
assess variation of a replicate (e.g. a triplicate), three individual aliquots of starting analyte are extracted and
amplified, and the variation from the mean value detected is determined as an indication of repeatability. Thus, it
is not acceptable to assess triplicate amplifications from one extraction. Likewise replicates from multiple runs
must be treated as individual samples. This process will result in estimates of intra- and inter-assay variability. In
a real-time PCR assay, the Ct-values produced from the replicated samples can be used to determine the inter-
run coefficient of variation (CV; see Chapter 1.1.3 Quality management in veterinary testing laboratories, Section
6.d Uncertainty).
It is important that the analyte to be detected in PCR be in the same matrix as test samples destined for use in
the assay. For example, if the assay is to be used for demonstrating freedom of an agent in a matrix known for
PCR-inhibitory activity (such as semen with extenders), it is particularly important to thoroughly evaluate
repeatability.
When new batches from new manufacturers of oligonucleotides or other reagents are introduced into the assay,
the repeatability of the assay needs to be re-established on each occasion.
3. Determination of analytical specificity and sensitivity
Analytical specificity is defined as the ability of an assay to distinguish the target agent from other infectious
agents. This ability is determined by analysing genetically related pathogens and clinical material obtained from
animals with diseases that may mimic that for which the assay is being designed. It is desirable to obtain field
samples from infected animals, but this may prove difficult or even impossible. In such cases viruses grown in
cell culture can be used. Acceptable cross-reactivity is largely dependent on the intended purpose of the test and
must be determined for each case. It is useful to perform ‘in silico’ studies as an adjunct to laboratory
assessment.
Analytical sensitivity (or limit of detection) is defined as the smallest amount of an agent detected by the assay,
and may be represented as number of genome copies, infectious dose, colony-forming units, plaque forming
units, etc. of the agent that can be detected and distinguished from a zero result. To determine analytical
sensitivity, an end-point dilution is used until the assay can no longer detect the target in question in more than
5% of the replicates. Cloned fragments of the PCR products in question can be used as standard samples, either
as DNA or for RNA targets, the RNA being transcribed in vitro into DNA. Estimates of analytical sensitivity can
vary substantially for the same assay when different sample matrices are used. When setting up a dilution series,
it is important to use a diluent that has qualities that are similar to the sample matrix, i.e. dilute positive semen in
negative semen and not in buffer.
E. ASSAY VALIDATION — PART 2

Performance characteristics (or assay parameters) give information about how a method functions under
specified conditions. Some typical performance characteristics are given in Chapter 1.1.4.

1. Determining assay performance characteristics
a) Reference animal populations

i) Negative reference animals
True negative samples, i.e. samples from animals that have had no possible exposure to the agent,
can sometimes be difficult to obtain. Often it is possible to collect samples from countries that have
eradicated the disease in question. It is important that the negative samples obtained be
representative of the samples that will be analysed, i.e. species, age, sex, breed, etc.
ii) Positive reference animals
It is generally problematic to find positive reference animals in sufficient numbers. Naturally infected or
experimentally infected animals are needed and their positive status is best demonstrated by isolation
of the agent. Before using experimentally infected animals, please see Chapter 1.1.4.
iii) Reference animal status determined by other assays
The term ‘gold standard’ is commonly used to describe any standard of comparison and should be
limited to methods that unequivocally classify animals as infected or uninfected. New PCR assays are
generally expected to outperform any already existing ‘gold standard’ method and thus the established
‘gold standard’ may not suitable to use as a comparison. This is especially true when demonstrating
that a negative reference animal is truly negative. Validation of molecular tests by comparing them to a
‘gold standard’ test may be complicated by the PCR being more sensitive, resulting in apparent
reduced specificity. To an extent this may be resolved by assessing sample derivation, clinical history
and sequencing any PCR products to confirm identity.
2. Threshold determination
Diagnostic sensitivity (DSe; proportion of known infected reference animals that test positive in the assay) and
specificity (DSp; proportion of known uninfected reference animals that test negative in the assay). The number
of reference samples required to determine estimates and allowable error of both DSe and DSp can be
calculated. To do this, a reasonable prediction of both DSe and DSp must be used. Generally, confidence in the
estimate is set at 95%. However, no formula can account for the numerous host/organism factors that can affect
the outcome of the test. The number of samples to determine estimates of DSe and DSp is outlined in Chapter
1.1.4. For a disease that is not endemic or widespread, it may be difficult initially to obtain the number of samples
to achieve a satisfactory confidence interval; but over time, accrual of additional data will enhance confidence in
the threshold. The use of spiked samples in PCR is a last resort because such samples might not be
representative of naturally infected samples. If samples from naturally infected animals are unavailable,
infections induced by means that mimic natural infections may provide samples that are useful. An example is
tick-borne infection induced by exposure to infected ticks.
It is not always possible to conform to suggested guidelines (e.g. OIE recommendations on test validation).
Faced with low numbers of samples for test evaluation, or for tests with no gold standard, one approach is to
introduce molecular tests as ‘partially validated’ and then add validation data if significant numbers of clinical
samples are tested. In this system, positives are confirmed by other means, such as isolation of the pathogen in
question or sequencing, and a sample of negatives is also confirmed as suitable (non-inhibitory) for PCR testing
using control genes. This principle of ‘on-going’ validation allows rapid introduction of new tests and reduces the
cost of validation. This process must be used under defined conditions. It is only applicable when there is sound
evidence from testing an appropriate range of known cultures, spiked samples (to provide analytical data) and
some clinical samples (to show that the target is available in particular tissues) that a test may be released as
partially validated (15).
F. ASSAY VALIDATION — PART 3
1. Establishing reproducibility of the assay
Reproducibility is an important parameter in assay precision. Reproducibility is determined in several laboratories

using the identical assay (protocol, reagents and controls). Each of at least three laboratories test the same
panel of samples (minimum of 20 samples), with identical aliquots going to each laboratory. This effort will yield
estimates of ruggedness of the assay. Reproducibility estimates for the assay are essential before it can be
considered valid for deployment to other laboratories.
Currently, reproducibility is rarely completely evaluated in veterinary diagnostic laboratories carrying out PCR
assays. Traditionally, many laboratories have used tests developed in-house, probably for practical reasons.
When possible, published standardised and validated methods, especially by OIE reference laboratories, ECRLs

or National Laboratories, should be followed. In addition inter-laboratory validation processes should be carried
out. This work will help to standardise assays, allowing harmonised diagnostic activity in various countries.
F. ASSAY VALIDATION — PART 4
1. Programme implementation
Reference laboratories play a major role in the implementation of new or validating existing molecular assays.
OIE Reference Laboratories ECRLs and National Laboratories are urged to assist in the implementation of
promising new assays for their disease of interest. An example is the assistance provided by the OIE and CDRL
to implement avian influenza molecular diagnostics in Europe.
2. Monitoring validity of assay performance
a) Interpretation of test results — factors affecting assay validity

A primary factor affecting interpretation of test results is the prevalence of the analyte in the target
population. A PCR assay that is highly precise and accurate, with estimates of DSe and DSp approaching
99%, may still provide false inferences (see Chapter 1.1.4). For nucleic acid assays, false-positive results
are of particular concern in low prevalence populations. In this instance, it may be necessary to confirm
PCR-positive results by sequence analysis of the amplified product to assist in correcting errors due to
nonspecific target or primer binding.
b) Maintenance of validation criteria

When the assay is used as a routine test, it is important to maintain the internal QC. The assay needs to be
consistently monitored for repeatability and accuracy. Reproducibility between laboratories (proficiency
testing) is recommended by the OIE to be completed at least twice a year (16). This testing is usually
administered by a reference laboratory that distributes panels of samples, receives the results from the
laboratories, analyses the data, and reports the results back to the laboratories. If the assay is to be applied
in another geographical region and/or population, it might be necessary to revalidate or document
equivalency under the new conditions. Revalidation or equivalency should be determined, if the test is
applied to a different sample matrix, e.g. validated on blood and used on another tissue, or validated for
cattle tissue and used on another species. Different extraction protocols may be needed if a different
species or tissues are tested; which possibly contain different inhibitory factors. This is especially true for
PCR assays as it is very common for point mutations to occur in many infectious agents (i.e. RNA viruses).
Mutations, which may occur within the primer or probe sites, can affect the efficiency of the assay and, by
doing so; the established performance criteria are no longer valid. It is also advisable to regularly confirm
the target sequence at the selected genomic regions for national or regional isolates of the infectious
agents. This is especially true for the primer sites, to ensure that they remain stable so that the validation of
the assay cannot be questioned. Validation and estimation of robustness may need to be repeated when
the test is transferred from the developing laboratory to the field as the conditions may be less than optimal
and the staff less experienced.
REFERENCES
1. BALLAGI-PORDÁNY A. & BELÁK S. (1996). The use of mimics as internal standards to avoid false negatives in
diagnostic PCR. Mol. Cell. Probes, 10, 159–164.
2. BELÁK S. & THORÉN P. (2001). Molecular diagnosis of animal diseases. Expert Rev. Mol. Diagn., 1, 434–444.
3. BELÁK S. (2005). The molecular diagnosis of porcine viral diseases: a review. Acta Vet. Hung., 53, 113–124.
(Review).
4. BELÁK S. (2007). Molecular diagnosis of viral diseases, present trends and future aspects. A view from the
OIE Collaborating Centre for the Application of Polymerase Chain Reaction Methods for Diagnosis of Viral
Diseases in Veterinary Medicine. Vaccine, 25, 5444-5452.
5. BRANSCUM, A.J., GARDNER I.A. & JOHNSON W.O. (2005). Estimation of diagnostic-test sensitivity and
specificity through Bayesian modelling. Prev. Vet. Med., 68, 145–163

6. BURNS M.J., NIXON G.J., FOY C.A. & HARRIS N. (2005). Standardisation of data from real-time quantitative
PCR methods - evaluation of outliers and comparison of calibration curves. BMC Biotechnol., 5, 31–44.
7. BUSTIN S.A. (2005). Real-time, fluorescence-based quantitative PCR: a snapshot of current procedures and
preferences. Expert Rev. Mol. Diagn., 5, 493–498.
8. BURKHARDT H.J. (2000). Standardization and quality control of PCR analyses. Clin. Chem. Lab. Med., 38,
87–91.
9. ELNIFRO E.M., ASHSHI A.M., COOPER R.J., & KLAPPER P.E. (2000). Multiplex PCR: optimization and application
in diagnostic virology. Clin. Microbiol. Rev., 13, 559–570.
10. JUNGKIND D. (2001). Automation of laboratory testing for infectious diseases using the polymerase chain
reaction – our past, our present, our future. J. Clin. Virol., 20, 1–6.
11. HUGGETT J., DHEDA K., BUSTIN S. & ZUMLA A. (2005). Real-time RT-PCR normalisation; strategies and
considerations. Genes Immun., 6, 279–284. (Review).
12. LAUERMAN L.H. (2004). Advances in PCR technology. Anim. Health Res. Rev., 5, 247–248. (Review).
13. LOUIE M., LOUIE L. & SIMOR A.E. (2000). The role of DNA amplification technology in the diagnosis of
infectious diseases. CMAJ., 163, 301–309.
14. MACKAY I.M., AARDE K.E. & NITSCHE A. (2002). Real-time PCR in virology. Nucleic Acids Res., 30, 1292–
1305.
15. SAWYER J., W AKELEY P., W EST D., FEARNLEY C., ANDERSON S., FLOWERS M., W EBSTER K., ERRINGTON J. &
W ILLIAMS R. (2006). Discussion 324-5, Practical experiences of moving molecular diagnostics into routine
use at the Veterinary Laboratories Agency. Dev. Biol. (Basel), 126, 89–97.
16. W ORLD ORGANISATION FOR ANIMAL HEALTH (OIE) (2002). OIE Guide 3: Laboratory Proficiency Testing. In: OIE
Quality Standard and Guidelines for Veterinary Laboratories: Infectious Diseases. OIE, Paris, France, 53–
63.
17. VILJOEN G.J., NELL H & CROWTHER J.R. (2005). Molecular Diagnostic PCR Handbook. IAEA-FAO, Springer,
Dordrecht, The Netherlands.
*
* *
NB: There is an OIE Collaborating Centre for the Biotechnology-based Diagnosis of Infectious Diseases in
Veterinary Medicine (see Table in Part 3 of this Terrestrial Manual or consult the OIE Web site for the most up-to-
date list: www.oie.int).

CHAPTER 1.1.6
LABORATORY METHODOLOGIES FOR BACTERIAL

ANTIMICROBIAL SUSCEPTIBILITY TESTING
SUMMARY
Historically, medical practitioners and veterinarians selected antimicrobials to treat bacterial

infectious diseases based primarily on past clinical experiences. However, with the increase in
bacterial resistance to traditionally used antimicrobials, it has become more difficult for clinicians to
empirically select an appropriate antimicrobial agent (15). As a result, in vitro antimicrobial
susceptibility testing (AST) of the relevant bacterial pathogens, from properly collected specimens,
should use validated methods. Thus, AST is an important component of prudent antimicrobial use
guidelines in animal husbandry worldwide and veterinarians in all countries should have these data
available for informed decision-making (1).
Although a variety of methods exist, the goal of in vitro antimicrobial susceptibility testing is to
provide a reliable predictor of how an organism is likely to respond to antimicrobial therapy in the
infected host. This type of information aids the clinician in selecting the appropriate antimicrobial
agent, aids in developing antimicrobial use policy, and provides data for epidemiological
surveillance. Such epidemiological surveillance data provide a base to choose the appropriate
empirical treatment (first-line therapy) and to detect the emergence and/or the dissemination of
resistant bacterial strains or resistance determinants in different bacterial species. The selection of
a particular AST method is based on many factors such as validation data, practicality, flexibility,
automation, cost, reproducibility, accuracy, and individual preference.
The use of genotypic approaches for detection of antimicrobial resistance genes has also been
promoted as a way to increase the speed and accuracy of susceptibility testing. Numerous DNA-
based assays are being developed to detect bacterial antibiotic resistance at the genetic level.
These methods, when used in conjunction with phenotypic analysis, offer the promise of increased
sensitivity, specificity, and speed in the detection of specific known resistance genes and can be
used in tandem with traditional laboratory AST methods.
INTRODUCTION
The spread of multiple antimicrobial-resistant pathogenic bacteria has been recognised by the World
Organisation for Animal Health (OIE), the Food and Agriculture Organisation (FAO) and the World Health
Organization (WHO) as a serious global human and animal health problem. The development of bacterial
antimicrobial resistance is neither an unexpected nor a new phenomenon. It is, however, an increasingly
troublesome situation due to the frequency with which new emerging resistance phenotypes are occurring among
many bacterial pathogens and even commensal organisms.
Historically, many infections could be treated successfully based on the clinician’s past clinical experience (i.e.
empirical therapy). However, this is becoming more the exception than the rule. Resistance has been observed
to essentially all of the antimicrobial agents currently approved for use in human and veterinary clinical medicine.
This, combined with the variety of antimicrobial agents currently available, makes the selection of an appropriate
agent an increasingly more challenging task. This situation has made clinicians more dependent on data from in
vitro antimicrobial susceptibility testing, and highlights the importance of the diagnostic laboratory in clinical
practice.
There is a number of antimicrobial susceptibility testing (AST) methods available to determine bacterial
susceptibility to antimicrobials. The selection of a method is based on many factors such as practicality, flexibility,
automation, cost, reproducibility, accuracy, and individual preference. Standardisation and harmonisation of AST
methodologies, used in epidemiological surveillance of antimicrobial drug resistance, are critical if data are to be

Chapter 1.1.6. — Laboratory methodologies for bacterial antimicrobial susceptibility testing
compared among national or international surveillance/monitoring programmes of OIE Member Countries. It is

essential that AST methods provide reproducible results in day-to-day laboratory use and that the data be
comparable with those results obtained by an acknowledged ‘gold standard’ reference method. In the absence of
standardised methods or reference procedures, susceptibility results from different laboratories cannot be
reliably compared. The method used to select samples for inclusion in antimicrobial resistance surveillance
programmes, as well as the methods use for primary bacterial isolation, are also important factors that should be
standardised or harmonised to allow direct comparison of data between different regions; consideration of these
issues is addressed in an OIE document (17).
As the science of AST has progressed, a greater understanding of the multiple factors that could affect the
overall outcome of susceptibility testing has become clearer. This chapter provides guidelines and
standardisation for AST methodologies, and interpretation of antimicrobial susceptibility test results.
1. Test requirements
In order to achieve standardisation of AST methods and comparability of AST results, the following requirements
apply:
i) the use of standardised AST methods and the harmonisation of AST test parameters (including choice of
antimicrobial agents and subsequent interpretive criteria) are essential,
ii) standardised AST methods, including all critical specifications and interpretive criteria, should be clearly
defined, documented in detail and used by all participating laboratories,
iii) all AST methods should generate accurate and reproducible data,
iv) all data should be reported quantitatively,
v) establishment of national or regional designated laboratories is essential for the coordination of AST
methodologies, interpretations and appropriate operational techniques used to ensure accuracy and
reproducibility (e.g. quality controls),
vi) microbiological laboratories should implement and maintain a formal quality management programme (see
Chapter 1.1.3 Quality management in veterinary testing laboratories),
vii) laboratories should have acquired a third party accreditation that includes the AST methodologies to be
used within the scope of that accreditation. The accreditation body should meet accepted international
Laboratory Accreditation Cooperation [ILAC]) standards and guidelines regarding the standards used for
the accreditation process. The accreditation standards used should include the requirement for participation
in proficiency testing programmes,
viii) specific bacterial reference/quality control strains are essential for determining intra- and inter-laboratory
quality control, quality assurance and proficiency testing.
2. Selection of antimicrobials for testing and reporting
Selecting the appropriate antimicrobials for susceptibility testing can be difficult given the vast numbers of agents
available. The following guidelines should be noted:
i) the FAO/OIE/WHO expert workshop on non-human antimicrobial usage and antimicrobial resistance
recommends creating a list of veterinary and human critically important antimicrobials for susceptibility
testing and reporting,
ii) selection of the most appropriate antimicrobials is a decision best made by each OIE Member Country in
consultation with the appropriate bodies and organisations,
iii) antimicrobials in the same class may have similar in-vitro activities against select bacterial pathogens. In
these cases, a representative antimicrobial should be selected that predicts susceptibility to other members
of the same class,
iv) certain microorganisms can be intrinsically resistant to particular antimicrobial classes; therefore it is
unnecessary and misleading to test certain agents for activity in vitro. The type of intrinsic resistance has to
be determined for these organisms via either the scientific literature or through testing,
v) the number of antimicrobials to be tested should be limited in order to ensure the relevance and practicality
of AST.
Periodic review of microorganisms that are currently predictably susceptible to certain antimicrobial agents is
recommended to ensure that emergent, unexpected resistance is detected. Emerging resistance may also be
suspected following poor response to a standard antimicrobial treatment regime.

3. Antimicrobial susceptibility testing methodologies
The following requirements should be respected:
i) bacteria subjected to AST must be isolated in pure culture from the submitted sample,
ii) standard reference methods should be used for identification so that the subject bacteria are consistently
and correctly identified to the genus and/or species level,
iii) bacterial isolates considered to be the most important and a sampling of other isolates, should be stored for
future analysis (either lyophilisation or cryogenic preservation at –70°C to –80°C).
The following factors influencing AST methods should be determined, optimised, and documented in a detailed
standard operating procedure:
i) once the bacterium has been isolated in pure culture, the optimum concentration of the inocula must be
determined to obtain accurate susceptibility results. Bacteria or other organisms used in AST testing should
be from a fresh culture,
ii) the composition and preparation of the agar and broth media used (e.g. pH, cations, thymidine or thymine,
use of supplemented media). Performance and sterility testing of media lots should also be determined and
documented as well as employed procedures,
iii) the content of antimicrobial in the carrier (antibiotics used in microtitre plates, disk, strip, tablet),
iv) composition of solvents and diluents for preparation of antimicrobial stock solutions,
v) growth and incubation conditions (time, temperature, atmosphere e.g. CO2),
vi) agar depth,
vii) number of concentrations tested per broth and agar dilution,
viii) the test controls to be used, including the reference organisms used,
ix) the subsequent interpretive criteria.
For these reasons, special emphasis has to be placed on the use of documented procedures and validated, well
documented methods, as sufficient reproducibility can be attained only through the use of such methodology.
4. Selection of antimicrobial susceptibility testing methodology
The selection of an AST methodology may be based on the following factors:
i) ease of performance,
ii) flexibility,
iii) adaptability to automated or semi-automated systems,
iv) cost,
v) reproducibility,
vi) reliability,
vii) accuracy,
viii) the organisms and the antimicrobials of interest in that particular OIE Member Country,
ix) availability of suitable validation data for the range of organisms to be susceptibility tested.
5. Antimicrobial susceptibility testing methods
The following three methods have been shown to consistently provide reproducible and repeatable results when
followed correctly (14, 15):
i) disk diffusion,
ii) broth dilution,
iii) agar dilution.

a) Disk diffusion method

Disk diffusion refers to the diffusion of an antimicrobial agent of a specified concentration from disks, tablets
or strips, into the solid culture medium that has been seeded with the selected inoculum isolated in a pure
culture (see section 3.i). Disk diffusion is based on the determination of an inhibition zone proportional to
the bacterial susceptibility to the antimicrobial present in the disk.
The diffusion of the antimicrobial agent into the seeded culture media results in a gradient of the
antimicrobial. When the concentration of the antimicrobial becomes so diluted that it can no longer inhibit
the growth of the test bacterium, the zone of inhibition is demarcated. The diameter of this zone of inhibition
around the antimicrobial disk is related to minimum inhibitory concentration (MIC) for that particular
bacterium/antimicrobial combination; the zone of inhibition correlates inversely with the MIC of the test
bacterium. Generally, the larger the zone of inhibition, the lower the concentration of antimicrobial required
to inhibit the growth of the organisms. However, this depends on the concentration of antibiotic in the disk
and its diffusibility.
Note: Disk diffusion tests based solely on the presence or absence of a zone of inhibition without regard to
the size of the zone of inhibition are not acceptable AST methodology.
• Considerations for the use of the disk diffusion methodology

Disk diffusion is straightforward to perform, reproducible, and does not require expensive equipment. Its
main advantages are:
i) low cost,
ii) ease in modifying test antimicrobial disks when required,
iii) can be used as a screening test against large numbers of isolates,
iv) can identify a subset of isolates for further testing by other methods, such as determination of MICs.
Manual measurement of zones of inhibition may be time-consuming. Automated zone-reading devices are
available that can be integrated with laboratory reporting and data-handling systems. The disks should be
distributed evenly so that the zones of inhibition around antimicrobial discs in the disc diffusion test do not
overlap to such a degree that the zone of inhibition cannot be determined. Generally this can be
accomplished if the discs are no closer than 24 mm from centre to centre, though this is dependent on disk
concentration and the ability of the antimicrobial to diffuse in agar.
b) Broth and agar dilution methods

The aim of the broth and agar dilution methods is to determine the lowest concentration of the assayed
antimicrobial that inhibits the growth of the bacterium being tested (MIC, usually expressed in mg/ml or
mg/litre). However, the MIC does not always represent an absolute value. The ‘true’ MIC is a point between
the lowest test concentration that inhibits the growth of the bacterium and the next lower test concentration.
Therefore, MIC determinations performed using a dilution series may be considered to have an inherent
variation of one dilution.
Antimicrobial ranges should encompass both the interpretive criteria (susceptible, intermediate and
resistant) for a specific bacterium/antibiotic combination and appropriate quality control reference
organisms.
Antimicrobial susceptibility dilution methods appear to be more reproducible and quantitative than agar disk
diffusion. However, antibiotics are usually tested in doubling dilutions, which can produce inexact MIC data.
Any laboratory that intends to use a dilution method and set up its own reagents and antibiotic dilutions
should have the ability to obtain, prepare and maintain appropriate stock solutions of reagent-grade
antimicrobials and to generate working dilutions on a regular basis. It is then essential that such
laboratories use quality control organisms (see below) to assure accuracy and standardisation of their
procedures.
• Broth dilution
Broth dilution is a technique in which a suspension of bacterium of a predetermined optimal or appropriate
concentration is tested against varying concentrations of an antimicrobial agent (usually serial twofold
dilutions) in a liquid medium of predetermined, documented formulation. The broth dilution method can be
performed either in tubes containing a minimum volume of 2 ml (macrodilution) or in smaller volumes using
microtitration plates (microdilution). Numerous microtitre plates containing prediluted antibiotics within the

wells are commercially available. The use of identical lots in microdilution plates may assist in the
minimisation of variation that may arise due to the preparation and dilution of the antimicrobials from
different laboratories. The use of these plates, with a documented test protocol, including specification of
appropriate reference organisms, will facilitate the comparability of results among laboratories.
Due to the fact that most broth microdilution antimicrobial test panels are prepared commercially, this
method is less flexible than agar dilution or disk diffusion in adjusting to the changing needs of the
surveillance/monitoring programme.
Because the purchase of antimicrobial plates and associated equipment may be costly, this methodology
may not be feasible for some laboratories.
• Agar dilution
Agar dilution involves the incorporation of varying concentrations of antimicrobial agent into an agar
medium, usually using serial twofold dilutions, followed by the application of a defined bacterial inoculum to
the agar surface of the plate. These results are often considered as the most reliable for the determination
of an MIC for the test bacterium/antimicrobial combination.
The advantages of agar dilution methods include:
i) the ability to test multiple bacteria, except bacteria that swarm, on the same set of agar plates at the
same time,
ii) the potential to improve the identification of MIC endpoints and extend the antibiotic concentration
range,
iii) the possibility to semi-automate the method using an inoculum-replicating apparatus. Commercially
produced inoculum replicators are available and these can transfer between 32 and 36 different
bacterial inocula to each agar plate,
Agar dilution methods also have certain disadvantages, for example:
i) if not automated, they are very laborious and require substantial economic and technical resources,
ii) once the plates have been prepared, they normally should be used within a week,
iii) the endpoints are not always easy to read nor is the purity of the inoculum easy to verify.
Agar dilution is often recommended as a standardised AST method for fastidious organisms (8), such as
anaerobes, Campylobacter and Helicobacter species.
c) Other bacterial AST and specific antimicrobial resistance tests

Bacterial antimicrobial MICs can also be obtained using commercially available gradient strips that diffuse a
predetermined antibiotic concentration. However, the use of gradient strips can be very expensive and MIC
discrepancies can be found when testing certain bacteria/antimicrobial combinations compared with agar
dilution results (2, 5).
Regardless of the AST method used, the procedures should be documented in detail to ensure accurate
and reproducible results, and appropriate reference organisms should always be tested every time AST is
performed in order to ensure accuracy and validity of the data.
The appropriate AST choice will ultimately depend on the growth characteristics of the bacterium in
question. In special circumstances, novel test methods and assays may be more appropriate for detection
of particular resistance phenotypes. For example, chromogenic cephalosporin-based tests (8) (e.g.
nitrocefin) may provide more reliable and rapid results for beta-lactamase determination in certain bacteria,
whereas inducible clindamycin resistance in Staphylococcus spp. may be detected using a disk diffusion
method employing standard erythromycin and clindamycin disks in adjacent positions and measuring the
resultant zones of inhibition (e.g. D-zone) (18).
Similarly, extended-spectrum beta-lactamase (ESBL) (8) activity in certain bacteria can also be detected by
using standard disk diffusion susceptibility test methods incorporating specific cephalosporins (cefotaxime
and ceftazidime) in combination with a beta-lactamase inhibitor (clavulanic acid) and measuring the
resulting zones of inhibition. Additionally, chloramphenicol resistance attributed to production of
chloramphenicol acetyl transferase can be detected in some bacteria via rapid tube or filter paper tests
within 1–2 hours (8). Also penicillin-binding protein 2a (PBP 2a) can be detected in methicillin resistant
staphylococci with a latex agglutination test (13).

d) Future directions in antimicrobial susceptibility/resistance detection

The use of genotypic approaches for detection of antimicrobial resistance genes has been promoted as a
way to increase the rapidity and accuracy of susceptibility testing (3). Numerous DNA-based assays are
being developed to detect bacterial antibiotic resistance at the genetic level. The newest and perhaps most
state-of-the-art approach is to predict antimicrobial resistance phenotypes via identification and
characterisation of the known genes that encode specific resistance mechanisms.
Methods that employ the use of comparative genomics, genetic probes, microarrays, nucleic acid
amplification techniques (e.g. polymerase chain reaction [PCR]), and DNA sequencing offer the promise of
increased sensitivity, specificity, and speed in the detection of specific known resistance genes (3, 4, 10).
Genotypic methods have been successfully applied to supplement traditional AST phenotypic methods for
other organisms including methicillin-resistant staphylococci, vancomycin-resistant enterococci, and
detection of fluoroquinolone resistance mutations (3, 4, 10). PCR methods have also been described for
beta-lactamases, aminoglycoside inactivating enzymes, and tetracycline efflux genes (4, 10).
Technological innovations in DNA-based diagnostics should allow for the detection of multiple resistance
genes and/or variants during the same test. The development of rapid diagnostic identification methods and
genotypic resistance testing should help reduce the emergence of antimicrobial resistance, by enabling the
use of the most appropriate antimicrobial when therapy is initiated. However, DNA techniques have to be
demonstrated to be complimentary to AST methods and results.
Additionally, new technological advances may facilitate the ability to probe bacterial species for large
numbers of antimicrobial resistance genes quickly and cheaply, thereby providing additional relevant data
for surveillance and monitoring programmes. However, despite the new influx of genotypic tests,
documented and agreed upon phenotypic AST methods will still be required in the near future to detect
emerging resistance mechanisms among bacterial pathogens.
6. Antimicrobial susceptibility breakpoints and zone of inhibition criteria

The objective of in-vitro AST is to predict how a bacterial pathogen may respond to the antimicrobial agent in
vivo. The results generated by bacterial in-vitro antimicrobial susceptibility tests, regardless of whether disk
diffusion or dilution methods are used, are generally interpreted and reported as resistant, susceptible or
intermediate to the action of a particular antimicrobial. No single formula for selection of optimal breakpoints has
been established. The process involves a review of existing data and is influenced by the subjectivity of
individuals tasked with selecting the appropriate breakpoints.
Generally, antimicrobial susceptibility breakpoints are established by national standards organisations,

professional societies or regulatory agencies. The relevant documents should be consulted. However, there can
be notable differences in breakpoints for the same antimicrobial agent within and among countries due to
differences between standards setting organisations and regulatory agencies and because of regional or national
decisions on dosing regimens (6).
As mentioned previously, antimicrobial susceptibility testing results should be recorded quantitatively:
i) as distribution of MICs in milligrams per litre or mg/ml,

ii) or as inhibition zone diameters in millimetres.
The following two primary factors enable a bacterium to be interpreted as susceptible or resistant to an
antimicrobial agent:
i) The development and establishment of quality control ranges (8), using diffusion when possible and dilution
testing, for quality control reference microorganisms.
Establishment of quality control ranges is essential for validating test results obtained using a specific AST
method. The allowable interpretive category ranges for the reference organisms should be established prior
to determining breakpoints for susceptibility or resistance. The use of reference organisms is a quality
control and quality assurance activity. However, it is only necessary to require the used of reference
organisms.
ii) The determination of the appropriate interpretive criteria regarding establishment of breakpoints (8).
This involves the generation of three distinct types of data:
• MIC population distributions of the relevant microorganisms,
• pharmacokinetic parameters and pharmacodynamic indices of the antimicrobial agent,
• results of clinical trials and experience.

The interpretation of the data involves creating a scattergram from the bacterial population distribution
(representative bacterial species), by plotting the zone of inhibition against the logarithm to the base 2 of the MIC
for each bacterial pathogen. The selection of breakpoints is then based on multiple factors, including regression
line analysis that correlates MICs and zone diameters of inhibition, bacterial population distributions, error rate
bounding, pharmacokinetics, and ultimately, clinical verification.
The development of a concept known as ‘microbiological breakpoints’, or ‘epidemiological cut-off points’, which is
based on the population distributions of the specific bacterial species tested, may be more appropriate for some
antimicrobial surveillance programmes. In this case, bacterial isolates that deviate from the normal wild-type
susceptible population would be designated as resistant, and shifts in susceptibility to the specific
antimicrobial/bacterium combination could be monitored (12). There is a great advantage in the recording of
quantitative susceptibility data in that such data may be analysed according to clinical breakpoints as well as by
using epidemiological cut-off values.
7. Antimicrobial susceptibility testing guidelines
A number of standards and guidelines are currently available for antimicrobial susceptibility testing and
subsequent interpretive criteria throughout the world (6). Amongst others, these include standards and guidelines
published by:
Clinical Laboratory and Standards Institute (CLSI/NCCLS, USA),

British Society for Antimicrobial Chemotherapy (BSAC, UK),
Comité de l’Antibiogramme de la Société française de Microbiologie (CASFM, France),
Swedish Reference Group for Antibiotics (SIR, Sweden),
Deutsches Institut für Normung (DIN, Germany),
Japanese Society for Chemotherapy (JSC, Japan),
Commissie richtlijnen gevoeligheidsbepalingen (CRG, the Netherlands).
At this time, only the CLSI/NCCLS has developed protocols for susceptibility testing of bacteria of animal origin
and determination of interpretive criteria (8). However, protocols and guidelines are available from a number of
standards organisations and professional societies (i.e. Clinical and Laboratory Standards Institute, British
Society for Antimicrobial Chemotherapy, Japan Society for Chemotherapy [JSC], Swedish Reference Group for
Antibiotics [SIR], Deutshes Institute für Normung, Comité de L’Antibiogramme de la Société Française de
Microbiologie, Werkgroep richtlijnen gevoeligheidsbepalingen, and others) for susceptibility testing for similar
bacterial species that cause infections in humans. It is possible that such guidelines can be adopted for
susceptibility testing for bacteria of animal origin, but each country must evaluate its own AST standards and
guidelines. Additionally, efforts focusing on both standardisation and harmonisation of susceptibility/resistance
breakpoints on an international scale are progressing. These efforts have primarily focused on the adoption of
the standards and guidelines of the CLSI, which provide laboratories with methods and quality control values
enabling comparisons of AST methods and generated data (8, 16). For those OIE Member Countries that do not
have standardised AST methods in place, the adoption of CLSI standards would be an appropriate initial step
towards acceptable methods and harmonisation.
As a first step towards comparability of monitoring and surveillance data, Member Countries should be
encouraged to strive for harmonised and standardised programme design (14). Data from countries using
different methods and programme design may otherwise not be directly comparable (7, 14). Notwithstanding this,
data collected over time in a given country may at least allow the detection of emergence of antimicrobial
resistance or trends in prevalence of susceptibility/resistance in that particular country (11). However, if results
achieved with different AST methods are to be presented side by side, then comparability of results must be
demonstrated and consensus on interpretation achieved.
Note: This will be best accomplished by the use of accurate and reliable documented AST methods used in
conjunction with monitoring of AST performance while using well characterised reference microorganisms among
participating laboratories.
8. Comparability of results
To determine the comparability of results originating from different surveillance systems, results should be
reported quantitatively including information on the performance of the methods, the reference organisms and
the antimicrobial.
AST data, consisting of cumulative and ongoing summary of susceptibility patterns (antibiograms) among
clinically important and surveillance microorganisms should be created, recorded and analysed periodically at
regular intervals (9). Data must also be presented in a clear and consistent manner so that both new patterns of
resistance can be identified and atypical findings confirmed or refuted. This data should be available on a central
data bank and published yearly.

Cumulative AST data will be useful in monitoring susceptibility/resistance trends in a region over time and
assessing the effects of interventions to reduce antimicrobial resistance.
9. Quality control and quality assurance
Adequate quality control/quality assurance systems should be established in AST performing laboratories:
i) quality control refers to the operational techniques that are used to ensure accuracy and reproducibility of
AST,
ii) quality assurance.
The following components should be determined and monitored:
i) precision of the AST procedure,

ii) accuracy of the AST procedure,
iii) qualifications, competence, and proficiency of the laboratory personnel, as well as the personnel that
interpret the results and those that are involved in monitoring of antimicrobial resistance,
iv) performance of the appropriate reagents.
The following requirements should be respected:
i) Strict adherence to specified and documented techniques in conjunction with quality control (i.e. assurance
of performance and other critical criteria) of media and reagents.
ii) Record keeping of:
• lot numbers of all appropriate materials and reagents,
• expiration dates of all appropriate materials and reagents,
• equipment calibration and monitoring,
• critical specifications for AST performance (reference results, time, temperature etc.).
iii) The appropriate reference microorganism(s) should always be used regardless of the AST method
employed.
iv) Reference microorganisms are to be obtained from a reliable source for example, from the American Type
®
Culture Collection (ATCC ), reliable commercial sources, or institutions with demonstrated reliability to store
and use the organisms correctly.
v) Reference microorganisms should be catalogued and well characterised, including stable defined
antimicrobial susceptibility phenotypes. Records regarding these reference organisms should include the
established resistant and susceptible ranges of the antimicrobials to be assayed, and the reference to the
method(s) by which these were determined.
vi) Laboratories involved in AST should use the appropriate reference microorganisms in all AST testing.
vii) Reference strains should be kept as stock cultures from which working cultures are derived and should be
obtained from national or international culture collections. Reference bacterial strains should be stored at
designated centralised or regional laboratories. Working cultures should not be subcultured from day to day
as this introduces contamination and the method of producing working cultures should ensure that stock
cultures are rarely used. This may be accomplished with the production of an intermediate stock of cultures
derived from the original cultures that are used to crate day-to-day working cultures.
viii) The preferred method for analysing the overall performance of each laboratory should test the working
stock of the appropriate reference microorganisms on each day that susceptibility tests are performed.
Because this may not always be practical or economical, the frequency of such tests may be reduced if the
laboratory can demonstrate that the results of testing reference microorganisms using the selected method
are reproducible. If a laboratory can document the reproducibility of the susceptibility testing methods used,
testing may be performed on a weekly basis. If concerns regarding accuracy, reproducibility, or method
validity emerge, the laboratory has a responsibility to determine the cause(s) and repeat the tests using the
reference materials. Depending on the cause(s), daily reference material use and any other corrective
action may be re-initiated.
ix) Reference microorganisms should be tested each time a new batch of medium or plate lot is used and on a
regular basis in parallel with the microorganisms to be assayed.

x) Appropriate biosecurity issues should be addressed in obtaining and dispersing microorganisms to

participating laboratories.
10. External proficiency testing
To ensure that reported antimicrobial susceptibility data are accurate; OIE Member Countries should initiate an
inter-laboratory proficiency testing programme. External proficiency testing can be carried out on a national
basis. Laboratories in Member Countries are also encouraged to participate in international inter-laboratory
comparisons (e.g. Enter-Net) (6). All bacterial species subjected to AST should be included.
Countries should appoint or establish designated reference or national laboratories that are responsible for:
i) monitoring the quality assurance programmes of laboratories participating in surveillance and monitoring of
antimicrobial resistance,
ii) characterising and supplying to those laboratories a set of reference microorganisms,
iii) creating, managing, and distributing samples to be used in external proficiency testing,
iv) creating a central database available on the internet (e.g. European Antimicrobial Resistance Surveillance
System [EARSS]) that contains the different susceptibility/resistance profiles for each bacterial species
under surveillance.
11. Conclusion
Although a variety of methods exist, the goal of in-vitro antimicrobial susceptibility testing is the same: to provide
a reliable predictor of how a microorganism is likely to respond to antimicrobial therapy in the infected host. This
type of information aids the clinician in selecting the appropriate antimicrobial agent, provides data for
surveillance, and aids in developing antimicrobial use policies.
In-vitro antimicrobial susceptibility testing can be performed using a variety of formats, the most common being
disk diffusion, agar dilution, broth macrodilution, broth microdilution, and a concentration gradient test (e.g.
E test®). Each of these procedures requires the use of specific testing conditions and methods, including media,
incubation conditions and times, and the identification of appropriate quality control organisms along with their
specific QC ranges. It is essential that AST methods provide reproducible results in day-to-day laboratory use
and that the data be comparable with those results obtained by an acknowledged ‘gold standard’ reference
method. In the absence of standardised methods or reference procedures, antimicrobial susceptibility/resistance
results from different laboratories cannot be reliably compared.
The use of genotypic approaches for detection of antimicrobial resistance genes has also been promoted as a
way to increase the rapidity and accuracy of susceptibility testing. Additionally, new technological advances may
facilitate the ability to probe bacterial species for large numbers of antimicrobial resistance genes quickly and
cheaply, thereby providing additional relevant data into surveillance and monitoring programs. Despite the new
influx of genotypic tests however, standardised phenotypic AST methods will still be required in the near future to
detect emerging resistance mechanisms among bacterial pathogens.
REFERENCES
1. ANTHONY F., ACAR J., FRANKLIN A., GUPTA R., NICHOLLS T., TAMURA Y., THOMPSON S., THRELLFALL E.J., VOSE D.,
VAN VUUREN M. & W HITE D.G. (2001). Antimicrobial resistance: responsible and prudent use of antimicrobial
agents in veterinary medicine. Rev. sci. tech. Off. int. Epiz., 20, 829–839.
2. BROWN D.F. & BROWN L. (1991). Evaluation of the E-test, a novel method of quantifying antimicrobial activity.
J. Antimicrob. Chemother., 26, 185–190.
3. CAI H.Y., ARCHAMBAULT M., GYLES C.L. & PRESCOTT J.F. (2003). Molecular genetic methods in the veterinary
clinical bacteriology laboratory: current usage and future applications. Anim. Health Rev., 4, 73–93.
4. CHEN S., ZHAO S., MCDERMOTT P.F., SCHROEDER C.M., W HITE D.G. & MENG J. (2005). A DNA microarray for
identification of virulence and antimicrobial resistance genes in Salmonella serovars and Escherichia coli.
Mol. Cell. Probes, 19, 195–201.
5. GE B., BODEIS S., W ALKER R.D., W HITE D.G., ZHAO S., MCDERMOTT P.F. & MENG J. (2002). Comparison of
Etest and agar dilution for in vitro antimicrobial susceptibility testing of Campylobacter. J. Antimicrob.
Chemother., 50, 487–494.

6. KAHLMETER G., BROWN D.F.J., GOLDSTEIN F.W., MACGOWAN A.P., MOUTON J.W., OSTERLUND A., RODLOFF A.,
STEINBAKK M., URBASKOVA P. & VATOPOULOS A. (2003). European harmonization of MIC breakpoints for
antimicrobial susceptibility testing of bacteria. J. Antimicrob. Chemother., 52, 145–148.
7. LEEGARD T.M., CAUGANT D.A., FROHOLM L.O. & HOIB, E.A. (2000). Apparent differences in antimicrobial
susceptibility as a consequence of national guidelines. Clin. Microbiol. Inf. Dis., 6, 290–293.
8. NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS (NCCLS) (2002). Document M31-A2.
Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from
Animals, Approved Standard, Second Edition. NCCLS document M31-A2. NCCLS, 940 West Valley Road,
Suite 1400, Wayne, Pennsylvania 19087-1898, USA, 80 pp.
9. NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS (NCCLS) (2002). Document M39-A. Analysis and
Presentation of Cumulative Antimicrobial Susceptibility Test Data; Approved Guideline. NCCLS document
M39-A. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA, 30 pp.
10. PERRETEN V., VORLET-FAWER L., SLICKERS P., EHRICHT R., KUHNERT P. & FREY J. (2005). Microarray-based
detection of 90 antibiotic resistance genes of gram-positive bacteria. J. Clin. Microbiol., 43, 2291–2302.
11. PETERSEN A., AARESTRUP F.M., HOFSHAGEN M., SIPILA H., FRANKLIN A. & GUNNARSSON E. (2003). Harmonization
of antimicrobial susceptibility testing among veterinary diagnostic laboratories in five Nordic countries.
Microb. Drug Resist., 9, 381–388.
12. RINGERTZ S., OLSSON-LILJEQUIST B., KAHLMETER G. & KRONVALL G. (1997). Antimicrobial susceptibility testing
in Sweden II. Species-related zone diameter breakpoints to avoid interpretive errors and guard against
unrecognised evolution of resistance. Scand. J. Infect. Dis. (Suppl.), 105, 8–12.
13. STEPANOVIC S., HAUSCHILD T., DAKIC I., AL-DOORI Z., SVABIC-VLAHOVIC M., RANIN L. & MORRISON D. (2006).
Evaluation of phenotypic and molecular methods for detection of oxacillin resistance in members of the
Staphylococcus sciuri group. J. Clin. Microbiol., 44, 934–937.
14. THRELFALL E.J., FISHER I.S.T., W ARD L., TSCHAPE H. & GERNER-SMIDT P. (1999). Harmonization of antibiotic
susceptibility testing for Salmonella: Results of a study by 18 national reference laboratories within the
European Union-funded Enter-Net group. Microb. Drug Resist., 5, 195–199.
15. W ALKER R.D. (2000). Antimicrobial susceptibility testing and interpretation of results. In: Antimicrobial
Therapy in Veterinary Medicine, Prescott J.F., Baggot J.D., Walker R.D., eds. Ames, IA, Iowa State
University Press, 12–26.
16. W HITE D.G., ACAR J., ANTHONY F., FRANKLIN A., GUPTA R., NICHOLLS T., TAMURA Y., THOMPSON S., THRELFALL
J.E., VOSE D., VAN VUUREN M., W EGENER H., & COSTARRICA L. (2001). Standardisation and harmonisation of
laboratory methodologies for the detection and quantification of antimicrobial resistance. Rev. Sci. Tech.
Off. int. Epiz., 20, 849–858.
17 W ORLD ORGANISATION FOR ANIMAL HEALTH (OIE) (2003). OIE International Standards on Antimicrobial
Resistance. OIE, Paris, France.
18. ZELAZNY A.M., FERRARO M.J., GLENNEN A., HINDLER J.F., MANN L.M., MUNRO S., MURRAY P.R., RELLER L.B.,
TENOVER F.C. & JORGENSEN J.H. (2005). Selection of strains for quality assessment of the disk induction
method for detection of inducible clindamycin resistance in staphylococci: a CLSI collaborative study. J.
Clin. Microbiol., 43, 2613–2615.
*
* *
NB: There is an OIE Reference Laboratory for Antimicrobial resistance (see Table in Part 3 of this Terrestrial
Manual or consult the OIE Web site for the most up-to-date list: www.oie.int).

CHAPTER 1.1.7.
BIOTECHNOLOGY IN THE DIAGNOSIS

OF INFECTIOUS DISEASES
AND VACCINE DEVELOPMENT
INTRODUCTION
Molecular biological methods have become increasingly applicable to the diagnosis of infectious
diseases and vaccine development. To become widely used the methods need to be easy, safe,
sensitive, reproducible and eventually automated to facilitate the evaluation of large numbers of
samples.
The purpose of this chapter is to provide general background information for the nonspecialist.
Two issues of the OIE Scientific and Technical Review are concerned with biotechnology and the
diagnosis of animal diseases, and may be consulted for a more detailed review (103, 104). The
following is an outline of the topics briefly reviewed in this chapter.
A. Detection of nucleic acids

1. Polymerase chain reaction (PCR) and real-time PCR
2. Diagnosis by restriction fragment length polymorphisms and related DNA-based approaches
3. Diagnosis by DNA probes and DNA microarray technology
4. Nucleic acid extraction
B. Detection of protein
1. Immunohistochemistry
2. Immunoblotting
3. Antigen-capture enzyme-linked immunosorbent assay (ELISA)
4. Proteomics
C. Antibody detection
1. Competitive ELISA (C-ELISA)
2. Production of antigens by recombinant DNA technology
D. Vaccines
1. Gene deletion vaccines – bacteria
2. Marker vaccines and companion diagnostic tests
3. Virus-vectored vaccines
4. DNA vaccines
5. Other developments in vaccine technology
A. DETECTION OF NUCLEIC ACIDS
1. Polymerase chain reaction (PCR) and real-time PCR
The PCR exploits natural DNA replication mechanisms and results in the in-vitro production of large quantities of
a desired sequence of DNA from a complex mixture of heterogeneous sequences (134). PCR can amplify a
selected region of 50 to several thousand base pairs into billions of copies. A detailed discussion on the
methodology and applications of PCR is given in Mullis et al. (93).
The amplification of DNA by the PCR is accomplished via a cyclic succession of incubation steps at different
temperatures. The target DNA is first heat-denatured to separate the two complementary strands to provide a
single-stranded template. Specific primers (short synthetic molecules of DNA complementary to both strands and

Chapter 1.1.7. — Biotechnology in the diagnosis of infectious diseases and vaccine development
flanking the target sequences) are then annealed to the single-stranded template at low temperature and
extended with DNA polymerase at an intermediate temperature. Once the polymerase has synthesised a new
strand of DNA, the product is separated from the template by heating to a higher temperature. These steps,
referred to as cycles, are repeated 20–40 times, resulting in amplification of target DNA sequences. The key to
the geometric amplification of target DNA sequences by the PCR is the selection of paired primers that, when
extended, will create additional reciprocal primer-annealing sites for primer extension in subsequent cycles. To
detect RNA (e.g. RNA viruses), a cDNA copy of the RNA must first be made using reverse transcriptase (RT).
The cDNA then acts as the template for amplification by the PCR. This technique is referred to as RT-PCR.
Any PCR product generated has, by definition, a characteristic size. Its identity is generally confirmed using DNA
probes (see below), or restriction digests, which can be used to provide RFLPs (see above). More commonly,
since the advent of automated cycle sequencing techniques, identification is via direct sequencing of the PCR
product. For example, sequencing has been used in the virulence typing of avian influenza A virus, in which
virulence-associated structural motifs at the haemagglutinin gene cleavage site are reliable indicators of high
pathogenicity in chickens (63). The sensitivity of a PCR may be enhanced by the use of a second set of primers
to amplify a sub-fragment from the PCR product of the first reaction. This technique is commonly referred to as
‘nested PCR’ and has been used to detect low levels of Anaplasma marginale in persistently infected cattle
(158). However, the use of nested PCR can increase the rate of false-positive results.
PCR is a highly sensitive procedure for detecting infectious agents in host tissues and vectors, even when only a
small number of host cells are infected. PCR can target and amplify a gene sequence that has become
integrated into the DNA of infected host cells. It can also target and amplify unintegrated viral gene sequences. It
is clear that PCR has a role in the testing of vaccines to detect contamination. However, it does not differentiate
between viable and nonviable organisms or incomplete pieces of genomic DNA, and this may complicate
interpretation of results and affect the applicability of PCR in this role.
PCR may prove to be very useful in the diagnosis of chronic-persistent infections, such as those caused by
retroviruses (bovine leukaemia virus, caprine arthritis/encephalitis virus, etc.). These diseases present serious
problems in terms of diagnosis and prevention since infected animals are a constant potential source for
transmission.
When PCR is used for diagnosis, a great deal of care is required to avoid contamination of the samples because
the exquisite sensitivity of the technique can easily lead to false-positive results. Multicentre studies have shown
that positive samples are detected consistently between laboratories, but that false positives are frequently
obtained with known negative samples, indicating the continuing presence of contamination problems (139).
Systems have been developed to deal with this problem, for example the dUTP-UNG system (d-uracil
triphosphate and uracil-N-glycosylase). This system uses an enzymatic reaction to specifically degrade PCR
products from previous PCR amplification (in which dUTP has been incorporated) without degrading native
nucleic acid templates (25). This, of course, does not exclude contamination of the sample with extraneous virus.
A new generation of robotic workstations is now available where PCR reactions may be set up with only a single
tube open at any one time. This greatly reduces the risk of contamination. It is also important to control for
potential ‘negative’ results caused by the presence of interfering substances in the PCR reaction mixture or
patient’s sample by the inclusion of a template known to produce a PCR product (25). Use of these precautions
allows the PCR to become a realistic option for the diagnostician.
To expand its utility in veterinary diagnostics and pathogen identifications, PCR has been extensively modified in
the past years. PCR using broadly conserved primers is designed for identification of classes of pathogens. The
best example is the use of sequences of the 16s rRNA gene, an evolutionarily conserved gene in bacterial
species (52). Using PCR primers that are complementary to these conserved sequence regions, one can
determine the presence of any bacteria of a desired class from the sample. It must be noted that a positive PCR
result needs to be further characterised by hybridisation with species-specific probes, analysis by restriction
enzyme digestion, or by sequencing. Similarly, consensus PCR is designed to use degenerate primers targeting
conserved sequence regions or motifs of a group of related pathogens (169). Use of degenerate primers
targeting the sequence regions of the herpesviral DNA polymerase gene has led to identification of many
previously unrecognised herpesviruses in various animal species (40, 76). On the other hand, multiplex PCR is
designed to use two or more primer pairs directed at pathogen-specific unique sequences within a single reaction
for simultaneous detection of multiple pathogens that are of interest (41). Multiplex PCR has the advantage of a
high degree of sensitivity and specificity. However, there have been reports that multiplexing can reduce
sensitivity compared with single reactions, because of competition. If it is important to have a very sensitive
assay, this should be considered.
Classical PCR methods for diagnosis of pathogens, both bacterial and viral, are now being complemented and in
some cases replaced with real-time PCR assays. Real-time PCR monitors the accumulation of PCR product
during the amplification reaction, thus enabling identification of the cycles during which near-logarithmic PCR
product generation occurs. In other words, the assay can be used to reliably quantify the DNA or RNA content in
a given sample. In contrast to conventional PCR, real-time PCR requires less manipulation, is more rapid than
conventional PCR techniques, has a closed-tube format therefore decreasing risk of cross-contamination, is

highly sensitive and specific, thus retaining qualitative efficiency, and provides quantitative information. In many
cases, the real-time PCR assays have proved to be more sensitive than existing reference methods (58, 175).
The recent development of portable real-time PCR machines and assays (128) raises the exciting prospect of
these techniques being used for rapid (less than 2 hours) diagnosis of disease outbreaks in the field.
Validation of PCR techniques is covered in Chapter 1.1.5 Validation and quality control of polymerase chain
reaction methods used for the diagnosis of infectious diseases. This chapter also discusses internal controls to
insure the validity of PCR results.
2. Diagnosis by restriction fragment length polymorphisms and related DNA-based

approaches
Serological tests that are commonly used to identify microorganisms may not distinguish between isolates of
closely related pathogens, whether they be viruses, bacteria, fungi or parasites. A DNA-based procedure will
offer the better discrimination that is often required and an appropriate starting point may be analyses for
restriction fragment length polymorphisms (RFLP).
The RFLP approach is based on the fact that the genomes of even closely related pathogens are defined by
variation in sequence. For example, the linear order of adjacent nucleotides comprising the recognition sequence
of a specific restriction enzyme in one genome may be absent in the genome of a closely related strain or isolate.
In practice the RFLP procedure consists of isolating the target pathogen, extracting DNA or RNA (with
subsequent reverse transcription to DNA) and then digesting the nucleic acid with one of a panel of restriction
enzymes. The individual fragments within the digested DNA are then separated within a gel by electrophoresis
and visualised by staining with ethidium bromide. Ideally each strain will reveal a unique pattern, or fingerprint.
Many different restriction enzymes may be considered at the outset of a new piece of work, so that analyses of
many molecular fingerprints from digestions with several individual restriction enzymes may be undertaken and
combination of the best set of results will allow a comprehensive differentiation between strains or isolates. A
good example of the application of this technique is the differentiation between rabies virus biotypes from dog or
vampire bat origin in Latin America (83).
Of greater utility to the study of pathogens is a modification to the basic RFLP technique whereby the polymerase
chain reaction (PCR) is incorporated as a preliminary step. The PCR method (described in more detail in Section
1 of this chapter) is used to amplify a specific region of the genome (known by the investigator to be variable in
sequence between pathogens), which then serves as the template DNA for the RFLP technique. This new
combination (PCR-RFLP) offers a much greater sensitivity for the identification of pathogens and is especially
useful when the pathogen occurs in small numbers or is difficult to culture, two features that characterise the
intestinal protozoan parasite Cryptosporidium spp. Both RFLP and, more especially, PCR-RFLP are immensely
useful for the genotyping of strains of Cryptosporidium as they can identify sources of human infection and
provide a commentary on their epidemiology and occurrence (16, 145). The involvement of specific strains or
types in a disease outbreak can be thus defined and the epidemiological tracing of isolates within a country or
between countries should be possible.
There are many other examples in which the RFLP/PCR-RFLP techniques are proving useful for discriminating
between genotypes; for example, the fungus Candida (32), the porcine reproductive and respiratory syndrome
virus (162) and the bacterium Helicobacter pylori (54).
The human pathogen Candida krusei provides a good illustration of the general application of a range of
molecular techniques for differentiating isolates. Dassanayake et al. (32) investigated the genetic diversity of
eleven oral isolates of C. krusei and identified five different genotypes by pulsed field gel electrophoresis (PFGE),
nine genotypes by RFLP using the enzyme HinfI, while DNA fingerprinting by the randomly amplified polymorphic
DNA approach (RAPD-PCR) revealed three, eight or eleven genotypes depending on the primers used.
The incorporation of PFGE facilitates the separation of large (up to megabase size) fragments of DNA and can
be a useful adjunct to the basic RFLP analysis. Jager et al. (66) used a combination of the rare-cutting restriction
enzyme NotI and PFGE to characterise 80 isolates of Coxiella burnetii derived from animals and humans in
Europe, USA, Africa and Asia. They distinguished 20 different restriction patterns and phylogenetic analysis of
the different RFLP patterns revealed evolutionary relationships among groups that corresponded to the
geographical origin of the isolates. No correlation between restriction group and the virulence of an isolate was
detected in this study, but similar approaches on some other pathogens have made such a connection. Grigg
and Boothroyd (53), for example, identified three restriction sites within the 35-fold-repetitive B1 locus that were
capable of discriminating type I (mouse-virulent) from type II or III (mouse-avirulent) strains of Toxoplasma
gondii.
RFLPs have clear value for use in epidemiological studies but more critical interpretation of RFLP data involves
the construction of databases to determine whether the RFLP profiles are linked to factors such as virulence,

host range and clinical significance. In practice, it is usual not to rely on one restriction site but to use sites from
several locations within the genome to classify the isolate. A continuing issue for veterinary diagnosticians is the
correct assessment of any molecular differences found between isolates of a pathogen as the loss or acquisition
of restriction endonuclease site(s) may not be associated with differences in the ability of the pathogen to cause
disease, i.e. an RFLP difference may not be functionally significant, except as a distinguishing feature.
Polymorphic RAPD markers that define individual strains, etc. may be sequenced and thence used as a
sequence-confirmed amplified region (SCAR). Thus conversion of an anonymous polymorphic marker to a SCAR
means that a single PCR may be done to more simply identify a specific genome. Lewin et al. (75) used the
approach to identify 19 unique multilocus genotypes among 29 strains of the protozoan, Leishmania donovani.
The techniques by which DNA from a pathogen may be detected and characterised continue to improve and
evolve. The ultimate discriminatory procedure is that of genome sequencing. Sequencing of a well characterised
portion of the genome is playing an important role in pathogen characterisation and epidemiological studies.
Sequencing the products amplified by PCR using degenerate primers targeting a gene common to the viruses in
the same family has become an important diagnostic tool, especially for identification of previously unrecognised
members in the family. Sequencing of degenerate PCR amplicons from the herpesviral DNA polymerase gene is
a good example (169). In a few cases the whole viral genome has been sequenced. For example, the outbreak
of severe acute respiratory syndrome (SARS) and the sequencing of the 29, 751-base genome of the associated
coronavirus (86) usefully revealed that the virus was only moderately related to other known coronaviruses,
including two human coronaviruses and did not closely resemble any of the three previously known groups of
coronaviruses. This degree of interrogation at the level of nucleic acid will not be available to study the majority of
pathogens. Techniques such as RFLP, PCR-RFLP, RAPD-PCR and SCAR analyses will continue to play a
central role in the identification of, and discrimination between, isolates of most pathogens.
3. Diagnosis by DNA probes and DNA microarray technology
Conventional DNA probing and microarray analysis are two sides of the same coin. Fundamental to both
processes is the binding (hybridisation) of DNA, derived from a sample suspected of containing a pathogen (the
‘unknown’), with highly characterised DNA derived in advance from a pathogen of interest (the ‘known’ DNA).
In conventional DNA probing the unknown DNA (or RNA), the target, is immobilised on a solid surface e.g. a
filter. The known DNA, made into a probe by labelling or tagging it in some way, is in the liquid phase and is
applied to the target. In microarray diagnosis it is the known DNA (large oligonucleotides or complementary DNA)
that is the target, immobilised on a glass slide, and the unknown DNA, in the liquid phase, that is labelled to
make a probe.
In conventional DNA probing the target can be nucleic acids extracted from clinical material or cultured cells and
either (a) added to filters (a dot or slot blot) or (b), less conveniently in a diagnostic context, transferred to a filter
after gel electrophoresis. The amount of pathogen in a clinical sample might be too low for detection.
Consequently one might amplify the nucleic acid by PCR or reverse transcription PCR (RT-PCR), the PCR
product being applied to a filter. In order to visualise a probe bound to its target, the probe can be labelled with a
radioactive nuclide or, more commonly and safely, ‘tagged’ non-radioactively. For example, biotin or psoralen–
biotin may be incorporated into the probe, bound probe being detected by addition of streptavidin linked to an
enzyme for subsequent generation of colour or light (chemiluminescence).
A microarray is so-called because it can comprise 20,000 or more different known DNAs, each DNA being
spotted onto glass slides, to form the array. Each spot is only around 10 μm in diameter. DNAs complementary to
parts of selected genes of pathogens can be used to make the arrays (17). However, if large numbers of
pathogens are to be investigated then it would be logistically easier to use large oligonucleotides. The microarray
that was used to identify the SARS virus as being a coronavirus had oligonucleotides comprising 70 nucleotides
(70-mer) (174). In microarray probing it is the sample from which a probe is made. Essentially nucleic acid is
extracted from a sample and an (RT-) PCR performed using random oligonucleotide primers. In this way part of
all the nucleic acids in the sample – both of host and pathogen origin – are amplified. These PCR products,
representative of every nucleic acid in the sample, are labelled with a fluorescent dye and applied to the
microarray. Under optimised conditions only the DNA derived from the pathogen will bind to the DNA on the
glass slide. If one is interested in detecting only a particular pathogen or group of related pathogens then
pathogen-specific oligonucleotides can be used to amplify these within the sample for probe production.
Microarrays for detecting pathogens can be designed for several levels of differentiation. In the case of
oligonucleotide target DNAs one might initially design oligonucleotides to be able to detect and differentiate
pathogens at the genus level. One would choose a number, perhaps 10 or so, of oligonucleotides with a high
degree of sequence conservation (consensus oligonucleotides) within a given genus, such that a probe made
from a field sample containing a member of that genus would be likely to hybridise to at least some of the
oligonucleotides, whilst not hybridising (or hybridising to a lesser degree) to those corresponding to related
genera, e.g. to differentiate Apthovirus (foot and mouth disease, FMDV) isolates from Enterovirus strains in the

Picornaviridae family. One could then select other sets of oligonucleotides, placed on the same array slide, able
to characterise a pathogen more specifically, e.g. to differentiate the seven types of FMDV, and potentially to
even further refinement at subtype level.
In conventional DNA probing the detection of a pathogen is limited by the number of probes used, whereas in
microarray analysis one is limited only by the number of target DNAs on the array. If a microarray has
1000 different oligonucleotides, then to achieve the same resolving power by conventional probing would require
1000 probes, 1000 separate probing reactions. The great advantage of microarray analysis in searching for
pathogens is that hundreds of pathogens can be looked for simultaneously when probing a single microarray
slide. Clearly microarray analysis has great potential when one is investigating diseases of unknown aetiology,
diseases where more than one pathogen might be present, and when subtyping is required. To enhance
sensitivity in pathogen detection, microarrays can be coupled with PCR amplifications. These PCRs are usually
designed to amplify one or more conserved genes, or multiple sequences, such as PCR using broadly conserved
primers, consensus PCR and multiplex PCR as mentioned in the above section. When one has a particular
pathogen in mind, then the use of a microarray would be less justifiable, since the production and hybridisation of
slides is relatively expensive. Instead, for these more simple cases, one might use pathogen/subtype specific
PCRs, followed by sequencing or restriction fragment analysis for confirmation.
If previous experience of biotechnology is indicative of the future, then one would expect microarray equipment
and reagents to become less expensive, leading to greater application of this technology in animal disease
diagnosis. It will assist in the search for hitherto undiscovered viruses or the characterisation of bacterial strains
in terms of virulence, anti-microbial sensitivity or other important markers. One of the main challenges faced
when using array-based approaches is the handling and analysis of the vast data sets that are generated.
4. Nucleic acid extraction
The main variants of molecular diagnostic assays described later in this section are wholly dependent upon the
availability of ‘clean’ nucleic acid mixtures to act as a template for the reactions. While it is relatively easy to
extract DNA from bacterial cultures or blood, it is technically more challenging to prepare suitable material from
fecal samples or abortion material. This stage is critical because if the target material has not been purified of
contaminants in the clinical sample, the assay stage is compromised and may yield false results (Chapter 1.1.5
Validation and quality control of polymerase chain reaction methods used for the diagnosis of infectious
diseases) There are a number of specialised methods for particular types of samples and tissues, some of which
are now commercially available either as manual or automated systems for robotic workstations. These are
destined to improve the reliability of nucleic acid extraction from different samples, but it remains a challenging
area.
B. DETECTION OF PROTEIN
1. Immunohistochemistry
As an adjunct to the isolation of causative organisms from tissue, immunohistochemistry is rapidly becoming a
standard tool in diagnostic laboratories for the identification of antigens associated with viral, bacterial and
protozoal microorganisms and transmissible spongiform encephalopathy (124). The in situ detection of antigens
in fixed tissues offers a number of advantages over other diagnostic techniques. These advantages are: (a)
convenience of sample submission; (b) safe handling of potential human pathogens; (c) retrospective studies of
stored specimens; (d) rapidity; and (e) the detection of nonviable organisms (55). Immunohistochemistry is also
used for the detection of abnormal prion protein (PrPSc) in brain tissue to confirm scrapie, bovine spongiform
encephalopathy and other transmissible spongiform encephalopathies, and has proved to be more sensitive than
the standard histopathological examination for diagnosis of these diseases (156). Demonstration of PrPSc in
lymphoid tissue biopsies, e.g. nictitating membrane, can also be used for the preclinical diagnosis of scrapie
(101). As the number of monoclonal antibodies (MAbs) to defined antigens increases, the use of
immunohistochemistry for the identification of organisms and other specific markers for autoimmunity and
neoplasia will increase. The limiting step in the process of immunohistochemistry is identifying a MAb/antigen
combination that will bind in formalin-fixed tissues. This may be overcome by using frozen sections or employing
antigen retrieval techniques (e.g. proteolytic enzyme digestion, microwaving) before immunostaining.
2. Immunoblotting
Immunoblotting combines the high resolution of gel electrophoresis with the specificity of immunochemical
detection and offers a means of identifying immunodominant epitopes recognised by antibodies from infected
animals or MAbs directed against the target agent. The immunoblotting procedure can be divided into six steps:
a) Preparation of the sample

b) Resolution of the antigen by gel electrophoresis

c) Transfer of the separated polypeptides to a membrane support (nitro-cellulose membrane)

d) Blocking nonspecific binding sites on the membrane
e) Incubation with detecting antibody
f) Detection of bound antibody
The choice of detecting antibody is critical. Polyclonal sera are composed of a range of antibodies reflecting the
full repertoire of the immune response to a particular complex antigen. They will therefore detect a number of
distinct polypeptides giving a characteristic ‘profile’ of reactivity. MAbs bind to only one epitope, therefore they
are useful in identifying highly specific polypeptides. After incubation with the detecting antibody, any antibodies
bound to specific protein bands are visualised using enzyme-labelled conjugated anti-species antisera and a
suitable substrate/chromogen.
Immunoblotting is performed chiefly in diagnostic laboratories to identify and/or characterise infectious agents
based on antigen specificity or to use known antigens to look for a specific serological response. False-positive
and false-negative results in other diagnostic assays can often be resolved by immunoblotting (89). As an
example of antigen detection, immunobloting is a major screening method for BSE and scrapie; it has been used
on millions of brain stem samples in Europe and elsewhere for the detection of prion protein (138). Revised
immunobloting techniques (e.g. SAF-Western blot) are used as confirmatory test methods (8) and to differentiate
between scrapie and BSE (146). Immunoblotting is also often used to determine the specificity of individual
MAbs. Individual purified polypeptides (or recombinant expressed proteins) may also be transferred to
nitrocellulose membranes by immunoblotting to examine the reactivity of test sera to individual proteins. This
characteristic profile of reactivity may be used to help distinguish between animals that have been vaccinated or
infected, such as the enzyme-linked immunoelectrotransfer blot (EITB), a western blot for FMD, which is widely
used in South America (10). The major factor affecting the success of an immunoblotting technique is the nature
of the epitopes recognised by the antibodies. Most high resolution gel techniques involve some form of
denaturation of the antigen. This destroys conformational determinants and allows only the detection of linear or
non-conformational epitopes. Most polyclonal antisera contain antibodies to both linear and conformational
epitopes, but MAbs are directed at single epitopes; thus if they target conformational epitopes, they will not react
with denatured protein.
To detect pathogens in tissue samples, which contain a large amount of protein, selective concentration of
antigen and highly sensitive detection methods are required. Good examples of these techniques have been
used for the detection of the scrapie prion protein (PrPSc) (91). Following the elimination of the normal prion
protein by proteninase digestion, the PrPSc is concentrated by sodium phosphotungstate precipitation or alcohol
precipitation, and the antigen-antibody complex on the filter or ELISA plate is detected by using a chemical
luminescence substrate and the use of a CCD camera or X-ray film to eliminate background emission (12, 133).
3. Antigen-capture enzyme-linked immunosorbent assay (ELISA)
The antigen-capture enzyme-linked immunosorbent assay (ELISA) facilitates detection of antigen from
pathogens directly from an animal prior to or during clinical disease. The ELISA commonly follows a sandwich
assay format using capture and detecting antibodies (either specific MAbs or polyclonal antibodies). Antigen from
the test sample is first captured by a specific MAb or polyclonal antibody bound to a solid-phase support and its
presence is detected through use of a second MAb or polyclonal antibody, which may either be radio- or more
generally, enzyme-labelled (conjugated). If the detecting antibody is not conjugated then an anti-species
conjugate (reactive to the detector antibody) is used. The capture antibody selects the target antigen from other
competing protein in sample suspensions and ensures that it is semi-concentrated to increase the chances of its
detection. The desired characteristics of the capture MAb are strong binding to the pathogen, recognition of a
conserved epitope highly specific for the target agent, and the ability to attach to an ELISA plate without loss of
reactivity. In addition, a second MAb recognising an epitope other than that recognised by the capture MAb that
is bound to the ELISA plate is often used as part of the indicator system. However, it may be difficult to identify
MAbs of comprehensive intra-typic reactivity and polyclonal antisera may be preferred to increase the likelihood
of reaction against all antigenic variants. Examples of antigen-capture ELISAs are the system for detection of
Anaplasma marginale in the blood of preclinical cattle (160), the use of antigen-capture ELISA on cattle blood
samples for the detection of bovine viral diarrhoea virus (88, 136), and the rapid detection of rinderpest and peste
des petits ruminants virus antigens in clinical samples (79). Respiratory syncytial antigen in nasal secretions was
captured using ELISA with MAbs directed against epitopes of the viral capsid (102). Several capture ELISAs for
the detection of prion protein have been extensively validated and are used as rapid screening methods
worldwide (42, 44, 91). Capture ELISAs are also widely used for chronic wasting disease testing in deer60) and
scrapie testing in sheep and goats (43). A related antigen capture method, the Priostrip test, which is a dipstick
method, is also used as a rapid BSE screening test (44). Related antigen-capture methods using
immunomagnetic beads are now important and well accepted methods for detecting certain bacterial infections,
including Listeria, Salmonella and Escherichia coli. The principle of this technology relies on immunomagnetic
separation, i.e. using small super-paramagnetic particles or beads coated with antibodies against surface

antigens of cells. Both intact bacteria and their soluble antigenic determinants can be detected after magnetic
extraction from the test sample, using a second antibody in a sandwich format. As solid surface-free binding, the
antigen-capture assays using immunomagnetic beads can enhance the kinetics of an antigen-antibody reaction.
As a result, both the nonspecific binding and the incubation time are reduced.
Validation of tests to detect antibody is addressed in Chapter 1.1.4 Principals of validation of diagnostic assays
for infectious disease.
4. Immunochromatography
Immunochromatograhy provides a convenient method for detection of antigens in several minutes without special
apparatus (3, 84). The sample is applied on one end of the filter and the microbeads (such as colloidal gold)
conjugated with antibody is applied. The antibody binds to microbead-antigen complex, is trapped on the second
antibody that is on the filter, and is easily visualised at the site where the second antibody was fixed.
5. Proteomics
The proteome is the total complement of proteins expressed within a cell, a tissue or an organism and
proteomics is the study of proteins, including their expression level, post-translational modification and interaction
with other proteins, on a large scale. Since not all proteins are expressed at all times, but are dependent on
physiological and environmental factors, proteomics can provide an excellent global view of disease processes at
the protein level. Because the application of proteomics to novel drug discovery promises huge economic
returns, companies all over the world have rapidly poured resources into this new research field (22).
Many methods used in proteomics, including two-dimensional gel electrophoresis (2DGE) and mass
spectrometry (MS) were established years ago. However recent advances in MS techniques, together with whole
genome sequencing and the development of powerful bioinformatics and robotics platforms, have revolutionised
protein identification. The general principle of proteomics is that proteins are separated, usually by 2DGE on
polyacrylamide gels, then protein spots are excised, digested with trypsin, and the resultant peptides analysed by
MS. The masses of these peptides are then compared to the predicted masses of peptides derived by
computational analyses of genome databases, resulting in gene identification. MS can also be used to deduce
the amino acid sequence of peptides and to characterise post-translational modifications such as glycosylation or
phosphorylation. 2DGE shows some drawbacks, particularly for the separation of hydrophobic proteins, and
other separation techniques based on liquid chromatography are now finding favour for some applications.
Nevertheless, 2DGE is the method of choice for creating quantitative maps of protein expression and many
thousands of proteins can be analysed in a short space of time.
Alterations in the proteome of body tissues or of fluids such as serum, urine or cerebro-spinal fluid can be
measured directly so changes that occur in a disease state can be accurately pinpointed. As well as identifying
molecules that may be targets for novel therapies, this approach is a very powerful tool for early-stage diagnosis
of disease. The best-established clinical applications of proteomics are so far in the identification of markers for
the early diagnosis of cancers, such as bladder cancers in urine (127). However, considerable research efforts
are also ongoing on other areas such as heart disease (68), Alzheimer’s disease (27) and insulin-dependent
diabetes (1).
The use of proteomics for the diagnosis of infectious disease is in its infancy but may prove to be of considerable
importance. For example, definitive diagnosis of chronic hepatitis B virus (HBV) infection still relies on liver
biopsy, but proteomic analysis of serum samples shows that the expression of at least seven serum proteins is
changed significantly in chronic HBV patients (57). Similarly, the ante-mortem differential diagnosis of
Creutzfeldt-Jakob disease (CJD) may be aided by proteomics since preliminary data show that seven proteins in
cerebro-spinal fluid (CSF) are differentially expressed between patients with variant or sporadic CJD (28).
An extremely useful application of proteomics to the diagnosis of infectious disease is in the identification of
novel diagnostic antigens by screening serum from infected and uninfected individuals against immunoblotted,
2DGE mapped proteomes of infectious agents. Using this type of approach with human sera, nine new potential
immunodiagnostic antigens were identified in Helicobacter pylori (50), over 80 antigens in Borrelia burgdorferi
that could potentially differentiate between patients with early or late symptoms of lyme disease (68) and seven
antigens of Toxoplasma gondii that could potentially differentiate between acute and latent toxoplasmosis (68).
Within the veterinary field, proteomics-based research projects are now underway and these will undoubtedly
yield novel diagnostic tools for the future. Proteome maps are being derived for a range of veterinary pathogens
including bacteria such as Brucella melitensis (92) and Streptococcus agalactiae (65), protozoa such as
Toxoplasma gondii (29), Eimeria tenella (21) and Trypanosoma brucei (130) and nematodes such as
Haemonchus contortus (179).

C. ANTIBODY DETECTION
1. Competitive enzyme-linked immunosorbent assay (C-ELISA)
Competitive ELISA (C-ELISA) is an immunoassay that can be used to detect or quantify antibody or antigen
using a competitive method. The C-ELISA for detetion of specific antibodies has largely replaced the indirect
ELISA for large-scale screening and sero-surveillance. The C-ELISA offers significant advantages over the
indirect assay since samples from many species may be tested without the need for species-specific enzyme-
labelled conjugates for each species under test. Many antigens are extremely difficult or time consuming to
purify. If used in an indirect assay, they would result in high background values due to nonspecific binding.
However, relatively crude antigens may be used in the C-ELISA provided the ‘detecting antibody” has the desired
specificity. The principle of a competitive assay for the detection of antibodies is competition between the test
serum and the detecting antibody. Specific binding of the detecting antibody is detected using an appropriate
anti-species conjugate. A reduction in the expected colour obtained is due to binding of antibodies in the test
serum, which prevent binding of the detecting antibody.
The detecting antibody may be polyclonal or monoclonal depending on the required specificity. MAbs directed
against highly conserved epitopes will give broadly reactive assays whereas those directed against highly
specific epitopes will result in a highly specific test. One of the early reports on the use of the C-ELISA was its
use in detecting anti-bluetongue virus antibody (4). This used an MAb against a highly conserved epitope on
bluetongue virus (BTV) P7 and allowed detection of antibodies to all 24 serotypes of BTV. The epitope was not
shared in any of the other closely related Orbivirus serogroups, therefore the test was also BTV-specific. The
specificity of the assay can therefore be tailored depending on the specificity of the detecting antibody. Sensitivity
of C-ELISA is improved using detecting antibody directly conjugated with an enzyme (77).
The C-ELISA format has been successfully used in the screening of large numbers of pig sera for classical swine
fever antibodies (177), the detection of antibody to malignant catarrhal fever virus in inapparently infected sheep,
deer and bison (77, 78) and antibodies to Babesia equi and B. caballi in persistently infected horses (71, 72). A
competitive ELISA for brucellosis, based on the immunodominent Brucella smooth lipopolysaccaride, has been
widely used as a screening test for brucellosis in bovine (98), caprine and ovine (96), porcine (97) and sea
mammals (99). More recently, a solid-phase C-ELISA was used for the large-scale serological surveillance
during the UK FMD outbreak in 2001 (107). This facilitated the testing of some 3 million sera over a period of less
than one year.
2. Production of antigens by recombinant DNA technology

Advances in molecular biology and genetics in the 1970s initiated the development of recombinant DNA
technology. Since then the impact of this technology is such that it plays a vital role in scientific research as well
as in the diagnosis and treatment of disease. Recombinant DNA technology simply refers to the transfer of a
gene from one organism into another – literally, the recombination of DNA from different sources. The objectives
of recombinant DNA technology include identifying genes, isolating genes, modifying genes, and re-expressing
genes in other hosts or organisms. These steps permit scientists and clinicians to identify new genes and the
proteins they encode, to correct endogenous genetic defects, and to manufacture large quantities of specific
gene products such as hormones, antigens for use in vaccines, and other proteins produced by biological agents
of interest. Of particular importance is the degree of specificity in diagnostic tests attainable by the use of
recombinant protein. One example is the use of ESAT-6/CFO-10, (immunogenic secreted antigens) present in
virulent Mycbacterium bovis and M. tuberculosis but not in avirulent BCG or most environmental mycobacteria,
for the diagnosis of tuberculosis in cattle and humans (24, 161). Overlapping peptides of M. tuberculosis antigens
ESAT-6 and CFP-10 increased specificity when they were used in the ELISPOT assay for gamma interferon
detection for the diagnosis of M. tuberculosis infection (61). This has the potential for providing a degree of
specificity in diagnosis not achievable with purified protein derivative (PPD), the bacterial extract currently used.
Native proteins are perhaps the ideal antigens, providing sequence-specific and surface structural epitopes.
Many current diagnostic tests require test antigens that need to be continuously produced from cell culture or
harvested from an infected animal. These antigen preparations are expensive and often have a short shelf-life,
with each new batch of antigen requiring standardisation. Natural proteins are rarely available in a completely
pure form, and antibodies often develop against contaminating polypeptides that can lead to false-positive
results. Recombinant DNA technology produces antigens that offer many advantages over antigens isolated from
other biological sources. These advantages include a high purity, high specific activity and since the protein is
synthesised in genetically modified laboratory-grown cells, each preparation of the protein product is identical to
the previous preparation, ensuring batch-to-batch consistency. When recombinant antigens are used in
combination with the C-ELISA format, purification of the recombinant antigen from the lysate may not be
necessary as the specificity of the C-ELISA resides mainly in the MAb used. An example of the procedure is the
cloning of the envelope genes of caprine arthritis/encephalitis lentivirus in a vaccinia expression vector (80).
Synthetic peptides can also be used as valuable antigens for veterinary laboratory diagnosis. The peptide-based
diagnostic tests rely on the selection of short fragments containing the most potent antigenic (linear) epitopes

that are recognised by specific antibodies induced by the whole viral proteins. In recent years, synthetic peptides
that mimic specific epitopes of infectious agents’ proteins have been used in diagnostic systems for various
human and animal diseases. Both recombinant proteins and synthetic peptides as antigens are useful for the
companion diagnostic tests in DIVA, differentiating infected from vaccinated animals. Marker vaccines carry at
least one less antigenic protein than the corresponding wild-type virus, which allows the serological tracing of
wild type strains in vaccinated individuals (59).
An outline of the procedure for the production of an antigen by recombinant DNA technology is as follows. The
identification of an antigen of potential diagnostic or scientific significance is achieved through the study of the
antibody response of the host to the proteins of the organism in question. Immunodominant antigens, defined
proteins of the organism against which the host responds with the highest potential diagnostic titre, are of
particular interest as they are major stimulants of cellular and humoral immunity against the disease of interest.
Antigen discovery studies are widely used to identify biologically relevant, immunodominant antigens for use in
generating MAbs as well as in vaccine development. Once a protein of interest has been identified, the gene
encoding the protein is generated using messenger RNA (mRNA) from the organism as a template for making
cDNA. This method of cloning the gene encoding the protein of interest requires a prior knowledge about the
gene sequence, either directly from the organism of interest or through the use of gene sequences from closely
related species. An alternative method, when gene sequence data is not available, is the generation of
recombinant libraries from the genomic DNA of the organism or from cDNA synthesised from mRNA. Fragments
of the recombinant libraries can be cloned into an expression system, which may be prokaryotic or eukaryotic,
and the gene library screened for expression of the protein.
There is a wide choice of expression systems. Protein may be expressed in bacteria, usually E. coli (121), yeast
(26), insect cells using baculovirus (147), or in eukaryotic cells by infection with appropriate viral vectors (143) or
by permanent transfection. Differences in glycosylation when prepared in bacterial, insect or mammalian cell
cultures can modify protein structure and its reactivity with antibody. Antigen may need to be extracted from the
cell or may be secreted. Purification is often, but not always, necessary. An upcoming trend in the production of
antigens for use in assays is in the development of synthetic peptide antigens. This allows antigens to be tested
as diagnostic reagents based on the gene sequence, without expression of the whole protein being necessary,
thus shortening the process. An example is the production of peptide antigens from two immunodominant
antigens, reported to be promising candidates as diagnostic reagents for the detection of M. bovis infection in
cattle (172). In recent years, the use of plants for a protein expression system has shown promise. For the
expression of candidate antigens in plants, plant viruses offer the advantages of speed of product development,
flexibility, and high levels of gene expression among others (48).
Genome sequences of hundreds of bacteria and thousands of viruses have already been determined. The
antigen gene can easily be cloned with PCR technology, using primers designed from the nucleotide sequence
of closely related species (126). The gene can also be expressed and its product can be purified using tag
peptide. The antigenicity of the gene products can then be determined. Systematic screening of the antigen gene
in silico, from genome sequence data, accelerates the development of diagnostic kits and vaccine (159).
D. VACCINES
1. Gene deletion vaccines — bacteria
During the past decade, recombinant DNA technology has made it possible to construct safer live vaccines. Live
attenuated bacterial vaccines confer better protection against challenge than killed vaccines (64). The reasons
for this improved protection are not yet clear, but one could be that live vaccines are able to express antigens in
vivo necessary for protection that killed vaccines preparations do not contain. Another reason could be that live
vaccines are able to stimulate antigen presenting cells (APC) in a manner in which killed vaccine preparations
are unable to. Most likely it is a combination of both, novel antigen expression and interaction with APC.
Generation of live attenuated bacterial vaccines relied mainly on the generation and selection of mutants by
serial passage in alternate animal hosts, prolonged culture in vitro, changes in temperature growth or chemical
modification, which resulted in undefined attenuations based on the accumulation of numerous genetic
mutations. In some cases, for unknown reasons, these mutants reverted to wild-type phenotype and therefore
could not be used as vaccines (144). In 1981 Hosieth & Stocker (62), using transposon technology, developed
Salmonella typhimurium strains with defined genetic mutations auxotrophic for aromatic aminoacids (Aro) that
were unable to survive in the immunocompetent host. These strains were able to confer protection against
virulent challenge in the murine model of salmonellosis and in several domestic species, although for unknown
reasons, not all the mutants were able to confer protection in the domestic species (141). In 1992, Jones et al.
(67) developed a live attenuated salmonella mutant using precise genomic excision of two genes involved in the
aromatic amino acid pathway, which resulted in an even lower probability of the strain reverting to wild-type
phenotype. This mutant proved to be a vaccine with relatively mild clinical secondary effects and able to confer
protection in cattle against virulent challenge at the age in which the host is more susceptible. This vaccine strain
has also been used as a delivery vector for guest antigens, which brings closer to reality the ideal single dose

multivaccine (170). Developments in molecular biology and a greater understanding of the host pathogen
interaction will permit the rational design of safer and more efficient vaccines with markers that will allow the
distinction between vaccinated and infected hosts. Although most of the developments described here focus on
salmonella, similar technologies are being applied to other bacterial pathogens. The technology has been used
in bovine tuberculosis; if cattle are vaccinated with BCG, the ESAT-6/CFO-10 peptide cocktail detects infected,
not vaccinated, animals in the IF gamma assay.
2. Marker vaccines and companion diagnostic tests
In animal health, one can either vaccinate animals in order to prevent a disease or try to eliminate the infection
through strict application of sanitary measures such as slaughtering of infected and in-contact animals. For
certain diseases for which no vaccine exists (e.g. African swine fever) and particularly for zoonotic infections (e.g.
Nipah virus infection of pigs), the systematic slaughtering of infected animals is the only available solution.
Diagnosis of infection is of paramount importance whatever the measures taken to fight the disease. Diagnosis
can be direct, through the detection and identification of the infectious agent using immunological or molecular
technologies, or indirect, based upon the detection of specific antibodies against the suspected infectious agent.
The latter methods have a major drawback in that one must wait until antibodies are synthetised by the animal
after infection and generally they do not allow distinction between a humoral immune response resulting from an
infection or a vaccination.
This problem can be overcome by adopting new approaches to vaccine development (105) using molecular
technologies that allow the production of marker vaccines associated with companion diagnostic tests. There are
currently two types, either based on the detection of a serological response against a protein whose gene has
been deleted in the vaccine strain (either used as a replicating vaccine or as an inactivated vaccine derived from
such a deleted virus vaccine strain), or on the detection of the serological response to virus nonstructural
proteins (purified inactivated vaccines). In the case of the deletion vaccines the gene coding for a non-essential
protein, the marker characteristic, is always linked with the detection test while in the case of subunit vaccines
(e.g. protein E2 of classical swine fever virus expressed in baculovirus) the choice of the marker test assay may
be linked to several other of the virus proteins. For harmonisation purposes, an agreed protein should be chosen
for the test (e.g. protein gE of pseudorabies virus). In the first type of marker vaccines, the marker must always
be negative since a positive marker, for instance provided through the insertion of a gene coding for a foreign
protein, is not suitable; such a vaccine will only show if the animal has been vaccinated but will not indicate if the
animal was also infected with the wild virus. Marker vaccine used with the intention of distinguishing a serological
response resulting from either vaccination or infection must always be associated with a companion diagnostic
test that can be used during a prophylactic campaign with the aim of eliminating the infectious agent. Previous
veterinary vaccines were mainly designed to prevent clinical signs in animals following an infection without taking
too much account of the epidemiological impact of vaccination on the excretion of wild virus following infection
and on its dissemination/circulation. If marker vaccines are used with the aim of eliminating a virus they must
have a clear impact on the epidemiology of the infection.
There can be problems with this approach, for example if wild virus multiplication is inhibited to the point that it
does not induce the synthesis of specific antibodies in all vaccinated animals. Therefore, most of the available
marker vaccines can only be used for herd certification and not for individual animal certification.
a) Marker vaccines with one gene deletion: the examples of pseudorabies and infectious bovine
rhinotracheitis
Pseudorabies in pigs and infectious bovine rhinotracheitis are two infections caused by herpesviruses that
become latent in an animal, even when it has already been vaccinated (111, 115, 116). The first marker
vaccine became available to prevent pseudorabies infection in pigs (165) following the development of an
attenuated strain of pseudorabies virus by Bartha in Hungary (7) that had a spontaneous deletion in the gE
glycoprotein. Analogous vaccines were later developed for infectious bovine rhinotracheitis.
As mentioned above, the herpesvirus responsible for infectious bovine rhinotracheitis becomes latent after
infection, whether or not the animal has been vaccinated. It does not matter if the vaccine is an inactivated
or an attenuated one, either way the animal becomes a latent carrier after infection with a wild virus.
Moreover, all the attenuated vaccine strains establish latency after vaccination, including gE deleted strains.
It should be borne in mind that attenuated vaccines produced with identical strains, deleted or not, are
generally more efficacious than their inactivated counterparts (18, 69, 70).
In an area where vaccination is prohibited, all animals serologically positive with regard to infectious bovine
rhinotracheitis virus must be considered as potentially infected and latent carriers of a wild virus. Similarly,
in an area where animals are vaccinated with a conventional (non-deleted) vaccine, either attenuated or
inactivated, it is impossible to distinguish between vaccinated and infected cattle and so if an elimination
programme is in place, all the seropositive animals must also be eliminated from the herd.

A solution is the use of a marked/deleted vaccine that allows the differentiation of antibody produced from
vaccine versus that produced due to infection. The deleted protein in the vaccine strain must have the
following characteristics:
1) Be a structural protein, in order to be able to produce inactivated vaccines;

2) Be non-essential in order to be able to produce the vaccine;
3) Not be an essential protective immunogen in order to still have an efficacious vaccine;
4) Induce a significant and long lasting humoral immune response when present in order to be used
(when deleted) as a marker;
5) Be present in all the wild virus strain;
6) Induce a humoral or cellular immune response by a pathogen in an already vaccinated animals.
If such a marker vaccine is used, whenever an animal is seropositive towards the deleted protein, it must be
seen as infected and eliminated. The gD protein of herpesviruses, being a major protective immunogen,
cannot be deleted but contrarily may be used to develop subunit vaccines. The main problem encountered
with the use of marker vaccines against infectious bovine rhinotracheitis is their inability to completely
prevent wild virus circulation when used within the framework of an elimination programme.
No available vaccine is able to induce sterile immunity for these diseases. As a consequence the
vaccination schedule must be more stringent than a conventional one designed merely to protect against
clinical signs in the herd. Vaccination must be repeated according to a strict schedule to reduce the
possibility of wild virus excretion and must, in addition, be associated with strict sanitary measures (79).
Within the framework of a coordinated virus elimination campaign, vaccination must prevent the excretion of
wild virus by naïve animals and prevent re-excretion by latently infected ones.
The efficacy of repeated vaccination using an inactivated gE negative vaccine administered intramuscularly
has been investigated under field conditions in the Netherlands. This study showed a significantly reduced
incidence of seroconversion against wild virus in the vaccinated group compared with the placebo injected
control animals. In addition, wild virus circulation, while not completely restricted, was nevertheless
significantly reduced (18) and in some circumstances even prevented (166).
b) Vaccination against classical swine fever with subunit vaccines

An elimination programme for classical swine fever has been set up within the European Union. Vaccination
using conventional vaccines is now prohibited and a slaughter policy is in place. This policy is challenged by
the existence of a strong antigenic relationship with other pestiviruses, such as the virus responsible for
bovine viral diarrhoea (BVD/MD), that impede serological diagnosis, the insidious circulation of hypovirulent
strains (14) and, last but not least, the presence of a wild reservoir in wild boar (Sus scrofa) in continental
Europe (5).
The classical, conventional, vaccines have a well proven efficacy (123) and even prevented the emergence
of asymptomatic carriers when they were of sufficient potency (15, 74). Live attenuated vaccines were more
efficacious than their inactivated counterparts in this respect (31) and they contributed greatly to the
elimination of the disease. Their one disadvantage was the creation of a population of serologically positive
animals, which is not acceptable if a slaughter policy is in place. Subunit vaccines have recently been
developed by expressing the E2 protein, a major immunogen of classical swine fever virus, either in a
baculovirus system (van Rijn, 1999 129 /id}) or in vaccinia or pseudorabies viruses (E1) (132, 168). The
baculovirus expressed E2 protein vaccine allows distinction between infected or vaccinated animals when
used with reliable companion diagnostic tests to detect the presence of specific antibodies directed against
other major immunogens of classical swine fever virus not present in the subunit vaccine, such as NS2
protein, a conserved virus protein. Unfortunately, inactivated vaccines are not sufficiently efficacious from
an epidemiological standpoint (37) when compared to the former conventional vaccines (35, 163).
Moreover, the companion diagnostic tests currently available are not fully reliable and therefore limit the use
of these subunit vaccines in the field.
c) Vaccination against foot and mouth disease using highly purified vaccines
Preventive vaccination has been prohibited in the European Union since 1991. This prohibition ended a 30-
year period of vaccination and consequently completely naïve cattle herds now exist in Europe (149). This
situation is particularly detrimental when the disease is accidentally reintroduced (39). The contingency plan
that has evolved to deal with unexpected outbreaks is mainly based on information and training of the
concerned partners in the European Union. In order to overcome the risks associated with the complete
susceptibility of European livestock, concentrated, highly purified virus antigen vaccine banks have been
established (135) and there is the possibility of using these as marker vaccines in case of an emergency
outbreak (34).

This is possible since, when highly purified vaccines are used, whenever an animal is found that is
seropositive to the nonstructural proteins (NSP) coded by the virus using an ELISA diagnostic test kit (33), it
must have been infected by a wild virus. The NSP are produced during the virus replication in the infected
animal and in cell culture. The NSP should be removed from FMDV antigen by purification during vaccine
production and appropriate testing of the antigens should be conducted to demonstrate absence of
seroconversion to NSP in vaccinated animals. The NSP are synthesised at the same level as the structural
proteins during infection and so produce a good humoral immune response. Infected animals become
seropositive to NSP and the antibodies to NSP are usually detected using ELISA diagnostic test kits or the
EITB. Unfortunately, the companion diagnostic tests currently available only permit certification of freedom
from FMD at the herd level and not the individual animal level. When virus multiplication occurs and are not
present in the extracellular virions used to produce purified inactivated vaccines.
d) Equine influenza as a special case

A similar approach as that used for FMD has been applied, in a different context, to equine influenza (110).
When carrying out studies on the duration of protective immunity with equine influenza inactivated vaccines,
it is useful to have a diagnostic tool that allows the exclusion of antibodies due to intercurrent infection of
the experimental animals by a wild influenza virus. A diagnostic test has been developed (13) based on the
serological response to a nonstructural protein coded by the virus.
3. Virus-vectored vaccines
Many virus species, including adenoviruses, herpesviruses and poxviruses, have been used as delivery systems
(vectors) for foreign antigens. The virus can be used simply as a vector, for example the vaccinia-rabies
recombinant virus, or as both a vector and a vaccine against the infection by the wild vector itself. An example of
a virus acting both as a vector and a self vaccine is the recombinant capripox virus expressing a peste des petits
ruminants virus antigen (11). A vector virus may undergo full multiplication cycle leading to the production of
progeny virus or abortive multiplication cycle without the production of progeny virus, such as in the case of the
avipoxvirus vector in mammalian species.
The most commonly used vectors are poxviruses and this chapter will therefore focus on the use of poxviruses
as vaccine vectors (117).
A number of features make poxvirus recombinants suitable as vaccines:
i) the stability of freeze-dried vaccine (28), its low cost, ease of manufacture and administration;
ii) the vaccine can be administrated by several routes (46) and in the case of vaccinia virus it has even been
shown that the virus can be administrated per os (this feature has been used for vaccinating wildlife) (113);
iii) the ability to induce both antibody and cytotoxic T cell responses against the foreign antigen with long
lasting immunity after a single inoculation (142, 143);
iv) the packing flexibility of the genome, which allows large amounts of the genome to be lost or deleted and
foreign DNA to be inserted in its place (at least 25 kb), thus enabling multivalent vaccines to be created
(118, 119, 142, 143);
v) the use of recombinant poxviruses as vaccines allows discrimination between naturally infected versus
vaccinated animal since the recombinant vaccine displays a defined subset of the antigens of the
pathogens concerned.
Within each genus of the Poxviridae family the members are antigenically related (90). This antigenic relationship
has raised an important question concerning the use of poxvirus-derived vectors as live vaccines, as pre-existing
immunity against the vector could reduce the success of a subsequent vaccination performed with a homologous
poxvirus vector (30, 73). To circumvent this problem, the use of different combinations of vectors and/or routes of
immunisation has been implemented (45, 125).
a) Vaccinia virus as a vector

The first recombinant vaccinia to be used in the field is the recombinant vaccinia-rabies vaccine (VRG) used
for oral vaccination of foxes against rabies. This was developed using the Copenhagen strain and tested in
many potential target species under laboratory conditions (20, 112, 155) before it was eventually used
under field conditions in 1987 (114) and proved to be safe and efficacious (20). It has been used on a large-
scale in several European countries that were, as a consequence, freed from rabies (19) as well as in North
America.

The safety of vaccinia virus can be enhanced by multiple gene deletions. This has been demonstrated by
the engineering of the NYVAC strain of vaccinia virus (152). The Copenhagen strain of vaccinia virus was
chosen as the vaccine substrate and based on the entire DNA sequence (49), on extensive knowledge of
virulence-related genes and on genes determining host range replication competency, unwanted genetic
information was deleted from the viral genome in a very precise manner. The resulting virus, named
NYVAC, has 18 open reading frames deleted compared to the parental strain. NYVAC is highly attenuated
as demonstrated in many animal studies. Intracranial inoculation of newborn and young adult mice
demonstrated a very favourable dose range compared with either the parental or other vaccinia strains.
Most significantly there is no dissemination of the virus in immunocompromised hosts. NYVAC has
dramatically reduced the ability to replicate in a variety of human tissue culture cells and is unable to
produce infectious particles in humans. Several animal and human trials have demonstrated the safety of
the NYVAC strain-derived vectors (108, 151, 176).
b) Avipoxvirus vectors
When considering the development of avipox-derived vectors for the production of vaccines for birds, the
use of attenuated strains is recommended in order to reduce the safety risk and the potential consequences
arising from environmental spread to other avian species. Attenuated derivatives of fowlpox virus, like
TROVAC, and canarypox virus, like ALVAC, have been extensively tested and their safety demonstrated in
a variety of species, including immunocompromised animals and human volunteers. These viruses can be
used under laboratory safety conditions level 1, the lowest category for recombinant organisms (109).
Despite the fact that their multiplication is restricted to avian species, attenuated strains of avipoxviruses
have been demonstrated to be efficacious and extremely safe vectors for mammals. Inoculation of avipox
based recombinants in mammalian cells results in expression of the foreign gene and inoculation into
mammalian species induces protective immunity without producing progeny viruses (153, 154). This
observation demonstrates that they have a significant safety advantage for human and animal use. As
immunisation can be achieved in the absence of productive replication, it eliminates the potential for
dissemination of the vector within the vaccinates and, therefore, the spread of the vector to non-vaccinated
contacts or to the general environment. Moreover, the use of this vector in species that are not a reservoir
of avipoxviruses renders the likelihood of recombination in vivo nil. Additionally, these vectors can be used
for vaccination of individuals with pre-existing immunity to vaccinia virus.
In the past decade, a great number of recombinant viruses have been produced using the attenuated
canarypox ALVAC strain as the parental strain. An impressive number of trials, both in humans and
animals, have demonstrated the safety and protective efficacy of vaccines using this vector.
4. DNA vaccines
DNA vaccination is the direct introduction into host cells of a bacterial plasmid DNA that expresses an antigenic
protein under the control of a eukaryotic cell promoter (129). As a consequence, the foreign antigen is expressed
within the host cell and can stimulate the induction of both humoral and cell-mediated immune responses. This
approach to vaccination has been effective against a wide-range of viruses, bacteria and parasites and not only
has many of the benefits of live vaccines but also has several advantages over more conventional approaches to
vaccination. For example, DNA vaccines encoding foreign genes are inexpensive and easy to produce; they
obviate the need for complex carrier organisms; the risks associated with live vaccines are absent; and the
impact of pre-existing immunity to the organism or vector on vaccine efficacy is circumvented. However, a
disadvantage of DNA vaccination is that, as the plasmid persists for a long time, there is a potential for
chromosomal integration with resulting cell transformation.
The immune response of DNA vaccines can be further improved by simultaneous inoculation of
immunostimulators, such as CpG motif sequences (122), plasmids expressing cytokines (178), plasmids
expressing co-stimulatory molecules (85), or even conventional adjuvants (167). Immunogenicity can also be
improved by first priming with a plasmid DNA vaccine expressing an immunogenic protein followed by
subsequent boosting with the protein or with a recombinant virus vector expressing the protein, the so-called
‘prime-boost’ approach (171).
Several DNA vaccines for veterinary use are currently being developed in cattle, pigs and poultry (100, 106, 167).
West Nile-Innovator® DNA is a novel vaccine for horses to aid in the prevention of viremia caused by the
potentially deadly West Nile virus; it represents a tremendous milestone in DNA science and technology. Delivery
of the DNA is either by intramuscular, intradermal or intranasal inoculation, particle-mediated intradermal delivery
using a gene gun in which the DNA is precipitated onto gold microspheres (82), or it can be accomplished using
attenuated intracellular bacteria, such as Shigella flexneri or Salmonella typhimurium (38). Live attenuated
bacterial vaccines allow vaccination via the mucosal surfaces and specific targeting to antigen presenting cells
located at the inductive sites of the immune system. While this latter approach has several advantages, there are
a number of safety issues that need to be addressed before this method of delivery is accepted. The
disadvantages of DNA vaccination should also be borne in mind: these include potential chromosomal

reintegration with resulting cell transformation as the plasmid persists in the host for a long time, although this
risk is low.
Another DNA vaccine strategy is based on the use of a DNA vector consisting of recombinant Semliki Forest
virus (SFV) cDNA under the control of a eukaryotic promoter and expressing a foreign gene (9). Unlike
conventional DNA vectors, the promoter is not directly driving the expression of the foreign antigen, but directs
the synthesis of a recombinant SFV replicon RNA transcript. Translation of this RNA molecule produces a SFV
replicase complex that allows replication of the RNA in the cell cytoplasm and results in high-level production of
the mRNA for the encoded foreign antigen. Since expression mediated by the SFV vector is transient and lytic,
there is less risk from possible chromosomal integration.
Applications of bacterial artificial chromosome (BAC) technology have opened new avenues for manipulation of
large DNA virus genomes, such as herpesviruses (2, 23). The use of BAC clones of herpesviruses is not only a
powerful tool for studying viral gene functions and pathogenesis (173), but also has great potential in herpesviral
vaccine development (94). Experimental studies using BAC clones as vaccines for herpesvirus infections have
delivered on their promise (120, 150, 157). This technology for the generation of novel herpesvirus vaccines will
have significant impact and application in veterinary medicine.
5. Other developments in vaccine technology
Subunit vaccines, which contain purified protein or glycoprotein components of a pathogen that have been
identified as carrying critical epitopes involved in inducing a protective immune response (6) have distinct safety
advantages and recent improvements in their production using recombinant DNA technology may facilitate their
more widespread use (36). Synthetic peptide vaccines have also been engineered (87), however, thus far they
have not been shown to be very effective in inducing protection against infectious diseases. There may be many
reasons why synthetic peptides may not induce protective immunity. For example, even so-called linear peptides
exhibit a degree of conformational flexibility so that they adopt a different structure from that of the parent
molecule and therefore induce antibodies of low avidity for the pathogen in question. A potential disadvantage of
using peptides that represent single antigenic sites to stimulate a protective antibody response is the possibility
of selecting for antigenic mutations in the pathogen.
A number of strategies have been developed for inducing cytotoxic T cell (CTL) responses using peptides, such
as coupling CTL epitopes to toxins that are able to invade eukaryotic cells or constructing virus-like particles
carrying foreign CTL epitopes (137, 140). However, the utility of this approach in outbred populations is limited by
the polymorphism of the major histocompatability complex molecules. Other virus-like particle vaccines that
involve self-assembling proteins that can be used to carry foreign antigens have been made from particles
produced from the TYA gene of the yeast retrotransposon Ty (47). A vaccine composed of empty virus-like
particles produced by expressing the four main structural proteins of bluetongue virus in baculovirus has been
shown to protect against challenge with bluetongue virus (131).
Another interesting approach is the development of ‘edible vaccines‘. Plants can be engineered to express a
number of foreign proteins and can express multiple transgenes at one time (148). The oral delivery of subunit
vaccines expressed in plants would be particularly suited to protect against intestinal pathogens. A disadvantage
would be that antigens delivered orally would be susceptible to proteolytic degradation. Moreover, oral delivery of
antigens tends to induce tolerance rather than active immunity. However, tolerance can be circumvented by
expression of a fusion protein composed of the antigen with the B subunit of the heat labile enterotoxin (LT-B) of
E. coli (56).
Cloning the whole viral genome has been made possible by reverse genetics, especially of negative-stranded
RNA viruses. By reverse genetics, the function of various NSPs, as well as the hidden function of virion proteins,
can be elucidated, thus enabling the construction of an RNA virus by recombinant technology. This also allows
the identification of the escape strategy the virus uses to avoid the host defence mechanisms, such as interferon
(51, 164). Reverse genetics also provides a novel approach for the attenuation of viruses by deleting anti-IFN or
cytokine functions of the virus (195). Reverse genetics can be applied to RNA viruses that have a relatively
simple genome structure, such as avian influenza virus. It has been observed that the presence of basic amino
acid residues at the cleavage site of the haemagglutinin gene assists the avian influenza virus to replicate within
the animal. Conversely, highly pathogenic avian influenza viruses, having this type of genetic structure, can be
attenuated by removing the basic amino acid residue from the site (81, 95).
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*
* *

CHAPTER 1.1.8.
PRINCIPLES OF VETERINARY
VACCINE PRODUCTION
SUMMARY
A reliable supply of pure, safe, potent, and effective vaccines is essential for maintenance of
animal health and the successful operation of animal health programmes. Immunisation of animals
with high quality vaccines is the primary means of control for many animal diseases. In other
cases, vaccines are used in conjunction with national disease control or eradication programmes.
The requirements and procedures described here are intended to be general in nature and to be
consistent with published standards that are generally available for guidance in the production of
veterinary vaccines. The approach to ensuring the purity, safety, potency, and efficacy of
veterinary vaccines may vary from country to country depending on local needs. However, proper
standards and production controls are essential to ensure the availability of consistent, high quality
products for use in animal health programmes.
As the pathogenesis and epidemiology of each disease varies, the role and efficacy of vaccination
as a means of control also varies from one disease to another. Some vaccines may be highly
efficacious, inducing an immunity that not only prevents clinical signs of the disease, but may also
prevent infection and reduce multiplication and shedding of the disease-causing agent. Other
vaccines may prevent clinical disease, but not prevent infection and/or the development of the
carrier state. In other cases, immunisation may be completely ineffective or only able to reduce the
severity of the disease. Thus the decision whether to recommend vaccination as part of an animal
disease control strategy requires a thorough knowledge of the characteristics of the disease agent
and its epidemiology, as well as the characteristics and capabilities of the various available
vaccines. There is also growing public interest in the beneficial implications for animal welfare of
the use of veterinary vaccines as a means of disease control. In any case, if vaccines are used,
successful performance requires that they be produced in a manner that ensures a uniform and
consistent product of high quality.
NOMENCLATURE
The nomenclature for veterinary biological products varies from country to country. For example, in the United
States of America (USA) the term ‘vaccine’ is used for products containing live or inactivated viruses or protozoa,
live bacteria, or nucleic acids. Products containing killed bacteria and other microorganisms are identified as
bacterins, bacterial extracts, conventional or recombinant subunits, bacterintoxoids, or toxoids, depending on the
type of antigen they contain. For example, products containing antigenic or immunising components of
microorganisms may be called ‘subunits’ or ‘bacterial extracts’, and those produced from the inactivation of toxins
are called ‘toxoids’. In the European Union (EU), Immunological Veterinary Medicinal Products are defined as
‘products administered to animals in order to produce active or passive immunity or to diagnose the state of
immunity’, see Directive 2001/82/EC, as amended by Directive 2004/28/EC. For this chapter, however, the term
‘vaccine’ will include all products designed to stimulate active immunisation of animals against disease, without
regard to the type of microorganism or microbial toxin from which they may be derived or that they contain. This
use is more consistent with international nomenclature. ‘Vaccine’ will not be used in this discussion in reference
to biological products recommended for passive immunisation, immunomodulation, treatment of allergies, or
diagnosis.
VACCINE TYPES OR FORMS

Vaccines may be prepared as live or inactivated (killed) products. Some live vaccines are prepared from low
virulence, mild, field isolates of a disease-causing agent that have been found to be safe and effective when

Chapter 1.1.8. — Principles of veterinary vaccine production
administered by an unnatural route or under other conditions where exposure to the microorganism will immunise
rather than cause disease. Other live vaccines are prepared from isolates of disease-causing agents that have
been modified by passage through laboratory animals, culture media, cell cultures, or avian embryos to select a
variant of reduced virulence. The development of recombinant DNA (rDNA) procedures has provided some
unique opportunities for vaccine production. Modified live vaccines may now be specifically produced by deletion
of virulence-related genes from a microorganism. Others are produced by the insertion of genes that code for
specific immunising antigens from a disease-causing microorganism into a nonvirulent vector microorganism.
Nucleic-acid-mediated vaccines containing plasmid DNA are being developed. The DNA is usually in plasmid
form and codes for immunising antigens from disease-causing microorganisms.
Killed products may contain: 1) Cultures of microorganisms that have been inactivated by chemical or other
means; 2) Inactivated toxins; or 3) Subunits (antigenic parts of microorganisms) that have been extracted from
cultures or that have been produced through rDNA procedures.
Both live and inactivated vaccines may be formulated with adjuvants designed to enhance their efficacy.
Frequently used adjuvants are typically water-in-oil emulsions (either single or double), made with mineral or
vegetable oil and an emulsifying agent. Other adjuvants, such as aluminium hydroxide gel or saponin, are also
used. In addition to these traditional adjuvants, vaccines are being developed that include additional ingredients
that induce immunomodulatory effects in the host animal and serve to enhance the efficacy of the product. These
ingredients may include immunogenic components of microorganisms such as killed bacteria, which stimulate
the immune response to other fractions contained in the vaccine, or cytokines, which may be used to regulate
specific aspects of the immune system and are included in rDNA constructs used in products manufactured
through biotechnology.
QUALITY ASSURANCE
The consistent production of pure, safe, potent, and efficacious vaccines requires quality assurance procedures
to ensure the uniformity and consistency of the production process. As production processes for vaccines provide
a great opportunity for variability, care must be taken to control variability to the greatest extent possible,
preferably using validated procedures, and to protect the product from contamination through all stages of
production.
Vaccine purity, safety, potency, and efficacy must be ensured by consistency in the production process.
Consistent product quality (batch-to-batch uniformity) must be built in at each stage. Final product testing is used
as a check to verify that the controls on the production procedures have remained intact and that the released
product meets the specification previously agreed with the licensing authority.
Regulatory authorities in different countries have developed various approaches to ensuring the quality of
vaccines. Although alike in their ultimate goal, these systems may vary in the emphasis given to control of the
production process (process standards) in comparison with control through testing of the final product
(performance standards). The control procedures selected should be those that best fit the conditions under
which vaccines are being produced and, where possible, comply with good manufacturing practice.
The control standards and procedures established for a product define the risk or possibility of producing and
releasing a product that is worthless, contaminated, dangerous, or harmful. The acceptable degree of risk may
depend on the benefits to be gained by having the product available to prevent disease losses. Thus standards
may justifiably vary from country to country or product to product, depending on local animal health conditions.
However, control authorities should strive to establish control standards and procedures that ensure a finished
product of the highest purity, safety, potency and efficacy possible.
The optimal quality assurance system should address both production procedures and final product testing in
proper balance. An absolutely fail-safe system that would result in no risk of releasing an unsatisfactory product
would probably be too expensive with regard to cost of production as well as control. Thus regulatory officials and
manufacturers of vaccines must select control procedures that are capable of ensuring an acceptable low level of
risk in relation to hazard. Such procedures, however, must not be burdensome to the extent that they inhibit the
development and availability of the products needed to provide proper preventative medical care at a cost that is
acceptable to the consumer.
PRODUCTION FACILITIES
Facilities used for the production of vaccines should be designed to protect the purity of the product throughout
the production process and to safeguard the health of the personnel. They must be constructed so that: 1) they
can be readily and thoroughly cleaned; 2) they provide adequate separation of preparation rooms; 3) they have
adequate ventilation; 4) they have ample clean hot and cold water and efficient drainage and plumbing; and
5) they have dressing rooms and other facilities for personnel that are accessible without passing through

biological product preparation areas. Facilities must be adequate to provide for all applicable production
functions, such as: storage of master seeds, ingredients, and other production materials; preparation of growth
media and cell cultures; preparation of glassware and production equipment; inoculation, incubation, and harvest
of cultures; storage of in-process materials; inactivation, centrifugation, addition of adjuvant, and formulation of
product; filling, desiccation, sealing of containers, labelling and storage of final product; quality control testing of
in-process materials and final product; and research and development.
Separate areas are generally required for different activities. All rooms and air-handling systems must be
constructed so as to prevent cross-contamination from other products and to prevent contamination by people or
equipment. Virulent or dangerous microorganisms must be prepared and stored in rooms separate from the
remainder of the establishment. In particular, challenge organisms must be completely separated from vaccine
strains. All equipment that comes into contact with product must be sterilised using validated procedures.
Production facilities have to be designed in such a way that contamination of the external environment is
prevented. Any material used during production has to be made safe before leaving the facility. If highly
contagious microorganisms are propagated, the exhaust air must be treated to prevent escape of infectious
agents. Personnel must follow safety procedures such as showering, and avoid contact with susceptible animals
after leaving the production facilities.
Although the quality and design of production facilities may vary significantly, they must always meet standards
considered to be appropriate for the vaccines that are to be produced. For example, the requirements for facilities
for the production of chicken embryo vaccines administered by oral, intranasal or intraocular routes in chickens
may not need to be quite as demanding as those for the production of cell culture vaccines administered
subcutaneously or intramuscularly.
FACILITIES PLAN
For each vaccine made in a facility, there should be a detailed production plan that describes where each step in
the production process will occur. This plan should be documented in a detailed standard operating procedure
(SOP) or by providing a building blueprint and accompanying blueprint legend. Each room in the establishment
should be uniquely identified, and all functions performed and all microorganisms involved should be specified for
each room. Disinfection procedures, monitoring of equipment and other procedures used in the operation of the
facilities to prevent contamination or errors during production should also be documented. This plan should be
updated as new products or microorganisms are added to the facility, or other changes or improvements in
procedures are developed.
DOCUMENTATION OF THE MANUFACTURING PROCESS

A detailed Outline of Production, a series of SOPs, or other documents should also be prepared to describe the
protocol for the manufacture and testing of each product produced in an establishment. Criteria and standards for
source materials should be clearly and accurately documented. Documentation should also address such things
as: the source, isolation, and passage (subculturing) history of each strain of microorganism; the source and
sequence of nucleic acid elements, or peptides included in products derived from biotechnology, including
plasmids or other vectors used in the construction of genetically modified microorganisms for use as master
seeds; methods for identifying the microorganisms and determining their virulence and purity; the medium or cell
culture system used for seed and production cultures, including the methods used to demonstrate that media are
free from contamination; the source of ingredients of animal origin; methods of media sterilisation; storage
conditions of cell lines and seed cultures; size and types of containers used for growth of cultures; methods for
preparing seed cultures and inoculating production cultures; time and conditions for incubation; observations
during growth; criteria and specifications for satisfactory harvest material; and harvest techniques. There should
be documentation on measures implemented by the firm to minimise the risk of transmissible spongiform
encephalopathies (TSE) agent contamination in ingredients of animal origin and procedures to insure that fetal
bovine serum is free of pestiviruses. It should also include: a description of all tests conducted to assess the
purity and quality of the product as it proceeds through the production process; each step in the formulation of
the final product; the tests used for assessing the purity, safety, potency, and other requirements of each
batch/serial of completed product; the specifications for finishing, including packaging and labelling with complete
indications and recommendations for use; and the expiry date established for the product.
Guidelines for the preparation of such documents for veterinary vaccines are published by competent control
authorities. This documentation is intended to define the product and to establish its specifications and
standards. It should serve along with the blueprints and blueprint legends (or production plan and SOPs) as a
uniform and consistent method of producing the product that should be followed in the preparation of each
batch/serial.

RECORD KEEPING
The producer should establish a detailed record-keeping system capable of tracking the performance of
successive steps in the preparation of each biological product. Records kept should indicate the date that each
essential step was taken, the name of the person who carried out the task, the identity and quantity of ingredients
added or removed at each step, and any loss or gain in quantity in the course of the preparation. Records should
be maintained of all tests conducted on each batch/serial. All records relevant to a batch/serial of product should
be retained for at least 2 years after the expiry date on the label, or in line with the requirements of the competent
control authority. In addition, a record should be maintained of all labels used on all products, with each label
identified as to its name, product number, product licence number, package size, and label identification number.
All labels printed should be accounted for. Records must be kept concerning sterilisation and pasteurisation
procedures. These are usually made by means of automatic recording devices. The manufacturer must also
keep complete records for all animals at the establishment, including health prior to being used for any tests,
results of tests performed, treatment administered, maintenance, necropsy, and disposal.
MASTER SEED
The objective of testing the master seed is to ensure vaccine safety, quality and efficacy. Safety should be tested
in an early stage. A master seed should be established for each microorganism used in the production of a
product to serve as the source of seed for inoculation of all production cultures. Working seeds and production
seeds may be prepared from the master seed by subculturing; generally the final production cultures should not
be more than five (sometimes ten) passages from the master seed. The number of passages should be
determined by data and designated in each case. Using a master seed and limiting the number of passages of
seed microorganism in this manner assists in maintaining uniformity and consistency in production. Records of
the source of the master seed should be maintained. For genetically modified microorganisms, the source of the
gene(s) for the immunogenic antigens and the vector microorganism should be identified. Furthermore, the gene
sequences introduced into the seed microorganism genome during construction of the modified seed should be
provided. The master seed should consist of a single uniform batch/serial of seed that has been mixed and filled
into containers as one batch/serial. Master seed should be frozen or desiccated and stored at low temperatures
such as –40°C or –70°C, or under other conditions found to be optimal for maintaining viability. Each master
seed should be tested to ensure its identity, safety and efficacy. Genetically modified seeds should also be tested
to ensure stability and safety of the inserted gene sequences. Purity should also be established by testing to
ensure freedom from extraneous bacteria, fungi, mycoplasma, and viruses.
MASTER CELL STOCKS

When cell cultures are used to prepare a product, a master cell stock (MCS) should be established for each type
of cell to be used. Records of the source of the master cell stock should be maintained. For each product, the
highest and lowest passage levels of cells that may be used for production should be established and specified
in the Outline of Production or SOP. Some control authorities do not permit more than 20–40 subcultivations.
Each MCS should be characterised to ensure its identity, and its genetic stability should be demonstrated when
subcultured from the lowest to the highest passage used for production. The karyotype of the MCS should be
shown to be stable with a low level of polyploidy. Freedom from oncogenicity or tumorogenicity should be
demonstrated by in-vivo studies in appropriate species using the highest cell passage that may be used for
production. Purity of MCSs should be established by testing to ensure freedom from extraneous bacteria, fungi,
mycoplasma, and viruses.
Primary cells
Primary cells are defined as a pool of original cells derived from normal tissue up to and including the tenth
subculture used in the production of biologicals. In the case of products for use in poultry, these cells are usually
obtained from specific pathogen free embryonating chicken eggs that have originated in an unvaccinated flock
subjected to intensive microbiological monitoring. Other primary cells are derived from normal tissue of healthy
animals and are tested for contamination with a wide variety of microorganisms as appropriate, including
bacteria, fungi, mycoplasmas, and cytopathic and/or haemadsorbing-inducing agents or other extraneous
viruses. The use of primary cells has an inherently higher risk of introducing extraneous agents compared with
the use of cell lines and should be avoided where alternative methods of producing effective vaccines exist.
Indeed, some control authorities only allow the use of primary cells in exceptional cases.
Embyronating eggs
Embyronating eggs are also commonly used in the production of biologicals. In almost all cases they should be
derived from specific pathogen free chicken flocks that have been intensively monitored for infectious agents and

have not been vaccinated. The route of inoculation of the egg and the choice of egg material to be harvested are
dependent on the particular organism that is being propagated.
INGREDIENTS
The specifications and source of all product ingredients should be defined in the Outline of Production, SOP, or
other appropriate documents. The Outline of Production must be approved by the National licensing agency. All
ingredients of animal origin that are not subject to a validated sterilisation procedure should also be tested to
ensure freedom from extraneous bacteria, fungi, mycoplasma, and viruses. Their country of origin should be
known. Measures should be implemented by the firm to avoid the risk of TSE agent contamination by ingredients
of animal origin. Some control authorities discourage the use of preservatives or (more importantly) antibiotics as
a means of controlling adventitious contamination during production and prefer the use of strict aseptic
techniques to ensure purity. However, they sometimes allow the use of preservatives in multidose containers to
protect the product during use. These control authorities usually limit any addition of antibiotics in the
manufacture of the product to cell culture fluids and other media, egg inocula, and material harvested from skin
or possibly other tissues. They normally permit the use of no more than three antibiotics in the same product.
Some control authorities prohibit the use of penicillin or streptomycin in vaccines administered by aerosol or
parenterally. If the antibiotics used are not recommended for use in the target species, they should be shown to
have no harmful effects in the vaccinated animals and not result in the contamination of food derived from
vaccinated animals.
SAFETY TESTS
The intrinsic safety of vaccines should be demonstrated early in the development stage and documented as part
of the licensing dossier. Safety studies during development and licensing for all products should include the
safety of a single dose, of an overdose and of repeated single doses. Additional data are derived for live vaccines
from the increase in virulence tests and by assessing risk to the environment and in-contact animals, as
discussed below. Safety should be demonstrated in each species for which the product is indicated. As a general
rule, overdose studies are required for all vaccines: ×10 for live and ×2 for inactivated vaccines (if this is not
practical, an indication of safety may be obtained from the results of the potency tests). For inactivated virus or
bacterial products, where host animals are used for potency testing, safety may be determined by measuring
local and systemic responses following vaccination and before challenge in the potency tests. Further evidence
concerning the safety of products is derived from field safety trials (discussed below). Vaccines derived through
biotechnology should be evaluated as discussed in the classification of biotechnology-derived vaccines and
release of live rDNA vaccines below.
INCREASE IN VIRULENCE TESTS

With live vaccines, there is concern that the organism might be shed from the host and transmitted to contact
animals, causing disease if it retains residual virulence or reverts to virulence. Therefore, all live vaccines should
be tested for virulence by means of passage studies. Vaccine organisms are propagated in vivo by inoculating a
group of target animals with master seed, in principle; this inoculation uses the natural route of infection for that
organism that is most likely to result in infection and reversion and, if possible, that represents a recommended
route of administration of the vaccine manufactured from this master seed. The vaccine organism is recovered
from tissues or excretions and is used directly to inoculate a further group of animals, and so on. After not less
than four passages, i.e. use of a total of five groups of animals (more for poultry products), the isolate must be
fully characterised, using the same procedures used to characterise the master seed. Regulatory authorities
opinion varies in whether or not it is acceptable to propagate in vitro between passages organisms that otherwise
cannot be passaged five times because of their degree of attenuation. The vaccine organism must retain an
acceptable level of attenuation after propagation in this way.
ASSESSING RISK TO THE ENVIRONMENT

The ability of each live vaccine to shed, to spread to contact target and non-target animals, and to persist in the
environment must be evaluated to provide information for assessing the risk of the vaccine to the environment,
taking into account human health. In some cases this may be done in conjunction with the increase in virulence
tests. These and additional considerations are especially important in the case of products based on
biotechnology or recombinant DNA techniques; more information about such products is provided in the sections
at the end of this chapter.

EFFICACY TESTS
The efficacy of veterinary vaccines should be demonstrated by statistically valid vaccination–challenge studies in
the host animal, using the most sensitive, usually the youngest, animals for which the product is to be
recommended. Data should support the efficacy of the vaccine in each animal species by each vaccination
regimen that is described in the product label recommendation, including studies on the onset of protection when
claims for onset are made in the product labelling and for the duration of immunity. The tests should be
performed under controlled conditions starting, wherever possible, with seronegative animals. Where validated
potency tests are available, target species vaccination–challenge studies may not be required if predictive
serological test results are available. The application of procedures to replace, reduce, and refine animal tests
(the ‘three Rs rule’) should be encouraged whenever possible.
Efficacy studies should be conducted with final product vaccine that has been produced at the highest passage
level from the master seed that is permitted in the Outline of Production, or other documentation of the
manufacturing process. This will have specified the minimum amount of antigen per dose that must be in the final
product throughout the entire authorised shelf-life. Where a range of antigen level per dose is permitted, the
antigen level per dose in the vaccine tested for efficacy must be at or below the minimum permitted amount. The
precise challenge method and the criteria for determining protection vary with the immunising agent and should
be standardised whenever possible.
Field efficacy studies may be used to confirm the results of laboratory studies or to demonstrate efficacy when
meaningful vaccination–challenge studies are not feasible. However, it is generally more difficult to obtain
statistically significant data to demonstrate efficacy under field conditions. Protocols for field studies are more
complex, and care must be given to establish proper controls to ensure the validity of the data. Even when
properly designed, field efficacy studies may be inconclusive because of uncontrollable outside influences. Some
problems include: a highly variable level of challenge; a low incidence of disease in nonvaccinated controls; and
exposure to other organisms causing a similar disease. Therefore, efficacy data from both laboratory and field
studies may be required to establish the efficacy of some products, as well as ‘posteriory’ field trials linked to
vaccinovigilance.
INTERFERENCE TESTS
For products with two or more antigenic components, tests must confirm that there is no interference between
individual components, that is, one component causing a decrease in the protective immunological response to
another component. Interference testing should be conducted for each combination product prior to approval.
A loss of potency may also result when residual inactivating agent in a killed liquid product used as a diluent for a
desiccated live fraction reduces the viability of the live organisms because of viricidal or bacteriocidal activity.
Each batch/serial of liquid killed vaccine that is to be used as a diluent for live vaccines must, therefore, be tested
for viricidal or bacteriocidal activity prior to release.
Consideration must also be given to possible interference between two different vaccines from the same
manufacturer recommended to be given to the same animal within a 2-week period.
CONSISTENCY OF PRODUCTION
Prior to marketing approval of any new product, each establishment should produce in its facilities three
consecutive production batches/serials of completed product to evaluate the consistency of production. These
batches/serials should be prepared according to the procedures described in the Outline of Production and
blueprints and legends, SOPs or other documentation of the manufacturing process and should therefore be
‘typical of production’. Some authorities require that the size of each of the three batches/serials should be at
least one-third the size of the average batch/serial that will be produced once the product is in production.
The manufacturer should test each of these batches/serials for purity, safety, and potency as provided in the
Outline of Production or other documentation of the manufacturing process. Applicable Standard Requirements
and test procedures, for example those described in CFR (Code of Federal Regulations) Title 9 part 113, in the
Annex to EU Directive 2001/82/EC (as amended), in the European Pharmacopoeia, or as described in this
Terrestrial Manual may be used. Satisfactory test results should be demonstrated for all three batches/serials
prior to approving the production of the product in the facilities and its release for marketing. Each subsequent
batch/serial should be tested in the same manner with satisfactory results prior to release for marketing.

STABILITY TESTS
Stability studies (based on an acceptable potency test) are required to establish the validity of the expiry date that
appears on the product package. Some authorities allow the use of accelerated stability tests to determine a
provisional expiry date for products, e.g. incubating at 37°C for 1 week for each year of dating. Such estimates
must be confirmed by periodic real-time potency tests on at least three different batches/serials through the
period of time indicated by the expiry date, and 3–6 months beyond. For products containing viable organisms,
testing should be done at release and at the approximate expiry date until a statistically valid record has been
established. For non-viable products, each batch/serial presented for licensing is tested at release and at
periodic intervals through, or past, the requested expiry date. If at the end of the dating period (shelf life)
specified, the product is tested and found still to be above the release quality, consideration can be given to
extending the designated shelf life, by request to the control authority. Stability testing also provides the
opportunity to test for residual moisture and for other important parameters, such as the stability of adjuvant
emulsions.
BATCH/SERIAL RELEASE FOR DISTRIBUTION

Prior to release, the manufacturer must test each batch/serial for purity, safety, and potency, as well as perform
any other tests described in the firm’s Outline of Production or other documentation of the manufacturing process
for that product. In countries that have national regulatory programmes that include official control authority re-
testing (check testing) of final products, samples of each batch/serial should also be submitted for testing in
government laboratories by competent authorities. If unsatisfactory results are obtained for tests conducted either
by the manufacturer or by competent authorities, the batch/serial should not be released. In such cases,
subsequent batches/serials of the product should be given priority for check testing by competent authorities.
1. Batch/serial purity test
Purity is determined by testing for a variety of contaminants. Tests to detect contaminants are performed on:
master seeds, primary cells, MCSs, ingredients of animal origin if not subjected to sterilisation (e.g. fetal bovine
serum, bovine albumin, or trypsin), and each batch/serial of final product prior to release.
Purity test procedures have been published, for example in CFR Title 9 part 113, in the annex to EU Directive
2001/82/EC (as amended), in the European Pharmacopoeia, or in this Terrestrial Manual, for the detection of
extraneous viruses, bacteria, mycoplasma and fungi, including for example: Salmonella, Brucella, chlamydial
agents, haemagglutinating viruses, avian lymphoid leukosis, pathogens detected by a chicken inoculation test,
chicken embryo inoculation test, lymphocytic choriomeningitis, cytopathic and haemadsorbing agents, and
pathogens detected by enzyme-linked immunosorbent assay, polymerase chain reaction, or the fluorescent
antibody technique. Procedures used to ensure that fetal or calf serum and other ingredients of bovine origin are
free of pestiviruses should be of high concern and well documented. Tests to be used to ensure purity vary with
the nature of the product, and should be prescribed in the Outline of Production or other documentation of the
manufacturing process. As tests for the detection of TSE agents in ingredients of animal origin have not been
developed, vaccine manufacturers should document in their Outlines of Production or SOPs the measures they
have implemented to minimise the risk of such contamination in ingredients of animal origin. This relies on three
principles: first, verification that sources of all ingredients of animal origin in production facilities are from
countries recognised as having the lowest possible risk of bovine spongiform encephalopathy; second, that the
tissues or other substances used are themselves recognised as being of low or nil risk of containing TSE agents;
third, where relevant, that the processes applied to the material have been validated for inactivation of TSE
agents. Methods of production should also document the measures taken to prevent cross contamination of low
risk materials by higher risk materials during processing.
2. Batch/serial safety test
Batch/serial safety tests are required for the release of each batch/serial and typical tests are described in CFR
Title 9 part 113, in the European Pharmacopoeia, in this Terrestrial Manual and elsewhere. Standard procedures
are given for safety tests in mice, guinea-pigs, cats, dogs, horses, pigs, and sheep and are generally conducted
using fewer animals than are used in the safety tests required for licensing. Batches/serials are considered
satisfactory if local and systemic reactions to vaccination with the batch/serial to be released are in line with
those described in the registration dossier and product literature. Some authorities do not permit batch/serial
safety testing in laboratory animals, requiring a test in one of the target species for the product.
3. Batch/serial potency test
Batch/serial potency tests, required for each batch/serial prior to release, are designed to correlate with the host
animal vaccination–challenge efficacy studies. For inactivated viral or bacterial products, potency tests may be

conducted in laboratory or host animals, or by means of quantitative in-vitro methods that have been validated
reliably to correlate in vitro quantification of important antigen(s) with in vivo efficacy. The potency of live vaccines
is generally measured by means of bacterial counts or virus titration. Recombinant DNA or biotechnology-based
vaccines should also be tested. Live genetically modified organisms can be quantified like any other live vaccine
by titration, and expressed products of recombinant technology are quantified by in vitro tests, which can be
easier to perform compared with tests on naturally grown antigens because of the in-process purification of the
desired product.
When testing a live bacterial vaccine for release for marketing, the bacterial count must be sufficiently greater
than that shown to be protective in the master seed immunogenicity (efficacy) test to ensure that at any time prior
to the expiry date, the count will be at least equal to that used in the immunogenicity test. When testing a live viral
vaccine for release, the virus titre must, as a rule, be sufficiently greater than that shown to be protective in the
master seed immunogenicity test in order to ensure that at any time prior to the expiry date, the titre will be at
least equal to that used in the immunogenicity test. Some control authorities specify higher bacterial or viral
content than these. It is evident that the appropriate release titre is primarily dependent on the required potency
and secondarily dependent on the rate of decay of the bacteria or viruses in the vaccine, as indicated by the
stability test.
Standard Requirements have been developed and published by competent authorities for potency testing several
vaccines. These tests can be found in CFR Title 9 part 113, in the European Pharmacopoeia, and in this
Terrestrial Manual.
OTHER TESTS
Depending on the form of vaccine being produced, certain tests may be indicated and should be provided as
appropriate in the Outline of Production or other documentation of the manufacturing process. These tests may
concern: the level of moisture contained in desiccated products, the level of residual inactivant in killed products,
the complete inactivation of killed products, pH, the level of preservatives and permitted antibiotics, physical
stability of adjuvants, retention of vacuum in desiccated products, and a general physical examination of the final
vaccine. Tests for these purposes may also be found in CFR Title 9 part 113, in EU Directive 2001/82/EC (as
amended), in the European Pharmacopoeia, or in this Terrestrial Manual.
SAMPLING
Samples should be selected from each batch/serial of product. The selector should pick representative final
containers from each batch/serial and store these samples at the storage temperature recommended on the
label. The producer should keep these reserve samples at the recommended storage temperature for a minimum
of 6 months after the expiry date shown on the label, so that they are available to assist in evaluating the cause
of any field problems reported from the use of the vaccine. The samples should be stored in a secure storage
area and be tamper-evident.
LABELLING
Standards for labelling products will vary from country to country; however, the label indications and all claims
that are made on the label should be supported by appropriate data that have been reviewed and approved by
competent authorities. It is recommended that all labels for veterinary vaccines be water-proof and contain the
following information, although for very small containers, the label may instead refer to the carton label or to an
enclosed package insert for some of the less prominent information:
1. The true name of the product, prominently lettered and with equal emphasis on each word;
2. The name and address of the producer (and also the importer for imported products);
3. The recommended storage temperature;
4. A statement that the product is ‘for veterinary (or animal) use only’. Full instructions for use, including all
required warnings;
5. For food animals, a statement indicating that the animals should not be vaccinated within a specified
number of days before slaughter. This will depend on the vaccine (e.g. type of adjuvant) and is not required
for all products;
6. The expiry date;
7. The batch/serial number by which to identify the product in the producer’s record of preparation;

8. The licence number for the product; in some countries this is replaced by the licence number of the
establishment/manufacturer;
9. The recoverable quantity and number of doses;
10. A statement that the entire contents of a multidose container should be used when the container is first
opened (or with appropriate holding time for certain products, as supported by data) and that any unused
portions should be disposed of in a proper manner;
11. A safety warning to the operator, if appropriate, e.g. accidental self-injection with oil emulsion vaccines.
12. Where it is allowed for an antibiotic to be added to a vaccine during the production process, the statement
“Contains (antibiotic name) as a preservative” or an equivalent statement indicating the antibiotic added
should appear on the carton or enclosures if used. If cartons are not used, such information should appear
on the final container label.
Labels may also include other factual statements that are not false or misleading. Special restrictions concerning
the use or handling of the product, when applicable, should also be indicated.
Similar information should also be given in a Product Data Sheet that is provided as a package insert. This will
also contain much more detail about method of use and possible adverse reactions.
FIELD TESTS (SAFETY AND EFFICACY)

All veterinary biological products administered to animals should be tested for safety and, if possible, for efficacy
in the field, using good clinical practice, before being authorised for general use. Field studies are designed to
demonstrate efficacy under working conditions and to detect unexpected reactions, including mortality, that may
not have been observed during the development of the product. Under field conditions there are many
uncontrollable variables that make it difficult to obtain good efficacy data, but demonstration of safety is more
reliable. The tests should be done on the host animal, at a variety of geographical locations, using appropriate
numbers of susceptible animals. The test animals should represent all the ages and husbandry practices for
which the product is indicated; unvaccinated controls must be included. The product tested should be one or
more production batches/serials. A protocol should be developed indicating the observation methods and the
recording methods.
INSPECTION OF PRODUCTION FACILITIES

Establishments that are approved to produce veterinary biologicals should be subject to in-depth inspections of
the entire premises by national competent authorities to ensure compliance with the Outline of Production and
blueprints and legends, SOPs, or other documentation of the manufacturing process. These inspections may
include such items as: personnel qualifications; record keeping; general sanitation and laboratory standards;
research activities on products being developed; production procedures; operation of sterilisers, pasteurisers,
incubators, and refrigerators; filling, desiccating, and finishing procedures; care and control of animals; testing
procedures; distribution and marketing; and product destruction. It is desirable to have good manufacturing
practice (for manufacturing) and good laboratory practice (for quality assurance testing). (See chapter 1.1.3
Quality management in veterinary testing laboratories, for guidelines.)
The inspectors should prepare a comprehensive report documenting the findings of the inspection and stating the
actions that the establishment must take to improve its production processes. The establishment should receive
a copy of the report. When necessary a follow-up inspection should be conducted to determine whether
appropriate action has been taken to correct deficiencies. Continued reassessment in this manner is needed to
ensure that production facilities continue to be operated in an acceptable manner.
UPDATING THE OUTLINE OF PRODUCTION

Before production procedures are changed, the corresponding Outline of Production or other documentation of
the manufacturing process should be changed. Establishments should have internal review procedures to
evaluate all changes in production before they are initiated. Changes should also be reviewed and approved by
competent authorities prior to their implementation. In cases where a significant production step is altered,
revisions may require additional data to support the purity, safety, potency, and/or efficacy of the product. In
countries with regulatory programmes that include check testing the final product at national laboratories,
revisions should entail testing of the new product by competent authorities.

PERFORMANCE MONITORING
Manufacturers are required to maintain an adverse reaction notification system and an effective mechanism for
rapid product recall. These should both be subject to audit by regulatory bodies. In many countries, the
manufacturer must notify all adverse reactions immediately to the regulatory authority, along with any remedial
action taken. An alternative used in some countries is that if at any time, there are indications that raise questions
regarding the purity, safety potency, or efficacy of a product, or if it appears that there may be a problem
regarding the preparation, testing or distribution of a product, the manufacturer must immediately notify the
regulatory authorities concerning the circumstances and the action taken.
After release of a product, its performance under field conditions should continue to be monitored by competent
authorities. Consumer complaints may serve as one source of information; however, such information needs to
be investigated to determine whether or not the reported observations are related to the use of the product. Users
of veterinary vaccines should be informed of the proper procedures for making their complaints. The
manufacturer of the product should be informed of all complaints received by competent authorities. Competent
authorities should also ascertain whether they have received other similar complaints for this product and, if so,
whether the manufacturer has taken appropriate action. Control laboratories may test samples of the batch/serial
of product involved, if necessary.
When the investigation is complete, a final report should be prepared and a summary of the findings sent to the
complainant and to the manufacturer. When it is determined that a product is causing serious problems,
immediate action should be taken to remove the product from the market and to notify animal health authorities.
ENFORCEMENT
National programmes established to ensure the purity, safety, potency, and efficacy of veterinary vaccines must
have adequate legal authority to ensure compliance with product registration conditions and other programme
requirements. The goal should be to obtain voluntary compliance with established regulatory requirements.
However, when violations occur, competent authorities must have adequate legal authority to protect animal and
human health. Authority for detention, seizure, and condemnation of products found to be worthless,
contaminated, dangerous, or harmful may be valuable for this purpose. Under such authority, product may be
detained for a period of time, and if during that time compliance cannot be achieved, competent authorities may
seek a court order or decree for seizure and condemnation.
The authority to remove or suspend establishment and/or product licenses, obtain injunctions, and stop the sale
of product is also needed. Civil penalties or criminal prosecution may also be necessary for serious or deliberate
violations.
LICENSING OF PRODUCTS DERIVED THROUGH BIOTECHNOLOGY

Recent advances in biotechnology have made possible the development and commercialisation of new biological
products with useful antigenic and diagnostic properties. Many such products have now been licensed or
approved, and more are being developed. Products of rDNA technology do not differ fundamentally from
conventional products. Therefore, existing laws and regulations are fully applicable to these new products.
CLASSIFICATION OF BIOTECHNOLOGY-DERIVED VACCINES

Each competent authority with power to regulate organisms and products derived from recombinant techniques
should ensure that the public health and the environment are protected from any potentially harmful effect. For
the purpose of evaluating licence applications, veterinary vaccines derived through rDNA technology may be
divided into three broad categories. The division is based on the products’ biological properties and on the safety
concerns they present.
Category I consists of nonviable or killed products that pose no risk to the environment and present no new or
unusual safety concerns. Such products include inactivated microorganisms, either whole or as subunits, created
by using rDNA techniques.
Category II products contain live microorganisms modified by adding or deleting one or more gene(s). Added
genes may code for marker antigens, enzymes, or other biochemical by-products. Deleted genes may code for
virulence, oncogenicity, marker antigens, enzymes, or other biochemical by-products. The licence application
must include a characterisation of the DNA segments added or deleted, as well as a phenotypic characterisation
of the altered organism. The genetic modifications must not result in any increase in virulence, pathogenicity, or

survivability of the altered organism in comparison with the wild-type form. It is important that the genetic
modification does not cause a deterioration in the safety characteristics of the organism.
Category III products make use of live vectors to carry recombinant-derived foreign genes that code for
immunising antigens. Live vectors may carry one or more foreign gene(s) that have been shown to be effective
for immunising target host animals. The use of DNA vaccines containing recombinant-derived foreign genes that
code for immunising antigens (plasmid DNA vaccines) constitutes a new approach to vaccine development. The
proper categorisation of this type of rDNA-derived product will be established as biological properties and safety
characteristics are determined. These new vaccines may find application in a wide variety of situations much as
conventional products have. Guidelines for the development, production, characterisation, and control of these
new products are still preliminary and subject to change as new data and knowledge are developed. Information
concerning the current thinking on regulatory guidelines may be found on the Internet at the following addresses:
http://www.cba.unige.it/VL/bio-info.html; http://www.aphis.usda.gov/vs/cvb;
http://www.orcbs.msu.edu/biological/biolsaf.htm; http://www.pestlaw.com/index.html;
http://www.emea.europa.eu/pdfs/vet/iwp/000798en.pdf
RELEASE OF LIVE rDNA PRODUCTS

The release of live rDNA microorganisms (Categories II and III) for field testing or general distributions as an
approved or licensed product may have a significant effect on the quality of the human and animal environment.
Before release is authorised, the manufacturers of the vaccine should conduct a risk assessment to evaluate the
impact on the human and animal environment. In the USA, for example, a procedure is adopted that could be
used as a model system in other countries. The European Union has adopted a similar system. It is performed as
follows:
A risk assessment is carried out that should contain the following information: the purpose and need for the
proposed action; the alternatives considered; a list of the government agencies, organisations, and persons
consulted; and the affected environment and the potential environmental consequences. The topics discussed
should include: the characteristics of the vaccine organism, human health risks, animal health risks for both
target and nontarget animals, persistence in the environment, and increase in virulence.
If the risk assessment results in a finding by competent authorities that the proposed release of the recombinant
vaccine into the environment for field trials or general distribution would not have a significant impact on the
environment, a notice should be published and distributed to the public announcing this and that the risk
assessment and findings are available for public review and comment. If no substantive comments are received
to refute the findings, competent authorities may authorise the field testing or grant the license or approval for
general distribution.
The preparation of a risk assessment and the findings made from the assessment may also include the
scheduling of one or more public meetings if a proposed action has ecological or public health significance. Such
meetings should be announced through a public notice. Interested persons should be invited to make
presentations, along with presentations by the producer of the product, and government personnel. The
transcripts of such meetings should become part of the public record.
If, in the course of preparing a risk assessment, competent authorities conclude that the proposed action may
have a significant effect on the human environment, an Environmental Impact Statement (EIS) should be
prepared. The EIS provides a full and fair discussion of the significant environmental impacts, and informs
decision-makers and the public of any reasonable alternatives that would avoid or minimise the adverse impacts.
(Environmental documents are considered in CFR Title 40 part 1508.) See also EU Directive 2001/18/EC and
http://www.emea.europa.eu/pdfs/vet/iwp/000404en.pdf
FURTHER READING
The following are some suggested texts that contain guidelines on aspects of vaccine production.
A. COUNCIL OF EUROPE (2005). European Pharmacopoeia, Fifth Edition. Editions of the Council of Europe,
Strasbourg, France.
B. ESPESETH D.A. (1993). Licensing Veterinary Biologics in the United States. The First Steps Towards an
International Harmonization of Veterinary Biologicals; and Free circulation of vaccines within the EEC. Dev.
Biol. Stand., 79, 17–25.

C. ESPESETH D.A. & GOODMAN J.B. (1993). Chapter 13. In: Licensing and Regulation in the USA. Vaccines for
Veterinary Application. Butterworth Heinemann, London, UK, 321–342.
D. EUROPEAN COMMISSION (2006). The Rules Governing Medicinal Products in the European Union. Eudralex.
Volumes 1–9. European Commission Enterprise and Industry DG; Directorate F – Consumer goods. Latest
versions only available at http://pharmacos.eudra.org/F2/eudralex/index.htm.
E. GAY C.G. & ROTH H.J. (1994). Confirming the safety characteristics of recombinant vectors used in
veterinary medicine: a regulatory perspective. Recombinant vectors in vaccine development. Dev. Biol.
Stand., 82, 93–105.
F. ROTH H.J. & GAY C.G. (1996). Specific safety requirements for products derived from biotechnology. In:
Veterinary Vaccinology, Pastoret P.-P., Blancou J., Vannier P. & Verschueren C., eds. Elseviers Science
Publishers B.V. Amsterdam, The Netherlands.
G. PASTORET P.P., BLANCOU J., VANNIER P. & VERSCHUEREN C., EDS (1997). Veterinary Vaccinology. Elsevier
Science, Amsterdam, The Netherlands.
H. UNITED STATES DEPARTMENT OF AGRICULTURE (USDA) (2000). Code of Federal Regulations, Title 9, Parts 1–
199. US Government Printing Office, Washington DC, USA.
1
I. USDA-APHIS -VETERINARY SERVICES-CENTER FOR VETERINARY BIOLOGICS (1999). Categories of Inspection for
Licensed Veterinary Biologics Establishments. Veterinary Services Memorandum No. 800.91. Center for
th
Veterinary Biologics, 510 S. 17 Street, Suite 104, Ames, Iowa 50010, USA.
J. USDA-APHIS-VETERINARY SERVICES-CENTER FOR VETERINARY BIOLOGICS (1999). Veterinary Biological Product

th
Samples. Veterinary Services Memorandum No. 800.59. Center for Veterinary Biologics, 510 S. 17 Street,
Suite 104, Ames, Iowa 50010, USA.
K. USDA-APHIS- VETERINARY SERVICES-CENTER FOR VETERINARY BIOLOGICS (1995). Guidelines for Submission of
Materials in Support of Licensure. Veterinary Biologics Memorandum No. 800.84. Center for Veterinary
th
Biologics, 510 S. 17 Street, Suite 104, Ames, Iowa 50010, USA.
L. USDA-APHIS-VETERINARY SERVICES-CENTER FOR VETERINARY BIOLOGICS (1995). Veterinary Biologics General

Licensing Considerations No. 800.200, Efficacy Studies. USDA-APHIS-Veterinary Biologics, 4700 River
Road, Riverdale, Maryland 20737, USA.
M. USDA-APHIS-VETERINARY SERVICES-CENTER FOR VETERINARY BIOLOGICS (1995). Veterinary Biologics General

th
Licensing Considerations No. 800.201, Back Passage Studies. Center for Veterinary Biologics, 510 S. 17
Street, Suite 104, Ames, Iowa 50010, USA.
N. USDA-APHIS-VETERINARY SERVICES (1964–1994). Standard Assay Methods, Series 100–900. National

Veterinary Services Laboratories, Ames, Iowa 50010, USA.
O. USDA-APHIS- VETERINARY SERVICES-CENTER FOR VETERINARY BIOLOGICS (1984). Basic License Requirements
th
for Applicants. Veterinary Biologics Memorandum No. 800.50. Center for Veterinary Biologics, 510 S. 17
Street, Suite 104, Ames, Iowa 50010, USA
P. USDA-APHIS-VETERINARY SERVICES-CENTER FOR VETERINARY BIOLOGICS (1988). Guidelines for the

Preparation and Review of Labeling Materials. Veterinary Services Memorandum No. 800.54. Center for
th
Veterinary Biologics, 510 S. 17 Street, Suite 104, Ames, Iowa 50010, USA.
*
* *
1 United States Department of Agriculture (USDA), Animal and Plant Health Inspection Services (APHIS). USDA-APHIS-
CENTER FOR VETERINARY BIOLOGICS HOME PAGE: http://www.aphis.usda.gov/vs/cvb/index.html

APPENDIX 1.1.8.1.
RISK ANALYSIS FOR BIOLOGICALS

FOR VETERINARY USE
GENERAL CONSIDERATIONS
All products, including biologicals for veterinary use, derived from animals have some capacity to transmit animal
disease. The level of this capacity depends on the inherent nature of the products, their source, the treatment
that they might have undergone, and the purpose for which they are intended. Biologicals for in vivo use in
particular will have the highest probability of exposure to animals and as such present the highest risk. Products
used for in vitro purposes can introduce disease into animal populations through deliberate or inadvertent use in
vivo, contamination of other biologicals, or spread by other means. Even products for diagnosis and research
have the potential for close contact with animals. Exotic micro-organisms, some highly pathogenic, which may be
held for research and diagnostic purposes in countries free from infection or the diseases they cause, could
possibly contaminate other biological products.
Veterinary Authorities of importing countries shall make available specific procedural requirements for approval
or licensing of biologicals for veterinary use. They may limit supply to registered institutions or in vitro use or for
non-veterinary purposes where such assurance cannot be provided.
*
* *

APPENDIX 1.1.8.2.
RISK ANALYSIS FOR VETERINARY VACCINES
INTRODUCTION
Risk analysis for veterinary vaccines has to be founded on the principles of quality assurance, which includes
quality control, in the production of veterinary vaccines. These recommendations are focused mainly on the risk
related to the contamination of vaccines by infectious agents particularly in regard to the risk of importing exotic
diseases. The major risk of introducing a disease into a country is through importation of live animals or animal
products and rarely through veterinary vaccines. Veterinary vaccines can however be contaminated by disease
agents if master seeds, strains, cell cultures, animals or ingredients of animal origin such as fetal calf serum used
in production are contaminated or if cross contamination occurs during the production process.
PRINCIPLES
Exporting countries and importing countries should agree on a system of classification of risks associated with
veterinary vaccines taking into account factors such as purification procedures which have been applied.
Exporting countries and importing countries should agree on risk analysis models to address specific issues and
products. Such risk analysis models should include a scientific risk assessment and formalised procedures for
making risk management recommendations and communicating risk. The regulation of veterinary vaccines
should include the use of either qualitative or quantitative models.
Risk analysis should be as objective and transparent as possible. Step risk and scenario tree methods should be
used in risk assessment whenever appropriate, as they identify the critical steps in the production and use of the
products where risks arise and help to characterise those risks.
The same conclusions about risk analysis may be reached by differing methods. Where methods may differ in
countries, the concept of equivalence should apply wherever possible and the methods should be validated to
ensure they are of comparable sensitivity.
MANUFACTURING PRACTICES
The manufacture of veterinary vaccines has special characteristics which should be taken into consideration
when implementing and assessing the quality assurance system. Due to the large number of animal species and
related pathogenic agents, the variety of products manufactured is very wide and the volume of manufacture is
often low; hence, work on a group basis is common. Moreover, because of the very nature of this manufacture
(cultivation steps, lack of terminal sterilisation, etc.), the products must be particularly well protected against
contamination and cross contamination. The environment must also be protected especially when the
manufacture involves the use of pathogenic or exotic biological agents and the worker must be particularly well-
protected when the manufacture involves the use of biological agents pathogenic to man.
These factors, together with the inherent variability of immunological products, means that the role of the quality
assurance system is of the utmost importance. It is important that vaccines should be manufactured in
accordance with a recognised codified system that includes specifications regarding equipment, premises,
qualification of personnel as well as quality assurance and regular inspections.
A commonly agreed system of facility inspection carried out by qualified and specialised inspectors must be in
place to assure confidence.

Appendix 1.1.8.2. — Risk analysis for veterinary vaccines
INFORMATION TO BE SUBMITTED WHEN APPLYING FOR REGISTRATION

IN THE IMPORTING COUNTRY
The manufacturer or Veterinary Authority of the exporting country should make available to the importing country
the pharmacopoeia it uses. For the importing country it is necessary to have documented both the quality control
methods used and the source of each batch of starting materials. The key steps of the manufacturing process of
veterinary vaccines should be described in detail to help risk analysis. Risk analysis has to be focused on the
quality and safety parts of the application file. Laboratory safety testing should cover target and non-target
organisms to obtain sufficient biological data. All test procedures used should correspond with the state of
scientific knowledge at the time and should be validated.
The description of the method of preparation of the finished product should include an adequate characterisation
of the substances needed to prepare the working seeds, the description of the treatments applied to starting
materials to prevent contamination, and a statement of the stages of manufacture at which sampling is carried
out for process control tests.
The results of control tests during production and on finished product, as well as the sensitivity of these tests,
have to be available for risk analysis. The stepwise procedures of the control tests should also be available.
CATEGORISATION OF VETERINARY VACCINES

To assist in risk analysis, countries should establish a system of categorisation of veterinary vaccines taking into
account criteria such as pathogens used as active ingredients, their inherent characteristics and the risk they
pose.
In case of live vectored vaccines, the safety of the vector to the targeted and non-targeted species and to human
beings must be assessed. Special attention should be paid to potential tissue tropism or host range modification
of the recombinant.
VACCINOVIGILANCE
Exporting countries and importing countries should ensure that a reliable system of vaccinovigilance (post
licensing monitoring) is established to identify, at the earliest stage, any serious problems encountered from the
use of veterinary vaccines. Vaccinovigilance should be ongoing and an integral part of all regulatory programmes
for veterinary vaccines, especially live vaccines.
RISK COMMUNICATION
Reliable data in support of applications submitted in importing countries should be provided by the manufacturer
or the Veterinary Authority of the exporting country. Relevant data on risk analysis, changes in animal health
situations and vaccinovigilance should be shared by Veterinary Authorities on a continuous basis.
*
* *

CHAPTER I.1.9.
TESTS FOR STERILITY AND FREEDOM FROM

CONTAMINATION OF BIOLOGICAL MATERIALS
INTRODUCTION
Sterility is defined as the absence of living organisms. It is achieved by heating, by filtration, by

treatment with ethylene oxide or by ionising irradiation, and by conducting any subsequent
processes aseptically. Freedom from contamination is defined as the absence of specified living
organisms. This may be achieved by selecting materials from sources shown to be free from the
specified organisms and by conducting subsequent procedures aseptically. Adequate assurance of
sterility and freedom from contamination can only be achieved by proper control of the primary
materials used and their subsequent processing and storage. Tests on the product are necessary
to check that this control has been achieved.
A. GENERAL PROCEDURES
1. Primary materials must be collected from sources shown to be free from contamination and handled in such
a way as to minimise contamination and the opportunities for any contaminants to multiply.
2. Materials that can be sterilised without their biological activities being affected unduly must be sterilised by
a method effective for the materials concerned. The method must reduce the level of contamination to be
undetectable, as determined by an appropriate sterility test (see paragraph B.3 below).
3. If a sterilisation process is used, it shall be validated to demonstrate its suitability and adequately controlled
to show that it has functioned properly on each occasion.
4. Materials that are not sterilised and those that are to be processed further after sterilisation must be
handled aseptically.
5. The environment in which any aseptic handling is carried out must be maintained in a clean state and
protected from external sources of contamination, and must be controlled to prevent internal contamination.
B. LIVING VIRAL VACCINES FOR ADMINISTRATION BY INJECTION

1 Materials of animal origin shall be (a) sterilised, or (b) obtained from healthy animals that, in so far as is
possible, should be shown to be free from pathogens that can be transmitted from the species of origin to
the species to be vaccinated, or any species in contact with them, or (c) the material shall be shown to be
free from such pathogens.
2. Seed lots of virus and of any continuous cell line used for virus growth shall be shown to be free from
bacteria, fungi, mycoplasmas, extraneous viruses and other pathogens that can be transmitted from the
species of origin to the species to be vaccinated or any species in contact with them. For the production of
avian vaccines and the quality control procedures for these vaccines, it is recommended that specific
pathogen-free embryonated chicken eggs be used.
3. Each batch of vaccine shall pass a test for sterility that is similar to published methods (1–3, 6).
4. Each batch of vaccine shall pass tests appropriate to prove that the vaccine is free from extraneous viruses.
(Such tests include tests in cell cultures susceptible to viruses of the species to be vaccinated, tests in
embryonated eggs, and, where necessary, tests in animals.)

Chapter 1.1.9. — Tests for sterility and freedom from contamination of biological materials
5. Some countries require that each batch of vaccine pass a test for freedom from mycoplasma. Suitable test
methods have been published (1, 2, 6).
6. Tests for freedom from certain specific bacteria may be required, e.g. tests for Salmonella, Mycobacterium
tuberculosis and M. paratuberculosis, Brucella spp. and Leptospira spp. (1, 2).
C. LIVING VIRAL VACCINES FOR ADMINISTRATION THROUGH DRINKING

WATER, SPRAY, OR SKIN SCARIFICATION
1. Paragraphs B.1, 2, 4, 5, and 6 apply.
2. A limited number of contaminating, nonpathogenic bacteria and fungi may be permitted (see Section J.2.5
below).
D. INACTIVATED VIRAL VACCINES

1. Paragraphs B.1, 2 and 3 apply.
2. Each batch of vaccine shall pass a test for inactivation of the vaccinal virus. This is done before addition of
preservative. The inactivation process and the tests used to detect live virus after inactivation must be
validated and shown to be suitable for their intended purpose.
3. Demonstration that the method of inactivation also inactivates representative pathogens may be required
unless the vaccine satisfies the conditions of paragraphs B.4 and B.5.
E. LIVING BACTERIAL VACCINES

1. Paragraphs B.1 applies.
2. Seed lots of bacteria shall be shown to be free from other bacteria as well as fungi and mycoplasmas.
3. Each batch of vaccine shall pass a test for purity carried out using solid media and ignoring the growth of
the vaccinal bacterium.
4. Some countries require that each batch of bacterial vaccine passes a test for freedom from mycoplasmas.
Suitable test methods have been published (ref. 6, and for avian mycoplasmas ref. 2).
F. INACTIVATED BACTERIAL VACCINES

1. Paragraphs B.1, B.3, and E.2 apply.
2. Each batch of vaccine shall pass a test for inactivation of the vaccinal bacterium. If appropriate, the test for
sterility may be used for this purpose.
G. SERA FOR ADMINISTRATION TO ANIMALS

1. Paragraph B.1 applies. Some countries require quarantine, health certification, and specific disease tests
be completed for all serum donor animals (1).
2. Paragraph B.2 or E.2 applies, as appropriate, if a virus or a bacterium is used in serum production.
3. Each batch of serum shall pass a test for sterility. Suitable test methods have been published (1, 2).
4. Each batch of serum shall pass tests appropriate to prove that the serum is free from extraneous viruses.
(Such tests include tests in cell cultures susceptible to viruses of the species to be treated, tests in
embryonated eggs and, where necessary, tests in animals.)

5. Some countries require that each batch of serum passes a test for freedom from mycoplasmas. Suitable
test methods have been published (ref. 6, and for avian mycoplasmas ref. 2).
H. DIAGNOSTIC AGENTS FOR ADMINISTRATION TO ANIMALS

1. Paragraphs B.1 and 3 apply.
2. Paragraphs B.2 and D.2 apply if a virus is used in the production of the diagnostic agent; E.2 and F.2 apply
if a bacterium is used.
I. EMBRYOS, OVA, AND SEMEN

Special precautions must be taken with relation to the use of embryos, ova and semen (4).
J. PROTOCOL EXAMPLES
1. General procedures
Materials used in the production of biological products should be sterilised and/or tested to ensure freedom from
contaminants before being used. Samples of the finished biological product should also be tested for bacterial,
fungal, or mycoplasmal contaminants.
The assays for bacteria, mycoplasma, fungi, and viruses described here are derived from various sources and
they are given as examples of methods that can be used with confidence.
2. Detection of bacteria and fungi
These assays describe the materials and methods that are used for the detection of bacteria and fungi by either
the membrane filtration method, or the direct inoculation of fluid media method used for materials that are
unsuitable for membrane filtration.
2.1. General procedure for detecting viable bacteria and fungi

Standard tests for detecting extraneous bacteria and fungi in raw materials, seed stocks, or final product
are: the membrane filtration test or the direct inoculation sterility test.
For the membrane filtration technique, a filter having a nominal pore size not greater than 0.45 µm and a
diameter of at least 47 mm should be used. Cellulose nitrate filters should be used if the material is
aqueous or oily; cellulose acetate filters should be used if the material is strongly alcoholic, oily or oil-
adjuvanted. Immediately before the contents of the container or containers to be tested are filtered, the filter
is moistened with 20–25 ml of Diluent A or B.
Diluent A – for aqueous products or materials: Dissolve 1 g peptic digest of animal tissue in water to make
1 litre, filter or centrifuge to clarify, adjust the pH to 7.1 ± 0.2, dispense into containers in 100 ml quantities,
and sterilise by steam.
Diluent B – for oil-adjuvanted products or materials: Add 1 ml polysorbate 80 to 1 litre Diluent A, adjust the
pH to 7.1 ± 0.2, dispense into containers in 100 ml quantities, and sterilise by steam.
If the biological being tested has antimicrobial properties, the membrane is washed three times after sample
application with approximately 100 ml of the appropriate diluent (A or B). The membrane is then transferred
whole to culture media, aseptically cut into equal parts and placed in media, or the media is transferred to
the membrane in the filter apparatus. If the test sample contains merthiolate as a preservative, fluid
thioglycollate medium (FTM) is used and the membranes are incubated at both 30–35°C and 20–25°C. If
the test sample is a killed biological without merthiolate preservative, FTM is used at 30–35°C and soybean
casein digest medium (SCDM) at 20–25°C. If the sample tested is a live viral biological, SCDM is used at
both incubation temperatures. Recently, it has been suggested that sulfite-polymyxin-sulfadiazine agar be
used to enhance the detection of Clostridium spp. when the membrane filtration technique is used (5).

If direct inoculation of culture media is chosen, a sterile pipette or syringe and needle are used to
aseptically transfer the biological material directly into liquid media. If the biological being tested has
antimicrobial properties, the ratio of the inoculum to the volume of culture medium must be determined
before the test is started. To determine the correct medium volume to negate antimicrobial activity,
100 colony-forming units (CFU) of the control microorganisms listed in Table 1 are used. If the test sample
contains merthiolate as a preservative, FTM is used in test vessels incubated at both 30–35°C and 20–
25°C. Growth should be clearly visible after an appropriate incubation time (see Section J.2.2). If the test
sample is a killed biological without merthiolate, or a live bacterial biological, FTM is used at 30–35°C and
SCDM at 20–25°C. If the test sample is a live viral biological, SCDM is used at both incubation
temperatures. If the inactivated bacterial vaccine is a clostridial biological, or contains a clostridial
component, the use of FTM with 0.5% added beef extract (FTMB) in place of FTM is preferred. It may also
be desirable to use both FTM and SCDM for all tests.
Table 1. Some American type culture collection 1 strains with their respective
medium and incubation conditions
Incubation
Medium Test microorganism Temperature (°C) Conditions
FTM Bacillus subtilis ATCC # 6633 30–35 Aerobic

FTM Candida krusei ATCC # 6258 20–25 Aerobic
SCDM Bacillus subtilis ATCC # 6633 30–35 Aerobic
SCDM Candida kursei ATCC # 6258 20–25 Aerobic
FTMB Clostridium sporogenes ATCC # 11437 30–35 Anaerobic
FTMB Staphylococcus aureus ATCC #6538 30-35 Aerobic
For both membrane filtration and direct inoculation sterility tests, all media are incubated for no fewer than
14 days. At intervals during incubation, and after 14 days’ incubation, the test vessels are examined for
evidence of microbial growth. Microbial growth should be confirmed by subculture and Gram stain.
2.2. Growth promotion and test interference

The sterility of the media should be confirmed by incubating representative containers at the appropriate
temperature for the length of time specified for each test.
The ability of the culture media to support growth in the presence and absence of product, product
components, cells, seeds, or other test material should be validated for each product to be tested, and for
each new batch or lot of culture media.
To test for ability to support growth in the absence of the test material, media should be inoculated with 10–
100 viable control organisms of the suggested American Type Culture Collection (ATCC) strains listed in
Table 1 and incubated according to the conditions specified.
To test for ability of the culture media to support growth in the presence of the test material, containers
should be inoculated simultaneously with both the test material (see Section J.2.3) and 10–100 viable
control organisms. The number of containers used should be at least one-half the number used to test the
product or product component. The test media are satisfactory if clear evidence of growth of the control
organisms appears in all inoculated media containers within 7 days. In the event that growth is evident, the
organism should be identified to confirm that it is the organism originally added to the medium. The sterility
test is considered invalid if any of the media show inadequate growth response, or if the organism
recovered is not the organism used to inoculate the material.
2.3. Number of items to be tested

The number of items in a batch determines the number of containers that should be tested for sterility. If the
batch size is not more than 100, then 10% or four containers, whichever is the greater, should be tested. If
the batch contains between 100 and 500 containers, then ten containers should be tested. If the batch has
more than 500 containers, then 2% or 20 containers, whichever is the lesser, should be tested. An
alternative is to test a maximum of 10 containers for all serials other than autogenous products.
1 American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA.

The amount of sterility test inoculum is dependent on the quantity of biological in each container. If the
quantity is less than 1 ml, then the entire contents are used for each medium. If the quantity is from 1 to
4 ml, then half the contents are used in each medium. If the quantity is from 4 to 20 ml, then 2 ml inoculum
per medium is used. If the quantity in each container is from 20 to 100 ml, then 10% of the contents are
used per medium. If the quantity per container is greater than 100 ml, then 10% or 50 ml, whichever is the
greater, is used to inoculate each medium.
2.4. Interpretation of sterility test results

If growth is found in any medium but it can be demonstrated by controls that the media or technique were
faulty, then the first test is declared invalid and may be repeated. If microbial growth is found in any of the
test vessels of the first test but there is no evidence invalidating it, then a retest may be conducted. The
minimum number of biological containers, test vessels, and membrane filters in a retest is double the
number used in the first test. If no growth is found in the first test or retest, the biological meets the
requirements of the test and is considered satisfactory for sterility. If microbial growth is found in any of the
retest vessels, the biological is considered unsatisfactory for sterility. If, however, it can be demonstrated by
controls that the media or technique of the retest were faulty, then the retest may be repeated.
2.5. General procedure for testing live viral vaccines produced in eggs and administered through
drinking water, spray, or skin scarification for the presence of bacteria and fungi
Each batch of final container biological should have an average contamination of not more than one
bacterial or fungal colony per dose for vaccines recommended for poultry, or ten colonies per dose for other
animals (see Section J.2.3 above to determine the number of samples to test). From each container
sample, each of two Petri dishes are inoculated with vaccine equal to ten doses if the vaccine is
recommended for poultry, or one dose if recommended for other animals. To each plate add 20 ml of brain–
heart infusion agar containing 0.007 IU (International Units) of penicillinase per ml. One plate should be
incubated at 30–35°C for 7 days and the other at 20–25°C for 14 days. Colony counts are made at the end
of each incubation period. An average colony count of all the plates representing a batch should be made
for each incubation condition. If the average count at either incubation condition exceeds one colony per
dose for vaccines recommended for poultry, or ten colonies per dose for vaccines recommended for other
animals in the initial test, one retest to rule out faulty technique may be conducted using double the number
of unopened final containers. If the average count at either incubation condition of the final test for a batch
exceeds one colony per dose for vaccines recommended for poultry, or ten colonies per dose for vaccines
recommended for other animals, the batch of vaccine should be considered unsatisfactory.
2.6. General procedure for testing seed lots of bacteria and live bacterial biologicals for purity
Each seed lot of bacteria or batch of live bacterial biological should be tested for purity by inoculation of
SCDM, which is incubated at 20–25°C for 14 days, and FTM, which is incubated at 30–35°C for 14 days
(see Section J.2.3 above to determine the number of samples to be tested and the amount of test inoculum
to be used). A sterile pipette or syringe and needle is used to aseptically transfer the quantity of biological
directly into the two types of culture medium. The minimum ratio of inoculum to culture medium is 1/15.
If the inoculum or growth of the bacterial vaccine renders the medium turbid so that the absence of atypical
microbial growth cannot be determined by visual examination, subcultures should be made from all turbid
tubes on day 3 through to day 11. Subculturing is done by transferring 0.1–1.0 ml to differential broths and
agar and incubating for the balance of the 14-day period. Microscopic examination by Gram stain should
also be done.
If no atypical growth is found in any of the test vessels when compared with a positive control included in
the test, the lot of biological may be considered satisfactory for purity. If atypical growth is found but it can
be demonstrated by control that the media or technique were faulty, then the first test may be repeated. If
atypical growth is found but there is no evidence invalidating the test, then a retest may be conducted.
Twice the number of biological containers and test vessels of the first test are used in the retest. If no
atypical growth is found in the retest, the biological is considered to be satisfactory for purity. If atypical
growth is found in any of the retest vessels, the biological is considered to be unsatisfactory for purity. If,
however, it can be demonstrated by controls that the media or technique of the retest were faulty, then the
retest may be repeated.
3. Detection of Mycoplasma contamination
3.1. General procedure for detecting Mycoplasma contamination

Each batch of live viral vaccine, each lot of master seed virus (MSV), each lot of primary and master cell
stock (MCS), and all ingredients of animal origin not steam sterilised should be tested for the absence of
mycoplasmas. Solid and liquid media that will support the growth of small numbers of test organisms, such
as typical contaminating organisms Acholeplasma laidlawii, Mycoplasma arginini, M. fermentans,

M. hyorhinis, M. orale, and M. synoviae should be used. The nutritive properties of the solid medium should
be such that no fewer than 100 CFU should occur with each test organism when approximately 100–
200 CFUs are inoculated per plate. An appropriate colour change should occur in the liquid media when
approximately 20–40 CFUs of each test organism are inoculated. The ability of the culture media to support
growth in the presence of product should be validated for each product to be tested, and for each new batch
or lot of culture media.
One sample of each lot of vaccine, MSV, etc., should be tested. Inoculate each of four plates of solid
medium with 0.25 ml of the sample being tested, and inoculate 100 ml of the liquid medium with 10 ml of
the sample. An alternative is to inoculate each of the plates with 0.1 ml and to inoculate 100 ml of liquid
medium with 1 ml of the sample being tested. Incubate two plates at 35–37°C aerobically (an atmosphere of
air containing 5–10% CO2 and adequate humidity) and two plates anaerobically (an atmosphere of nitrogen
containing 5–10% CO2 and adequate humidity) for 28 days. On day 3 or day 4 after inoculation, subculture
0.25 ml from the liquid media on to two plates of solid media. Incubate one plate aerobically and the second
anaerobically at 35–37°C until day 28 of the test. Repeat the subculture procedure on day 6, 7, or 8 and
again on day 13 or 14. An alternative method is to subculture on days 3, 5, 10, and 14 on to a plate of solid
medium. All the subculture plates are incubated for 10 days except for the 14-day subculture, which is
incubated for 14 days. Observe the liquid media every 2–3 days and, if any colour change occurs,
subculture immediately.
3.2. Interpretation of Mycoplasma test results

At the end of the incubation period (day 28), examine all the inoculated solid media microscopically for the
presence of mycoplasma colonies. The test sample passes the test if the growth of mycoplasma colonies
has occurred on the positive controls, and if growth has not occurred on any of the solid media inoculated
with the test material. If at any stage of the test, more than one plate is accidentally contaminated with
bacteria or fungi, or is broken, the test is invalid and should be repeated. If mycoplasma colonies are found
on any agar plate, the test should be repeated once to confirm the mycoplasma contamination. Twice the
volume (0.5 ml) of biological material being tested may be used in the retest. If mycoplasma colonies are
found on any of the agar plates of the retest, the test sample should be considered unsatisfactory because
of mycoplasma contamination. Some mycoplasmas cannot be cultivated, in which case the MSV and MCS
have to be tested using an indicator cell line (Vero cells), DNA staining, or polymerase chain reaction (PCR)
methods.
4. Detection of Salmonella contamination
Each batch of live virus biological made in eggs should be free from contamination with Salmonella. This testing
must be done before bacteriostatatic or bactericidal agents are added. Five samples of each batch should be
tested; 5 ml or one-half of the container contents, whichever is the lesser, of the sample should be used to
inoculate 100 ml of tryptose broth and tetrathionate broth. The inoculated broths should be incubated for 18–
24 hours at 35–37°C. Transfers from these broths should be made on to MacConkey and Salmonella–Shigella
agar, incubated for 18–24 hours, and examined. If no growth typical of Salmonella is noted, the agar plates
should be incubated an additional 18–24 hours and again examined. If colonies typical of Salmonella are
observed, further subculture on to suitable differential media should be made for positive identification. If
Salmonella is found, the batch of biological is unsatisfactory.
5. Detection of viruses in biological materials
Biological materials subject to viral contamination that cannot be sterilised before use, such as ingredients of
animal origin (for example, serum), primary cells, line cells or viral seed stocks, should be tested before they are
used. Assays are described to detect viral contaminants by cytopathic effects (CPE), haemadsorption,
haemagglutination, fluorescent antibody techniques and other suitable methods, e.g. PCR and enzyme-linked
immunosorbent assay. All biological materials should be specifically tested for pestiviruses. Avian materials and
vaccines should be inoculated on to primary avian cell cultures, eggs and/or chicks for the detection of avian
viruses. In addition to examining for CPE and cellular abnormalities in these inoculated cells/eggs/chicks, tests
for haemadsorbing and haemagglutinating viruses should also be included.
Cells shall be tested in the following manner. On day 0, primary or frozen cells to be tested are seeded on
75 cm2 (or similar) flasks; 7 days later, at least two 75 cm2 flasks are prepared. On day 14, one flask is used to
test the cells for cytopathology, haemadsorption, and fluorescent antibody staining (procedures follow). The other
flask is passaged a second time, and on day 21 is subjected to three freeze–thaw cycles. An alternative method
is to freeze–thaw the cells at 26 days instead of 21 days. After the third freeze–thaw cycle, the cells are
centrifuged at 2000 g for 10 minutes, and the supernatant is used to inoculate appropriate virus-sensitive cells,
i.e. cells susceptible to viruses that may be present in the species of animal from which the cells were obtained,
cells susceptible to viruses that may occur in the animals in which the material is going to be used and cells
susceptible to pestiviruses. These cells are then passed twice at 7-day intervals, and tested for cytopathology,
haemadsorption and by fluorescent antibody staining.

Ingredients of animal origin are tested on both African green monkey kidney (Vero) cells and on a cell line or
primary cells derived from the same species as the ingredient under test. Cells are inoculated using 75 cm2
flasks with 3.75 ml of test material in 25 ml of media or 15% of the test material, whichever is the lesser. The
cells are passaged two or three times at 7-day intervals, and tested for cytopathology, haemadsorption and
fluorescent antibody staining. The cells should be observed for cytopathology every 2 to 3 days, and prior to
each subculture, throughout the incubation period.
MSV are tested on Vero cells, cell lines or primary cells of the species for which the product is intended, and cell
lines or primary cells of the species in which the product is prepared (if different from the intended species).
For each cell type required for testing, 1 ml of the test MSV is thawed or reconstituted and neutralised with the
addition of 1 ml monospecific antiserum. The serum must be shown to be free from antibodies against any of the
contaminants for which the test is intended. Antisera should also be tested for nonspecific inhibiting affects At
least two cell types are always required, so a minimum of 2 ml of MSV and 2 ml of antiserum are required. The
antiserum is allowed to neutralise the MSV at room temperature for 1 hour. Of the MSV/antiserum mixture, 2 ml
is then inoculated on to a 75 cm2 flask of the appropriate cells. If the MSV is known to be high-titred or is a
difficult agent to neutralise, or if the blocking serum is known to be low-titred, the blocking antiserum can be
added to the growth medium at a final concentration of 1–5%. The cells should be passaged at least twice over a
14-day period, and the final culture is examined for cytopathology, haemadsorption and by fluorescent antibody
staining.
The May–Grünwald–Giemsa staining procedure is usually used to detect cytopathology caused by extraneous
viruses. Monolayers are usually prepared on two-chambered tissue culture slides and incubated for 7 days. The
plastic wells of the slides are removed leaving the rubber gasket attached to the slide. The slides are rinsed in
warm Dulbecco’s phosphate buffered saline (PBS), fixed in alcohol and placed on a staining rack. The slides are
stained for 15 minutes at room temperature with May–Grünwald stain diluted 1/5 with absolute methanol. The
May–Grünwald stain is removed by inverting the slides. The slides are then stained for 20 minutes with Giemsa
stain diluted 1/15 in deionised water. The Giemsa stain is removed by inverting the slides and rinsing them in
deionised water for 10–20 seconds. The slides are air-dried, and paraffin oil and a cover-slip are applied. The
May–Grünwald–Giemsa stain will differentially stain DNA and RNA nucleoproteins. DNA nucleoproteins stain
red-purple, while RNA nucleoproteins stain blue. The monolayers are examined with a conventional microscope
for the presence of inclusion bodies, an abnormal number of giant cells, or other cytopathology attributable to a
viral contaminant. The inoculated monolayers are compared with the noninoculated monolayers. If specific
cytopathology attributable to an extraneous virus is found, the test material should be considered unsatisfactory.
Testing to detect extraneous viruses that produce haemadsorption in infected cells is usually carried out on
monolayers of the second passage of test-material-inoculated cell cultures and noninoculated cell cultures. The
monolayers are usually on 75 cm2 plastic flasks. Guinea-pig, chicken, and any other blood for use in this assay is
collected in an equal volume of Alsever’s solution. The blood may be stored at 4°C for up to 7 days if it is washed
several times in Alsever’s solution before storage in an equal volume of Alsever’s. Just prior to use, the stored
erythrocytes are again washed by adding 5 ml of blood in Alsever’s solution to 45 ml of calcium- and magnesium-
free PBS and centrifuging in a 50 ml centrifuge tube at 500 g for 10 minutes. The supernatant is removed by
suction and the erythrocytes are suspended in PBS and recentrifuged. This washing procedure is repeated at
least twice until the supernatant is clear. Erythrocytes from each species are combined by adding 0.1 ml of each
type of packed blood cells to 100 ml of PBS. The erythrocytes from different species may be kept separate or
combined, as desired. To each flask, add 5 ml of the erythrocyte suspension, and incubate the flasks at 4°C for
30 minutes. The monolayers are washed twice with PBS and examined for haemadsorption. If no
haemadsorption is apparent, 5 ml of the erythrocyte suspension is added to each flask, the flasks are incubated
at 20–25°C for 30 minutes, rinsed as before, and examined for haemadsorption. Separate flasks may be used for
each incubation temperature if desired. Monolayers are examined for the presence of haemadsorbtion both
grossly (using an illuminated glovebox) and microscopically. It is important to compare the noninoculated
monolayers with the test monolayers to detect nonspecific haemadsorption that may occur with some cell types.
The use of calcium- and magnesium-free PBS and fresh erythrocytes should prevent most nonspecific
haemadsorption from occurring. If specific haemadsorption attributable to an extraneous agent is found, the test
material should be considered to be unsatisfactory.
Tests to detect extraneous viruses by fluorescent antibody usually use monolayers of the second passage of
test-material-inoculated cell cultures and noninoculated cell cultures. The monolayers are usually on eight-
chamber tissue culture slides. One positive control slide (consisting of eight monolayers) is made for each
antiviral conjugate by inoculating each monolayer with approximately 100 TCID50 (50% tissue culture infective
dose) of the appropriate virus. Three groups of monolayers are stained with each antiviral conjugate. They are
Group 1 – the second passage of test-material-inoculated cell cultures; Group 2 – the second passage of the
noninoculated cell cultures; and Group 3 – the second passage of noninoculated cell cultures (for the production
of positive control cell cultures). At the time of staining, the plastic walls of the slides are removed, leaving the
rubber gasket attached to the slide. The slides are rinsed in Dulbecco’s PBS, fixed for at least 10 minutes in
acetone at 4°C, and dried. Approximately 0.1 ml of each conjugate is placed on each well of one slide from
Groups 1, 2, and the corresponding positive control slide from Group 3. The slides are incubated in a humidified

chamber at 37°C for 30 minutes, rinsed once in Dulbecco’s PBS, and placed in a container of Dulbecco’s PBS
for 10 minutes. The slides are rinsed thoroughly in deionised water and dried. All slides are examined for
fluorescence attributable to each specific virus. The three slides from each group with the same conjugate are
compared. If the slide prepared from cells inoculated with test material shows any evidence of specific viral
fluorescence, the MSV should be considered unsatisfactory.
REFERENCES
1. CODE OF FEDERAL REGULATIONS (2007). Animals and Animal Products, Title 9, Parts 1–199. The Office of the
Federal Register, National Archives and Records Administration. US Government Printing Office,
Washington DC, USA.
2. COUNCIL OF EUROPE (2006). European Pharmacopoeia, Fifth Edition. Editions of the Council of Europe,
Strasbourg, France. ISBN 92-871-4587-3.
3. EUROPEAN UNION (1999). The Rules Governing Medicinal Products in the European Union. Eudralex, Vols
4–9. Office Publications of the European Communities, Luxembourg.
4. HARE W.C.D. (1985). Diseases transmissible by semen and embryo transfer techniques. OIE Technical
Series No. 4. OIE, Paris, France. ISBN 92-9044-164-1.
5. TELLEZ S., CASIMIRO R., VELA A.I., FERNANDEZ-GARAYZABAL J.F., EZQUERRA R., LATRE M.V., BRIONES V.,
GOYACHE J., BULLIDO R., ARBOIX M. & DOMINGUEZ L. (2005). Unexpected inefficiency of the European
pharmacopoeia sterility test for detecting contamination in clostridial vaccines. Vaccine, 24, 1710–1715.
6. W ORLD HEALTH ORGANIZATION (WHO) (1998). WHO Expert Committee on Biological Standardization. World
Health Organization Technical Report Series, Report No. 858. World Health Organization, Geneva,
Switzerland. ISBN 92-4-120878-3.
FURTHER READING
Details of methods and culture media will be found in the following books, and also in commercial catalogues.
A. BARROW G.I. & FELTHAM R.K.A., eds. (1993). Cowan and Steel’s Manual for the Identification of Medical
Bacteria, Third Edition. Cambridge University Press, Cambridge, UK.
B. CODE OF FEDERAL REGULATIONS (2003) Subchapter E. Viruses, serums, toxins, and analogous products;
organisms and vectors. In: Code of Federal Regulation, Animals and Animal Products, Title 9, Parts 101–
124. The Office of the Federal Register, National Archives and Records Administration. US Government
Printing Office, Washington, DC, USA, 557–737.
C. COLLINS C.H., LYNE P.M. & GRANGE J.M., eds. (1995). Collins and Lyne’s Microbiological Methods, Seventh
Edition. Butterworth Heinemann, Oxford, UK.
D. MURRAY P.R., ed. (2003). Manual of Clinical Microbiology, Eighth Edition. American Society for Microbiology
Press, Washington DC, USA.
*
* *

1 APPENDIX 1.1.9.1.
2 RISK ANALYSIS FOR BIOLOGICALS

3 FOR VETERINARY USE OTHER THAN VACCINES
4 INTRODUCTION
5 For the purpose of this chapter, the term ‘biologicals’ means ‘biologicals for veterinary use other than veterinary
6 vaccines’.
7 CATEGORISATION OF BIOLOGICALS
8 Categorisation provides a means of facilitating risk analysis for the international trade in biologicals.
9 The categorisation system should take into account the source, the nature and the stated purpose of the
10 biologicals. By conducting generic risk analyses, and by developing generic certification and quality assurance,
11 continued supply of products can be made available without the need for repeated risk assessments that are
12 expensive and consume significant resources. Once made, the risk assessment can be linked to appropriate
13 manufacturing and testing parameters. Categories of biologicals for veterinary use into which generic risk
14 assessments could apply may include (not in order of risk):
15 1. synthetic material;
16 2. amino acids, alcohols, esters, sugars and vitamins;
17 3. cosmetics;
18 4. plant extracts and processed biochemicals of plant origin;
19 5. products derived by microbial fermentation;
20 6. diagnostic, analytical and immunochemical kits for in-vitro use;
21 7. material of human origin;
22 8. therapeutics;
23 9. implantables of animal origin;
24 10. antibodies and immunoglobulins;
25 11. deoxyribonucleic acid (DNA), ribonucleic acid (RNA), restriction enzymes and other products of molecular
26 biology;
27 12. cell-lines and hybridomas;
28 13. animal proteins, hormones, enzymes, albumins, tissue extracts and culture media containing animal
29 material;
30 14. animal serum;
31 15. micro-organisms (conventional or genetically modified);
32 16. probiotics;
33 17. preserved specimens, microscope slides and smears.
34 All of these materials may contain pathogens depending on their source and processing procedures.

35 INFORMATION TO BE SUBMITTED WHEN APPLYING FOR AN IMPORT LICENCE

36 When undertaking risk analysis for biologicals, Veterinary Authorities should follow the Terrestrial Manual. The
37 manufacturer or the Veterinary Authority of the exporting country should make available detailed information, in
38 confidence if necessary, on the source of the materials used in the manufacture of the product (e.g. substrates).
39 They should make available details of the method of manufacture (and where appropriate inactivation) of the
40 substrates and component materials, the quality assurance procedures for each step in the process, final product
41 testing regimes, and the pharmacopoeia with which the product must conform in the country of origin. They
42 should also make available challenge organisms, their biotypes and reference sera, and other means of
43 appropriate product testing.
44 RISK ANALYSIS PROCESS
45 Risk analysis should be as objective and transparent as possible and should be performed in accordance with
46 Section 2 of the Terrestrial Code, and certification in line with Section 5 of the Terrestrial Code. Of necessity,
47 assessment of the country and commodity factors and risk reduction measures will be based largely on
48 manufacturers’ data. These data depend on quality assurance at all stages of manufacture, rather than on testing
49 of the final product alone.
50 Domestic exposure may be influenced by the approved usage of the product. Veterinary Authorities may place
51 limits on usage of some products (e.g. restricting usage to institutions of appropriate biosecurity).
52 BIOCONTAINMENT
53 Suitable biocontainment may be necessary for many forms of biologicals. In particular, the importation of exotic
54 micro-organisms should be carried out in accordance with Chapter 1.1.2 Biosafety and biosecurity in the
55 veterinary microbiology laboratory and animal facilities.
56 *
57 * *

CHAPTER 1.1.10.
GUIDELINES FOR INTERNATIONAL

STANDARDS FOR VACCINE BANKS
INTRODUCTION
Emergency vaccination is one of several measures that may be deployed to control outbreaks of
disease as it provides a valuable adjunct to the application of the essential zoosanitary measures.
These include rapid diagnosis, tracing, movement control and disinfection, and may also include
slaughter of infected and in-contact animals.
The terms ‘emergency vaccine’ and ‘emergency vaccination’ can have different connotations, but
are usually applied to differentiate between routine, prophylactic (preventive) vaccination and
emergency vaccination, the latter being applied as an immediate response to an outbreak of
disease. Emergency vaccination may be applied in a number of different circumstances and in a
number of different ways, including the following:
a) Against an outbreak of disease in a country that is normally free of this disease and that does
not normally vaccinate against this disease. It may be applied as Ring Vaccination or
Barrier Vaccination, outside of and around a focus of the disease to inhibit outward spread.
b) Against an outbreak of disease in a neighbouring country or region when emergency Barrier
Vaccination may be applied along the border in the country or region that is at risk.
c) As a complimentary measure in a stamping-out policy, when emergency vaccination is
applied to the animal population around an outbreak location, usually within the protection
zone in which outbreaks have occurred, by so-called Suppressive or Dampening Down
Vaccination. This is a form of ring vaccination that is followed by killing of the vaccinated
animals.
d) Against an outbreak of disease in a country that does normally vaccinate but where
emergency vaccine is applied to boost existing immunity.
e) Against an outbreak of disease in a country that does normally practice preventative
vaccination, but where the vaccine(s) employed do not provide protection against the strain
involved in the outbreak.
Criteria that determine the successful application of emergency vaccination include rapid access to
vaccine(s) that (i) contain virus strain(s) of sufficient antigenic relatedness to the outbreak strain(s)
(ii) are of the required type of vaccine formulation (iii) have acceptable safety and potency (iv) have
appropriate availability, including quantity and immediacy of supply and (v) meet considerations of
cost. The evident need to hold strategic reserves, or banks, of such valuable commodities is best
exemplified by foot and mouth disease (FMD) vaccines. They are specified in contingency plans
for use in an FMD outbreak and have led to an escalation in the establishment of national and
international FMD vaccine reserves for use all over the world (3), providing assurance that vaccine
would be readily available and at the disposal of the country requiring it.
Emergency FMD vaccines are normally formulated to a higher potency than its conventional
counterpart and there are banks who stipulate a requirement of at least 6 PD50 (50% protective
dose) per dose for cattle in contrast to the minimal statutory requirement of 3PD50. Higher potency
can be achieved by simply increasing the antigen payload per dose and its benefits can include
rapidity, magnitude and duration of the protective response (1, 4). However, conventional vaccines
may also be used in an emergency, particularly when vaccine of appropriate strain composition is
immediately available or where revaccination might be desired in an already pre-immune
population.

Chapter 1.1.10. — Guidelines for international standards for vaccine banks
The concept of vaccine banks, exemplified by FMD, and the increased reliance on such banks is
indicative of it being a very practical adjunct to other control measures that could usefully be
adopted for a number of other diseases such as bluetongue, classical swine fever and avian
influenza.
DEFINITION OF A VACCINE BANK

Strategic reserves, or banks as they are more commonly referred, are of two types. They may hold the final end
product, a ready-to-use formulated vaccine, and/or the antigen component, which can be stored for a very long
time for subsequent formulation into vaccine as and when required. The latter has been more commonly adopted
for FMD because of the economic benefits, and this avoids constantly replacing vaccines that exceed their shelf-
life. Stockpiles of antigens, or ready-to-use vaccine, will be referred to as vaccine banks in this chapter.
TYPES OF VACCINE BANKS

A country may hold its own national bank and/or it may be part of a larger group of countries that have drawing
rights and share a bank such as exemplified by the North American or European Union FMD vaccine banks.
Such consortiums may share a common geographical region, or have similar disease status and approach to
control. The bank may be held on the territory of one or several of its members or be retained by the
manufacturer, and, if held as antigen, would be formulated for use either by the manufacturer, or in a dedicated
facility maintained by the bank members. However, in the latter case, the recent increasing demands by licensing
authorities to require the same standards of independent manufacturing facilities as those of the commercial
sector with a marketable product, is making this option very difficult. In the case of an antigen bank, a contract
between the authorities and the vaccine manufacturer (formulation and filling) has to clearly define the details of
formulation of the vaccine, e.g. time between reception of order and delivery, availability of buffers and vials, etc.
The location of stored antigens is of vital importance since the need to formulate vaccine may require antigen to
be returned to the original manufacturer, incurring a delay in supply. Even if the antigens are held by the
commercial sector, delay following a request for the supply of emergency vaccine might still occur if the
manufacturer is currently in the middle of production of a product. The time to produce the vaccine should be
about 48–72 hours. Delays in the production and despatch of emergency vaccine to control an outbreak may
lead to wider spread of the disease and further difficulty in its control. Formulated vaccine would of course allow
for immediate access. However, beside the wasteful and uneconomic implications resulting from regular
replacement of the vaccine, it may not always contain the most suitable strain to deal with an outbreak.
The economic benefits of sharing a bank are obvious, but they also provide potential to stockpile greater doses
and a wider number of vaccine strains, and reduce the problem of deciding on the introduction of narrow
spectrum vaccine strains. Collaboration between vaccine banks would also be an economic way of increasing
the amount of emergency vaccine available. Care would be required to ensure that collaborating banks operate
to the same standards that drawing rights were clearly defined and that regular contact was maintained between
banks to confirm the safety, efficacy and availability of the vaccines. Issues related to regulatory compliance
would also need to be addressed at an early stage to ensure that vaccine produced from the bank would be
authorised for use in any of the participating countries.
SELECTION OF VACCINES FOR A BANK

Depending on the disease and the likely contingency requirements, a range of vaccine strains may be required.
Disease control authorities in consultation with the vaccine bank administrators must decide upon the vaccine
strains that should be held and on what basis they should be stored (i.e. as a separate antigen component for
subsequent formulation, or as a ready to use formulation). The value of any vaccine bank is very much
dependant upon the appropriateness of what it holds for field application, particularly in respect to diseases that
are made up of several serotypes and have wider strain variation. The potential of an outbreak not adequately
covered by a banked vaccine must be alleviated by continuous monitoring of the global disease situation and
recognition that additional vaccine strains may need to be included in the banks portfolio or, in the case where no
suitable vaccine strain is available, developed speedily for subsequent inclusion.
The world as an interdependent community that encompasses rapid and extensive movement of people, animals
and animal products and the increasing awareness of the potential to deliberately introduce disease through bio-
terrorism heightens the risk of an incursion and makes prediction of specific threat difficult. To improve the
process of vaccine selection, a continuous exchange of information and increased co-operation and collaboration
between different international, regional and national laboratories, vaccine manufacturers and the

vaccine/antigen banks authorities should be encouraged. Risk analysis studies should be done to classify the
virus strains to be stored with the priority level of high, medium and low. Close liaison with national and
international reference laboratories is therefore recommended as some laboratories already provide periodic
recommendations on strains that should be included in FMD vaccine banks. In the context of the risk of bio-
terrorism, disease control authorities may consider it pertinent to restrict the information released relating to the
storage of specific stockpiles of antigens and/or vaccines.
QUANTITIES OF VACCINE REQUIRED IN A BANK

The decision as to how many doses of vaccine are required is complex and problematic, embracing questions of
serotypes, strains, use of mono or polyvalent vaccines, and type of formulation. Factors bearing on the decision
include the type of disease, the different circumstances and ways of applying emergency vaccination (items a to
e) described in the introduction), number, species and location of livestock that are to be protected, geographical
considerations, knowledge of the current and predicted global epidemiological situation, and the analyses of risks
of introduction and spread of disease, together with cost–benefit studies. In determining the supply of emergency
vaccines, decisions on the quantity of the product inevitably involve a compromise between the cost of purchase
and the likely number of doses required. The minimum vaccine requirement might therefore be based on the
number of doses that could be distributed and applied in the first week of vaccination, the expectation would be
that additional supplies could by then have been procured, either from other banks or from commercial sources.
For example, 500,000 bovine doses of different FMD vaccine strains were routinely maintained by the
International FMD Vaccine Bank (IVB), and drawing rights by member countries varied from 100,000 to 500,000
bovine doses. Nevertheless, this would soon be exhausted if used in an area of high livestock density.
ACQUISITION OF VACCINES FOR A BANK

According to both the type of vaccine bank and the disease concerned, the acquisition of the appropriate
vaccine(s) or antigen(s) will depend on whether they are available from the commercial sector or government
institutions or produced in-house.
Regulatory concerns on existing, or potential, immunological veterinary medicinal products (IVMPs) and the
advisability to use approved, authorised medicines, will predispose a bank to acquire, or maintain, its vaccines
and antigens selectively. It is recommended that appropriately licensed manufacturers that have the necessary
Marketing Authorisation (MA) and internationally accepted standards of Good Manufacturing Practice (GMP),
modern quality assurance (QA) and Qualified Person (QP) product release should be used as authorised
sources.
This has certainly been exemplified in recent years by FMD vaccine banks in which there has been a strong
preference for purchasing and holding antigens/vaccines within the commercial manufacturing sector and thus
avoiding the expense and difficulties of maintaining a dedicated ‘licensed’ facility compliant with GMP for the
purpose of formulation in an emergency.
Disease control authorities should consider the option of requesting a tender for antigens/vaccine from more than
one supplier, particularly where regulatory considerations are of paramount importance, and they may wish to
seek advice from appropriate licensing authorities on the necessary standards required. Request for tenders can
then ensure not only a competitive price but a veterinary medicinal product manufactured to an acceptable level
of quality. It should also establish suppliers that can produce the desired vaccines/antigens and dose amounts
within a specified time period that meet necessary, or indeed mandatory, tests of compliance such as safety and
efficacy.
Where the requirement is to hold antigens/vaccines at a site other than at the principle site of manufacture,
disease control authorities may wish to consider only accepting them after they have been shown to have passed
the necessary acceptance testing procedures such as safety and/or efficacy. Alternatively, if the antigen/vaccine
has to be located in the bank prior to completion of any acceptance testing, then the antigen/vaccine should be
stored apart and labelled as quarantined material until the testing shows full compliance to the vaccine banks
requirements.
REGULATORY STANDARDS — SAFETY, EFFICACY AND QUALITY

Regulatory requirements for a veterinary medicinal product must be considered by any country wishing to have
the necessary authorisation to use emergency vaccine in an outbreak situation. For example, all veterinary
medicinal products that are placed on the market in the European Union (EU) must hold a marketing
authorisation and the EU lays down the requirements for such authorisations. The EU also has emergency

provisions under Articles 7 and 8 that permit release of a vaccine without an authorisation in the country requiring
it. However, a more recent EU Directive 2003/85/EC on current and future policy on control of FMD places more
emphasis on the use of vaccines as part of a vaccinate-to-live policy. This makes the issue of an authorised
product even more essential, particularly where vaccinated animals are intended for the food chain and require
the support of agencies responsible for human health. Therefore, it is important that licensed products be used;
unlicensed products are very much a last resort.
Quality, safety and efficacy are therefore all the more important and these will vary depending on the disease.
Where particular immunologicals are covered by individual monographs in official Pharmacopoeias (e.g. FMD
vaccine in the European Pharmacopoeia – Monograph 63) then the standards for Safety, Efficacy, Sterility and
Quality are laid down. For the other case where the immunological comes under the Pharmacopoeia general
section on Vaccines for Veterinary Use then those minimum standards should apply, and disease control
authorities may wish to add, to the minimum standards, other individual requirements. These standards might
include antigen strain identity, freedom from adventitious agents, innocuity, absence of toxicity, quantity of
antigen payload per dose, safety, potency and sterility, and manufacture in officially approved quality assured
(QA) good manufacturing practice (GMP) premises.
Any adjuvant or pharmacologically active ingredient used in a formulation must also conform to the necessary
guideline requirements including residues in food-producing species.
Differentiating between animals that have been vaccinated and animals that have either recovered from infection
or that have acquired sub-clinical infection post-vaccination may also be an important issue, as is the case for
FMD. The detection of antibodies to non-structural polyproteins (NSPs) such as 3ABC of FMDV has been shown
to be a sensitive and specific method to differentiate between infection and vaccination. This relies on
manufacturing methods whereby the NSP component can be reduced to a level that will not cause detectable
sero-conversion following vaccination making purity of vaccine an important consideration.
STORAGE OF VACCINES/ANTIGENS IN A BANK

It is important that the areas of storage chosen to hold emergency antigens/vaccines are suitable in the context
of the required national or internationally accepted standards of GMP. This is usually covered when a bank is
held in a ‘licensed’ and routinely inspected vaccine plant. However, if the bank is located outside a nominated
vaccine formulation facility, regulatory considerations again may be of paramount importance and Disease
Control Authorities may wish to seek advice from appropriate licensing authorities on the necessary standards
required.
If the vaccine bank is associated with a laboratory, or other facility, where pathogens are handled, this should be
completely independent of the bank storage facilities, and maintenance and monitoring personnel should obey a
quarantine period before entering the bank.
Appropriate storage of antigens/vaccines in an emergency reserve will be very much dependent on the disease
to which they are targeted to. The antigen may be a chemically inactivated or killed virus, for example such as
that used in FMD vaccines, or it may be an attenuated vaccine such as that exemplified by Bluetongue vaccines.
The antigens themselves may be concentrated and held at ultra-low temperature, over liquid nitrogen for
example, or may be a freeze dried commodity where low temperature is not necessarily important. Whatever the
method of storage, it is vitally important that they are optimally maintained and routinely monitored in order to
have some assurance that they will be efficacious when needed. Managers of vaccine banks should therefore
ensure that the necessary arrangements are in place to monitor their reserves on a routine basis and to include,
where necessary and at appropriate time intervals, a testing regime to ensure integrity of the antigen component
or acceptable potency of the final product. For example, 24-hour storage temperature may be recorded as well
as periodic inspection of the bottles containing the antigen for cracks or leakage. In this context, managers may
wish to also consider the possibility of independent testing, or of greater reliance on overseeing/auditing of the
manufacturer’s test procedures.
The need for routine testing of stocks for stability is evident, and therefore depositories of antigens/vaccines
should include a large number of small samples that are representative of the larger stock for such purposes
stored side by side with it.
Whilst not directly related to the establishment, storage and operation of vaccine or antigen banks, Countries
should nevertheless recognise the importance of contingency planning to ensure that the stored vaccine, if
required, is distributed and administered in an efficient and prompt manner. They should make certain that the
necessary cold-chain facilities are available, that vaccination protocols are defined in advance, that vaccination
teams are established and trained appropriately and that all the other necessary documentation, equipment,
reagents and clothing is stockpiled to sufficient levels to support any potential vaccination campaign. Therein the
benefits of also performing periodic exercises and simulations should not be overlooked.

It would be advisable for member countries to monitor the literature published on important advances that are
being made in subjects relating to vaccine bank technology. Ongoing research does lead to improvements of
product, equipment, manufacture and distribution and therein more efficient and practical use of Banks. In this
context there has been a recent study examining methods of prolonging the storage of fully formulated vaccine
by a novel formulation procedure (2).
REFERENCES
1. BARNETT P.V., KEEL P., REID S., ARMSTRONG R.M., STATHAM R.J., VOYCE C., AGGARWAL N. & COX S.J. (2004).
Evidence that high potency foot-and-mouth disease vaccine inhibits local virus replication and prevents the
‘carrier’ state in sheep. Vaccine, 22, 1221–1232.
2. BARNETT P.-V. & STATHAM R.-J. (2002). Stratified and cryogenically stored (SACS) vaccines, a new concept
in emergency foot-and-mouth disease vaccine formulation and storage. Vaccine, 20, 2060–2064.
3. FORMAN A.J. & GARLAND A.J.M. (2002). Foot and mouth disease: the future of vaccine banks. Rev. sci. tech.
Off. Int. Epiz., 21, 601–612.
4. RWEYEMAMU M.M., BLACK L., BOGE A., THORNE A.C. & TERRY G.M. (1984). The relationship between the 140S
antigen dose in aqueous foot-and-mouth disease vaccines and the serum antibody response of cattle. J.
Biol. Standard., 12, 111–120.
*
* *

CHAPTER 1.1.11.
THE ROLE OF OFFICIAL BODIES IN

THE INTERNATIONAL REGULATION OF
VETERINARY BIOLOGICALS
SUMMARY
The official control of veterinary biologicals is vested in various national and regional organisations
that differ in their approach to ensuring the quality, safety and efficacy of the products. International
harmonisation of regulations concerning biological products did not begin until well after those
concerning chemically defined products. The first biological products for veterinary use were not
manufactured and distributed until the end of the nineteenth century. They were often produced
under unsophisticated conditions, and distributed or sold without any control other than those of
their manufacturers. Later, each manufacturer developed its own standards. In Europe, these were
subject to State controls as early as 1895 for certain diagnostic products (e.g. mallein, tuberculin)
or vaccines. Gradually the conditions for international harmonisation of standards evolved,
beginning with the comparative testing of products being issued by different European laboratories.
It was only in the second half of the twentieth century that national laws covering veterinary
biologicals were imposed. These demanded that precisely defined techniques be followed before
biological products for veterinary use could be licensed. This was followed by considerable efforts
to harmonise these national regulations, first at the regional level (notably in Europe and the
Americas) then at the global level, notably by the Office International des Épizooties (OIE) with the
publication of the first edition of the OIE Manual of Standards for Diagnostic Tests and Vaccines in
1989.
World-wide harmonisation of standards for veterinary biologicals will be of help to Chief Veterinary
Officers who must follow the instructions given in the OIE International Animal Health Code, as
they apply to all biological products for use in international trade. It will also be of assistance to
vaccine producers, who have expressed their wish for world-wide harmonisation of registration
rules so as to simplify and facilitate marketing of their products. Evidently, it will also be of interest
to farmers and to consumers, who would benefit from the fact that the safety and efficacy of the
products that they use would have been assured to a uniformly high level.
The different sections of this chapter will review and compare regulations from the regions of the
world that have made most progress in this field and will describe current attempts at harmonising
these regulations on an international scale.
Note: In this chapter the term ‘veterinary biological’ will be taken to include vaccines for use in
animals, antisera for use in animals, and in-vivo diagnostic preparations.
A. REGULATION OF VETERINARY BIOLOGICALS: PRESENT SITUATION
1. In Japan
1.1. Introduction
Medicinal products that are exclusively used for animals, including veterinary biologicals, are under the
jurisdiction of the Ministry of Agriculture, Forestry and Fisheries, and securing their quality, efficacy and safety is
stipulated in the Pharmaceutical Affairs Law (1). Since 1972, registration procedures have been developed with
the aim of rationalising the examination procedure and facilitating the acquisition of approval. These procedures
are stipulated in the Pharmaceutical Affairs Law and other related regulations. Consequently, a speedy and

Chapter 1.1.11. —The role of official bodies in the international regulation of veterinary biologicals
simple examination procedure has been achieved with emphasis on the assurance of quality, safety and efficacy.
The Food Safety Commission was established in the Cabinet Office, Government of Japan, in July 2003. In the
case of approval examination, re-examination and re-evaluation, all veterinary vaccines, except products of dogs
and cats, must comply with the Food Safety Basic Law.
1.2. Regulations governing the approval and quality assurance of veterinary biologicals
a) Application for approval and licence
A person intending to release veterinary biologicals on the market shall obtain the license for marketing
approval holders and the marketing approval for each biological from the Minister of Agriculture, Forestry
and Fisheries. The application for the marketing approval should be submitted with designated appended
documents, such as those on clinical studies. Of the latter, the safety studies and clinical studies using the
target animal species should have been performed in compliance with GLP (Good Laboratory Practice) and
GCP (Good Clinical Practice). A marketing approval holder shall comply with the standard of GQP (Good
Quality Practice) and the GVP (Good Vigilance Practice).
A licence to manufacture veterinary biologicals is issued by the Minister of Agriculture, Forestry and
Fisheries and must be renewed every 5 years. Conformity to GMP (Good Manufacturing Practice) is
stipulated as one of the conditions for obtaining or renewing the licence to manufacture.
b) National assay
After receiving a licence, each batch of the veterinary biological must be examined by the National
Veterinary Assay Laboratory according to the procedures of the Assay Standard for Veterinary Biological
Products (3, 9). A marketing approval holder must apply it to the national assay. Each product for marketing
must include an official identification stamp on the container or the package as a seal.
c) Re-examination and re-evaluation

Re-examination is performed on newly approved veterinary biologicals. Usually a field assessment of the
veterinary vaccines is conducted over a period of 6 years following initial approval of the veterinary
vaccines. During this investigation, the efficacy and the safety are re-examined.
Re-evaluation is performed on availability of approved products after marketing by order of the Minister of
Agriculture, Forestry and Fisheries. This may happen when it is suspected that a veterinary biological does
not conform to the latest standards of veterinary biological products.
d) Minimum requirement of veterinary biological products

The examinations provide information about the consistency of the manufacturing process and the quality of
the product: manufacturing methods, properties of strains used for manufacturing, methods of quality
control, methods of storage and shelf life, according to the standards given in the ‘Minimum Requirement of
Veterinary Biological Products’ (2). Any product that does not conform to these product standards cannot be
manufactured, imported or marketed.
e) Cases of rejection of approval

When the quality of the veterinary biological that has been submitted for approval is found to be
unsatisfactory, or its adverse effects are marked as compared with its indications, the product is judged to
be of little value and approval is not given.
f) Cancellation of approvals
At the time of granting approval to market, the quality, safety and efficacy of the product are carefully
examined with reference to the latest available technology. However, if scientific knowledge acquired since
the granting of approval indicates that there could be a health hazard associated with the product, re-
examination and re-evaluation are performed and an order of ‘cancellation of approval’ may be made.
1.3. Procedure for marketing approval

When a person intends to market veterinary biologicals, an application for approval to market the veterinary drug
must be submitted on a designated form to an official in charge of veterinary biologicals at the Department of
Animal Hygiene of each Prefecture. If the documentation is satisfactory, the application for approval to market,
together with appended documents, are sent to and reviewed by the Secretariat of the Ministry of Agriculture,
Forestry and Fisheries. At that time, a hearing may be conducted if necessary. The application is then discussed
in the Pharmaceutical Affairs Sub-council, Pharmaceutical Affairs and Food Sanitation Council, and if any
problems are not found, notice of approval to market the veterinary product is sent to the applicant.

2. In the European Union
2.1. Introduction
The pharmaceutical legislation of the European Union (EU), which has evolved over a 30-year period, covers
both medicinal products for human and veterinary use. Harmonisation of requirements in the area of veterinary
medicines began in 1981 with the adoption of Directives 81/851/EEC and 81/852/EEC, laying down common
requirements for manufacturing and marketing authorisations, based on the evaluation of the quality, safety, and
efficacy of the product. These Directives, and subsequent veterinary and human pharmaceutical legislation, were
consolidated into Directive 2001/82/EC and 2001/83/EC for veterinary and human products, respectively. A
series of detailed guidelines were first published in 1994 entitled ‘Rules Governing Medicinal Products in the EU’
(7). These have since been updated and describe in detail the legal basis for obtaining marketing authorisations,
how dossiers should be compiled and how they should be assessed. These rules serve as extremely useful
reference publications for any authority that is setting up a system for authorisation of veterinary biologicals. The
rules were formally adopted and applied specifically to veterinary biologicals from 1993. Many additional
measures were taken to further harmonise the procedures and the criteria for the evaluation of veterinary
medicinal products, such as framework requirements and interpretive guidelines for their testing, principles and
guidelines of GMP, and a Community procedure for the evaluation of high-technology products. However,
granting of authorisations remained at the national level. As a consequence, although applications were
evaluated on the basis of these harmonised criteria and procedures, and in some cases simultaneously by the
authorities of the Member States, there were differences in the decisions reached by the Member States on
individual products. This was why in 1990 the Commission proposed a new system for marketing authorisation
for medicinal products, which was adopted by the Council of Ministers in 1993 and entered into force on
1 January 1995.
One of the first consequences was the creation of the European Medicines Evaluation Agency (EMEA) in
London, United Kingdom (UK).
New legislation for veterinary products (Regulation 726/2004 and Directive 2004/28/EC) was published in May
2004. This legislation, for the main part, came into force in 2005 and resulted in a number of changes aimed at
strengthening public and animal health, and environment protection by reinforcing requirements and controls.
Directive 2001/82/EC already stated that the competent authorities cannot grant a marketing authorisation (MA)
without having conducted a benefit–risk analysis. The document in the MA dossier must “demonstrate that the
benefit bound to efficacy outweighs potential risk”. But the relation between benefit and risk was not defined in
that Directive. The new Directive gives the definition of the “risk–benefit balance”: an evaluation of the positive
therapeutic effects of the veterinary medicinal product in relation to the risk”.
The risks concern:

• The animal;
• The user of medicinal product;
• The consumer liable to ingest animal food containing medicinal products residues;
• The environment.
With the revision of the Directive, if no medicinal product is available for 3 consecutive years, its MA is secluded.
Before the revision, the MA was renewed every 5 years. Now, a single renewal is required. The
pharmacovigilance is reinforced.
Title IV of regulation 726/2004 related to responsibilities and administrative structure of the European Agency
has come into force in April 2004 in order to face the consequences of the enlargement of the EU.
2.2. The role of the European Medicines Evaluation Agency

In 1995, a new European system for the authorisation of medicinal products came into force. After 10 years of
cooperation between national registration authorities at the EU level and 4 years of negotiations, the Council of
the EU adopted, in June 1993, three directives and one regulation, which together form the legal basis of the
system (5).
The EMEA was established by Council Regulation 2309/93/EEC of 22 July 1993. (OJ No. L 214, 24.8.1993), and
London, UK, was chosen as its location by decision made by the Heads of State and Government on 29 October
1993. This agency formulates opinions and, apart from the administrative staff and the management board, is
composed of two scientific committees, the CHMP (Committee for Human Medicinal Products) in charge of
medicinal products for humans and the CVMP (Committee for Veterinary Medicinal Products) in charge of
medicinal products for use in animals.
The CVMP is responsible for the evaluation of applications for marketing authorisation for products derived from
biotechnology, for productivity enhancers, new chemical entities and other innovative new products. In addition,

the CVMP makes recommendations regarding MRLs (maximum residue limits) for substances used in food-
producing animals. To support its activities, the CVMP relies on a pool of experts put at the disposal of the
agency by the EU Member States. These experts may participate in any of the CVMP working parties. Among the
working parties, the Immunologicals Working Party (CVMP/IWP) advises the CVMP on general policy issues
such as the elaboration and revision of guidelines on immunological products. A scientific advice working party
foreseen in regulation 726/2004 has been created. The aim of this working party is to advise applicants during
the development phase of a veterinary medicinal product. The guidelines for the testing of veterinary medicinal
products are contained within ‘The Rules Governing Medicinal Products in the EU’, last published by the
European Union in 1999 (7). New guidelines, and revisions of old guidelines, are no longer produced in hardcopy
form and may be accessed on the EMEA web site at www.emea.eu.int and/or the DG Enterprise web site at
pharmacos.eudra.org.
2.3. Present European procedures for marketing authorisation

Four registration procedures for human and veterinary medicinal products have become available through this
new legislation:
1. The centralised procedure allows a unique marketing authorisation (MA) to be obtained and made available
in all the Member States. This applies to high technology products defined in the annex to the Regulation. It
is optional for innovative medicinal products. This procedure was extended to veterinary vaccines covering
animal diseases that are subject to Community prophylactic measures.
2. The national procedure allows an MA to be obtained for a medicinal product in a single country or in a
country that will be the origin of a mutual recognition procedure.
3. The mutual recognition procedure: applications for authorisation of a product may still be obtained in a
single Member State (the ‘Reference Member State’) by means of a national procedure. The same ‘Rules
Governing Medicinal Products in the EU’ apply. Following approval in the Reference Member State,
applications may be made, if desired, to other ‘Concerned’ Member States for identical authorisations to be
granted on the basis of ‘mutual recognition’.
4. The new decentralised procedure is the addition of the national one and the mutual recognition procedure,
i.e. it is based upon the principle of mutual recognition of national authorisations. At the beginning of this
procedure, all Member States are associated; this is immediately followed by a mutual recognition
procedure.
The most important change is the compulsory aspect of arbitrage in the case of a disagreement between
Member States during the mutual recognition or the decentralised procedures. If a Member State considers that
there is a serious risk to public health, a pre-arbitrage procedure must be carried out. In such a situation, an MA
holder cannot remove his/her demand. The arbitrage allows a decision to be made on whether there is a “serious
risk” with the use of the medicinal product. Finally, the decision (to grant or refuse the MA) is harmonised
throughout the community.
2.4. Manufacturing authorisation and batch release control

In accordance with Directive 2001/82/EC, authorisation is also required for the manufacture of veterinary
medicinal products, including immunologicals. This directive provides for regular inspections and stipulates that
manufacture must be supervised by a ‘qualified person’, who certifies that each batch is in conformity with the
approved specifications for the product. For the implementation of these requirements, the Commission has
adopted Directive 91/412/EEC relating to the principle and guidelines of GMP, and published a detailed guide on
GMP developed by a group of pharmaceutical inspectors from the Member States.
The new directive also establishes GMP for active starting materials for medicinal products. This provision is
reinforced through the provision of the opportunity for Member States to carry out inspections of active materials
destined for the manufacturers of veterinary medicinal products.
Manufacturers are required to have the services of a qualified person at their disposal to certify that each batch
of product has been manufactured and checked in accordance with the conditions for marketing authorisation.
This is a basic requirement of the pharmaceutical legislation. In the case of batches imported from third
countries, each batch has to undergo a full qualitative and a quantitative analysis of at least the active
ingredients in the first Member State of import into the EU, under the supervision of a qualified person. Not until
this control by the qualified person has been carried out can a batch circulate within the EU without further
control. In the special case of immunological veterinary medicinal products, an additional step may be
introduced. Directive 90/677/EEC allows those Member States that consider it necessary to ask for the
submission of samples of each production batch of the bulk and/or finished product for examination by a control
laboratory before that batch is placed on the market. This official batch release does not relinquish the
requirement of batch control by the qualified person. Except in specially justified circumstances, batch release

carried out by one national control laboratory must be recognised without repetition by the other Member States.
To ensure the smooth operation of this provision, an administrative information exchange procedure has been
agreed between the Competent Authorities. Although all Member States do not require official batch release for
veterinary immunologicals, it was felt by all that they had to be involved in this information exchange scheme.
Discussions are in progress to establish a harmonised system of batch release in all Member States of the EU.
2.5. The role of the European Pharmacopoeia

The past 30 years have seen profound changes in the organisation and regulation of medicinal products in
European countries (4). Thirty years ago, each country had its own regulations, and among them the European
countries had two-thirds of the world’s pharmacopoeias. The European Pharmacopoeia Convention has now
been signed by 35 Member States 1; moreover 17 European and non-European countries 2, and the World Health
Organisation (WHO) have observer status. Close relations are maintained with the licensing authorities of the
European Economic Area, where integration is developing through contact with the EMEA and the
implementation of common directives and guidelines on medicines for human and veterinary use. In 1990, the
European Pharmacopoeia co-founded, with the Japanese Pharmacopoeia and the United States (US)
Pharmacopoeia, the Pharmacopoeial Discussion Group (PDG); this group is working assiduously for
harmonisation at the world level, and it participates in the International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) programme. A parallel
programme for veterinary medicinal products, the International Cooperation on Harmonisation of Technical
Requirements for Registration of Veterinary Medicinal Products (VICH), has recently turned its attention to
harmonising the requirements for certain types of tests routinely performed on extraneous agents (see Section
C.4 below). The European Pharmacopoeia defines the minimum acceptable standards for products to be
authorised within the European Union because compliance with monographs is a mandatory requirement within
Directive 2001/82/EC. This requires that products must comply with the relevant specific monograph where one
exists or with the general monographs where one does not.
The European Pharmacopoeia also contains a department called the ‘European Department for the Quality of
Medicines’ (EDQM). This creates, maintains and distributes the international standard reagents referred to in
monographs of the European Pharmacopoeia. To date there are few standards for veterinary biologicals, but the
EDQM is becoming increasingly active in this area and it is anticipated that several more will be available in the
near future.
3. In the United States of America
3.1. Introduction
In the United States of America (USA), veterinary biologics or veterinary biological products are defined as all
viruses, sera, toxins (excluding substances that are selectively toxic to microorganisms, e.g. antibiotics), or
analogous products at any stage of production, shipment, distribution, or sale, that are intended for use in the
treatment (prevention, diagnosis, management, or cure) of diseases of animals and that act primarily through the
direct stimulation, supplementation, enhancement, or modulation of the immune system or immune response.
The term biological products includes, but is not limited to, vaccines, bacterins, allergens, antibodies, antitoxins,
toxoids, immunostimulants, certain cytokines, antigenic or immunising components of live organisms, and
diagnostic components that are of natural or synthetic origin or that are derived from synthesising or altering
various substances or components of substances such as microorganisms, genes or genetic sequences,
carbohydrates, proteins, antigens, allergens, or antibodies.
3.2. Legal basis

The Virus/Serum/Toxin Act of 1913 (the ‘VST Act’), as amended, 21 U.S.C. Sections 151 to 159, provides the
legal authority for the regulation of immunologicals and biologicals for animal use in the USA. The regulatory
programme implementing the requirements of the VST Act is administered by the Center for Veterinary Biologics
(CVB), Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture
(USDA). Administrative regulations, duly promulgated and with effect of law, are published in Title 9, Code of
Federal Regulations, Parts 101 to 118 (6). In addition, APHIS has issued programme guidance in CVB Notices,
Veterinary Services Memoranda, Veterinary Biologics General Licensing Considerations, and the Veterinary
Biologics Program Manual. These may be accessed on the CVB web site at www.aphis.usda.gov/vs/cvb.
1 Austria, Belgium, Bosnia Herzegovina, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former
Yugolsav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Portugal, Romania, Serbia and Montenegro, Slovak Republic, Slovenia, Spain,
Sweden, Switzerland, Turkey, United Kingdom and the European Union; Member States must apply the standards of the
European Pharmacopoeia.
2 Albania, Algeria, Australia, Brazil, Canada, the People’s Republic of China, Georgia, Israel, Poland, Madagascar,
Malaysia, Morocco, Senegal, Syria, Tunisia, Ukraine, United States of America; Observer States do not have to apply the
European Pharmacopoeia standards. Some of them apply the standards on a voluntary basis.

The VST Act requires that products governed by the Act that enter channels of commerce be ‘not worthless,
contaminated, dangerous or harmful’. The regulatory scheme implementing these standards is structured to
require manufacturers of these products to apply for licences prior to marketing, and to place certain evidentiary
responsibilities on those applicants, i.e. manufacturers are required to demonstrate through the submission of
certain information, research data, and test results that their products are ‘pure, safe, potent and efficacious’. The
APHIS programme for immunologicals and biologicals for animal use regulates the manufacture and release of
products on to the market through a system of licensing, inspection, testing and post-marketing surveillance that
ensures that the statutory and regulatory standards are met.
3.3. Licensing and initial inspection

Any person or firm seeking to manufacture in the USA an immunological or biological for animal use must obtain
from APHIS both a licence to manufacture at a specified facility (Establishment Licence), and a licence for every
particular product to be manufactured (Product Licence). These licence requirements apply whether the product
is to be released on to the US market or is to be exported to markets abroad. Typically, an applicant will request
a facility licence at the same time as the licence for the first product. Once the facility licence and one product
licence have been obtained, a firm that seeks to manufacture and market new products needs only to apply for
additional product licences. A person or firm located overseas that seeks to market its product in the USA must
also apply for marketing authorisation. In the case of an imported product, however, the authorisation is termed a
‘permit’ rather than a ‘licence’.
To obtain a facility licence, the applicant must submit for approval the blueprint (that is, the architect’s plan of the
buildings) and blueprint legends for the facility. APHIS reviews these blueprints and legends to ensure that the
facility will operate in a manner consistent with GMP. If the applicant subsequently makes any physical or
operational changes to the facility, revised blueprints and legends must be submitted immediately.
To obtain a product licence, the applicant must establish the purity and identity of all master seeds and master
cell stocks that will be used in the manufacture of the product, and must submit for approval a detailed outline of
production. The outline of production includes not only the details of the method of product manufacture, but also
a description of the procedures for collecting and submitting samples and for releasing batches. The applicant
must also provide information regarding the professional and technical credentials of company personnel, and
must identify a qualified individual (termed under US regulations as the ‘government liaison’) who acts as the
official contact with CVB during the licensing process, and who is subsequently responsible for the submission of
the firm’s test reports in conjunction with the release of the product on to the market. The applicant is required to
submit test data that demonstrate that the product produced in accordance with the outline is pure, safe, potent
and efficacious. The applicant must submit to CVB laboratories samples of three consecutive batches of the
product so that the results of the applicant’s tests of the product can be confirmed.
Finally, before the facility or product licences are issued, the applicant’s premises are subject to a
comprehensive inspection by APHIS examiners. The inspection ensures that the facility is operating in a manner
consistent with GMP by confirming that the establishment is configured in the manner set out in the approved
blueprints and legends, that the production line is set up and operating in accordance with the approved outline
of production, and that records are adequately kept and maintained for each step in production. The inspection
also confirms that the applicant follows procedures consistent with GLPs, that the in-process and final product
testing programme is conducted properly and appropriately documented, that the sampling is conducted
properly, and that adequate procedures for determining and documenting the release of the product on to the
market are in place.
3.4. Post-licensing inspection

Once a firm has been issued facility and product licences, APHIS will routinely conduct thorough follow-up
inspections of the facility to ensure that the licensee continues to operate the establishment in accordance with
the programme regulations and in the manner represented at the time of licensing. Post-licensing inspections are
conducted unannounced periodically. If the licensee proposes any significant changes to the facility or to the
method of production of a licensed product, APHIS retains the right to conduct a special inspection prior to
approving the changes.
3.5. Testing
Each licensee is responsible for thoroughly testing all of its production processes and each serial (or lot) of every
product prior to release on to the market. The type and amount of testing required depends on the particular
product, but is determined and approved by the regulatory authority prior to the issuing of the product licence. A
qualified individual employed by the licensee (‘government liaison’) is responsible for selecting the samples to be
tested, for monitoring the licensee’s testing programme, and for certifying the test results to the regulatory
authority.
At the same time that the firm selects its samples for its own in-house testing, it also selects samples to be
submitted to the CVB laboratories. The CVB retains the right to conduct confirmatory testing. CVB then selects a

percentage of the samples submitted for confirmatory testing to verify the accuracy and proficiency of the
manufacturer’s tests. The testing is conducted prior to marketing authorisation for each serial. By regulation, CVB
policy stipulates that it is required to put its selected samples on test within 14 days of the date on which the
samples are received; ordinarily, samples are put on test sooner than the 14-day limit so that the testing of
production by the firm and the CVB proficiency testing programme are effectively being conducted at the same
time.
When the firm receives the results of its own tests, the government liaison submits those results to the regulatory
authority along with a batch release form, initiating the release procedure. If the batch has not been selected as
part of the proficiency testing programme, or if it has been selected but the CVB tests confirm the company’s test
results, the release form is counter-signed by the regulatory authority completing the release procedure. If either
the company tests or the proficiency tests indicate that the batch may be unsatisfactory, the batch is not eligible
for release.
If the licensee makes a proposal to modify its facility or its operation in any way that could affect the purity,
safety, potency or efficacy of the product, the regulatory authority may require the licensee to provide data
demonstrating the purity, safety, potency and efficacy of the product as well as to submit samples of the product
to CVB’s laboratories for confirmatory testing.
3.6. Post-marketing surveillance

CVB operates a post-marketing surveillance programme to monitor the performance of products on the market.
Under this programme, CVB typically learns of any problems relating to product quality through consumer reports
or complaints, although the programme regulations also place an obligation on the licensee to inform CVB of any
problem that comes to its attention regarding the purity, safety or potency of the product. CVB has the legal
authority to intervene in the marketplace where there are serious concerns with respect to the purity, safety,
potency or efficacy of the product.
B. COMPARISON OF EUROPEAN UNION AND UNITED STATES REGULATIONS

Veterinary biologicals must meet certain basic criteria, regardless of the Regulatory Agency overseeing their
production. These criteria include:
• Safety: the product must be safe in the target species and, if live, in species exposed to shed organisms;
• Efficacy: the product should be effective according to claims indicated on the label;
• Quality: includes purity, potency and consistency;
• Purity: the product must be free from contaminating agents;
• Potency: each batch of product should be formulated, and tested, to ensure effectiveness and
reproducibility of activity as demonstrated in the registration data.
Although, on a global basis, agencies and regulations differ, all strive to ensure that products offered to the end-
consumer conform to these basic standards.
The EU uses a complete system that is a combination of GMP, including validated processes and specifications
of materials, together with production methods that ensure the quality of the final product. In-process and batch
controls (tests) constitute additional guarantees of the quality of IVMPs. The safety tests are conducted under
GLP and the field efficacy tests under GCP. The USA defines acceptable manufacturing processes in the outline
of production and detailed facility description (blueprints and blueprint legends), and relies on inspection and
confirmatory testing to achieve the same goal. Although different, both systems are designed to allow only pure,
safe, potent, and effective biologicals to be released to the consumer.
In addition to the data provided by the applicant, expert reports have to be included in the EU marketing
authorisation application file (dossier). Each main section of the EU dossier, including analytical, safety and
efficacy, must be reviewed by an independent expert. The assessment of the expert is included in the marketing
authorisation file. No such system of third-party review exists under the USDA registration system with the
exception of certain biotechnologically derived products.
There are many procedural differences between the EU and the USA. Harmonisation between the two systems
should be established where possible, on the recognition of equivalence for tests and procedures that are
performed to assess a vaccine and that ensure quality, safety and efficacy of the product. Mutual recognition
agreements (MRAs) covering veterinary biologicals have been signed between the EU and Australia and
between the EU and New Zealand. These MRAs are now at an operational stage. Progress on MRAs between
the EU and the USA, regarding veterinary biologicals, is likely to take longer to achieve.

C. THE ROLE OF INTERNATIONAL ORGANISATIONS

Most nations have a range of official acts that regulate the sale and use of veterinary biologicals. Almost all of
these acts stipulate ‘minimum requirements’ for quality, safety and efficacy of veterinary biologicals (mostly
vaccines), to be tested at independent laboratories, usually under State supervision. These acts and tests may
differ from one country to another, and they involve costs and restrictions for producers, users and testers.
Many of the vaccines described in this Terrestrial Manual are produced and/or used in countries that do not
currently apply regimens of authorisation and testing as stringent as those described in this chapter.
Nevertheless, it is useful to be aware of the regulations operating in different regions and, therefore, the testing
and inspection that is likely to have been carried out there on a veterinary biological.
The idea of harmonising this testing to simplify and reduce costs on a regional, or even world scale, is not new,
and much has been accomplished during the past 20 years. The purpose of this section is to review the current
situation by describing the role of international organisations in the regulation of veterinary vaccines.
In this section the term ‘international organisation’ refers to those concerned with animal health on a world-wide
scale (OIE, the Food and Agriculture Organization of the United Nations [FAO] and the WHO).
1. The role of the OIE (World Organisation for Animal Health)
The OIE was founded in Paris in 1924 as the world organisation for animal health, and comprises 172 Member
Countries in the year 2008. The principal aims of the OIE are: to ensure transparency in the global animal
disease and zoonosis situation, to collect, analyse and disseminate scientific veterinary information, to provide
expertise and encourage international solidarity in the control of animal diseases, within its mandate under the
Agreement on Sanitary and Phytosanitary Measures (SPS Agreement) of the World Trade Organization (WTO),
to safeguard world trade by publishing health standards for international trade in animals and animal products, to
improve the legal framework and resources of national Veterinary Services and to provide a better guarantee of
the safety of food of animal origin and to promote animal welfare through a science-based approach (13).
Within the OIE there are four Specialist Commissions: the Terrestrial Animal Health Standards Commission,
which deals with the Terrestrial Animal Health Code, the Biological Standards Commission, the Scientific
Commission for Animal Diseases and the Aquatic Animal Health Standards Commission (including diseases of
molluscs and crustaceans). In addition, there are three Working Groups: the Working Group on Wildlife
Diseases, the Working Group on Animal Production Food Safety and the Working Group on Animal Welfare.
Among the Specialist Commissions, the one most closely connected with standardisation is the Biological
Standards Commission. This Commission establishes standards for diagnostic methods (including diagnostic
preparations) and for vaccines. Its terms of reference reflect the Commission’s obligation to participate in the
standardisation of biological products, including vaccines used for prophylactic purposes. The Biological
Standards Commission is responsible for the preparation of the OIE Manual of Diagnostic Tests and Vaccines for
Terrestrial Animals (11), and the organisation of Reference Laboratories for many of the diseases on the OIE
List.
However, full standardisation of vaccine testing can be achieved only when the necessary standards have been
devised. It is hoped to reach the goal of standardisation and wide availability of standards through the
participation of OIE Reference Laboratories. The functions and responsibilities of experts at the over 170 OIE
Reference Laboratories include the provision of a centre of excellence in a designated activity; standardisation of
methods; preparation, storage and distribution of standard antisera, antigens and other reagents.
Among the OIE Collaborating Centres, three may be involved at some stage in veterinary vaccine control and/or
harmonisation: the Collaborating Centre for Veterinary Medicinal Products in Fougères (France), the
Collaborating Centre for ELISA (enzyme-linked immunosorbent assay) and Molecular Techniques in Animal
Disease Diagnosis in Vienna (Austria), and the Collaborating Centre for the Diagnosis of Animal Diseases and
Vaccine Evaluation in the Americas in Ames (USA).
In 1994, following discussions with the International Technical Consultation on Veterinary Drug Registration
(ITCVDR), the OIE set up an Ad hoc Group on the Harmonisation of Veterinary Medicines, which was the first
step towards the creation of the VICH (International Cooperation on Harmonisation of Technical Requirements
for Registration of Veterinary Products) (see Section C.4 below).
In May 2003, the OIE International Committee adopted a resolution entitled OIE Procedure for Validation and
Certification of Diagnostic Assays (Test Methods) for Infectious Animal Diseases. This resolution requires the
OIE Director General to make provisions to establish an OIE registry for assays with levels of validation specified.
Fitness for purpose should be used as a criterion for validation.

2. The role of the Food and Agriculture Organization of the United Nations
FAO, established in 1945, is responsible for agricultural development and food production. The Animal
Production and Health Division (‘AGA’) within the Agriculture Department is concerned with livestock
development, and it includes the Animal Health Service (‘AGAH’), the main role of which is to assist Member
Countries in the control of animal diseases, with the aim of improving livestock production as an integral
component of general social, economic and agricultural development. FAO’s involvement in testing veterinary
biologicals is primarily through its technical assistance system to Member Countries to set up and even execute
independent quality control of vaccines and other biologicals. One example is FAO’s assistance to the AU
(African Union) in setting up a system for continental testing of veterinary vaccines, especially against rinderpest
and contagious bovine pleuropneumonia, by the Pan African Veterinary Vaccine Center (PANVAC). FAO also
commissions, at the request of Member Countries, initiatives for either quality assurance of vaccines and other
biologicals or expert consultations on this subject, or publication of manuals on the production and quality control
of vaccines. Furthermore, two auxiliary services can be asked to intervene on matters concerning these products,
namely Codex Alimentarius and the Division of Nuclear Techniques in Food and Agriculture. The latter is
operated jointly by FAO and the International Atomic Energy Agency (IAEA) based in Vienna (Austria). It has an
Animal Production and Health Section, which assists veterinary services and research institutes in developing
countries to establish radio-immunoassay (RIA) and ELISA techniques. Linked to this activity is a quality
assurance scheme under which laboratories in receipt of FAO/IAEA ELISA kits are required to routinely monitor
internal quality controls and to periodically (once or twice a year) test a batch of unknown samples, and to
forward the results to IAEA. The overall aim is to provide assurance to all concerned that the results being
generated through the use of such internationally standardised and validated kits can be relied upon as correct.
3. The role of the World Health Organization
Currently WHO is not directly involved in preparing international reference preparations (i.e. antigens or
antibodies) for purely veterinary use, but has developed and still retains in the National Institute for Biological
Standards and Control, Potter’s Bar (UK) some materials related to purely veterinary diseases (e.g. Newcastle
disease live vaccine, classical swine fever serum). WHO wishes to retain a role in this area in instances where
the veterinary reference preparations and guidance documents have a direct relevance to human health (8, 10,
15, 16). This involves zoonotic and potentially zoonotic agents and other infectious agents of animal origin that
are potential contaminants of biological products, whether these are vaccines produced in cell cultures or organs
for xenotransplantation. At the meeting of the Expert Committee on Biological Standardization in October 1998, a
review of currently retained international standards and reference preparations for veterinary medicine was
carried out and a list of candidates for discontinuation, replacement and revision was suggested (8). The Expert
Committee however decided to defer taking action on preparation of veterinary reference materials pending an
evaluation by WHO with its partners in the veterinary field of the need for these various biological products. In
addition, the present day topicality of certain preparations, especially veterinary vaccines against known
zoonoses (e.g. anthrax, brucellosis) adopted and/or revised in the 1960s and 1970s, also needs to be evaluated.
The format of the list of Requirements for Biological Substances published as an Appendix to each report of the
Expert Committee on Biological Standardization was revised in 1998 and should facilitate the retrieval of
information and achieve the aim of improved transparency.
4. The role of VICH
4.1. What is VICH?
a) Short description of VICH

VICH is a trilateral (EU-Japan-USA) programme aimed at harmonising technical requirements for veterinary
product registration. Its full title is the International Cooperation on Harmonisation of Technical
Requirements for Registration of Veterinary Medicinal Products. VICH was officially launched in April 1996.
b) Background and history

The initiative to begin the harmonisation process came in 1983 when the first International Technical
Consultation on Veterinary Drug Registration (ITCVDR) was held. Since then a series of government and
industry initiatives have been developed, culminating in the formation of the VICH.
The Codex Alimentarius formed a Committee on Residues of Veterinary Drugs in Foods in 1985. Standard
requirements for veterinary product registration were adopted in Europe in 1981.

The US Food and Drug Administration and the European Commission have held regular bilateral meetings
for the last decade to discuss common areas of interest. This has involved mutual exchange of guidelines
for consultation.
The first International Conference on Harmonisation of Technical Requirements for Registration of

Pharmaceuticals for Human Use (ICH) was held in Brussels in November 1991. The meeting brought
together regulators and industry representatives from the USA, the EU and Japan to address quality, safety
and efficacy requirements in the three regions.
Meetings on harmonisation of veterinary biologicals were held in Ploufragan, France, in January 1992, in
Arlington, USA, in 1994 and in Singapore in 1995.
In January 1993 the GHOST (Global harmonisation of standards) discussion document was published by
FEDESA 3. It set out a programme for the international harmonisation of registration requirements for
veterinary pharmaceuticals and biologicals.
Following discussions at ITCVDR and the OIE conferences, the OIE set up an ad hoc group on
harmonisation of veterinary medicinal products in 1994.
c) The creation and scope of VICH

Preparatory work for the establishment of VICH was carried out by this OIE ad hoc group. During 1994 and
1995, two meetings were held at which the scope of veterinary harmonisation was discussed and the
membership and objectives of the VICH proposed.
On the subject of food safety standards, it was decided that the VICH should complement the work of
Codex and JECFA 4. Issues related to GLP and GMP that are already the subject of mutual agreements will
not normally come within the remit of the VICH. Issues related to biologicals were considered appropriate to
fall within the scope of VICH.
Fundamental to the selection of priority topics for consideration by the VICH was the discussion document
prepared by COMISA for the Steering Committee. This report:
• assesses those ICH guidelines which could be adapted to the VICH programme;
• defines in detail areas of non-harmonisation between the EU, the US and Japan and provides a series
of ‘concept papers’ on key topics; and
• puts forward preliminary suggestions for priority topics.
With all the ground-breaking work completed, the Steering Committee of the VICH held its first meeting in
April 1996, at which the membership and the working procedures were agreed and a work programme
established.
d) The objectives of VICH

The objectives of the VICH are along the same lines as those of the ICH.
• Establish and implement harmonised regulatory requirements for veterinary medicinal products in the
VICH regions, which meet high quality, safety and efficacy standards and minimise the use of test
animals and costs of product development.
• Provide a basis for wider international harmonisation of registration requirements.
• Monitor and maintain existing VICH guidelines, taking particular note of the International Conference on
Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH)
work programme and, where necessary, update these VICH Guidelines.
• Ensure efficient processes for maintaining and monitoring consistent interpretation of data requirements
following the implementation of VICH guidelines.
3 FEDESA: Fédération Européenne de la Santé Animale. In 2002, FEDESA became the International Federation for
Animal Health (IFAH).
4 JECFA: Joint FAO/WHO Expert Committee on Food Additives.

• By means of a constructive dialogue between regulatory authorities and industry provide technical
guidance enabling response to significant emerging global issues and science that impact on regulatory
requirements within the VICH regions.
e) Progress toward achieving the VICH objectives

• For veterinary immunologicals there is an ongoing programme of harmonisation in a number of areas
including target animal safety studies, reversion to virulence and tests for the presence of Mycoplasma.
To date only a relatively small number of VICH guidelines have been developed for veterinary
biologicals and it is worth emphasising the difficulties in reaching agreement on veterinary biologicals
between the three regions.
• The only two adopted VICH Guidelines for veterinary biologicals are testing of residual formaldehyde
adopted May 2003 and testing of residual moisture also adopted in May 2003. Several other Guidelines
that apply to veterinary biologicals and all other veterinary medicinal products have also been adopted.
Following an in-depth reflection held by all parties concerned by VICH under the auspice of OIE, the second
phase of VICH for 2006–2010 was publicly launched during a public conference “VICH3” held in
Washington in May 2005.
More information is available at: http://vich.eudra.org.
CONCLUSION
At the moment, there is a clear intention to achieve greater international harmonisation of regulatory
requirements for veterinary biologicals (14). Progress has already been achieved through international
organisations to allow fair competition in the marketing of veterinary products. Although past efforts by
international organisations have not resulted in a level of harmonisation sufficient to facilitate international trade,
they have laid the groundwork for current efforts. National authorities recognise the advantages of harmonisation
and are now committed to working toward this goal.
The efforts of international organisations have made the goal of harmonisation possible and have resulted in an
organisation and process for proceeding toward this goal. Success in achieving this goal will depend on the
willingness of participating national authorities to work together and accept the compromises that will be
necessary to resolve the difficult scientific and policy issues.
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9. MAKIE H. (1998). The activities of veterinary vaccine control laboratories. Rev. sci. tech. Off. Int. Epiz., 17,
578–584.
10. MESLIN F.-X., KAPLAN M.M. & KOPROWSKI H. (1996). Laboratory Techniques in Rabies. World Health
Organization, Geneva, Switzerland, 476 pp.
11. OFFICE INTERNATIONAL DES EPIZOOTIES (OIE) (2004). Manual of Diagnostic Tests and Vaccines for Terrestrial
Animals (mammals, birds and bees), Fifth Edition. Office International des Epizooties, Paris, France.
12. PASTORET P.P. & FALIZE F. (1998). Licensing procedures for immunological veterinary medicinal products in
the European Union. Adv. Vet. Med., 41, 595–607.
13. TRUSZCZYNSKI M. & BLANCOU J (1992). The role of the Office International des Epizooties in the
standardisation of biologicals. In: Symposium on the First Steps Towards an International Harmonization of
Veterinary Biologicals and Free Circulation of Vaccines within the EEC, Ploufragan, 1992. Dev. Biol. Stand.,
79, 95–98.
14. VANNIER P., TRUSZCYNSKI M. & ESPESETH D. (1997). Implementing International harmonisation. In: Veterinary
Vaccinology, Pastoret P.P., Blancou J., Vannier P. & Verschueren C., eds. Elsevier Science, Amsterdam,
the Netherlands, 712–717.
15. W ORLD HEALTH ORGANIZATION (WHO) EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION (1992). Forty-
second Report. WHO Technical Report Series No. 822, World Health Organization, Geneva, Switzerland,
84 pp.
16. W ORLD HEALTH ORGANIZATION (WHO) EXPERT COMMITTEE ON RABIES (1992). Eighth Report. WHO Technical
Report Series No. 824, World Health Organization, Geneva, Switzerland, 75 pp.
*
* *

PART 2
OIE LISTED DISEASES AND OTHER DISEASES OF

IMPORTANCE TO INTERNATIONAL TRADE

SECTION 2.1.
MULTIPLE SPECIES
CHAPTER 2.1.1.
ANTHRAX
SUMMARY
Definition of the disease: Anthrax is primarily a disease of herbivorous animals, although all
mammals, including humans, and some avian species can contract it. Mortality can be very high,
especially in herbivores. The aetiological agent is the spore-forming, Gram-positive rod-shaped
Bacillus anthracis. The disease has world-wide distribution and is a zoonosis.
Description of the disease: The disease is mediated by exotoxins. Peracute, acute, subacute
and, rarely, chronic forms of the disease are reported. Ante-mortem clinical signs may be virtually
absent in peracute and acute forms of the disease. Subacute disease may be accompanied by
progressive fever, depression, inappetence, weakness, prostration and death. Acute, subacute,
and chronic disease may show localised swelling, fever; and in chronic disease the only sign may
be enlarged lymph glands.
Identification of the agent: Bacillus anthracis is readily isolated in relatively high numbers from
blood or tissues of a recently dead animal that died of anthrax, and colony morphology of
B. anthracis is quite characteristic after overnight incubation on blood agar. The colony is relatively
large, measuring approximately 0.3–0.5 cm in diameter. It is grey-white to grey, non-haemolytic
with a rough, ground-glass appearance and has a very tacky, butyrous consistency. The
vegetative cells of B. anthracis are large, measuring 3–5 µm in length and approximately 1 µm in
width. Ellipsoidal central spores, which do not swell the sporangium, are formed at the end of the
exponential cell growth phase. The cells stain strongly Gram positive, and long chains are often
seen in vitro, while paired or short chains are seen in vivo. Visualisation of the encapsulated bacilli,
usually in large numbers, in a blood smear stained with polychrome methylene blue (M’Fadyean
reaction) is fully diagnostic.
Serological tests: Antibody detection in serum from infected animals is rarely used for diagnostic
purposes and is essentially a research tool. The predominant procedure today is the enzyme-
linked immunosorbent assay (ELISA).
Requirements for vaccines and diagnostic biologicals: The most widely used livestock anthrax
vaccine developed by Max Sterne in 1937, is a live, non-encapsulated, spore former held in
suspension. In Russia and Eastern Bloc countries, an equivalent type of vaccine is used (strain
55). The Pasteur vaccine is no longer used in Italy. A new vaccine, Carbosap, has been developed
that retains both plasmids and exhibits very low virulence. A list of producers is given in the World
Health Organization anthrax guidelines.
A. INTRODUCTION
Anthrax, an acute bacterial disease of primarily herbivores, is transmissible to humans. The aetiological agent,
Bacillus anthracis, is a Gram-positive spore-forming rod-shaped bacterium. Anthrax is known by many names
around the world including charbon, woolsorters disease, ragpickers disease, malignant carbuncle, and
malignant pustule.

Chapter 2.1.1. — Anthrax
Animals become infected by ingesting spores or possibly by being bitten by flies that have fed on an infected
animal or carcass. Infected animals are usually found dead as death can occur within 24 hours. A careful post-
mortem examination of recently dead animals may show any number of lesions, none of which is pathognomonic
or entirely consistent. To avoid environmental contamination, post-mortem examinations of carcasses of animals
suspected to have died of anthrax is discouraged. Lesions most commonly seen are those of a generalised
septicaemia often accompanied by an enlarged spleen having a ‘blackberry jam’ consistency and poorly clotted
blood. Haemorrhage from the nose, mouth, vagina and/or anus at death is not a common sign.
Gram-positive rod-shaped Bacillus anthracis is the only obligate pathogen within the genus Bacillus. Most of the
other species of Bacillus are common ubiquitous environmental saprophytes, although a number, notably
B. cereus, B. licheniformis and B. subtilis, are occasionally associated with food poisoning in humans and with
other clinical manifestations in both humans and animals.
More than 95% of human anthrax cases take the cutaneous form and result from handling infected carcasses or
hides, hair, meat or bones from such carcasses. Bacillus anthracis is not invasive and requires a lesion to infect.
Protection for veterinarians and other animal handlers involves wearing gloves, and other protective clothing
when handling specimens from suspected anthrax carcasses and never rubbing the face or eyes. The risk of
gastrointestinal anthrax may arise if individuals eat meat from animals infected with anthrax.
The risk of inhaling infectious doses becomes significant in occupations involving the processing of animal by-
products for manufacturing goods (industrial anthrax). These include the tanning, woollen, carpet, bone
processing, and other such industries, where the potential for aerosolisation of substantial numbers of spores
increases the risk of exposure to infectious doses.
B. DIAGNOSTIC TECHNIQUES
1. Identification of the agent
Demonstration of encapsulated B. anthracis in smears of blood or tissues from fresh anthrax-infected carcasses
and growth of the organism on blood agar plates is relatively uncomplicated and within the capability of most
bacteriology laboratories. Difficulty may be encountered in the case of pigs and carnivores in which the terminal
bacteraemia is frequently not marked, or in animals that received antibiotics before death.
Recovery of B. anthracis from old decomposed carcasses, processed specimens (bone meal, hides), or
environmental samples (contaminated soil) is also often difficult, requiring demanding and labour-intensive
procedures.
a) Culture and identification of Bacillus anthracis

i) Fresh specimens
Bacillus anthracis grows readily on most types of nutrient agar, however, 5–7% horse or sheep blood agar
is the diagnostic medium of choice. Blood is the primary clinical material to examine. Swabs of blood, other
body fluids or swabs taken from incisions in tissues or organs can be spread over blood agar plates. After
overnight incubation at 37°C, B. anthracis colonies are grey-white to grey, 0.3–0.5 mm in diameter, non-
haemolytic, with a ground-glass moist surface, and very tacky when teased with an inoculating loop. Tailing
and prominent wisps of growth trailing back toward the parent colony, all in the same direction, are
sometimes seen. This characteristic has been described as a ‘medusa head’ appearance. Confirmation of
B. anthracis should be accomplished by the demonstration of a capsulated, spore-forming, Gram-positive
rod in blood culture. Absence of motility is an additional test that can be done.
The susceptibility of B. anthracis to the gamma bacteriophage was first described by Brown & Cherry in
1955 (3). The phage is available from various national central veterinary laboratories and anthrax reference
laboratories. The procedure for the test is simply to streak a lawn on a blood or nutrient agar plate, or
portion of a plate (several tests can be done on one plate) with the suspect organism and place a 10–15 µl
drop of the phage suspension on one side of the streaked area and place a 10-unit penicillin disk to the
other side. Allow the drop of phage suspension to soak in and incubate the plate at 37°C. A control culture
should be included; the Sterne vaccine or the NCTC strain 10340 can be used for this. If the culture is
B. anthracis, the area under the phage will be devoid of bacterial growth, due to lysis, and a clear zone will
be seen around the penicillin disk after overnight incubation. (Note: phage-resistant B. anthracis isolates
are encountered occasionally; similarly, there are a few reports in the literature of penicillin-resistance.)
ii) Capsule visualisation

Virulent encapsulated B. anthracis is present in tissues and blood and other body fluids from animals that
have died from anthrax. The bacteria should be looked for in smears of these specimens that have been
dried, fixed and stained with polychrome methylene blue (M’Fadyean’s reaction). The capsule stains pink,

whereas the bacillus cells stain dark blue. The cells are found in pairs or short chains and are often square-
ended (the chains are sometimes likened to a set of railway carriages – so-called ‘box-car’ appearance).
Gram and Giemsa stains do not reveal the capsule. The capsule is not present on B. anthracis grown
aerobically on nutrient agar or in nutrient broths, but can be seen when the virulent bacterium is cultured for
a few hours in a few millilitres of blood (defribrinated horse or sheep blood seems to work best).
Alternatively, the capsule is produced when the virulent B. anthracis is cultured on nutrient agar containing
0.7% sodium bicarbonate and incubated in the presence of CO2 (20% is optimal, but a candle jar works
well). The agar is prepared by reconstituting enough nutrient agar base powder for 100 ml of agar in 90 ml
of water. Autoclave and cool to 50°C in a water bath. Add 10 ml of a filter-sterilised (0.22–0.45 µm filter) 7%
solution of sodium bicarbonate. Mix and pour into Petri dishes. The encapsulated B. anthracis will form
mucoid colonies and the capsule can be visualised by making thin smears on microscope slides, fixing, and
staining with polychrome methylene blue.
iii) Other specimens

Identification of B. anthracis from old, decomposed specimens, processed materials, and environmental
samples, including soil, is possible but these samples often have saprophytic contaminants that outgrow
and obscure B. anthracis on nonselective agars. The following procedure is suggested:
a) The sample is blended in two volumes of sterile distilled or deionised water and placed in a water bath
at 62.5 ± 0.5°C for 30–60 minutes.
b) Tenfold dilutions to 10–2 or 10–3 are then prepared. From each dilution, 10–100 µl are plated on to
blood agar and optionally 250–300 µl on to PLET agar (polymyxin, lysozyme, EDTA [ethylene diamine
tetra-acetic acid], thallous acetate) (7, 11). All plates are incubated at 37°C.
c) Blood agar plates are examined for typical colonies as previously described after overnight incubation,
and the PLET plates are examined after 40–48 hours. Confirmation of the identity of suspect colonies
as B. anthracis is done as described above.
PLET medium (7, 11) is prepared by using heart-infusion agar base (DIFCO) made up to the manufacturer’s
instructions with the addition of 0.25–0.3 g/litre EDTA and 0.04 g/litre thallous acetate. (NOTE: thallous
acetate is poisonous and should be handled with care.) The mixture is autoclaved and uniformly cooled to
50°C before adding the polymyxin at 30,000 units/litre and lysozyme at 300,000 units/litre. After mixing
thoroughly, the agar is dispensed into Petri dishes.
Reports of procedures for direct detection of B. anthracis in soils and other environmental specimens using
the polymerase chain reaction (PCR) are emerging. None of these has become routinely applicable at the
present time.
Animal inoculation may be considered for recovery of B. anthracis if all other methods fail. Examples of
when this might occur are specimens from animals that received antibiotic therapy before death or
environmental samples containing sporostatic chemicals. Due to the increasing concern to eliminate the
use of animals for biological testing, this approach should be used as a last resort and only if justified. Adult
mice or guinea-pigs are the animals of choice. If the samples involved are soils, the animals should be
pretreated, the day before testing, with both tetanus and gas gangrene antiserum. The samples are
prepared as described for culturing, including heat-shocking at 62.5°C for 15 minutes. Mice are injected
subcutaneously with 0.05–0.1 ml; guinea pigs are inoculated intramuscularly with up to 0.4 ml (0.2 ml in
each thigh muscle). Any B. anthracis present will result in death in 48–72 hours and the organism can be
cultured from the blood as described above.
b) Immunological detection and diagnosis

It needs to be borne in mind that B. anthracis is antigenically very closely related to B. cereus, which is
considered a ubiquitous component of the environmental microflora. The only unshared antigens that lend
themselves to differentiating these two species by immunological approaches are the anthrax toxin
antigens, produced during the exponential phase of growth, and the capsule of B. anthracis. This places
considerable constraints on the extent to which immunological methods can be used in routine detection
methodology.
i) Ascoli test
In 1911, Ascoli (1) published a procedure for the detection of thermostable anthrax antigen in animal tissue
being used for by-products. This uses antiserum raised in rabbits to produce a precipitin reaction. The test
lacks high specificity, in that the thermostable antigens of B. anthracis are shared by other Bacillus spp.,
and is dependent on the probability that only B. anthracis would proliferate throughout the animal and
deposit sufficient antigen to give a positive reaction. Nowadays, it appears to be used in Eastern Europe
only.

To perform the Ascoli test, put approximately 2 g of sample in 5 ml of saline containing 1/100 final
concentration of acetic acid and boil for 5 minutes. The resultant solution is cooled and filtered through filter
paper. A few drops of rabbit antiserum (see preparation below) are placed in a small test tube. The filtrate
from the previous step is gently layered over the top of the antiserum. A positive test is the formation of a
visible precipitin band in under 15 minutes. Positive and negative control specimen suspensions should be
included.
Antiserum is prepared in rabbits by the subcutaneous inoculation of Sterne anthrax vaccine on days 1 and
14. On days 28 and 35, the rabbits receive 0.5 ml of a mixture of several strains of virulent B. anthracis not
exceeding 105 colony-forming units (CFU)/ml suspended in saline. Alternatively, the live virulent bacteria
can be inactivated by prolonged suspension in 0.2% formalised saline, but the antigen mass needs to be
increased to 108–109 CFU/ml. The suspension should be checked for inactivation of the B. anthracis before
animal inoculation by culture of 0.1 ml into 100 ml of nutrient broth containing 0.1% histidine and, after
incubation at 37°C for 7 days, subculture on to blood or nutrient agar. The dose regimen for the formalised
suspension after initial vaccination on days 1 and 14 is increasing doses of 0.1, 0.5, 1, and 2 ml given
intravenously at intervals of 4–5 days. Following either procedure, a test bleed at 10 days after the last
injection should determine whether additional 2 ml doses should be administered to boost the precipitin
titre.
ii) Immunofluorescence
While some success has been achieved with immunofluorescence for capsule observation in the research
situation (4), it does not lend itself to routine diagnosis.
c) Confirmation of virulence with the polymerase chain reaction

Full confirmation of virulence can be carried out using the PCR. The following instructions are taken from
ref. 11. Template DNA for PCR can be prepared from a fresh colony of B. anthracis on nutrient agar by
suspension of a loop of growth in 25 µl sterile deionised (or distilled) water and heating to 95°C for
20 minutes. Following cooling to approximately 4°C, and brief centrifugation, the supernatant can be used
for the PCR reaction.
Suitable primers (2, 5) for confirming the presence of the pX01 and pX02 plasmids are given in the table
below.
Target Primer ID Sequence 5’–3’ Product size Concentration
Protective PA 5 TCC-TAA-CAC-TAA-CGA-AGT-CG 596 bp 1 mM

antigen (PA) 3048–3029
PA 8 GAG-GTA-GAA-GGA-TAT-ACG-GT
2452–2471
Capsule 1234 CTG-AGC-CAT-TAA-TCG-ATA-TG 846 bp 0.2 mM
1411–1430
1301 TCC-CAC-TTA-CGT-AAT-CTG-AG
2257–2238
PCR can be carried out in 50 µl volumes using the above primers, 200 µM each of dATP, dCTP, dTTP and
dGTP, 1.5 mM MgCl2 and 2.5 units of ampli Taq DNA polymerase™ 1, all in NH4 buffer, followed by the
addition of 5 µl of template DNA. A 2% agarose gel has been found to work best with these small
fragments.
Alternatively, ‘Ready-To-Go™’ beads are available from Pharmacia Biotech 2. These are premixed,
predispensed, dried beads, stable at room temperature, containing all the necessary reagents, except
primer and template, for performing 25 µl PCR reactions. The template can be added in a 2.5 µl volume.
The following PCR cycle can be used: 1 × 95°C for 5 minutes; 30 × 95°C for 0.5 minute followed by 55°C for
0.5 minute followed by 72°C for 0.5 minute; 1 × 72°C for 5 minutes; cool to 4°C.
It should be noted that, in use for some years now in an anthrax reference facility, the primers given in the
table above have proved successful for confirming the presence or absence of pXO1 and/or pXO2 in pure
cultures of isolates from animal (including human) or environmental specimens or samples. They are
unsuitable, however, for direct detection of B. anthracis in such specimens or samples. A choice of
1 This product is available from Applied Biosystems;

(https://products.applied biosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catlD=601622
2 Uppsala, Sweden, product number 27-9555-01

alternatives can be found in reference 6 and 9. For the rare possibility that an isolate may lack both pXO1
and pXO2, a chromosomal marker should also be run; primers for these are also supplied in references 6
and 9.
C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS

The most widely used vaccine for prevention of anthrax in animals was developed by Sterne in 1937 (10). He
derived a rough variant of virulent B. anthracis from culture on serum agar in an elevated CO2 atmosphere. This
variant, named 34F2, was incapable of forming a capsule and was subsequently found to have lost the pX02
plasmid, which codes for capsule formation. It has become the most widely used strain world-wide for animal
anthrax vaccine production. In Central and Eastern Europe, an equivalent pX02– derivative, Strain 55, is the
active ingredient of the current livestock vaccine. A list of manufacturers of anthrax vaccine for use in animals is
given in Appendix V of reference 11.
The following information concerning preparation of the anthrax vaccine for use in animals is based on
references 8 and 12. Generalised procedures are given; national regulatory authorities should be consulted in
relation to Standard Operating Procedures that may pertain locally.
1. Seed management
a) Characteristics of the seed

Anthrax vaccine production is based on the seed-lot system. A seed lot is a quantity of spores of uniform
composition processed at one time and maintained for the purpose of vaccine preparation. Each seed lot is
no more than three passages from the parent culture and must produce a vaccine that is efficacious and
safe for use in animals. It is recommended that a large seed lot be prepared from the parent strain and
preserved by lyophilisation for future production lots. The parent culture can be purchased 3. The seed lot is
acceptable for anthrax vaccine if a vaccine prepared from the seed lot or a suspension harvested from a
culture derived from a seed lot meets the requirements for control of final bulk with respect to freedom from
bacterial contamination, safety and efficacy (immunogenicity).
b) Preparation of the master seed

Seed lots are cultured on solid media formulated to promote sporulation of the organism (see Section C.2
below). The solid medium formula given in reference 8 is: 50 g tryptic digest of casein; 10 g yeast extract;
0.1 g CaCl2.6H2O; 0.01 g FeSO4.7H2O; 0.05 g MgSO4.7H2O; 0.03 g MnSO4.4H2O; 5.0 g K2HPO4; 1.0 g
KH2PO4; 22 g agar; 1000 ml deionised or distilled water. The ingredients are dissolved in the water with the
appropriate amount of heating; the solution is adjusted to pH 7.4, distributed into Roux bottles (120 ml per
bottle) or other appropriate container, sterilised by autoclaving and cooled in the horizontal position. After
the agar has solidified, excess liquid should be removed aseptically and the bottles left in an incubator
(37°C) for at least 2 days to dry and to check the sterility.
Volumes of 2 ml of vaccine seed from a reference laboratory should be spread across the agar in Roux
bottles, which should be incubated at 37°C until at least 80% sporulation is apparent by microscopic
examination of aseptically extracted loopfuls (at least 72 hours). The growth is harvested with 10 ml per
bottle of sterile deionised or distilled water and checked for purity. After washing three times in sterile
deionised or distilled water with final suspension, also in sterile deionised or distilled water, sterilised
lyophilisation stabiliser is added and the suspension is dispensed into lyophilisation vials and freeze-dried.
c) Preparation and testing of the working seed

Reconstitute a vial of seed stock and inoculate several slants (approximately 10 ml) of sporulation (casein
digest) agar. Incubate at 37°C for 72 hours and store in a refrigerator. Test the slants for purity by culture on
to nutrient agar plates and in nutrient broth (0.1 ml in 100 ml of nutrient broth). The latter should be
subcultured on to nutrient agar after incubation at 37°C for 7 days and should be a pure culture of
B. anthracis. A sample of the broth culture should also be checked for lack of motility.
Volumes of seed needed for a production run should be calculated on the basis of harvesting the spores
from each slant with 10 ml of sterile deionised or distilled water and using this to inoculate five Roux bottles.
3 National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6
3QG, United Kingdom.

d) Safety of the seed lot

Not less than 5 × 109 culturable spores should be injected subcutaneously into each of three healthy, 1–2-
year-old, unvaccinated sheep, which must survive an observation period of at least 10 days.
e) Immunogenicity of the seed lot

At least 10 healthy guinea-pigs, 300–500 g in weight should be inoculated with 5 × 106 viable spores and
observed for 21 days. At least 80% of the animals should survive. The immunised animals, together with
three unimmunised controls, should then be challenged with 10 median lethal doses (LD50) of the strain
17 JB of B. anthracis. During a 10-day observation period, none of the immunised animals should succumb
to the challenge while all the controls should die from anthrax. The test should be repeated if one of the
immunised animals dies.
2. Method of production
a) Preparation of vaccine concentrate

Roux bottles with casein digest agar are prepared as for the master seed in Section C.1.b above. One Roux
bottle can be expected to yield about 2000 doses of vaccine. Each Roux bottle is inoculated with 2 ml of
working seed suspension and incubated at 37°C with porous plugs for several days until small loopfuls of
culture from randomly selected bottles show at least 90% of the organisms to be in sporulated forms when
examined in wet mounts by phase contrast (phase bright spores) or following staining for spores. The
growth from each bottle is then harvested with 20 ml of physiological saline. Tests for contaminants should
be carried out by subculture to nutrient agar plates and inoculation of 100 ml nutrient broth with 0.1 ml of
harvested spores followed by subculture to nutrient agar after 7 days at 37°C and by tests for motility.
Acceptable harvests (i.e. those showing no evidence of contaminants) are pooled.
b) Glycerination
Twice the volume of sterile, pure, neutral glycerol should be added to the bulk pool. Saponin (0.1% final
concentration) may also be added at this point if it is to be included as an adjuvant. Mix thoroughly (the
inclusion of sterilised glass beads may be helpful). Carry out a purity test as before and hold for 3 weeks at
ambient temperature to allow lysis of any vegetative bacteria, determine the viable spore count and store
under refrigeration thereafter.
c) Determining titre and dilution for use

The number of culturable spores in the product is then calculated by spreading tenfold dilutions on nutrient
agar plates. The suspension is diluted so that the final bulk contains the number of culturable spores
desired. The diluent should contain the same proportions of saline, glycerol and (if being included) saponin
as present in the vaccine concentrate. The vaccine should contain not less than 1 × 107 viable spores per
dose for cattle, buffaloes and horses, and not less than 1–5 × 106 viable spores per dose for sheep, goats
and pigs.
d) Safety
Safety testing is performed on two healthy sheep or goats and consists of inoculating subcutaneously twice
the recommended vaccination dose. The animals are observed for 10 days. The final bulk passes the test if
no systemic reactions develop and if not more than a transient oedema is observed at the injection site. If
the test is carried out in sheep only, a progressive oedema indicates that the vaccine may be unsuitable for
goats.
e) Filling the containers

Distribution of aliquots of vaccine into single and multidose containers is performed as outlined in World
Health Organization Technical Report No. 363 series entitled General Requirements for Manufacturing
Establishments and Control Laboratories (Requirements for Biological Substances No. 1), 1965, 16–17.
Basically, the final bulk is distributed to containers in an aseptic manner in an area not used for production,
and any contamination or alteration of the product must be avoided. The vaccine may be lyophilised after
distribution into appropriate dosage containers. Containers are sealed as soon as possible with a material
that is not detrimental to the product and that is capable of maintaining a hermetic seal for the life of the
vaccine.
3. In-process control
Purity tests consist of microscopic examination of stained smears with culture and motility tests as described in
Section C.2.a.

4. Batch control and tests on the final product
a) Sterility
The vaccine is a live culture of B. anthracis spores; sterility does not apply, but the batches must be tested
for freedom from contamination (see Chapter 1.1.9 Tests for sterility and freedom from contamination of
biological materials).
b) Efficacy
Efficacy or immunogenicity is tested on the final bulk as follows: at least ten healthy 300–500 g guinea-pigs
are inoculated with a sheep dose of the vaccine. The guinea-pigs are observed for 21 days, and at least
80% of the animals must survive the observation period. Surviving immunised guinea-pigs and three non-
vaccinated controls are challenged with an appropriate dose of virulent B. anthracis. A recommended
challenge is 200 LD50 of the Pasteur II strain (17JB), which is available from the same source as the Sterne
34F2 vaccine strain. If, by 10 days after challenge, all vaccinated guinea-pigs survive and control animals
die, the final bulk is deemed to be satisfactory. If any vaccinated animals die during the post-challenge
observation period from a cause other than anthrax, and death is not associated with the vaccine, the test
may be repeated.
c) Dose
The recommended dose for cattle and horses is a minimum of 1 × 107 culturable spores; for sheep, goats
and pigs, it is 1–5 × 106 culturable spores. The vaccine should contain these spores in an appropriate
volume, e.g. 1 × 107/ml.
d) Duration of immunity
Most experts agree that immunity is good for at least 1 year and it is recommended that an annual booster
be given. Horses may be slow to develop immunity following initial vaccination; some manufacturers
therefore recommend a two-dose initial vaccination, administered 1 month apart, followed by a single
annual booster.
e) Stability
As there is no generally acceptable test for stability of anthrax vaccines, it is recommended that, in each
filling lot, the number of culturable spores be determined before and after holding at an appropriate
temperature for an appropriate period. There should be no evidence of a fall in the number of culturable
spores.
f) Preservatives and storage

Bacillus anthracis spores are stable in unlyophilised or lyophilised vaccine and preservatives are not
required. Storage under refrigeration is recommended (4°C).
g) Precautions (hazards)
The vaccine has been shown to cause disease in some goats and llamas; this may be related to the
saponin adjuvant. The vaccine is not recommended for use in pregnant animals, nor in animals destined for
slaughter within 2–3 weeks of vaccination. Local regulations may specify other time periods in some
countries or regions, but there is no scientific reason for regarding meat from clinically healthy animals as
unfit for human handling or consumption after a holding period of 2 weeks following vaccination. Concurrent
administration of antibiotics to vaccinated animals is contraindicated as the antibiotic will interfere with the
vaccine. Antibiotics should not be given for several days before and after vaccination. Leftover vaccine,
empty vials, and equipment used for vaccinating are contaminated with the live spores and should be
autoclaved, disinfected, or incinerated. Accidental human inoculation is treated by expressing as much of
the inoculum as possible from the injection site and washing the wound thoroughly with soap and water.
Medical attention should be sought if infection develops.
5. Tests on the final product
a) Safety
Every batch of vaccine will be tested for safety as described in Section C.2.d.
b) Potency
Every batch of vaccine will be tested for potency, as described in Section C.4.b.

6. Propagation of the diagnostic ‘gamma’ bacteriophage
Anthrax-specific phages were first isolated in the 1950s, and the specifically named gamma phage was first
reported in 1955 and quickly became the standard diagnostic phage for anthrax. Gamma phage belongs to a
family of closely related anthrax phages (11). On occasion a phage-negative B. anthracis or phage-positive
B. cereus is encountered. The phage must be used in conjunction with other tests for confirmation, yet it is a
useful and reliable test.
Phage suspensions may be obtained from central veterinary laboratories or central public health laboratories.
The phage can be propagated and concentrated by the following protocol. Store phage at 2–4°C and do not
freeze phage as it will quickly become non-viable.
Stage one
i) Spread a blood agar (BA) plate of the Sterne vaccine strain of B. anthracis. Incubate overnight at 37°C.
ii) Inoculate approximately 10 ml of nutrient broth (NB) with growth from the BA plate and incubate at 37°C for
approximately 4 hours or until just cloudy, then refrigerate.
iii) Spread 100 µl of the culture from step ii on three pre-dried BA plates and incubate at 37°C for 30–
60 minutes.
iv) Spread 100 µl of the phage suspension to be amplified over the same plates. Incubate at 37°C overnight.
v) Harvest the phage-lysed growth on the BA plate in 5 ml of NB followed by a second ‘wash’ of 5 ml NB.
Incubate at 37°C overnight.
vi) Filter (0.45 µm) and count by dropping 20 µl drops (three drops per dilution) of tenfold dilutions of the filtrate
in saline onto lawns of the B. anthracis culture prepared as in step iii.
Stage two
This is essentially the same procedure as Stage one, only using the filtrate from step vi to harvest the phage from
the plates.
vii) Prepare three Sterne strain lawns on BA, as in step iii. Incubate at 37°C for 30–60 minutes.
viii) Spread 100 µl phage from step vi. Incubate at 37°C overnight.
ix) To 9 ml of filtrate from step vi, add 1 ml of 10× concentrated NB.
x) Harvest the phage from step viii with 5 ml of the solution from step ix, followed by a second 5 ml wash with
the rest of the solution from step ix.
xi) Add 10 ml of 1× NB.
xii) Incubate at 37°C overnight, filter and count.
Stage three
xiii) Inoculate 100 ml of brain–heart infusion broth with approximately 2.5 ml of the culture from step ii. Incubate
on a rotary shaker at 37°C until just turbid.
xiv) Add the 20 ml of filtrate from step xii and continue incubation overnight.
xv) The resultant filtrate is checked for sterility and titrated in ten-fold dilutions on lawns of the vaccine strain as
in step vi to determine the concentration of the phage. This should be of the order of 108–109 plaque
forming units per ml.
7. Polychrome methylene blue (M’Fadyean’s stain)
Polychrome methylene blue is prepared as follows: 0.3 g of methylene blue is dissolved in 30 ml of 95% ethanol;
100 ml of 0.01% potassium hydroxide (KOH) is mixed with the methylene blue solution. Ideally, this should be
allowed to stand exposed to the air, with occasional shaking, for at least 1 year to oxidise and mature. Addition of
K2CO3 (to a final concentration of 1%) hastens the ‘ripening’ of the stain, but before it is regarded as
diagnostically reliable, its efficacy should be established by testing it in parallel with an earlier, functional batch of
stain on bona fide samples. It has now been found that stains that give positive reactions with cultures of
B. anthracis cultured artificially in horse blood sometimes do not give positive results in the field.
In making smears for staining, only small drops of blood or tissue fluid are needed and a thin, small smear is
best. After fixing and drying, a small (approximately 20 µl) drop of stain is placed on the smear and spread over it

with an inoculating loop. After 1 minute, the stain is washed with water, blotted, air-dried and observed initially
using the ×10 objective lens under which the short chains appear like short hairs; once found, these can be
observed under oil immersion (×1000) for the presence of the pink capsule surrounding the blue/black-staining
bacilli. To avoid laboratory contamination, the slide and blotting paper should be autoclaved or left for some
hours in a 10% sodium hypochlorite solution.
REFERENCES
1. ASCOLI A. (1911). Die Präzipitindiagnose bei Milzbrand. Centralbl. Bakt. Parasit. Infeckt., 58, 63–70.
2. BEYER W., GLOCKNER P., OTTO J. & BOHM R. (1996). A nested PCR and DNA-amplification-fingerprinting
method for detection and identification of Bacillus anthracis in soil samples from former tanneries. Salisbury
Med. Bull., No. 87, Special Suppl., 47–49.
3. BROWN E.R. & CHERRY W.B. (1955). Specific identification of Bacillus anthracis by means of a variant
bacteriophage. J. Infect. Dis., 96, 34–39.
4. EZZELL J.W. & ABSHIRE T.G. (1996). Encapsulation of Bacillus anthracis spores and spore identification.
Salisbury Med. Bull., No 87, Special Suppl., 42.
5. HUTSON R.A., DUGGLEBY C.J., LOWE J.R., MANCHEE R.J. & TURNBULL P.C.B. (1993). The development and
assessment of DNA and oligonucleotide probes for the specific detection of Bacillus anthracis. J. Appl.
Bacteriol., 75, 463–472.
6. JACKSON P.J., HUGH-JONES M.E., ADAIR D.M., GREEN G., HILL K.K., KUSKE C.R., GRINBERG L.M., ABRAMOVA,
F.A. & KEIM P. (1998). PCR analysis of tissue samples from the 1979 Sverdlovsk anthrax victims: The
presence of multiple Bacillus anthracis strains in different victims. Proc. Natl Acad. Sci. USA, 95, 1224–
1229.
7 KNISELY R.F. (1966). Selective medium for Bacillus anthracis. J. Bacteriol., 92, 784–786.
8. MISRA R.P. (1991). Manual for the Production of Anthrax and Blackleg Vaccines. Food and Agriculture
Organisation of the United Nations (FAO) Animal Production and Health Paper 87, FAO, Rome, Italy.
9. RAMISSE V., PATRA G., GARRIGUE H., GUESDON J.L. & MOCK M. (1996). Identification and characterization of
Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosal
DNA. FEMS Microbiol. Lett., 145, 9–16.
10. STERNE M. (1937). The effect of different carbon dioxide concentrations on the growth of virulent anthrax
strains. Onderstepoort J. Vet. Sci. Anim. Ind., 9, 49–67.
11. TURNBULL P.C.B., BOEHM R., COSIVI O., DOGANAY M., HUGH-JONES M.E., LALITHA M.K. & DE VOS V. (1998).
Guidelines for the Surveillance and Control of Anthrax in Humans and Animals. WHO/EMC/ZDI/98.6. World
Health Organization, Geneva, Switzerland.
12. W ORLD HEALTH ORGANIZATION EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION (1967). Requirements for
Anthrax Spore Vaccine (Live – for Veterinary Use) (Requirements for Bioiogical Substances No. 13). World
Health Organization (WHO) Technical Report Series No. 361. WHO, Geneva, Switzerland.
FURTHER READING
A. LOGAN N.A. & TURNBULL P.C.B. (1998). Bacillus and recently derived genera. In: Manual of Clinical
Microbiology, Seventh Edition, Murray P.R., Baron E.J., Pfaller M.A., Tenover F.C. & Yolken R.H., eds.
American Society for Microbiology, Washington DC, USA, 357–369.
B. QUINN C.P. & TURNBULL P.C.B. (1998). Anthrax. In: Topley & Wilson’s Microbiology and Microbial Infections,
Vol. 3, Ninth Edition, Collier L., Balows A. & Sussman M., eds. Arnold, London, UK, 799–818.
C. TURNBULL P.C.B., (ED.) (1996). Proceedings of the International Workshop on Anthrax. Salisbury Med. Bull.,
Special Supple. No. 87, 139 pages.

D. TURNBULL P.C.B. (1998). Anthrax. In: Zoonoses. Biology, Clinical Practice, and Public Health Control.
Palmer S.R., Soulsby E.J.L. & Simpson D.I.H., eds. Oxford University Press, Oxford, UK, 3–16.
E. W HITFORD H.W. & HUGH-JONES M.E. (1994). Anthrax. In: Handbook Series in Zoonoses, Second Edition,
Beran G.W., editor-in-chief. CRC Press, Boca Raton, Florida, USA, 61–82.
*
* *
NB: There are OIE Reference Laboratories for Anthrax (see Table in Part 3 of this Terrestrial Manual or consult
the OIE Web site for the most up-to-date list: www.oie.int).

CHAPTER 2.1.2.
AUJESZKY’S DISEASE
SUMMARY
Aujeszky’s disease, also known as pseudorabies, is caused by an alphaherpesvirus that infects the
central nervous system and other organs, such as the respiratory tract, in a variety of mammals
except humans and the tailless apes. It is associated primarily with pigs, the natural host, which
remain latently infected following clinical recovery (except piglets under 2 weeks old, which die from
encephalitis). The disease is controlled by containment of infected herds and by the use of vaccines
and/or removal of latently infected animals.
A diagnosis of Aujeszky’s disease is established by detecting the agent (virus isolation, polymerase
chain reaction [PCR]), as well as by detecting a serological response in the live animal.
Identification of the agent: Isolation of Aujeszky’s disease virus can be made by inoculating a
tissue homogenate, for example of brain and tonsil or material collected from the nose/throat, into a
susceptible cell line such as porcine kidney (PK-15) or SK6, or primary or secondary kidney cells.
The specificity of the cytopathic effect is verified by immunofluorescence, immunoperoxidase or
neutralisation with specific antiserum. The viral DNA can also be identified using PCR; this can be
accomplished using the real-time PCR techniques.
Serological tests: Aujeszky’s disease antibodies are demonstrated by virus neutralisation, latex
agglutination or enzyme-linked immunosorbent assay (ELISA). A number of ELISA kits are
commercially available world-wide. An OIE international standard serum defines the lower limit of
sensitivity for routine testing by laboratories that undertake the serological diagnosis of Aujeszky’s
disease.
Since about 1990, it has become possible to distinguish between antibodies resulting from natural
infection and those from vaccination by use of gene-deleted vaccines.
Requirements for vaccines and diagnostic biologicals: Vaccines, either gene-deleted attenuated
or inactivated virus vaccines should prevent or at least limit the excretion of virus from the infected
pigs. More recently, these conventional vaccines have been supplemented by rDNA-derived gene-
deleted or naturally deleted live Aujeszky’s disease virus vaccines. The virus used in these new
vaccines, sometimes referred to as marker vaccines, lacks a specific glycoprotein (gG, gE, or gC).
A. INTRODUCTION
Aujeszky’s disease, also known as pseudorabies, is caused by an alphaherpesvirus, a member of the family
Herpesviridae. The virus infects the central nervous system and other organs, such as the respiratory tract, a
variety of mammals except humans and the tailless apes. It is associated primarily with pigs, the natural host,
which remain latently infected following clinical recovery (except piglets under 2 weeks old, which die from
encephalitis). The disease is controlled by containment of infected herds and by the use of vaccines and/or
removal of latently infected animals.
Whereas isolation of the Aujeszky’s disease virus will assist in a provisional diagnosis in the case of lethal forms of
Aujeszky’s disease or clinical disease in pigs, other techniques and serological tests are required for diagnosis of
latent infections. Many affected animals, however, except pigs, do not live long enough to produce any marked
serological response.

Chapter 2.1.2. — Aujeszky’s disease
a) Virus isolation
The diagnosis of Aujeszky’s disease can be confirmed by isolating PRV from the oro-pharyngeal fluid, nasal
fluid (swabs) or tonsil swabs from living pigs, or from samples from dead pigs or following the presentation of
clinical signs such as encephalitis in herbivores or carnivores. For post-mortem isolation of PRV, samples of
brain, tonsil, lung and spleen are the preferred specimens. In cattle, infection is usually characterised by a
pruritus, in which case a sample of the corresponding section of the spinal cord may be required in order to
isolate the virus. In latently infected pigs, the trigeminal ganglion is the most consistent site for virus isolation,
although latent virus is usually non-infective unless reactivated, making it difficult to recover in culture.
The samples are homogenised in normal saline or cell culture medium with antibiotics and the resulting
suspension is clarified by low speed centrifugation at 900 g for 10 minutes. The supernatant fluid is used to
inoculate any sensitive cell culture system. Numerous types of cell line or primary cell cultures are sensitive to
PRV, but a porcine kidney cell line (PK-15) is generally employed. The overlay medium for the cultures
should contain antibiotics (such as: 200 IU/ml penicillin; 100 µg/ml streptomycin; 100 µg/ml polymyxin; and
3 µg/ml fungizone).
PRV induces a cytopathic effect (CPE) that usually appears within 24–72 hours, but cell cultures may be
incubated for 5–6 days. The monolayer develops accumulations of birefringent cells, followed by complete
detachment of the cell sheet. Syncytia also develop, the appearance and size of which are variable. In the
absence of any obvious CPE, it is advisable to make one blind passage into further cultures. Additional
evidence may be obtained by staining infected cover-slip cultures with haematoxylin and eosin to
demonstrate the characteristic herpesviral acidophilic intranuclear inclusions with margination of the
chromatin. The virus identity should be confirmed by immunofluorescence, immunoperoxidase, or
neutralisation using specific antiserum.
The isolation of PRV makes it possible to confirm Aujeszky’s disease, but failure to isolate does not
guarantee freedom from infection.
b) Identification of virus by the polymerase chain reaction

The polymerase chain reaction (PCR) can be used to identify PRV genomes in secretions or organ samples.
Many individual laboratories have established effective protocols, but there is as yet no internationally agreed
standardised approach.
The PCR is based on the selective amplification of a specific part of the genome using two primers located at
each end of the selected sequence. In a first step, the complete DNA may be isolated using standard
procedures (e.g. proteinase K digestion and phenol–chloroform extraction) or commercially available DNA
extraction kits. Using cycles of DNA denaturation to give single-stranded DNA templates, hybridisation of the
primers, and synthesis of complementary sequences using a thermostable DNA polymerase, the target
sequence can be amplified up to 106-fold. The primers must be designed to amplify a sequence conserved
among PRV strains, for example parts of the gB or gD genes, which code for essential glycoproteins, have
been used (10, 29).
The amplified product may be identified from its molecular weight as determined by migration in agarose gel,
with further confirmation where possible by Southern hybridisation using a complementary probe. Recent
techniques involve liquid hybridisation using enzyme-labelled probes, which give a colour reaction after
incubation with the appropriate substrate. More recent techniques include the use of fluorescent probes
linked to an exonuclease action and real-time monitoring of the evolution of product, enabling simultaneous
amplification and confirmation of the template DNA thus increasing the rapidity and specificity of the PCR
assays.
In all cases, the main advantage of PCR, when compared with conventional virus isolation techniques, is its
rapidity; with the most modern equipment, the entire process of identification and confirmation can be
completed within one day, However, because of the nature of the test, many precautions need to be taken to
avoid contamination of samples with extraneous DNA from previous tests or from general environmental
contamination in the laboratory (see Chapter 1.1.9 Tests for sterility and freedom from contamination of
biological materials). This may limit the value of the test for many laboratories unless care is taken to avoid
DNA carry-over contamination.

2. Serological tests
Any serological technique used should be sufficiently sensitive to give a positive result with the OIE International
Standard Reference Serum. This serum can be obtained from the OIE Reference Laboratory for Aujeszky’s
Disease in France (see Table given in Part 3 of this Terrestrial Manual). For international trade purposes, the test
should be sensitive enough to detect the standard serum diluted 1/2.
Virus neutralisation (VN) has been recognised as the reference method for serology (4, 28), but for general
diagnostic purposes it has been widely replaced by the enzyme-linked immunosorbent assay (ELISA) because of
its suitability for large-scale testing (2, 12, 16, 18). The tests can be performed on a variety of matrices (e.g. serum,
whole blood, milk) but the preferred matrix is serum.
A latex agglutination test has also been developed and can be used for screening for antibodies. Kits for the test
are commercially available.
a) Virus neutralisation (a prescribed test for international trade)

VN in cell culture can be performed in several ways, which vary according to the length of incubation of the
virus/serum mixtures (e.g. 1 hour at 37°C or 24 hours at 4°C), and the presence or absence of complement.
Most laboratories use a reaction period of 1 hour at 37°C in the absence of complement, because this is easy
and rapid. However, the sensitivity can be improved by increasing the incubation period to 24 hours at 4°C,
which facilitates the detection of antibody levels 10–15 times lower than in the 1-hour method. For
international trade purposes, the test method should be validated as being sensitive enough to detect the OIE
Standard Reference Serum diluted 1/2.
VN cannot be used to differentiate antibodies of vaccinal origin from those caused by natural infection. It is
one of the two tests available that complies with the requirement in the OIE Terrestrial Animal Health Code
chapter when it refers to ‘a diagnostic test to the whole virus’.
Cells: Cells susceptible to infection with PRV are used; they may be cell lines (e.g. PK-15, SK6, MDBK), or
primary or secondary cell cultures.
Cell culture medium: The medium depends on the type of cells. For example, the medium for PK-15 cells is
Eagle’s minimal essential medium (MEM) + 10% fetal bovine serum and antibiotics (100 IU/ml penicillin and
100 µg/ml streptomycin, or alternatively, 50 µg/ml gentamycin).
Maintenance of the cells: The cells are cultured in cell culture vessels of, for example, 75 cm2. They are
trypsinised once or twice per week. For weekly trypsinisation, the cells are cultured in 50 ml of medium, with a
multiplication rate of 5. For two trypsinisations a week, the cells are cultured in 30 ml of medium, with a
multiplication rate of 3.
For trypsinisation, the growth medium is removed once the cell sheet is complete. The cell sheet is washed
with about 5 ml of recently thawed trypsin/ethylene diamine tetra-acetic acid (EDTA) (0.25%) in an isotonic
buffer. The washing fluid is discarded and the preparation is washed again, retaining only a few drops of
trypsin. The container is placed in an incubator at 37°C for 5–10 minutes until the cells have become
detached. Once the sheet is detached and the cells are well separated, they are suspended in 90 ml of
growth medium, and this suspension is distributed into three 75 cm2 cell culture bottles.
Virus: A suitable strain of PRV, such as the Kojnok strain, or NIA-3 strain, is stored at a temperature of
–70°C or below, or in freeze-dried form at 4°C.
Preparation of stock virus suspension: The culture fluid is removed from a cell culture bottle containing a
complete cell sheet. About 1 ml of stock virus suspension of known titre (about 107 TCID50/ml [50% tissue
culture infective dose]) is added, and the bottle is incubated at 37°C for 1 hour. Then, 30 ml of culture
medium is added and the bottle is again incubated at 37°C. The bottle is examined frequently until there is
about 75% cell destruction (after about 36–48 hours). It is then frozen at a temperature of –20°C or lower in
order to disrupt the cells.
The bottle is then thawed and shaken vigorously. Medium is collected and centrifuged at 1500 g for
15 minutes. The supernatant fluid is divided into portions (of about 0.5 ml) in small tubes that are labelled
(date and virus reference) before being stored at a temperature of –70°C or lower until required.
Titration of the stock virus suspension: Titration of the stock suspension is performed by the method of Reed
& Muench or that of Kärber, and the titre is expressed per 50 µl and per ml.

The VN test requires an internal quality control serum with a known titre of neutralising antibody to PRV (it
must be calibrated against an international standard serum or a secondary standard prepared from that
serum), and a negative control serum (from a specific antibody free pig, e.g. from an official Aujeszky’s
disease free herd). The test sera themselves should be of good quality. Serum should be separated from the
coagulum without delay, thereby preventing toxicity.
There are qualitative and quantitative procedures for VN, both of which are described below.
• Qualitative technique
i) Complement in the serum samples is destroyed by heating in a water bath at 56°C for 30 minutes.
ii) Each undiluted serum is placed in three wells, at 50 µl per well, of a 96-well cell-culture grade microtitre
plate.
iii) 50 µl of virus suspension containing 100 TCID50 (or 2 × 103 TCID50/ml), obtained by diluting stock virus
suspension of known titre with MEM, is added to each well.
iv) The plate is shaken and placed in an incubator for 1 hour at 37°C (CO2 optional).
v) 150 µl of cell suspension containing about 150,000 cells/ml is added to each well.
vi) The plate is covered (for incubation in CO2), or a plastic sheet is sealed carefully around the edges of
the plate (for incubation in air). The plate is shaken lightly to obtain an even distribution of cells at the
bottom of the wells, and placed in the incubator at 37°C (CO2 optional).
vii) Controls: Each set of plates must include the following controls:
Virus control: This is to verify the amount of virus actually used for the test. The virus dose used for virus
neutralisation (target titre 100 TCID50/50 µl) is diluted with MEM at 1/10, 1/100 and 1/1000. Of each
dilution, 50 µl is placed in at least eight wells, to which 50 µl of medium is added before the wells are
incubated for 1 hour at 37°C. The cell suspension is added in the same way as for the sera under test.
Cell control: 150 µl cell suspension and 100 µl MEM are placed in each of at least two wells.
Positive serum control: A serum of known PRV neutralising antibody titre is used. Five dilutions are
prepared in the same way as for the sera under test: a dilution corresponding to the serum titre, two-fold
and four-fold dilutions, and 1/2 and 1/4 dilutions (equivalent to T, T/2, T/4, 2T and 4T, where T is the
serum titre, i.e. undiluted serum for the qualitative test). To 50 µl of positive control sample dilutions, add
50 µl of virus suspension containing 100 TCID50/50 µl. The cells are incubated and the cell suspension
is added in the same way as for the sera under test.
Serum control: This is to verify the absence of a toxic effect of the sera on the cells. Wells containing
50 µl of each serum are incubated for 1 hour at 37°C in the presence of 50 µl of medium. Then, 150 µl
of cell suspension is added in the same way as for the sera under test.
Negative serum control: This is done in the same way as for sera under test.
viii) Reading the results: An inverted-image microscope (×100) is used to examine the wells for toxic effects
and CPEs after 48 and 72 hours. The controls must give the following results if the tests are to be
considered valid:
Virus control: The titre of the viral suspension should be between 30 and 300 TCID50/50 µl.
Cell control: The cell sheet must be intact.
Positive serum control: The titre obtained must be equal to the predicted titre, within one dilution.
Serum control: Examination for a CPE should take into account a possible toxic effect on cells.
Negative serum control: A CPE should be present.
ix) For the sera under test, the following results may be seen: presence of a CPE in three wells = negative
result; absence of a CPE in three wells on day 3 = positive result; presence of a CPE in one well but not
in the others = doubtful result, test must be repeated; small plaques indicating a CPE on day 3 =
doubtful result, test must be repeated; toxicity in serum control and test wells = unreadable result, test
must be repeated (NB replacement of medium with fresh medium after 16 hours’ incubation will reduce
the toxicity without affecting the titre of specific antibody).
x) Interpretation of the results: This test is capable of detecting the presence or absence of neutralising
antibody to PRV. It is incapable of distinguishing vaccinated animals from infected animals.
The technique described (VN for 1 hour at 37°C) can give false-negative and false-positive results. The
sensitivity can be increased (leading to fewer false negatives) by adopting a method based on
neutralisation involving 24 hours of contact between virus and serum at 4°C, before the addition of cells.

A qualitative technique such as this one, which employs undiluted serum (1/2 final dilution), can give a
false-positive result in certain cases due to nonspecific neutralisation of the virus. This problem can be
addressed by carrying out a confirmatory test using the quantitative technique (see below).
• Quantitative technique
This is similar to the qualitative procedure, but each serum is used both undiluted and in a series of dilutions.
Depending on the desired precision, the purpose of testing and the expected titre, two wells are used for each
dilution of serum, and a greater or smaller range of dilutions. Ideally, the procedure may be described for a
range of dilutions reaching an initial maximum of 1/256.
i) Complement in the serum samples is destroyed by heating in a water bath at 56°C for 30 minutes.
ii) 50 µl of MEM is added to wells A3 to A6 of a 96-well cell-culture grade microtitre plate.
iii) 50 µl of undiluted serum is added to wells A1 to A3, and continued for wells in rows B, C, etc., with other
serum samples.
iv) Using a multichannel pipette, the contents of wells in row 3 are mixed, then 50 µl is transferred to row 4,
and so on to row 6 or further to a predetermined row, using the same nozzles. The 50 µl portions
remaining after the last row is discarded.
v) Controls are set up as described for the qualitative technique.
vi) 50 µl of MEM is added to row 1 instead of virus: this is a control row of sera. Viral suspension is
deposited in the wells of the other rows. Subsequent manipulations are the same as described for the
qualitative technique.
vii) Reading the results: The neutralising titre of a serum is expressed by the denominator of the highest
initial dilution that brings about complete neutralisation of the CPE of the virus in 50% of the wells.
Neutralisation at any dilution (even undiluted, equivalent to a final dilution of 1/2) is considered to be
positive. If the serum shows neutralisation only when undiluted (with growth of virus and CPE at the 1/2
and subsequent dilutions), it would be advisable to apply alternative tests (ELISA or latex agglutination)
to provide confirmation of the result, or to request another sampling of the animal, at least 8 days after
the first.
b) Enzyme-linked immunosorbent assay (a prescribed test for international trade)

The sensitivity of the ELISA is generally superior to that of the VN test using 1-hour neutralisation without
complement. Some weak positive sera are more readily detected by VN tests using 24-hour neutralisation,
while others are more readily detectable by ELISA.
ELISA kits, which are available commercially, use indirect or competitive techniques for measuring antibody
levels. They differ in their mode of preparation of antigen, conjugate, or substrate, in the period of incubation
and in the interpretation of the results. Their general advantage is that they enable the rapid processing of
large numbers of samples. This can also be automated and the results analysed by computer. Some of these
kits make it possible to differentiate between vaccinated and naturally infected animals when used with a
‘matching’ vaccine (6, 21, 22). Alternatively, non-commercial ELISA protocols may be adopted (2, 18)
provided they are shown to detect the OIE International Standard Reference Serum as positive at a dilution of
1/2 (the minimum sensitivity for international trade purposes). It is recommended to use a kit or in-house
assay that has been validated to this standard by external quality control tests by an independent laboratory.
A suitable test protocol for whole virus antibodies is presented below (18).
• Preparation of antigen
i) A cell line sensitive to PRV is used, such as PK-15 or fetal pig testis. It must be free from extraneous
viruses, such as bovine viral diarrhoea virus. The cells should be split and seeded into fresh 75 cm2
flasks the day before inoculation. A suitable medium such as MEM, without serum, is used to overlay the
cultures.
ii) Virus inoculated, and control uninoculated flasks are processed in parallel throughout. A suitable well
characterised strain of PRV is used, e.g. Kojnock strain. When a confluent cell monolayer has
developed (approximately 24 hours after seeding), it is inoculated with 108 TCID50 PRV in 5 ml medium;
and 5 ml medium (without virus) is placed in control flasks. The cultures are left for adsorption for
30 minutes at 37°C, and then overlaid with 20 ml medium.
iii) When CPE is just beginning, the supernatant medium is discarded and 4 ml KCl (4 mM solution) and
glass beads are added. The flasks are shaken gently to detach cells.
iv) Cells are washed by centrifuging three times at 770 g in 4 mM KCl. The pellet is resuspended in 4 mM
KCl with 0.2% Triton X-100 (1 ml per flask) by applying 60 strokes with a glass homogeniser

v) The cell homogenate is layered on to 0.25 mM sucrose in 4 mM KCl and centrifuged for 10 minutes at
770 g.
vi) The pellet is resuspended in antigen-diluting buffer, pH 9.6 (0.1 M Tris, 2 mM EDTA, 0.15 mM NaCl) at
1/50 the volume of the original culture medium. It may then be stored in small aliquots at –70°C. Antigen
is stable in this form for 2 years.
• Coating microtitre plates

i) Virus antigen and control (no virus) antigen are diluted in diluting buffer, pH 9.6 (see above) to a dilution
predetermined in chequerboard titrations.
ii) 200 µl of antigen is dispensed into each well of 96-well ELISA-grade plates, coating alternate rows with
PRV positive and control antigen. Incubation is for 18 hours at 4°C.
iii) The plates are washed three times with washing solution (Tween 20, 0.5 ml/litre).
iv) Coated plates are stored at –20°C or –70°C. They are stable for several months.
• Test procedure
i) Test serum samples are diluted 1/30 in PBS/Tween buffer, pH 7.2 (137 mM NaCl, 9.5 mM phosphate
buffer, 0.5 ml/litre Tween 20).
ii) Diluted samples are added to virus and control antigen coated wells, and incubated at 37°C for
30 minutes.
iii) The plates are washed three times with washing solution (0.5 ml/litre Tween 20).
iv) Protein A/peroxidase conjugate is added to all wells at a predetermined dilution in PBS/Tween buffer,
pH 7.2 (see above), with added bovine serum albumen fraction V (10 g/litre), and the plates are
incubated at 37°C for 30 minutes.
v) The plates are washed three times with washing solution (0.5 ml/litre Tween 20).
vi) A suitable chromogen/substrate mixture, such as tetra methyl benzidine (TMB)/hydrogen peroxide, is
added to each plate.
vii) The reaction is stopped with 2 M sulphuric acid. The absorbance is read at 492 nm.
The test must be fully validated using known positive and negative sera, and calibrated against the OIE
International Standard Reference Serum. All tests must include positive and negative internal controls,
including a weak positive that, when diluted at the appropriate dilution for the test, has equivalent activity to a
1/2 dilution of the OIE International Standard Reference Serum. For further details see reference 18 and
Chapter 1.1.4 Principles of validation of diagnostic assays for infectious diseases. Commercial ELISA kits
also have to be validated in the setting in which they are going to be used.
As well as testing sera, the ELISA can be adapted to test filter paper disks that have been moistened with a
small quantity of blood obtained by puncturing a superficial vein. This technique makes it convenient to
collect blood samples from large numbers of pigs (3, 19). The disks are air-dried before shipment to the
laboratory.
Requirements for the detection of gE antibodies by ELISA in pigs destined for slaughter, that are to be
introduced into zones free from Aujeszky’s disease, have been defined by several control authorities. The
OIE Terrestrial Animal Health Code specifies circumstances in which gE-specific tests may be used. The gE
ELISAs can also be adapted to test blood on filter paper disks.

Aujeszky’s disease may be controlled by the use of vaccines containing either modified live or inactivated virus
antigens. In addition, these conventional vaccines have been supplemented by recombinant DNA-derived gene-
deleted or naturally deleted live PRV vaccines. These vaccines, sometimes referred to as marker vaccines, are
made with a virus that lacks a specific glycoprotein (most commonly gE-, although gG- or gC-deleted vaccines
1
have also been described ). At least one commercially available vaccine has dual deletions. These gene-deleted
marker vaccines have the advantage over conventional whole virus vaccines that it is possible to distinguish
noninfected vaccinated animals from those with field infection. This is done by testing for the antibodies directed
against the protein coded for by the deleted gene, which will be absent in noninfected marker-vaccinated pigs but
1 The nomenclature for the genes changed several years ago, but the old designation is still in the literature. The old and the
new nomenclature is: g11 – gB; g111 = gC; gp50 = gD; g1 = gE; gX = gG; gp63 = gl.

present in field-infected pigs. Therefore, in countries with infected pigs, where the eradication of Aujeszky’s
disease is planned, these marker vaccines are the vaccines of choice. Standards applicable to the manufacture of
live and inactivated virus vaccines are described. For marker vaccines, the tests should include demonstrable
absence of a serological response in vaccinated pigs to the protein coded for by the deleted gene, and in addition
a demonstrable response to the same protein in vaccinated pigs that become infected by field virus.
Guidelines for the production of veterinary vaccines are given in Chapter 1.1.8 Principles of veterinary vaccine
production. The guidelines given here and in Chapter 1.1.8 are intended to be general in nature and may be
supplemented by national and regional requirements.
1. Seed management
Vaccines are made using a seed-lot system in which a master seed virus (MSV) is prepared from a suitable
strain of Aujeszky’s disease virus. A number of strains are used for vaccine manufacture. The antigen in an
inactivated vaccine can be one of a number of wild-type strains, or the naturally deleted Bucharest virus, or
rDNA-derived gene-deleted virus. Modified live conventional vaccines and rDNA-engineered vaccines use
numerous strains, such as Bartha (8, 9, 11, 15, 17, 23, 27), or are derived from Aujeszky’s original isolate or
from other field isolates, such as the NIA-3 strain.
It is recommended that for differentiating between infected and vaccinated animals, deleted strains should be
used.
A virus identity test (using either a fluorescent antibody test, neutralisation test, [constant serum/decreasing
virus method], or any other suitable identity test) must be conducted on the MSV.
The MSV must be shown to be free from mycoplasma, bacteria, fungi, cytopathogenic or haemadsorbing
viruses, porcine parvovirus, cytopathic and noncytopathic ovine and bovine pestiviruses and other extraneous
agents, such as circovirus, as determined by culturing and by fluorescent antibody procedures or others, such
as PCR.
b) Method of culture
Most of the cell lines used to propagate PRV are continuous lines, such as the PK-15 line. A master cell stock
(MCS) is established at a specified passage level. The MCS and the highest passage level (MCS × n)
intended for use in the preparation of a biological product is specified in an Outline of Production. Both MCS
and MCS × n are monitored by a variety of procedures to characterise the cell line and to ensure freedom
from adventitious agents. The extraneous agents to be detected are generally defined in monographs and/or
guidelines (e.g. European Pharmacopoeia, US Code of Federal Regulations, EU guidelines, etc.). In general,
the type of agents to be looked for is founded on a risk analysis depending on the history of the viral strain
and cells on which the vaccinal strain was isolated and on which it is cultivated. The MCS must be monitored
for species of origin. A minimum of 50 mitotic cells should be examined at both the MCS and MCS × n
passage levels. The modal number in the MCS × n must not exceed 15% of the modal number of the MCS.
Any marker chromosomes in the MCS must also be present in the highest cell passage.
If there is evidence that the cell line may induce malignancies in the species for which the product is
intended, the cell line is tested for tumorgenicity and oncogenicity.
c) Validation as a vaccine
i) Purity
The MSV must be shown to be free from mycoplasma, bacteria, fungi, cytopathogenic or
haemadsorbing viruses, porcine parvovirus, cytopathic and noncytopathic ovine and bovine pestiviruses
and other extraneous agents, such as circovirus, as determined by culturing and by fluorescent antibody
procedures or others, such as PCR.
ii) Stability tests

Tests have to be carried out to verify the shelf life proposed by the manufacturer. These tests must
always be real-time studies; they must be carried out on a sufficient number of batches (at least three)
produced according to the described production process and on products stored in the final container,
and normally include biological and physicochemical stability tests. The manufacturer has to provide the
results of analyses that support the proposed shelf life under all proposed storage conditions. Usually,
the proposed shelf life corresponds to the period for which the product is considered to be stable minus
3 months.

iii) Safety tests

Local and general reactions must be examined. When a live vaccine is used, it is necessary to
differentiate the exact safety properties of the vaccinal strain from those of the finished product if this
includes an adjuvant.
Objective and quantifiable criteria to detect and measure adverse reactions should be used; these
would include temperature changes, weight gain, litter size, reproductive performance, etc., of
vaccinated and control groups. The tests must be performed by administering the vaccine to the pigs in
the recommended dose and by each recommended route of administration.
In general, safety is tested initially under experimental conditions. When the results of these preliminary
tests are known, it is necessary to increase the number of animals vaccinated in order to evaluate the
safety of the vaccine under practical conditions.
• Laboratory testing
All tests must be carried out on pigs that do not have antibodies against Aujeszky’s disease virus or
against a subunit of the virus.
a. General effects
1. Live vaccines
Intranasal tests and vaccination of 3–5-day-old piglets are very useful for ascertaining the degree of
safety of a strain. At least five piglets should be used.
It is also essential to assess the properties of a vaccine, especially live ones, in the target animals under
normal conditions of use and at the youngest age intended for vaccination, e.g. fattening pigs, which are
generally vaccinated when they are between 9 and 12 weeks old, and pregnant sows when this use of
the vaccine is claimed by the manufacturer and is authorised. No clinical signs, including significant
thermal reactions (data have to be recorded before vaccination and 6 hours, 24 hours and 48 hours
later, then on a daily basis during the observation period) , should be observed after vaccination. These
assays have to be performed on at least ten vaccinated pigs, with unvaccinated pigs as controls.
Reversion to virulence following serial passage must be examined. Primary vaccination is done by the
intranasal route. Series of at least five passages in piglets are made. No fewer than two fully susceptible
animals must be used for each passage.
The object of these assays is to test the genetic stability of live vaccine strains. The tests appear to be
less necessary when a genetically modified live strain is concerned, especially if it is produced by gene
deletion.
It is recommended to test for possible excretion of the vaccine strain. For this purpose, no fewer than
14 piglets, 3–4 weeks old each receive one dose of vaccine by the recommended route and at the
recommended site (except for vaccines administered by the intranasal route). Four unvaccinated piglets
are kept as controls. Suitably sensitive tests for the virus are carried out individually on the nasal and
oral secretions of vaccinated and in-contact pigs as follows: nasal and oral swabs are collected daily
from 1 day before vaccination to 10 days after vaccination. Vaccine strains that are isolated from the
nasal/oral secretion collected from pigs in which the vaccine was administered by the parenteral route
are not recommended.
The ability of the Aujeszky’s disease vaccine strain to spread from a vaccinated pig to unvaccinated
ones (lateral spread) must be tested by using the recommended route of administration that presents
the greatest risk of spreading (except for vaccines administered by the intranasal route). A repetition of
the assays (four times) is necessary as this phenomenon is difficult to detect. Four piglets should be
used each time for vaccination and placed in contact, 1 day later, with two unvaccinated piglets. It may
also be necessary to examine the spread of the strain to non target species that may be susceptible to
the vaccine strain.
Live attenuated vaccine strains are tested with regard to their general effects by administering to 5–10-
day-old piglets ten times the field dose. This administration of an overdose makes it possible to detect
reactions not produced under normal conditions of use. Such reactions may be produced inadvertently
when large numbers of animals are vaccinated. If vaccines are administered by the intranasal route, the
manufacturer has to indicate clearly that the vaccine will spread from vaccinated pigs to unvaccinated
ones.
2. Inactivated vaccines
It is essential to test inactivated vaccines in the target animals under normal conditions of use for
fattening pigs and for sows when this use is claimed by the manufacturer and authorised (26). As
described previously, it is fundamental to use objective and quantifiable criteria to detect and to
measure adverse reactions, such as temperature changes, weight performance, litter size, reproductive

performance, etc., on vaccinated and control groups. The tests must be performed by administering the
vaccine in the recommended dose and by each recommended route of administration to the pigs for
which it is intended.
Pigs or sows are usually observed until there is no further evidence of vaccine reaction. The period of
observation must not be fewer than 14 days from the day of administration. This period has to be
extended when, for example, the vaccine is used in pregnant sows and it is necessary to assess the
possible effects of the vaccine on reproductive performance. In this case, the period of observation lasts
the full duration of the pregnancy.
Control authorities generally request vaccination with a double dose so that adverse reactions, which
may be at the limit of detection when a single dose is administered, are more likely to be detected.
b. Local reactions
Local reactions are often associated with the use of inactivated vaccines, as these side-effects can be
induced by adjuvants, particularly oil adjuvants (24). However, some Aujeszky’s disease live vaccines
are mixed with different adjuvants, which modify what has been observed in the past.
Local reactions are mainly inflammatory and can be more or less complicated (necrotic or suppurative),
depending on the nature of the adjuvants used and the aseptic conditions of the vaccination. Oil
adjuvants can induce a variety of effects including muscular degeneration, granuloma, fibrosis and
abscessation. In addition to the nature of the oil used (the intensity of the reaction is reduced when
metabolisable oils are used in the vaccine), the type of emulsion used (water/oil, oil/water,
water/oil/water) induces these reactions to a greater or lesser extent. In consequence, it is necessary to
observe the site of injection not only from the outside, but also by dissection after slaughter, especially
for growing and finishing pigs.
• Field testing
Field trials are necessary to assess the safety of an Aujeszky’s disease vaccine in a large number of
pigs or sows. In Europe (7), tests must be carried out in each category of animals for which the vaccine
is intended (sows, fattening pigs). At least three groups of no fewer than 20 animals each are used with
corresponding groups of no fewer than 10 controls. The rectal temperature of each animal is measured
at the time of vaccination, and 24 and 48 hours later. At slaughter, the injection site must be examined
for local reactions. If the vaccine is intended to be used in sows, reproductive performances have to be
recorded. Field trials are supplemented by laboratory studies of efficacy correlated to vaccine potency.
iv) Efficacy tests

• Laboratory trials
All tests must be carried out on pigs that do not have antibodies against Aujeszky’s disease virus or
against a subunit of the virus, except that some tests may be done using maternally immune animals.
a. Assessment of passive immunity

To test the efficacy of vaccines, it is important to mimic the natural infection conditions (1). PRV infection
gives rise to important losses of young piglets from nonimmune sows. Thus, when vaccinating sows, the
main goal is to protect the young piglets through passive immunity conferred by the colostrum ingested
immediately after birth, with the secondary objective of preventing abortion.
To measure this passive immunity and the protection induced by vaccinating the sows, experimental
models have been established. The sows are vaccinated according to the vaccinal protocol during
pregnancy. When the piglets are, for example, 6–10 days old they are given an intranasal challenge
exposure with a virulent PRV strain. It is preferable to use a strain titrated in median lethal doses (LD50).
Pigs should be inoculated by the nasal route, 102 pig LD50 per pig in 1 ml. The efficacy of the vaccine is
assessed by comparing clinical signs, but also and more importantly, mortality in piglets from
unvaccinated dams with that observed in piglets from vaccinated sows.
Piglets from vaccinated sows can be found to have 80% protection against mortality compared with
those from the control sows. In order for the results to be significant, it is recommended that eight
vaccinated sows and four control sows be used (subject to satisfactory numbers of piglets from each
sow).
b. Assessment of active immunity

1. Clinical protection
Several criteria can be considered when measuring active immunity induced by vaccinating pigs.
Generally, pigs are vaccinated at the beginning of the growing period, i.e. when they are between 9 and

12 weeks old. Laboratory trials are performed by challenging pigs at the end of the finishing period,
when they weigh between 80 and 90 kg.
In general, at least three criteria, such as rectal temperatures, weight losses and clinical signs, along
with mortality, are used to measure the clinical protection of pigs after vaccination and challenge (5).
The antibody titres have little predictive value for the efficacy of the vaccines. Weight loss compared
between the vaccinated and control groups is the most reproducible and reliable parameter when the
challenge conditions are well standardised. The measure of the difference in weight gain or loss
between the two groups of pigs and, in the interval of time between challenge (day 0 and day 7), has a
very good predictive value for the efficacy of the vaccines (14). Significant results can be obtained when
weight performances are compared between one group of at least eight vaccinated pigs and another
group of eight unvaccinated control pigs
For challenge, it is usually preferable to use a high titre of a virulent strain, as this makes it possible to
obtain a more marked difference between vaccinated and control pigs. On the basis of previous work, a
challenge dose with at least 106 TCID50/ml virulent strain having undergone not more that three
passages on primary cells can be sufficient, but a higher titre (107.5 TCID50/ml) is recommended. The
oro-nasal route should be used to challenge the pigs by introducing the virulent strain in an
appropriately high volume (≥4 ml).
This method of evaluating the efficacy of PRV vaccines is now well tested and has made it possible to
establish an objective index for determining the efficacy of a vaccine. This index, which compares the
relative weight losses between vaccinated and control pigs, can also be used for potency testing
batches before release and for batch efficacy testing. However, the value of the cut-off index will be
different as the conditions of the assay will not be identical. The influence of passively acquired,
maternally derived antibodies on the efficacy of a vaccine must be evaluated adequately.
2. Virulent virus excretion

Additionally, it is desirable that vaccines should prevent or at least limit viral excretion from infected pigs
(13, 20, 25). When a control programme against Aujeszky’s disease is based on large-scale
vaccination, it is essential to choose the vaccines or the vaccinal scheme that best limits the replication
of virulent virus in infected pigs. Several assays have been performed to compare vaccines on that
basis.
Generally, the pigs are vaccinated and challenged at different periods. It is better, but more time-
consuming, to infect pigs at the end of the finishing period. To measure the virus excretion, nasal swabs
(taken at 10 cm depth in the nostrils) are taken daily from each pig from the day before challenge to at
least 12 days after challenge. The swabs can be weighed before the sampling and immediately after to
calculate the exact weight of collected mucus. Medium is then added to each tube containing a swab.
The virus is titrated from the frozen and thawed medium.
Different indexes can be used to express the quantity of virulent virus excreted by pigs, taking into
consideration the duration and the level of viral excretion, and the number of pigs excreting virulent
virus.
3. Duration of immunity
It is recommended that any claims regarding the onset and duration of immunity should be supported by
data from trials. Assessment of duration of immunity can be based on challenge trials or, as far as it is
possible, on immunological and serological tests.
• Field trials
In general terms, it is extremely difficult to assess vaccine efficacy in animal populations. In order to do
this, it would be necessary to vaccinate the animals in the absence of the pathogen that the vaccine
protects against, then to await the moment of infection and to compare the effects of infection in
vaccinated animals (or the offspring of vaccinated dams) with the effects in the unvaccinated animals of
the same age, in the same building and in the same batch as the vaccinated animals (or those protected
passively). As all these conditions are difficult to achieve in the field, field trials are certainly more
appropriate to safety testing than to efficacy testing.
2. Method of manufacture
Only MSV that has been established as pure, safe and immunogenic may be used as seed for a vaccine product.
Cells from the MCS are propagated in a variety of growth media. All batches of vaccine must be from the first to
the twentieth passage of MCS.

It is necessary to carry out tests at each critical step of the manufacturing process. The control tests are also
carried out on intermediate products with a view to verifying the consistency of the production process and the final
product.
4. Batch control
It is essential to differentiate the tests that are carried out on a routine basis to release batches of final product
from those that are performed to define the biological properties of a vaccine. The trials carried out for batch
release are not the same as the ones carried out once only to determine the safety and efficacy of a vaccine. The
batch release controls are always short-term trials, as inexpensive as possible, and not always carried out in pigs.
Their purpose is mainly to attest to the reproducibility of the quality of the finished product, which has to be in
compliance with the quality initially defined in the application for marketing authorisation.
a) Sterility and purity tests

Tests must be carried out for sterility and freedom from contamination (see Chapter 1.1.9).
Each batch of PRV vaccines must be tested for freedom from extraneous viruses. Using a minimum amount
of a monospecific antiserum, the live vaccinal strain is neutralised and inoculated into cell cultures known to
be sensitive to viruses pathogenic for pigs. No CPE and no haemadsorbing agents should be detected. The
vaccines have to be free from pestiviruses.
b) Inactivation
For inactivated vaccines, inactivation must be checked using two passages in the same type of cell culture as
used in the production of the vaccine. Tests can be carried out by vaccinating susceptible animals such as
rabbits.
c) Identity
Where necessary, a specific test for virus identification should be carried out.
d) Safety
Safety of live vaccines is tested by administering ten doses of the reconstituted vaccine by the route stated on
the leaflet to each of at least two piglets of the minimum age recommended for vaccination that are free from
PRV antibodies. Two piglets of the same origin and age are kept as controls. No abnormal local or systemic
reaction should occur. The weight curve of the vaccinated piglets must not differ significantly from that of the
controls.
For inactivated vaccines, safety is tested by injecting two doses into piglets under the same conditions as
described previously.
e) Potency
The potency of the vaccine must be demonstrated using a suitable method, the results of which have to be
correlated with the efficacy tests described previously.
In this kind of test, the most difficult point is to determine an acceptability threshold for using or rejecting the
batch according to the results that are obtained.
Virus content tests should be carried out using each of at least three containers. The virus titre of the vaccine
must be determined and must normally not be higher than 1/10 of the dose at which the vaccine has been
shown to be safe, and not lower than the minimum release titre.
f) Preservatives
If no preservative is included in the final product, the manufacturer must demonstrate that the product
remains acceptable for its recommended period of use after opening the vial.
The efficacy of preservatives in multidose containers must be demonstrated. The concentration of the
preservative in the final filled vaccine and its persistence throughout shelf life must be checked.

All information about possible adverse reactions induced by the vaccine must be indicated. Any putative risk
for human health if the user is accidentally given a small quantity of the product has to be indicated. The
manufacturer should indicate all the conditions of use of the vaccine: mixing, reconstitution, storage, asepsis,
length of needle, route of administration and health status of the vaccinated animals.
a) Safety
Every batch of vaccine must be tested for safety, as described in Section C.4.d.
b) Potency
Every batch of vaccine must be tested for potency, as described in Section C.4.e.
REFERENCES
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(Aujeszky’s disease) virus on pigs with maternal immunity from vaccinated sows. Am. J. Vet. Res., 39, 1282–
1285.
2. BANKS M. (1983). Rapid ELISA for Aujeszky’s disease eradication. Vet. Rec., 113, 94–95.
3. BANKS M. (1985). Detection of antibodies to Aujeszky’s disease virus in whole blood by ELISA-disc. J. Virol.
Methods, 12, 41–45.
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antibody. In: Current Topics in Veterinary Medicine and Animal Science 17. Aujeszky’s Disease, Wittmann G.
& Hall S.A., eds. Martinus Nijhoff, The Hague, The Netherlands, 41.
5. DE LEEUW P.W. & VAN OIRSCHOT J.T. (1985). Vaccines against Aujeszky’s disease: evaluation of their efficacy
under standardized laboratory conditions. Vet. Q., 7, 780–786.
6. ELOIT M., FARGEAUD D., VANNIER P. & TOMA B. (1989). Development of an ELISA to differentiate between
animals either vaccinated with or infected by Aujeszky’s disease virus. Vet. Rec., 124, 91–94.
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inactivatum (inactivated Aujeszky’s disease vaccine for pigs). Monograph 0745: Vaccinum morbi Aujeszky ad
suem vivum cryodesiccatum ad usum parenterale (Freeze-dried Aujeszky disease live vaccine for pigs for
parenteral administration). Council of Europe, Strasbourg, France.
8. KIT S. (1988). Safety and efficacy of genetically engineered Aujeszky’s disease vaccines. In: Vaccination and
Control of Aujeszky’s Disease, Van Oirschot J.T., ed. Kluwer Academic Publishers, The Netherlands, 45–55.
9. MARCHIOLI C.C., YANCEY R.J., W ARDLEY R.C., THOMSEN D.R. & POST L.E. (1987). A vaccine strain of
pseudorabies virus with deletions in the thymidine kinase and glycoprotein genes. Am. J. Vet. Res., 48,
1577–1583.
10. MENGELING W.L., LAGER K.M., VOLZ D.M. & BROCKMEIER S.L. (1992). Effect of various vaccination procedures
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Res., 53 (11), 2164–2173.
11. MCFERRAN J.B. & DOW C. (1975). Studies on immunisation of pigs with the Bartha strain of Aujeszky’s disease
virus. Res. Vet. Sci., 19, 17–22.
12. MOENNING V., W OLDESENBERT P., FEY H.R., LIESS B., DOPOTKA H.D. & BEHRENS F. (1982). Comparative
evaluation of ELISA and neutralization test for the diagnosis of Aujeszky’s disease. In: Current Topics in
Veterinary Medicine and Animal Science 17. Aujeszky’s Disease, Wittmann G. & Hall S.A., eds. Martinus
Nijhoff, The Hague, The Netherlands, 51.
13. PENSAERT M.B., DE SMET K. & DE W AELE K. (1990). Extent and duration of virulent virus excretion upon
challenge of pigs vaccinated with different glycoprotein-deleted Aujeszky’s disease vaccines. Vet. Microbiol.,
22, 107–117.

14. STELLMANN C., VANNIER P., CHAPPUIS G., BRUN A., DAUVERGNE M., FARGEAUD D., BUGAUD M. & COLSON X. (1989).
The potency testing of pseudorabies vaccines in pigs. A proposal for a quantitative criterion and a minimum
requirement. J. Biol. Stand., 17, 17–27.
15. TOMA B. (1979). Obtention et caracterisation d’une souche thermosensible de virus de la maladie d’Aujeszky
(souche ALFORT 26). Rec. Med. Vet., 155, 131–137.
16. TOMA B. (1982). Serological diagnosis of Aujeszky’s disease using enzyme-linked immunosorbent assay
(ELISA). In: Current Topics in Veterinary Medicine and Animal Science 17. Aujesky’s Disease, Wittmann G. &
Hall S.A., eds. Martinus Nijhoff, The Hague, The Netherlands, 65.
17. TOMA B., BRUN, A., CHAPPUIS G. & TERRE J. (1979). Propriétés biologiques d’une souche thermosensible
(ALFORT 26) de virus de la maladie d’Aujeszky. Rec. Med. Vet., 155, 245–252.
18. TOMA B. & ELOIT M. (1986). Pseudorabies virus antibodies (Aujeszky’s disease). In: Methods of Enzymatic
Analysis X, Antigens and Antibodies 1, Bergmeyer V.C.H., ed. D-6940 Weinheim, Germany.
19. TOMA B., ELOIT M. & TILMANT P. (1986). Sérodiagnostic de la maladie d’Aujeszky: utilisation de prélèvements
de sang sur papier filtre. Rec. Med. Vet., 162, 1111–1117.
20. VAN OIRSCHOT J.T. & GIELKENS A.L.J. (1984). Intranasal vaccination of pigs against pseudorabies: absence of
vaccinal virus latency and failure to prevent latency of virulent virus. Am. J. Vet. Res., 45, 2099–2103.
21. VAN OIRSCHOT J.T. & OEI H.L. (1989). Comparison of two ELISAs for detecting antibodies to glycoprotein I of
Aujeszky’s disease virus. Vet. Rec., 125, 63–64.
22. VAN OIRSCHOT J.T., RZIHA M.J., MOONEN, P.J.L.M., POL J.M. & VAN ZAANE D. (1986). Differentiation of serum
antibodies from pigs vaccinated or infected with Aujeszky’s disease virus by a competitive immunoassay. J.
Gen. Virol., 67, 1179–1182.
23. VAN OIRSCHOT J.T., TERPSTRA C., MOORMANN R.J.M., BERNS A.J.M. & GIELKENS A.L.J. (1990). Safety of an
Aujeszky’s disease vaccine based on deletion mutant strain 783 which does not express thymidine kinase
and glycoprotein I. Vet. Rec., 127, 443–446.
24. VANNIER P. (1986). Immunisation de porcs charcutiers contre la maladie d’Aujeszky avec deux vaccins à
adjuvants huileux. Etude des réactions locales. Rec. Med. Vet., 162, 37–44.
25. VANNIER P., HUTET E., BOURGUEIL E. & CARIOLET R. (1991). Level of virulent virus excreted by infected pigs
previously vaccinated with different glycoprotein deleted Aujeszky’s disease vaccines. Vet. Microbiol., 29,
213–223.
26. VANNIER P., TILLON J.P., TOMA B., DELAGNEAU J.F., LOQUERIE R. & PRUNET P. (1976). Protection conférée au porc
par un nouveau vaccin huileux à virus inactivé contre la maladie d’Aujeszky. Conséquences pratiques. J.
Rech. Porc Fr., 281–290.
27. VISSER N. & LUTTICKEN D. (1988). Experiences with a gI-/TK-modified live pseudorabies virus vaccine: strain
Begonia. In: Vaccination and Control of Aujeszky’s Disease, Van Oirschot J.T., ed. Kluwer Academic
Publishers, The Netherlands, 37–44.
28. W ITTMANN G. & LEITZKE I.I. (1985). Die Beeinflussung des Aujeszkyvirus-neutralizationstests durch
verschiedene Testbedingungen. Dtsch. Tierarztl. Wochenschr., 92, 262–266.
29. YOON H.-A., EO S.-K., ALEYAS A.G., CHA S.-Y., LEE J.-H., CHAE J.-S., JANG H.-K., CHO J.-G. & SONG H.-J. (2006).
Investigation of pseudorabies virus latency in nervous tissues of seropositive pigs exposed to field strain. J.
Vet Med. Sci., 68 (2), 143–148.
*
* *
NB: There are OIE Reference Laboratories for Aujeszky’s disease (see Table in Part 3 of this Terrestrial Manual or
consult the OIE Web site for the most up-to-date list: www.oie.int).

CHAPTER 2.1.3.
BLUETONGUE
SUMMARY
Bluetongue virus (BTV) infection involves domestic and wild ruminants such as sheep, goats,
cattle, buffaloes, deer, most species of African antelope and various other Artiodactyla as
vertebrate hosts. This noncontagious, insect-borne viral Infection is inapparent in the vast majority
of infected animals but causes fatal disease in a proportion of infected sheep, deer and wild
ruminants. Although cattle rarely show clinical signs, they are important in the epidemiology of the
disease due to the prolonged viraemia in the absence of clinical disease. Clinical signs range from
mild to severe and vary not only between species but between breeds and within the flock or herd.
BT disease is caused by fever and vascular permeability and includes hyperaemia and congestion,
facial oedema and haemorrhages, and erosion of the mucous membranes. However in mild cases
of the disease, a transitory hyperaemia and slight ocular and nasal discharge may be observed. In
very severe cases the tongue may show hyperaemia, become oedematous and protrude from the
mouth, or become cyanotic. Hyperaemia may extend to other parts of the body, particularly the
coronary band of the hoof, the groin, axilla and perineum. There is often skeletal and cardiac
muscle degeneration. Wool breaks may occur. Sheep may become lame as a result of laminitis
and skeletal myopathy. A similar severe disease of wild ruminants is caused by epizootic
haemorrhagic disease virus (EHDV), which, like BTV, is a member of the Orbivirus genus, but is
classified in a separate species.
Identification of the agent: BTV is a member of the Orbivirus genus of the family Reoviridae, one
of 20 recognised species in the genus. The BTV species, or serogroup, contains 24 recognised
serotypes. The orbiviral species are differentiated by immunological tests that detect viral proteins
that are conserved within each, and hence are distinguishable by serogrouping tests. However
there may be considerable cross-reaction between related species, and this is the case with the
BT and EHD serogroups. The serotype of individual viruses in each species is identified on the
basis of neutralisation tests and different strains within a serotype are identified by sequence
analysis. Complete BTV particles are double-shelled icosahedral double-stranded RNA virus. The
outer layer contains two proteins, one of which, VP2, is the major determinant of serotype
specificity. The inner shell and core contains two major and three minor proteins and ten double-
stranded RNA genetic segments. VP7 located in the inner shell is the major core protein
possessing the species or serogroup-specific antigens. Virus identification traditionally requires
isolation and amplification of the virus in embyonating hen eggs tissue culture or inoculations of
susceptible ruminants and the subsequent application of serogroup- and serotype-specific tests.
Reverse-transcription polymerase chain reaction (RT-PCR) technology has permitted rapid
amplification of BTV RNA in clinical samples, and RT-PCR-based procedures are now available.
These procedures can augment the classical virological techniques to provide information on virus
serogroup, serotype and topotype.
Serological tests: Serological responses in ruminants appear some 7–14 days after BTV infection
and are generally long-lasting. Historically, tests such as agar gel immunodiffusion and indirect
enzyme-linked immunosorbent assay (ELISA) were used to detect BTV species-specific
antibodies, but have the major drawback of being unable to consistently distinguish between
antibodies to viruses in the BTV and EHDV species. A monoclonal antibody-based competitive
ELISA has solved this problem and competitive ELISAs to specifically detect anti-BTV antibodies
are recommended. Procedures to determine the serotype-specificity of antibodies in sera are more
complex and time-consuming because they assess whether the sera inhibit the infectivity of panels
of known virus serotypes in neutralisation tests.

Chapter 2.1.3. — Bluetongue
Requirements for vaccines and diagnostic biologicals: Vaccination is used in several countries
to limit direct losses, minimise the circulation of BTV and allow safe movement of animals. For
many years South Africa has used live, attenuated vaccines that are serotype-specific. Live
attenuated vaccines are produced by adapting BTV field isolates to growth in vitro through serial
passages in tissue culture or in embryonating chicken eggs. Stimulation of a strong antibody
response by these vaccines is directly correlated with their ability to replicate in the vaccinated
host. Live attenuated vaccines are cheap to produce in large quantities, they generate protective
immunity after a single inoculation and have proven effective in preventing clinical BT disease.
Adverse consequences are depressed milk production in lactating sheep, and abortion/embryonic
death and teratogenesis in offspring from pregnant females that are vaccinated during the first third
of gestation. Another risk associated with the use of live attenuated vaccines is their potential for
spread by vectors, with eventual reversion to virulence and/or reassortment of vaccine virus genes
with those of wild-type virus strains. The frequency and significance of these events remain poorly
defined but natural and local dissemination of vaccine strains has already been documented in
Europe. The fact that attenuated viruses are teratogenic makes determination of transmissibility
very important. Vaccine efficacy, teratogenic potential and transmissibility should be tested. Hence
inactivated or recombinant vaccines would be preferred if effective. The inactivated vaccines are
not teratogenic and have been used under government supervision since 2004 in the management
of recent European outbreaks.
A. INTRODUCTION
Midges of certain species in the genus Culicoides (the insect host) (39) transmit bluetongue virus (BTV) among
susceptible ruminants, having become infected by feeding on viraemic animals (the vertebrate host). After a
replication period of 6–8 days in the insect’s salivary gland the virus can be transmitted to a new vertebrate host
during feeding. Infected midges remain infective for life. The central role of the insect in BT epidemiology ensures
that distribution and prevalence of the infection is governed by ecological factors, such as high rainfall,
temperature, humidity and soil characteristics, which favour insect survival (6). In many parts of the world
therefore, infection has a seasonal occurrence (43). It is accepted that BTV does not establish persistent
infections in ruminants, and that survival of BTV in the environment is associated with insect factors (20, 23).
Globally the distribution of BTV can be considered on the basis of epidemiological systems (episystems) based
on the vector species present and their natural history (40).
The vertebrate hosts for BTV include both domestic and wild ruminants, such as sheep, goats, cattle, buffaloes,
deer, most species of African antelope and other Artiodactyla such as camels Although antibodies to BTV, and in
some cases virus antigen, have been demonstrated in some carnivores, felids, black and white rhinoceroses and
elephants, the roll of non-ruminant species in the disease in the wild is not known. The outcome of infection
ranges from inapparent in the vast majority of infected animals, especially cattle, to fatal in a proportion of
infected sheep, goats, deer and some wild ruminants (43). However clinical disease has been observed in cattle
infected with BVT8 in Europe. Some breeds of sheep are more susceptible to disease than others, with the result
that in some countries BTV infections of livestock can occur unobserved, and be detected only by active
surveillance (9). Epizootic haemorrhagic disease virus (EHDV) can produce a disease in wild ruminants with
clinical manifestations identical to those observed in response to BTV infection.
Clinical signs of disease in sheep vary markedly in severity, influenced by husbandry factors as well as by breed
(43). In severe cases there is an acute febrile response characterised by hyperaemia and congestion, leading to
oedema of the face, eyelids and ears, and haemorrhages and erosions of the mucous membranes. The tongue
may show intense hyperaemia and become oedematous, protrude from the mouth and, in severe cases become
cyanotic. Hyperaemia may extend to other parts of the body particularly the coronary band of the hoof, the groin,
axilla and perineum. There is often severe muscle degeneration. Breaks in the wool may occur associated with
pathology in the follicles. A reluctance to move is common, or even torticolis in severe cases. In fatal cases the
lungs may show interalveolar hyperaemia, severe alveolar oedema and the bronchial tree may be filled with froth.
The thoracic cavity and pericardial sac may contain several litres of plasma-like fluid. Most cases show a
distinctive haemorrhage near the base of the pulmonary artery (43).
Taxonomically, BTV is classified as a species or serogroup in the Orbivirus genus in the family Reoviridae, one of
20 recognised species in the genus that also includes EHD and African horse sickness (AHS) (31). Within
species, individual members are differentiated on the basis of neutralisation tests, and 24 serotypes of BTV have
been described. There is significant immunological cross-reactivity between members of the BT and EHD viruses
(31).
BTV particles are composed of three protein layers. The outer layer contains two proteins, VP2 and VP5. VP2 is
the major neutralising antigen and determinant of serotype specificity. Removal of the outer VP2/VP5 layer

leaves a bi-layered icosahedral core particle that is composed of two major proteins, VP7 and VP3, three minor
proteins and the ten species of double-stranded RNA. VP7 is a major determinant of serogroup specificity and
the site of epitopes used in competitive enzyme-linked immunosorbent assay (C-ELISA) to detect anti-BTV
antibody (30). VP7 can also mediate attachment of BTV to insect cells (46).
Genetic sequencing of BTVs allows differentiation and analysis of strains separately from serotype (16, 27, 36,
45). Even for strains within the one serotype it is possible to identify the likely geographical origin (16, 35). Such
studies have led to the detection of international movements of BTV strains. Natural movements of vectors by
climatic forces are believed to result in intercontinental movement of BTV. Identification of apparent associations
between some genotypes of virus and some vector species has resulted in a concept of viral-vector ecosystems
(9, 23, 40). A more complete understanding of such epidemiological aspects may further facilitate international
trade in ruminants.
1. Identification of the agent (a prescribed test for international trade)
a) Virus isolation
The same diagnostic procedures are used for domestic and wild ruminants. A number of virus isolation
systems are in common use, but generally the most practical method is by inoculation of embryonated
chicken eggs (ECE). Inoculation of sheep may also be a useful approach if the titre of virus in the sample
blood is very low, as may be the case several weeks after virus infection. Attempts to isolate virus in
cultured cells in vitro may be more convenient, but the success rate is frequently much lower than that
achieved with in vivo systems (15). Cell culture has proven to be a more sensitive technique for isolation of
EHDV.
• Isolation in embryonated chicken eggs

i) Blood is collected from febrile animals into an anticoagulant such as EDTA (ethylamine diamine tetra-
acetic acid), heparin or sodium citrate, and the blood cells are washed three times with sterile
phosphate buffered saline (PBS). Washed cells are resuspended in PBS or isotonic sodium chloride
and either stored at 4°C or used immediately for attempted virus isolation.
ii) For long-term storage where refrigeration is not possible blood samples are collected in oxalate–
phenol–glycerin. If samples can be frozen, they should be collected in buffered lactose peptone or
10% dimethyl sulphoxide (41) and stored at –70°C or colder. The virus is not stable for long periods at
–20°C.
iii) In fatal cases, spleen and lymph nodes are the preferred organs for virus isolation attempts. Organs
and tissues should be kept and transported at 4°C to a laboratory where they are homogenised in PBS
or isotonic saline, and used as described below, for blood cells.
iv) Washed blood cells are re-suspended in distilled water or sonicated in PBS and 0.1 ml amounts
inoculated intravascularly into 5–12 ECE that are 9–12 days old. This procedure requires practice.
Details are provided by Clavijo et al. (7).
v) The eggs are incubated in a humid chamber at 32–33°C and candled daily. Any embryo deaths within
the first 24 hours post-inoculation are regarded as nonspecific.
vi) Embryos that die between days 2 and 7 are retained at 4°C and embryos remaining alive at 7 days are
killed. Infected embryos may have a haemorrhagic appearance. Dead embryos and those that live to
7 days are homogenised as two separate pools. Whole embryos, after removal of their heads, or
pooled organs such as the liver, heart, spleen, lungs and kidney are homogenised and the debris is
removed by centrifugation.
vii) Virus in the supernatant may be identified either directly by antigen-capture ELISA (17) or reverse
transcription polymerase chain reaction (RT-PCR), or indirectly by antigen-detection methods such as
immunofluorescence or immunoperoxidase after further amplification in cell culture, as described in
the next section.
viii) If no embryos are killed following inoculation of sample material, an inoculum made from the first egg
passage material may be repassaged in ECE or in cell culture.
• Isolation in cell culture

Virus isolation may be attempted in bluetongue virus susceptible cell cultures such as mouse L, baby
hamster kidney (BHK-21), African green monkey kidney (Vero) or Aedes albopictus clone C6/36 (AA). The

efficiency of isolation is often significantly lower following inoculation of cultured cells with diagnostic
samples compared with that achieved in ECE. Highest recovery rates are achieved by primary isolation of
virus in ECE, followed by passage in AA cells for further replication of virus. Additional passages in
mammalian cell lines such as BHK-21 or Vero are usually performed. A cytopathic effect (CPE) is not
necessarily observed in AA cells but appears in mammalian cells. Cell monolayers are monitored for the
appearance of a CPE for 5 days at 37°C in 5% CO2 with humidity. If no CPE appears, a second passage is
made in cell culture. The identity of BTV in the culture medium of cells manifesting a CPE may be confirmed
by a number of immunological methods described below, including antigen-capture ELISA, immuno-
fluorescence, immunoperoxidase, virus neutralisation (VN) tests, or by RT-PCR.
• Isolation in sheep
i) Sheep are inoculated with washed cells from 10 ml to 500 ml of blood, or 10–50 ml tissue suspension.
Inocula are administered subcutaneously in 10–20 ml aliquots. Large volumes may aid in the virus
isolation attempts and should be administered intravenously.
ii) The sheep are held for 28 days and checked daily for pyrexia and weekly for antibody response using
serological tests such as the C-ELISA as described below. Sheep blood collected at 7 to 14 days post-
inoculation will usually contain the isolated virus, which can be stored viable at 4°C or –70°C.
b) Immunological methods
• Serogrouping of viruses
Orbivirus isolates are typically serogrouped on the basis of their reactivity with specific standard antisera
that detect proteins, such as VP7, that are conserved within each serogroup. The cross-reactivity between
members of the BT and EHD serogroups raises the possibility that an isolate of EHDV could be mistaken
for BTV on the basis of a weak immunofluorescence reaction with a polyclonal anti-BTV antiserum. For this
reason, a BT serogroup-specific MAb can be used. A number of laboratories have generated such
serogroup-specific reagents (2, 21). Commonly used methods for the identification of viruses to serogroup
level are as follows.
i) Immunofluorescence
Monolayers of BHK or Vero cells on chamber slides (glass cover-slips) are infected with either tissue
culture-adapted virus or virus in ECE lysates. After 24–48 hours at 37°C, or after the appearance of a
mild CPE, infected cells are fixed with agents such as paraformaldehyde, acetone or methanol, dried
and viral antigen detected using anti-BTV antiserum or BTV-specific MAbs and standard
immunofluorescent procedures.
ii) Antigen capture enzyme-linked immunosorbent assay
Viral antigen in ECE and culture medium harvests (17), infected insects (28) and sheep blood may be
detected directly. In this technique, virus derived proteins are captured by antibody adsorbed to an
ELISA plate and bound materials detected using a second antibody. The capture antibody may be
polyclonal or a serogroup-specific MAb. Serogroup-specific MAbs and polyconal antibody raised to
baculovirus-expressed core particles have been used successfully to detect captured virus (17).
iii) Immunospot test
Small volumes (2 µl) of infected cell culture supernatant or lysed or sonicated infected cells are
adsorbed to nitrocellulose and air-dried. Nonspecific binding sites are blocked by incubation in a
solution containing skim milk protein. After incubation with a BTV serogroup-reactive MAb, bound
antibody is detected using horseradish peroxidase-conjugated anti-mouse IgG (14).
• Serotyping by virus neutralisation

Neutralisation tests are type specific for the currently recognised 24 BTV serotypes and can be used to
serotype a virus isolate or can be modified to determine the specificity of antibody in sera. In the case of an
untyped isolate, the characteristic regional localisation of BTV serotypes can generally obviate the need to
attempt neutralisation by all 24 antisera, particularly when endemic serotypes have been identified.
There is a variety of tissue culture-based methods available to detect the presence of neutralising anti-BTV
antibody. Cell lines commonly used are BHK, Vero and L929. Four methods to serotype BTV are outlined
briefly below. BTV serotype-specific antisera generated in guinea-pigs or rabbits have been reported to
have less serotype cross-reactivity than those made in cattle or sheep. It is important that antiserum
controls be included.
i) Plaque reduction
The virus to be serotyped is diluted to contain approximately 100 plaque-forming units (PFU), and
incubated with either no antiserum or with dilutions of individual standard antisera to a panel of BTV

serotypes. Virus/antiserum mixtures are added to monolayers of cells. After adsorption and removal of
inoculum, monolayers are overlaid with agarose. The neutralising antibody titres are determined as the
reciprocal of the serum dilution that causes a fixed reduction (e.g. 90%) in the number of PFU. The
unidentified virus is considered serologically identical to a standard serotype if the latter is run in
parallel with the untyped virus in the test, and is similarly neutralised.
ii) Plaque inhibition
Tests are performed in 90 mm diameter Petri dishes containing confluent cell monolayers that are
infected with approximately 5 × 104 PFU standard or untyped virus. After adsorption and removal of
inoculum, monolayers are overlaid with agarose. Standard anti-BTV antisera are added to individual
filter paper discs and placed on the agarose surface. Dishes are incubated for at least 4 days. A zone
of virus neutralisation, with concomitant survival of the cell monolayer, will surround the disc containing
the homologous antiserum.
iii) Microtitre neutralisation
Approximately 100 TCID50 (50% tissue culture infective dose) of the standard or untyped virus is
added in 50 µl volumes to test wells of a flat-bottomed microtitre plate and mixed with an equal volume
of standard antiserum diluted in tissue culture medium. Approximately 104 cells are added per well in a
volume of 100 µl, and after incubation for 4–6 days, the test is read using an inverted microscope.
Wells are scored for the degree of CPE observed. Those wells that contain cells only or cells and
antiserum, should show no CPE. In contrast, wells containing cells and virus should show convincing
CPE. The unidentified virus is considered to be serologically identical to a standard BTV serotype if
both are neutralised in the test to a similar extent.
iv) Fluorescence inhibition test
This rapid and simple neutralisation assay (5) requires varying concentrations of an unknown virus
and standard concentrations of reference antisera. Virus isolates grown in cell culture are serially
diluted starting and mixed with individual reference antisera in the wells of a Lab-Tek slide for 1 hour
prior to addition of cells. After incubation for 16 hours, cells are fixed and probed by an
immunofluorescent procedure using a BT serogroup-specific MAb. The serotype of the virus is
indicated by the specificity of the antiserum causing the largest reduction in the number of fluorescent
cells.
c) Reverse-transcription polymerase chain reaction (a prescribed test for international trade)

Primer-directed amplification of viral nucleic acid has revolutionised BT diagnosis (8, 27, 44). RT-PCR
techniques have allowed the rapid identification of BT viral nucleic acid in blood and other tissues of
infected animals. RT-PCR-based diagnostics should be interpreted with caution. The RT-PCR procedure
will detect virus-specific nucleic acid, but this does not necessarily indicate the presence of infectious virus
(24). RT-PCR can also be used to ‘serogroup’ Orbiviruses and may ultimately be possible to ‘serotype’ BTV
within a few days of receipt of a clinical sample, such as infected sheep blood (29). Traditional approaches,
which rely on virus isolation followed by virus identification serologically, may require up to 4 weeks to
generate information on serogroup and serotype.
Oligonucleotide primers used so far have been derived from RNA 7 (VP7 gene) (44), RNA 6 (NS1 gene)
(8), RNA 3 (VP3 gene) (36), RNA 10 (NS3 gene) (4) and RNA 2 (VP2 gene) (27). The size of the amplified
transcripts is usually small – in the order of several hundred nucleotides – but can also be a full-length
gene. In the procedure described in detail below, a 101-nucleotide stretch of RNA 6 is amplified. Primers
derived from the more highly conserved genes, such as VP3, VP7 and NS1, may be used for serogrouping
(i.e. will react with all members of the BT serogroup), while primers for which the sequence was determined
from VP2 gene sequences provide information on virus serotype. A multiplex RT-PCR assay that depends
on the size of the amplified products has been used to identify the five North American BTV serotypes, both
alone and in mixtures, in a single reaction (18).
The nucleic acid sequence of cognate BTV genes may differ with the geographical area of virus isolation
(16). This has provided a unique opportunity to complement studies of BTV epidemiology by providing
information on the potential geographical origin of virus isolates, a process termed genotyping or
topotyping. Thus, determination of the nucleic acid sequence of portions of RNA may provide information on
where the virus came from. It appears likely that sequencing of BTV isolates from other parts of the world
may permit finer discrimination of geographical origin. However, the relationship between sequence and
geographical origin may not be straightforward. This sequencing information is important and all data
regarding BTV segment sequences should be made widely available by submitting the data to officially
recognised web sites.
http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ and
http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/btv_sequences.htm.
http://eubtnet.izs.it/btnet/index.htm

The web sites provide phylo-genetic tree analyses of BTV isolates based on the sequence of RNA
segments. These compiled data will provide a resource for epidemiological studies, the identification of new
isolates and the design of new primers for further RT-PCR and possibly serotype-specific assays for BTV.
It has been observed that BTV nucleic acid can be detected by RT-PCR from the blood of infected calves
and sheep at least 30 days, and sometimes over 90 days, after the virus can be isolated The presence of
virus-specific nucleic acid does not necessarily indicate the presence of infectious virus.
The capacity of RT-PCR assays to detect very small numbers of nucleic acid molecules means that such
tests are exquisitely sensitive to contamination by extraneous nucleic acids. The latter may include any
primers in use in the laboratory or previously amplified polynucleotides. It is critical therefore to have a
‘clean’ area containing all equipment necessary for reagent and test preparation and a separate area with
its own equipment for amplification. Impervious gloves should be worn and changed frequently at all stages
of the procedure, particularly after working with sample RNA or amplified DNA. This will help protect
reagents and samples from contamination by ubiquitous RNases and other agents and from cross-
contamination by DNA. The possibility of false positives, due to sample contamination, highlights the
importance of sequencing RT-PCR products to determine, for example, if the amplified sequence is
identical to or different from that of the positive control. False negatives, due for example to poor sample
quality or inappropriate primers, may be identified following the failure to amplify both BTV and a host gene,
such as globin, from extracts of infected cells. This is covered in more detail in Chapter 1.1.5 Validation and
quality control of polymerase chain reaction methods used for the diagnosis of infectious diseases.
There are many RT-PCR assays currently in use that use different extraction methods, reverse
transcriptases, amplification enzymes, primers and conditions. Technology is changing rapidly. Also the
genetic diversity of the BTV genes makes the choice and validation of RT-PCR assays conditional on its
application in a regional setting. This is also the case for real-time PCR, which will be developed for use in
routine molecular diagnosis of BTV in the future. Therefore the procedures listed below are examples only.
Increasingly it will be more important to maintain diagnostic testing under accreditation to an international
standard such as ISO/ISEC 17025 and participate in proficiency testing. Systems for offering proficiency
testing for RT-PCR tests are being developed in a number of countries.
The RT-PCR assay described here involves three separate procedures. In the first, BTV RNA is extracted
from blood using a chaotropic agent such as guanidine thiocyanate (GuSCN) to denature protein and
release viral RNA. A number of commercial kits are available for this purpose and the protocol below
describes the use of one such kit, IsoQuick (Orca Research, Bothell, Washington, United States of America
[USA]). The reagents provided with the kit are numbered and their use is indicated in the protocol below.
Other kits are available and one, TRIZOL (Life Technologies, Grand Island, New York, USA), is particularly
useful for the extraction of viral nucleic acid from spleen or blood clots. Operators should follow the
procedures specified in each kit and use reagent solutions either provided or recommended for the kit of
their choice. The second procedure is the denaturation of viral double-stranded RNA and reverse
transcription to generate cDNA, which is amplified by RT-PCR. In the procedure described below, the
SuperscriptTM Preamplification System (Life Technologies) is used to transcribe viral RNA, and reagents
from Perkin-Elmer are used for the RT-PCR. Equivalent kits and reagents are available from other sources.
The final step of the process is the analysis of the RT-PCR product by electrophoresis. Procedures used to
determine the sequence of the amplified product are not described here.
• Extraction of viral RNA

i) Whole blood is collected from test and uninfected control animals in EDTA tubes and centrifuged at
800–1000 g for 10 minutes. The plasma is aspirated and the red blood cells (RBCs) are gently
resuspended in sterile PBS. RBCs are pelleted by centrifugation at 1000 g for 10 minutes and the
supernatant is removed.
ii) Next, 400 µl of test RBCs is added to each of four 1.7 ml microcentrifuge tubes, and 400 µl of control
RBCs is added to each of two microcentrifuge tubes. An equal volume of RNase-free water is added to
each tube and the tubes are vortexed briefly to mix and lyse the cells. Two tubes containing test RBCs
are frozen at –70°C for repository purposes and the extraction is continued in duplicate.
iii) Lysed test and control RBCs are centrifuged at 12,000–16,000 g for 10 minutes and the supernatant is
discarded. Next, 800 µl RNase-free water is added and the tubes are vortexed and centrifuged again
at the same speed for 10 minutes. The supernatant is removed and the RBC pellet is drained.
iv) A small volume of BTV (e.g. 5 µl containing from 103 to 107 PFU) is added to one of two control RBC
pellets. This is the positive control. The other control RBC pellet remains as the negative control.
v) Next, 75 µl of sample buffer (IsoQuick reagent A) is added to each pellet, and the pellets are then
vortexed vigorously, followed by the addition of 125 µl of the GuSCN-containing lysis solution
(IsoQuick reagent 1). The mixture is vortexed vigorously for 30 seconds.

vi) Before use the extraction matrix provided with the kit (IsoQuick reagent 2 plus dye 2A) is shaken
vigorously and 500 µl is added to the sample lysates. Then, 400 µl extraction buffer (IsoQuick reagent
3) is added and the tubes are vortexed for 10 seconds.
vii) The tubes are incubated at 65°C for 10 minutes, vortexed briefly after 5 minutes and centrifuged at
12,000 g for 5 minutes.
viii) The aqueous phase (500 µl) is transferred to a new microcentrifuge tube and an equal volume of
extraction matrix (IsoQuick reagent 2) is added. The tubes are vortexed for 10 seconds and
centrifuged at 12,000 g for 5 minutes.
ix) The aqueous phase (330 µl) is transferred to a new microcentrifuge tube and a 10% volume (33 µl) of
sodium acetate (IsoQuick reagent 4) and 365 µl isopropanol are added. After gentle mixing, the tubes
are placed at –20°C for from 20 minutes to 1 hour.
x) The RNA is pelleted by centrifugation at 12,000 g for 10 minutes. The supernatant is decanted and
1.0 ml 70% ethanol is added and mixed gently. After centrifugation at 12,000 g for 5 minutes, the
supernatant is decanted and 1.0 ml 100% ethanol is added. The tubes are stored at –70°C until ready
for use in the RT-PCR.
• Reverse-transcription polymerase chain reaction

i) RNA in ethanol is centrifuged at 12,000 g for 5 minutes. The ethanol is decanted and the tubes are
inverted and allowed to drain. The pellet, which may not be obvious, must not be allowed to dry out
because this makes resuspension difficult. A dry pellet is also likely to fall out of the inverted tube.
ii) Next, 12 µl RNase-free water is added to each tube, mixed and heated at 65°C for 5–10 minutes. The
samples are placed in ice.
iii) In a ‘clean’ biohazard hood, stock solutions containing 200 pmol/µl of primers A, B, C and D are
prepared in RNase-free water and stored at –70°C.
First stage RT-PCR primers (to amplify RNA 6 from nucleotide 11 to 284)
Primer A: 5’-GTT-CTC-TAG-TTG-GCA-ACC-ACC-3’
Primer B: 5’-AAG-CCA-GAC-TGT-TTC-CCG-AT-3’
Nested RT-PCR primers (to amplify RNA 6 from nucleotide 170 to 270)
Primer C: 5’-GCA-GCA-TTT-TGA-GAG-AGC-GA-3’
Primer D: 5’-CCC-GAT-CAT-ACA-TTG-CTT-CCT-3’
iv) Primer stock solutions are diluted to a concentration of 15–20 pmol/µl. Primers for the first stage RT-
PCR reaction are prepared by mixing equal volumes of A and B. Primers for the nested RT-PCR
reaction are prepared by mixing equal volumes of C and D. Small aliquots of pooled primer mixes are
frozen at –20°C.
v) RT-PCR reaction tubes are labelled and, for first stage synthesis, 4.0 µl of primer (A + B) mix is added
to each tube. The tubes are held on ice.
vi) In a ‘clean’ fume hood methylmercuric hydroxide is diluted to 50 mM (1/20 dilution) and 2-
mercaptoethanol is diluted to 350 mM (1/40 dilution) in RNase-free water. Methylmercuric hydroxide
and 2-mercaptoethanol are considered to be extremely and highly toxic, respectively. Use both
chemicals with extreme care and dispose of them and pipette tips as required by safety regulations.
Alternative methods using heat denaturation have been described (22, 29).
vii) Next, 4 µl of test and positive and negative control RNA samples (step ii) are added to 4 µl of the
primer mix in RT-PCR tubes (45).
viii) To each RT-PCR tube 2.0 µl of the 1/20 dilution of methylmercuric hydroxide is added with gentle
mixing and allowed to sit at room temperature for 10 minutes prior to adding 2.0 µl of the 1/40 dilution
of 2-mercaptoethanol. For safety reasons, some laboratories use formamide instead of
methylmercuric hydroxide for double-stranded RNA denaturation. However, for optimum sensitivity,
methylmercuric hydroxide is preferred.
ix) In a ‘clean’ hood a cDNA mix is prepared containing the following reagents in sufficient volume for the
number of samples being tested. The amount given is per sample and the reagents are contained in
the SuperscriptTM Preamplification System (Life Technologies).
10 × SuperscriptTM buffer (200 mM Tris/HCl, pH 8.4, and 500 mM KCl) 2.0 µl
MgCl2 (25 mM) 2.0 µl
dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP) 1.25 µl
Dithiothreitol (DTT) (0.1 M) 2.0 µl
Reverse transcriptase (200 units/µl) 0.75 µl
x) Then, 8.0 µl of the mix is added to each RT-PCR tube to a final volume of 20.0 µl.

xi) The RT-PCR tubes are placed in a thermal cycler, such as GeneAmpTM PCR System 9600, which is
programmed for reverse transcription as follows:
Hold 44°C 50 minutes
Hold 4°C Forever
xii) The tubes are removed from the thermal cycler and 1.0 µl RNase H and a wax bead are added to
each tube. The cycler is programmed as follows:
Hold 4°C Forever
xiii) In a ‘clean’ hood a first stage amplification mix is prepared containing the following reagents and in a
volume sufficient for the number of samples being tested. All these reagents except water are
available from Perkin-Elmer. The amount given is per sample.
RNase-free water 62.0 µl
10 × PCR Perkin-Elmer buffer (100 mM Tris/HCl, pH 8.3, and 500 mM KCl) 7.0 µl
MgCl2 (25 mM) 7.0 µl
dNTP mix (2.5 mM each dATP, dCTP, dGTP, dTTP) 4.0 µl
Taq DNA polmerase (5 units/µl) 0.85 µl
xiv) The first stage mix is removed from the ‘clean’ area to the thermal cycling area and 80 µl is overlaid in
each sample tube. The wax layer must not be pierced. Each tube should now contain 101 µl.
xv) The tubes are placed in the thermal cycler, which is programmed as follows (correct for GeneAmp
PCR System 9600 – programmes for other thermal cyclers would need to be determined) for first
stage amplification:
One cycle: Hold 95°C 3 minutes
Hold 58°C 20 seconds
40 cycles: Hold 95°C 20 seconds
One cycle: Hold 95°C 20 seconds
Hold 4°C Forever
xvi) RT-PCR reaction tubes are prepared for the nested reaction in a ‘clean’ hood 15 minutes before
cycling is complete, and held on ice:
Rnase-free water 17 µl per tube
Nested primer mix (C+D) 4.0 µl per tube
Wax bead
xvii) When first stage amplification is complete, the tubes are removed from the thermal cycler and placed
in a biological safety cabinet (not the ‘clean’ hood). Then, 1.5 µl of the first stage product is transferred
to the corresponding nested RT-PCR tube containing primer, water and a wax bead.
xviii) The tubes are placed in the thermal cycler, which is programmed as follows for wax layer formation:
Hold 4°C Forever
xix) In a ‘clean’ hood the nested mix of the following reagents is prepared in sufficient volume for the
number of samples being tested. The reagents used are the same as in the first stage (step xii). The
amount given is per sample.
RNase-free water 17.0 µl
10 × PCR buffer 5.0 µl
MgCl2 3.5 µl
dNTP mix 4.5 µl
Taq DNA polymerase 0.5 µl
xx) The nested mix is removed from the ‘clean’ hood to the thermal cycler and 30 µl is overlaid into each
sample tube. Each tube should now contain 52 µl.
xxi) The tubes are placed in the thermal cycler, which is programmed as follows for nested amplification.
After completion, the tubes are held at 4°C or at –20°C until electrophoresis:

One cycle: Hold 95°C 3 minutes

40 cycles: Hold 95°C 20 seconds
One cycle: Hold 95°C 20 seconds
Hold 4°C Forever
• Electrophoretic analysis of RT-PCR product

i) First, 1 × TBE buffer (0.045 mM Tris/borate, pH 8.6, and 1.5 mM EDTA) is prepared from a ×10 stock
solution. For the Bio-Rad Wide Mini-Sub cell system, 700 ml buffer is prepared (100 ml for the gel and
600 ml for the tank buffer).
ii) A 3% solution of NuSieve 3/1 agarose (FMC Bioproducts, Rockland, Maine, USA) or an equivalent is
prepared in TBE buffer. The solution is boiled until the agarose is completely dissolved, and then
allowed to cool to 40°C. Ethidium bromide is added to a concentration of 0.5 µg/ml to both the agarose
and the tank buffer. Ethidium bromide is a mutagen and is toxic. Gloves, protective clothing, and eye-
wear must always be worn.
iii) The ends of the electrophoresis tray are taped and the agarose solution is poured. The comb is
inserted and the agarose is allowed to solidify on a level surface for 30–60 minutes. The comb and the
tape are gently removed from the electrophoresis tray.
iv) Pour the tank buffer into the electrophoresis apparatus and insert the tray with the agarose so that the
buffer covers the agarose.
v) Test and positive and negative control samples are prepared for electrophoresis in 0.65 ml
microcentrifuge tubes as follows:
Gel-loading solution (Cat. G-2526, Sigma, St Louis, Missouri, USA) 5.0 µl
Amplified DNA from each of the RT-PCR tubes and an extra tube
is set up for a DNA ladder 15.0 µl
Gel-loading solution (Cat. G-2526, Sigma, St Louis, Missouri, USA) 5.0 µl
100 base-pair ladder (Cat. 15268-019, Life Technologies,
Grand Island, New York, USA) 1.0 µl
vi) Samples are loaded into the appropriate wells in the gel and run at 65–75 volts for 1–1.5 hours or until
the dye has travelled about half the length of the gel. The gel is transferred to a transilluminator and
photographed for a permanent record. Use protective eye-wear to visualise the gel bands.
vii) BT-positive samples will have a band of 101 base pairs. For the test to be valid, the positive control
must show a band of the correct size, and the negative and ‘no RNA’ controls show no band. Samples
are considered to be positive if there is a band of the same size as the positive control. Duplicate
samples should show the same reaction. If there is disparity, the test should be repeated.
viii) A destaining bag (Ameresco, Solon, Ohio, USA) is placed in the tank buffer overnight to remove the
ethidium bromide. The buffer can then be poured down the drain and the destaining bag, after reuse
10–15 times, should be placed in a properly identified ethidium bromide waste container and ultimately
incinerated.
Anti-BTV antibodies generated in infected animals can be detected in a variety of ways that vary in sensitivity and
specificity. Both serogroup-specific and serotype-specific antibodies are elicited and if the animal was not
previously exposed to BTV, the neutralising antibodies generated are specific for the serotype of the infecting
virus. Multiple infections with different BTV serotypes lead to the production of antibodies capable of neutralising
serotypes to which the animal has not been exposed.
a) Complement fixation
A complement fixation test to detect BTV antibodies was widely used until 1982, when it was largely
replaced by the AGID test although the CF test is still used in some countries.
b) Agar gel immunodiffusion (a prescribed test for international trade)

The AGID test to detect anti-BTV antibodies is simple to perform and the antigen used in the assay
relatively easy to generate. Since 1982, the test has been the standard testing procedure for international

movement of ruminants. However, one of the disadvantages of the AGID used for BT is its lack of specificity
in that it can detect antibodies to other Orbiviruses, particularly those in the EHD serogroup. Thus AGID
positive sera may have to be retested using a BT serogroup-specific assay. The lack of specificity and the
subjectivity exercised in reading the results have encouraged the development of ELISA-based procedures
for the specific detection of anti-BTV antibodies. The preferred format, a C-ELISA is described in the
Section B.2.c.
• Test procedure
i) A 2.8 mm thick layer of 0.9% agarose in 0.85% NaCl is prepared and circular wells, 4.0 mm in
diameter and 2.4 mm apart, are cut out with six wells arranged around a central well.
ii) Viral antigen is prepared by generating a crude soluble preparation from BHK or Vero cells infected
with a single BTV serotype 24–48 hours previously. Antigen can be concentrated by precipitation or
ultrafiltration.
iii) A reference positive serum and three test sera are placed in alternate wells in a six-well pattern
surrounding antigen in a central well and the plates are incubated at 20–25°C in a humid environment
for 24 hours.
iv) A series of precipitin lines form between the antigen and known positive sera and lines generated by
strong positive test sera will join up with those of the positive controls. With weak positive samples the
control lines bend toward the antigen and away from the test sample well, but may not form a
continuous line between the control test wells. With negative samples, the precipitin lines will continue
into the sample wells without bending toward the antigen.
v) All weak positive samples and other samples that produce questionable results should be repeated
using wells that are 5.3 mm in diameter placed 2.4 mm apart or retested using the C-ELISA as
described below.
c) Competitive enzyme-linked immunosorbent assay (a prescribed test for international trade)

The BT competitive or blocking ELISA was developed to measure BTV-specific antibody without detecting
cross-reacting antibody to other Orbiviruses (1, 4, 21, 32, 37). The specificity is the result of using one of a
number of BT serogroup-reactive MAbs, such as MAb 3-17-A3 (2) or MAb 20E9 (21) or MAb 6C5F4D7 (26).
The antibodies were derived in a number of laboratories, and although different, all appear to bind to the
amino-terminal region of the major core protein VP7. In the C-ELISA, antibodies in test sera compete with
the MAbs for binding to antigen. The following procedure for the C-ELISA has been standardised after
comparative studies in a number of international laboratories.
• Test procedure
There are several test procedures described; this is an example of one BT ELISA procedure.
i) First, 96-well microtitre plates are coated at 4°C overnight or at 37°C for 1 hour with 50–100 µl of
either tissue culture-derived sonicated cell culture antigen (2) or the major core antigen VP7
expressed in either baculovirus (33) or yeast (26) and diluted in 0.05 M carbonate buffer, pH 9.6.
ii) The plates are washed five times with PBST (0.01 M PBS containing 0.05% or 0.1% Tween 20,
pH 7.2).
iii) Next, 50 µl of test sera is added in duplicate at a single dilution, either 1/5 (1) or 1/10 (21) in PBST
containing 3% bovine serum albumin (BSA).
iv) Immediately, 50 µl of a predetermined dilution of MAb diluted in PBST containing 3% BSA is added to
each well. MAb control wells contain diluent buffer in place of test serum.
v) Plates are incubated for 1 hour at 37°C or 3 hours at 25°C, with continuous shaking.
vi) After washing as described above, wells are filled with 100 µl of an appropriate dilution of horseradish
peroxidase-labelled rabbit anti-mouse IgG (H+L) in PBST containing 2% normal bovine serum.
vii) Following incubation for 1 hour at 37°C, the conjugate solution is discarded and plates are washed five
times using PBS or PBST. Wells are filled with 100 µl substrate solution containing 1.0 mM ABTS
(2,2’-azino-bis-[3-ethylbenzothiazoline-6-sulphonic acid]), 4 mM H2O2 in 50 mM sodium citrate, pH 4.0,
and the plates are shaken at 25°C for 30 minutes. (Other substrates may be used and the reaction
continued with shaking for an appropriate length of time to permit colour development.)
viii) The reaction is stopped by addition of a stopping reagent, such as sodium azide.
ix) After blanking the ELISA reader on wells containing substrate and stopping reagent, the absorbance
values are measured at 414 nm. Results are expressed as per cent inhibition and are derived from the
mean absorbance values for each sample by the following formula.

% inhibition = 100 – [(Mean absorbance test sample)/(Mean absorbance MAb control) × 100].
NB: Some laboratories prefer to use a negative control serum that has previously been shown to have
a percentage inhibition of zero as an alternative to the MAb control.
x) Percentage inhibition values >50% are considered to be positive. Inhibition between 40% and 50% is
considered to be suspicious. The results of the test sera duplicates can vary as long as the results do
not lie either side of the positive cut -off.
xi) Strong and weak positive sera and a negative serum should be included on each plate. The weak
positive should give 60–80% inhibition and the negative should give less than 40% inhibition.

Both live attenuated and killed BTV vaccines are currently available for use. Recombinant vaccines based on
various approaches are under development but none has been licensed and these vaccines will not be
addressed here. In South Africa live attenuated vaccines have been used for over 40 years and are known to
induce an effective and lasting immunity (11). Live attenuated vaccines are produced by adapting BTV field
isolates to growth in vitro through serial passages in tissue culture or in embryonated chicken eggs. Stimulation
of a strong antibody response by these vaccines is directly correlated to their ability to replicate in the vaccinated
host. Live attenuated vaccines are cheap to produce in large quantities; they generate protective immunity after a
single inoculation and have proven effective in preventing clinical BT disease in the areas where they are used
(10). However, live attenuated BTV vaccines suffer from a variety of documented or potential drawbacks,
including under-attenuation, the impact of which may vary with different breeds of sheep. Potential adverse
consequences are depressed milk production in lactating sheep, and abortion/embryonic death and
teratogenesis in offspring when used on pregnant females in the first period of pregnancy. Another risk
associated with the use of live attenuated vaccines is that of their potential for spread by vectors, with eventual
reversion to virulence and/or reassortment of modified live virus (MLV) genes with those of wild-type virus strains.
The frequency and significance of these events remain poorly defined but natural and local dissemination of
vaccine strains has already been documented in the USA and Europe (25, 42). Therefore inactivated vaccines, if
effective, are preferred. Virus inactivation eliminates risks of vector transmission, reversion to virulence, fetal
abnormalities and the possibility of viral reassortment.
1. Seed management
a) Characteristics of seed
For live, attenuated vaccines the master or primary virus seed is prepared from a single plaque of serially
passaged, attenuated BTV. Vaccine viruses have been attenuated by either passage in ECE, tissue culture
cells or a combination of both. Each primary seed virus lot should also be tested for transmissibility and
reversion to virulence prior to vaccine manufacture. Samples of vaccine prepared from secondary seed
virus at the maximum permitted passage level should be tested in sheep for avirulence, safety and
immunogenicity.
For killed vaccines the issues of attenuation do not apply, and the approach adopted has been to use field
strains of low passage level with the intent of achieving high antigenicity.
Primary seed virus must be free of contaminating bacteria, viruses, prions, fungi and mycoplasmas,
particularly pestivirus contamination. For the latter, particular attention should be paid to the fetal bovine
serum used in cell cultures, as it may be contaminated. Seed viruses must be shown to have the desired
serotype specificity.
BTV seed lot viruses should be sequenced and the data made available to relevant databases (34).
Secondary seed lots, which are used as inocula for vaccine production, are usually not more than three
passages beyond the primary seed lot.
Although the first attenuated BT vaccines were propagated in ECE subsequently cell cultures have been
used for tissue culture adaptation and serial passage. These include primary bovine embryo, lamb and fetal
lamb kidney cells, and the continuous BHK cells. Cell cultures must be thoroughly checked for the presence
of contaminating viruses.
BTV for inactivated vaccines is produced in large-scale suspension cell systems that have been shown to
be susceptible to bluetongue virus.

Attenuated BT vaccines must be safe and efficacious, and a brief description of appropriate tests for these
parameters is given below. In addition, attenuated viruses should not revert to virulence during replication in
vaccinated animals or be transmitted from such animals by insect vectors. The latter criterion is very
important because insect-mediated transmission of attenuated virus from vaccinated to nonimmune
animals, with the subsequent replicative steps in each host species, increases the possibility of reversion to
virulence. Although tests for reversion to virulence and transmissibility are rarely, if ever performed, a brief
description of what may be necessary is outlined.
There is a variation in bluetongue susceptibility between breeds of sheep; it is important that sheep that that
have been proven to be susceptible to infection with BTV be used for vaccine validation.
i) Safety
All vaccines must be safety tested. Safety tests for attenuated vaccines do not address the issue of
their teratogenicity. Attenuated virus vaccines are teratogenic and should not be administered to
pregnant sheep during the first half of pregnancy as this may cause fetal abnormalities and embryonal
death (12, 20).
Demonstration of avirulence is necessary for live, attenuated vaccines. A number of sheep,
seronegative by an appropriate, sensitive serological test (that will reliably detect antibodies even in
vaccinated animals), are inoculated with the primary seed stock. Temperatures are noted twice daily.
The animals are monitored at regular intervals over a period of 28 days for clinical signs and any local
or systemic reactions to ensure avirulence and innocuity. Blood samples removed at regular intervals
can be used to measure level of viraemia and antibody responses. The test shall be valid if all of the
vaccinated sheep show evidence of virus replication and do not display signs of disease other than
mild transient illness. In South Africa, a clinical reaction index is calculated for each animal between
days 4 and 14 and must be below a specific standard value.
ii) Efficacy
Vaccinated and unvaccinated sheep should be challenged with virulent homologous serotype. It is
recommended that the challenge model use wild type virus preferably passaged only in ruminant
animals and with no ECE or cell culture passages. Passage in such an isolation system results in viral
cultures that might induce clinical bluetongue disease that is milder than the natural disease (12).
Animals are monitored for clinical signs of BT, rectal temperatures are taken twice daily and blood
samples removed at regular intervals to measure viraemia and antibody responses. Unvaccinated
control sheep should show clinical signs of BT and viraemia. Unvaccinated control sheep should show
clinical signs of BT. However, it is difficult to be certain of the appearance of clinical disease following
inoculation of sheep with certain BTV serotypes and isolates, and consequently, evidence of infection
of unvaccinated control sheep may rest on the appearance of a temperature rise of at least 1.7°C over
the pre-challenge mean and a viraemia. As a further evidence of infection pre- and post-vaccination
sera are checked for the presence of neutralising antibody.
iii) Transmissibility
Transmissibility is an issue with live attenuated vaccines but not with killed vaccines. Procedures to
determine if attenuated virus can be transmitted by insects that feed on vaccinated, viraemic sheep
are difficult to perform and analyse statistically, and consequently, this criterion of vaccine validation is
rarely sought. Laboratory data indicate that laboratory-adapted viruses can be transmitted by insect
vectors (13, 31, 39). A suitable procedure to determine attenuated virus transmissibility requires that
sheep be vaccinated and, during viraemia, that they be exposed to competent, uninfected Culicoides,
which are then permitted to feed on uninfected animals that are monitored for the presence of BTV
and anti-BTV antibody. Due to the fact that the titre of attenuated virus in the blood of vaccinated
sheep is low, very large numbers of Culicoides would be needed and only a small proportion of these
would become infected and live long enough to feed on and potentially transmit the virus to other
uninfected sheep. It is difficult to design a laboratory experiment that takes account of the large
numbers of vaccinated sheep and insects that would be present in field situations. Current data
indicate that during viraemia and in contrast to wild-type virus, laboratory-adapted strains of BTV may
be found in the semen of bulls and rams (19, 38). A recent study of semen from rams vaccinated with
BTV2 live attenuated vaccine showed that even if BTV was not detected in the semen, the vaccine
caused a decrease in the quality of the semen (3).
A suitable procedure to determine attenuated virus transmissibility requires that sheep be vaccinated
and, during viraemia, that they be exposed to competent, uninfected Culicoides, which are then
permitted to feed on uninfected animals that are monitored for the presence of BTV and anti-BTV
antibody. Due to the fact that the titre of attenuated virus in the blood of vaccinated sheep is low, very
large numbers of Culicoides would be needed and only a small proportion of these would become
infected and live long enough to feed on and potentially transmit the virus to other uninfected sheep. It
is difficult to design a laboratory experiment that takes account of the large numbers of vaccinated

sheep and insects that would be present in field situations. Although virus titres in blood less than
103TCID50/ml have traditionally been considered a “safe” threshold, authentic instances of insects
acquiring BTV from animals with viremic titres less than 103TCID50/ml have been reported. Given the
complex interaction of BTV, Culicoides vectors and animal hosts in the life cycle of infection, virus
titres induced by live attenuated vaccine should be kept to an absolute minimum specially if field
transmission of vaccine strains is a concern.
Current data indicate that during viraemia and in contrast to wild-type virus, laboratory-adapted strains
of BTV may be found in the semen of bulls and rams (19, 38). The implications of these observations
for virus transmissibility are unclear.
iv) Reversion to virulence
Validation studies confirm that attenuated viruses do not revert to virulence in vaccinated sheep.
However, if attenuated viruses can be transmitted from vaccinated animals, reversion to virulence
during a number of sheep-insect replication cycles becomes a distinct prospect. The only appropriate
way to monitor for reversion to virulence under these circumstances is to compare the virulence of the
vaccine virus with that which had been subject to several sheep–insect replication cycles as described
above. As indicated, this is difficult to achieve. Consequently, the effect of a number of sheep–insect
passages on the virulence of attenuated viruses has not been determined. In South Africa, it is
accepted that if blood from vaccinated animals during the viraemic stages is serially passaged three
times in sheep without reversion to virulence, the chances of reversion in the field will be infinitely
small.
Attenuation of field isolates of BTV was first achieved by serial passage in ECE. Because of the concern about
transmission of the egg propagated attenuated virus, it has been recommended that animals receiving vaccines
produced in ECE should not be moved internationally (34). More recently, it is clear that passage in cultured cells
will also result in attenuation of virulence. No studies have been done to precisely relate passage number and
extent of attenuation for individual virus isolates or serotypes. To prepare attenuated virus, field isolates are
adapted to cell culture and passaged in vitro up to 40 times or more. Ideally, a number of plaque-purified viruses
are picked at this stage and each is examined to determine the level of viraemia they generate and their ability to
elicit a protective immune response in vaccinated sheep. The most suitable virus is one that replicates to low titre
but generates a protective immune response, and this may represent the source of vaccine primary seed stock
virus.
BTV for killed vaccines is produced in large-scale suspension cell systems under aseptic and controlled
conditions. Cell lines adapted for large scale industrial cultures are used, and these are proven to be free from
contaminating microorganisms. When the viral suspension virus reaches its maximum titre, cell disruption is
performed and the culture is clarified and filtered. Subsequently inactivation is performed according to processes
adopted by the manufacturer, such as by addition of binary ethyleneimine (BEI) or other inactivants. The process
must comply with legislation relevant for the intended market, be validated to ensure complete inactivation and
be supported by the appropriate documentation. The inactivation process should not significantly alter the
immunogenic properties of the viral antigens. Purification is carried out by chromatography. The inactivated virus
is then concentrated by ultrafiltration and stored. The inactivated, chromatography-purified and concentrated BTV
antigens are made into vaccine by dilution in a buffer solution and addition of adjuvants.
All ingredients of animal origin, including serum and cells must be checked for the presence of viable bacteria,
viruses, fungi or mycoplasmas.
Virus concentration of attenuated vaccines is assessed by infectivity and ELISAs.
For inactivated vaccines, during inactivation of the virus, timed samples are taken at regular intervals for the
purpose of monitoring the rate and linearity of the inactivation process. Virus titres in the samples are determined
by inoculation of BHK-21 or other appropriate cell cultures. At the end of the inactivation process, the vaccine is
checked to ensure that there is no live virus.
4. Batch control
a) Sterility
Every batch of vaccine should be tested for the presence contaminant viruses of viable bacterial, fungal or
mycoplasmal contamination. For example, in South Africa a pool of ten randomly selected ampoules are

inoculated into soya broth and thioglycollate broth, and incubated at room temperature and 37°C,
respectively, for 14 days. If contaminated, the batch is disqualified.
b) Safety
Every batch of attenuated vaccine is safety tested in newborn and adult mice, guinea-pigs and sheep. If any
adverse reactions or significant signs are noted, the test is repeated. Any increase in the body temperature
of the target animal that is above the level expected for the particular strain of attenuated virus under test
should be regarded as symptomatic. If the results are unsatisfactory after a second attempt, the batch is
disqualified.
Safety testing of inactivated vaccines is conducted in sheep to ensure side effects are not observed.
c) Potency
Each batch is tested by inoculation of susceptible sheep. Prevaccination, and 21- and 28-day post-
vaccination sera are tested by VN assay to determine neutralising antibody levels. To be passed, the
antibody titre must be equal to or higher than a set standard based on international vaccine standards.
Studies with live attenuated BTV vaccine have shown that antibodies in sheep may appear before day 10
post-vaccination, reach a maximum approximately 4 weeks later and persist for well over a year. There is a
temporal relationship between the increase in neutralising antibody titre and clearance of virus from the
peripheral circulation. Live attenuated BTV vaccines have been in use for over 40 years and are known to
induce an effective and lasting immunity (43). Many serotypes of BTV may be present in endemic areas of
South Africa, and polyvalent vaccines are used. The inclusion of 15 serotypes in the vaccine means that an
effective immune response is not generated to all serotypes, presumably because of the antigenic mass of
individual serotype-specific antigens is small. In an attempt to broaden the response, vaccination is
repeated annually (11).
Initial studies with inactivated vaccines show that antibody against BTV can be detected by day 7 post-
vaccination and increase in titre to days 14–21. A second dose of vaccine boosts the titre. Data to
demonstrate the expected duration of immunity is under development.
e) Stability
Procedures have been developed for attenuated vaccines. Stability should be tested over a period of
2 years. Vaccines in liquid and lyophilised forms are deemed to have shelf lives of 1 and 2 years,
respectively. Each batch of vaccine is subjected to an accelerated shelf-life test by storing it at 37°C for
7 days. It is then titrated and evaluated according to a set standard, as determined in the initial testing of
the vaccine.
Inactivated vaccines have been used to the present time in emergency situations where shelf life has not
been an issue. Requirements and procedures for routine commercial use have not been developed.
f) Precautions (hazards)
Attenuated vaccines should be used in the cooler months when the Culicoides population and its typical
activity are at the lowest level. They should not be used in ewes during the first half of pregnancy and in
rams 2 months before the breeding season.
a) Safety
See C.4.b.
b) Potency
See C.4.c.
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risk. In: Bluetongue, African Horsesickness and Related Orbiviruses, Walton T.E. & Osburn B.I., eds. CRC
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*
* *
NB: There are OIE Reference Laboratories for Bluetongue (see Table in Part 3 of this Terrestrial Manual or

CHAPTER 2.1.4.
ECHINOCOCCOSIS/HYDATIDOSIS
SUMMARY
Diagnosis of echinococcosis in dogs or other susceptible carnivores relies on the demonstration of

adult cestodes of the Echinococcus genus or their eggs in their faeces or small intestine. Recently,
coproantigen and copro-DNA assays have proven useful for safe, fast and accurate diagnosis. In
intermediate hosts, diagnosis depends on detection of the larval cyst form that can infect almost
any organ, particularly the liver and lungs.
Identification of the agent: At present, five species of the genus Echinococcus are regarded as
taxonomically valid. These are E. granulosus, E. multilocularis, E. oligarthrus, E. vogeli and
E. shiquicus. Echinococcus oligarthrus and E. vogeli occur less frequently than the first two
species. Until recently E. shiquicus had been discovered only in a specific region of the People’s
Republic of China. These five species are morphologically distinct in both adult and larval stages.
A number of intraspecific variants have been described for E. granulosus, which exhibit
morphological and biological characteristics, and these can reliably be differentiated by DNA
analysis. Some of the E. granulosa genotypes have been recommended for elevation to species
status.
Larval forms of Echinococcus can usually be visually detected in organs. Special care has to be
taken for a specific diagnosis of E. granulosus in instances where Taenia hydatigena in sheep is
also a problem. Histological examination may confirm the diagnosis after formalin-fixed material is
processed by conventional staining methods. The presence of a periodic-acid-Schiff positive,
acellular laminated layer with or without an internal cellular, nucleated germinal membrane can be
regarded as a specific characteristic of metacestodes of Echinococcus. The identification of larval
E. multilocularis in rodents and other hosts is possible by macroscopic or microscopic examination
and by DNA detection using the polymerase chain reaction (PCR).
The small intestine is required at necropsy for the detection of adult Echinococcus spp. in wild or in
domestic carnivores. The technique of carrying out surveys with the use of arecoline has been
generally adopted for determining the prevalence of E. granulosus in dogs. Handling infected
material presents a risk to the operator of contracting a potentially fatal disease. Significant
progress is being made in the development of immunological tests for the diagnosis of intestinal
Echinococcus infections by use of coproantigen detection. The technique has been used
successfully in surveys of E. granulosus in dogs and is currently used in surveys for
E. multilocularis in populations of dogs, foxes, and cats. Coproantigen detection is possible in
faecal samples collected from dead or living animals or from the environment.
PCR/DNA methods for the detection of E. multilocularis and more recently E. granulosus in
definitive hosts have now been established in specialised laboratories as diagnostic techniques.
Serological tests: Antibodies directed against oncosphere, cyst fluid and protoscolex antigens
can be detected in the serum of infected dogs and sheep, but this approach is presently of limited
practical use as it does not distinguish between current and previous infections. Cross-reactivity
between Echinococcus and Taenia species also may occur.
Requirements for vaccines and diagnostic biologicals: Progress has been made in the
development of an effective vaccine against infection with the larval stage of E. granulosus in
sheep and cattle.

Chapter 2.1.4. — Echinococcosis/Hydatidosis
A. INTRODUCTION
Species under genus Echinococcus are small tapeworms of carnivores with larval (metacestode) stages known
as hydatids proliferating asexually in various mammals including humans. There were four morphologically
distinct species in this genus until recently when Echinococcus shiquicus was added to the previously known
species: E. granulosus, E. multilocularis, E. oligarthus, and E. vogeli. Discovered in the Shiqu County, the
Qinghai-tibet plateau region of western Sichuan, the People’s Republic of China (57, 58), E. shiquicus is
morphologically distinct both in adult and larval stages from other species.
A number of interspecific and intraspecific variants have been described for E. granulosus. Some genotypes of
E. granulosus exhibit characteristic features that would justify the recognition as separate species according to
some authors. Recently other species and genotypes of Echinoccocus have been proposed (51). Further studies
are needed to define the full range of genetic diversity (32, 37, 43, 50). Echinococcus granulosus has a global
distribution; E. multilocularis occurs in wide areas of the Northern Hemisphere, E. shiquicus is found in the
People’s Republic of China and E. oligarthrus and E. vogeli are confined to Central and South America. All five
species are infective to humans causing various forms of echinococcosis. Human cystic echinococcosis, caused
by E. granulosus and alveolar echinococcosis, caused by E. multilocularis, are important public health threats in
many parts of the world (56).
Table 1. Useful characteristics for identification of Echinococcus species. Source: Xiao et al. (58)
E. granulosus E. multilocularis E. oligarthus E. vogeli E. shiquicus
Distribution Cosmopolitan Holoarctic region Neotropical region Neotropical region Tibet plateau
Definitive Host Dogs Foxes Wild felids Bush dog Tibetan fox
Intermediate Host Ungulates Microtine rodents Neotropical Neotropical Plateau pika

rodents rodents
Adult:
Body length (mm) 2.0–11.0 1.2–4.5 2.2–2.9 3.9–5.5 1.3–1.7
No. segments 2–7 2–6 3 3 2–3
Length of large 25.0–49.0 24.9–34.0 43.0–60.0 49.0–57.0 20.0–23.0

hooks (µm)
Length of small 17.0–31.0 20.4–31.0 28.0–45.0 30.0–47.0 16.0–17.0

hooks (µm)
No. testes 25–80 16–35 15–46 50–67 12–20
Position of genital
pore
Near to middle Anterior to middle Anterior to middle Posterior to Near to upper edge
a. Mature segment middle
Posterior to Anterior to middle Near to middle Anterior to middle
b. Gravid segment middle Posterior to
middle
Gravid uterus Branching Sac-like Sac-like Tubular Sac-like

laterally
Metacestode Unilocular cysts Multilocular cysts Polycystic cysts in Polycystic cysts in Unilocular cysts in
in viscera in viscera muscles viscera viscera
• Echinococcus granulosus
The parasite is transmitted between the domestic dog and a number of domestic ungulate species. The
dog/sheep cycle is most important. Sylvatic definitive and intermediate hosts also occur, e.g. wolf/cervid. The
adult varies between 2 and 11 mm in length and usually possesses from two to seven segments, averaging from

three to four segments. The penultimate segment is mature, and the genital pore normally opens posterior to the
middle in both mature and gravid segments. The last (gravid) segment is usually more than half the length of the
entire worm. There are rostellar hooks of various sizes on the protoscolex in two rows. The size of the hooks
varies between 25 to 49 µm in the first row, and between 17 and 31 µm in the second row. The gravid uterus has
well-developed sacculations.
The larval stage is a fluid-filled bladder or hydatid cyst that is unilocular, although communicating chambers also
occur. Growth is expansive, and endogenous daughter cysts may be produced. Individual bladders may reach up
to 30 cm in diameter and occur most frequently in liver and lungs, but may develop in other internal organs. The
infection with this stage is referred to as cystic echinococcosis.
The strain specificities of E. granulosus in domestic cycles include, dog/sheep in the Mediterranean region,
South America (Argentina, Brazil, Chile, Peru and Uruguay), Africa (Ethiopia, Kenya and Sudan), the Middle East
and Levant regions, Russia, Central Asia (Kazakhstan, Kyrgyzstan and Uzbekistan), Mongolia, the People’s
Republic of China, Oceania and the United Kingdom; dog/horse in Belgium, Ireland and the United Kingdom;
dog/cattle in Belgium, Germany, South Africa and Switzerland; dog/swine in Poland; and dog or wolf/reindeer in
sub-Arctic regions of Norway, Finland and Alaska. The status of dog/camel strains requires further elucidation.
This strain has recently been identified in human cases in Argentina, Nepal, the People’s Republic of China and
Iran (3, 19, 44, 59). To date, all genotypes of E. granulosus except the dog/horse (G4) and the Finnish cervid
(G10) strains have been found to infect humans.
• Echinococcus multilocularis
The parasite is transmitted primarily between wild definitive hosts (e.g. Vulpes vulpes, V. ferrilata, V. corsac,
Alopex lagopus, Canis latrans) and small arvicolid rodents (voles and lemmings). The adult varies between
1.2 and 4.5 mm in length and usually possesses from two to six segments, with an average of four to five. The
penultimate segment is characteristically mature, and the genital pore is anterior to the midline in both mature
and gravid segments. The gravid uterus is sac-like. On the rostellum, the larger hooks of the first row vary in size
between 24.9 and 34.0 µm and the smaller hooks of the inner row between 20.4 and 31.0 µm.
The metacestode is a multivesicular structure consisting of conglomerates of small vesicles, usually not
exceeding a few millimetres in diameter. Unlike E. granulosus, the larval mass often contains a semisolid rather
than a fluid matrix. It proliferates by exogenous budding, which results in infiltration of tissues. Infection with this
stage is commonly referred to as alveolar echinococcosis. There is no clear evidence for distinct strains or
genotypes of E. multilocularis, though regional variations at the continental scale have been described (56).
This zoonotic parasite is found in the Northern Hemisphere, and its life cycle is mainly maintained in wildlife (25).
The sylvatic cycle involves foxes and many species of wild rodents. Coyotes, raccoon dogs, wolves, wild cats,
domestic dogs and cats (20, 27), however, may serve as definitive hosts while pigs, horses, primates and
humans can be infected as intermediate hosts (25).
• Echinococcus oligarthrus
The parasite typically uses neptropical wild felids as definitive hosts (e.g. Felis concolor, F. jaguarundi) and large
rodents (e.g. Dasyprocta sp., Cuniculus paca) as intermediate hosts. The adult varies between 2.2 and 2.9 mm
in length, and normally possesses three segments, the penultimate of which is mature. The genital pore is
anterior to the middle in mature segments and approximately at the middle in gravid segments. The gravid uterus
is sac-like.
The metacestode is polycystic and fluid-filled with a tendency to become septate and multichambered. The
rostellar hooks of the protoscolex vary in length between 25.9 and 37.9 µm. The hooks are described in more
detail in the next section and compared with those of E. vogeli. The single cyst may reach a diameter of
approximately 5 cm. Predilection sites are internal organs and muscles. To date, there have only been a few
reports of human disease. The parasite appears not to mature in dogs.
• Echinococcus vogeli
The parasite typically uses the South American bush dog (Speothus venaticus) as a wild definitive host, but the
domestic dog is susceptible, as are large rodents (e.g. Cuniculus paca) as intermediate hosts. The adult varies
between 3.9 and 5.5 mm in length, and usually has three segments, the penultimate of which is mature. The
genital pore is situated posterior to the middle in both the mature and gravid segments. The gravid uterus has no
lateral sacculations and is characterised by being relatively long and tubular in form, compared with the other
segments, which are sac-like.
The metacestode is similar to that of E. oligarthrus. It has been reported that the two species can be
distinguished by comparing differences in the dimensions and proportions of the rostellar hooks on the
protoscolex. The hooks of E. oligarthrus vary in length between 25.9 and 37.9 µm (average 33.4 µm) and

between 22.6 and 29.5 µm (average 25.45 µm) for large and small hooks, respectively. Those of E. vogeli vary
between 19.1 and 43.9 µm (average 41.64 µm) and between 30.4 and 36.5 µm (average 33.6 µm) for the large
and small hooks, respectively. Also the hook-guard for E. oligarthrus divides the hook 50:50, compared with
30:70 for E. vogeli.
Echinococcus vogeli is a zoonotic agent with approximately 100 human cases reported in South America. The
infection caused by the larval stage of this species is commonly referred to as polycystic echinococcosis.
• Echinococcus shiquicus
The parasite was found in the Tibetan fox (Vulpes ferrilata) its definitive host and the plateau pika (Ochotona
curzoniae), the intermediate host. In most species of Echinococcus, the gravid segment is connected to a mature
segment; however, a strobila consisting of only two segments (a gravid segment directly attaching to a premature
segment) is unique to this species (56). The adult stage is morphologically similar to E. multilocularis but differs
by its smaller hooks, fewer segments, upper position of genital pore in the premature segment and fewer eggs in
the gravid segment. It is easily distinguishable from E. granulosus by its shorter length, branchless gravid uterus
and anterior position of genital pore in the gravid segment. The adult measures 1.3 to 1.7 mm.
The metacestode is found in the liver and is essentially a unilocular minicyst containing fully developed brood
capsules; however, oligovesicular forms have also been observed. It is differentiated from E. granulosus having
no daughter cysts appearing within the fertile cyst (56).
A detailed description of echinococcosis in humans and animals can be found in the WHO/OIE Manual on
echinococcosis (56).
In the intermediate host, diagnosis depends on the detection of the larval cyst form, which can occur in almost
any organ, but particularly in the liver and lungs. The diagnosis of echinococcosis in dogs or other carnivores
requires the demonstration of the adult cestodes of Echinococcus spp. in their faeces or the small intestine or the
detection of specific coproantigens or coproDNA.
Investigators carrying out these procedures are exposed to the risk of infection and severe disease, which must
be minimised by appropriate procedures. Infective material can be decontaminated by freezing at –80°C (core
temperature) for 48 hours, or –70°C for 4 days or by heating to 70°C for 12 hours (41, 45). Face masks,
disposable gloves and an apron must be worn. Chemical disinfection is not reliable, although sodium
hypochlorite may destroy a proportion of eggs (8). Contaminated material must be destroyed by heat; hot water,
at temperature of 85°C or above, is very effective. The decontamination of laboratories can be achieved at
reduced humidity (40%) combined with increased room temperature (30°C) for at least 48 hours.
a) Diagnosis of Echinococcus eggs in environmental samples

• Faecal samples (22, 49)
This is a concentration method in which a saturated solution is used to separate Echinococcus eggs from
faeces. A faecal sample of 0.5–2 g is mixed with water or 0.3% Tween 20 in 1% formalin (42) in a 10–15-ml
test tube and centrifuged (1000 g for 10 minutes) once or twice until the supernatant is clear. Sediment is
mixed with either zinc sulphate 33% (1.18 sp. Gr.) or sucrose solution (1.27 sp. Gr.) and centrifuged at
1000 g for 5–10 minutes. The test tube is filled to the top and a cover-glass is placed on the tube. The
cover-glass is examined microscopically 2–16 hours later.
• Soil samples (35)

A 20-g soil sample is placed in a 50-ml conical tube to which is added 40 ml of 0.05% Tween 80. The
mixture is stirred vigorously and sieved through a 100-µm mesh. The suspension is centrifuged at 1000 g
for 5 minutes and the supernatant is discarded. The remaining procedure follows the concentration method
used for faecal sample examination.
b) Diagnosis of larval echinococcosis

• Necropsy
Whereas surveillance for E. granulosus in domestic animals may take place in licensed slaughter houses,
that for Echinococcus sp. in wildlife must be done by field surveys. Specimens should be preserved by

removal of tissue and fixation in 4% formal saline or kept cool at +4°C and deep-frozen at –20°C for
subsequent examination. When undertaking surveillance work with E. granulosus in intermediate hosts, it is
vitally important that data are stratified and reported according to the age of animals slaughtered.
Prevalence rates are strongly age dependent (53) and reports from abattoirs that may slaughter only young
animals will substantially under-represent the true situation. This is because older animals may be heavily
infected even when animals have very few larvae.
Larvae can be observed in many organs, but in large animals, such as sheep and cattle, palpation or
incision should be done. Pigs, cattle, sheep and goats may be infected with larval Taenia hydatigena, and it
is sometimes difficult to differentiate between these two parasites when they occur in the liver. In wild
animals, such as ruminants and rodents, several other larval cestodes should be considered for differential
diagnosis.
Formalin-fixed material can be stained by conventional histological techniques. The presence of a periodic-
acid-Schiff (PAS) positive acellular laminated layer, underlying a connective tissue layer, and with or without
an internal cellular, nucleated germinal membrane can be regarded as a specific characteristic of the
metacestodes of Echinococcus spp. The presence of protoscoleces within brood-capsules or in hydatid
sand is also diagnostic for the genus. Genotyping of E. granulosus or E. multilocularis is usually done on
DNA derived from protoscoleces or larval tissue material that is frozen, refrigerated or preserved in 90%
ethanol.
c) Diagnosis of adult parasites in carnivores

• Necropsy
Necropsy is invariably employed in studies of echinococcosis in wildlife and is useful if domestic dogs are
humanely culled. It should be emphasised that it is necessary to isolate and identify the adult
Echinococcus, because under normal conditions of faecal examination, the eggs of Echinococcus cannot
be differentiated from those of Taenia spp. The eggs of E. granulosus and E. multilocularis can now be
identified and differentiated from other taeniid eggs by polymerase chain reaction (PCR).
The small intestine is removed as soon as possible after death, and tied at both ends. If the material is not
frozen or formalin fixed (4–10%), it should be examined quickly, as the parasite can be digested within
24 hours. Formalin does not kill eggs. The fresh intestine is divided into several sections and immersed in
0.9% saline at 37°C for examination. Worms adhering to the intestinal wall may be observed and counted
by means of a hand lens (for E. granulosus and E. vogeli). For accurate counts, the unfixed intestine is best
divided into four or six sections, opened up and immersed in 0.9% saline at 37°C for 30 minutes to release
the parasites. The contents are washed into another container for detailed examination, and the intestinal
wall is scraped with a spatula. All material is boiled and washed by sieving to eliminate most of the
particulate material and to make it noninfectious. The washed intestinal contents and scrapings are placed
on a black tray, and the worms are counted with the aid of a hand lens or stereoscopic microscope.
Echinococcus granulosus is usually found in the first third of the small intestine of dogs and E. multilocularis
in the mid/posterior sections.
Necropsy is considered to be the most reliable form of diagnosis for E. multilocularis in definitive hosts. It is
an inexpensive method for determining the prevalence in a population and the best way to determine worm
burden (15). Carcasses or intestines of definitive hosts for examination should be deep frozen at between
–70°C and –80°C for 3–7 days before necropsy to kill any eggs. Eggs of E. multilocularis are resistant to
freezing to –50°C.
Methods for quantitative determination of E. multilocularis in definitive host’s intestine.
• Sedimentation and counting technique (SCT; 16, 21)

This technique can be regarded as the ‘gold standard’ for assessing the sensitivity and specificity of other
techniques.
i) The small intestine is incised longitudinally and cut into 20 cm long segments or into 5 pieces of
approximately the same length. These pieces are transferred to a glass bottle containing 1 litre
physiological saline (0.9% NaCl) solution.
ii) The glass bottle is shaken vigorously for a few seconds and the pieces of intestine are removed. The
superficial mucosal layer is stripped by exerting pressure between thumb and forefinger to dislodge
attached helminths.
iii) The glass bottle is left for 15 minutes for sedimentation to occur; the supernatant is then decanted.
The glass bottle is refilled with physiological saline solution. This procedure is repeated 2–6 times until
the supernatant is cleared of coloured particles.

iv) The sediment fraction is examined in small portions of about 5–10 ml in rectangular plastic or Petri
dishes with a counting grid (9 × 9 cm) in transmission light under a stereomicroscope at a
magnification of ×120.
v) If up to 100 worms are found, the entire sediment fraction is checked; if higher numbers are present,
the total worm burden is calculated from the count of one subsample.
• Intestinal scraping technique (IST; 12, 17)

i) Deep mucosal scrapings are taken at nearly equal distances from the small intestine using
microscope slides (75 × 25 × 1 mm). Five mucosal scrapings from proximal, middle and posterior
thirds of the small intestine (total 15) are recommended. Adherent materials are transferred to a
square plastic Petri dish.
ii) Scrapings are squashed between slides and examined under a stereoscopic light microscope (×120).
Three slides are placed in one plastic dish and examined. Echinococcus multilocularis is usually found
in the second half of the small intestine.
• ‘Shaking in a vessel’ technique (SVT; 15)

i) A plastic vessel (1 litre), which has a plastic screw-on lid with a central hole 6–7 cm in diameter is
used. The hole is covered with a high-grade steel mesh (mesh size 500 µm) fixed into the remaining
plastic ring with a hot soldering iron. Silicone is applied to seal the edges of the steel mesh.
ii) The longitudinally opened small intestine is transferred to the vessel with all its contents; the vessel is
closed with the lid and filed with water.
iii) The vessel is inverted and shaken; the water is decanted. Vessel is refilled with water, and the
process is repeated until the decanted water is clear.
iv) The half-filled vessel is opened and the intestines are removed. The intestines are stripped between
the thumb and forefinger to dislodge parasites stuck to the mucosa into the vessel.
v) The vessel is closed again, refilled and shaken one last time draining as much water from it as
possible.
vi) The remaining sediment is filled into a 1 litre plastic jug and stored at 4°C. For prolonged storage, a
0.9% NaCl solution is added to the sediment to prevent the parasites from shrinking.
vii) For analysis, the materials are placed into small glass Petri dishes and scanned along engraved lines
using the stereomicroscope as above.
• Preserving specimens
Intact worms are fragile and for morphological studies are best handled in normal saline with a Pasteur
pipette. They are washed free of other material and left for approximately 30 minutes for all movement to
cease. After removal of the fluid, cold 5–10% formalin (5°C) or FAA fixative (95% ethanol [80 ml], 37–
40% formaldehyde [10 ml], and glacial acetic acid [5 ml]) is added and the worms are left for a further
12 hours. For staining, the worms are washed in water for 15 minutes and transferred to Mayer’s
paracarmine (carminic acid [1.0 g], aluminium chloride [0.5 g], calcium chloride [4.0 g], and 70% ethanol
[100 ml]) for 12–24 hours. Excess stain is removed by immersion in 0.5–1.0% hydrochloric acid solution for
a few seconds. Dehydration is accomplished by serial passage in ascending concentrations of alcohol (41,
50, 70, 85, 95, and 100%) for at least 15 minutes in each, with two changes in 100%. The alcohol is
removed by xylol (10 minutes) and cleared with methyl salicylate or creosote. Prior to mounting in any
suitable medium such as balsam, picolyte, etc., the specimens should be returned to the xylol for a few
minutes. Persons involved in such examinations should receive serological screening for anti-Echinococcus
serum-antibodies at least once a year (56).
Recently, some methods have been developed with the aim of simplifying and improving epidemiological
investigations in final host populations and of allowing diagnosis in living animals. These methods include
the detection of coproantigens and PCR DNA detection (see below).
d) Arecoline surveys and surveillance

Arecoline has been used to perform surveys of tapeworm infections in dog populations. Its use as a control
agent has now been superseded by praziquantel. Arecoline is a parasympathomimetic agent. Its action
results in sweating, and stimulation of salivary, lachrymal, gastric, pancreatic, and intestinal glands. It
increases intestinal tonus and the mobility of smooth muscle, and this effect is responsible for purgation.
The liver is the principal site of detoxification. Arecoline also has a direct action on the worm itself, by
causing paralysis, but not death, and thereby making it relax its hold on the intestinal wall. Thus, it must be
administered by the oral route. The accompanying purgation carries the worms out with the faeces. It is
particularly suitable for baseline surveys of E. granulosus, however, 15–25% of dogs may not purge.

Arecoline may also be used to purge dogs infected with E. multilocularis. In animals, arecoline purgation
has been useful; again, the recovered tapeworms are identified morphologically. Products containing
arecoline are no longer available as an anthelmintic, but can be obtained from chemical supply companies.
As it has side-effects, old, infirm and pregnant animals should be excluded from treatment. A dose of
4 mg/kg should result in purgation in under 30 minutes. Walking and abdominal massage of recalcitrant
cases or enema for constipated dogs may avoid the use of a second dose (2 mg/kg), which should be given
only sparingly.
Dogs that are purged successfully may produce at least two motions; the first will be formed faeces and can
be ignored (or collected for laboratory tests as described later), but the mucus that follows may be
productive. This can be divided into several samples and each examined separately, but this method is not
recommended as the worms will be difficult to detect. Preferably, the mucus sample (about 4 ml) is diluted
with 100 ml of tap water, covered with a thin layer of 1 ml of kerosene (paraffin) and boiled for 5 minutes.
The kerosene prevents foaming and reduces the smell.
Investigators carrying out these procedures are exposed to risk of infection and severe disease. Personnel
should wear whole body coveralls, boots, disposable gloves and a face mask. Coveralls should be boil
washed after use, and boots disinfected in 10% sodium hypochlorite solution. The purge should be boiled
as soon as possible after collection. Dogs may continue to pass eggs, proglottides and worms after the first
purge, therefore, they should remain tethered for 2 hours after purgation and given access to drinking
water. After arecoline testing, the area of ground used to tether dogs should be sprayed with kerosene and
flamed.
e) Coproantigen tests
An alternative to arecoline testing, based on a faecal antigen-detection antibody sandwich enzyme-linked
immunosorbent assay (ELISA), has been developed and has shown particular promise as coproantigens
can be detected shortly after infection (10–14 days) and the level declines rapidly following expulsion of the
worms. The sensitivity and specificity of the test have been estimated at 70% and 98%, respectively (2, 5, 8,
12, 13).
Both qualitative and quantitative results can be obtained from arecoline testing, which is most useful for
base-line epidemiological studies on the comparative rates of infection with Taeniidae in dogs. Further
studies may show that the coproantigen test may be more cost-effective than arecoline testing during
routine surveillance of E. granulosus in the dog population (6).
ELISAs for specific coproantigen have now been developed that have sufficient specificity and sensitivity to
replace arecoline testing for detecting Echinococcus in dogs and other definitive hosts (8). When testing for
genus-specific Echinococcus coproantigens (against necropsy as a gold standard), specificity is around
98% and overall sensitivity approximately 70%; however, when mean worm burdens are >50–100,
sensitivity approaches 100% (2, 8, 9, 13). Dogs, dingoes, foxes and wolves have been screened
successfully for coproantigen ELISAs and, importantly, E. multilocularis worm infestations are also
detectable in red foxes and domestic dogs (13, 46). When the capture ELISA uses either anti-ES or anti-
somatic proglottid antibodies to E. granulosus, the sensitivity for E. multilocularis infection may be reduced,
though genus specificity remains intact. Polyclonal- or monoclonal-antibody-based ELISAs for
coproantigens exhibit high sensitivity and specificity to E. granulosus (~80%), even though they were
developed for E. multilocularis (11, 44). However, for low worm burdens (<50), the sensitivity of the
E. multilocularis coproantigen ELISA is below that of the mucosal smear method at necropsy (11).
The exact nature of Echinococcus antigens released in faeces for coproantigen detection has not been fully
characterised. However, their stability in 5% formal saline after boiling and susceptibility to periodate
treatment suggest involvement of large (>150 KDa) of carbohydrate antigen(s) with α-D-mannose, α-D-
glucose, β-galactose and N-acetyl-β-glucosamine residues (8, 18).
Coproantigens can be detected prior to release of eggs by Echinococcus worms, and therefore are not
related to egg antigen(s) (13, 44). This has the advantage of detection of prepatent infections. Furthermore,
coproantigen levels return to the preinfection baseline within 5 days of anthelminthic treatment of infected
dogs (13). More importantly, it reduces the biohazardous risk of exposure of personnel to potentially
infective eggs during purgation or necropsy (26).
For detection of E. multilocularis infection of foxes, necropsy is time-consuming. Coproantigen testing by

ELISA offers a specific practical alternative. Fox faecal samples should be taken at post-mortem from the
rectum rather than from the small intestine. Echinococcus coproantigens are also stable in fox or dog

faeces left at 20°C for 1 week and in frozen dog faeces. Coproantigen testing has also been successfully
used to evaluate he efficacy of deworming wild foxes infected with E. multilocularis using praziquantel-laced
bait, which proved to be a successful combination of eliminating the source of infection (24).
• Coproantigen test procedure (Echinococcus granulosus) (2, 9)

i) The faecal sample (collected per rectum or from the ground) is mixed with an equal volume of
phosphate buffered saline (PBS), pH 7.2, containing 0.3% Tween 20 (PBST), in a capped 5 ml
disposable tube. This is shaken vigorously and centrifuged at 2000 g for 20 minutes at room
temperature. Faecal supernatants can be tested immediately or stored at –20°C or lower.
Supernatants that appear very dark or viscous are still acceptable for use.
ii) A 96-well ELISA microtitre plate (Immulon #4, Thermo Electron Corporation) is coated with optimal
concentration (typically 5 µg per ml) of a protein A purified IgG fraction of rabbit anti-E. granulosus
proglottid extract (2) in 0.05 M bicarbonate/carbonate buffer, pH 9.6 (100 µl per well). The plate is
covered and incubated overnight at 4°C.
iii) The wells are rinsed three times in PBST with 1 minute between washes; 100 µl of the same buffer is
added to each well, and the plate is incubated for 1 hour at room temperature.
iv) The PBST is discarded and 50 µl of neat fetal calf serum is added to all wells. This is followed by the
addition of 50 µl per well of faecal sample supernatants is added (in duplicate wells). The plate is
incubated at room temperature for 1 hour with clingfilm seal covering the plate.
v) The wells are rinsed as in step iii, but the contents are discarded into a 10% bleach (hypochlorite)
solution.
vi) An optimal dilution concentration of around 1 µg/ml of an IgG rabbit anti-E.-granulosus proglottid
extract peroxidase conjugate (2) in PBST is prepared and 100 µl per well is added to all wells. The
plate is incubated for 1 hour at room temperature (22–24°C).
vii) The wells are rinsed as in step iii.
viii) Next, 100 µl per well of tetramethyl benzidene substrate (TMB, KPL Labs) is added and the plate is left
in the dark for 20 minutes at room temperature (22–24°C).
ix) Absorbance of wells is read at 630 nm. The enzyme-substrate reaction is stopped by adding 100 µl of
1 M phosphoric acid (H3PO4) to each well. The colour turns from blue to yellow if positive.
x) Laboratories should establish their own end-point criteria using standard positive and negative
samples. Standards can also be obtained from the OIE Reference Laboratories (see Table given in
Part 3 of this Terrestrial Manual). Usually, the positive to negative threshold is taken as 3 standard
deviations above the mean absorbance value of control negatives, or against a reference standard
control positive using absorbance units equivalence.
• Coproantigen test procedure (Echinococcus multilocularis) (39, 42)

Sandwich ELISA using a monoclonal antibody EmA9 raised against adult E. multilocularis somatic antigen
(28).
i) 0.5 g of each faecal sample is placed in a centrifuge tube and a 1% formalin solution containing 0.3%
Tween 20 is added to a total volume of 15 ml.
ii) After adequate mixing, the faecal solution is centrifuged at 1200 g for 10 minutes at room temperature.
A supernatant fraction is used for the coproantigen detection assay.
iii) Flat-bottomed microtitre plates (Immulon 600, Greiner, Germany) are coated with 50 µl/well of 1 µg/ml
rabbit IgG directed against adult E. multilocularis excretory/secretory (ES) products in 0.05 M
NaHCO3/Na2CO3 buffer (pH 9.6) and are left overnight at 4°C.
iv) The plates are washed three times with 250 µl/well PBS (pH 7.4) containing 0.05% Tween 20 (PBST),
and blocked using 100 µl/well 1% bovine serum albumin (BSA) in PBS for 1 hour at room temperature
(22–24 C).
v) The plates are washed three times (with the wash disinfected with 10% bleach) and 50 µl of faecal
supernatant is added to each well and the plates are incubated for 2 hours.
vi) The plates are again washed four times and 0.5 µg/ml of the biotinylated monoclonal antibody in 0.5%
BSA/0.5% casein in PBST is added to each well and the plates are incubated for 1 hour.
vii) The plates are washed four times and streptavidine-biotinylated horseradish peroxidase complex
(Amersham Life Science), diluted 1/1000 in 0.5% BSA/0.5% cCasein in PBST is added to each well
and the plates are incubated for 1 hour.

viii) The plates are washed five times and 100 µl/well of substrate solution (20 mg of phenylenediamine
(Wako) in 50 ml of 0.1 M citric phosphate buffer with 10 µl of H2O2) is added.
ix) The plates are shaken immediately and placed in a 37°C incubator for 30 minutes. The reaction is
stopped by adding 50 µl/well of 4 N H2SO4. The optical densities (OD) of the plates are read at
490 nm.
x) The cut-off value is calculated as the mean OD value plus 3 standard deviations of samples from
uninfected animals.
This procedure was also used in a sandwich ELISA for E. granulosus coproantigen detection (45). In 2008,
a latex agglutination test and immunochromatography in-house kit using EmA9 became available for
coproantigen detection (23, 41).
f) DNA recognition methods

Definitive hosts: Copro-DNA has proven to be of value for the diagnosis of Echinococcosis in animal
definitive hosts. DNA isolation from the faeces, however, is laborious.
PCR is a technically demanding and expensive technique. It is currently used mainly for confirmatory
testing of coproantigen-positive samples or for identification of taenid eggs recovered from faeces. Table 2
presents the different PCR primers used for identification of copro-DNA from faeces in definitive hosts of
genus Echinococcus.
Differential diagnosis of E. granulosus and E. multilocularis infections in definitive hosts may be achieved by
specific detection of PCR-amplified DNA from E. multilocularis eggs present in faeces (4, 32). In practice, it
is recommended to screen definitive hosts (e.g. foxes) using the coproantigen test and confirm with the
PCR DNA test. In Europe, transmission of E. multilocularis generally occurs in regions where E. granulosus
is not endemic or appears very infrequently. In other regions, including parts of the Near East (Turkey and
Iran), Central Asia, Russia and the People’s Republic of China, these two species may occur together (10).
Further evaluation of E. multilocularis infection is required to investigate intermittent shedding and duration
of shedding of parasite DNA. Recently PCR has been developed for the detection of copro-DNA for
E. granulosus and for genotypic differentiation. (36, 55)
As PCR is generally used as a confirmatory test, it is suggested to concentrate the taeniid eggs by
sequential sieving and an in-between concentration method step. DNA isolation from these eggs can be
achieved using a simplified protocol of the alkaline lysis method combined with a commercial kit with no
need for organosolvent extractions (30).
Table 2. PCR primers used for copro-DNA detection (modified from Mathis & Deplazes [34])
Primer designation: primer sequences (5’–3’) Ref. Target, comments
E. multilocularis 4 U1 sRNA gene: may yield non-specific

products when used with metacestode material
GTG-AGG-CGA-TGT-GTG-GTG-ATG-GAG-AGA-AGG containing host DNA (unpublished observation)
CAA-GTG-GTC-AGG-GGC-AGT-AG
Mitochondrial 12S RNA gene; used in two-tube
nested PCR
Outer primers: 14 Mitochondrial 12S RNA gene; modified from

ref. 14 for use in one-tube nested PCR
(P60 forward)
TTA-AGA-TAT-ATG-TGG-TAC-AGG-ATT-AGA-TAC-CC
(P375 reverse)
AAC-CGA-GGG-TGA-CGG-GCG-GTG-TGT-ACC
Inner primers:
(Pnest forward)
ACA-ATA-CCA-TAT-TAC-AAC-AAT-ATT-CCT-ATC
(Pnest reverse)
ATA-TTT-TGT-AAG-GTT-GTT-CTA

Table 2 cont. PCR primers used for copro-DNA detection (modified from Mathis & Deplazes [34])
Outer primers: 54 Mitochondrial 12S RNA gene; modified from

ref. 14 for use in single PCR
(Em-1)
TAA-GAT-ATA-TGT-GGT-ACA-GGA-TTA-GAT-ACC-C
(Em-2)
GGT-GAC-GGG-CGG-TGT-TGT-A
Inner primers:
(Em-3)
ATA-TTA-CAA-CAA-TAT-TCC-TAT-C
(Em-4)
ATA-TTT-TGT-AAG-GTT-GTT-CTA
(EM-H15) 48 NADH dehydrogenase subunit 1 (ND1) of

CCA-TAT-TAC-AAC-AAT-ATT-CCT-ATC mtDNA; cleavage with enzyme Cfo1 distinguish
E. multilocularis from E. granulosus
(EM-H17)
GTG-AGT-GAT-TCT-TGT-TAG-GGG-AAG
E. multilocularis and E. granulosus 38 Repeated sequences from E. granulosus.

‘sheep strain’; yields banding pattern upon
ND1 electrophoresis
(NDfor2-)
Mitochondrial 12SRNA gene; specific for
AGT-TTC-GTA-AGG-GTC-CTA-ATA E. granulosus ‘sheep strain’
(NDrev2-)
Amplify a fragment of the coxI genespecific fo
CCC-ACT-AAC-TAA-CTC-CCT-TTC E. granulosus
E. granulosus 1
(Eg1121a)
GAA-TGC-AAG-CAG-CAG-ATG
(Eg1122a)
GAG-ATG-AGT-GAG-AAG-GAG-TG
(Eg1f) 47
CATTAATGTATTTTGTAAAGTTG
(Eg1r)
CAC-ATC-ATC-TTA-CAA-TAA-CAC-C
(EgO/DNA-IM1) 40
forward
TCA-TAT-TTG-TTT-GAG-KAT-YAG-TKC
reverse
GTA-AAT-AAM-ACT-ATA-AAA-GAA-AYM-AC

Table 2 cont. PCR primers used for copro-DNA detection (modified from Mathis & Deplazes [34])
E. multilocularis, E. granulosus and Taeniid spp. 52

(JB11) Cestodes
AGA-TTC-GTA-AGG-GGC-CTA-ATA
(JB12)
AC-CAC-TAA-CTA-ATT-CAC-TTT-C
(60.for.-mod) Cestodes
ATG-TGG-TAC-AGG-ATT-AGA-TAC-CC
(375.rev.-mod)
GGT-GAC-GGG-CGG-TGT-GTA-CC
(Eg1f) 52 Echinococcus granulosus (sheep strain)
CAT-TAA-TGT-ATT-TTG-TAA-AGT-TG
(Eg1r)
CAC-ATC-ATC-TTA-CAA-TAA-CAC-C
(EM-H15) 52 E. multilocularis
CCA-TAT-TAC-AAC-AAT-ATT-CCT-ATC
(EM-H17)
GTG-AGT-GAT-TCT-TGT-TAG-GGG-AAG
(Cest1) 52 E. multilocularis
TGC-TGA-TTT-GTT-AAA-GTT-AGT-GAT-C
(Cest2)
CAT-AAA-TCA-ATG-GAA-ACA-ACA-ACA-AG
(Cest4) 52 E. granulosus
GTT-TTT-GTG-TGT-TAC-ATT-AAT-AAG-GGT-G
(Cest5) 52 Taenia spp.
GCG-GTG-TGT-ACM-TGA-GCT-AAA-C
(Cest3) 52 Taenia spp. (Sequencing primer for the

267 bp amplicon of the multiplex PCR)
YGA-YTC-TTT-TTA-GGG-GAA-GGT-GTG
(Cest5)
GCG-GTG-TGT-ACM-TGA-GCT-AAA-C
(Cest5seq)
GAT-TCT-TTT-TAG-GGG-AAG-G
Intermediate hosts: DNA hybridisation methods are not currently used for the detection of E. granulosus in
livestock intermediate hosts. Molecular methods are, however, important in identification of isolates or
strains of E. granulosus for epidemiological purposes (37).
For the identification of small, degenerated or calcified lesions of E. multilocularis in intermediate or

aberrant hosts, PCR is of great value (33).
a) Intermediate hosts
Immunological tests, useful in humans, are less sensitive and specific in livestock and at present cannot
replace necropsy (8, 30).

b) Definitive hosts
An extensive programme has been initiated to develop immunodiagnostic tests to control canine
echinococcosis. Following ingestion of a cyst, dogs will be exposed at the intestinal level to various antigens
during the establishment of the parasite and its development and oogenesis. Specific antibodies against
oncosphere and protoscolex antigens can be readily detected in the serum of infected dogs. This
methodology has not reached a practical stage as it does not differentiate between current and previous
infections and false positives may occur with infections of Taenia species.
1. Intermediate hosts
Application of an effective vaccine to reduce hydatid infection in livestock would be likely to have a substantial
impact on the rate of transmission of the disease to humans (29). As E. granulosus belongs to the Taeniid family,
many aspects of its immunological relationship with its intermediate host are similar to that occurring in Taenia
species. Moreover, it was considered that the vaccine development approach used in Taenia species such as
the native host-protective antigens of T. ovis would also be successful for E. granulosus. Using recombinant DNA
technology, an oncosphere antigen vaccine EG95 was shown to be capable of inducing a high level of protection
against experimental challenge infection with E. granulosus eggs in sheep (31).
The EG95 vaccine has been licensed by the University of Melbourne and AgResearch New Zealand to a
commercial group in the People’s Republic of China (29).
2. Definitive hosts
While considerable research has been undertaken with crude antigens to protect dogs from echinococcosis, only
limited evidence has been demonstrated so far. Recent studies using recombinant protoscolex antigens,
however, look encouraging (7). Basic research on canine mucosal immunology and Echinococcus infection is
required for progress.
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34. MATHIS A. & DEPLAZES P. (2006). Copro-DNA tests for diagnosis of animal taeniid cestodes. Parasitol. Int.,
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35. MATSUO K. & KAMIYA H. (2005). Modified sugar centrifugal flotation technique for recovering Echinococcus
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*
* *
NB: There are OIE Reference Laboratories for Echinococcosis/Hydatidosis (see Table in Part 3 of this Terrestrial

CHAPTER 2.1.5.
FOOT AND MOUTH DISEASE
SUMMARY
Foot and mouth disease (FMD) is the most contagious disease of mammals and has a great
potential for causing severe economic loss in susceptible cloven-hoofed animals. There are seven
serotypes of FMD virus, namely, O, A, C, SAT 1, SAT 2, SAT 3 and Asia 1. Infection with one
serotype does not confer immunity against another. FMD cannot be differentiated clinically from
other vesicular diseases, including swine vesicular disease, vesicular stomatitis and vesicular
exanthema. Laboratory diagnosis of any suspected FMD case is therefore a matter of urgency.
Typical cases of FMD are characterised by a vesicular condition of the feet, buccal mucosa and, in
females, the mammary glands. Clinical signs can vary from mild to severe and fatalities may occur,
especially in young animals. In some species the infection may be subclinical, e.g. African buffalo
(Syncerus caffer). The preferred tissue for diagnosis is epithelium from unruptured or freshly
ruptured vesicles or vesicular fluid. Where collecting this is not possible, blood and/or
oesophageal–pharyngeal fluid samples taken by probang cup in ruminants or throat swabs from
pigs provide an alternative source of virus. Myocardial tissue or blood can be submitted from fatal
cases, but vesicles are again preferable if present.
It is vital that samples from suspected cases be transported under secure conditions and according
to international regulations. They should only be dispatched to authorised laboratories.
Diagnosis of FMD is by virus isolation or by the demonstration of FMD viral antigen or nucleic acid
in samples of tissue or fluid. Detection of virus-specific antibody can also be used for diagnosis
and antibodies to viral nonstructural proteins (NSPs) can be used as indicators of infection,
irrespective of vaccination status.
Identification of the agent: The demonstration of FMD viral antigen or nucleic acid is sufficient for
a positive diagnosis. Due to the highly contagious nature and economic importance of FMD, the
laboratory diagnosis and serotype identification of the virus should be done in a laboratory that
meets the OIE requirements for Containment Group 4 pathogens.
Complement fixation (CF) has been replaced in most laboratories by the enzyme-linked
immunosorbent assay (ELISA), as it is more specific and sensitive and is not affected by pro- or
anti-complement factors. If the sample is inadequate or the diagnosis remains uncertain, sample
materials should be inoculated on to susceptible cell cultures or into 2–7-day old unweaned mice
to amplify any live virus that may be present. The cultures should preferably be of primary bovine
(calf) thyroid, but pig, lamb or calf kidney cells, or cell lines of comparable sensitivity may be used.
Once a cytopathic effect (CPE) is complete in the cultures, the fluids can be used in CF tests
ELISAs or by reverse transcription polymerase chain reaction (RT-PCR). Similar tests can be
performed on homogenised suspensions of the dissected musculo-skeletal tissues of any mice
that die.
Nucleic acid recognition tests, such as the RT-PCR are being used increasingly as rapid and
sensitive diagnostic methods. Electron microscopic examination of lesion material is sometimes
used to differentiate FMD from disease caused by other viruses.
Serological tests: The demonstration of specific antibodies to structural proteins in nonvaccinated

animals, where a vesicular condition is present, is sufficient for a positive diagnosis. This is
particularly useful in mild cases or where epithelial tissue cannot be collected. Tests for antibodies
to some NSPs of FMD virus are useful in providing evidence of previous or current viral replication
in the host, irrespective of vaccination status. NSPs, unlike structural proteins, are highly

Chapter 2.1.5. — Foot and mouth disease
conserved and therefore are not serotype specific and as a consequence, the detection of these
antibodies is not serotype restricted.
Virus neutralisation (VN) tests and ELISAs for antibodies to structural proteins are used as
serotype-specific serological tests. VN tests depend on tissue cultures and are therefore more
prone to variability than ELISAs; they are also slower and subject to contamination. ELISAs for
antibodies have the advantage of being faster, and are not dependent on cell cultures. The ELISA
can be performed with inactivated antigens, thus requiring less restrictive biocontainment facilities.
Requirements for vaccines and diagnostic biologicals: Inactivated virus vaccines of varying
composition are available commercially. Typically, virus is used to infect a suspension or
monolayer cell culture and the resulting preparation is clarified, inactivated with ethyleneimine and
blended with adjuvant. Many FMD vaccines are multivalent to provide cover against the different
serotypes likely to be encountered in a given field situation.
The finished vaccine must be shown to be free from residual live virus. This is most effectively
done using in-vitro tests on concentrated inactivated virus preparations prior to formulation of the
vaccine and freedom from live virus is subsequently confirmed during in-vivo and/or in-vitro tests
on the finished product. Challenge tests are also conducted in vaccinated cattle to establish a
PD50 (50% protective dose) value or protection against generalised foot infection (PGP), although
a serological test is considered to be satisfactory where a valid correlation between the amount of
antigen present in the vaccine, the observed protection, and the specific antibody response has
been established.
FMD vaccine production facilities should also meet the OIE requirements for Containment Group 4
pathogens.
Diagnostic and reference reagents are available from the OIE Reference Laboratories for FMD or
the FAO (Food and Agriculture Organization of the United Nations) World Reference Laboratory for
FMD. The Institute for Animal Health Pirbright Laboratory has dual designations as both the FAO
World Reference Laboratory and as an OIE Reference Laboratory for FMD.
A. INTRODUCTION
Foot and mouth disease (FMD) is caused by a virus of the genus Aphthovirus, family Picornaviridae. There are
seven serotypes of FMD virus, namely O, A, C, SAT 1, SAT 2, SAT 3, and Asia 1, that infect cloven-hoofed
animals. Infection with any one serotype does not confer immunity against another. Within serotypes, many
strains can be identified by biochemical and immunological tests.
In Africa, FMD viruses are maintained by cattle and African buffalo (Syncerus caffer) and they are usually the
most common host. Available evidence indicates that although other domestic and wild species become infected,
they are unable to maintain the infection for more than a few months in the absence of cattle or African buffalo.
Elsewhere in the world cattle are usually the main reservoir, although in some instances the viruses involved
appear to be specifically adapted to domestic pigs or sheep and goats. It is probable that these adapted viruses
are able to modify their adaptation and affect other species if given the opportunity. However, the pig-adapted
Cathay strain of FMD virus apparently does not infect large ruminants in the field or experimentally and requires
cells of porcine origin for primary isolation. Wildlife outside Africa has not, so far, been shown to be able to
maintain FMD viruses. The evidence indicates that infection of deer in the past was derived from contact, direct
or indirect, with infected domestic animals.
Of the domesticated species, cattle, pigs, sheep, goats and buffalo are susceptible to FMD (30). In addition,
many species of cloven-hoofed wildlife, such as deer, antelope and wild pigs may become infected, although,
apart from the African buffalo they have not been shown to play a significant role in the epidemiology of FMD.
Strains of FMD virus that infect cattle have been isolated from wild pigs and deer. For the diagnosis of FMD in
wild species, procedures similar to those described for farm animals can be applied.
Infection of susceptible animals with FMD virus leads to the appearance of vesicles on the feet, in and around
the oral cavity, and on the mammary glands of females. Coronary band lesions may give rise to growth arrest
lines whose progress down the side of the hoof can be used to indicate the time since infection occurred. In
severe infections of the feet, hooves may be shed. Mastitis is a common sequel of FMD in dairy cattle. Vesicles
can also occur at other sites, such as inside the nostrils and at pressure points on the limbs – especially in pigs.
The severity of clinical signs varies with the strain of virus, the exposure dose, the age and breed of animal, the
host species and its degree of immunity (44). The signs can range from a mild or inapparent infection to one that

is severe. Death may result in some cases. Mortality from a multifocal myocarditis is most commonly seen in
young animals: myositis may also occur in other sites.
On premises with a history of sudden death in young cloven-hoofed livestock, close examination of adult animals
may often reveal the presence of vesicular lesions if FMD is involved. The presence of vesicles in fatal cases is
variable.
In animals with a history of vesicular disease, the detection of FMD virus in samples of vesicular fluid, epithelial
tissue, oesophageal–pharyngeal (OP) sample, milk, or blood is sufficient to establish a diagnosis. Diagnosis may
also be established by the isolation of FMD virus from the blood, heart or other organs of fatal cases. A
myocarditis may be seen macroscopically (the so-called “tiger heart”) in a proportion of fatal cases.
FMD virus can replicate and be excreted from the respiratory tract of animals. Airborne excretion of virus occurs
during the acute phase of infection. FMD viruses may occur in all the secretions and excretions of acutely
infected animals including expired air. Transmission is generally effected by direct contact between infected and
susceptible animals or, more rarely, exposure of susceptible animals to the excretions and secretions of acutely
infected animals. Following recovery from the acute stage of infection, infectious virus disappears from all
secretions and excretions with the exception of OP fluids from some ruminants, where live virus may continue to
be recovered. Animals in which the virus persists in the OP for more than 28 days after infection are referred to
as carriers. Pigs do not become carriers. Circumstantial evidence indicates, particularly in the African buffalo,
that carriers are able, on rare occasions, to transmit the infection to susceptible animals with which they come in
close contact: the mechanism involved is unknown. The carrier state in cattle usually does not persist for more
than 6 months, although in a small proportion it may last up to 3 years. In African buffalo individual animals have
been shown to harbour the virus for at least 5 years, but it is probably not a lifelong phenomenon. Within a herd
of buffalo, the virus may be maintained for 24 years or longer. There is no information on the duration of the
carrier state in another domestic buffalo, the swamp buffalo of East Asia. Domestic buffalo, sheep and goats do
not usually carry FMD viruses for more than a few months.
Due to the highly contagious nature and economic importance of FMD, the laboratory diagnosis and serotype
identification of the virus should be done in a facility that meets the requirements for Containment Group 4
pathogens as outlined in Chapter 1.1.2 Biosafety and biosecurity in the veterinary microbiology laboratory and
animal facilities. Countries lacking access to such a specialised national or regional laboratory should send
specimens to an OIE FMD Reference Laboratory. Vaccine production facilities should also meet the
requirements for Containment Group 4 pathogens.
Diagnostic and standard reagents are available in kit form or as individual items from OIE Reference
Laboratories for FMD. The use of inactivated antigens in the enzyme-linked immunosorbent assay (ELISA), as
controls in the antigen-detection test or to react with test sera in the liquid-phase blocking or solid-phase
competitive ELISA, reduces the disease security risk involved compared to the use of live virus. Reagents are
supplied freeze-dried or in glycerol or non-glycerinated but frozen and can remain stable at temperatures
between +1°C and +8°C, –30°C and –5°C and –90°C and –50°C, respectively, for many years. The International
Atomic Energy Agency has produced a manual that includes a recommended test and quality control protocols.
For laboratory diagnosis, the tissue of choice is epithelium or vesicular fluid. Ideally, at least 1 g of epithelial
tissue should be collected from an unruptured or recently ruptured vesicle, usually from the tongue, buccal
mucosa or feet. To avoid injury to personnel collecting the samples, as well as for animal welfare reasons, it is
recommended that animals be sedated before any samples are obtained.
Epithelial samples should be placed in a transport medium composed of equal amounts of glycerol and 0.04 M
phosphate buffer, pH 7.2–7.6, preferably with added antibiotics (penicillin [1000 International Units (IU)],
neomycin sulphate [100 IU], polymyxin B sulphate [50 IU], mycostatin [100 IU]). If 0.04 M phosphate buffer is not
available, tissue culture medium or phosphate buffered saline (PBS) can be used instead, but it is important that
the final pH of the glycerol/buffer mixture be in the range pH 7.2–7.6. FMD virus is extremely labile in low pH and
buffering of the transport media is critical for successful sample collection. Samples should be kept refrigerated
or on ice until received by the laboratory.
Where epithelial tissue is not available from ruminant animals, for example in advanced or convalescent cases,
or where infection is suspected in the absence of clinical signs, samples of OP fluid can be collected by means of
a probang (sputum) cup (or in pigs by swabbing the throat) for submission to a laboratory for virus isolation or
reverse-transcription polymerase chain reaction (RT-PCR). Viraemia may also be detected by examining serum
samples by means of RT-PCR or virus isolation. For the collection of throat swabs from pigs, the animal should
be held on its back in a wooden cradle with the neck extended. Holding a swab in a suitable instrument, such as
an artery forceps, the swab is pushed to the back of the mouth and into the pharynx.

Before the collection of OP samples from cattle or large ruminants (e.g. buffaloes), 2 ml transport fluid
(composed of 0.08 M phosphate buffer containing 0.01% bovine serum albumin, 0.002% phenol red, antibiotics
[1000 units/ml penicillin, 100 units/ml mycostatin, 100 units/ml neomycin, and 50 units/ml polymyxin], and
adjusted to pH 7.2) should be added to a container of around 5 ml capacity capable of withstanding freezing
above solid carbon dioxide (dry ice) or liquid nitrogen (39).
An OP sample is collected by inserting a probang over the tongue into the oro-pharyngeal area and then passing
it vigorously backwards and forwards 5–10 times between the first portion of the oesophagus and the back of the
pharynx. The purpose is to collect oro-pharyngeal fluid and especially superficial epithelial cells from these areas,
including the proximal part of the oesophagus, the walls of the pharynx, the tonsillar crypts and the surfaces of
the soft palate. If the sample does not contain adequate cellular debris the actions may be repeated.
After collection of OP fluid by probang, the contents of the cup should be poured into a wide-necked transparent
bottle of around 20 ml capacity. The fluid is examined, and should contain some visible cellular material. Of this,
2 ml is then added to the 2 ml of transport fluid, ensuring that cellular material is transferred; the mixture is
shaken gently and should have a final pH of around pH 7.6. Samples contaminated with ruminal contents may be
unsuitable for culture. Samples seen to contain blood are not entirely satisfactory. Repeat sampling can be done
after the mouth and throat of the animal have been rinsed with water or PBS. Where several animals are to be
sampled the probang must be cleaned and disinfected between each animal. This is done by washing the
probang in tap water, then immersing it in a suitable disinfectant (e.g. 0.5% [w/v] citric acid in tap water) and then
rinsing off the disinfectant well with water before sampling the next animal.
OP samples from small ruminants are collected by putting 2 ml of transport fluid into a wide-necked bottle of
about 20 ml capacity and, after collection, rinsing the probang cup in this transport fluid to discharge the OP
sample. This is then transferred to a container of about 5 ml capacity for transport. The small container should be
capable of withstanding freezing above solid carbon dioxide or liquid nitrogen (39).
Samples of OP fluid should be refrigerated or frozen immediately after collection. If they are to remain in transit
for more than a few hours, they should preferably be frozen by being placed either above solid carbon dioxide or
liquid nitrogen. Before freezing, the containers should be carefully sealed using airtight screw caps or silicone.
This is particularly important when using solid carbon dioxide, as introduction of CO2 into the OP sample will
lower its pH, inactivating any FMD virus that may be in the samples. Glass containers should not be used
because there is a risk that they will explode on defrosting in the event of liquid nitrogen leaking into them.
Samples should reach the laboratory preferably in a frozen state or, if this is not feasible, under refrigeration.
Special precautions are required when sending perishable suspect FMD material both within and between
countries. The International Air Transport Association (IATA), Dangerous Goods Regulations (DGR) has explicit
requirements for packaging and shipment of diagnostic specimens by all commercial means of transport. These
are summarised in Chapter 1.1.1 Collection and shipment of diagnostic specimens.
A range of sample types including epithelium, OP samples and serum may be examined by virus isolation or RT-
PCR. By contrast, ELISA is suited to the examination of epithelial suspensions, vesicular fluids or cell culture
supernatants, but is insufficiently sensitive for the direct examination of OP samples or serum.
a) Virus isolation
The epithelium sample should be taken from the PBS/glycerol, blotted dry on absorbent paper to reduce the
glycerol content, which is toxic for cell cultures, and weighed. A suspension should be prepared by grinding
the sample in sterile sand in a sterile pestle and mortar with a small volume of tissue culture medium and
antibiotics. Further medium should be added until a final volume of nine times that of the epithelial sample
has been added, giving a 10% suspension. This is clarified on a bench centrifuge at 2000 g for 10 minutes.
Once clarified, such suspensions of field samples suspected to contain FMD virus are inoculated onto cell
cultures or into unweaned mice. Sensitive cell culture systems include primary bovine (calf) thyroid cells
and primary pig, calf or lamb kidney cells. Established cell lines, such as BHK-21 (baby hamster kidney)
and IB-RS-2 cells, may also be used but are generally less sensitive than primary cells for detecting low
amounts of infectivity (19). The sensitivity of any cells used should be tested with standard preparations of
FMD virus. The use of IB-RS-2 cells aids the differentiation of swine vesicular disease (SVD) from FMD (as
SVD virus will only grow in this cell type) and is often essential for the isolation of porcinophilic strains, such
as O Cathay. The cell cultures should be examined for cytopathic effect (CPE) for 48 hours. If no CPE is
detected, the cells should be frozen and thawed, used to inoculate fresh cultures and examined for CPE for
another 48 hours. Unweaned mice are an alternative to cell cultures and should be 2–7 days old and of
selected inbred strains. Some field viruses may require several passages before they become adapted to
mice (58). In the case of OP fluids, pre-treatment with an equal volume of chloro- fluoro- carbons may
improve the rate of virus detection by releasing virus from immune complexes.

• Enzyme-linked immunosorbent assay
The preferred procedure for the detection of FMD viral antigen and identification of viral serotype is the
ELISA (28, 53). This is an indirect sandwich test in which different rows in multiwell plates are coated with
rabbit antisera to each of the seven serotypes of FMD virus. These are the ‘capture’ sera. Test sample
suspensions are added to each of the rows, and appropriate controls are also included. Guinea-pig antisera
to each of the serotypes of FMD virus are added next, followed by rabbit anti-guinea-pig serum conjugated
to an enzyme. Extensive washing is carried out between each stage to remove unbound reagents. A colour
reaction on the addition of enzyme substrate and chromogen indicates a positive reaction. With strong
positive reactions this will be evident to the naked eye, but results can also be read spectrophotometrically
at an appropriate wavelength. In this case, an absorbance reading greater than 0.1 above background
indicates a positive reaction; the serotype of FMD virus can also be identified. Values close to 0.1 should be
confirmed by retesting or by amplification of the antigen by tissue culture passage and testing the
supernatant once a CPE has developed. A suitable protocol is given below. Other protocols are available
with slightly different formats and interpretation criteria (3, 6).
Depending on the species affected and the geographical origin of samples, it may be appropriate to
simultaneously test for SVD virus or vesicular stomatitis (VS) virus. Ideally a complete differential diagnosis
should be undertaken in all vesicular conditions.
Rabbit antiserum to the 146S antigen of each of the seven serotypes of FMD virus (plus SVD virus or VS
virus if required) is used as a trapping antibody at a predetermined optimal concentration in
carbonate/bicarbonate buffer, pH 9.6.
Control antigens are prepared from selected strains of each of the seven types of FMD virus (plus SVD
virus or VS virus if appropriate) grown on monolayer cultures of BHK-21 cells (IB-RS-2 cells for SVD or VS
virus). The unpurified supernatants are used and pretitrated on ELISA plates. The final dilution chosen is
that which gives an absorbance at the top of the linear region of the titration curve (optical density
approximately 2.0), so that the five-fold dilutions of the control antigens used in the test give two additional
lower optical density readings from which the titration curve can be derived. PBS containing 0.05% Tween
20 and phenol red indicator is used as a diluent (PBST).
Guinea-pig antisera prepared by inoculating guinea-pigs with 146S antigen of one of the seven serotypes of
FMD virus (plus SVD virus if required) and preblocked with normal bovine serum (NBS) is used as the
detecting antibody. Predetermined optimal concentrations are prepared in PBS containing 0.05% Tween
20, and 5% dried, nonfat skimmed milk (PBSTM).
Rabbit (or sheep) anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase and preblocked
with NBS is used at a predetermined optimum concentration in PBSTM. As an alternative to guinea-pig or
rabbit antisera, suitable monoclonal antibodies (MAbs) can be used coated to the ELISA plates as capture
antibody or peroxidase-conjugated as detecting antibody.
• Test procedure
i) ELISA plates are coated with 50 µl/well rabbit antiviral sera in 0.05 M carbonate/bicarbonate buffer,
pH 9.6. Rows A to H receive, respectively, antisera to serotypes O, A, C, SAT 1, SAT 2, SAT 3, Asia 1
and SVD virus or VS virus (optional).
ii) Leave overnight at 4°C in a stationary position or place on an orbital shaker set at 100–120 revolutions
per minute in a 37°C incubator for 1 hour.
iii) Prepare test sample suspension (with 10% original sample suspension or undiluted clarified cell
culture supernatant fluid).
iv) The ELISA plates are washed five times in PBS.
v) On each plate, load wells of columns 4, 8 and 12 with 50 µl PBST. Additionally, add 50 µl of PBST to
wells 1, 2 and 3 of rows A to H on plate 1. To well 1 of row A of plate 1 add 12.5 µl of control antigen
type O, to well 1 of row B add 12.5 µl of control antigen type A; continue in this manner for control
antigen of types C, SAT 1, SAT 2, SAT 3, Asia 1 and SVDV or VS (if appropriate) in order to well 1,
rows C to H. Mix diluent in well 1 of rows A to H and transfer 12.5 µl from well 1 to 2 (rows A to H), mix
and transfer 12.5 µl from well 2 to 3, mix and discard 12.5 µl from well 3 (rows A to H) (this gives a
five-fold dilution series of each control antigen). It is only necessary to change pipette tips on the
micropipette between antigens. The remainder of the plate can be loaded with the test sample(s). Add
50 µl of sample one to wells 5, 6 and 7 of rows A to H, the second sample is placed similarly in
columns 9, 10 and 11, rows A to H.
If more than two samples are to be tested at the same time, the other ELISA plates should be used as
follows:

Dispense 50 µl of the PBST to the wells (rows A to H) of columns 4, 8 and 12 (buffer control columns).
Note that the control antigens are not required on these plates. These test samples may be added in
50 µl volumes in rows A to H to columns 1, 2, 3; 5, 6, 7; 9, 10, 11, respectively.
vi) Cover with lids and place on an orbital shaker at 37°C for 1 hour.
vii) Wash the plates by flooding with PBS – wash three times as before and empty residual wash fluid.
Blot the plates dry.
viii) Transfer 50 µl volumes of each guinea-pig serum dilution to each plate well in the appropriate order,
e.g. rows A to H receive, respectively, antisera to serotypes O, A, C, SAT 1, SAT 2, SAT 3, Asia 1 and
SVD virus or VS virus (optional).
ix) Cover the plates with lids and replace on the orbital shaker. Incubate at 37°C for 1 hour.
x) The plates are washed again three times, and 50 µl of rabbit anti-guinea-pig immunoglobulin
conjugated to horseradish peroxidase is added to each well. The plates are incubated at 37°C for
1 hour on a rotary shaker.
xi) The plates are washed again three times, and 50 µl of substrate solution, containing 0.05% % H2O2
plus orthophenylene diamine or a suitable alternative chromogen, is added to each well.
xii) The reaction is stopped after 15 minutes by the addition of 50 µl of 1.25 M sulphuric acid. The plates
are read at 492 nm on a spectrophotometer linked to a computer.
• Complement fixation test

In general, the ELISA is preferable to the complement fixation (CF) test because it is more sensitive and it is
not affected by pro- or anti-complementary factors. If ELISA reagents are not available, the CF test may be
performed as follows:
Antisera to each of the seven types of FMD virus are diluted in veronal buffer diluent (VBD) in 1.5-fold
dilution steps from an initial 1/16 dilution to leave 25 µl of successive antiserum dilutions in U-shaped wells
across a microtitre plate. To these are added 50 µl of 3 units of complement, followed by 25 µl of test
sample suspension(s). The test system is incubated at 37°C for 1 hour prior to the addition of 25 µl of 1.4%
standardised sheep red blood cells (SRBC) in VBD sensitised with 5 units of rabbit anti-SRBC. The
reagents are incubated at 37°C for a further 30 minutes and the plates are subsequently centrifuged and
read. Appropriate controls for the test suspension(s), antisera, cells and complement are included. CF titres
are expressed as the reciprocal of the serum dilution producing 50% haemolysis. A CF titre ≥36 is
considered to be a positive reaction. Titre values of 24 should be confirmed by retesting an antigen that has
been amplified through tissue culture passage.
c) Nucleic acid recognition methods

RT-PCR can be used to amplify genome fragments of FMD virus in diagnostic materials including
epithelium, milk, serum and OP samples (7, 13). RT combined with real-time PCR has a sensitivity
comparable to that of virus isolation (2, 51) and automated procedures enhance sample throughput (52).
Specific primers have been designed to distinguish between each of the seven serotypes. In situ
hybridisation techniques have been developed for investigating the presence of FMD virus RNA in tissue
samples (63). These techniques are only in use in specialised laboratories, although simplified systems for
potential field-use are under development (18).
Agarose gel-based RT-PCR assay

The procedure used at the OIE Reference Laboratory at Pirbright is described (50). The RT-PCR assay
consists of the three successive procedures of (i) extraction of template RNA from the test or control sample
followed by (ii) RT of the extracted RNA, (iii) PCR amplification of the RT product and (iv) detection of the
PCR products by agarose gel electrophoresis.
Test procedure
®
i) Add 200 µl of test sample to 1 ml of TRIzol Reagent in a sterile tube. Store at –70°C until required for
RNA extraction.
ii) Transfer 1 ml of the solution from i) into a fresh, sterile tube containing 200 µl of chloroform. Vortex
mix for about 10–15 seconds and leave at room temperature for 3 minutes.
iii) Centrifuge for 15 minutes at 20,000 g.
iv) Transfer 500 µl of the aqueous phase into a fresh, sterile tube containing 1 µl of glycogen (20 mg/ml)
and add 500 µl of iso-propyl-alcohol (propan-2-ol). Vortex mix for a few seconds.
v) Leave at room temperature for 10 minutes then centrifuge for 10 minutes at 20,000 g.

vi) Discard the supernatant fluid from each tube and add 1 ml of 70% ethanol. Vortex mix for a few
seconds.
vii) Centrifuge for 10 minutes at 20,000 g.
viii) Carefully remove the supernatant fluid from each tube taking care not to dislodge or lose any pellet at
the bottom of the tube.
ix) Air dry each tube at room temperature for 2–3 minutes.
x) Re-suspend each pellet by adding 20 µl of nuclease-free water to the tube.
xi) Keep the extracted RNA samples on ice if the RT step is about to be performed. Otherwise store at
–70°C.
xii) For each sample to be assayed, add 2 µl of random hexamers (20 µg/ml) and 5 µl of nuclease-free
water into a sterile 0.5 ml microcentrifuge tube. It is recommended to prepare the dilution in bulk for
the total number of samples to be assayed but allowing for one extra sample.
xiii) Add 5 µl of RNA from the extraction procedure described above to give a volume of 12 µl in each tube.
Mix by gently pipetting up and down.
xiv) Incubate at 70°C for 5 minutes.
xv) Cool at room temperature for 10 minutes.
xvi) During the 10-minute incubation period, prepare the RT reaction mixture described below for each
sample. Prepare the reaction mixture in bulk in a sterile 1.5 ml microcentrifuge tube for the number of
samples to be assayed plus one extra sample.
First strand buffer, 5× conc. (4 µl); bovine serum albumin (acetylated), 1 mg/ml (2 µl); dNTPs, 10 mM
mixture each of dATP, dCTP, dGTP, dTTP (1 µl); DTT, 1 M (0.2 µl); Moloney Murine Reverse
Transcriptase, 200 U/ µl (1 µl).
xvii) Add 8 µl reaction mix to the 12 µl of random primer/RNA mix. Mix by gently pipetting.
xviii) Incubate at 37°C for 45 minutes.
xix) Keep the RT products on ice if the PCR amplification step is about to be performed, otherwise store at
–20°C.
xx) Prepare the PCR mix described below for each sample. It is recommended to prepare the mix in bulk
for the number of samples to be tested plus one extra sample.
Nuclease-free water (35 µl); PCR reaction buffer, 10× conc (5 µl); MgCl2, 50 mM (1.5 µl); dNTPs,
10 mM mixture each of dATP, dCTP, dGTP, dTTP (1 µl); primer 1, 10 pmol/µl (1 µl); primer 2,
10 pmol/µl (1 µl); Taq Polymerase, 5 units/µl (0.5 µl).
xxi) Add 45 µl of PCR reaction mix to a well of a PCR plate or to a microcentifuge tube for each sample to
be assayed followed by 5 µl of the RT product to give a final reaction volume of 50 µl.
xxii) Spin the plate or tubes for 1 minute in a suitable centrifuge to mix the contents of each well.
xxiii) Place the plate in a thermal cycler for PCR amplification and run the following programme:
94°C for 5 minutes: 1 cycle;
94°C for 1 minute, 55°C for 1 minute, 72°C for 2 minutes: 30 cycles;
72°C for 7 minutes: 1 cycle.
xxiv) Mix a 20 µl aliquot of each PCR reaction product with 4 µl of staining solution and load onto a 1.5%
agarose gel. After electrophoresis a positive result is indicated by the presence of a 328 bp band
corresponding to FMDV sequence in the 5’ untranslated region of the genome.
Stock solutions
®
i) Nuclease-free water, TRIzol Reagent, chloroform, glycogen, iso-propyl-alcohol (propan-2-ol), ethanol,
random hexanucleotide primers, First strand buffer, BSA (acetylated), dNTPs, DTT, Moloney Murine
Reverse Transcriptase, PCR reaction buffer (10×), MgCl2 and Taq Polymerase are commercially
available.
ii) Primers at a concentration of 10 pmol/µl: Primer 1 sequence 5’-GCCTG-GTCTT-TCCAG-GTCT-3’
(positive strand); Primer 2 sequence 5’-CCAGT-CCCCT-TCTCA-GATC-3’ (negative strand).
Real-time RT-PCR assay

The procedure used at the OIE Reference Laboratory at Pirbright is described. The real-time RT-PCR assay
uses the same procedures of extraction of total RNA from the test or control sample followed by RT of the

extracted RNA as for the conventional agarose gel-based procedure. Automated extraction of total nucleic
acid from samples followed by automated pipetting programmes for the RT and PCR steps (52) can be
used as an alternative to the manual procedures described above. PCR amplification of the RT product is
performed by a different procedure. Detection of the PCR products in agarose gels is not required following
real-time amplification.
i) Take the RT products from step xix (see above).

ii) Prepare the PCR mix described below for each sample. Again it is recommended to prepare the mix in
bulk for the number of samples to be tested plus one extra sample: nuclease-free water (6 µl); PCR
reaction master mix, 2× conc. (12.5 µl); real-time PCR forward primer, 10 pmol/µl (2.25 µl); real-time
®
PCR reverse primer, 10 pmol/µl (2.25 µl); TaqMan probe, 5 pmol/µl (1 µl).
iii) Add 24 µl PCR reaction mix to a well of a real-time PCR plate for each sample to be assayed followed
by 1 µl of the RT product to give a final reaction volume of 25 µl.
iv) Spin the plate for 1 minute in a suitable centrifuge to mix the contents of each well.
v) Place the plate in a real-time PCR machine for PCR amplification and run the following programme:
95°C for 15 seconds, 60°C for 1 minute: 50 cycles.
vi) Reading the results: Assign a threshold cycle (CT) value to each PCR reaction from the amplification
plots (a plot of the fluorescence signal versus cycle number; different cut-off values may be
appropriate for different sample types; 51). The CT values used to assign samples as either FMDV
positive or negative should be defined by individual laboratories using appropriate reference material.
For example at the OIE Reference Laboratory at Pirbright, negative test samples and negative controls
should have a CT value at >50.0. Positive test samples and positive control samples should have a CT
value <40. Samples with CT values falling within the range 40–50 are designated “borderline” and can
be re-tested. Strong positive FMD samples have a CT value below 20.0 (51).
Stock solutions for real-time PCR assay

i) Nuclease-free water and real-time PCR reaction master mixes are available from commercial
suppliers.
ii) Either of the two following primers and probe sets can be used for real-time PCR of FMDV:
5’UTR (51) Forward primer: CACYT YAAGR TGACA YTGRT ACTGG TAC; Reverse primer: CAGAT
®
YCCRA GTGWC ICITG TTA and TaqMan probe: CCTCG GGGTA CCTGA AGGGC ATCC.
3D (18) Forward primer: ACTGG GTTTT ACAAA CCTGT GA; Reverse primer: GCGAG TCCTG
®
CCACG GA and TaqMan probe: TCCTT TGCAC GCCGT GGGAC.
Molecular epidemiology
The molecular epidemiology of FMD is based on the comparison of genetic differences between viruses.
Dendrograms showing the genomic relationship between vaccine and field strains for all seven serotypes
based on sequences derived from the 1D gene (encoding the VP1 viral protein) have been published.
Reverse-transcription PCR (RT-PCR) amplification of FMD virus RNA, followed by nucleotide sequencing,
is the current preferred option for generating the sequence data to perform these comparisons. Many
laboratories have developed techniques for performing these studies, and reference laboratories hold
databases containing over 3000 partial sequences.
The recommended method is to:
i) Extract FMD virus RNA directly from epithelial suspensions or from a low cell culture passage.
ii) Perform an RT-PCR of the complete 1D gene (or if only part of the 1D gene, then the 3’ end of the
gene is more useful).
iii) Determine the nucleotide sequence of the PCR product (or at least 170 nucleotides [preferably 420 for
the SAT types] at the 3’ end of the gene).
A protocol, complete with primer sequences, is available from the OIE Reference Laboratories on request
or can be downloaded from the following World Wide Web URLs:
http://www.iah.bbsrc.ac.uk/virus/picornaviridae/aphthovirus/fmd.htm
http://bvs.panaftosa.org.br/textoc/SerManDid17.pdf

Serological tests for FMD are performed in support of four main purposes namely: 1) to certify individual animals
prior to import or export (i.e. for trade); 2) to confirm suspected cases of FMD; 3) to substantiate absence of
infection; 4) to demonstrate the efficacy of vaccination. For substantiating freedom from infection, different
approaches are required according to whether the population has been vaccinated or not and if vaccination has
been used, whether this has been applied as an emergency application or as part of an ongoing programme of
vaccination. Different tests and different interpretations of test results will be appropriate according to the above-
mentioned purposes and the validation of the selected procedure must take account of the purpose. For
example, test cut-offs may be set at a different threshold for herd-based serosurveillance than is appropriate for
certifying freedom from infection for individual animals for the purposes of international trade.
Serological tests for FMD are of two types; those that detect antibodies to viral structural proteins (SP) and those
that detect antibodies to viral nonstructural proteins (NSPs).
The SP tests are serotype-specific and detect antibodies elicited by vaccination and infection; examples are the
virus neutralisation (VN) test (32), the solid-phase competition ELISA (SPCE; 41, 46) and the liquid-phase
blocking ELISA (LPBE; 35, 36). These tests are serotype-specific and are highly sensitive, providing that the
virus or antigen used in the test is closely matched to the strain circulating in the field. They are the prescribed
tests for trade and are appropriate for confirming previous or ongoing infection in non-vaccinated animals as well
as for monitoring the immunity conferred by vaccination in the field. The VN test requires cell culture facilities, the
use of live virus and takes 2–3 days to provide results. The ELISA tests are blocking- or competition-based
assays that use serotype-specific polyclonal or monoclonal antibodies, are quicker to perform and are not
dependent on tissue culture systems and the use of live viruses. Low titre false-positive reactions can be
expected in a small proportion of the sera in either ELISA test. An approach combining screening by ELISA and
confirming the positives by the VN test minimises the occurrence of false-positive results. Reference sera to
standardise FMD SP serological tests for some serotypes and subtypes are available from the Reference
Laboratory at Pirbright.
The detection of antibody to the NSPs of FMD virus can be used to identify past or present infection with any of
the seven serotypes of the virus, whether or not the animal has also been vaccinated. Therefore the tests can be
used to confirm suspected cases of FMD and to detect viral activity or to substantiate freedom from infection on a
population basis. For certifying animals for trade, the tests have the advantage over SP methods that the
serotype of virus does not have to be known. However, there is experimental evidence that some cattle,
vaccinated and subsequently challenged with live virus and confirmed persistently infected, may not be detected
in some anti-NSP tests, causing false-negative results (17). These assays measure antibody to NSPs using
antigens produced by recombinant techniques in a variety of in-vitro expression systems. Antibody to the
polyproteins 3AB or 3ABC are generally considered to be the most reliable indicators of infection (42). In animals
seropositive for antibody to 3AB or 3ABC, antibody to one or more of the other NSPs can aid in the final
interpretation of the test (14, 42). However, lack of vaccine purity may affect diagnostic specificity as the
presence of NSPs in some vaccine preparations may result in misclassification in animals that have been
repeatedly vaccinated. Procedures for evaluating vaccine purity are covered in Section D of this chapter.
International standard sera for NSP testing of cattle have been developed and are available from the OIE
Reference Laboratory, Panaftosa, PAHO/WHO. In the future, standard sera will also be made available for sheep
and pigs. Bovine serum panels have been established to compare the sensitivity of NSP tests at OIE Reference
Laboratories.
a) Virus neutralisation test (a prescribed test for international trade)

The quantitative VN microtest for FMD antibody is performed with IB-RS-2, BHK-21, lamb or pig kidney cells
in flat-bottomed tissue-culture grade microtitre plates.
Stock virus is grown in cell monolayers and stored at –20°C after the addition of 50% glycerol. (Virus has
been found to be stable under these conditions for at least 1 year.) The sera are inactivated at 56°C for
30 minutes before testing. The control standard serum is 21-day convalescent or post-vaccination serum. A
suitable medium is Eagle’s complete medium/LYH (Hank’s balanced salt solution with yeast lactalbumin
hydrolysate) with hepes buffer and antibiotics.
The test is an equal volume test in 50 µl amounts.
• Test procedure
i) Starting from a 1/4 dilution, sera are diluted in a twofold, dilution series across the plate, using at least
two rows of wells per serum , preferably four rows, and a volume of 50 µl.

ii) Previously titrated virus is added; each 50 µl unit volume of virus suspension should contain about
100 TCID50 (50% tissue culture infective dose) within an accepted range (e.g. 32–320 TCID50).
iii) Controls include a standard antiserum of known titre, a negative serum, a cell control, a medium
control, and a virus titration used to calculate the actual virus titre used in the test.
iv) Incubate at 37°C for 1 hour with the plates covered.
v) A cell suspension at 106 cells/ml is made up in medium containing 10% bovine serum (specific
antibody negative) for cell growth. A volume of 50 µl of cell suspension is added to each well.
vi) Plates are sealed with pressure-sensitive tape and incubated at 37°C for 2–3 days. Alternatively, the
plates may be covered with loosely fitting lids and incubated in an atmosphere of 3–5% carbon dioxide
at 37°C for 2–3 days.
vii) Microscope readings may be feasible after 48 hours. The plates are finally fixed and stained routinely
on the third day. Fixation is effected with 10% formol/saline for 30 minutes. For staining, the plates are
immersed in 0.05% methylene blue in 10% formalin for 30 minutes. An alternative fixative/stain
solution is naphthalene blue black solution (0.4% [w/v] naphthalene blue black, 8% [w/v] citric acid in
saline). The plates are rinsed in tap water.
viii) Positive wells (where the virus has been neutralised and the cells remain intact) are seen to contain
blue-stained cells sheets; the negative wells (where virus has not been neutralised) are empty. Titres
are expressed as the final dilution of serum present in the serum/virus mixture where 50% of wells are
protected (38). The test is considered to be valid when the amount of virus used per well is in the
range log10 1.5–2.5 TCID50, and the positive standard serum is within twofold of its expected titre.
ix) Interpretation of tests can vary between laboratories in regard to the negative/positive cut-off
threshold. Laboratories should establish their own criteria by reference to standard reagents that can
be obtained from the OIE Reference Laboratory at Pirbright. In general, a titre of 1/45 or more of the
final serum dilution in the serum/virus mixture is regarded as positive. A titre of less than 1/16 is
considered to be negative. For certification of individual animals for the purposes of international trade,
titres of 1/16 to 1/32 are considered to be doubtful, and further serum samples may be requested for
testing; results are considered to be positive if the second sample has a titre of 1/16 or greater. For the
purposes of herd-based serosurveillance as part of a statistically valid serological survey, a cut-off of
1/45 may be appropriate. Cut-off titres for evaluating immunological protection afforded by vaccination
have to be established from experience of potency test results with the relevant vaccine and target
species.
b) Solid-phase competition enzyme-linked immunosorbent assay (a prescribed test for international

trade)
Rabbit antiserum to the 146S antigen of one of the seven types of FMD virus is used as the trapping
antibody at a predetermined optimal concentration in carbonate/bicarbonate buffer, pH 9.6.
Antigens are prepared by inactivating viruses propagated in cell culture with ethyleneimine using the
procedures described for vaccine manufacture. The final dilution chosen is that which, after addition of an
equal volume of diluent, gives an absorbance on the upper part of the linear region of the titration curve
(optical density approximately 1.5). PBS containing 0.05% Tween 20, 10% normal bovine serum and 5%
normal rabbit serum and phenol red indicator is used as a diluent (blocking buffer).
Guinea-pig antisera, prepared by inoculating guinea-pigs with 146S antigen of one of the seven serotypes
and preblocking with normal bovine serum, is used as the detecting antibody. Predetermined optimal
concentrations are prepared in blocking buffer PBS containing 0.05% Tween 20, and 5% dried, nonfat
skimmed milk (PBSTM).
Rabbit (or sheep) anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase and preblocked
with NBS is used as conjugate at a predetermined optimum concentration in PBSTM blocking buffer.
Test sera are diluted in PBST blocking buffer.
The solid-phase competitive ELISA is more specific but as sensitive as the liquid-phase blocking ELISA (41,
46). Methods have been described for the development of secondary and working standard sera (33) and
for charting assay performance (34).
• Test procedure
i) ELISA plates are coated with 50 µl/well rabbit anti-FMD virus antigen diluted in carbonate/bicarbonate
buffer, pH 9.6, and left overnight in a humid chamber at 4°C.
ii) The ELISA plates are washed three times with PBS.

iii) Then 50 µl of the FMD virus antigen diluted in blocking buffer is added to each well of the ELISA
plates. (Blocking buffer: 0.05% [w/v] Tween 20, 10% [v/v] normal bovine serum, 5% [v/v] normal rabbit
serum.) The plates are covered and placed on an orbital shaker at 37°C for 1 hour, with continuous
shaking.
iv) After washing three times with PBS, 40 µl of blocking buffer is added to each well, followed by 10 µl of
test sera (or control sera), giving an initial serum dilution of 1/5.
v) Immediately 50 µl of guinea-pig anti-FMD virus antiserum diluted in blocking buffer is added, giving a
final serum dilution of 1/10.
vi) The plates are covered and incubated on an orbital shaker at 37°C for 1 hour.
vii) After washing three times with PBS, 50 µl of anti-guinea-pig Ig conjugate (pre-blocked by incubation
for 1 hour at room temperature with an equal volume of NBS) diluted in blocking buffer is added. The
plates are covered and incubated for 1 hour at 37°C on an orbital shaker.
viii) After washing three times with PBS, 50 µl of substrate solution, containing 0.05% H2O2 plus
orthophenylene diamine or a suitable alternative chromogen, is added to each well.
ix) The reaction is stopped after 10 minutes by the addition of 50 µl of 1 M sulphuric acid. The plates are
read at 492 nm on a spectrophotometer linked to a computer.
x) Controls: On each plate two wells are used for conjugate control (no guinea-pig serum), four wells
each for strong and weak positive sera, two wells for negative sera, and four wells for 0% competition
(no test sera).
xi) Interpretation of the results: A percentage of inhibition is calculated for each well, either manually or
using a suitable computer programme (100 – [optical density of each test or control value/mean optical
density of the 0% competition] × 100%), representing the competition between the test sera and the
guinea-pig anti-FMD virus antisera for the FMD virus antigen on the ELISA plate. Laboratories should
validate the assay in terms of the cut-off value above which sera should be considered positive in
relation to (i) the particular serotypes and strains of virus under investigation (ii) the purpose of testing
(iii) the population under test, using the methods described in Chapter 1.1.4 Principles of validation of
diagnostic assays for infectious diseases. At the OIE Reference Laboratory at Pirbright, for serotype
O, for all species, for the purposes of demonstrating freedom from infection in a naïve population,
greater than 60% inhibition is considered positive (46). For maximum sensitivity, for example when
certifying individual animals for international trade, an inconclusive range may be set between 40 and
60%.
c) Liquid-phase blocking enzyme-linked immunosorbent assay (a prescribed test for international

trade)
Antigens are prepared from selected strains of FMD virus grown on monolayers of BHK-21 cells. The
unpurified supernatants are used and pretitrated in a twofold dilution series but without serum. The final
dilution chosen is that which, after addition of an equal volume of diluent (see below), gives an absorbance
on the upper part of the linear region of the titration curve (optical density approximately 1.5). PBS
containing 0.05% Tween 20 and phenol red indicator is used as a diluent (PBST). The other reagents used
in the test are the same as those in the solid-phase blocking ELISA. An example of the test procedure is
described below. Temperature and incubation times can vary depending on the protocol.
• Test procedure
i) ELISA plates are coated with 50 µl/well rabbit antisera to the 14S antigen being tested for and left
overnight in a humid chamber at room temperature.
ii) The ELISA plates are washed three times with PBS.
iii) In U-bottomed multiwell plates (carrier plates) 50 µl of a duplicate, twofold series of each test serum is
prepared, starting at 1/8. To each well, 50 µl of a constant dose of viral antigen that is homologous to
the rabbit antisera used to coat the plates is added and the mixtures are left overnight at 4°C, or
incubated at 37°C for 1 hour. The addition of the antigen increases the final serum dilution to 1/16.
iv) Then 50 µl of serum/antigen mixtures is transferred from the carrier plates to the rabbit-serum coated
ELISA plates and the plates are incubated at 37°C for 1 hour on a rotary shaker.
v) After washing, 50 µl of guinea-pig antiserum homologous to the viral antigen used in the previous step
(iv) (pre-blocked with normal bovine serum and diluted in PBST containing 5% skimmed milk powder)
is added to each well. The plates are then incubated at 37°C for 1 hour on a rotary shaker.
vi) The plates are washed and 50 µl of rabbit anti-guinea-pig immunoglobulin conjugated to horseradish
peroxidase (pre-blocked with normal bovine serum and diluted in PBST containing 5% skimmed milk
powder) is added to each well. The plates are incubated at 37°C for 1 hour on a rotary shaker.
vii) The plates are washed again three times and 50 µl of substrate solution, containing 0.05% H2O2 plus
orthophenylene diamine or a suitable alternative chromogen, is added to each well.

viii) The reaction is stopped after 15 minutes by the addition of 50 µl of 1 M sulphuric acid. The plates are
read at 492 nm on a spectrophotometer linked to a computer.
ix) Controls: A minimum of four wells each of strong positive, weak positive and negative bovine
reference sera at a final dilution of 1/32 should be included on each plate together with an equivalent
number of reaction (antigen) control wells containing antigen in diluent alone without serum. For end-
point titration tests, duplicate twofold dilution series of positive and negative homologous bovine
reference sera should be included on at least one plate of every run.
x) Interpretation of the results: Antibody titres are expressed as the 50% end-point titre, i.e. the dilution at
which the reaction of the test sera results in an optical density equal to 50% inhibition of the median
optical density of the reaction (antigen) control wells (38). The median is calculated as the mean of two
mid-values of the reaction control wells, eliminating from the calculation the highest and lowest values
(alternatively, the mean value can be used after setting suitable tolerance limits to control for inter-well
variation). In general sera with titres greater than or equal to 1/90 are considered to be positive. A titre
of less than 1/40 is considered to be negative. For certification of individual animals for the purposes
of international trade, titres of greater than 1/40, but less than 1/90 are considered to be doubtful, and
further serum samples may be requested for testing; results are considered to be positive if the
second sample has a titre of 1/40 or greater. For the purposes of herd-based serosurveillance as part
of a statistically valid serological survey, a cut-off of 1/90 may be appropriate. Cut-off titres for
evaluating immunological protection afforded by vaccination have to be established from experience of
potency test results with the relevant vaccine and target species.
d) Nonstructural protein antibody tests

Antibody to expressed recombinant FMD virus NSPs (e.g. 3A, 3B, 2B, 2C, 3ABC) can be measured by
different ELISA formats or immunoblotting. These ELISAs either use purified antigens absorbed directly to
microplates or use polyclonal or monoclonal antibodies to trap specific antigens from semi-purified
preparations (14, 20, 42, 59). The index screening method used in Panaftosa is described in detail below.
Other indirect and competitive ELISAs detecting bovine antibodies to 3ABC have been shown to have
equivalent diagnostic performance characteristics (17). This same study corroborates preliminary data from
Panaftosa that suggests that the diagnostic performance characteristics of these tests are similar in cattle,
sheep and pigs.
• Indirect enzyme-linked immunosorbent assay

• Preparation of recombinant antigens (see Section B.2.d Enzyme-linked immunoelectrotransfer blot
assay below)
• Test procedure
i) Microplates are coated overnight at 4°C with 1 µg/ml of the fusion antigen 3ABC in carbonate/
bicarbonate buffer, pH 9.6 (100 µl per well). Antigen 3ABC was expressed and purified as indicated for
the EITB (enzyme-linked immunoelectrotransfer blot) tests (45).
ii) The plates are washed six times with PBS, pH 7.2, supplemented with 0.05% Tween 20 (PBST).
iii) Test sera (100 µl per well) are added in a 1/20 dilution in blocking buffer consisting of PBS, 0.05%
Tween 20, 5% nonfat dry milk, 10% equine sera and 0.1% Escherichia coli lysate. Each plate includes
a set of strong and weak positive and negative controls calibrated against the International Standard
Sera described below.
iv) The plates are incubated for 30 minutes at 37°C and washed six times in PBST.
v) Horseradish-peroxidase-conjugated rabbit anti-species IgG is diluted optimally in the blocking buffer,
added at 100 µl per well and the plates are incubated for 30 minutes at 37°C.
vi) After six washings, each well is filled with 100 µl of 3’3’, 5’5’-tetramethylbenzidine plus 0.004% (w/v)
H2O2 in phosphate/citrate buffer, pH 5.5.
vii) The reaction is stopped after 15 minutes of incubation at room temperature by adding 100 µl of 0.5 M
H2SO4. Absorbance is read at 450 nm and at 620 nm for background correction.
viii) Interpreting the results: Test results are expressed as per cent positivity relative to the strong positive
control [(optical density of test or control wells/optical density of strong positive control) × 100] or
alternatively as a test to control (T/C) index relative to a cut-off (i.e. threshold positive) control. Profiling
the NSP antibody reactivity levels in herds along with age/vaccination stratification aids interpretation
of herd infection status in vaccinated populations (15). Test cut-off values, with or without suspicious
zones, need to be determined with consideration to the purpose of testing and the intended target
population. Inconclusive results may be followed up using confirmatory tests. In case of multi-
vaccinated animals, EITB is the recommended approach, whereas, in animals that have received only
one or two vaccinations, inconclusive results are resolved and positive results confirmed by retesting
with a second NSP ELISA (taking account of the conditional dependence of the two tests). This must

take into account the overall test system sensitivity and specificity when designing the serosurveillance
programme. Although not a prescribed test for trade, NSP ELISAs may be a valuable adjunct in
circumstances where the serotype or subtype of virus in the originating country is not known.
• Enzyme-linked immunoelectrotransfer blot assay (EITB)

The EITB assay has been widely applied in South America as a confirmatory test for the above-described
index screening method. Further information is available from the OIE Reference Laboratory, Panaftosa,
PAHO/WHO.
• Preparation of test strips containing the recombinant antigens

i) The five bioengineered FMD virus NSPs 3A, 3B, 2C, 3D and 3ABC are expressed in E. coli C600 by
thermo-induction. The 3D polypeptide is expressed in its complete form (45), whereas the rest of the
proteins are obtained as fusions to the N-terminal part of the MS-2 polymerase gene (60).
ii) The expressed polymerase is purified over phosphocellulose, followed by poly(U) Sepharose columns.
The fused proteins 3A, 3B, 2C and 3ABC are purified by sequential extraction of the bacterial extracts
with increasing concentrations of urea. The 7M fraction containing the fusion proteins is further purified
on a preparative 10% SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis). The
fusion protein band is excised from the gel and electroeluted (45).
iii) A mixture containing 20 ng/ml of each one of the purified recombinant polypeptides is separated on
12.5% SDS-PAGE and electrophoretically transferred to nitrocellulose (45).
• Test procedure
i) The required amount of test strips should be assessed, taking into account that for each nitrocellulose
sheet, which defines one transferred gel, a positive, a weakly positive, a cut-off and a negative control
serum should be assayed. In general, 24 nitrocellulose strips, each 3 mm wide, should result from a
gel.
ii) A volume of 0.8 ml of saturation buffer (50 mM Tris/HCl, pH 7.5; 150 mM NaCl; 0.2% Tween 20;
5% nonfat dry milk; and 0.05% bacterial E. coli lysate) is added to each well. The antigen-coated strips
are blocked by placing the trays on a rocker and agitating for 30 minutes at room temperature (20–
22°C).
iii) A dilution of 1/200 of test sera and of each of the controls is added to the appropriate trough. The
strips must be completely submerged and facing upwards, and maintained in that position during the
whole process.
iv) Strips are incubated for 60 minutes on a rocker at room temperature.
v) Liquid is removed from the trays, and each test strip is washed three times with washing solution
(50 mM Tris/HCl, pH 7.5; 150 mM NaCl; and 0.2% Tween 20) by agitation for 5 minutes.
vi) The alkaline-phosphatase-conjugated rabbit anti-bovine solution is added to each test well, and the
strips are incubated with shaking for 60 minutes at room temperature.
vii) The liquid is removed from the trays and each test strip is washed three times with washing solution as
above.
viii) Substrate solution (0.015% bromochloroindolylphosphate/0.03% nitroblue tetrazolium) is prepared in
substrate buffer (100 mM NaCl; 5 mM MgCl2; and 100 mM Tris/HCl, pH 9.3), and is added to each test
well.
ix) Strips are incubated by placing the test tray on the orbital mixer and agitating until the cut-off control
shows five distinct, discernible bands. Strips are washed with running deionised water and air-dried.
x) Interpreting the results: The EITB may be scanned with a densitometer but visual reading, although
more subjective, is considered suitable as well. Individual control sera are run that exhibit minimal but
consistent staining for each of the five antigens. A test sample is considered positive if antigens 3ABC,
3A, 3B and 3D (±2C) demonstrate staining densities equal to or higher than that of their appropriate
controls. A sample is considered negative if two or more antigens demonstrate densities below their
control sera. Test samples not fitting either profile are considered indeterminate.
C. VACCINE MATCHING TESTS
1. Introduction
The selection of vaccine strains has recently been reviewed (48). Vaccination against one serotype of FMDV
does not cross-protect against other serotypes and may also fail to protect fully or at all against other strains of

the same serotype. The most direct and reliable method to measure cross-protection is to vaccinate relevant
target species and then to challenge them by exposure to the virus isolate against which protection is required.
This will take account of both potency and cross-reactivity. However, such an approach is slow and expensive,
and the use of animals for such studies should be avoided where possible by the use of in vitro alternatives.
A variety of in vitro serological methods can be used to quantify antigenic differences between FMDV strains and
thereby estimate the likely cross-protection between a vaccine strain and a field isolate. Genetic characterisation
and antigenic profiling can also reveal the emergence of new strains for which vaccine matching may be required
and, conversely, may indicate that an isolate is similar to one for which vaccine matching information is already
available.
Appropriate vaccine strain selection is an important element in the control of FMD and is necessary for the
application of vaccination programmes in FMD-affected regions as well as for the establishment and
maintenance of vaccine antigen reserves to be used in the event of new FMD incursions.
Vaccine potency also contributes to the range of antigenic cover provided by a vaccine. A highly potent vaccine
that stimulates a strong immune response may give greater protection against a heterologous virus than an
equally cross-reactive vaccine that stimulates a weaker immune response. Furthermore, booster doses of
vaccine can increase efficacy and the subsequent breadth of antigenic cover provided by a given vaccine,
although the onset of full protection may be delayed.
2. Selection of field viruses for vaccine matching
Serological matching of field isolates to vaccine strains requires that isolates have been serotyped and adapted
to growth in cell cultures. The serotype is usually determined by ELISA or CFT using type-specific serological
reagents, although methods based on monoclonal antibodies or genetic typing may also be used. BHK or IB-RS-
2 cell cultures are usually used for in vitro virus replication. For vaccine matching, preferably, at least two isolates
should be evaluated from any outbreak and inconsistent results should be followed up to determine whether this
is due to genuine antigenic differences or is an artefact of testing.
Viruses can be selected based on epidemiological information, for instance isolation at different stages of an
epidemic, from different geographic locations or from different hosts (4). Field evidence for a suspected lack of
vaccine efficacy, as shown by reduced apparent protection, is an important criterion for vaccine matching.
Antigenic profiling by CFT or ELISA, or sequence analysis of the VP1 gene, are suitable approaches for selecting
representative virus isolates for vaccine matching. Antigenic profiling is performed by CFT using panels of
hyperimmune guinea-pig sera raised against epidemiologically relevant field isolates (16) or by ELISA using
panels of well-characterised monoclonal antibodies (5).
3. Selection of vaccine strains to be matched
The serotype of the virus, the region of origin and any information on the characteristics of the field isolate may
give indications as to the vaccine strains most likely to provide an antigenic match. The availability of reagents for
matching to particular vaccine strains may limit the extent of testing that is possible. Antigenic characterisation
has two purposes; first, to chose the most effective vaccine strain for use in a particular circumstance and,
second, to monitor, on an ongoing basis, the suitability of vaccine strains maintained in strategic antigen
reserves.
4. Choice of vaccine matching test
The serological relationship between a field isolate and a vaccine virus (‘r’ value) can be determined by CFT,
ELISA or VNT (40, 49, 55). One way testing is recommended (r1) with a vaccine antiserum, rather than two way
testing (r2) which also requires an antiserum against the field isolate to be matched. Due to the inherently low
repeatability of the assays used, tests need to be repeated to be confident of the results (56). In vitro
neutralisation may be more relevant to in vivo protection than other measures of virus-antibody interaction,
although non-neutralising antibodies may also be protective (43). Advantages of ELISA are that the test is rapid
and uses smaller volumes of post-vaccination sera, which are often available in only limited quantities. ELISA
and CFT are recommended to be used as screening methods, whereas VNT or the expected percentage of
protection (EPP) method provides more definitive results. For either VNT or ELISA, post-vaccination sera should
be derived from at least five cattle 21–30 days after immunisation. The titre of antibody to the vaccine strain is
established for each serum. Sera are used individually or pooled, after excluding low responders. The CFT
method uses guinea-pig sera raised against vaccine strains.
A more thorough evaluation is provided by the EPP method (4), which measures the reactivity of a panel of post-
vaccination antisera using either VNT or ELISA and relates the serological titres to the probability of protection,
established through correlation tables associating antibody titres with protection against the relevant vaccine

strain. These correlation tables derive from previously performed vaccine-specific challenge tests. However, the
requirement for a panel of antisera and accompanying challenge test data for the vaccine in question currently
cannot be met for a wide range of vaccine strains.
a) Vaccine matching by ELISA

This test uses an antiserum raised against a vaccine strain. The blocking ELISA titres of this reference
serum against antigens prepared from the homologous vaccine strain and are compared with the
corresponding titres of the serum against a field isolate to determine how antigenically ‘similar’ the field
virus is to the vaccine virus.
The test procedure is similar to that of the liquid phase blocking ELISA (see Section B.2.c). Additional
biological reagents are: 21–30 day post-vaccination bovine vaccine sera (inactivated at 56°C for 45–
60 minutes); the homologous vaccine strain; and the test virus, a field isolate of the same serotype as the
vaccine strain.
• Test procedure
i) Grow the field isolate and the vaccine strain in BHK or IB-RS-2 cells. The number of cell culture
passages should be kept to a minimum (normally less than four) to avoid selection of antigenic
variants unrepresentative of those in the original material. A sufficient quantity of virus should be
present if cell cultures show CPE within 24 hours of inoculation.
ii) Harvest and titrate the vaccine and field viruses using a panel of trapping rabbit antisera and detector
guinea pig antisera raised against the same or closely related vaccine strains. If necessary, the virus
antigens may be inactivated prior to use using binary ethyleneimine.
iii) Select the optimum trapper/detector combination and the working dilution of the field virus. This should
not be less than 1/6. If there is no suitable trapper/detector combination then a back-titration of the
antigen stock must be performed to confirm that sufficient virus is present. If it is confirmed that the
field virus is present at high tire, this indicates that none of the available vaccine strains are suitable.
iv) Titrate 21–30 day post-vaccination serum of a chosen vaccine strain against the field isolate and the
homologous vaccine strain. The titre against the vaccine strain should not fluctuate more than twofold
either side of the running mean value for the virus stock.
v) To determine the serum titre, calculate the average optical density (OD) of 24 antigen control wells
without blocking serum. This represents the maximum OD value for the test, i.e. the 100% control
value. Divide this by 2 to determine the 50% inhibition value. Score wells with blocking serum positive
if the OD is less than or equal to 50% and negative if the OD value is greater than this. The end-point
is defined as the dilution at which half of the wells show 50% inhibition or more (i.e. identify the dilution
at which one out of the two duplicate wells has an OD less than 50% of the antigen control). If the end-
point falls between two dilutions, it is taken as the mid-point between these dilutions as estimated by
the Spearman–Kärber method.
Derive the ‘r’ value, the relationship between the field and the vaccine strain, as:
r1 = reciprocal titre of reference serum against field virus
reciprocal titre of reference serum against vaccine virus
At least two consistent results are needed for acceptance.
vi) Interpretation of the results: for r1 values derived by ELISA the following guidelines are used for
interpretation (29):
0.4–1.0: Close relationship between field isolate and vaccine strain. A potent vaccine containing the
vaccine strain is likely to confer protection.
0.2–0.39: The field isolate is antigenically related to the vaccine strain. The vaccine strain might be
suitable for use if no closer match can be found provided that a potent vaccine is used and animals
are preferably immunised more than once.
<0.2: The field isolate is only distantly related to the vaccine strain and the vaccine strain is unlikely to
protect against challenge with the field isolate.
b) Vaccine matching by two-dimensional neutralisation test

This test also uses an antiserum raised against a vaccine strain. The titres of this serum against 100 TCID50
of the homologous vaccine strain and the same dose of a field isolate are compared to determine how
antigenically ‘similar’ the field virus is to the vaccine strain.

The procedure is similar to that of the microtitre plate virus neutralisation test (see Section B.2.a). Additional
biological reagents are: 21–30 day post-vaccination bovine vaccine sera (inactivated at 56°C for 45–
60 minutes); the homologous vaccine strain; and the test virus, a field isolate of the same serotype as the
vaccine strain.
i) Field isolates are passaged on cell cultures until adapted to give 100% CPE in 24 hours. Passages
should be kept to a minimum. When adapted, determine the virus titre (log10 TCID50/ml) by end-point
titration.
ii) For each test and vaccine virus a chequerboard titration is performed of virus against vaccine serum
along with a back-titration of virus alone. Cells are added and incubated at 37°C for 48–72 hours after
which time CPE is assessed.
iii) Antibody titres of the vaccine serum against the vaccine strain and field isolate for each virus dose
used are calculated using the Spearman-Kärber method. The titre of the vaccine serum against
100 TCID50 of each virus can then be estimated by regression. The relationship between the field
isolate and the vaccine strain is then expressed as an 'r' value as described for vaccine matching by
ELISA.
iv) Interpretation of the results: in the case of neutralisation, r1 values greater than 0.3 indicate that the
field isolate is sufficiently similar to the vaccine strain that use of the vaccine is likely to confer
protection against challenge with the field isolate (54). Conversely, values less than 0.3 suggest that
the field isolate is so different from the vaccine strain that the vaccine is unlikely to protect. In these
cases, either the field isolate should be examined against alternative vaccine strains or, rarely, it will
be necessary to adapt a suitable field isolate to become a new vaccine strain.
v) Tests should always be repeated more than once. The confidence with which ‘r’ values can be taken
to indicate differences between strains is related to the number of times that the examination is
repeated. In practice, a minimum of at least three repetitions is advised.
c) Vaccine matching by CFT

The relationship between a field isolate and a vaccine strain can also be determined by complement fixation
using a guinea-pig antiserum raised against the relevant vaccine strain.
CFT 50% titres of this reference serum against antigens prepared from the homologous vaccine strain and
a field isolate are compared to determine how antigenically ‘similar’ the field virus is to the homologous
vaccine virus.
i) Field isolates are passaged on cell cultures until adapted to give 100% CPE in 24 hours. Passages
should be kept to a minimum. When adapted, the virus titre that fixes 2.5 CFU50 (50% complement
fixing units) is determined.
ii) A relationship is established by titration of the guinea-pig antisera through a twofold dilution series
against 2.5 CFU50 of the homologous and heterologous antigens in veronal buffer diluent (VBD) or
borate saline solution (BBS) placed in separate tubes. Four haemolysis units of complement are then
added to each reaction.
iii) The test system is incubated at 37°C for 30 minutes prior to the addition of 2% of standardised sheep
red blood cells (SRBC) in VBD or BSS sensitised with rabbit anti SRBC. Reagents are incubated at
37°C for a further 30 minutes and the tubes are subsequently centrifuged and read.
iv) The CFT 50% titres are calculated by the Spearman-Kärber method and an ‘r’ value is derived from
the relationship between the reactivity of the field isolate and the vaccine strain, as:
r1 = Reciprocal titre of hyperimmune serum against field virus
Reciprocal titre of hyperimmune serum against vaccine virus
v) Interpretation of the results: in the case of CFT, r1 values greater than 0.25 indicate that the field
isolate is sufficiently similar to the vaccine strain and that use of the vaccine is likely to confer
protection against challenge with the field strain (3).
d) Expected percentage of protection

The expected percentage of protection (EPP) estimates the likelihood that cattle would be protected against
a challenge of 10,000 infective doses after a single or boosted vaccination.
i) Individual sera are required from 16 or 30 18–24 month-old cattle at 30 days post-vaccination and
30 days post-re-vaccination, using a full dose of the vaccine strain to be matched.

ii) This panel of sera is tested for antibody titres to the homologous FMD vaccine strain and the field
isolate to be matched using VNT or LPB-ELISA (see Sections B.2.a and B.2.c).
iii) If necessary, the antigens used in the ELISA may be inactivated prior to use using binary
ethyleneimine.
iv) The EPP is determined from the serological titre obtained, for each individual serum, by reference to
predetermined tables of correlation between serological titres and clinical protection. The mean EPP is
then calculated from the EPP for each individual serum.
v) The clinical protection data are derived from previously performed experiments carried out on
hundreds of cattle that have been immunised using the vaccine strain in question and challenged with
a homologous virus (similar to the PGP potency tests described in Section D.4.b). Each animal is
scored as protected or not and tables of correlation based on logistic regression models are
established between antibody titre and clinical protection.
vi) An EPP <75% (when sera from a group of 16 re-vaccinated animals are used) and <70% (when sera
from a group of 30 re-vaccinated animals are used) is an indication that the vaccines will give a low
protection against the field strain (47).
D. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS

The control of FMD is usually a national responsibility and, in many countries, the vaccine may be used only
under the control of the Competent Authority.
supplemented by national and regional requirements. Varying requirements relating to quality, safety and efficacy
apply in particular countries or regions in order for manufacturers to obtain an authorisation or licence for a
veterinary vaccine. Where possible, manufacturers should seek to obtain such a license or authorisation for their
FMD vaccines as independent verification of the quality of their product.
Virulent FMD virus must be used to produce FMD vaccine; consequently, the FMD vaccine production facility
should operate under the appropriate biosecurity procedures and practices. The facility should meet the
requirements for Containment Group 4 pathogens as outlined in Chapter 1.1.2 of this Terrestrial Manual.
Routine vaccination against FMD is used in many countries or zones recognised as free from foot and mouth
disease with vaccination and in countries where the disease is endemic. In contrast, a number of disease-free
countries have never vaccinated their livestock but have preferred the use of strict movement controls and culling
of infected and contact animals when outbreaks have occurred. Nevertheless, many disease-free countries
maintain the option to vaccinate and have their own strategic reserves of highly concentrated inactivated virus
preparations. Such antigen reserves offer the potential of supplying formulated vaccine in an ‘emergency’ at
short notice (26).
FMD vaccines may be defined as a fixed formulation containing defined amounts (limits) of one or more
chemically inactivated cell-culture-derived preparations of a seed virus strain blended with a suitable adjuvant/s
and excipients. The vaccines are formulated for their specific purpose and in the case of vaccines destined for
use in swine, oil adjuvants are preferred. Oil adjuvanted vaccines can also be used in ruminants and may have
advantages in terms of less interference from maternal antibody and a longer duration of immunity. FMD
vaccines may be classified as either ‘standard’ or ‘higher’ potency vaccines. Standard potency vaccines are
formulated to contain sufficient antigen to ensure that they meet the minimum potency level required
(recommended at Section D.4.b as 3 PD50 [50% protective dose]). Higher potency vaccines are formulated with
an increased amount of antigen such that the potency is in excess of the minimum requirement to provide
particular features such as a more rapid onset of immunity and a wider spectrum of immunity against relevant
field viruses. Higher potency vaccines are thus well suited for emergency use. Live FMD vaccines are not
acceptable due to the danger of reversion to virulence and as their use would prevent the differentiation of
infected from vaccinated animals.
Because of the presence of multiple serotypes of the virus, many FMD vaccines are multivalent and it is common
practice to prepare vaccines from two or more different virus strains. In certain areas, it may be advisable to
include more than one virus per serotype to ensure broad antigenic coverage against prevailing viruses.
1. Seed virus management
a) Characteristics of the seed virus

Selection of master seed viruses (MSVs) should ideally be based on their ease of growth in cell culture,
virus yield, stability and broad antigenic spectrum (57). MSVs should be characterised and distributed by

the official control laboratories in regions where such laboratories exist; they should be selected in
accordance with the epidemiological importance of each variant.
Where no suitable established vaccine strain exists, new vaccine strains are derived through the
establishment of MSVs from local field isolates by adapting them to growth in suspension or monolayer
cells by serial passage. In order to remove the risk of contaminating lipid-enveloped viruses, it is
recommended that putative MSVs undergo a validated organic solvent treatment prior to, or during,
adaptation. It is preferable to keep the number of passages in cell culture to a minimum as there is
evidence of antigenic ‘drift’ of FMD virus during this procedure.
c) Validation as a vaccine strain

MSVs must be antigenically and, if possible genetically, characterised and proven to be pure and free from
all extraneous agents listed by the appropriate licensing authorities. Homology should be established with
the original candidate isolates and effectiveness against the circulating strains from which they were
developed should be proven. This often encompasses a number of methods, the most reliable being in vivo
cross protection assays. Alternatively, in vitro tests (preferably virus neutralisation) can also be used, which
require the availability of post-vaccination sera against these master seeds. Seed viruses may be stored at
–20°C if glycerinated or at a lower temperature (e.g. –70°C) if not glycerinated. Working seed viruses may
be expanded in one or a few more passages from the master seed stock and used to infect the final cell
culture at an approximate rate of 1 PFU (plaque-forming unit) per 100 cells. Whenever possible, the exact
source of the isolate should be recorded and should include details such as the location, species and the
type of material from which the virus was derived. The in-vitro passage history of the virus should be
recorded. Consideration should also be given to minimising the risk of transmission of transmissible
spongiform encephalopathy agents (TSEs) by ensuring that TSE risk materials are not used as the source
of the virus or in any of the media used in virus propagation.
The recommended method of virus propagation for antigen production is the growth of FMD virus in large-scale
suspension cultures or monolayers using cell lines under sterile conditions. Primary cell culture may be
acceptable for vaccine production in some countries but only if the method of production is entirely compliant with
Good Manufacturing Practice, a validated procedure is applied to ensure inactivation of all possible extraneous
agents and adequate in-process and final products tests are in place to ensure consistency and safety of the
final product. It is essential that all pipework and vessels be thoroughly sterilised ensuring that no areas in the
system harbour microorganisms. In addition to general considerations of sterility, it is important to note that the
virus is vulnerable to attack by proteolytic enzymes, such as those produced by microorganisms (22). Control of
pH and temperature are also critical because of the acid and temperature lability of the virus (21). Optimum
temperature for cell, virus growth and inactivation, normally around 37°C and 26°C, respectively, should be
precisely controlled. During other stages of manufacture, the temperature should be reduced to 4–6°C. Virus
should be maintained at approximately pH 7.6 and should never be below pH 7.0.
A suitable strain of the virus is used to infect a suspension or monolayers of an established cell line, such as
BHK. Such cell cultures should be proven to be free from contaminating microorganisms. It is common practice
to keep stocks of BHK cells over liquid nitrogen and revive as necessary. On revival, they are expanded in
nutrient medium to a volume and cell density appropriate to seeding the main culture. As an approximation, the
main culture is seeded to give an initial density of 0.2–0.5 × 106 cells/ml, which is allowed to multiply to 2–
5 × 106 cells/ml before being infected with virus.
When the virus has reached its maximum titre, which is variously determined by infectivity, CF or other tests, the
culture is clarified, often by chloroform treatment followed by centrifugation and filtration. The virus is
subsequently inactivated by addition of ethyleneimine, usually in the form of binary ethyleneimine (BEI). This is
usually prepared by dissolving, to a concentration of 0.1 M, 2-bromoethylamine hydrobromide in 0.2 N sodium
hydroxide solution, and incubating at 37°C for 1 hour (9, 10). The BEI formed is then added to a virus suspension
held at 26°C, to give a final concentration of 3 mM. Inactivation is usually continued for 24 hours, followed by a
second dose of BEI for a further 24 hours. The time period for BEI treatment and temperature used for
inactivation must be validated for the actual conditions and equipment used. After inactivation any residual BEI in
the harvest can be neutralised by adding sodium thiosulphate solution to a final concentration of 2%. To
decrease the likelihood of live virus failing to contact the EI at the second application, it is essential to transfer the
vessel contents immediately to a second sterile vessel where inactivation is allowed to go to completion at
48 hours.
The inactivated virus may be concentrated by ultrafiltration, polyethylene glycol precipitin or polyethylene oxide
adsorption (1, 62) concentrated inactivated virus may be purified further by procedures such as chromatography.
These concentrated antigens can be kept at –70°C or lower temperatures for many years, if necessary, and
made into vaccine when required by dilution in a suitable buffer and addition of adjuvants (24).

Conventional FMD vaccines are usually formulated as aqueous or oil adjuvanted. The aqueous vaccine, which is
most commonly used for cattle is prepared by adsorbing the virus on to aluminium hydroxide gel, one of the
adjuvant constituents of the final vaccine blend. Other components of the final blend include antifoam, phenol red
dye (if permitted by the country requiring vaccine), lactalbumin hydrolysate, tryptose phosphate broth, amino
acids, vitamins and buffer salts. A second adjuvant, saponin, derived from the South American tree Quillaja
saponaria mollina, is also incorporated, as well as a preservative such as merthiolate or chloroform.
Oil-adjuvanted vaccines are usually formulated using mineral oils, such as Marcol and Drakeol, as adjuvants.
These preparations offer a number of advantages over the standard aluminium hydroxide/saponin vaccine, not
least of which is their efficacy in pigs. They are widely used for vaccinating cattle in South America because of
the longer duration of immunity. The mineral oil is usually premixed with an emulsifying agent, such as mannide
monooleate, before the addition of a proportion, or all, of the aqueous phase of the vaccine, and emulsified by
use of a colloid mill or continuous mechanical or flow ultrasonic emulsifier. More complex double emulsions
(water/oil/water) may be produced by emulsifying once more in an aqueous phase containing a small amount of
detergent such as Tween 80 (37).
A further alternative are the ‘ready-to-use’ oil adjuvants. Oils containing esters of octadecenoic acid and
2,5 anhydro-d-mannitol, for example, readily form double or mixed emulsions (water/oil/water) that are both
stable and of low viscosity, without the requirement of sophisticated emulsification equipment (11, 26). When
using novel components, including adjuvants, in any vaccine it is important to take into account that its status
with regard to residues in products derived from food producing species must be assessed to ensure that
adequate assurance can be giving to licensing authorities in relation to safety for consumers. This requirement
limits considerably the choice of adjuvants for use in food producing species.
In general, virus titres reach optimum levels within about 24 hours of the cell culture being infected. The time
chosen to harvest the culture may be based on a number of assays; for instance cell death. Virus concentration
may be assessed by infectivity test, sucrose density gradient (23) or serological techniques. It is preferable to
use a method for measuring antigenic mass, such as sucrose density gradient analysis, as well as one that
measures infectivity, as the two properties do not necessarily coincide and the different methods may
complement one another.
During inactivation of the virus, timed samples should be taken at regular intervals for the purpose of monitoring
the rate and linearity of the inactivation process. Virus titres in the samples are determined by inoculation of cell
cultures proven to be highly susceptible to FMD virus, e.g. BHK or bovine thyroid cells. Such cultures permit the
testing of statistically meaningful samples under reproducible conditions. The log10 infectivities of the timed
samples are plotted against time, and the inactivation procedure is not considered to be satisfactory unless at
least the latter part of the slope of the line is linear and extrapolation indicates that there would be less than one
infectious particle per 104 litres of liquid preparation at the end of the inactivation period.
a) Safety
Tests for innocuity (non-infectivity) are most effectively carried out on the bulk, concentrated, inactivated
viral harvest (see Sections D.3 and D.5.b, below). Although it may be possible to confirm innocuity by
testing virus eluted from the vaccine, this is not universally applicable to all formulations and is not as
reliable as testing concentrated antigens. For example, saponin influences greatly the elution of FMD virus
from aluminium hydroxide/saponin vaccines (25). If the elution procedure is appropriate to a particular
formulation, then it may be validated by seeding parallel samples of vaccine with trace amounts of live virus
(12).
For the purposes of gaining regulatory approval, a trial batch of vaccine should be tested for local and
systemic toxicity by each recommended route of administration in an in-vivo test in an appropriate number
of cattle (27). Double dose and repeat dose tests using vaccines formulated to contain the maximum
permitted amount and number of antigens should be conducted using a similar protocol described below for
batch safety tests.
b) Potency
Cattle of at least 6 months of age, obtained from areas free from FMD that have not previously been
vaccinated against FMD and are free from antibodies to the different types of FMD virus should be used.
Three groups of no fewer than five cattle per group should be vaccinated by the route recommended by the
manufacturer. The vaccine should be administered at different doses per group by injecting different
volumes of the vaccine. For example, if the label states that the injection of 2 ml corresponds to the
administration of 1 dose of vaccine, a 1/4 dose of vaccine would be obtained by injecting 0.5 ml, and a

1/10 dose would be obtained by injecting 0.2 ml. These animals and a control group of two nonvaccinated
animals are challenged either 3 weeks (aqueous) or up to 4 weeks (oil) after vaccination with a suspension
of bovine virus that is fully virulent and appropriate to the virus types in the vaccine under test by inoculating
the equivalent of a total of 10,000 ID50 (50% bovine infectious dose) intradermally into two sites on the
upper surface of the tongue (0.1 ml per site). Animals are observed for at least 8 days. Unprotected animals
show lesions at sites other than the tongue. Control animals must develop lesions on at least three feet.
From the number of animals protected in each group, the PD50 (50% bovine protective dose) content of the
vaccine is calculated. There are a variety of methods for calculating PD50 (31), but procedures based on the
Kärber (38) method are generally preferred. The vaccine should contain at least 3 PD50 per dose for cattle,
when employed for routine prophylactic use, although 6 PD50 per dose is more commonly preferred. In
some cases, vaccine of high potency will prevent the development of local tongue lesions at the site of
challenge.
For the PGP test (percentage of protection against generalised foot infection) a group of 16 FMD-
seronegative cattle of at least 6 months of age, with the same characteristics described for the PD50 test,
are vaccinated with a full vaccine dose by the route recommended by the manufacturer. These animals and
a control group of two nonvaccinated animals are challenged 4 weeks or more after vaccination with the
challenge strain, which is a suspension of bovine virus that is fully virulent and appropriate to the virus types
in the vaccine under test by inoculating a total of 10,000 BID50 (50% bovine infectious dose), intradermally
into at least two sites on the upper surface of the tongue. Unprotected animals show lesions at sites other
than the tongue within 7 days after inoculation. Control animals must develop lesions on at least three feet;
for routine prophylactic use, the vaccine should protect at least 12 animals out of 16 vaccinated. Animals
are observed at 7–8 days after challenge (61).
Potency tests in other target species, such as sheep, goats or buffalo are either different or not yet
standardised. In general, a successful test in cattle is considered to be sufficient evidence of the quality of a
vaccine to endorse its use in other species. Under circumstances where a vaccine is produced for use
primarily in a species other than cattle, it may be more appropriate to potency test the vaccine in that same
species. With respect to African or Asiatic buffalo (Bubalus bubalis) and sheep, due to the often inapparent
nature of the disease in these species, potency results from a cattle test may be a more reliable indicator of
vaccine quality than attempting a potency test reliant on the detection of clinical signs in these other
species.
A similar protocol to the cattle test can be adopted for potency testing FMD vaccines in pigs using three
groups of five pigs, not less than 2 months old and free from antibodies neutralising the different serotypes
of FMD virus. One group is vaccinated with the full pig dose recommended by the manufacturer, one group
receives a reduced dose e.g. 1/4 dose, and a third group receives a further reduced dose e.g. 1/16 dose of
the vaccine. Traditionally, the response to oil vaccine is allowed longer to develop, and not until day 28 after
vaccination are the three groups, plus two unvaccinated control pigs challenged. However, depending on
the formulation, this interval could be reduced to that used in the cattle test. It is important that the different
dose groups are individually separated from each other during the trial and that animals are removed as
soon as they appear to be developing generalised FMD to avoid excessive challenge to those remaining.
Challenge is by intradermal injection into the heel bulbs of one foot with 10,000 TCID50 (0.2 ml), as
calculated by growth in a suitable pig cell culture, of a virulent challenge virus homologous to a strain used
in the vaccine and that normally results in generalised disease in the pig. Alternatively, the challenge virus
may be administered into one site in the muscular part of the neck behind the ear, using a dose of virus
known to cause generalised disease by this route. The animals are observed daily for 10 days after
challenge for clinical signs of FMD. Both control animals should develop clinical signs on more than one
foot. From the number of animals protected in each group, the PD50 content of the vaccine is calculated.
There are a variety of methods for calculating PD50 (31), including procedures based on Kärber. The
vaccine should contain at least 3 PD50 per dose for pigs. Likewise, a similar protocol to the PGP test in
cattle can be adopted for pigs using a group of 16 animals vaccinated with a full vaccine dose and two non-
vaccinated controls. Challenge is by intradermal injection into the heel bulb of one foot with 10,000 TCID50
(0.2 ml) of a virulent challenge virus homologous to the strain used in the vaccine and that is known to
induce clinical signs in pigs.
Indirect tests, including measurement following vaccination of virus neutralising antibodies in cell culture, or
ELISA antibodies, or serum-protecting antibodies in suckling mice, may be used to assess the potency of a
vaccine provided that a statistical evaluation has established a satisfactory correlation between the results
obtained by the test on the relevant vaccine serotype and the potency test in cattle (60). For example, the
expected percentage of protection is used to analyse the sera of a group of at least 16 vaccinated cattle
and to express the probability of an animal being protected by measuring neutralising, ELISA or protecting
antibodies. In a single group of animals given a full dose of vaccine, the mean individual expected
percentage protection should be equal to or greater than 75% when 16 animals are used or 70% when
30 animals are used in the experimental group.
The presence of more than one serotype in a vaccine does not diminish the induction of antibodies against
another serotype or the correlation of antibody titre with protection.

c) Purity: testing for antibody against nonstructural proteins

The OIE Terrestrial Animal Health Code stipulates that a criterion for regaining FMD free status following an
outbreak, if vaccine is used, is to test the vaccinated animals for antibody against NSP. Likewise, countries
wishing to be recognised as FMD free with vaccination must demonstrate the absence of virus circulation by
showing that vaccinated animals are free from antibody to NSPs arising as a result of infection.
Consequently, FMD antigens used to formulate vaccines that may be used in these circumstances should
be purified to reduce the NSP content. With current manufacturing techniques it is possible to exclude the
majority of NSPs so that they induce little, if any, NSP specific antibody. Under these circumstances,
detection of NSP antibodies can provide evidence that vaccinated animals have been exposed to FMD
virus. Vaccine manufacturers may wish to exploit this potential by including a claim that their vaccines do
not induce antibody to one or more NS proteins and can be used in conjunction with an appropriate
diagnostic test. In addition to providing supporting documentation on the processes involved in such
purification, manufacturers should demonstrate lack of immunogenicity against NS proteins as part of the
licensing procedure in order to make such a claim on their product literature. A test method that can be
used is to vaccinate an appropriate number of calves, preferably with at least a double dose of a trial blend
of the vaccine containing the maximum number and amounts of antigen permitted on the authorisation
(these calves may be the same as those used for the safety test described in Section D.4.a of this chapter).
Calves should be vaccinated at least three times over a period of 3–6 months and then tested 30–60 days
after the last vaccination for the presence of antibody to NSPs using the tests described in Section B.2.d of
this chapter. Negative results in these NSP assays support claims that the vaccine does not induce
antibody to NSPs.
At the batch level, confirmation of vaccine purity can be shown by demonstrating a lack of increase in
reactivity against NS proteins of the sera from the animals used in the potency test obtained 30 days after
primovaccination and before challenge, when compared with the sera of the same animals prior to
vaccination.
In order to establish a satisfactory level of immunity it is usual to give a primary course consisting of two
inoculations, 2–4 weeks apart, followed by revaccination every 4–12 months. The frequency of
revaccination will depend on the epidemiological situation and the type and quality of vaccine used. Where
access to the animals is difficult, it is preferable to use oil adjuvanted vaccine at 4 months and 1 year of
age, followed by annual revaccination. Wherever possible, vaccine manufacturers should demonstrate the
duration of immunity for their specific formulation in each species for which it is indicated.
For calves born of vaccinated dams, the first vaccination should be delayed as long as possible to allow
decline of maternal antibody. This period should not be extended beyond 4 months, as by this age a high
proportion can be expected to respond effectively to vaccination. For calves born to non-vaccinated dams,
the first vaccination may be administered from as early as 1 week of age for some vaccines (8).
e) Stability
The shelf life of conventional FMD vaccines is usually 1–2 years at 4°C (maximum range 2–8°C), but they
are temperature labile and should never be frozen nor stored above a target temperature of 4°C. The
stability of all vaccines, but particularly oil emulsion vaccines, should be demonstrated as part of the shelf
life determination studies for authorisation.
f) Preservatives
The most commonly used preservatives are chloroform and merthiolate. The latter is used at a final
concentration of 1/30,000 (w/v).
Current FMD vaccines are innocuous and present no toxic hazard to the user. Care must be taken to avoid
self-injection with oil-emulsion vaccines.
5. Batch control
a) Sterility
The bulk inactivated antigen, the adjuvants, the dilution buffers and the final formulated product should all
undergo sterility testing. This may be carried out directly with components of the vaccine and the final
product, but the preferred method is to collect any contaminating microorganisms by membrane filtration of
the material to be examined and to detect any organisms present by incubation of the membranes with
culture media. The latter procedure allows the removal of preservatives, etc., which may inhibit the
detection of microorganisms. Guidelines on techniques and culture media, which allow the detection of a

wide range of organisms, are described in the European Pharmacopoeia 2008 (27; also refer to Chapter
1.1.9 Tests for sterility and freedom from contamination of biological materials).
b) Innocuity
The test for innocuity is an in-process test that should be carried out for every batch of antigen. Following
inactivation, a sample of each batch of inactivated antigen representing at least 200 doses should be tested
for freedom from infectious virus by inoculation of sensitive monolayer cell cultures, preferably of the same
origin as those used for the production of antigen. It may be preferable to concentrate the antigen to do this,
in which case it must be shown that the concentrated material does not interfere with the sensitivity or
reading of the assay. The cell sheets are examined daily over a period of 3 days, after which the spent
medium is transferred to fresh monolayers and the original monolayers are replenished with fresh medium.
Using this method, traces of live virus can be amplified by the passage procedure and detected on the
basis of CPE observed. Two to three passages of the original virus preparation are commonly used. A
variant on this method is to freeze–thaw the old monolayers to release intracellular virus, which can be
detected by further passage.
c) Safety
This final product batch safety test is conducted to detect any abnormal local or systemic adverse reactions.
The test may also confirm innocuity but is not as sensitive as the in- vitro tests described above. For the
purposes of batch release, each of at least two healthy seronegative cattle is inoculated by the
recommended route of administration with double the recommended dose of vaccine. The animals are
observed for local and systemic reactions to vaccination for no fewer than 14 days. Should any of the
animals develop clinical signs of FMD, the vaccine fails the safety test. Equally, any undue toxicity
attributable to the vaccine should be assessed and may prevent acceptance of the batch. Ideally, vaccines
prepared for species other than cattle should also be safety tested in the species for which they are
intended, administering a double dose of vaccine according to the manufacturer’s recommended route and
dose volume. The animals should be examined daily for a minimum of 14 days for evidence of toxicity or
signs of FMD.
d) Potency
Potency is only examined on the final formulated product (see Section D.5.b). Antigen load can be used as
an indirect indicator of potency, provided a correlation has previously been established between antigen
load, serological response and protection against challenge, and provided that a suitable alternative test
measuring the serological response to immunisation has been carried out with satisfactory results.
6. Storage and monitoring of antigen concentrates
The process of storing concentrated antigens at ultra-low temperature for later formulation into FMD vaccine is
becoming an increasingly popular option for vaccine manufacture. It not only forms the basis for the storage of
antigens in a strategic reserve for emergency purposes (see Chapter 1.1.10 Guidelines for International
Standards for Vaccine Banks), but allows the manufacturer immediate access to many different antigen strains
which can be rapidly formulated and dispatched to the customer. Such stockpiling minimises delays subsequent
to an order, particularly where a multivalent vaccine is requested. Another advantage of this procedure is that
much of the quality testing can be completed well in advance of shipment.
a) Pre-storage criteria
It is necessary to state that antigens have to be controlled using standards indicated in Sections D.1–4.
Special attention should be paid to the following:
- freedom from extraneous agents;
antigens should be proven free from all extraneous agents listed by the appropriate licensing authorities.
- sensitivity of the cell line used to detect residual virus;
Cells used to test for absence of residual live virus are not suitable if use of an amount of virus
corresponding to 1 µg of 146S antigen gives a titre of less than 106 CC ID50 (27).
- emergency procedures for provisional acceptance of new Master Seed Virus (MSV), and subsequent
release of formulated vaccines.

In the case of incursion in a region of a new strain that is antigenically distinct from existing vaccine strains,
it may be necessary to develop a new vaccine strain from a representative field isolate. Before the new
MSV can be accepted, full compliance should be demonstrated with the relevant guidelines to demonstrate
freedom from all extraneous agents listed by the appropriate licensing authorities using both general and
specific tests, and to establish homology to the original candidate isolates. The time taken to raise the
specific antisera necessary to neutralise the new strain for use in the general tests for detection of
extraneous agents and to conduct other specific tests that require specialised techniques may be lengthy.
Therefore, in emergency situations where there is insufficient time to complete full testing of the MSV,
provisional acceptance of the new strain should be based on a risk analysis of the possibility of
contamination of the antigen produced from the new MSV with extraneous agents. This risk assessment
should take into account that a validated procedure to inactivate enveloped viruses must be used when
establishing the MSV and that the virus is inactivated using a chemical inactivant with first order kinetics.
Further assurance is provided by the requirement for the kinetics of inactivation to be monitored and
recorded for each production batch.
In order to accelerate the release of batches of vaccine formulated to contain new vaccine strains, it may be
acceptable for batch potency testing to be carried out using a vaccine formulated using an intermediate
antigen lot pending production of all of the batches of antigen that are intended to constitute the final
antigen lot. This will allow the potency of antigen derived from a new MSV to be determined whilst the
manufacturer continues to build up stocks of this new antigen.
b) Storage criteria
Facilities
It is important that all aspects of the storage of antigen concentrate conform fully to internationally accepted
standards of Good Manufacturing Practice.
Containment of stored antigens

The dose numbers or volumes stored are an important consideration, particularly where a reserve is shared
between Member Countries and there is variation in number of doses perceived to be needed by each
member in an emergency. It may be advisable to store antigen concentrates in user-friendly units to allow
better usage of storage space and capability in producing smaller vaccine batches. One to two litre sized
containers can accommodate in excess of 30,000 bovine doses. Where the requirement is for a large
stockpile of a particular vaccine strain that can only be produced from several separate production runs,
vaccine bank managers must consider the need to either formulate each lot into a representative final blend
for testing purposes or mixing the individual batches, at some convenient point, for ease of formulating
and/or testing.
The type of container used to hold antigen concentrate is important. Under ultra-low temperature conditions
it is important to use containers made from materials that do not become brittle and fragile, a good example
being fluoropolymer based moulded bottles. Polyfluoro-alkoxy (PFA) based bottles, for example, have a
temperature resistance range of between –270°C and +250°C.
Labelling of stored antigens

Although there are national and international guidelines on the required labelling of veterinary medical
products, there are no such guidelines for emergency stored materials such as the antigen component of a
vaccine, as these are essentially regarded in regulatory terms as ‘in process’ materials. Under ultra-low
temperature conditions, the method of labelling must be of a durable nature. From experience, wire tagging
bottles is the most preferred option using a metal tag sizeable enough to allow the necessary detail. Such
detail should include the antigen/vaccine strain, batch number, date received and should also include an
individual container or stock number. This information should be clear to read and marked on the tag using
an indelible marker pen. Aluminium metal tags have been used for such purpose and these can be
obtained with different colour coatings to allow better identification and accessibility, particularly when
different antigen strains are housed in the same container. Metal tags also allow information to be
permanently engraved.
Monitoring
It is vitally important that antigen concentrates are optimally maintained and routinely monitored in order to
have some assurance that they will be efficacious when needed. Therefore arrangements should be made
to monitor these antigen concentrates on a routine basis and to include where necessary, and at
appropriate time intervals, a testing regime to ensure integrity of the antigen component or acceptable
potency of the final product. For example, storage temperature monitoring is normally undertaken and
recorded in FMD vaccine banks, as well as periodic inspection of the bottles containing the antigen for
cracks or leakage. Depending on type, volume and how they are stored, there may also be value in

weighing antigen deposits annually to ensure they have not lyophilised. Some FMD vaccine banks have
incorporated physico-chemical tests like sucrose density gradient analyses to monitor virus integrity and
hence stability and some have also carried out in-vivo tests. However, since it has been shown that the
shelf-life of FMD antigen concentrates are likely to be well in excess of 15 years when stored at ultra-low
temperature, a physico-chemical approach would appear sufficient.
The following timetable of tests is proposed as suitable for validation and re-validation of stored antigens.
Time Test
On receipt (year 0) and every 5 years 146S quantification*
thereafter
Potency test in cattle that may rely on serological
techniques where potency has been adequately
correlated with immunogenicity for the antigen
concerned or, at the discretion of the bank holder, may
be a ‘truncated’ test** to demonstrate that the minimum
potency of the vaccine remains greater than the
minimum requirement; however, truncation may
underestimate vaccine potency
Years 2 and 4, and immediately before 146S quantification
formulation if the need arises
Every 5 years Evaluation of all data for the preceding 5 years to
assess need to replace antigen
* Other physiochemical tests such as SDS-PAGE have been used to evaluate integrity of VP1 but are
not sufficiently validated for routine use.
** In a truncated test all animals in the next lower volume group are assumed to have not been
protected. The test therefore gives an artificially low PD50 value but reduces the number of animals
required.
To support these testing requirements for depositories of antigen, concentrates should include a number of
small samples that are representative of the larger stock. Small aliquots/stocks of FMD antigen have usually
consisted of a volume representing approximately one milligram of antigen.
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serological data. J. Biol. Stand., 12, 225–229.
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59. SORENSEN K.J., MADSEN K.G., MADSEN E.S., SALT J.S., NQUINDI J. & MACKAY D.K.J. (1998). Differentiation of
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proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus. Arch. Virol., 143, 1461–
1476.
60. STREBEL K., BECK E., STROHMAIER D. & SCHALLER H. (1986). Characterisation of foot-and-mouth disease virus
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61. VIANNA FILHO Y.L., ASTUDILLO V., GOMES I., FERNANDEZ G., ROZAS C.E.E., RAVISON J.A. & ALONSO A. (1993).
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protection against generalisation. Vaccine, 11–14, 1424–1428.
62. W AGNER G.G., CARD J.L. & COWAN K.M. (1970). Immunochemical studies of foot-and-mouth disease virus.
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*
* *
NB: There are OIE Reference Laboratories for Foot and mouth disease (see Table in Part 3 of this Terrestrial

CHAPTER 2.1.6.
HEARTWATER
SUMMARY
Heartwater (also known as cowdriosis) is an acute, fatal, non-contagious, infectious, tick-borne

rickettsial disease of ruminants caused by Ehrlichia ruminantium (formerly Cowdria ruminantium)
and transmitted by Amblyomma ticks. It occurs in nearly all sub-Saharan countries of Africa, in its
neighbouring islands, and also in the Caribbean, threatening the American mainland. The disease
can cause high mortality (up to 90%) in susceptible domestic ruminants. Goats and sheep are
more susceptible than cattle, and European breeds are generally more susceptible than
indigenous African breeds.
Clinically, the disease most commonly presents as an acute form characterised by a sudden high
fever, depressed demeanour, nervous signs and a high mortality. Hydropericardium and
hydrothorax and lung oedema are commonly associated post-mortem signs. Acute and peracute
clinical forms of the disease occur: In the former, there are high death rates without many clinical
manifestations and in the latter, there is a higher recovery rate.
Recovered animals become carriers of infection. Certain wild animals can play a role as reservoir;
Rusa deer, white tailed deer, and springbok are susceptible to this infection and can experience
high mortality.
Identification of the agent: The specific diagnosis of heartwater is based on the observation of
colonies of E. ruminantium in capillary endothelial cells of the brain. In the absence of adequate
tools, a piece of cerebellum can easily be removed with a curette through the foramen magnum
after cutting off the head, while a sample of cerebral cortex can be obtained through a hole made
in the skull with a hammer and a large nail. Brain smears are prepared by crushing to a paste and
spreading thinly a small piece of cerebral or cerebellar cortex between two microscope slides. The
capillaries are spread out in a single cell layer by drawing one slide across the other. The smears
are air-dried, fixed with methanol and stained with Giemsa. With fast stains, the smears can be
fixed and stained in less than 1 minute. The colonies (clusters) are reddish-purple to blue, and very
often close to the nucleus of the infected endothelial cell. They can be scanty and difficult to find,
particularly in peracute cases, but they are always present in the brain of a ruminant that died from
heartwater, if not treated with drugs. Colonies are not likely to be detected in animals that were
treated with antibiotics. The colonies are still visible 2 days after death in a brain that has been
stored at room temperature (20–25°C) and up to 34 days in a brain that has been stored in a
refrigerator at 4°C.
Fresh whole blood collected from suspect animals can be inoculated intravenously into a
susceptible sheep or goat. The development of clinical signs and the demonstration of
E. ruminantium in the brain of the inoculated animal during the febrile reaction are diagnostic for
heartwater. Due to animal welfare concerns, this method should be avoided.
Ehrlichia ruminantium can be isolated from the blood of an infected host using cultivation on
ruminant endothelial cells. When a cytopathic effect consisting of plaques of cell lysis appears, the
presence of characteristic morulae is confirmed by staining the cell monolayer with eosin–
methylene blue or by immunofluorescence or immunoperoxidase techniques using a specific
antiserum.
DNA probes and especially the more sensitive polymerase chain reaction (PCR) techniques are
available to reveal the presence of E. ruminantium in the blood of animals with clinical signs, and in
the tick vectors, to a lesser extent in the blood or bone marrow of carrier animals. Apart from

Chapter 2.1.6. — Heartwater
diagnosis, PCR is widely used for research on the E. ruminantium genome and for epidemiological
studies.
Serological tests: Serological tests available include indirect fluorescent antibody tests, enzyme-
linked immunosorbent assays (ELISAs) and Western blot. However, when the whole
E. ruminantium is used as antigen, cross-reactions with Ehrlichia spp. occur in all of these tests.
Serology has limited diagnostic applications.
One recently developed ELISA uses a recombinant antigen expressed as a partial fragment of the
recombinant major antigenic protein 1 (MAP1) antigens –the MAP1-B ELISA. This test has shown
a dramatic improvement in specificity compared with previous tests. Although this test is more
specific, it still detects cross-reacting antibodies to other Ehrlichial organisms. Hence, definitive
proof of heartwater must rely on epidemiological evidence and additional molecular testing
indicating the presence of the organism. This ELISA has made the interpretation of serological
results more reliable in regions where Ehrlichia infections occur in ruminants. It can help to monitor
experimental infections and to measure the immune response of immunised animals, whose pre-
immunisation serological history is known. Serology has very limited diagnostic use as clinically
infected animals remain sero-negative during the febrile reaction and sero-convert after they
recover from the infection.
Serology is also not an effective import test. Prior to importation of animals from a heartwater
endemic region, it is important to study the epidemiological data to try to establish that the herd
and the resident ticks are not infected; in addition repeated PCR testing should be carried out to
demonstrate that the pathogenic agent is not present in the herd.
Requirements for vaccines and diagnostic biologicals: The immunisation against heartwater
by the ‘infection and treatment’ method using infected blood is still in use in some countries. A first-
generation vaccine consisting of inactivated purified elementary bodies of E. ruminantium
emulsified in Montanide ISA 50 adjuvant has given promising results in experimentally controlled
conditions and has demonstrated significant protection in the field. An additional isolate,
Welgevonden, has been attenuated and shown to confer good protection, and significant
protection has also been obtained using DNA vaccination. However, none of these new
experimental vaccines has been fully validated under field conditions. Field trials have revealed
that antigenic diversity is important in formulating effective vaccines, and further investigations are
critical for the delivery of any vaccine in the field.
A. INTRODUCTION
Heartwater (cowdriosis) is a rickettsial disease of domestic and wild ruminants caused by Ehrlichia ruminantium
(formerly Cowdria ruminantium) and transmitted by Amblyomma ticks (3, 8, 36). It is also known by the synonyms
malkopsiekte (Afrikaans), péricardite exsudative infectieuse (French), hidrocarditis infecciosa (Portuguese),
idropericardite dei ruminanti (Italian), and a variety of names in different African languages (5). Ehrlichia
ruminantium is classified in the order Rickettsiales and in the family Anaplasmataceae, together with the genera
Anaplasma. Although ruminants remain the primary target of the pathogen, in South Africa a possible canine
E. ruminantium infection has been reported (1), and, more recently, E. ruminantium has been strongly suspected
in several cases of rapidly fatal encephalitis in humans (14). However, in all cases, evidence of E. ruminantium
infection was based on molecular detection. Isolation and characterisation of the infectious agent is necessary
before E. ruminantium can be considered an emerging pathogen in species other than ruminants and especially
in humans.
Heartwater occurs in nearly all the sub-Saharan countries of Africa where Amblyomma ticks are present and in
the surrounding islands: Madagascar, Reunion, Mauritius, Zanzibar, the Comoros Islands and Sao Tomé. The
disease is also reported in the Caribbean (Guadeloupe, Marie-Galante and Antigua) (34), from where it threatens
the American mainland. All domestic and wild ruminants can be infected, but the former appear to be the most
susceptible. Indigenous domestic ruminants are usually more resistant to the disease. Wild animals could play a
role as reservoir, but Rusa deer, white tailed deer, springbok, chital, timor deer, which are used in wildlife
farming, seem to be the main wild ruminant species in which heartwater can have a significant economic impact
(36).
The average natural incubation period is 2–3 weeks, but can vary from 10 days to 1 month. In most cases,
heartwater is an acute febrile disease, with a sudden rise in body temperature, which may exceed 41°C within 1–

2 days after the onset of fever. It remains high for 4–5 weeks with small fluctuations and drops shortly before
death
Fever is followed by inappetence, sometimes listlessness, diarrhoea, particularly in cattle (4), and dyspnoea
indicative of lung oedema. Nervous signs develop gradually. The animal is restless, walks in circles, makes
sucking movements and stands rigidly with tremors of the superficial muscles. Cattle may push their heads
against a wall or present aggressive or anxious behaviour. Finally, the animal falls to the ground, pedalling and
exhibiting opisthotonos, nystagmus and chewing movements. The animal usually dies during or following such an
attack.
Subacute heartwater with less pronounced signs, and peracute heartwater with sudden death, can also occur,
according to the breed of ruminant and the strain of E. ruminantium involved.
The most common macroscopic lesions are hydropericardium, hydrothorax, pulmonary oedema, intestinal
congestion, oedema of the mediastinal and bronchial lymph nodes (4), petechiae on the epicardium and
endocardium, congestion of the brain, and moderate splenomegaly.
A tentative diagnosis of heartwater is based on the presence of Amblyomma vectors, nervous signs, and
presence of transudates in the pericardium and thorax on post-mortem examination. When making a diagnosis
based on clinical signs, the following other diseases should be considered: bovine cerebral babesiosis and
theileriosis, anaplasmosis, botulism, haemonchosis in small ruminants, rabies and poisoning.
During the febrile reaction, E. ruminantium can be readily isolated in culture from blood or plasma; however, it is
difficult to detect these organisms in a blood smear. Typical colonies of E. ruminantium can be observed in brain
smears made after death and this represents a definitive diagnosis for heartwater.
Opening the cranium is not necessary. An alternative method (42) is to cut off the head in front of the first
cervical vertebra. Then, introduce a curette through the foramen magnum, between the medulla and the
meninges. The curette is turned over towards the brain and removed with a piece of cerebellum. Another method
consists of making a hole in the skull with a hammer and a large nail and aspirating a sample of brain cortex with
a needle attached to a syringe. These methods also lessen the danger to the operator in cases where the
nervous signs have been caused by rabies.
In the live animal, a brain biopsy may be obtained aseptically and harmlessly after local anaesthesia, although
with difficulty; appropriate restraint must be used especially with large and horned animals. Colonies of Ehrlichia
are observed during the febrile period. This method is useful for experimental studies, but not suitable for routine
diagnosis.
Colonies of E. ruminantium are still present 48 hours after death in a brain that has been stored at room
temperature (20–25°C) and for up to 34 days in a brain that has been stored in a refrigerator at 4°C (5).
A small fragment of grey matter (approximately the size of a match head) is placed on a microscope slide,
crushed to a paste consistency by another slide and, while maintaining pressure, the slides are drawn over each
other lengthwise to produce a single layer of cells. The slides are air-dried, fixed in methanol, stained with
Giemsa diluted with Sörensen buffer (2.54 g KH2PO4; 8.55 g Na2HPO4.H2O; q.s. to 5 litres with distilled water),
pH 7.2, and washed with tap water. Fast Giemsa stains (DiffQuick, RAL555, Field’s stain, CAM’s Quick stain)
give quicker results, but the colour contrast is usually poorer. Some ‘fast’ stains do provide excellent contrast,
e.g. Hema 3 stain.
The slides are examined under a microscope at a low magnification (×10 objective) to locate the cerebral
capillaries. An oil-immersion lens with a magnification of at least ×50 is useful for identifying the colonies of
rickettsiae. Experience is required E. ruminantium colonies must be differentiated from other haemoparasites
(Babesia bovis), certain blood cells (thrombocytes, granulocytes), normal subcellular structures (mitochondria,
mast cell granules), or stain artefacts (stain precipitates), etc. The specificity of the reading can be improved by
staining formalin-fixed brain sections using immunoperoxidase techniques.
Ehrlichia ruminantium colonies are formed from clusters of granules (0.2–0.5 µm), sometimes arranged in the
shape of a ring or a horseshoe (1–3 µm), that are placed close to the nucleus inside the endothelial cell. The
granules can be scanty, particularly in peracute cases, but they are always present in the brain of an animal that
died from heartwater. However, if the animal has been treated with doxycyclin or oxytetracyclin 48 hours before,
the granules of Ehrlichia tend to fuse making the diagnosis very difficult, and sometimes impossible.

Fresh whole blood collected from suspect animals can be inoculated intravenously into a susceptible sheep or
goat. The development of clinical signs and the demonstration of E. ruminantium in the brain of the inoculated
ruminant are diagnostic for heartwater.
Transmission electron microscopy has been used to demonstrate that the E. ruminantium organisms develop
inside a vacuole-like structure, which is surrounded by a membrane in the endothelial cell’s cytoplasm (39). Each
organism is enclosed by a double membrane. Within the vacuole-like structure, E. ruminantium electron-dense
forms (elementary bodies), as well as intermediate reticulate forms, are identified.
a) Isolation of Ehrlichia ruminantium using in-vitro culture

Although numerous cell lines have been shown to support growth of E. ruminantium, isolation is not the first
choice of test for a rapid diagnosis of cowdriosis as isolation is a labour intensive and time-consuming
laboratory procedure. For a rapid diagnosis, polymerase chain reaction (PCR)/molecular diagnosis is
preferable. However, E. ruminantium isolation should be encouraged for typing the strains present in a
region for the purpose of vaccination programmes. Ehrlichia ruminantium can be isolated from the blood of
reacting animals by cultivation on ruminant endothelial cells (45). Endothelial cells from umbilical cord,
aorta, or the pulmonary artery of different ruminant species (cattle, goat, sheep) are used most often for
isolation, although other endothelial cell types (brain capillaries, circulating endothelial cells, etc.) have been
described for the routine culture of the microorganism. Endothelial cell lines from sable, eland, buffalo, kudo
and bush pig can also be used to grow E. ruminantium. No standard cell line has yet been designated for
isolation.
Isolation procedure
i) The blood of the reacting animal (optimal time for detection of the organism is the second or third day
of febrile reaction) is collected in anticoagulant (heparin or sodium citrate, not ethylene diamine tetra-
acetic acid) and diluted 1/2 in the culture medium consisting of Glasgow minimal essential medium
(MEM) supplemented with 10% inactivated fetal bovine serum, 2.95 mg/ml tryptose phosphate broth,
200 mM L-glutamine, and antibiotics if necessary (penicillin 100 international units/ml, streptomycin
100 µg/ml).
ii) The culture medium is poured off the endothelial cell monolayer, and infective blood (approximately
2 ml for a 25 cm2 flask) is added. The flask is incubated at 37°C on a rocking platform for 2 hours.
iii) After incubation, the blood is poured off and the monolayer is gently washed three times with culture
medium prewarmed at 37°C.Fresh culture medium is added and the flask is incubated at 37°C. The
medium is changed twice weekly.
(The use of plasma instead of blood is more efficient when taken from an animal with a febrile reaction
>41°C. In this case, steps ii and iii above may be replaced with the following:
Seed 4 ml of plasma (smaller inoculum can be used if there is a limited amount of plasma
available) on to a susceptible endothelial cell culture and incubate for 1 hour at 37°C on a
rocking platform.
Wash off plasma with growth medium and then add 5 ml of growth medium (per 25 cm2 flask)
and observe for development of cytopathic effect.)
iv) The monolayer is inspected regularly for the appearance of small plaques of cell lysis. The first
plaques generally appear after about 2 weeks. Passaging on uninfected cell monolayers is performed
when the lysis reaches 80% of the cell layer. The remaining cells are stained with eosin/methylene
blue or Giemsa or DiffQuick and examined microscopically for the presence of E. ruminantium
morulae. Alternatively, cells can be stained by an indirect fluorescent antibody (IFA) test or an
immunoperoxidase test using an E. ruminantium-specific antiserum; the immunoperoxidase test is not
commonly used.
b) Isolation of Ehrlichia ruminantium using in-vivo culture

It is feasible to assess the presence of heartwater in a herd, a region or a country, or to isolate a strain of
E. ruminantium by inoculating blood or tick homogenate into a susceptible animal. However, due to the
animal welfare concerns, this method is not recommended. Blood from individual animals, or pooled blood,
is injected slowly at a dose of 10–100 ml intravenously into a susceptible sheep or goat. Blood as an
inoculum, to determine infection status of herd, will be infective if there are clinically infected donor animals
present; however, the method will rarely detect infection in carrier/recovered animals. Another method
consists of collecting and homogenising adult Amblyomma ticks, and after centrifuging the homogenate and
then inoculating the resulting supernatant into susceptible hosts. This method can be more sensitive than
blood from suspect animals (especially if blood is from recovered animals) because the concentration of
E. ruminantium is higher in the tick than in the blood. However, the tick infection rate in the field is variable
and sometimes as low as 1% (6). In this case, to detect an infection as many ticks as possible should be

used; at least 100 ticks are needed. In both cases, the inoculum, to which 10% dimethyl suphoxide (final
concentration) has been added, can be stored in liquid nitrogen for several years. Note that inoculation of
tick homogenates into susceptible animals may cause anaphylaxis, which can be prevented by the
simultaneous administration of adrenaline. The development of clinical signs and the detection of circulating
rickettsiae by molecular methods and/or the demonstration of E. ruminantium in the brain of the inoculated
ruminant, on the second or third day of fever, are diagnostic for heartwater. In addition, confirmation could
be accomplished by in-vitro isolation on endothelial cells using plasma from the inoculated animals.
2. Molecular methods
a) Detection of Ehrlichia ruminantium using DNA probes

A genomic DNA fragment pCS20 specific for E. ruminantium has been cloned and used as a nucleic acid
probe (24, 50). It recognises all strains of E. ruminantium tested to date. This probe, designated pCS20,
readily detects infection in clinically ill animals and experimentally infected Amblyomma ticks (18, 20, 24,
51). However, it is not sufficiently sensitive to detect most carrier animals or low level infections in ticks (35,
36). The pCS20 probe proved nevertheless to be more sensitive than 16S and MAP1 (major antigenic
protein 1) probes for the detection of E. ruminantium in ticks when hybridised on a PCR-amplified product of
the homologous DNA fragment (2).
b) Detection of Ehrlichia ruminantium using PCR and nested PCR

Two primers –AB128 (5’-ACT-AGT-AGA-AAT-TGC-ACA-ATC-TAT-3’) and AB129 (5’-TGA-TAA-CTT-GGT-
GCG-GGA-AAT-CCT-T-3’) – have been designed from the DNA sequence of the pCS20 probe (24) for use
in a PCR. These primers amplify a 279 base pair DNA fragment which is specific only for E. ruminantium.
Hybridisation of the amplified PCR products to a labelled pCS20 probe, as an additional step, resulted in a
350-fold more sensitive assay than using the nucleic acid probe to detect E. ruminantium directly in DNA
extracted from ticks. Low levels of infection in animals and in ticks fed on carrier animals are detected by
PCR, while a hybridisation reaction with the pCS20 probe alone (without PCR first) usually remains negative
(37). Experimentally, the detection limit of the conventional PCR assay was found to be between 10 and
102 organisms, whereas it was between 1 and 10 organisms after PCR/hybridisation. The PCR/
hybridisation has been shown to detect 37 strains from all endemic areas with a specificity of 98%.
However, the sensitivity of the PCR assay is variable, ranging from 88 to 97% with tick samples containing
107 to 104 organisms, and dropping to 61% and 28% with samples containing 103 and 102 organisms,
respectively (35). Consequently, the rate of 86% of ticks testing positive when fed on a clinically reacting
animal dropped to 21% when fed on carrier animals due to a lower rickettsemia in such animals. The
PCR/hybridisation assay has been used widely to define the epidemiology of heartwater in southern Africa.
Two nested PCR assays have been developed to enhance detection of low levels of rickettsemia and to do
away with the hybridisation step (30, 43). Both use the pCS20 region as the target sequence. The Semu et
al. assay uses two external primers U24 (5’-TTT-CCC-TAT-GAT-ACA-GAA-GGT-AAC-3’) and L24 (5’-AAA-
GCA-AGG-ATT-GTG-ATC-TGG-ACC-3’) and then the AB 128 and AB 129 for the nested reaction. The
sensitivity of detection of this assay is one gene copy of the pCS20 fragment or 1 organism. The other
nested PCR assay (30) uses a pair of external primers comprises the AB128 sense primer together with an
anti-sense primer called AB130. These amplify a 413 bp fragment used as a template in a second round
PCR using AB128 and AB129 as internal primers. The use of AB128 and AB129 primers avoids the need to
repeat a full evaluation of the test specificity. The nested PCR shows a hundred-fold improvement in
sensitivity compared with a simple PCR, and an average detection limit of 6 organisms. The direct
implication of this was an increase in the detection rate in wild ticks of from 1.7% to 36% in an
epidemiological study in the Caribbean. The detection limit is comparable to that of the PCR/hybridisation
method, which is nevertheless much more complex and time-consuming to perform. The pCS20 nested
PCR allowed regular detection of E. ruminantium organisms from ticks, blood, brain and lungs from infected
animals, whether the samples were processed fresh, or after freezing or preservation in 70% ethanol. One
drawback of the nested PCR is that extreme care needs to be exercised to prevent introduction of
contamination due to repeated opening of the tubes containing the first PCR reaction when conducting the
nested reaction.
A nested PCR targeting the entire map1 polymorphic gene has been developed in parallel in order to type
the strains by restriction fragment length polymorphism or sequencing of the amplification fragment directly
from the pathological samples testing positive in the pCS20 nested PCR (30). An additional nested PCR
targets the polymorphic map1 gene and can be used to type circulating heartwater strains for vaccine
selection and disease management. PCR amplicons are analysed by restriction fragment length
polymorphisms or sequencing. A high genetic diversity of E. ruminantium is observed in the field may
influence the formulation of vaccines and needs to be further investigated. The map1 nested PCR performs
well although with a slightly lower sensitivity than the pCS20 nested PCR. Its detection limit was evaluated
at around 60 organisms and only 91% of samples testing positive in the pCS20 nested PCR also tested
positive in the map1 nested PCR; some positives of low intensity found using the pCS20 nested PCR were
negative in the map1 PCR.

Primers 32F1 and 32R1 designed from the sequence of the MAP1 gene of E. ruminantium as well as
additional primer sets designed to target the MAP1, MAP2, gltA, and 16SrDNA genes of E. ruminantium
have been used in PCR to detect the pathogen in tick, blood and bone marrow of carrier sheep and wild
African ungulates, but these methods has not been widely evaluated and used.
Although the PCR methods have proved highly effective in detecting infection in ticks or in animal samples
during the clinical phase of the disease or after death, only limited studies have been done to evaluate their
value in healthy carrier ruminants. Ehrlichia ruminantium can easily be demonstrated in the blood of
infected animals just before the onset of the febrile period and for a few days after recovery (24, 43), but
after that period, its detection is sporadic and appears to be dependant on the rickettsemia levels. In one
study in Zimbabwe only between 3.3 and 26.7% of cattle, and 23.3% of goats were found to be positive,
while data from ticks collected in the same area would suggest that given the age of the cattle or goats, they
should have all been exposed or infected with E. ruminantium (19). A comparison of the indirect MAP1-B
ELISA and the pCS20 PCR/hybridisation assay, to evaluate their respective detection sensitivity levels over
a period of 8 weeks (tests performed every 2 weeks), was done on 15 cattle located in Zimbabwe on a
heartwater-endemic farm where tick control was minimal and the infection pressure was high (44). The
E. ruminantium tick infection rate on this farm was between 10 and 12%. The data demonstrated that the
pCS20-PCR assay was more reliable in detecting infection in blood of these cattle than detection of
antibodies by the indirect MAP1-B ELISA. These cattle were not always PCR positive or positive for
antibodies at every testing time and some cattle were negative by PCR throughout the study. These data
suggest that the rickettsemia levels fluctuate from high to low, and that the PCR detects infection when the
levels are high. Hence detecting carrier/recovered animals is less reliable than detecting clinically infected
animals. This highlighted the fact, that for determination of infection status of sub-clinical animals, it is
advisable to repeatedly test the blood of such animals for E. ruminantium by the pCS20-PCR assay.
Whether the absence of detection in most carrier animals is due to an insufficient sensitivity of the PCR
methods for detecting very low rickettsemia, or is due to an intermittent release of organisms in the
circulation, is not fully understood. A useful technique for confirming the status of a suspected carrier
animal, whose blood is PCR negative, is to feed batches of naive ticks on the animal and then test the ticks
by a pCS20 semi-nested PCR. It is not known whether ticks act simply by concentrating circulating
organisms, or also by amplifying their number or even by inducing release of micro-organisms in the
circulation during feeding.
c) Detection of Ehrlichia ruminantium using the reverse line blot technique

The reverse line blot technique (RLB) has been used for the simultaneous detection and identification of
Anaplasma and Ehrlichia species known to occur in ruminants on the basis of differences in the small
subunit rRNA gene (3). Primers 16S8FE and B-GA1B-new were designed from conserved domains and
used to amplify a 492–498 bp fragment of the 16S rRNA gene spanning the variable V1 region. Species-
specific oligonucleotide probes were designed in this V1 loop to allow species-specific detection of
E. ruminantium, E. ovina, E. sp. strain Omatjenne, Anaplasma marginale, A. centrale, A. bovis, A. ovis and
A. phagocytophilum. One oligonucleotide probe cross-reactive with all species (catch-all probe) was also
designed to serve as control in case a PCR product does not hybridise to any of the species-specific
probes. In the method, the species-specific probes are covalently linked to the hybridisation membrane,
which is hybridised with the PCR product obtained using primers 16S8FE and B-GA1B-new. PCR products
obtained from all above-mentioned microorganisms were shown to bind with specific oligonucleotide probes
only. No PCR product was detected and no hybridisation occurred when the PCR-RLB was applied to
Theileria annulata, Babesia bigemina or mammalian DNA. Similarly, negative control ticks were always
negative in the RLB assay whereas it was possible to detect Ehrlichia ruminantium infection in 15–70% of
ticks fed on experimentally infected or long-term carrier sheep. In Mozambique, E. ruminantium could also
be detected in the blood of 12 sentinel small ruminants placed in the field with the infected animals; mixed
infection was detected in five of the infected sentinel animals, thus demonstrating the usefulness of the
method for detecting multiple infections. However, the sensitivity of the assay has not yet been determined
and there is a need to further validate the technique in large epidemiological studies.
d) Detection of Ehrlichia ruminantium using real-time PCR

Two real-time PCR (QPCR) tests have been described for the detection and quantitative determination of
E. ruminantium organisms. In a first test, a 182 bp fragment from the non-polymorphic map1-1 gene was
amplified and detection carried out using the SYBR Green method (41). DNA from six different isolates was
successfully amplified. The detection limit mentioned was higher than 0.1 organism/µl, but this finding was
not subjected to an in-depth investigation. Counting E. ruminantium under the microscope after Giemsa
staining does not give very precise results. The method does not significantly improve the detection
sensitivity of a nested PCR, although it does allow E. ruminantium organisms to be quantified. In addition to
limited laboratory validation, the QPCR method was used in only one study aimed at following the
E. ruminantium kinetics in the blood of experimentally infected sheep. Ehrlichia ruminantium was detected
only during the hyperthermia reaction period. QPCR thus does not improve detection of asymptomatic
carriers compared with nonquantitative PCR.

A second SYBR Green-based real-time PCR has been described and fully validated for use in the
characterisation of E. ruminantium replication and release kinetics in endothelial cell cultures and its
subsequent use to control the mass production process in bio-reactors (40). The product is an 873 bp
fragment from the map1 gene. The external standard for quantifying E. ruminantium is a pCI-neo plasmid
containing one copy of the map1 target sequence, and is a more precise method of quantifying the
organisms than the method described previously where the standard is based on the counting of
E. ruminantium bodies under the microscope. The dynamic quantitative range allows accurate
measurements to be taken in samples containing 102 to 108 gene copies. The method was successfully
applied to four different isolates but has not been validated for use on diagnostic samples.
• Reading the results

As E. ruminantium is an obligate intra-cellular bacteria that cannot be cultivated in acellular media and its
isolation is complex and takes several weeks, molecular detection techniques are the best methods for the
diagnosis of cowdriosis. PCR proves to be easier to perform and more sensitive than DNA probes. With all
PCRs, however, care must be taken to ensure that no cross-contamination occurs between samples.
Negative and positive controls must be included in each test. As heartwater serology has several limitations
(see Section B.3), the PCR could be used to help confirm if seronegative animals, originating from an
endemic area, are not infected, prior to translocating them to a heartwater-free area that has the risk of
becoming infected, because of the presence of potential vectors. However, despite interesting experimental
results in detecting subclinical carriers, there is not enough available information on the reliability of carrier
detection by PCR; more extensive field studies need to be conducted to recommend the best protocol of
detecting carrier animals. It is nevertheless clear from the Zimbabwe study in cattle that detection of
infection in carrier hosts is going to be difficult and will require repeated testing to confirm status of infection
(44). The current results obtained with the PCR, the nested PCR, the RLB assay and more recently the
QPCR, show that the direct detection of E. ruminantium in the blood is only reliable during and around the
febrile phase of the disease. PCR-based methods appear to be more reliable in detecting infection in ticks,
and this could have epidemiological value in determining the geographical distribution of E. ruminantium. In
addition, when necessary in endemic areas, the inclusion of testing (originally naive) ticks fed on a suspect
animal would greatly improve the sensitivity of carrier detection when serology and PCR on blood have
failed. The procedure is nevertheless not convenient for routine diagnostic laboratories as it requires the
maintenance of tick colonies and the capacity to experimentally infect animals.
Various serological tests for diagnosing heartwater have been described: an IFA test with E.-ruminantium-
infected endothelial cell culture as antigen (CIFA test), indirect ELISA, a competitive ELISA (C-ELISA), and a
Western blot. The IFA test using E.-ruminantium-infected mouse peritoneal macrophages (MIFA) (10) is no
longer used.
The major drawback of all of these tests is the detection of false-positive reactions due to common antigenic
determinants between the E. ruminantium MAP1 (11) and similar proteins in several Ehrlichia species (23, 43).
Almost all of these tests are no longer used to study the epidemiology or for diagnosis. The CIFA test is still used
in some places, but care must be taken when interpreting the results because of the problem of false-positive
reactions.
To minimise the problem of cross-reactions with Ehrlichia, two ELISAs based on a recombinant MAP1 antigen
have been developed. The first is an indirect ELISA that uses an immunogenic region of the MAP1 protein (called
MAP1-B) and gives far fewer cross-reactions with Ehrlichia spp. (MAP1-B ELISA) (49). The second is a
competitive ELISA that uses the MAP1 gene cloned in a baculovirus and monoclonal antibodies (MAbs) raised
against the MAP1 protein (MAP1 C-ELISA) (12). Both tests have dramatically improved specificity, but they still
show some reactivity with high titre sera against E. canis, E. chaffeensis and an unclassified white-tailed deer
agent. The MAP 1-B ELISA has been the most extensively used and will be described in detail. The MAP1-B
ELISA does detect antibodies to E. muris (Mahan S.M., pers. comm.) an Ehrlichial agent that is very closely
related to E. ruminantium; this agent is found in white tailed deer in Georgia USA and is transmitted by
Amblyomma americanum ticks (15). Hence serology as a diagnostic tool for detecting of individual animals
exposed specifically to E. ruminantium is unreliable. Serology should be considered at a herd level taking into
consideration the epidemiological environment and, if necessary, be complemented by molecular techniques.
a) Indirect fluorescent antibody test with infected endothelial cell tissue culture as antigen (CIFA
test) (29)
To prepare the antigen, an E. ruminantium strain is cultivated in ruminant endothelial cell cultures. When
most cells are lysed, the remaining adherent cells are scraped and mixed with the supernate. The cells are
centrifuged three times with phosphate buffered saline (PBS) at 200 g for 10 minutes. Of the washed cell
suspension, 10 µl are placed in every well of an immunofluorescence slide. The antigen slides are dried,
fixed in acetone and stored at –20°C.

• Test procedure
i) The sera to be tested are diluted 1/20 or a higher dilution in PBS, added to the antigen wells and
incubated for 30 minutes in a humid chamber at 37°C.
ii) The slides are then washed in PBS buffer for 15 minutes.
iii) The appropriate anti-species conjugate, usually diluted 1/60, is added to cover the wells. The slides
are incubated again for 30 minutes at 37°C.
iv) After a second washing, the slides are mounted in glycerine buffer under a cover-slip and examined
under a fluorescence microscope.
v) Control positive and negative sera are included on each slide.
b) MAP1-B enzyme-linked immunosorbent assay (43, 49)

Using the vector pQE9, the PCR fragment MAP1-F2R2, which encodes the amino acids 47–152 of the
MAP1 protein including the immunogenic region MAP1-B, is expressed in Escherichia coli M15[pREP4] as
a fusion protein containing six additional histidine residues. The recombinant MAP1-B is purified using Ni2+-
NTA agarose (nitrilotriacetic acid agarose) under denaturing conditions as described by the manufacturer.
The antigen is preserved at 4°C and each batch is titrated.
The antigen is diluted at 0.5 µg/ml in 0.05 M sodium carbonate buffer, pH 9.6, and immobilised on to
polystyrene plates by incubation for 1 hour at 37°C, and stored at 4°C until use. However, in initial trials, an
antigen concentration of 2 µg/ml reduced background noise and improved specificity (data not shown, 43).
• Test procedure
i) Plates are blocked for 30 minutes by adding 100 µl per well of 0.1 M PBS, pH 7.2, supplemented with
0.1% Tween 20 and 3% non fat dry milk (PBSTM).
ii) The plates are washed three times with PBS supplemented with 0.1% Tween 20 (PBST) and twice
with distilled water.
iii) 100 µl of test serum diluted 1/100 in PBSTM is added in duplicate to wells, which are then incubated
for 1 hour at 37°C.
iv) Plates are washed three times in PBST and twice in distilled water.
v) Horseradish-peroxidase-conjugated anti-species IgG optimally diluted in PBSTM is added at 100 µl
per well and the plate is incubated for 1 hour at 37°C.
vi) After washing as in step iv, each well is filled with 100 µl of 0.1 M citrate buffer, pH 5.5, containing
0.5 mg/ml orthophenylene-diamine and 3 µl/ml of 9% H2O2.
vii) The reaction is stopped after 30 minutes of incubation at room temperature (20–25°C) by adding 50 µl
of 2 N H2SO4. Absorbance is read at 495 nm. Positive and negative controls are included in each
plate.
c) MAP1 competitive enzyme-linked immunosorbent assay (32)

Recombinant MAP1 antigen is prepared as follows: 8-day-old Trichoplusia ni insect larvae are infected by a
baculovirus expressing the map1 gene and moribund larvae are homogenised (10% [w/v]) in PBS
supplemented with 0.001% (v/v) Triton X-100.
Anti-MAP1 MAb is prepared as follows: spleen cells of BALB/C mice previously inoculated with larval
homogenate are fused to SP2/0 cells. Supernatant fluids from hybridoma cell cultures are screened for
reactivity with MAP1 by immunoblotting and immunoperoxidase methods. A reactive cell culture is
subcloned, isotyped and subsequently used for ascites production.
After a further 1/800 (v/v) dilution in PBS, the antigen is immobilised on to polystyrene plates (Nunc-Immuno
Plates PolySorp) by incubation overnight at 4°C, and stored at –70°C.
• Test procedure
i) Prior to use, the plates are blocked for 30 minutes by adding 100 µl per well of PBS, pH 7.2,
supplemented with 0.05% Tween 20 and 5% nonfat dry milk.
ii) Plates are washed three times with PBS/Tween, 50 µl/well of test serum diluted 1/50 in PBS
supplemented with 0.05% Tween 20 and 1% nonfat dry milk (PBSTM) is added in duplicate and the
plates are incubated for 30 minutes at 37°C.

iii) Without an intervening washing step, 75 µl/well of the MAb diluted 1/4000 (v/v) in PBSTM is added
and the plates are incubated for another 30 minutes at 37°C.
iv) Plates are washed three times in PBS/Tween and horseradish-peroxidase-conjugated anti-mouse IgG
optimally diluted in PBSTM is added at 50 µl per well. The plate is incubated for 1 hour at 37°C.
v) After three washings as before, 100 µl of 0.1 M citrate buffer, pH 5.5, containing 0.5 mg/ml O-
phenylene diamine and 3 µl/ml of 9% H2O2 are added to each well. After 30 minutes of incubation at
room temperature in the dark, the reaction is stopped by adding 50 µl of 2 N H2SO4 and the
absorbance is read at 495 nm. Positive and negative controls are included in each plate.
• Reading the results

All serological tests based on non-recombinant E. ruminantium antigens, such as CIFA, ELISAs, and
Western blot, are still used for experimental studies but are no longer used for sero-epidemiological studies.
The tests have been compared and applied to known positive and negative sera to E. ruminantium (9). No
false-positive reactions were observed with any of the tests against known negative sera. There is good
correlation among tests, but the specificity of all five tests is low because cross-reactions occur with certain
Ehrlichia spp.
The interpretation of results of the various tests applied to field surveys is thus difficult in areas where
E. ruminantium infections occur in ruminants, which is probably the case in most of the heartwater-endemic
regions of Africa. This situation has also been demonstrated in farms without Amblyomma but infected with
tick species not known to be vectors of E. ruminantium (13, 49).
Both the MAP1-B ELISA and the MAP1 C-ELISA have shown a high specificity after evaluation in
3000 ruminant sera (goat, sheep and cattle) collected from 14 A.-variegatum-infested islands of the Lesser
Antilles, among which only three are known to be infected by E. ruminantium (32). Overall specificity
calculated from the 11 heartwater-free islands was 98.5% and 99.4% for the MAP1 C-ELISA and the MAP1-
B ELISA, respectively. Although a few false-positive sera are still found, these tests are likely to solve much
of the specificity problems of the earlier serological tests. However, high seroprevalence in vector-free
areas of Zimbabwe or South Africa has also been reported although not explained (it may be caused by a
cross-reacting agent not transmitted by Amblyomma) and should be kept in mind when interpreting the
results.
Evaluating the sensitivity of the tests is more problematic as it would require knowledge of the exact status
of a high number of animals sampled in the field. As mentioned before there is currently no simple
technique available to confirm if an animal is infected. Experimentally, the sensitivity of the C-ELISA in
goats was reported to be 91.6–95.4% for the MAP1-B ELISA, and 96.3–96.9% for the MAP1 C-ELISA (32).
However, in another study the sensitivity averaged 95% for cut-off values set at 31% and 26.6% of the
positive control serum for sheep and goat sera, respectively (31). Indeed, calculations are based on a
limited number of experimentally inoculated animals in a period of time soon after inoculation, when almost
all the animals are still positive. Sensitivity in cattle is even lower and several reports show that after
infection most of the animals become seronegative again in less than 6 months and some animals never
seroconvert (21, 43). This observation is in line with the difference in antibody prevalence observed
between small ruminants and cattle in epidemiological surveys that cannot be explained by a lower risk of
infection of the latter. For example, in Zimbabwean farms situated in endemic areas, more than 90% of
goats presented antibodies in their serum compared with only 33% of cattle maintained in the same
conditions (21). Similar observations were made in the Caribbean. In addition, some areas of Zimbabwe,
which was labelled heartwater-free, had a large number of goats positive for MAP1-B antibodies; this further
complicates the sero-diagnosis of heartwater (13).
Serological tests are useful for the assessment of heartwater antibody response in vaccinated animals. The
tests should not be used to screen animals for importation into heartwater-free areas. Antibodies are
maintained at detectable levels in naturally infected domestic ruminants for a few months only and
circulating antibodies disappear more rapidly in cattle than in small ruminants. It is thus possible that
serologically negative animals may be carriers of infection. Serology should therefore only be regarded as a
diagnostic method to be applied at the herd level and not at the individual animal level (38). When
interpreting diagnostic serology results, other epidemiological parameters must be considered.
Molecular methods, such as PCR assay, could potentially help in detecting carrier animals, but this
approach has still significant drawbacks (see Section B.2 Molecular methods).

No commercial vaccines are available at present. The only method of immunisation against heartwater remains
the ‘infection and treatment’ method using infected blood followed by treatment of reacting animals with

tetracycline (4). This method is still in use in several areas, but it is likely to be replaced soon by preparations
using attenuated or inactivated organisms, which have given promising research results.
1. Inactivated vaccine preparations
Vaccination with preparations of inactivated E. ruminantium elementary bodies emulsified in oil adjuvants was
shown to be possible following the demonstration that susceptible goats can be protected by inactivated Ehrlichia
in Freund’s adjuvant (27). This vaccine also protected against challenge in sheep (16) using different strains of
E. ruminantium (17), and in cattle (47) using the same strain as in goats. A first generation vaccine preparation of
inactivated Ehrlichia in Montanide ISA 50 oil adjuvant was shown to be similarly effective to the Freund’s
adjuvant preparation on laboratory challenge of immunised goats and sheep (28).
In initial vaccine trials, animals were immunised with two subcutaneous injections of 250–1000 µg of antigen
(depending on trial) emulsified (50/50) in Montanide ISA 50 adjuvant in a volume of 2 ml. It has recently been
shown in goats in experimental conditions that the vaccine dose can be lowered to 35 µg of antigen without
decreasing the effect on protection (48). From the initial description, this represents a 28-fold reduction in the
dose of vaccine from 1 mg to 35 µg of E. ruminantium without any modification to the protective effect. The
process for the mass production of E. ruminantium has been developed in parallel (25). Critical parameters have
been determined and optimised for the production of E. ruminantium in endothelial cells in stirred-tank
bioreactors. Using serum-free medium in such bioreactors, E. ruminantium production yields reached a 6.5-fold
increase compared with conventional methods. Using 2-litre bioreactors and the estimated efficient 30 µg
vaccine dose, the cost estimation for one vaccine dose was around 0.11 euro, which makes it affordable in
countries with limited resources. Efficacy trials conducted with vaccine preparations entirely produced using the
mass production and purification process followed by preservation of the product in various solutions (NaCl
versus PBS) and at different temperatures (–20°C, 4°C) have demonstrated that the efficacy of the vaccine is
maintained after the entire mass production and preservation process (26).
In Zimbabwe, field trials of the inactivated vaccine emulsified in ISA 50 adjuvant have also demonstrated
protection of sheep against natural tick challenge (17). In larger field trials conducted in East and South Africa, a
significant reduction in mortality has been achieved in cattle, goats and sheep using either a prototype strain
from Zimbabwe (Mbizi strain) or a local strain from the experimental sites (22). However, in three out of four sites,
the vaccine prepared from the local isolate was less effective than the prototype Mbizi vaccine, strongly
suggesting an inadequate coverage of the antigenic repertoire of isolates present in each site. Lack of cross-
protection between E. ruminantium isolates due to disparities of antigenic composition is well established, but the
complexity of the E. ruminantium population structure in the field has been underestimated. It has recently been
demonstrated in large field evaluation trials carried out in several farming systems in West Africa that, in limited
geographical areas, more than 10 genotypes with differing cross-protection capacities can be present and have a
significant influence on protection with inactive vaccine preparations (unpublished data).
The Mbizi strain inactivated vaccine is being developed commercially by Onderstepoort Biological Products in
South Africa (Mahan S.M., pers. comm.). These inactivated vaccines do not prevent infection but do prevent or
reduce death of vaccinated animals when exposed to live virulent challenge. The advantage however is that
several field strains can be incorporated to make the vaccine more widely cross-protective.
A major challenge remains the characterization of the extent of strain diversity in a region to be covered by an
appropriate formulation of the vaccine. This knowledge will also be essential for new generation vaccines that will
be developed in the future.
2. Attenuated vaccine preparations
Infection of ruminants with live E. ruminantium strains induces a strong long-lasting protection against an
homologous isolate. This is the basis for infection and treatment using virulent isolates. Isolates of attenuated
virulence that do not necessitate treatment of animals would be ideal but a limited number of such attenuated
isolates are available. An attenuated Senegal isolate has been obtained and shown to confer 100% protection
against an homologous lethal challenge, but very poor protection against a heterologous challenge. The Gardel
isolate, which gives a significant level of cross-protection with several isolates (although far from complete), has
also been attenuated. Recently, a third isolate named Welgevonden from South Africa has been attenuated and
shown to confer complete protection against four heterologous isolates under experimental conditions (46). The
main drawback of attenuated vaccines is their extreme lability, which necessitates their storage in liquid nitrogen
and their distribution in frozen conditions. In addition, they have to be administered intravenously.
3. Recombinant vaccine preparations
Several reports show partial protection of mice using map1 DNA vaccination and an improvement of protection
by vaccination following a prime (plasmid) – boost (recombinant MAP1) protocol (33). However, protection of

ruminants has never been demonstrated using this strategy. In opposition, significant protection of sheep was
reported against homologous and heterologous experimental challenge following plasmid vaccination using a
cocktail of four ORFs (open reading frames) from the 1H12 locus in the E. ruminantium genome (7). No further
results have been described since then. Recombinant vaccines will probably not be available in the near future.
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35. PETER T.F., BARBET A.F., ALLEMAN A.R., SIMBI B.H., BURRIDGE M.J. & MAHAN S.M. (2000). Detection of the
agent of heartwater, Cowdria ruminantium, in Amblyomma ticks by PCR: validation and application of the
assay to field ticks. J. Clin. Microbiol., 38, 1539–1544.
36. PETER T.F., BURRIDGE M.J. & MAHAN S.M. (2002). Ehrlichia ruminantium infection (heartwater) in wild
animals. Trends Parasitol., 18, 214–218.
37. PETER T.F., DEEM S.L., BARBET A.F., NORVAL R.A.I., SIMBI B.H., KELLY P.J. & MAHAN S.M. (1995).
Development and evaluation of PCR assay for detection of low levels of Cowdria ruminantium infection in
Amblyomma ticks not detected by DNA probe. J. Clin. Microbiol., 33, 166–172.
38. PETER T.F., O’CALLAGHAN C.J., MEDLEY G.F., PERRY B.D., SEMU S.M. & MAHAN S.M. (2001). Population-based
evaluation of the Ehrlichia ruminantium MAP 1B indirect ELISA. Exp. Appl. Acarol., 25, 881–897.
39. PIENAAR J.G. (1970). Electron microscopy of Cowdria (Rickettsia) ruminantium (Cowdry, 1926) in the
endothelial cells of the vertebrate host. Onderstepoort J. Vet. Res., 37, 67–78.
40. PEIXOTO C.C., MARCELINO I., VACHIERY N., BENSAID A., MARTINEZ D., CARRONDO M.J.T. & ALVES P. (2005).
Quantification of Ehrlichia ruminantium by real time PCR. Vet. Microbiol., 107, 273–278.
41. POSTIGO M., BELL-SAKYI L., PAXTON E. & SUMPTION K. (2002). Kinetics of experimental infection of sheep with
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in tissue culture endothelial cell lines from wild African mammals. J. Wildl. Dis., 34, 297–304.
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47. TOTTE P., MCKEEVER D., MARTINEZ D. & BENSAID D. (1997). Analysis of T-cell responses in cattle immunised
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50. W AGHELA S.D., RURANGIRWA F.R., MAHAN S.M., YUNKER C.E., CRAWFORD T.B., BARBET A.F., BURRIDGE M.J. &
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*
* *
NB: There is an OIE Reference Laboratory for Heartwater (see Table in Part 3 of this Terrestrial Manual or

CHAPTER 2.1.7.
JAPANESE ENCEPHALITIS
SUMMARY
Japanese encephalitis virus is a mosquito-borne flavivirus that causes encephalitis, principally in

horses. It also infects humans, and causes abortions in pigs. Pigs act as amplifiers of the virus,
and birds can also be involved in its spread. The disease has been observed in large parts of Asia
and recently in the western Pacific region.
A definitive diagnosis of Japanese encephalitis in horses depends on the isolation of virus from
dead or sick animals. As the virus is difficult to isolate, clinical, serological and pathological
findings are useful in diagnosis.
Identification of the agent: For virus isolation, brain material is collected from sick or dead horses
that have demonstrated the clinical signs of encephalitis. Isolation procedures include the
inoculation of mice and cell cultures. A suspension of brain tissue is inoculated intracerebrally into
2–4-day-old mice. If the mice show neurological signs followed by death within 14 days, then virus
identification can be carried out by cell culture. The virus can also isolated in cell cultures made
from chicken embryos, porcine or hamster kidney cells, the African green monkey kidney (Vero)
cell line, the MDBK (Madin-Darby bovine kidney) cell line, and mosquito cell lines. Identification of
the virus isolated in mice or in tissue cultures is confirmed by serological or nucleic acid detection
methods such as reverse-transcription polymerase chain reaction assay.
Serological tests: Antibody assay is a useful technique for determining the prevalence of infection
in a horse population, and also for diagnosing Japanese encephalitis in diseased individuals. The
assay methods include virus neutralisation (VN), haemagglutination inhibition, and complement
fixation tests. There is serological cross reactivity with other flaviviruses, such as West Nile, which
can confuse the diagnosis. The plaque reduction VN test is the most specific and can be used to
differentiate Japanese encephalitis virus infection from other flavivirus infections.
Requirements for vaccines and diagnostic biologicals: There is an inactivated vaccine

prepared from a virus suspension derived from infected mouse brains or cell cultures.
A. INTRODUCTION
Japanese encephalitis (JE) is a disease of horses caused by a mosquito-borne flavivirus that elicits clinical signs
of encephalitis in infected animals and can be fatal (9, 12). It also infects humans, and causes stillbirths and
abortions in pigs. Pigs act as amplifiers of the virus, and birds can also be involved in its spread. JE virus (JEV)
is widespread in eastern, south-eastern and southern Asian countries and has recently spread to western India
and to the western Pacific region including the eastern Indonesian archipelago, New Guinea and Northern
Australia (17). Only a single serotype of JEV has been identified, although antigenic and genetic differences
among JEV strains have been demonstrated by several techniques including complement fixation,
haemagglutination inhibition, neutralisation tests using polyclonal or monoclonal antibodies (1, 2, 10, 11, 15) and
oligonucleotide fingerprints of viral RNA (3, 13). Based on the 240-nucleotide sequence analysis of the viral
premembrane (prM) region, JEV strains are classified into four distinct genotypes (5, 6). Recently the envelope
(E) gene analysis was shown to be good representative for the phylogenetic analysis of JEV. To date, five
genotypes of JEV have been described based on the phylogenetic analysis of the viral E gene (18, 20, 21).
The definitive diagnosis of Japanese encephalitis in horses depends on the isolation of the causal virus. The
isolation rate of virus from diseased or dead horses is usually very low, which may be due to the instability of the

Chapter 2.1.7. — Japanese encephalitis
virus under certain environmental conditions, and also to the presence of antibody in infected animals. Clinical,
serological and pathological findings are of assistance in diagnosis. Diagnosis is also possible by the detection of
specific IgM and IgG antibodies in cerebrospinal fluid by enzyme linked immunosorbant assay (ELISA) methods
(4). Viral nucleic acid has been detected in the brain of infected horses by reverse transcription polymerase chain
reaction (RT-PCR) (16).
The specimens collected for virus isolation are portions of the corpus striatum, cortex or thalamus of the brain of
affected horses. The virus can also be isolated from blood and spinal cord samples. All materials should be
refrigerated immediately after collection and frozen to –80°C if specimens are to stored for more than 48 hours.
Any potentially infected materials must be handled following containment level 3 procedures (see Chapter 1.1.2
Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities), to prevent the risk of
human infection. Humans may be infected by direct contact of infectious material with broken skin or mucous
membranes, accidental parenteral inoculation or aerosol. Diagnosticians collecting samples should also take the
appropriate precautions. A human vaccine is available and at risk field veterinarians and laboratory workers
should be vaccinated.

Samples of brain and spinal cord are homogenised in a 10% suspension in buffered saline, pH 7.4, containing
calf serum (2%) or bovine serum albumin (0.75%), streptomycin (100 µg/ml) and penicillin (100 units/ml). The
calf serum should be free from Japanese encephalitis antibodies. The suspension is centrifuged at 1500 g for
15 minutes, and the supernatant fluid is removed for inoculation: 0.02 ml is inoculated intracerebrally into 2 to
4-day-old mice. The inoculated mice are kept under clinical observation for 14 days. No clear clinical signs may
develop, but anorexia becomes evident by the disappearance of the white milk spot on the abdomen. The skin
then changes colour from pinkish to dark red, and convulsions develop immediately before the mice die. Brains
of dead or moribund mice are collected and stored at –80°C for a further passage.
To identify the virus, sucrose/acetone-extracted antigen is prepared from the infected mouse brains of a second
passage in mice as described in Section B.2.b.1. This antigen is checked for its ability to agglutinate the red
blood cells (RBCs) of 1-day-old chickens or of geese at different pH levels between pH 6.0 and 7.0, at intervals
of pH 0.2, according to the method described (8). Briefly, RBC suspensions of 1/24 dilution are prepared in the
diluent with different pH values. In a 96-well plate with a U-shaped bottom, 25 µl volumes of the extracted antigen
is diluted serially. Then, 25 µl of the diluted RBCs is added to each well. The plate is incubated at 37°C for
1 hour, and the haemagglutination result is read. If the antigen is able to haemagglutinate red blood cells, it is
used in a haemagglutination inhibition (HI) test using a Japanese encephalitis antiserum.
Primary cultures of chicken embryo, African green monkey kidney (Vero), baby hamster kidney (BHK) cells, or
the C6/36 mosquito cell line (a cloned cell line from Aedes albopictus) may be used for virus isolation. The
specimens, such as brain and blood taken from animals suspected of being infected, and the brain suspension
from mice after inoculation, are inoculated onto the cell cultures. Monoclonal antibodies specific to flavivirus and
Japanese encephalitis virus are used to identify the virus in the indirect fluorescent antibody test (16). RT-PCR
assay can also be used for identification of JEV in clinical specimens or cell culture fluid using appropriate
primers specific for JEV (7, 14, 16, 19).
Serological tests are useful to determine the prevalence of infection in an animal population, the geographical
distribution of the virus, and the degree of antibody production in vaccinated horses. If serology is to be used for
the diagnosis of the disease in individual horses, it should be remembered that horses in an endemic area may
have been inapparently infected with the virus or may have been immunised with a vaccine. Diagnosis requires a
significant rise in antibody titre in paired sera collected during the acute and convalescent phases. The specificity
of each serological test should also be considered. A latex agglutination test to detect swine antibodies to
Japanese encephalitis has recently been described (22).
In some regions of the world, there is a need to perform additional tests for related viruses before an unequivocal
diagnosis of Japanese encephalitis can be made. For example, in Australia antigenically closely related viruses
of Murray Valley encephalitis and Kunjin virus occur. Recent expansion of the distribution of West Nile virus in
North America, where St Louis encephalitis virus was known to be endemic, further illustrates the flexibility of
flaviviruses to adapt to new environments. The presence of antibody to these other flaviviruses can make
serological diagnosis of Japanese encephalitis difficult. There is some cross reactivity with other flaviviruses on
all the tests; the plaque reduction virus neutralisation test is the most specific.
a) Virus neutralisation
The plaque reduction test using chicken embryo primary cultures, African green monkey kidney (Vero) cells
or baby hamster kidney (BHK) cells is sensitive and the most specific serological procedure available. The
cross reaction with other flaviviruses is minimal; however, if an animal has a high titre to another flavivirus,
such as West Nile, there may be a low neutralising antibody titre to Japanese encephalitis.

Japanese encephalitis virus (Nakayama strain or JaGAr-01 strain) is propagated by intracerebral

inoculation in 1-day-old mice. Brains are collected from moribund or dead mice and a 10% suspension is
prepared in phosphate buffered saline (PBS), pH 7.2, containing 10% fetal calf serum. The suspension is
centrifuged at 5000 g for 20 minutes at 4°C. The supernatant is stored in aliquots at –80°C. The
supernatant fluid of virus infected cell cultures could also be used.
• Test procedure
i) Inactivate sera for 30 minutes in a water bath at 56°C.
ii) Make twofold serial dilutions of the sera in cell culture medium, starting with a 1/10 dilution, in a 24-
well (17 mm in diameter) flat-bottomed microplate or test tubes.
iii) Dilute stock virus in cell culture medium to make 100 plaque-forming units (PFU)/0.2 ml.
iv) Mix one volume of each diluted serum with an equal volume of diluted virus. Include culture medium,
negative serum control and positive serum controls in each plate.
v) Incubate for 90 minutes at 37°C.
vi) Add 200 µl of the virus/serum mixture to each well of BHK-21 cell monolayer in 24-well culture plates.
vii) Incubate the plates in a CO2 atmosphere for 90 minutes at 37°C.
viii) Remove the inoculum and add 1 ml of overlay medium (1.5% carboxymethyl cellulose, 1% fetal calf
serum in Eagle’s medium).
ix) Incubate the plates in a CO2 atmosphere for 4 days at 37°C.
x) After removing the culture fluid, fix the plates in a solution containing 2.5% potassium dichromate, 5%
glacial acetic acid and 5% formalin for 30 minutes at room temperature. Wear rubber gloves when
handling the fixing solution.
xi) Stain the plate in 0.1% crystal violet solution for 30 minutes at room temperature.
xii) Discard the stain and rinse the cells with tap water.
xiii) Air dry the cells and count the plaques.
xiv) Estimate the serum dilution that reduces the number of plaques by 50% or more of the control without
serum.
b) Haemagglutination inhibition
The HI test is widely used for the diagnosis of Japanese encephalitis, but has cross-reactivity with other
flaviviruses. For this test, the sera must first be treated with acetone or kaolin, and then adsorbed with
homotypic RBCs to remove any nonspecific haemagglutinins. The RBCs of geese or of 1-day-old chickens
are used at the optimum pH (6.6–7.0). The test should be conducted with the treated sera and 8 units of
standard antigen; this is commercially available in some countries.
• Haemagglutination (HA)
• Preparation of virus antigen
1. Sucrose–acetone extraction of antigen from infected suckling mouse brains (SMB)
i) Homogenise infected SMB with 4 volumes of 8.5% sucrose.
ii) Add the homogenate drop-wise to 20 times its volume of cold acetone.
iii) Centrifuge (500 g for 5 minutes), then remove the supernatant.
iv) Resuspend the sediment with the same volume as above of cold acetone, and keep in an ice bath for
1 hour.
v) Centrifuge (500 g for 5 minutes), then remove the supernatant.
vi) Pool the sediment with cold acetone in a single tube.
vii) Centrifuge (500 g for 5 minutes), then remove the supernatant.
viii) Spread the sediment inside the tube and vacuum dry for 1–2 hours.
ix) Dissolve the dry sediment with saline: 0.4 volume of original homogenate.
x) Centrifuge (8000 g for 1 hour, 4°C). The supernatant is ready for use.
2. Infected fluid of Aedes albopictus, clone C6/36, cell line

i) Harvest the infected fluid after incubation of the infected cultures at 28°C for 1 week.
ii) Centrifuge (1000 g for 15 minutes). The supernatant is ready for use.

• Preparation of goose red blood cells
1. Solutions
Acid-citrate-dextrose (ACD): 11.26 g sodium citrate (Na3C6H5O7.2H2O); 4.0 g citric acid (H3C6H5O7.H2O);
11.0 g dextrose (C6H12O6); distilled water to a final volume of 500 ml. Autoclave at 10 lb (1.7 unit pressure)
for 10 minutes.
Dextrose-gelatine-veronal (DGV): 0.58 g veronal (Barbital); 0.60 g gelatine; 0.38 g sodium veronal (sodium
barbital); 0.02 g (0.026 g) CaCl2 (for CaCl2.2H2O); 0.12 g MgSO4.7H2O; 8.50 g NaCl; 10.0 g dextrose;
distilled water to a final volume of 1000 ml. Autoclave at 10 lb (1.7 unit pressure) for 10 minutes (five times
stock volume is easier to prepare).
2. Blood collection
1.5 ml of ACD + 8.5 ml of blood (0.5 ml of ACD + 2.8 ml of blood).
3. Washing (sterile)
i) Total blood + 2.5 volume of DGV. Centrifuge (500 g for 15 minutes), then remove the supernatant.
ii) Resuspend the sedimented RBCs in three volumes (total blood) of DGV.
iii) Centrifuge (500 g for 15 minutes), then remove the supernatant. Repeat steps 2 and 3 twice more
(total four spin cycles).
iv) Transfer the final RBC suspension to a flask with aluminium foil cover.
4. Adjusting the RBC concentration
i) 0.2 ml of the RBC suspension + 7.8 ml of 0.9% NaCl (1/40 dilution).

ii) Read the optical density (OD)490 in a spectrophotometer with 10 mm tube.
iii) Adjust the RBC stock so that 1/40 dilution gives 0.450 of OD490. (Final volume = Initial volume ×
absorbance OD490/0.450.)
iv) Store the RBC stock in a refrigerator for up to 3 weeks.
v) Before use, resuspend the RBCs gently and dilute 1/24 in virus-adjusting diluent (VAD).
• Antigen dilution
1. Stock solutions (should be kept at 4°C): 1.5 M NaCl: 87.7 g NaCl and distilled water to a final volume of
1000 ml; 0.5 M boric acid: 30.92 g H3BO3 and hot distilled water to a final volume of 700 ml (dissolve boric
acid and cool down); 1 N NaOH: 40.0 g NaOH and distilled water to a final volume of 1000 ml; borate saline
(BS), pH 9.0: 80 ml 1.5 M NaCl, 100 ml 0.5 M H3BO3, 24 ml 1.0 N NaOH, and distilled water to a final
volume of 1000 ml; 4% bovine albumin: 4 g bovine albumin fraction V (Armour), 90 ml BS, pH 9.0, adjust
pH to 9.0 with 1 N NaOH, and BS, pH 9.0, to make a final volume of 1000 ml.
2. Antigen diluent: 0.4% bovine albumin/borate saline (BABS): 10 ml 4% bovine albumin, pH 9.0, and 90 ml
BS, pH 9.0.
3. Twofold serial dilution of antigen with BABS on U-bottom microtitre plate.
• Addition of goose red blood cells
1. Stock solutions
1.5 M NaCl
0.5 M Na2HPO4: 70.99 g Na2HPO4 (for Na2HPO4, 12 H2O: 179.08 g), and distilled water to a final volume of
1000 ml.
1.0 M NaH2PO4: 138.01 g NaH2PO4.H2O (for Na2PO4, 2H2O: 156.01 g), and distilled water to a final
volume of 1000 ml.

2. Working solution: virus adjusting diluent (VAD)
VAD 1.5 M NaCl 0.5 M Na2HPO4 1.0 M NaH2PO4

6.0 100 32 184
6.2 100 62 160 Add distilled
6.4 100 112 144 water to a
6.6 100 160 120 final volume of
6.8 100 192 104 1000 ml
7.0 100 240 80
Values of VADs are not the pH of each VAD, but the pH after each VAD is mixed with
an equal volume of BABS, pH 9.0.
3. Procedures
i) 1 volume of stock goose RBCs + 23 volumes of VAD (1/24 dilution).
ii) Add 25 µl of diluted RBCs to each well on microtitre plate containing diluted antigen (25 µl/well).
iii) Incubate at 37°C for 1 hour, then read the result.
++ Complete agglutination (uniformly thin pellicle of RBCs following the curvature of the well
bottom)
+ Partial agglutination (a ring associated with a rough or thinner pellicle)
± Minimal agglutination (a button on a thin or scattered pellicle)
– Negative agglutination (clearly defined button with no RBC film)
End point is the last dilution (highest dilution) in which ++ or + is observed.
Titre: the reciprocal of the end point dilution.
• Haemagglutination inhibition
• Preparation of test sera
1. Blood collection and separation of the sera
i) Incubate blood specimen at 37°C for 1 hour and then at 4°C overnight. If the test must be performed
immediately, incubating the sample for 2–3 hours at 37°C can replace the overnight incubation.
ii) Centrifuge (2000 g for 15 minutes) to separate the serum from the clot.
iii) Heat inactivate at 56°C for 30 minutes.
iv) Store at –20°C if not processed immediately.
2. 2-mercaptoethanol treatment (perform this step when IgM antibody titres should be determined)
i) Place 50 µl of the sera into two small test tubes.
ii) Add 150 µl of 0.13 M 2-mercaptoethanol in PBS into one test tube, and 15 µl PBS into another tube.
iii) Incubate at 37°C for 1 hour, then cool in an ice bath.
3. Acetone extraction
i) Place 2.5 ml of cold acetone into each tube. Apply rubber stoppers and extract for 5 minutes in an ice
bath.
ii) Centrifuge cold (1500 g for 5 minutes), then remove the supernatant.
iii) Repeat steps i and ii once more.
iv) Spread the sediment inside tubes and vacuum dry at room temperature for 1 hour.
v) Add 0.5 ml of BS, pH 9.0, to each tube. Apply rubber stoppers. Dissolve the sediment overnight at 4°C
to make 1/10 dilution of the sera.

4. Kaolin extraction as an alternative to acetone extraction

i) 25% acid-washed kaolin (Fischer) in BS, pH 9.0.
ii) 1 volume of sera + 4 volumes of BS + 5 volumes of 25 % kaolin.
iii) Extract at room temperature for 20 minutes with occasional shaking.
iv) Centrifuge (1000 g for 30 minutes). The supernatant is 1/10 dilution of the sera.
5. Adsorption with goose RBCs

i) To each treated serum add 1/50 volume of packed goose RBCs.
ii) Adsorb for 20 minutes in an ice bath.
iii) Centrifuge (800 g for 10 minutes). The supernatant is ready for the HI test (1/10 dilution).
• Haemagglutination inhibition test

1. Primary haemagglutination titration of antigen
Dilute the antigen to make 8 units/50 µl.
2. Serial twofold dilution of test sera on microtitre plate

Serum–antigen reaction
Add 25 µl of diluted antigen into each well containing diluted test sera. Place the remainder of the antigen in
empty wells and incubate at 4°C overnight.
3. Secondary haemagglutination titration of the antigen

i) Serially dilute the prepared antigen (8 units/50 µl) twofold in a 25 µl system.
ii) Add 25 µl of BABS to each well to make 50 µl/well.
4. Addition of goose RBCs

i) Dilute RBC stock (1/24) in VAD.
ii) Distribute 50 µl into each well containing 50 µl of serum antigen mixture or secondary titration of
antigen.
iii) Incubate at 37°C for 1 hour then read the result.
Serum HI titre: the reciprocal of the highest dilution of the test sera showing complete inhibition of HA.
5. Interpretation of the results

Four-fold difference between the titre in the acute and convalescent sera is considered to be a significant
rise or fall and is diagnostic of infection with a virus antigenically related to that used in the test.
c) Complement fixation
Complement fixation (CF) is sometimes used for serological diagnosis. The antigen for this test is extracted
with acetone/ether from the brains of inoculated mice.
• Antigen preparation
i) Extract and weigh the brains of the inoculated dead mice.
ii) Add to the brains 20 volumes of cold acetone, kept at –20°C, and homogenise.
iii) Centrifuge the suspension at 5000 g for 5 minutes at 4°C, and remove the supernatant.
iv) Add to the pellet the same volume of cold acetone as used in step ii above, and mix well.
v) Extract with acetone by keeping the pellet at –20°C for 20 minutes, and repeat the centrifugation
described in step iii above.
vi) Repeat steps iv and v.
vii) Repeat steps iv and v, but this time use cold acetone/ether (equal volume mixture).
viii) Repeat steps iv and v twice using cold ether.
ix) Remove the supernatant by aspirator and spread the pellet over the centrifuge tube.

x) Vacuum dry for 1–2 hours.

xi) Dissolve the pellet in cold saline (2 ml/g of brain) and keep at 4°C overnight.
xii) Centrifuge at 5000 g for 1 hour. The supernatant is the antigen.
• Test procedure
i) Heat-inactivate the test sera at 1/4 dilution in gelatin–veronal buffer.
ii) Serially dilute the sera twofold in a 96-well microtitre plate (25 µl).
iii) Add 25 µl of 4 units of antigen and mix by vibration.
iv) Add 50 µl of 2 units of complement (pooled fresh guinea-pig serum).
v) Mix by vibration and incubate at 4°C for 18 hours.
vi) Leave the microtray at room temperature for 15 minutes.
vii) Add 25 µl of sensitised sheep RBCs to each well.
viii) Mix by vibration and incubate at 37°C for 30 minutes, then read the result.
ix) The highest dilution of test sera showing no haemolysis is the titre of the sera by CF test. A rise or
drop of four-fold or more in the titre is considered to be significant.
The vaccine for Japanese encephalitis in horses is prepared by the inactivation of a virus suspension derived
from infected mouse brains or cell cultures.
1. Seed management

The Beijing-1 strain of Japanese encephalitis virus is used for vaccine production in Japan. The strain must
be lethal for mice when inoculated intracerebrally, and must be able to grow in a primary culture of porcine
kidney. This strain has the capacity to haemagglutinate the RBCs of geese, 1-day-old chickens or pigeons.
The virus must be able to be neutralised by a standard antiserum to Japanese encephalitis virus.
The original and seed viruses should be grown in mouse brains or cell cultures. The passage levels should
not exceed three more than the original virus and two more than the seed virus.
Sara, I’m not sure about the above change. Are you marking these changes for the author to review after
you get MC comments included? If not, let me know and I will try to remember to add with MC comments
The vaccine product from this strain provides immunity to encephalitis in equines and prevents stillbirths in
pregnant sows.
It is recommended that the original and seed viruses be maintained below –70°C, or below 5°C after
lyophilisation.
The virus is grown in the brains of 3–4 week old mice or in a monolayer culture. The cultures should be tested to
confirm that they do not contain adventitious agents (Chapter 1.1.9 Tests for sterility and freedom from
contamination of biological materials). Seed virus is inoculated intracerebrally into mice. The brains of those mice

that show severe clinical signs of encephalitis are collected. These brains are homogenised in PBS, centrifuged
at 1500 g for 30 minutes, and the supernatant fluid is processed as the virus suspension.
Seed virus is inoculated into cell cultures and the fluids are later harvested separately from each batch when
virus replication is at its maximum. This fluid is filtered, or centrifuged at 1500 g for 30 minutes, and the
supernatant fluid is processed as the virus suspension.
Formalin (0.5%) is added to the suspension to inactivate any live virus; this is considered to be the ‘undiluted
virus suspension’. Adjuvant may be added to enhance its immunogenicity.
The virus suspension should be examined for bacterial contamination by culture techniques and for virus
infectivity by intracerebral mouse inoculation or inoculation into cell cultures. The inactivated undiluted virus
suspension should be re-examined for contamination by culture and by microscopy after staining, and should be
checked by intracerebral mouse inoculation to ensure complete inactivation by the formalin.
4. Batch control
a) Sterility
Tests for sterility and freedom from contamination of biological materials may be found in Chapter 1.1.9.
b) Safety
Ten 3-day-old mice are inoculated intracerebrally with 0.02 ml of the final product, and observed for 14 days
to ensure (by the absence of any deaths) the complete inactivation of live virus.
a) Sterility
b) Safety
See Section C.4.b.
c) Formalin determination
The formalin concentration should be less than 0.2% (v/v) by general quantification procedures.
d) Potency
The final product must be checked for immunogenicity by mouse protection tests. The product is diluted
one part to ten parts of PBS; 30 mice aged 2–3 weeks are inoculated intraperitoneally with 0.1 ml of the
diluted product twice at 3-day intervals. There should be an equivalent uninoculated control group. All mice
are challenged intraperitonally with graded doses of live virus 8 days following the first inoculation, and
observed for 14 days. The survival rate should be more than 40% in the immunised group and the mortality
rate in the control group should be more than 90%. The titre of challenge virus should be not less than
103 LD50 (50% lethal dose) per 0.2 ml.
e) Stability
The final product must be shown to be fully effective for 12 months when stored at 4°C.
REFERENCES
1. ALI A. & IGARASHI A. (1997). Antigenic and genetic variations among Japanese encephalitis virus strains
belonging to genotype 1. Microbiol. Immunol., 41, 241–252.
2. BANERJEE K. (1986). Certain characteristics of Japanese encephalitis virus strains by neutralization test.
Indian J. Med. Res., 83, 243–250.

3. BANERJEE K. & RANADIVE S. N. (1989). Oligonucleotide fingerprint analysis of Japanese encephalitis virus
strains of different geographical origin. Indian J. Med. Res., 89, 201–216.
4. BURKE D.S., HISALAK A. & USSERY M.A. (1982). Japanese encephalitis. In: Proceedings of International
Seminar on Viral Diseases in SE Asia and the Western Pacific, Mackenzie J.S., ed. Academic Press,
Sydney, Australia, 537–540.
5. CHEN W.R., TES R.B. & RICO-HESSE R. (1990). Genetic variation of Japanese encephalitis virus in nature. J.
Gen. Virol., 71, 2915–2922.
6. CHEN W.R., TES R.B. & RICO-HESSE R. (1992). A new genotype of Japanese encephalitis virus from
Indonesia. Am. J. Trop. Me. Hyg., 47, 61–69.
7. CHUNG Y.J., NAM J.H., BAN S.J. & CHO H.W. (1996). Antigenic and genetic analysis of Japanese encephalitis
viruses isolated from Korea. Am. J. Trop. Me. Hyg., 55, 91–97.
8. CLARKE D.H. & CASALS I. (1958). Techniques for haemagglutination with arthropod-borne viruses. Am. J.
Trop. Med. Hyg., 7, 561–573.
9. FENNER F.J., GIBBS E.P.J., MURPHY F.A., ROTT R., STUDDERT M.J. & W HITE D.O. (1992). Flaviviridae. In:
Veterinary Virology, Second Edition. Academic Press, New York, USA, 441–455.
10. HALE J. H. & LEE L.H. (1954). A serological investigation of six encephalitis viruses isolated in Malaya. Br. J.
Exp. Pathol., 35, 426–433.
11. HASEGAWA H., YOSHIDA M., FUJITA S. & KOBAYASHI Y. (1994). Comparison of structural proteins among
antigenically different Japanese encephalitis virus strains. Vaccine, 12, 841–844.
12. HOKE C.H. JR & GINGRICH J.B. (1994). Japanese encephalitis. In: Handbook of Zoonoses, Second Edition,
Beran G.W., ed. CRC Press, Boca Raton, Florida, USA, 59–69.
13. HORI H., MORITA K. & IGARASHI A. (1986). Oligonucleotide fingerprint analysis on Japanese encephalitis virus
strains isolated in Japan and Thailand. Acta Virol., 30, 353–359.
14. JAN L.R., YUEH Y.Y., W U Y.C., HORNG C.B., & W ANG G.R. (2000). Genetic variation of Japanese encephalitis
virus in Taiwan. Am. J. Trop. Me. Hyg., 62, 446–452.
15. KIMURA-KURODA J. & YASUI K. (1986). Antigenic comparison of envelop protein E between Japanese
encephalitis virus and some other flaviviruses using monoclonal antibodies. J. Gen. Virol., 67, 2663–2672.
16. LIAN W.C., LIAU M.Y. & MAO C.L. (2002). Diagnosis and genetic analysis of Japanese encephalitis virus
infected in horses. J. Vet. Med. [B] Infect. Dis. Vet. Public Health, 49, 361–365.
17. MACKENZIE J.S. (2005). Emerging zoonotic encephalitis viruses: lessons from Southeast Asia and Australia.
J. Neurovirol., 11, 434–440.
18. SOLOMON T., NI H., BEASLEY D.W.C., EKKELENKAMP M., CARDOSA M.J. & Barrett A.D.T. (2003). Origin and
evolution of Japanese encephalitis virus in Southeast Asia. J. Virol., 77, 3091–3098.
19. TANAKA M. (1993). Rapid identification of flavivirus using the polymerase chain reaction. J. Virol. Methods,
41, 311–322.
20. UCHIL P.D. & SACHIDANANDAM V. (2001). Phylogenetic analysis of Japanese encephalitis virus: envelope
gene based analysis reveals a fifth genotype, geographic clustering, and multiple introductions of the virus
into the Indian subcontinent. Am. J. Trop. Med. Hyg., 65, 242–251.
21. W ILLIAMS D.T., W ANG L.F. DANIELS P.D. & MACKENZIE J.S. (2000). Molecular characterization of the first
Australian isolate of Japanese encephalitis virus, the FU strain. J. Gen. Virol., 65, 2471–2480.
22. XINGLIN J., HUANCHUN C., QIGAI H., XIANG W., BIN W., DEXIN Q. & LIURONG F. (2002) The development and
application of the latex agglutination test to detect serum antibodies against Japanese encephalitis virus.
Vet. Res. Commun., 26, 495–503.
*
* *

CHAPTER 2.1.8.
LEISHMANIOSIS
SUMMARY
Leishmaniosis is not a single entity but comprises a variety of syndromes due primarily to at least
16 species and subspecies of Leishmania. Dogs are commonly affected by L. infantum and
L. chagasi (now regarded as synonyms), but canine infections with L. tropica, L. major and
L. braziliensis have also been reported. In humans, the clinical spectrum ranges from
asymptomatic infections to those with high mortality, with three distinct forms being classically
described: visceral (VL), cutaneous (CL) and mucocutaneous (MCL). The vectors of these
diseases are phlebotomine sandflies belonging to the genera Phlebotomus and Lutzomyia.
Identification of the agent: When clinical signs and characteristic lesions are present in affected
humans and animals, the demonstration of the parasites in stained smears of splenic, bone
marrow and lymph node aspirates, of skin scrapings, and in tissue biopsies, gives a positive
diagnosis. If the infection is low grade, detection of parasites is possible only by attempting in-vitro
or in-vivo isolation or by polymerase chain reaction (PCR). As there are very few morphological
differences among various species, any isolated Leishmania organism must be identified by
molecular, biochemical and/or immunological methods. Several centres throughout the world are
presently using isoenzyme, DNA and antigen characterisation to identify the agent.
Serological tests: Serology is the preferred method for diagnosis of canine leishmaniosis and VL,
even during the early stages of the disease. In subclinical forms, seropositive cases are confirmed
by parasitological diagnosis or PCR. Serology is of less value for CL and MCL. Of the several
serological techniques available, the indirect fluorescent antibody test and the enzyme-linked
immunosorbent assay are the most suitable. Serodiagnostic antigens need to be prepared in the
laboratory, though some commercial products are under evaluation.
Delayed hypersensitivity test: The leishmanin skin test is useful for determining the distribution
of human infections, distinguishing immune from nonimmune cases. The test is positive in CL,
MCL and cured VL, but negative in active VL.
Requirements for vaccines and diagnostic biologicals: There is no effective vaccine available
at present for use in dogs or humans. Leishmanin is no longer available commercially and needs
to be standardised.
A. INTRODUCTION
Leishmaniosis is caused by the vector-borne protozoan parasite, Leishmania. Various forms of clinical
manifestations of human leishmaniosis have been described and divided into three entities: visceral
leishmaniosis (VL, kala azar), cutaneous leishmaniosis (CL, oriental sore, uta, pian bois, chiclero’s ulcer) and
mucocutaneous leishmaniosis (MCL, espundia) (50). In the New World 1, leishmanioses are caused by
L. braziliensis complex (MCL and CL), L. mexicana complex (CL), L. peruviana (CL) and L. infantum (VL and CL);
in the Old World, the aetiological agents are L. donovani (VL), L. infantum (VL and CL), L. tropica (CL), L. major
(CL) and L. aethiopica (CL). Leishmania infantum and L. chagasi have been found to be identical by biochemical
genotyping and should be regarded as synonyms (29). The diseases are mainly zoonoses with two exceptions,
that of CL due to L. tropica in urban areas of Near and Middle East, and that of VL due to L. donovani the Indian
sub-continent (northern India, Nepal and Bangladesh). Canine leishmaniasis (CanL) is a chronic viscero-
cutaneous disease caused by L. infantum (= L. chagasi), for which the dog acts as the source reservoir. In some
instances, parasites belonging to L. braziliensis complex, L. major and L. tropica have been isolated from this
1 In this chapter, the term ‘New World’ refers to the Americas, and the term ‘Old World’ refers to Europe, Africa and Asia.

Chapter 2.1.8. — Leishmaniosis
host (31, 40). The vectors of leishmanioses are phlebotomine sandflies belonging to the genera Lutzomyia (New
World) and Phlebotomus (Old World).
Clinical examination of suspected cases, parasitological diagnosis and immunodiagnosis are the routine
methods available for the diagnosis of leishmaniosis. However, the demonstration of the parasite is the only way
to confirm the disease conclusively. In VL and CanL, isolation and identification of the parasite from biopsies
(lymph node, bone marrow, and spleen aspirate) coupled with molecular and immunodiagnostic tests are
recommended. Parasitological diagnosis is necessary for confirmation of CL (through lesion scraping or needle
aspiration from the edge of the lesions) as neither clinical examination nor serology is adequate. Smears of
biopsy material are stained with Giemsa stain and examined microscopically at ×600–1000 magnification.
Material should also be cultured in appropriate media at 22–26°C.
Morphological characteristics of amastigotes (in humans and mammalian hosts) and promastigotes (in
phebtomine sand flies and in cultures) are the following:
• Amastigote: small intracellular rounded or oval body, 1.5–3 × 2.5–6.5 µm in size, found in vacuoles within
the cytoplasm of the macrophages. There is no free flagellum. The organism has a relatively large nucleus
and a kinetoplast consisting of a rod-like body and a dot-like basal body;
• Promastigote: elongated extracellular organism, body size 15–20 × 1.5–3.5 µm with a single flagellum 15–
28 µm long, arising close to the kinetoplast at the anterior. The nucleus is situated centrally.
The choice of the isolation and culture methods will depend on the immediate circumstances and on the
technical capability and experience of the laboratory staff (45). In-vitro isolation offers certain advantages over
the in-vivo methods: cultures become positive more rapidly (5–30 days compared with months for lesions to
appear on an animal) and the materials are less expensive. However, for in-vitro isolation, the techniques used
should be carried out under strictly sterile conditions, which is rarely feasible in the field. Unfortunately, there is
still no ‘universal’ culture medium in which all the different leishmanias will grow easily, and it is almost
impossible to predict which medium will be best suited to the growth of a particular isolate of Leishmania.
Individual laboratories have to find the most suitable medium among biphasic blood agar media and tissue
culture media supplemented with fetal calf serum (14). When attempting primary isolation of unknown organisms,
a blood agar-based medium should be used – preferably NNN medium (Novy, McNeil and Nicolle), otherwise
brain–heart infusion (BHI) agar medium or EMTM (Evan’s modified Tobie’s medium) should be used. For bulk
cultivation of established isolates, suitable media are reported in Section B.1.a (see ref. 14 for media
composition). The organisms from patients with VL and MCL can be very difficult to cultivate. The parasites
sometimes die when subcultured, even when the initial isolation is successful. This seems especially common
when the initial isolation has been into a rich medium. Often this can be overcome if subcultures are made into
less nutritionally rich media, such as NNN, or one of the semisolid media such as ‘sloppy Evans’ or semisolid
Locke blood agar.
Hamster (Mesocricetus auratus) is the most commonly used animal for in-vivo isolation. Tissue suspensions or
aspirates are inoculated intradermally into the nose and/or feet in the case of detection of dermotropic parasites.
When the material is suspected to be infected with parasites causing VL, the inoculation should preferably be
made by the intraperitoneal route. The resulting infection becomes apparent, weeks or months later, by the
development of a nodule or ulcer at the site of inoculation, and in case of viscerotropic parasites, the infection
becomes apparent, some months later, by massive infection of internal organs. The examination of Giemsa-
stained smears of hamster tissue suspension/aspirate will show amastigotes. BALB/c mice are commonly used
for the diagnosis of L. major.
Several techniques are now being used in many centres to identify the different Leishmania species, subspecies
or strains.
a) Isoenzyme characterisation, also known as MLEE (multi-locus enzyme electrophoresis), is the reference
method for species identification (23, 39, 45, 50), although this technique requires cultivation of a large
number of parasites (5 × 109–1 × 1010). The principles of enzyme electrophoresis are as follows: soluble
enzymes are extracted from the organisms grown in media for bulk cultivation (BHI medium,
MEM/FCS/EBLB [minimal essential medium/fetal calf serum/Evans’ blood lysate broth] medium,
Schneider’s Drosophila medium). A small amount of the extract is then placed in an inert supporting
substance, the matrix, containing a buffer at a fixed pH. The matrix is usually starch gel, but it could equally
well be absorbent cellulose acetate, acrylamide or agarose. The pH of the buffer in the matrix is usually
chosen so that the isoenzymes are negatively charged. A direct current is passed through the matrix carried
by the ions in the buffer. When electrophoresis is completed, most proteins will have moved in the matrix

towards the anode, depending on the amount of negative charge. If stained at this stage with a general
protein stain, many bands will be seen. However, the high substrate and cofactor specificity of enzymes
make it possible to stain only these proteins. Hence, the electrophoretic mobility of one particular enzyme
can be compared among several organisms. The stained matrix with its collection of stained isoenzyme
bands is known as a zymogram. Normally one or more extracts from reference organisms, in which the
enzyme banding patterns are well documented, are included in the gel to aid the interpretation of results.
Most enzymes used for characterisation purposes are stained by methods incorporating a dehydrogenase
reaction. At least 12 enzymes should be examined; organisms showing identical zymograms are classified
into zymodemes of a given species.
b) The monoclonal antibody (MAb) technique is applied to the analysis and classification of Leishmania
species and subspecies (20). For the production of the antibodies, BALB/c mice are immunised with
membrane preparations from either promastigotes or amastigotes. Antibody-secreting hybridoma cultures
are then selected and cloned by limiting dilutions. Specificity to Leishmania strains is assessed through
immunofluorescence or immunoradiometric assays. This analysis should be quantitative, as the amount of
the same surface antigen may vary among Leishmania species. Monoclonal antibodies have also been
used in immunohistochemical techniques applied to tissue biopsies.
c) DNA hybridisation probes are a very specific tool the principle of which is to allow labelled, single-stranded
nuclear or kinetoplast DNA sequences from well characterised standard strains to find and hybridise with
homologous DNA sequences from or within unknown Leishmania isolates (19, 44). Only complementary
DNA sequences will form double-stranded DNA, which can be detected by autoradiography if the probe is
radiolabelled, or by immunoenzymatic reaction. These techniques are sensitive enough to identify 102–
103 organisms spotted on to nylon filters. Much fewer parasites (<10) are required for identification through
the in situ hybridisation technique.
d) Polymerase chain reaction (PCR)-based methods are available for diagnosis and/or identification of
Leishmania from different types of human and canine samples. Essentially, techniques developed either to
detect organisms from fresh or frozen, formalin-fixed and paraffin-embedded biopsies, or to identify
established isolates of Leishmania include: (a) digestion of material with proteinase K and DNA extraction;
(b) standard PCR amplification using oligonucleotide sequences (primers) selected from the small-subunit
rRNA gene (28), kinetoplast DNA minicircles (25) or other highly repetitive genomic DNA sequences (9, 36);
(c) analysis of amplification products by 1–2% agarose gel. To increase sensitivity, a nested or semi-nested
PCR using internal primers from the above sequences can be performed. In human VL, PCR has a
sensitivity comparable with that of culture-based methods, but gives results much faster. In CanL, the
diagnostic efficacy of PCR as compared with serology depends on the natural course of the disease, the
sensitivity being highest shortly after infection (37). In American CL and MCL, PCR appears to be
consistently more sensitive than any previously recommended method of diagnosis (13). Different
techniques have been described that improve both sensitivity and specificity of the method, such as the
PCR-RFLP (restriction fragment length polymorphism) analysis in which the PCR products are digested by
appropriate restrictions enzymes and the resulting restriction fragment pattern is analysed for species or
strain identification (30, 46). Real-time PCR methods, which allow the continuous monitoring of the
accumulation of PCR products during amplification, have been described and are available commercially.
They can be more sensitive than conventional PCR, and are mainly addressed to study the kinetics of
infection and monitoring therapeutic response (3, 7). In addition, real-time PCR has been reported to be
useful for evaluating infections in less invasive samples such as blood (15).
Several serological tests are used for detecting anti-leishmanial antibodies. Sensitivity values reported below for
each test, however, apply only to individuals who are not immunocompromised. A high percentage of patients
with VL co-infected with human immunodeficiency virus (HIV) have been reported to be seronegative for anti-
leishmanial antibodies (18).
a) Indirect fluorescent antibody test

The indirect fluorescent antibody (IFA) test is widely used because it is easy to perform. The test is genus
specific, although significant cross-reactions have been reported in individuals infected with Trypanosoma
cruzi. For these subjects, serological tests based on specific recombinant Leishmania antigens would be
more appropriate (see Section B.2.b and d below). In Chagas’ disease-free areas, the IFA test for the
diagnosis of clinical VL or CanL has a sensitivity of 96% and specificity of 98%, which is similar to the
ELISA. Although amastigotes from frozen sections or smears of infected organs can be used as antigen,
cultured promastigotes represent the commonest antigen source.
i) Harvest 3–4 ml of the liquid media of a 3-day-old culture showing flourishing promastigote growth (see
Section B.1 for culture media).

ii) Wash the organisms three times with phosphate buffered saline (PBS), pH 7.2–7.4, by centrifugation
at 350 g for 15 minutes at room temperature.
iii) Resuspend the final cell pellet in PBS and adjust the promastigote concentration to approximately 4 ×
106/ml with the aid of a haemocytometer.
iv) Distribute 30 µl of the promastigote suspension on to each circle of a multispot slide and allow to dry
at room temperature.
v) Fix the promastigotes in cold acetone for 10 minutes, then put the slides into a plastic box and keep in
a deep freezer (–35°C) for no longer than 2–3 months.
• Test procedure
i) Wash the frozen antigen-coated slides in PBS and allow to dry at room temperature.
ii) Inactivate the sera for 30 minutes in a water bath at 56°C.
iii) Make doubling dilutions of test sera from 1/80 to 1/10,240 for human VL, and from 1/40 to 1/5120 for
CanL. Positive and negative control sera, at dilutions of 1/80 and 1/160 for human VL, and of 1/40 and
1/80 for CanL, are also included in the test. No standard sera are available, but internal standards
should be prepared and titrated.
iv) Distribute 30 µl of diluted serum samples on to each slide circle and incubate for 30 minutes at 37°C.
v) Remove the serum samples by vigorous washing in PBS, followed by immersion of the slides in PBS
for 10 minutes. Allow the slides to dry.
vi) Distribute 30 µl of diluted fluorescein isothiocyanate (FITC)-conjugated anti-immunoglobulin on to
each slide circle and incubate for 30 minutes at 37°C. FITC-conjugated anti-human and anti-dog
immunoglobulins are commercially available. Follow the instructions for the appropriate dilution.
vii) Repeat step v and mount with a cover-slip in a few drops of PBS/glycerol (50% [v/v] of each).
viii) Read the slides under a fluorescent microscope. The highest dilution showing fluorescent
promastigotes is taken to be the antibody titre. In human VL, the threshold titre usually ranges from
1/80 to 1/160, while in CanL it ranges from 1/40 to 1/160. As IFA test performance may vary in
different laboratories, it is better for each laboratory to define its own threshold titre using defined
positive and negative reference sera.
b) Enzyme-linked immunosorbent assay

The ELISA can be carried out on serum or on a measured volume of blood. The blood is collected by
needle-prick on to suitable absorbent paper strips and allowed to dry. The sample is eluted and tested at a
single dilution previously determined to give an acceptable sensitivity and specificity. This test can be used
for seroepidemiological surveys under field conditions.
In the classical method, the antigen is prepared as follows: promastigotes harvested from cultures are
washed four times with PBS, pH 7.2, at 1000 g for 15 minutes. The packed promastigotes are resuspended
in twice their volume of distilled water, and then sonicated at medium amplitude in an ice bath. The
suspension is left at 4°C overnight to allow the proteins to come into solution. After a final centrifugation at
4000 g for 10 minutes to eliminate the cellular debris, the overlay, representing the concentrated soluble
antigen, is dispensed into vials and stored at –20°C until required. For use in the test, it is reconstituted with
PBS to the predetermined optimal protein concentration (around 20 µg/ml) as measured by Lowry’s
method. The ELISA is useful for the diagnosis of Old and New World leishmanioses. There is little or no
cross-reaction with other diseases and, according to the Leishmania strain used, sensitivity can range from
86% to 99%.
A version of the ELISA called the Falcon assay screening test and enzyme-linked immunosorbent assay
(FAST-ELISA) and which uses antigen-coated beads, is considered to be a sensitive, specific and field-
adaptable test for visceral CanL with comparable sensitivity and specificity to the IFA test and ELISA.
Whole blood or plasma can be evaluated quickly without the use of a microscope or spectrophotometer (1).
A detergent-soluble promastigote antigen has been used in ELISA instead of the crude lysate, for the
diagnosis of CanL. The detergent was Triton X-100 and the proteic extract was protected with protease
inhibitors. Using this method, ELISA sensitivity increased to 99.5%, while its specificity was comparable
with that of the IFA test (97%) (26).
The ELISA methods described above are all based on crude antigenic preparations. More recently, a
recombinant antigen from a cloned protein of L. infantum, called rK39, has been reported to be highly
reactive to sera from human and canine visceral leishmaniosis cases when run in an ELISA format. Using
25–50 ng of the antigen, 99% specificity and sensitivity was consistently found for immunocompetent

human patients with clinical VL and for dogs with parasitologically proven disease (2, 41). In HIV-positive
patients, K39-ELISA showed higher sensitivity (82%) than the IFA test (54%) (24). The K39 antigen, which
shows remarkable stability and reproducibility, is now produced commercially.
c) Direct agglutination test

The direct agglutination test (DAT) has been described for the diagnosis of VL and CanL. After test
improvement, DAT has been validated as a specific and sensitive assay for field investigations (4, 10, 35).
The antigen consists of promastigotes harvested from cultures, washed in PBS, pH 7.2, treated with 0.4%
trypsin (for 45 minutes at 37°C and then washed again), and stained with 0.02% Coomassie brilliant blue.
Twofold serial dilutions of serum in PBS are made in V-bottomed microtitre-plate wells; 50 µl of antigen
preparation is added to each well, and the plate is then carefully shaken by hand and left for 18 hours at
room temperature. The test is read visually against a white background. Positive reactions are indicated by
a clear sharp-edged blue spot.
A modified DAT for detection of specific anti-leishmanial antibodies in canine reservoir hosts is considered
to be highly suitable for wide-scale epidemiological and ecological field work and diagnosis of CanL, having
100% sensitivity and 98.9% specificity (21, 22). The reliability of the test was improved by treating the test
sera with 0.2 M 2-mercaptoethanol and incubating them at 37°C.
d) Rapid immunochromatographic assay (dipstick or strip-test)

A rapid immunochromatographic assay using rK39 as antigen (K39 dipstick or strip-test, commercially
available) has been evaluated in different endemic settings of VL. The nitrocellulose membrane of the test
kit holds an absorbent pad at one end, a band of immobilised anti-protein A antibody (used to detect IgG) at
the other (control region), and a band of rK39 antigen in the middle (test region). A protein-A-colloidal gold
conjugate is used as the immunochromatographic detection reagent. One small drop (20 µl) of the serum to
be examined is placed on the absorbent pad before two large drops (100 µl) of test buffer are added to the
pad, and the mixture is allowed to migrate up the strip by capillary action. After 2–10 minutes, the result is
positive if two distinct red lines appear (one in the test region and another in the control region), it is
negative when no red line appears in the test region, and it is invalid if the control line fails to appear.
In clinical cases of human VL, the K39 dipstick showed 100% sensitivity and 93% specificity in India (43),
90% sensitivity and 100% specificity in Brazil (11), and 100% sensitivity and specificity in the Mediterranean
basin (8). In parasitologically proven CanL, in both asymptomatic and symptomatic cases, the sensitivity of
the K39 dipstick was 97% and the specificity 100% (34).
3. Delayed hypersensitivity test
Delayed hypersensitivity is an important feature of all forms of human leishmaniosis and can be measured by the
leishmanin test, also known as the Montenegro reaction (27). The leishmanin skin test has no value for the
diagnosis of CanL. Leishmanin is a killed suspension of whole (0.5–1 × 107/ml) or disrupted (250 µg protein/ml)
promastigotes in pyrogen-free saline containing phenol. A delayed reaction develops and is read at 48–72 hours.
The false-positive reaction rate in otherwise healthy people is approximately 1%, but this can be higher in areas
where there is a background of leishmaniosis, as many of the healthy population may show quite high rates of
leishmanin sensitivity. Although there is complete cross-reactivity among all strains of Leishmania, although
heterologous antigens often give smaller reactions, which may be caused by difficulty in standardisation. The
leishmanin test is used in the clinical diagnosis of CL and MCL. In VL it will only measure past infections because
during active disease, a complete anergy is found. Leishmanins are not available commercially.
1. Vaccine
There is no effective vaccine available for prophylactic immunisation against leishmaniosis. Until now, the only
dependable vaccination against Leishmania has been limited to the protection of humans from both L. tropica
and L. major by prior syringe-induced infection with L. major organisms. The promastigotes are injected into the
arm or other parts of the body. The living promastigotes used must either be freshly extracted from cultures or
may be preserved in liquid nitrogen. The infection is allowed to run a natural course and after recovery, the
individual is firmly immune to subsequent infection with both Leishmania species. This type of immunisation has
been practised on a limited scale in hyperendemic areas of CL (due to L. major) in Israel, Iran and the former
Union of Soviet Socialist Republics (42). Leishmania major causes cross-protection against L. tropica, but the
reverse is probably not true. However, this species cannot be considered to be totally safe and this type of
immunisation should be used only for humans moving into high-risk areas. Moreover, it is not beneficial in highly
endemic areas as individuals contract infection long before this type of preparation confers protection (i.e.

approximately 3 months after vaccination). Standardisation and quality control of such vaccines, presently not
available, are needed.
At present, a number of promising anti-leishmanial vaccines are under development (12, 16). Among the first-
generation vaccines, the glycoprotein-enriched fraction of L. donovani known as ‘fucose-mannose ligand’ (FML),
developed in Brazil, represents the first licensed veterinary vaccine against CanL. Field studies showed about
80% clinical protection conferred by the antigen administered with QuilA saponin as adjuvant (5), and also good
immunotherapeutic efficacy when used in sick dogs (6). Killed Leishmania organisms mixed with a low
concentration of BCG as adjuvant have undergone phase I–II and phase III trials for immunisation against CL
Leishmania agents in humans and against VL in humans and in dogs, with limited success (32, 33).
Second-generation vaccines, most of which are at predevelopment stage, consist of genetically reconstructed
Leishmania parasites incapable of producing disease, recombinant molecules or their corresponding DNAs, or
recombinant organisms carrying leishmanial genes and expressing parasite antigens. A chimeric antigen
generated from three recombinant Leishmania antigens screened for their ability to elicit cellular immune
responses (known as Leish-111f), entered Phase I clinical testing in healthy volunteers in January 2003 (38). The
same polyproteinic antigen, administered with monophosphoryl lipid A – stable emulsion (MPL-SE) or Adjuprime
as adjuvants, failed to protect dogs from L. infantum infection in a phase III trial (17).
2. Immunodiagnostic antigens
Neither the leishmanin used for skin tests nor the antigens commonly employed in serodiagnosis in
leishmaniosis are internationally standardised (the recombinant K39 antigen, which is virtually standardised, is
patent-protected and is not widely available). The leishmanin test is group-specific, not species-specific, and the
leishmanin prepared from one clinical type of leishmaniosis will cause the development of delayed
hypersensitivity to the same or other clinical types. Similarly, serological cross-reactions are common among
leishmanial species.
a) Leishmanin
The leishmanin test is described in Section B.3. Sterility, safety and potency tests are required for
leishmanin preparations.
b) Antigens for serological tests

Commercial antigens for the IFA tests and ELISAs have been produced are still under evaluation. The main
reason for unsatisfactory results with these antigens is the poor stability of leishmanial antigens. They can
be obtained in the laboratory by growing a Leishmania strain in a suitable culture medium. For the IFA test
and the DAT, crude particulate antigens, i.e. intact promastigotes, are required, whereas for ELISAs a
soluble form of the antigen is needed.
3. Seed management

Strains of Leishmania species used to prepare biological products should be identified at species and
subspecies level by appropriate identification tests given in Section B.1. Once the organisms have been
isolated and established in the laboratory, they must be assigned an International Code (45, 50). This Code
should consist of four elements separated by oblique strokes: (a) the type of host from which the strain was
isolated (M for Mammalia and I for Insecta followed by three letters indicating the generic name of the host);
(b) the country where isolation was made, indicated by a two-letter code; (c) the year of isolation indicated
by the last two digits, and (d) the original laboratory code given to the isolate (for example,
MHOM/IN/80/DD8). The parasites must be free from contaminating organisms and should be capable of
yielding a product that conforms to the norms. Standard strains are available on request from the World
Health Organization (WHO) Collaborating Centres in Madrid (Spain), Montpellier (France) and Jerusalem
(Israel). A list of Identification Centres has been published by WHO (50).
The strain of the parasite used for preparing leishmanin should be capable of producing a product that
conforms to national/international norms. It should be free from ingredients causing toxic or allergic
reactions. There is no single specific antigen standardised for use in serodiagnostic tests, but when these
antigens are prepared in the laboratory, they must be standardised for their sensitivity depending on the
requirement. For the preparation of leishmanin as well as serodiagnostic antigens, the organisms should be
grown in a suitable culture medium (such as those recommended in Section B.1 for Leishmania isolation
and bulk cultivation). Normally, good growth of parasites is obtained 7 days after inoculation, and care must

be taken that leishmanial stocks are not lost by overgrowth of the flagellates, which may occur after
approximately 10 days.
c) Cryopreservation
Promastigote cultures and tissue infected with amastigotes may easily be conserved in the living state at
low temperatures. Both forms can be cryopreserved for years at low temperatures in mechanical freezers
(–70°C), in solid carbon dioxide containers (–76°C), or in liquid nitrogen containers (–196°C) (45). A sterile
cryoprotectant is required – glycerol, to give a final concentration of 7.5–10%, or dimethyl sulphoxide
(DMSO), to a final concentration of 5–7.5%. The cryoprotected samples are transferred to the sterile
containers in which they are to be frozen. These may be 2 ml plastic freezing tubes with airtight screw-caps,
hard glass, heat-sealed ampoules, or glass/plastic capillaries. A slow cooling rate (approximately
1°C/minute) is essential for the cryopreservation of Leishmania. This can be obtained by cooling samples to
4°C and keeping them at this temperature for a minimum of 1 hour; they are then transferred to a –20°C
freezer and left for 24 hours, then removed to a –70°C freezer for at least 24 hours. They can be
permanently stored at this temperature, or else transferred into liquid nitrogen or solid carbon dioxide. If
possible, a programmable freezing unit should be used. When the cryopreserved material is required, the
sample is taken out and thawed rapidly in a water bath at 37°C.
d) Validation
Cultures for leishmanin or serodiagnostic antigens should be checked for sterility before use. Leishmanin is
stored at 4°C and serodiagnostic antigens at –20°C or –70°C until required. The latter should be
reconstituted with PBS, pH 7.2, before use. Viable Leishmania cultures can be kept at –70°C for 3–4 years
or at –196°C indefinitely. Because of nonavailability of suitable vaccine, it has not been possible to validate
the currently developed immunising agents. Live or attenuated promastigotes of L. major used in some
areas are far from being satisfactory. Leishmanin should be tested for allergenicity in guinea-pigs before
use. Serodiagnostic antigens should be tested for their efficacy and sensitivity by proper standardisation for
a particular test. If a batch of antigen has not been used for a long time, it should be rechecked before
being used in the test.
As standardised immunodiagnostic antigens are not available commercially, they need to be prepared in the
laboratory. Workers in the laboratory can be at risk of laboratory acquired infection, especially by injection.
Appropriate biosafety precautions are therefore essential to minimise the risks (see Chapter 1.1.2 Biosafety and
biosecurity in the veterinary microbiology laboratory and animal facilities).
a) Leishmanin
Leishmania species are grown, preferably in blood-free liquid media such as Schneider’s Drosophila
medium and RPMI (Rosewell Park Memorial Institute) medium, in order to avoid blood–antigen
contamination. The promastigotes are harvested during the log phase, washed four times in pyrogen-free
saline at 1000 g for 15 minutes, and resuspended in pyrogen-free saline containing 0.5% phenol (w/v) to
obtain a final concentration of 0.5–1 × 107/ml. Leishmanin can also be made with disrupted promastigotes
obtained as above and sonicated. The filtrate is adjusted to a final protein concentration of 250 µg/ml with
pyrogen-free saline containing Tween 80 (0.0005% [v/v]) and phenol (0.28% [w/v]).
b) Antigens for serological tests

Methods of antigen preparation for various tests are given in Section B.2.
One or more batches of leishmanin should be tested in guinea-pigs by allergic test. Sensitivity and specificity of
the leishmanin should preferably be determined by performing the test in appropriate animal models (different
inbred mice according to the Leishmania species), or in patients who have recovered from leishmanial infections,
and in an unexposed control population.
6. Batch control
The WHO has suggested guidelines for the production of leishmanin (48, 49). It is recommended that the source
material be controlled by using isoenzyme analysis to type the Leishmania strains used in preparing leishmanin.
a) Sterility
Each filling lot should be tested for bacterial and mycotic sterility according to WHO (47). Absence of live
leishmaniae is checked by inoculating one sample of each lot in an appropriate blood–agar medium, which

is then incubated at 23°C for at least 15 days. One sample is injected intradermally (for dermotropic
leishmaniae) or intraperitoneally (for viscerotropic leishmaniae) in mice or hamsters. These animals are
observed during a period of 30–90 days.
b) Safety
Samples from each filling lot should be tested for abnormal toxicity by appropriate tests in guinea-pigs and
mice. For each lot, five mice weighing 17–22 g and two guinea-pigs weighing 250–350 g are injected
subcutaneously and intraperitoneally with one human dose of the product. The animals are then observed
for at least 7 days for death or signs of disease.
c) Potency
The leishmanin is tested on animal models (according to the Leishmania species involved) that have been
previously infected by the same strain used for leishmanin production. Lots of at least five infected animals
and control animals are injected intradermally into one of the posterior footpads with 50 µl of leishmanin.
After 2–3 days, all the infected animals should show a significant enlargement of the footpad compared with
control animals.
a) Safety
See Section C.6.b.
b) Potency
See Section C.6.c.
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*
* *

CHAPTER 2.1.9.
LEPTOSPIROSIS
SUMMARY
Leptospirosis is a transmissible disease of animals and humans caused by infection with any of the
pathogenic members of the genus Leptospira. Laboratory diagnosis of leptospirosis can be
complex and involves tests which fall into two groups. One group of tests is designed to detect
anti-leptospiral antibodies and the other group of tests is designed to detect leptospires, leptospiral
antigens, or leptospiral nucleic acid in animal tissues or body fluids. The particular testing regimen
selected depends on the purpose of testing (e.g. herd surveys or individual animal testing) and on
the tests or expertise available in the area.
Identification of the agent: The isolation or demonstration of leptospires in:
a) the internal organs (such as liver, lung, brain, and kidney) and body fluids (blood, milk,
cerebrospinal, thoracic and peritoneal fluids) of clinically infected animals gives a definitive
diagnosis of acute clinical disease or, in the case of a fetus, chronic infection of its mother.
b) the kidney, urine, or genital tract of animals without clinical signs is diagnostic only of a
chronic carrier state.
Isolation of leptospires from clinical material and identification of isolates is time-consuming and is
a task for specialised reference laboratories. Isolation followed by typing from renal carriers is
important and very useful in epidemiological studies to determine which serovars are present
within a particular group of animals, an animal species, or a geographical region.
The demonstration of leptospires by immunochemical tests (immunofluorescence and

immunohistochemistry) is more suited to most laboratory situations. However, the efficacy of these
tests is dependent on the number of organisms present within the tissue, and these tests lack the
sensitivity of culture. Unless specially prepared reagents are used, immunochemical tests do not
identify the infecting serovar and results must be interpreted in conjunction with serological results.
Reagents for immunofluorescence are best prepared with high IgG titre anti-leptospire sera, which
are not available commercially. Rabbit leptospiral-typing serum or monoclonal antibodies can be
used for immunohistochemistry and are available from leptospiral reference laboratories.
Genetic material of leptospires can be demonstrated in tissues or body fluids using a variety of
assays based on the polymerase chain reaction (PCR), either in real-time or traditional formats.
PCR assays are sensitive, but quality control procedures and sample processing for PCR are
critical and must be adjusted to the tissue, fluid and species being tested. Like immunochemical
tests, PCR assays do not identify the infecting serovar.
Serological tests: Serological testing is the most widely used means for diagnosing leptospirosis,
and the microscopic agglutination test (MAT) is the standard serological test. Antigens selected for
use in the MAT should include representative strains of the serogroups known to exist in the
particular region plus those known to be maintained elsewhere by the host species under test.
The MAT is used to test individual animals and herds. As an individual animal test, the MAT is very
useful for diagnosing acute infection: a four-fold rise in antibody titres in paired acute and
convalescent serum samples is diagnostic. To obtain useful information from a herd of animals, at
least ten animals, or 10% of the herd, whichever is greater, should be tested and the vaccination
history of the animals documented.

Chapter 2.1.9. — Leptospirosis
The MAT has limitations in the diagnosis of chronic infection in individual animals and in the
diagnosis of endemic infections in herds. Infected animals may abort or be renal/genital carriers
with MAT titres below the widely accepted minimum significant titre of 1/100 (final dilution).
Enzyme-linked immunosorbent assays (ELISAs) can also be useful for detection of antibodies
against leptospires. Numerous assays have been developed and are primarily used for the
detection of recent infections and the screening of experimental animals for use in challenge
studies. Animals that have been vaccinated against the serovar of interest may be positive in some
ELISAs, thus complicating interpretation of the results.
Requirements for vaccines and diagnostic biologicals: Vaccines for veterinary use are most
often suspensions of one or more serovars of Leptospira spp. inactivated in such a manner that
immunogenic activity is retained. While a range of experimental vaccines based on cellular
extracts have been tested, commercial vaccines are, with few exceptions, whole cell products. The
leptospires are grown in suitable culture media, which often contain serum or serum proteins. If
used, serum or serum proteins should be removed from the final products. Vaccines may contain
suitable adjuvants.
A. INTRODUCTION
Leptospirosis is a transmissible disease of animals and humans caused by infection with the spirochete
Leptospira. All the pathogenic leptospires were formerly classified as members of the species Leptospira
interrogans, however the genus has recently been reorganised and pathogenic leptospires are now identified in
17 named species and four genomospecies of Leptospira (14, 61, 71, 105). There are more than 200 distinct
leptospiral serovars recognised and these are arranged in 23 serogroups (52, 98).
The use, interpretation, and value of laboratory diagnostic procedures for leptospirosis vary with the clinical
history of the animal or herd, the duration of infection, and the infecting serovar. Acute leptospirosis should be
suspected in the following cases: sudden onset of agalactia (in adult milking cattle and sheep); icterus and
haemoglobinuria, especially in young animals; meningitis; and acute renal failure or jaundice in dogs. Chronic
leptospirosis should be considered in the following cases: abortion, stillbirth, birth of weak offspring (may be
premature); infertility; chronic renal failure or chronic active hepatitis in dogs; and cases of periodic ophthalmia in
horses. Two major chronic microbiological sequelae of leptospiral infection present particular diagnostic
problems: the localisation and persistence of leptospires in the kidney and in the male and female genital tract.
Chronically infected animals may remain carriers for years to life and serve as reservoirs of the infection for other
animals and humans.
The demonstration of leptospires in blood and milk of animals showing clinical signs suggestive of acute
leptospirosis is considered to be diagnostic. However, isolation from blood is not often successful because
bacteraemia is transient and not always accompanied by clinical signs. Dogs are often treated with antibiotics
before samples are collected for testing for Leptospira, which further decreases the likelihood of identifying the
agent in blood. The demonstration of generalised leptospiral infection in a range of organs taken at necropsy is
also considered to be diagnostic. However, if the animal lives long enough or has been treated with antibiotics, it
may be difficult to detect intact organisms systemically; immunohistochemistry can be particularly helpful in
identifying residual leptospiral antigen in these cases. Demonstration of leptospires in the genital tract, kidneys,
or urine only must be interpreted with full consideration of the clinical signs and serological results as these
findings may merely indicate that the animal was a carrier.
Failure to demonstrate leptospires in the urine of an animal does not eliminate the possibility that the animal is a
chronic renal carrier, it merely indicates that the animal was not excreting detectable numbers of leptospires at
the time of testing. Collection of urine following treatment of the animals with a diuretic enhances the chances of
detecting the organism (63). In important cases involving individual animals (e.g. clearing an infected stallion to
return to breeding), negative tests on three consecutive weekly urine samples has been considered to be good
evidence that an animal is not shedding leptospires in the urine.

The demonstration of leptospires in body fluids or internal organs (usually kidney, liver, lung, brain, or adrenal
gland) of aborted or stillborn fetuses is considered to be diagnostic of chronic leptospirosis of the mother, and is
evidence of active infection of the fetus.
In experienced hands, the isolation of leptospires is the most sensitive method of demonstrating their presence,
provided that antibiotic residues are absent, that tissue autolysis is not advanced, that tissues are processed for
culture rapidly after collection, and – in the case of urine – at a suitable pH. If tissues or fluids cannot be
transported promptly to the laboratory for leptospiral culture, the sample should be kept at 2–5°C to prevent
overgrowth with other bacteria and autolysis of tissue samples. Liquid culture medium or 1% bovine serum
albumin (BSA) solution containing 5-fluorouracil at 100–200 µg/ml should be used as transport medium for the
submission of samples.
Culture should be carried out in a semisolid (0.1–0.2% agar) medium containing BSA and either Tween 80 (e.g.
Tween 80/BSA medium or EMJH) (44) or containing BSA and a combination of Tween 80 and Tween 40 (30).
Contamination may be controlled by the addition of a variety of selective agents, e.g. 5-fluorouracil (48), nalidixic
acid (49), fosfomycin (64), and a mixture of rifamycin, polymyxin, neomycin, 5-fluorouracil, bacitracin, and
actidione (1). However, use of selective agents may reduce the chances of isolation when there are only small
numbers of viable leptospires, and some strains of leptospires will not grow in selective media containing multiple
antibiotics. Addition of 0.4–1% rabbit serum to semisolid culture medium enhances the chances of isolating
fastidious leptospiral serovars.
Cultures should be incubated at 29 ± 1°C for at least 16 weeks, and preferably for 26 weeks (30). The time
required for detection of a positive culture varies with the leptospiral serovar and the numbers of organisms
present in the sample. Less fastidious serovars (e.g. Pomona and Grippotyphosa) may result in positive cultures
as soon as 7–10 days after inoculation; other serovars (e.g. Hardjo and Bratislava) may take much longer.
Cultures should be examined by dark-field microscopy every 1–2 weeks. It is important to use a 100 watt light
source and a good quality dark-field microscope.
Leptospires may also be demonstrated by a variety of immunochemical staining techniques, e.g.

immunofluorescence (10, 31), and various immunohistochemical techniques (5, 33, 79, 81, 99, 106). These are
useful in diagnosing infection in pathological material that is unsuitable for culture or where a rapid diagnosis is
required. As the success of these techniques is dependant on the number of organisms present, they are less
suitable for diagnosing the chronic carrier state, where the numbers of organisms may be very low or localised.
Leptospires do not stain satisfactorily with aniline dyes, and silver-staining techniques lack sensitivity and
specificity, although they are a useful adjunct for histopathological diagnosis (7).
Polymerase chain reaction (PCR)-based assays are now used in some diagnostic and many reference
laboratories for the detection of leptospires in tissues and body fluids of animals. A variety of primer sets for the
conduct of PCR assays have been described (3, 6, 13, 37, 39, 44, 50, 51, 54, 59, 60, 66, 84, 93, 97, 103) with
some primers only specific for the genus Leptospira and others designed to identify only pathogenic species.
These assays do not identify the infecting serovar, although some primer sets may permit further identification to
the species or serovar level if the PCR amplicons are sequenced. This further analysis is not a routine diagnostic
method. Many of the PCR primer sets have been designed and evaluated for use in human rather than animal
specimens and general agreement about the PCR primers to be used for testing of animal samples is lacking.
Therefore, the individual laboratory is generally responsible for the validation of the particular assay they use for
the tissue, fluid, and species being tested. PCR assays can be quite sensitive, but lack of specificity (i.e. false-
positive results) can be a problem. Presence of amplification inhibitors in clinical samples can cause false-
negative results, particularly in animal specimens that may be compromised by contamination with faeces or
autolysis. Quality control of PCR assays used for diagnosis of leptospirosis requires careful attention to
laboratory design and workflow to prevent contamination of reagents, and use of appropriate control samples
(29, 57). In addition, sample processing for PCR is critical and must be suited to the tissue, fluid, and species
being tested. A procedure for the preparation of urine samples for PCR using magnetic beads coated with anti-
leptospiral antibody shows promise in enhancing the detection of pathogenic leptospires in urine (88).
The identification of leptospiral isolates is a task for specialised reference laboratories. For complete
identification, a combination of procedures is used to determine: 1) if the isolate is a pathogen or a saprophyte;
2) the species of Leptospira to which the isolate belongs; and 3) the serogroup and serovar of the isolate. A pure
leptospiral culture may be identified as belonging to a pathogenic or saprophytic species by a variety of tests: the
ability to infect animals; the relative resistance to 8-azaguanine; lipase activity; salt and temperature tolerance
(46, 47); PCR-assay-based amplification of 23S rDNA (102); and G+C content of DNA (46).
New leptospiral species have been identified based on DNA–DNA hybridisation analysis (14, 71, 105). In most
cases, the type strain for each serovar was used in these analyses; for a few serovars, clinical isolates have also
been tested to determine the new species designations. Different isolates belonging to a single serovar usually
belong to the same species, but this is not always the case. Species identification of field isolates is still
cumbersome but can be done by sequence analysis of the 16S rDNA, by genetic analysis of the 16S or 23S
ribosomal RNA genes (17, 53, 61, 68, 70, 100, 101), by multilocus sequence typing (4, 77), by sequencing the
DNA gyrase subunit B encoding gene (82), or by PCR using species-specific ompL1 primer sets (73).

Strains belonging to Leptospira can be differentiated to the serogroup level by cross-agglutination reactions (28).
Further differentiation to the serovar level was traditionally by cross-agglutination absorption, although for most
isolates this is now being done using less time-consuming methods: factor analysis (28), monoclonal antibodies
(MAbs) (89, 90), restriction endonuclease analysis (43, 56, 91, 92), and various other molecular strategies (17,
21, 26, 38, 61, 69, 70, 72, 75, 77, 78, 82, 83, 107, 108). However, genetic-based tests may not always give the
same results as the cross-agglutination absorption test.
Serological testing is the laboratory procedure most frequently used to confirm the clinical diagnosis, to
determine herd prevalence, and to conduct epidemiological studies. Leptospiral antibodies appear within a few
days of onset of illness and persist for weeks or months and, in some cases, years. Unfortunately, antibody titres
may fall to undetectable levels while animals remain chronically infected. To overcome this problem, sensitive
methods are needed to detect the organism in urine or the genital tract of chronic carriers.
A wide variety of serological tests, which show varying degrees of serogroup and serovar specificity, have been
described. Two tests have a role in veterinary diagnosis: the microscopic agglutination test (MAT) and the
enzyme-linked immunosorbent assay (ELISA).
a) Microscopic agglutination test

The MAT using live antigens is the most widely used serological test. It is the reference test against which
all other serological tests are evaluated and is used for import/export testing. For optimum sensitivity, it
should use antigens representative of all the serogroups known to exist in the region in which the animals
are found and, preferably, strains representing all the known serogroups. The presence of a serogroup is
usually indicated by frequent reaction in serological screening but can only be definitively identified by
isolation of a serovar from clinically affected animals. The sensitivity of the test can be improved by using
local isolates rather than reference strains, but reference strains assist in the interpretation of results
between laboratories.
The specificity of the MAT is good; antibodies against other bacteria usually do not cross-react with
Leptospira to a significant extent. However, there is significant serological cross-reactivity between serovars
and serogorups of Leptospira and an animal infected with one serovar is likely to have antibodies against
the infecting serovar that cross-react with other serovars (usually at a lower level) in the MAT. Therefore,
serology cannot be used to definitively identify the infecting serovar in an individual infection or outbreak –
this requires isolation of the agent. However, in areas where the serovars of Leptospira present have been
well described by isolation studies, serological examination of the infected animal(s) may suggest, but not
definitively identify, the infecting serovar. In addition, animals that have been vaccinated against
leptospirosis may have antibodies against the serovars present in the vaccine used. Therefore, it is
particularly important to consider the vaccination history of the animals under test. The two methods for
carrying out the test have been described in detail (36, 62).
The strains selected should be grown in liquid leptospiral culture medium (e.g., EMJH, Tween 80 BSA, or
other suitable medium) at 29 ± 1°C and the culture should be at least 4 days old, but no more than 8 days.
Live cultures with densities of approximately 2 × 108 leptospires per ml are to be used as the antigens. The
culture density can be determined by counting the cells directly using a bacterial counting chamber and
dark-field microscopy. Alternatively, cell counts can be estimated by measuring transmittance in a
spectrophotometer with a 400 nm filter or by nephelometry. If indirect methods are used, direct bacterial cell
counts should be correlated with the readings on the specific instrument being used. The number of
antigens to be used is determined and a screening test may be performed with a 1/50 serum dilution (or a
different starting dilution based on the purpose of the test). A volume of each antigen, equal to the diluted
serum volume, is added to each well, making the final serum dilution 1/100 in the screening test. The
microtitration plates are incubated at 29 ± 1°C for 2–4 hours. The plates are examined by dark-field
microscopy.
The endpoint is defined as that dilution of serum that shows 50% agglutination, leaving 50% free cells
compared with a control culture diluted 1/2 in phosphate buffered saline. The result of the test may be
reported as the endpoint dilution of serum (e.g. 1/100 or 1/400) or as a titre that is the reciprocal of the
endpoint serum dilution (e.g. 100 or 400). Many laboratories perform a screening test at a final serum
dilution of 1/100 and then retest sera with titres of ≥100 to determine an endpoint using doubling dilutions of
sera beginning at 1/100 through to 1/12,800 or higher.
Identity of antigens is a crucial factor in conducting the MAT. Antigens should be evaluated for identity,
using hyperimmune rabbit sera, MAbs, or a molecular method that confirms passages over time, preferably
each time the test is run, but at least twice a year. Hyperimmune rabbit serum for this purpose can be
obtained from a reference laboratory or prepared using a protocol such as that given by the Subcommittee
on the Taxonomy of Leptospira (45). Briefly, healthy rabbits weighing 3–4 kg that lack detectable anti-

leptospiral antibodies are selected. Each rabbit is given an intravenous injection in a marginal vein of the
ear with a well-growing live or formalin-treated cloned culture with a density of approximately 2 ×
108 leptospires/ml. The culture should be grown in Tween 80 BSA medium or another appropriate medium.
Five injections of 1 ml, 2 ml, 4ml, 6 ml, and 6 ml each are given at 7-day intervals. One week following the
final injection, the homologous antibody titre is determined by MAT. If the titre is ≥1/12,800, the rabbit is
anaesthetised and bled by cardiac puncture 7 days later (i.e. 14 days after the final injection). If the titre is
<1/12,800, a further injection of 6 ml of culture can be given; 7 days after this injection the homologous titre
is again determined. Unless the titre is ≥1/12,800 the procedure should be repeated with another rabbit.
Two rabbits are used to prepare each antiserum. If the titres are satisfactory in both rabbits, the sera may
be pooled. To preserve potency, it is preferable to freeze-dry the antiserum in 2 ml volumes and store it at
2–5°C. Alternatively, the serum can be stored in 2 ml volumes at –15 to –20°C. All animal inoculations
should be approved and conducted according to the relevant standards for animal care and use. Other
immunisation protocols may be considered based on the intended use of the antiserum.
Purity of antigens used in the MAT should be checked regularly by culture on blood agar and in
thioglycolate broth. Stock cultures of antigens may be stored at –70 to –80°C or in liquid nitrogen. There
may be a low survival rate of leptospires after lyophilisation. Repeated passage of antigens in liquid medium
results in a loss of antigenicity. In this case, a new liquid culture should be derived from the stock culture.
As an individual animal test, the MAT is very useful in diagnosing acute infection; the demonstration of a
four-fold change in antibody titres in paired acute and convalescent serum samples is diagnostic. In
addition, a diagnosis of leptospirosis is likely based on the finding of very high titres in an animal with a
consistent clinical picture. The test has limitations in diagnosis of chronic infection in individual animals,
both in the diagnosis of abortion (32) and in the identification of renal or genital carriers (30). This is
particularly true with the host-adapted leptospiral infections, e.g. serovar Hardjo infection in cattle: when a
titre of 1/100 or greater is taken as significant, the sensitivity of the test is only 41%, and even when the
minimum significant titre is reduced to 1/10, the sensitivity of the test is only 67% (30). The demonstration of
antibodies in fetal blood is diagnostic, but the titres are often very low, i.e. 1/10, requiring a modified testing
procedure for most laboratories.
As leptospirosis is a herd problem, the MAT has much greater use as a herd test. To obtain useful
information, Cole et al. (20) suggested that samples be taken from at least ten animals, or 10% of the herd,
whichever is the greater. In a study of Hardjo infection in cattle, Hathaway et al. (42) found that a 10-cow
sample usually indicated the presence or absence of infection in a herd. Increasing the sample size
markedly improved epidemiological information, investigations of clinical disease, and public health
tracebacks.
In making a serological diagnosis of leptospirosis, the infecting serovar and the clinical condition involved
must be fully considered. In the case of serovar Pomona-induced abortion in cattle, a high titre is commonly
found at the time of abortion because the clinical incident occurs relatively soon after infection. Abortion in
cattle due to serovar Hardjo is a chronic event; in this case, the serological response at the time of abortion
is more variable, with some animals seronegative and others showing high titres. Cattle may experience a
drop in milk production during the acute phase of Hardjo infection and this clinical sign is associated with
high titres. Vaccination history must also be considered in the interpretation of MAT results as widespread
vaccination contributes significantly to the number of seropositive animals and may mask the presence of
chronic infections in the herd – particularly with serovar Hardjo.
b) Enzyme-linked immunosorbent assays

ELISAs for detection of anti-leptospiral antibodies have been developed using a number of different antigen
preparations, assay protocols and assay platforms, including plate tests and dipstick tests. Information
regarding the surface antigens of Leptospira has been reviewed (25). In general, ELISAs are quite
sensitive, but lack the serovar specificity of the MAT. An ELISA that measures canine IgG and IgM against
various leptospiral serovars has been developed and evaluated in Europe (40, 41). Anti-leptospiral IgM is
detectable in this assay as early as 1 week after infection, before agglutinating antibodies are present. IgG
antibodies are detectable in infected dogs beginning 2 weeks after infection and persist for long periods of
time. Therefore, dogs with acute leptospirosis have high IgM titres and relatively low IgG titres; dogs that
are vaccinated or have had previous leptospiral infections have high IgG titres but low IgM titres. Similar
assays to detect anti-leptospiral bovine, porcine, and ovine antibodies have also been developed (2, 19, 24,
58, 74, 85–87, 94, 104). The major identified role of ELISA in livestock species is the use of an IgM ELISA
for identification of recent infections (24) and for screening herds in regions where vaccination for
leptospirosis is not practiced. A total-Ig ELISA is useful in identification of fully susceptible animals suitable
for experimental challenge work (34). ELISAs have also been developed for use in milk from individual cows
or in bulk tank milk for the detection of serovar Hardjo antibodies. These tests have been helpful in
identifying Hardjo-infected herds. However, herds that are vaccinated against serovar Hardjo will also be
positive in these various ELISAs decreasing their usefulness in regions where vaccination is a routine

practice. New ELISAs have been developed based on detection of antibodies against surface proteins or
lipoproteins of Leptospira (12, 27, 55, 65, 67) but these tests are not yet widely available.

Leptospiral vaccines for veterinary use are suspensions of one or more strains of pathogenic Leptospira
inactivated in such a manner that immunogenic activity is retained. While experimental vaccines based on
cellular extracts have been tested (9), commercial vaccines are, with few exceptions, whole-cell products. The
leptospires are grown in suitable culture media that may contain serum or serum proteins. If used, serum or
serum proteins should be removed from the final product. Vaccines may contain suitable adjuvants.
1. Seed management

Proper selection of vaccine production strains is of utmost importance. Immunity induced by vaccination is
largely serovar specific (18). A vaccine should be formulated for use in a particular animal species in a
particular geographical region. It should contain only those serovars – and preferably those genotypes –
that cause problems in the animal species, or that are transmitted by the animal species to other species in
the region. Strains selected for use as master seed culture should be cloned on solid medium to ensure the
absence of saprophytic Leptospira contaminants and uniformity of the culture.
Suitable strains should be further selected by their ability to grow to high yields under batch culture
conditions.
b) Methods of culture
Each component strain to be included in the final vaccine should be grown separately in liquid medium;
preferably in a protein-free (8, 80) or low-protein medium (8).
The volume of each master seed culture should be amplified by growth for 2–10 days at 29°C ±1°C in a
series of subcultures until a volume sufficient for use as a production seed culture is achieved. Cultures
should be aerated and agitated as required.
Each subculture of the master seed culture should be checked for purity and for satisfactory growth. Purity
can be checked by inoculating a loopful of culture into blood agar plates or into thioglycolate broth for
incubation at 35–37°C for 2–5 days, and by examining a Gram-stained smear of culture sediment. Growth
can be checked by dark-field microscopy. Each production seed culture should also be checked against its’
homologous rabbit antiserum (28) to ensure purity and homology. MAbs may also be used for this purpose.
There is a large volume of literature describing the efficacy of leptospiral vaccines. In most cases, vaccines
provide significant protection against disease produced by homologous challenge under field conditions.
Vaccines are less efficacious at preventing infection in animals and a percentage of vaccinated animals will
become infected with the relevant serovar and may shed the organism in their urine despite a lack of clinical
signs of disease.
Efficacy trials and vaccine validation must be conducted in the target species for the vaccine. The vaccine
should be administered as recommended on the label, and immunity should be tested by challenge with
virulent field strains of each serovar by natural routes of infection, i.e. by conjunctival and/or vaginal
challenge. Validation studies have often been conducted with challenge of immunity by intravenous or
intramuscular injections of leptospires. Vaccines validated in this way have not always been shown to be
protective against field challenge, which occurs by exposure of mucous membranes of the eye, mouth, and
genital tract to leptospires. Most notably, commercial leptospiral vaccines containing serovar Hardjo have
not always protected cattle from conjunctival or field challenge with serovar Hardjo (11). A draft monograph
for the efficacy testing of serovar Hardjo vaccines has been prepared and specifies the use of more natural
routes of challenge (35).

Manufacture is carried out by batch culture in appropriately sized fermentor vessels. These should be equipped
with ports for the sterile addition of seed culture, air, and additional medium. They should also have sampling
ports so that the purity and growth of the production culture can be monitored.
Ideally, low-protein or protein-free media are used for production. However, some strains require the presence of
animal protein to achieve suitable yields; this is usually supplied as BSA. All media components that are not
degraded by heat should be heat sterilised. This reduces the risk of contamination by water-borne saprophytic
leptospires that are not removed by filter sterilisation.
After addition of the seed culture, the growth of the production culture is monitored at frequent intervals for the
start of log-phase growth. Once this is observed, the vessel is then agitated and aerated. The final yield can often
be improved by the addition of more Tween 80 to the culture when log-growth is first observed to be slowing
down. Adequate growth may require up to 10 days of incubation at 29 ± 1°C.
Inactivation is usually by the addition of formalin, but phenol, merthiolate, and heat inactivation have also been
used.
After the appropriate inactivation period, the culture may be concentrated and extraneous protein material may
be removed by ultrafiltration. Suitable volumes of the various strains to be included in the final vaccine can then
be blended, and adjuvant and preservative added, if appropriate.
During production, daily or twice daily subsamples should be taken and monitored for growth of leptospires and
absence of contaminants. Growth is monitored either by counting leptospires in a counting chamber under dark-
field microscopy or by a nephelometer. The absence of contamination can be monitored by the microscopic
examination of Gram-stained preparations of centrifuged culture.
Immediately prior to inactivation, a sample should be taken for checking against its homologous antibody in a
MAT. The inactivated culture must be checked for freedom from viable leptospires. This is done by inoculating
aliquots of inactivated culture into an appropriate growth medium, such as the medium of Johnson & Harris (47),
incubating at 29 ± 1°C for at least 4 weeks, and examining weekly by dark-field microscopy for the presence of
viable leptospires.
After blending, the levels of free inactivating agents, minerals present in adjuvants (such as aluminum), and
preservative (such as thiomersal) must be within prescribed limits.
4. Batch control
a) Sterility
Selected samples of the completed vaccine should be tested for the absence of viable bacteria and fungi
(16, 22, 23, 95). Tests for sterility and freedom from contamination of biological materials may be found in
Chapter 1.1.9.
b) Safety
Samples of completed product should be tested for safety. Methods for this have been described elsewhere
(15, 22, 96). The test should be carried out for each route of inoculation indicated on the label and in two
healthy animals of each category (e.g. pregnant animals, young stock) for which the vaccine is intended.
The animals must be susceptible to the serovars used in the vaccine and their sera must be free from
agglutinating antibodies to those serovars. Each animal is given an injection of the vaccine by the
recommended route with twice the recommended dose, as stated on the label. The animals are observed
for 14 days and should show no adverse local or systemic effects attributable to the vaccine.
c) Potency
Samples of completed vaccine should be tested for potency in hamsters or guinea-pigs. Potency is usually
measured by the vaccine’s ability to prevent the death of the animal when challenged with between 10 and
10,000 LD50 (50% lethal dose). With some serovars that are not hamster or guinea-pig lethal, such as
serovar Hardjo, potency is measured against prevention of renal infection when the animals are challenged
with between 10 and 10,000 ID50 (50% infectious dose) or by induction of a suitable antibody titre in rabbits.

An example protocol is to inject 1/40 dog dose of the vaccine into each of ten healthy hamsters no more
than 3 months old. After 15–30 days, each vaccinated hamster, and each of ten unvaccinated hamsters of
the same age, is injected intraperitoneally with a suitable quantity of a virulent culture of leptospires of the
serovar used to make the vaccine (or a suspension of liver or kidney tissue collected from an experimentally
infected animal). In the case of bivalent vaccines, each serovar is tested separately. For the vaccine to pass
the test, at least 8/10 of the vaccinated animals should remain in good health for 14 days after the death of
the controls. Other protocols may apply to cattle and pig vaccines, which contain as many as five or six
components.
In-vitro potency tests for leptospiral vaccines are being developed based on quantifying the protective
antigen in the vaccine using MAbs in a capture ELISA (76). These assays are being standardised using
reference vaccines and correlation with existing hamster or antibody-based potency assays and target–host
efficacy data.
Duration of immunity should be determined in the animal species for which the vaccine is intended using
natural routes of challenge (11). Duration of immunity should not be estimated based on the duration of
MAT titres in vaccinated animals as protection against clinical disease may be present with very low titres.
Vaccinal immunity should persist for at least 6 months or longer depending on the label claim.
e) Stability
When stored under the prescribed conditions, the vaccines may be expected to retain their potency for 1–
2 years. Stability should be assessed by determining potency after storage at 2–5°C, room temperature,
and 35–37°C.
5. Tests on the final project
a) Safety
See Section C.4.b.
b) Potency
See Section C.4.c.
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*
* *
NB: There are OIE Reference Laboratories for Leptospirosis (see Table in Part 3 of this Terrestrial Manual or

CHAPTER 2.1.10.
NEW WORLD SCREWWORM

(Cochliomyia hominivorax) AND
OLD WORLD SCREWWORM (Chrysomya bezziana)
SUMMARY
The New World screwworm 1 (NWS), Cochliomyia hominivorax (Coquerel), and the Old World
screwworm1 (OWS), Chrysomya bezziana Villeneuve, are both obligate parasites of mammals
during their larval stages. Both species are in the subfamily Chrysomyinae of the family
Calliphoridae of the order Diptera (true flies). Larvae feeding on the skin and underlying tissues of
the host cause a condition known as wound or traumatic myiasis, which can be fatal. Infestations
are generally acquired at sites of previous wounding, due to natural causes or to animal husbandry
practices, but they may also occur in the mucous membranes of body orifices.
Female flies are attracted to wounds at the edges of which each female lays an average of
175 (OWS) to 340 (NWS) eggs. The larvae emerge within 12–24 hours and immediately begin to
feed, burrowing head-downwards into the wound. After developing through three larval stages
(instars) involving two moults, the larvae leave the wound and drop to the ground into which they
burrow to pupate. The duration of the life-cycle off the host is temperature dependent, being
shorter at higher temperatures, and the whole cycle may be completed in less than 3 weeks in the
tropics.
Treatment is generally effected by application of organophosphorus insecticides into infested
wounds, both to kill larvae and to provide a residual protection against reinfestation. Preventive
measures include the spraying or dipping of susceptible livestock with organophosphorus
compounds and, more recently, use of avermectins (especially doramectin) as subcutaneous
injections to animals ‘at risk’. Strict control of the movement of animals out of affected areas also
acts as a preventive measure.
Identification of the agent: The larvae of NWS and OWS can be easily confused with each other
and with the larvae of other agents of myiasis. Accurate diagnosis involves the identification of
larvae extracted from the deepest part of an infested wound. The mature, third instar larvae are
most reliable for this purpose and those of NWS can be identified by their darkly pigmented dorsal
tracheal trunks extending from the twelfth segment forward to the tenth or ninth. This pigmentation
is unique to the larvae of NWS among the species encountered in wound myiasis. Confirmation of
OWS relies on the recognition of a characteristic combination of spinulation, the number of lobes
on the anterior spiracles (4–6), and pigmentation of secondary tracheal trunks.
In the adult stage, species in the genus Cochliomyia can be separated from other genera involved
in wound myiasis by confirmation of a body colour that is usually a metallic blue/green with three
dark longitudinal stripes always present on the thorax. The separation of NWS from the very
similar C. macellaria and the identification of adult OWS are discussed in this chapter.
Serological tests: At present there are no applicable serological tests, nor are they indicated in
the identification of this disease. However, serology may have a future role in studies of the
prevalence of myiasis.
Requirements for vaccines and diagnostic biologicals: There are no vaccines or biological
products available except for the use of sterilised male flies in the sterile insect technique (SIT). In
this technique, vast numbers of sterilised male flies are sequentially released into the environment,
1 In this chapter, the term ‘New World’ refers to the Americas and the term ‘Old World’ refers to Europe, Africa and Asia.

Chapter 2.1.10. — Screwworm (Cochliomyia hominivorax and Chrysomya bezziana)
where their matings with wild females produce infertile eggs, leading to an initial population
reduction and, progressively, eradication.
A. INTRODUCTION
The New World screwworm fly (NWS), Cochliomyia hominivorax (Coquerel), and the Old World screwworm fly
(OWS), Chrysomya bezziana Villeneuve, are species of two genera of the subfamily Chrysomyinae of the
Dipteran family Calliphoridae (blowflies). Both species are obligate parasites of mammals including humans and,
rarely, birds. Despite being in different genera and geographically separated, the two species have evolved in
remarkable parallel. They have almost identical life histories because they fill identical parasitic niches in their
respective geographical zones. The following discussion will relate to both species, except where indicated.
Unlike most other species of blowflies, adult female screwworms do not lay their eggs on carrion. Instead, they
lay them at the edges of wounds on living, injured mammals or at their body orifices. Virtually any wound is
attractive, whether natural (from fighting, predators, thorns, disease, and/or tick and insect bites) or man made
(from shearing, branding, castrating, de-horning, docking, and/or ear-tagging). Commonly infested natural
wounds are the navels of newborn animals and the vulval and perineal regions of their mothers, especially if
traumatised. If eggs are deposited on mucous membranes, the larvae can invade undamaged natural body
openings such as the nostrils and associated sinuses, the eye orbits, mouth, ears, and genitalia.
Within 12–24 hours of the eggs being laid, larvae emerge and immediately begin to feed on the wound fluids and
underlying tissues, burrowing gregariously head-downwards into the wound in a characteristic screwworm
fashion. As they feed, tearing the tissue with their hook-like mouthparts, the wound is enlarged and deepened,
resulting in extensive tissue destruction. Infested wounds often emit a characteristic odour, which can be the first
indication that at least one animal in a group is infested. Although the odour is not always apparent to humans, it
is obviously highly attractive to gravid females (19), which lay further batches of eggs so increasing the extent of
the infestation. A severe infestation that is left untreated may result in the death of the host.
Screwworm larvae pass through three stages (or instars), separated by cuticular moults that facilitate rapid
growth, and they reach maturity about 5–7 days after egg hatch. They then stop feeding and leave the wound,
falling to the ground into which they burrow and pupariate. The pupa develops within the puparium, a barrel-
shaped protective structure formed by hardening and darkening of the cuticle of the mature larva. On completion
of development, adult flies usually emerge from the puparium in the morning and work their way up to the soil
surface, where they extend their wings for hardening prior to flight. Males become sexually mature and able to
mate within 24 hours, but the ovaries of females need to mature over 6–7 days, and females only become
responsive towards males and mate when about 3 days old. About 4 days after mating, female flies are ready to
oviposit. They seek a suitable host and lay their eggs, all oriented in the same direction, like a tiled roof, firmly
attached to each other and to the oviposition substrate. The numbers of eggs laid per batch vary depending on
many factors (e.g. fly strain, disturbance during oviposition), but the average first batch has in the order of
175 eggs for OWS and 340 for NWS (43). Following the first egg batch, further batches are laid at intervals of 3–
4 days (51). Adult flies live on average for 2–3 weeks in the field during which time they feed at flowers, and the
females also take in protein, e.g. from serous fluids at animal wounds.
The rate of development of the immature stages is influenced by environmental and wound temperatures, being
slower at low temperatures, although true diapause does not occur. This effect is most pronounced in the off-host
pupal stage, which can vary from 1 week to 2 months’ duration depending on the season (24). Thus, the
complete life cycle of NWS may take 2–3 months in cold weather (36), whereas in temperate conditions with an
average air temperature of 22°C, it is completed in about 24 days (22), and in tropical conditions averaging 29°C
it is completed in about 18 days (51).
The degree to which NWS and OWS can tolerate cold has had a major influence on their distributions, best
documented for NWS. Historically, the range of NWS extended from the southern states of the United States of
America (USA), through Mexico, Central America, the Caribbean islands and northern countries of South
America to Uruguay, northern Chile and northern Argentina (22). This distribution contracted during the winter
months but expanded during the summer months, producing a seasonality at its edges and year round
populations in the central areas – the New World tropics. Use of the sterile insect technique (SIT) in major
programmes has resulted in eradication of NWS from the USA (6), Mexico (17), Curacao, Puerto Rico, and the
Virgin Islands and, in Central America, from Guatemala, Belize, El Salvador, Honduras, Nicaragua and, in 2000,
Costa Rica (55). The Central American eradication programme is continuing in Panama, where sterile flies were
first released in July 1998. The ultimate objective is to establish a barrier zone in Panama that will become the
future northern limit of NWS in the Americas. A NWS eradication programme was also officially launched in
Jamaica in July 1998, as part of a plan to eradicate the species from the entire Caribbean. This programme has
encountered severe setbacks due to a complex combination of management and technical difficulties, but is
ongoing although with an uncertain future (12, 53). Although NWS is a New World species, in 1988, it was
detected in Libya in North Africa where it threatened to become firmly established. However, it was eradicated in

1991 by an intensive SIT campaign (14, 26). The threat of spread of screwworms aided by modern rapid
transport systems is ever present, necessitating constant vigilance from quarantine and other front-line animal
health and medical officers in unaffected areas. Imported cases of NWS have been reported recently in Mexico,
USA, and even in the United Kingdom (30).
The distribution of OWS is confined to the Old World, as the name suggests, throughout much of Africa (from
Ethiopia and sub-Saharan countries to northern South Africa), the Gulf countries, the Indian subcontinent, and
South-East Asia (from southern China [People’s Rep. of] through the Malay Peninsula and the Indonesian and
Philippine islands to Papua New Guinea) (22, 43, 47, 56). OWS was reported from Hong Kong for the first time in
2000, infesting dogs, and a first human case was reported in 2003 (35). OWS myiasis has also been reported
from Algeria (1), in a local shepherd, but in the absence of other reported cases, particularly animal cases, a
continuing presence there seems unlikely and the original case could have been a misidentification. The situation
in the Gulf area and surrounding regions is dynamic with recent reports confirmed from Iran (34) and Iraq (2).
Epidemics of traumatic myiasis can follow introductions into such areas, especially where the livestock owners
and veterinarians are unfamiliar with OWS (38). The climatic requirements of the two screwworm species are
very similar and their potential distributions, if unrestrained, would overlap considerably (47).
Organophosphorus insecticides such as dichlofenthion, fenchlorphos, and in particular, coumaphos are

recommended for the treatment of wounds infested with OWS and NWS (16, 37, 45). They have the effect of
expelling the larvae, which die on the ground. To provide residual protection against reinfestation, they must be
applied at 2–3-day intervals until the wound has healed. The contents of individual wound treatment sachets, e.g.
5 g of 5% coumaphos wettable powder, should be either sprinkled directly on to a wound or, more effectively,
brushed into the wound as a paste after mixing with ordinary cooking oil (33 ml). Organophosphorus compounds
may also be applied as aerosol sprays, in which marker dyes and bacteriostats are included, or as dusts that are
puffed into the wound from plastic squeeze bottles. Dichlorfenthion is used in South America as a 1% aerosol to
treat NWS cases and is also effective against OWS (37). Any larvae that die in the wound should be removed to
prevent sepsis. Close attention should always be paid to the manufacturers’ safety instructions.
Direct prevention of screwworm infestation can be achieved by spraying or dipping of livestock with coumaphos
(0.25% aqueous suspension of 50% wettable powder) or other organophosphorus insecticides at the maximum
concentration prescribed for external parasite control. The effects of such treatment are twofold: firstly, the
treatment kills larvae directly and provides residual protection; secondly, the treatment kills ticks and other
external parasites, which means that there are fewer wounds available as sites for oviposition. Synthetic
pyrethroids have potential for control of screwworm larvae in wounds, but there have been few reported trials of
their effect on screwworms (e.g. Permethrin versus NWS; ref. 39). Dipping or spraying of a group of animals
would be indicated if any member of the group was found to be infested, or if animals were traversing or leaving
an infested area, or following wound-inducing animal husbandry practices, e.g. shearing.
A single subcutaneous injection of ivermectin (200 µg/kg) was effective against OWS in preventing navel strike of
newborn calves (37) and scrotal strike of castrated calves (44). Ivermectin also prevented re-strike of treated
wounds of adult cattle. Cattle treated with a sustained-release bolus of ivermectin developed no OWS myiasis
from 14 to 102 days after treatment (54). However, because of the negative effects on dung-breeding fauna, it
was recommended that boluses be reserved for use in containing outbreaks of OWS. Early results suggested
that ivermectin may be ineffective against NWS (Mackley & Brown, in ref. 17), but more recent studies
demonstrated that it can produce a significant reduction in the incidence of navel and scrotal myiasis due to
NWS (7, 28). Although results of ivermectin trials show variation, results of doramectin trials are overwhelmingly
positive (18). There has been an increasing number of publications reporting that a subcutaneous injection of
doramectin (200 µg/kg) was up to 100% effective as a NWS prophylactic, preventing infestation of artificial
wounds, umbilical or castration wounds of calves, and infestation of post-parturient cows, for up to 12–14 days
post-treatment (4, 32, 33). This doramectin treatment does not reduce egg-laying and, therefore, is efficient
because gravid adults are not repelled and driven towards untreated animals. Effectiveness depended on factors
such as cattle breed and degree of challenge. In one comparative trial, doramectin and ivermectin, both at
200 µg/kg subcutaneous injection, gave 100% and 50% protection, respectively, against NWS myiasis,
experimentally induced 2 hours after treatment (31). Doramectin also provided complete protection for 21 days
and partial protection (56%) at 28 days post-treatment (31). In another, larger, comparative trial, doramectin had
a mean efficacy of 94.6% (range 53.3–100%) compared with 43.7% (range 0–100%) for ivermectin (10).
Abamectin (subcutaneous injection, 200 µg/kg) gave good, but not 100%, prevention of post-castration myiasis
by NWS (3). Pour-on formulations of moxidectin, eprinomectin and doramectin gave poor protection against
OWS myiasis (54) when compared with injectable formulations of doramectin against NWS. There are early
indications that fipronil (a phenyl-pyrazole) might be effective as a preventive of post-castration myiasis of cattle.
Topical application of 10 mg/kg bodyweight of a 1% fipronil solution did not prevent oviposition by NWS, but it
reduced the proportion of bulls developing active myiasis over the critical 10-day post-castration period, when
most ovipositions occurred, from 65% in untreated controls to just 3% in treated animals (25). Similarly, topical
application of an insect growth regulator (IGR), dicyclanil, to castration wounds in cattle gave good protection
(>90%) against NWS myiasis (5). IGRs are very specific to insects and, therefore, are less hazardous in the
environment than many other groups of insecticides. Spinosad, a formulation of products derived from the
fermentation of a bacterium with low mammalian and avian toxicity, was effective in treating and preventing
myiasis due to NWS and OWS when applied as an aerosol spray (41).

Indirect prevention of screwworm flies infestation includes the avoidance of wounding procedures at the times of
year when screwworm are numerous, the careful handling of livestock to minimise wounding, the removal of
sharp objects (e.g. wire strands) from livestock pens, and the use of measures to reduce other wound-causing
parasites, in particular ticks, e.g. by dipping and by insecticide impregnated ear-tags.
To prevent the spread of the screwworms beyond present limits, strict observation of the requirements for
international trade, as set out in the OIE Terrestrial Animal Health Code, is necessary.
Identification of the eggs and first instar larvae of the agents of myiasis based on morphology is difficult, and,
because these stages are relatively short lived and seldom encountered during the collection of specimens from
infested wounds, they will not be considered further here.
Larvae collected for diagnosis should be removed from the deepest part of the wound to reduce the possibility of
collecting non-screwworm species, which may infest the shallower parts of the wound. Living specimens should
first be examined for pigmentation of the dorsal tracheal trunks (Figures 1 and 4) and then be preserved in 80%
ethanol and returned to the laboratory for examination under a dissecting microscope at up to ×50 magnification
(for further techniques see references 13, 21, 42, 56). If larvae are placed directly into most preservative
solutions they contract and darken. However, optimal preservation of larvae, in their natural extended state, can
be made by killing them in boiling water (15–30 seconds immersion) before storage in 80% ethanol. This killing
method had no negative effect on subsequent extraction of mitochondrial DNA, amplified by polymerase chain
reaction (PCR) (20), but it might impact other molecular techniques and this should be borne in mind.
Second instar larvae: Second instars have only two spiracular slits in each of the posterior spiracular plates
compared with the three slits of third instars (Figures 2 and 3). Second instars of NWS can be diagnosed by the
presence of dark pigmentation of the dorsal tracheal trunks, for over half their length in the terminal segment.
Other species have less extensive pigmentation of the dorsal tracheal trunks, for example, these trunks are
pigmented for no more than one-third of their length in the twelfth segment of OWS. The anterior spiracles of
second instar NWS have from seven to nine branches compared with about four branches in OWS (23). More
positive identification may be gained by rearing living, immature larvae to third instars. This can be done on the
standard meat medium used for large-scale rearing of NWS before the introduction of gel diets, i.e. in the
proportion of 1 litre water, 1.3 kg ground horse or beef meat, 50 g dried bovine blood, and 1.5 ml formalin (49),
mixed and maintained at 35–38°C and 70% relative humidity. For simply rearing up larvae for identification, the
exact meat and blood types are not essential, and more readily available fresh blood could be used instead of
dried blood.
Third instar larvae: Third instars of both NWS and OWS have a robust, typical maggot shape, with a cylindrical
body from 6 to17 mm long and from 1.1 to 3.6 mm in diameter, pointed at the anterior end (24, 42). Fully mature
larvae of both NWS and OWS develop a reddish-pink tinge over the creamy white colour of younger larvae. Both
screwworm species have prominent rings of spines around the body and these spines appear large and
conspicuous under a microscope when compared with most non-screwworm species, the longest averaging
130 µm. In NWS the spines can be either single or double pointed, but in OWS they are always single pointed
and thorn-like (Figure 2). The anterior spiracles of NWS each have from six to eleven well separated branches,
but usually from seven to nine (Figure 2). In OWS, the anterior spiracles each have from three to seven
branches, but usually from four to six (Figure 2). The latter character should not be used on its own to identify
OWS, because third instars of the obligate myiasis-causing species Wohlfahrtia magnifica (Diptera:
Sarcophagidae), whose distribution overlaps that of OWS in the Middle East, have similarly branched anterior
spiracles. Hence, in using any identification key, such as that in Figure 1, it is essential that each specimen be
taken through the whole key to avoid misidentifications. On the posterior face of the terminal segment of both
NWS and OWS, the posterior spiracular plates all have a darkly pigmented, incomplete peritreme partially
enclosing three straight, slightly oval-shaped slits, which point towards the break in the peritreme. These
diagnostic features are illustrated in Figure 3. Of greatest diagnostic value are the dorsal tracheal trunks, which
extend forwards from the posterior spiracular plates and are darkly pigmented up to the tenth or ninth segment in
NWS (Figure 1; see also refs 13, 15, 18, 21, 22, 42, 56 for identification keys). This feature is seen most easily in
living larvae. Those in preservative may need dissection to remove opaque tissues covering the trunks. The
dorsal tracheal trunks of OWS are darkly pigmented only in the twelfth segment. However, in OWS the
secondary trachea branching off the dorsal tracheal trunks are pigmented from the twelfth segment forwards to at
least the tenth segment (confirmed in specimens throughout the range, from Malaysia, Bahrain and Zimbabwe;
M.J.R. Hall, unpublished). Conversely, in NWS these secondary trachea are not pigmented, only the dorsal
trachea are. Hence, the tracheal pigmentation appears almost reversed between the two screwworm species
(Figure 4).

Remove larva from wound and examine gross surface structure
‘Hairy’ larva with obvious body processes ‘Smooth’ larva, with spine bands but no obvious body
processes except on last segment
Chrysomya albiceps, C. rufifacies, C. varipes
Posterior spiracles almost concealed in deep cavity on Posterior spiracles not in cavity but clearly exposed on
posterior ‘face’ of last segment posterior ‘face’ of last segment
Sarcophagidae
Peritreme of posterior spiracle closed Peritreme of posterior spiracle open
Muscidae and Lucilia/Calliphora species
Dorsal tracheal trunks darkly pigmented forwards from the Dorsal tracheal trunks not darkly pigment except possibly in
12th to the 10th or even 9th segment posterior half of 12th segment
Cochliomyia hominivorax
Anterior spiracle with 46, rarely 7, lobes Anterior spiracle with nine or more lobes
Chrysomya bezziana Other species of Chrysomya, Cochliomyia, Phormia or

Protophormia
Fig. 1. Identification key for the diagnosis of third instar larvae of Cochliomyia hominivorax and Chrysomya
bezziana from cases of wound myiasis. To avoid misidentifications, it is essential that the key is worked through
from the first step for each specimen.

as
as
Fig. 2. Head and first two thoracic segments of third instar larvae of Cochliomyia hominivorax (left, viewed by
scanning electron microscopy, inset is the anterior spiracle of Chrysomya bezziana) and of Chrysomya bezziana
(right, viewed by compound light microscopy, note the thorn-like spines and that this slide preparation has been
cleared using 10% KOH so that the anterior spiracles on both sides of the first thoracic segment are visible);
as = anterior spiracle.
Fig. 3. Characteristics of third instar larvae of Cochliomyia hominivorax: (A) whole larva, lateral aspect;
(B) posterior face of terminal segment; (C) posterior spiracular plate; a = anterior spiracle; b = button adjacent to
opening in peritreme; p = peritreme; sl = spiracular slit; sp = spines. (After Laake et al. [24].)
Adult: Adult flies needed for identification purposes are often collected using wind-oriented traps (8) and sticky
traps (42) baited with a synthetic odour, swormlure-4 (29). A modified bucket-trap and newly developed attractant
(‘Bezzilure’) is being developed for surveillance of OWS in Australia (Rudolf Urech, pers. comm.). Alternative
sampling systems, using electrocuting grids or sticky surfaces at odour-baited visual targets, have been used for
research purposes (19). Identification of adult flies is seldom required for the diagnosis of myiasis, because the
larval stages are those most apparent to livestock owners and veterinary personnel. However, a brief description
follows.
i) NWS: The body length is usually 8–10 mm long and has a deep blue to blue-green metallic colour, with
three dark longitudinal stripes on the dorsal surface of the thorax. This combination of colour and pattern is
not shared by any other species commonly involved in wound myiasis except the secondary screwworm of
the New World, Cochliomyia macellaria (Fabricius). These two Cochliomyia species can be separated by
the presence of black setulae on the fronto-orbital plates of the head of NWS compared with only light
yellow hairs on the fronto-orbital plates of C. macellaria. The fifth (=fourth visible) abdominal tergite of NWS
has only a very slight lateral pollinose dusting, whereas that of C. macellaria has a dense dusting,
producing a pair of distinct, lateral, silvery-white spots. In addition, females of NWS have a dark brown-
black basicosta, whereas those of C. macellaria have a yellow basicosta (Figure 5; see also refs 11, 15, 24,
42).
ii) OWS: The body is up to 10 mm long and has a metallic blue, bluish-purple or blue-green colour, i.e. it is
very similar to NWS, but without the thoracic stripes. The lower squama (s in Figure 5) also differs from

NWS, being distinctly covered with fine hairs over its entire upper surface in OWS and other Chrysomya
species, whereas in NWS it is hairless above, except near the base. Adults of OWS can be distinguished
from other Chrysomya found in cases of myiasis by the combination of black-brown to dark-orange-
coloured anterior thoracic spiracles (rather than pale yellow, creamy, or white), with waxy-white, lower
squamae (rather than blackish-brown to dirty-grey) (42, 56).
NWS OWS
DT
ST
Fig. 4. Dorsal tracheal trunks of third instar larvae of Cochliomyia hominivorax (left) and Chrysomya bezziana (right)
dissected forwards from the posterior spiracles (top) to ninth abdominal segment (bottom).
Note that the pigmentation of the main dorsal trunks (DT) and the smaller secondary trunks (ST)
is almost reversed between the species.
Fig. 5. Characteristics of adult Cochliomyia hominivorax; note longitudinal thoracic stripes;

b = basicosta; p = fronto-orbital plate, indicated from above on whole Cochliomyia hominivorax and laterally on
head of typical calliphorid fly; s = lower squama, surface hairless except at base; v = stem vein with
hairs on dorsal posterior surface.

In addition to the standard morphological techniques discussed previously, more recent techniques for
identification of screwworms and their geographical origins include cuticular hydrocarbon analysis (9), analysis of
mitochondrial DNA (20, 27, 50), the complete 16,022 base-pair sequence of which is known for NWS, and use of
random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) (40). Problems with identification of
larvae or adults from cases of myiasis can be referred to the Food and Agriculture Organisation of the United
Nations Collaborating Centre on Myiasis-Causing Insects and Their Identification 2.
No standardised serological tests are presently available, nor are they indicated for diagnosis of this disease.
However, experimental studies have shown that serological techniques have potential value in future
investigations of the prevalence of screwworm infestations in animal populations to detect antibodies to
screwworm post-infestation (52).

There are no biological products such as vaccines, available currently. However, research towards development
of potential vaccines is being conducted (48). The only proven method of eradication of NWS relies on a
biological technique, the sterile insect technique, SIT (17, 26), which has also been applied experimentally to
OWS (46). In this technique, male flies sterilised in their late pupal stage by gamma or x-ray irradiation are
sequentially released into the wild in vast numbers. Any of their matings with wild females result in infertile eggs
only, leading to a progressive population reduction and, eventually, eradication. In operational situations, SIT is
supported by the insecticide treatment of screwworm-infested wounds in livestock, by strict control of livestock
movement, by the quarantining of infested animals and by an active publicity campaign. SIT is very expensive
because of the cost of continuous production and aerial dispersion of sterile flies. Historically, it has been
considered cost effective only when used as an eradication strategy in situations where the geography would
favour such a programme (e.g. references 14, 26). For many years there was only one New World sterile
screwworm production facility, located at Tuxtla Gutiérrez in the south of Mexico. However, a second facility
opened in Panama 3 in late 2006. An experimental facility to produce sterile OWS opened in Malaysia in 1998 4.
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th
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*
* *
NB: There is an OIE Reference Laboratory for New world screwworm (Cochliomyia hominivorax) (see Table in
Part 3 of this Terrestrial Manual or consult the OIE Web site for the most up-to-date list: www.oie.int).

CHAPTER 2.1.11.
PARATUBERCULOSIS
(Johne’s disease)
SUMMARY
Paratuberculosis (Johne’s disease) is a chronic enteritis of ruminants caused by Mycobacterium

avium subsp. paratuberculosis (M. paratuberculosis) (49).
Identification of the agent: The diagnosis of paratuberculosis is divided into two parts: the
diagnosis of clinical disease and the detection of subclinical infection. The latter is essential for
control of the disease at the farm, national or international level.
Diagnosis of paratuberculosis is made on clinical grounds confirmed by the demonstration of

M. paratuberculosis in the faeces by microscopy, culture, or by the use of DNA probes and the
polymerase chain reaction. Diagnosis is made at necropsy by the finding of the pathognomonic
lesions of the disease in the intestines, either grossly with the demonstration of typical acid-fast
organisms in impression smears of the lesions or histologically, and by isolation of
M. paratuberculosis in culture.
The detection of subclinical infection depends on the detection of specific antibodies by serology,
or culture of M. paratuberculosis from faeces or tissues collected at necropsy, or the demonstration
of cell-mediated responses. The choice of test depends on the circumstances and the degree of
sensitivity required at individual animal or herd level.
Cultures of M. paratuberculosis may be obtained from faeces or tissues, after treatment to

eliminate contaminants, by inoculation into artificial media with and without the specific growth
factor – mycobactin – that is essential for the growth of M. paratuberculosis.
Serological tests: Control of paratuberculosis is difficult because of the prolonged course of

infection, the predominantly subclinical nature of the disease and lack of tests for accurate detection
of subclinically infected animals.
The serological tests commonly used for paratuberculosis in cattle are complement fixation (CF),
absorbed enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID).
Sensitivity and specificity are often determined by reference to results of faecal culture, which itself
has unknown sensitivity in subclinically infected cattle. When used to confirm diagnosis of
paratuberculosis in cows with typical clinical signs, some tests, for example CF and absorbed
ELISA, perform very well.
Requirements for vaccines and diagnostic biologicals: Vaccines for paratuberculosis may be
live attenuated or killed bacteria either incorporated with an adjuvant or lyophilised and adjuvanted
on reconstitution. Bacterial counting is difficult and bacterial content of vaccines may be based on
weight, while vaccine potency may be judged by batch tests for sensitising ability in guinea-pigs.
Vaccine safety or abnormal toxicity may also be tested in guinea-pigs.
For diagnostic skin tests, Johnin and avian tuberculin are purified protein derivatives (PPD) of a
heat-treated culture of M. paratuberculosis or M. avium, respectively. Johnin is standardised for
content of PPD by chemical assay and its biological activity is identified in guinea-pigs sensitised
with M. paratuberculosis. Avian tuberculin activity is determined in guinea-pigs sensitised with
M. avium by comparison with a reference preparation calibrated in international units.

Chapter 2.1.11. — Paratuberculosis (Johne’s disease)
A. INTRODUCTION
Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is an organism first observed by Johne
& Frothingham in 1895. Mycobacterium avium subsp. paratuberculosis causes paratuberculosis or Johne’s
disease, an intestinal granulomatous infection. First recognised in cattle, then in sheep and later in goats,
paratuberculosis is found most often among domestic and wild ruminants and has a global distribution. The
disease has also been reported in horses, pigs, deer and alpaca, and recently in rabbits, stoat, fox and weasel
(3, 14). Under natural conditions, the disease in cattle spreads by ingestion of M. paratuberculosis from the
contaminated environment. The disease persists after the introduction of infected animals. Infection can be
spread vertically to the fetus (25) and semen can be infected with the organism (46). The primary source of
infection in calves is milk from infected cows or milk that is contaminated with the faeces of diseased cattle.
The identification of M. paratuberculosis is based on its mycobactin requirement and its pathogenicity in the host.
Mycobactin dependence has long been used as a taxonomic characteristic for M. paratuberculosis because most
mycobacteria are able to make mycobactin for themselves. Mycobacterium avium subsp. paratuberculosis,
M. silvaticum and some primary isolates of M. avium lack this capacity, however, and require mycobactin to grow
in the laboratory. Thus, the mycobactin requirement is not confined to M. paratuberculosis; this characteristic
exists to various degrees within the M. avium group (48).
Clinical signs of paratuberculosis are a slowly progressive wasting and diarrhoea, which is intermittent at first,
becoming progressively more severe until it is constantly present in bovines (13). Diarrhoea is less common in
small ruminants.
Early lesions occur in the walls of the small intestine and the draining mesenteric lymph nodes, and infection is
confined to these sites at this stage. As the disease progresses, gross lesions occur in the ileum, jejunum,
terminal small intestine, caecum and colon, and in the mesenteric lymph nodes. Mycobacterium avium subsp.
paratuberculosis is present in the lesions and, terminally, throughout the body. The intestinal lesions are
responsible for a protein leak and a protein malabsorption syndrome, which lead to muscular wasting. Clinical
signs usually first appear in young adulthood, but the disease can occur in animals at any age over 1–2 years.
Within a few weeks of infection, a phase of multiplication of M. paratuberculosis begins in the walls of the small
intestine. Depending on the resistance of the individual, this infection is eliminated or the animal remains infected
as a healthy carrier. The proportion of animals in these categories is unknown. A later phase of multiplication of
the organisms in a proportion of carriers leads to the extension of lesions, interference with gut metabolism and
clinical signs of disease. Subclinical carriers excrete variable numbers of M. paratuberculosis in the faeces. In
most cases larger numbers of organisms are excreted as clinical disease develops.
Delayed-type hypersensitivity (DTH) is detectable early in the infection and remains present in a proportion of the
subclinically infected carriers, but as the disease progresses, DTH wanes and may be absent in clinical cases.
Serum antibodies are detectable later than DTH. They may also be present in carriers that have recovered from
infection. Serum antibodies are present more constantly and are of higher titre as lesions become more
extensive, reflecting the amount of antigen present. In sheep, there may be a serological response that is more
likely to be detected in multibacillary than in the paucibacillary form of the disease.
Other mycobacterial diseases and infections, including mammalian and avian tuberculosis, cause DTH and the
presence of serum antibodies. It follows therefore that these diseases need to be differentiated from
paratuberculosis, both clinically and by the use of specific diagnostic tests. Exposure to environmental
saprophytic mycobacteria may also sensitise livestock, resulting in nonspecific DTH reactions.
Animals vaccinated against paratuberculosis develop both DTH and serum antibodies. Vaccination is an aid to
the prevention of clinical disease, but does not necessarily prevent infection. It also interferes with programmes
for the diagnosis and control of bovine tuberculosis. Thus, if it is necessary to attempt a diagnosis of infection in
vaccinates, only tests to detect M. paratuberculosis in the faeces can be used (21).
In individual animals, especially from a farm in which the disease has not previously been diagnosed, a tentative
clinical diagnosis must be confirmed by laboratory tests. However, a definitive diagnosis may be warranted on
clinical grounds alone if the clinical signs are typical and the disease is known to be present in the herd.
Confirmation of paratuberculosis depends on the finding of either gross lesions with the demonstration of typical
acid-fast organisms in impression smears or microscopic pathognomonic lesions and the isolation in culture of
M. paratuberculosis.
To diagnose the presence of paratuberculosis in an individual clinically suspect animal, a number of laboratory
tests can be used including: faecal smears, faecal and tissue culture, DNA probes using faeces or tissues,
serology, necropsy and histology.

Herd tests to detect subclinical infection are carried out to determine the prevalence of the infection, usually so
that control measures can be instituted. As no test is 100% sensitive or specific, control of the disease by the
disposal of positive reactors depends on repeated tests at 6-month or yearly intervals over a number of years
and the elimination of reactors to serological tests or faecal shedders; the removal of offspring from female
reactors is also considered to be prudent. Even these procedures are not always successful without changes in
hygiene and livestock management to reduce the transmission of infection within a herd (2).

a) Necropsy
Paratuberculosis cannot be diagnosed on superficial examination of the intestines for signs of thickening.
The intestines should be opened from the duodenum to the rectum to expose the mucosa. There is not
always a close correlation between the severity of clinical signs and the extent of intestinal lesions. The
mucosa, especially of the terminal ileum, is inspected for pathognomonic thickening and corrugation. Early
lesions are seen by holding the intestine up to the light, when discrete plaques can be visualised. Mucosal
hyperaemia, erosions and petechiation have been observed in deer with paratubeculosis. The earliest
lesions are thickening and cording of lymphatic. The mesenteric lymph nodes are usually enlarged and
oedematous. Smears from the affected mucosa and cut surfaces of lymph nodes should be stained by
Ziehl–Neelsen’s method and examined microscopically for acid-fast organisms that have the morphological
characteristics of M. paratuberculosis. However, acid-fast organisms are not present in all cases. Diagnosis
is therefore best confirmed by the collection of multiple intestinal wall and mesenteric lymph node samples
into fixative (10% formol saline) for subsequent histology. Both haematoxylin-and-eosin-stained sections
and Ziehl–Neelsen-stained sections should be examined. The typical lesions of paratuberculosis consist of
infiltration of the intestinal mucosa, submucosa, Peyer’s patches and the cortex of the mesenteric lymph
nodes with large macrophages, also known as epithelioid cells, and multinucleate giant cells, in both of
which clumps or singly disposed acid-fast bacilli are usually, but not invariably, found.
b) Bacteriology (microscopy)
Ziehl–Neelsen-stained smears of faeces or intestinal mucosa are examined microscopically. A presumptive
diagnosis of paratuberculosis can be made if clumps (three or more organisms) of small (0.5–1.5 µm),
strongly acid-fast bacilli are found. The presence of single acid-fast bacilli in the absence of clumps
indicates an inconclusive result. The disadvantages of this test are that it does not differentiate among other
mycobacterial species and only a small proportion of cases can be confirmed on microscopic examination
of a single faecal sample.
c) Bacteriology (culture)
The isolation of M. paratuberculosis from an animal provides the definitive diagnosis of infection with the
organism. Although culture is technically difficult and time-consuming to carry out, it is the only test that
does not produce false-positive results (100% specificity).
The faecal culture is the best test available for the diagnosis of paratuberculosis in live animals. It is
believed that the faecal culture method involving the double incubation method for decontamination of
samples and cultivation on solid media detects about 30–40% of infected cattle (56). The faecal culture is
able to detect most animals in advanced stages of the disease but identifies only a few animals in early
stages of infection (56). It will detect infected animals 6 months or more before they develop clinical signs,
and during the clinical stage its sensitivity approaches 100%. The culture of bovine and caprine tissues for
M. paratuberculosion is more sensitive than histopathological examination.
There are several culture methods, which vary with respect to media and sample processing protocols. The
cultivation of M. paratuberculosis is always performed using special media supplemented with mycobactin
J 1,
Mycobacterium avium subsp. paratuberculosis organisms are vastly outnumbered by other bacteria or fungi
in faecal and intestinal tissue specimens. The successful isolation of M. paratuberculosis from such
samples depends on efficient inactivation of these undesirable organisms. The optimal method of
decontamination must have the least inhibitory effect on growth of M. paratuberculosis. Routine
decontamination protocols were shown to decrease the number of M. paratuberculosis organisms isolated
per sample by about 2.7 log10 and 3.1 log10 for faeces and tissues, respectively (34).
There are two basic methods in use for the conventional culture of M. paratuberculosis on solid media: the
method using oxalic acid and NaOH for decontamination and Löwenstein–Jensen (LJ) medium for growth,
and the method using hexadecylpyridinium chloride (HPC) for decontamination in combination with
Herrolds’s egg yolk medium (HEYM) for growth. Both media contain mycobactin. Although it has been
1 Mycobactin can be obtained commercially (mycobactin J) from Allied Monitor, P.O. Box 71, 201 Golden Drive, Fayette,
MO 65248, United States of America, or Symbiotic Society, 299 av. Jean Jaurès, 69007 Lyon, France.

published that HEYM supports growth of bovine isolates of M. paratuberculosis significantly better than LJ
(33), recent studies have shown that certain strains grow better on LJ or Middlebrook media (9).
In addition, there is a technique of radiometric culture where growth in liquid medium BACTEC™ 12B
(Middlebrook 7H12) supplemented with egg yolk and mycobactin is measured by the release of radioactive
14CO from palmitate, as a consequence of bacterial metabolism. This method reduces the time required
2
for results and is considered more sensitive than the conventional culture methods on solid media for the
detection of both ovine and bovine stains of M. paratuberculosis (11, 58). The decontamination protocol
involving double incubation of faecal samples in HPC and mixture of antibiotics may further improve culture
sensitivity (11). However, as the BACTEC™ system is radiometrically based, it is not feasible for use in
some laboratories and has been phased out in others. The evaluation of the usefulness of alternative
culture systems based on liquid media such as MGIT (Becton Dickinson) ESPII (Difco) and MB/BacT Alert
(Organon Teknika) that do not use radioactive material for the detection of M. paratuberculosis is currently
in progress.
Primary colonies of M. paratuberculosis on solid media may be expected to appear any time from 5 weeks
to 6 months after inoculation (7). Sheep strains, including the uncommon, bright yellow pigmented types,
grow less well than cattle strains on commonly used media such as HEYM or LJ, and primary cultures
should not be discarded as negative without prolonged incubation. The solid medium Middlebrook 7H10
and liquid medium BACTEC™ 12B both supplemented with egg yolk and mycobactin are excellent for
cultivation of ovine strains of M. paratuberculosis (58).
Primary colonies of the cattle strain of M. paratuberculosis on HEYM are very small, convex
(hemispherical), soft, non-mucoid and initially colourless and translucent. Colony size is initially pinpoint, It
may remain at 0.25–1 mm, and tend to remain small when colonies are numerous on a slope. Colony
margins are round and even, and their surfaces are smooth and glistening. The colonies become bigger
more raised, opaque, off-white cream to buff or beige coloured as incubation continues. Older isolated
colonies may reach 2 mm. The colonial morphology changes with age from smooth to rough, and from
hemispherical to mammilate (7, 47).
On modified 7H10 medium, colonies of the cattle strain are less convex than those on HEYM, especially in
aged cultures, They are pinpoint to approximately 1 mm in diameter and, being buff coloured, are only
slightly lighter than the media. Compared with colonies of cattle strains on HEYM, those on 7H10 are more
difficult to detect. Colonies of the sheep strain of M. paratuberculosis on modified 7H10 are convex, soft,
moist, glistening, off-white to buff, and very similar to the colour of the media. Colonies are typically
between pinpoint and 0.5 mm, but can reach 1 mm, and rarely 1.5 mm if few colonies occur on a slope (7).
Saprophytic mycobacteria may have a similar appearance on either medium but are often evident after 5–
7 days (7).
For identification of M. paratuberculosis, small inocululum of suspect colonies should be subcultured on the
same medium with and without mycobactin, to demonstrate mycobactin dependency. Mycobactin is present
in the cell wall of the organism, and heavy inoculum may contain enough mycobactin to support the growth
of a mycobactin-independent mycobacterium on medium that contains no mycobactin.
• Media
Examples of suitable media are:
i) Herrold’s egg yolk medium with mycobactin (28)

For 1 litre of medium: 9 g peptone; 4.5 g sodium chloride; 2.7 g beef extract; 27 ml glycerol; 4.1 g
sodium pyruvate; 15.3 g agar; 2 mg mycobactin; 870 ml distilled water; six egg yolks (120 ml); and
5.1 ml of a 2% aqueous solution of malachite green. Measure the first six ingredients and dissolve by
heating in distilled water. Adjust the pH of the liquid medium to 6.9–7.0 using 4% NaOH, and test to
ensure the pH of the solid phase is 7.2–7.3. Add the mycobactin dissolved in 4 ml ethyl alcohol.
Autoclave at 121°C for 25 minutes. Cool to 56°C and aseptically add six sterile egg yolks 2 and sterile
malachite green solution. Blend gently and dispense into sterile tubes.
It is permissible to add 50 mg chloramphenicol, 100,000 U penicillin and 50 mg amphotericin B.
2 Use fresh eggs not more than 2 days old from a flock that is not receiving antibiotics. With a brush, scrub the eggs with
water containing a detergent. Rinse with water and place the eggs in 70° alcohol for 30 minutes. Dry by inserting between
two sterile towels. With sterile rat-tooth forceps, crack one end of the eggshell, making a hole of approximately 10 mm,
and remove the egg white with the forceps and gravity. Make the hole larger and break the yolk. Mix the egg yolk by
twirling the forceps, and remove the yolk sac. Pour the mixed egg yolk into media.

ii) Modified Dubos’s medium (41)

For 1 litre of medium: 2.5 g Difco casamino acids; 0.3 g asparagine; 2.5 g anhydrous disodium
hydrogen phosphate; 1 g potassium dihydrogen phosphate; 1.5 g sodium citrate; 0.6 g crystalline
magnesium sulphate; 25 ml glycerol; 50 ml of a 1% solution of Tween 80; and 15 g agar. Dissolve
each salt in distilled water with minimum heat and make up to 800 ml. Add mycobactin in alcoholic
solution at 0.05% (2 mg dissolved in 4 ml ethyl alcohol), heat the medium to 100°C by free-steaming,
and then sterilise by autoclaving at 115°C for 15 minutes. Cool to 56°C in a water bath, add antibiotics
(100,000 U penicillin; 50 mg chloramphenicol; and 50 mg amphotericin B) and serum (200 ml of
bovine serum sterilised by filtering through a Seitz ‘EX’ pad and inactivated by heat at 56°C for
1 hour). The medium is kept thoroughly mixed and then dispensed into sterile tubes. An advantage of
this medium is that it is transparent, which facilitates the early detection of colonies.
iii) Modified Middlebrook 7H10 (7)
To prepare this medium 19 g Middlebrook 7H10 agar (Difco), 1g Casitone and 5 ml Glycerol are
resuspended in 900 ml water, autoclaved at 121°C for 15 minutes and cooled to 58°C. Using an
aseptic technique, the following additional ingredients are combined adding the egg yolk last: 50 ml
PANTA PLUS (Becton Dickinson), 25 ml Mycobactin J solution (50 µg/ml), 100 ml ADC enrichment
(Difco), 250 ml egg yolk. The mixture is thoroughly mixed using a slow swirling action and 10-ml
volumes are dispensed into sterile tubes to form slopes. After a sterility check, media are stored at
4°C.
iv) BACTEC 12B vials (7)
The following supplements are added to each vial to give final concentrations of 0.8–1 µg/ml
Mycobactin J and a minimum of 16–17% egg yolk in a final volume of 5–6 ml. For the 6-ml volume,
0.1 ml Mycobactin J (50 µg/ml), 0.1 ml PANTA PLUS, 1 ml egg yolk and 0.8 ml water are added. For
the 5-ml volume, 0.1 ml Mycobactin J (50 %g/ml), 0.1 ml PANTA PLUS and 0.8 ml egg yolk are added.
v) Middlebrook 7H9, 7H10 and 7H11 media (Difco)
This media enhanced with mycobactin in the same proportion as for Herrold’s medium can also be
used. The advantage of this media is that it is transparent, which facilitates the early detection of
colonies.
vi) Löwenstein–Jensen medium with or without mycobactin (20).
• Sample preparation
• Processing tissue specimens
Chemical preservatives should not be used. The tissues can be frozen at –20°C.
To avoid contamination, the faeces should be rinsed from portions of intestinal tract before shipment
to the laboratory.
i) Digestion/sedimentation method for decontamination of tissues
Approximately 4 g of mucosa from the ileocaecal valve or 4 g of mesenteric node are placed in a
sterile blender jar containing 50 ml of trypsin (2.5%). The mixture is adjusted to neutrality using 4%
NaOH and pH paper, and stirred for 30 minutes at room temperature on a magnetic mixer. The
digested mixture is filtered through gauze. The filtrate is centrifuged at approximately 2000–3000 g for
30 minutes. The supernatant fluid is poured off and discarded. The sediment is resuspended in 20 ml
of 0.75% HPC and allowed to stand undisturbed for 18 hours at room temperature. The particles that
settle to the bottom of the tube are to be used as the inoculum and are removed by pipette without
disturbing the supernatant fluid. Alternatively, other methods of decontamination can be used, such as
treatment with 5% oxalic acid.
ii) Double incubation method for decontamination of tissues (7)
About 2 g of tissue sample (trimmed of fat) is finely chopped using a sterile scalpel blade or scissors
and homogenised in a stomacher for 1 minute in 25 ml 0.75% HPC. Allow the sample to stand so that
foam dissipates and larger pieces of tissue settle. Pour tissue homogenate into a centrifuge tube
taking care to avoid carry over of fat or large tissue pieces. Allow to settle for 30 minutes then take
10 ml of the suspension from just above the sediment to a 30 ml tube and incubate for 3 hours at
37°C. Centrifuge for 30 minutes at 900 g, discard supernatant fluid and resuspend pellet in 1 ml
antibiotic cocktail containing 100 µg of each of vancomycin, amphotericin and nalidixic acid (VAN).
Incubate overnight at 37°C. Use the suspension to inoculate media as described below.
iii) Inoculation of culture media and incubation
Approximately 0.1 ml of inoculum is transferred to each of three slants of Herrold’s medium containing
mycobactin and to one slant of Herrold’s medium without mycobactin. The inoculum is distributed
evenly over the surface of the slants. The tubes are allowed to remain in a slanted position at 37°C for

approximately 1 week with screw caps loose. The tubes are returned to a vertical position when the
free moisture has evaporated from the slants. The lids are tightened and the tubes are placed in
baskets in an incubator at 37°C.
The egg in Herrold’s medium contributes sufficient phospholipids to neutralise the bactericidal activity
of residual HPC in the inoculum. The other media (Modified Dubos and Middlebrook) do not have this
property. Other treatments can be used for sample decontamination, for example oxalic acid at 5%.
HPC is relatively ineffective in controlling the growth of contaminating fungi. Amphotericin B
(fungizone) was found to control effectively fungal overgrowth of inoculated media (31). Fungizone
may be incorporated in the Herrold’s medium at a final concentration of 50 µg per ml of medium. Due
to loss of antifungal activity, storage of Herrold’s medium containing fungizone should be limited to
1 month at 4°C.
The slants are incubated for at least 4 months and observed weekly from the sixth week onwards.
• Processing faecal specimens

No chemical preservative is used. The faecal specimens can be frozen at –70°C.
i) Suspension and decontamination of faeces
1 g of faeces is transferred to a 50 ml tube containing 20 ml of sterile distilled water. The mixture is
shaken for 30 minutes at room temperature. The larger particles are allowed to settle for 30 minutes.
The uppermost 5 ml of faeces suspension is transferred to a 50 ml tube containing 20 ml of HPC. The
tube is inverted several times to assure uniform distribution and allowed to stand undisturbed for
18 hours at room temperature.
ii) Inoculation of culture media
0.1 ml of the undisturbed sediment is transferred to each of four slants of Herrold’s medium, three with
mycobactin and one without mycobactin. A smear may be made from the sediment and stained by the
Ziehl–Neelsen method.
iii) Incubation and observation of slants
The same as for tissue specimens.
Variations in the above methods have been described (4, 24, 30, 35, 40, 54, 55). The sensitivity of culture
may be enhanced using liquid media and with centrifugation rather than sedimentation techniques. The
double incubation method described by Whitlock et al. (55) assists with decontamination of the inoculum
(43) and offers higher sensitivity than the sedimentation or filtration protocols (11). The double incubation
method involves mixing 2 g faeces with 15 ml saline or water followed by sedimentation for 30 minutes and
transferring (avoiding fibrous matter) the top 5ml of the suspension to 25 ml of 0.9% HPC in half-strength
brain–heart infusion (Difco). After incubating at 37°C for 16–24 hours, the mixture is centrifuged at 900 g for
30 minutes (room temperature), the supernatant is discarded and pellet resuspended in 1 ml VAN. The
mixture is incubated for 24–72 hours at 37°C and used to inoculate media as described above (7).
d) DNA probes and polymerase chain reaction

DNA probes are being developed that offer a means of detecting M. paratuberculosis in diagnostic samples
and of rapidly identifying bacterial isolates (12, 29). They have been used to distinguish between
M. paratuberculosis and other mycobacteria.
McFadden et al. have identified a sequence (26, 27), termed IS900, which is an insertion sequence specific
for M. paratuberculosis. It has been reported that a small number of isolates other than M. paratuberculosis
have produced amplified products the same size as expected from M. paratuberculosis. A restriction
enzyme digest may be applied to positive IS900 products to confirm that their sequence is consistent with
M. paratuberculosis (6).
The identifications of new DNA sequences considered to be unique to M. paratuberculosis; ISMav2, f57,
and ISMap02 sequences, offer additional tools for rapid identification of this organism using the polymerase
chain reaction (PCR) technology (44, 45, 51). The restriction enzyme analysis of IS1311, an insertion
sequence common to M. avium and M. paratuberculosis can be used to distinguish between these species
and for typing of ovine, bovine and bison strains of M. paratuberculosis (38, 57).
The use of IS900 as a DNA probe for specific identification of M. paratuberculosis in faecal samples from
cattle by PCR has been reported (52). Commercial diagnostic PCR tests for the detection of
M. paratuberculosis in milk and faecal samples have been developed.

The serological tests commonly used for paratuberculosis in cattle are complement fixation (CF), enzyme-linked
immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) (42) corresponding to humoral immunity,
and the gamma interferon assay corresponding to cellular immunity.
a) Complement fixation test

The CF test has been the standard test used for cattle for many years. The CF test works well on clinically
suspect animals, but does not have sufficient specificity to enable its use in the general population for
control purposes. Nevertheless, it is often demanded by countries that import cattle. A variety of CF test
procedures are used internationally. An example of a microtitre method for performing the CF test is as
follows:
i) The antigen is an aqueous extract of bacteria from which lipid has been removed (strain
M. paratuberculosis 316F). Mycobacterium avium D9 may also be used.
ii) All sera are inactivated in the water bath at 60°C for 30 minutes and diluted at 1/4, 1/8 and 1/16. A
positive control serum and a negative control serum should be included on each plate. The following
controls are also prepared: antigen control, complement control and haemolytic system control.
iii) Reconstituted, freeze-dried complement is diluted to contain six times H50 (50% haemolysing dose) as
calculated by titration against the antigen.
iv) Sheep erythrocytes, 2.5%, are sensitised with 2 units of H100 haemolysin.
v) All dilutions and reagents are prepared in calcium/magnesium veronal buffer; 25 µl volumes of each
reagent are used in 96-well round-bottom microtitration plates.
vi) Primary incubation is at 4°C overnight and secondary incubation is at 37°C for 30 minutes.
vii) Reading and interpreting the results: Plates may be left to settle or centrifuged and read as follows: 4+
= 100% fixation, 3+ = 75% fixation, 2+ = 50% fixation, 1+ = 25% fixation and 0 = complete haemolysis.
The titre of test sera is given as the reciprocal of the highest dilution of serum giving 50% fixation. A
reaction of 2+ at 1/8 is regarded as positive. Results should be interpreted in relation to clinical signs
and other laboratory findings.

The ELISA is, at present, the most sensitive and specific test for serum antibodies to M. paratuberculosis in
cattle. Its sensitivity is comparable with that of the CF test in clinical cases, but is greater than that of the CF
test in subclinically infected carriers. The specificity of the ELISA is increased by M. phlei absorption of
sera. The absorbed ELISA, designed by Yokomizo et al. (61, 62) and modified by Milner et al. (32), was
developed into a commercial kit by Cox et al. (8).
The ELISA detects about 30–40% cattle identified as infected by culture of faeces on solid media (56).
Similarly to the culture methods, the sensitivity of the ELISA depends on the level of M. paratuberculosis
shedding in faeces and the age of animals. A large study recently performed in Australia showed that the
actual sensitivity of the ELISA in 2-, 3- and 4-year-old cows was 1.2%, 8.9% and 11.6%, respectively, but
remained between 20 and 30% in older age-groups (22). The overall actual sensitivity for all age-groups
was calculated to be about 15% (22, 56).
In small ruminants the commercially available ELISA had a specificity of 98.2–99.5% (95% confidence
intervals [CI]) and detected 35–54% (95% CI) of animals with histological evidence of infection (18). In
another study the estimated specificity of an in-house ELISA was 99% and its sensitivity measured against
histological results was 21.9% (37).
The absorbed ELISA combines the sensitivity of ELISA with the added specificity of an absorption step.
Sera to be tested are diluted with buffer containing soluble M. phlei antigen prior to testing in an indirect
ELISA. This procedure eliminates nonspecific cross-reacting antibodies. In early versions, sera were
absorbed with whole M. phlei, which were removed by centrifugation prior to testing.
A microtitre plate format has been developed in which M. paratuberculosis antigen is coated on to 96-well
plates. Samples are diluted in sample diluent containing M. phlei to remove cross-reacting antibodies. On
incubation of the diluted sample in the coated well, antibody specific to M. paratuberculosis forms a
complex with the coated antigens. After washing away unbound materials from the wells, horseradish
peroxidase (HPRO)-labelled anti-bovine immunoglobulin is added. This reacts with immunoglobulins bound
to the solid-phase antigen. The rate of conversion of substrate is proportional to the amount of bound
immunoglobulin. Subsequent colour, measured (at 450 nm) spectrophotometrically is proportional to the
amount of antibody present in the test sample.

The antigen used to coat the ELISA plates is available commercially.
An anti-bovine IgG labelled with HRPO is used as conjugate. The substrate chromogen solution is hydrogen
peroxide tetramethyl benzidine. A solution of 0.5 M H2SO4 is used to stop the reaction when the
absorbance of the positive control serum reaches a predetermined point.
Several absorbed ELISA kits are commercially available. The method and test materials needed, the
interpretation of the results and calculations are fully described in the instructions accompanying the
commercial kit. It has recently been reported that several commercially available ELISAs have similar
sensitivities and specificities (5). Some commercial kits offer an option of testing milk samples. The ELISA
on bovine and caprine milk has been found to have specificity similar to that of the serum ELISA, but less
sensitive than the blood test (17, 36).
c) Agar gel immunodiffusion test

The AGID test is useful for the confirmation of the disease in clinically suspect cattle, sheep and goats (39).
It has been reported that in small ruminants in New Zealand and Australia the AGID offers slightly higher
sensitivity and specificity than that obtained by the ELISAs (15, 18, 37). The reported specificity and
sensitivity of the AGID measured against histological results were 99–100% (95%CI) and 38–56% (95%
CI), respectively (18).
The antigen employed is a crude protoplasmic extract of laboratory strain M. avium 18 (formally
M. paratuberculosis 18) prepared by disruption of cells in a hydraulic press cell fractionator. Disrupted cells
are centrifuged at 40,000 g for 2 hours to remove cell wall debris, and the supernatant fraction is retained
and lyophilised. This antigen is resuspended in water at a concentration of 10 mg/ml.
Agarose is dissolved in barbital buffer, pH 8.6, containing sodium azide, to give a final agarose
concentration of 0.75%. Agarose may be poured into Petri dishes or on to glass slides. Wells are cut in a
hexagonal pattern. Wells are 4 mm in diameter, 4 mm apart, and the agar should be 3–4 mm deep. Antigen
is added to centre wells. Test, positive and negative control sera are added to alternate peripheral wells.
Plates are incubated in a humid chamber at room temperature. Gels are examined for precipitation lines
after 24 and 48 hours’ incubation. The appearance of one or more clearly definable precipitation line(s),
showing identity with that of a control positive serum, before or at 48 hours, constitutes a positive test result.
Absence of any precipitation lines is recorded as a negative test result. Nonspecific lines may occur.
Several variations of the method are in use.
3. Tests for cell-mediated immunity
The detection of a systemic cell-mediated response precedes detectable antibody production. Animals that are
minimally infected frequently fail to react on serological testing but may react positively to tests that measure cell-
mediated immunity.
a) Gamma interferon assay

The assay is based on the release of gamma interferon from sensitised lymphocytes during an 18–36-hour
incubation period with specific antigen (avian purified protein derivative [PPD] tuberculin, bovine PPD
tuberculin or johnin) (60). The quantitative detection of bovine gamma interferon is carried out with a
sandwich ELISA that uses two monoclonal antibodies to bovine gamma interferon. A commercial diagnostic
test based on the detection of gamma interferon has been developed for the diagnosis of bovine
tuberculosis. The method and test materials needed are fully described in the instructions accompanying
the commercial kit. This test has not been validated by the manufacturer (Prionics, Switzerland) for the
diagnosis of paratuberculosis. As such, results derived from this assay are frequently difficult to interpret
because there is no agreement with respect to the interpretation criteria and types and amounts of antigens
used to stimulate blood lymphocytes. In cattle the reported specificity of the test varied from 94% to 67%
depending on the interpretation criteria (23).
b) Delayed-type hypersensitivity
The skin test for delayed-type hypersensitivity (DTH) is a measure of cell-mediated immunity, but has
limited value. The test is carried out by the intradermal inoculation of 0.1 ml of antigen into a clipped or

shaven site, usually on the side of the middle third of the neck. In the past, avian PPD tuberculin 3 or johnin
was used for this purpose as it was believed that avian tuberculin and johnin are of comparable sensitivity
and specificity. The skin thickness is measured with calipers before and 72 hours after inoculation.
Increases in skin thickness of over 2 mm should be regarded as indicating the presence of DTH. It should
be noted that positive reactions in deer may take the form of diffuse plaques rather than discrete
circumscribed swellings, thus making reading of the test more difficult. The presence of any swelling should
be regarded as positive in this species. However, sensitisation to the M. avium complex is widespread in
animals, and neither avian tuberculin nor johnin are highly specific (19). Furthermore the interpretation of
the skin test results is complicated by the lack of agreement with respect to interpretation criteria. In a
recent study in which johnin (ID-Lelystad, Lelystad, The Netherlands) was used to test cattle, the skin test
specificity was 88.8% at the cut-off value of ≥2 mm, 91.3% at the cut-off value of ≥3 mm and 93.5% at the
cut-off value of ≥4 mm (23). The effect of these cut-off values on the sensitivity has not been determined.
The performance of this test may also be significantly affected by minor antigenic differences that occur in
different batches of antigen (23). Further research is required to increase the value of the skin test.

Vaccines: Vaccines used against paratuberculosis are: live, attenuated, incorporated with oil and pumice;
lyophilised, live, attenuated, which may be adjuvanted with, for example, oil after reconstitution; and heat-killed
bacterins. Vaccines may be prepared from one strain of M. paratuberculosis 316F or 2E (Weybridge) or
M. paratuberculosis 3 and 5 or II (Canadian strains), or as many as three strains may be used. The information
below applies to a live, attenuated vaccine adjuvanted with oil and pumice (10, 50, 59). Vaccination may cause a
reaction at the site of injection. Vaccination may also interfere with eradication programmes based on
immunological testing and elimination of animals identified as infected and can interfere with the interpretation of
DTH skin tests for bovine tuberculosis.
Diagnostic products: Johnin PPD is a preparation of the heat-treated products of growth and lysis of
M. paratuberculosis. Avian tuberculin PPD is a preparation of heat-treated products of growth and lysis of
M. avium D4ER or TB 56. Details of avian tuberculin PPD are in Chapter 2.3.6 Avian tuberculosis. These two
preparations are used, by intradermal injection, to reveal DTH as a means of identifying animals infected or
sensitised with M. paratuberculosis.
Guidelines for the production of veterinary vaccines are given in Chapter 1.1.8. Principles of veterinary vaccine
1. Seed management

Vaccine: Seed strains should be of a prevalent type, which may be checked by biotyping or genetic
analysis. They should have been demonstrated to be innocuous when administered by the recommended
route of vaccination to intended target species.
Johnin: Strains of M. paratuberculosis used to prepare seed cultures should be identified by biotyping or
genetic tests. They should be shown to be free from contaminating organisms.
Vaccine: Seed cultures may be made on potato slants partly immersed in a suitable medium, such as
Reid’s synthetic medium 4 (53). Cultures may be stored lyophilised. Active cultures are normally incubated
at 37°C.
3 Avian tuberculin can be obtained from VLA Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom, or
from Symbiotic Society, 299 av. Jean Jaurès, 69007 Lyon, France.
4 L-Asparagine, 5.0 g
Potassium dihydrogen phosphate (KH2PO4, anhydrous), 2.0 g
Magnesium sulphate (MgSO4-7H2O), 1.0 g
Ammonium citrate ([NH4]3 C6H5O7), 2.0 g
Sodium chloride, 2.0 g
Ferric ammonium citrate, 0.075 g
Dextrose monohydrate B.P., 10 g
Glycerol B.P. (48 ml), 60 g
Distilled water to 1000 ml

Johnin: The culture substrate should be shown to be capable of producing a product free from substances
known to cause toxic or allergic reactions. A suitable medium for seed culture is that of Reid, solidified with
1.75% agar, in screw cap tubes. Cultures may also be stored lyophilised.
Vaccine: Purity tests should be carried out on seed cultures and final harvest by stained smears.
The vaccine should be used as part of a control programme and will not on its own provide complete
protection against disease caused by M. paratuberculosis (59). There is usually good control of clinical
disease, but subclinical infection persists in vaccinated herds, albeit at a reduced level. Vaccine should be
administered to animals in early life only, e.g. calves in their first month of life. It should be inoculated
subcutaneously and causes a small inflammatory swelling. This is gradually replaced by a cold, painless,
fibro-caseous nodule, which varies in size and which may persist for years. Vaccination has been used to
control the disease in sheep and goats, including older animals. In order to get the best results from
vaccination, management practices to control the disease should also be in place.
The use of vaccines may interfere with the outcome of diagnostic skin tests for tuberculosis, and this should
be remembered when planning a control programme (16).
Johnin: Cultures should be checked by staining smears for the presence of contaminating organisms.
To test for lack of sensitising effect, three guinea-pigs that have not previously been treated with any
material that could interfere with the test, are each injected intradermally on each of three occasions at 5-
day intervals, with 0.01 mg of the preparation under test in a volume of 0.1 ml. Each guinea-pig, together
with each of three control guinea-pigs that have not been injected previously, is injected intradermally 15–
21 days after the third injection with the same dose of the same johnin. The reactions of the two groups of
guinea-pigs should not be significantly different when measured 24–48 hours later.
Vaccine: For vaccine batches, the organisms may be grown on a liquid synthetic medium, such as Reid’s
synthetic medium. The organisms grow as a pellicle on the liquid surface. To ensure a good surface area, it is
convenient to use vessels such as conical flasks containing one-third of their nominal volume of liquid medium.
These flasks may be seeded directly from potato slant cultures, but with some strains, one or more passages on
liquid medium may be necessary to ensure adequate pellicle growth for the final, vaccine batch passage. Such
passaging should usually take place at 2-week intervals as longer periods may result in over-maturation and
sinking of the pellicle. Incubation is at 37°C.
To prepare the vaccine, the pellicle growth from 2-week-old cultures of each strain to be included may be
separated from the liquid medium by decantation, filtration and pressing between filter paper pads. The moist
M. paratuberculosis culture is blended with an adjuvant, such as liquid paraffin, olive oil and pumice (10).
Johnin: Johnin for skin test diagnosis is a PPD prepared from one or more strains of M. paratuberculosis
(available from VLA Weybridge or CDI, Lelystad, the Netherlands). It may be prepared by the following method.
Mycobacterium paratuberculosis strains are grown as a pellicle on liquid Reid’s medium. Production cultures are
usually inoculated from liquid seeding cultures rather than directly from seed on solid medium (Reid’s synthetic
medium). Production cultures are incubated at 37°C for 10 weeks.
At the end of the incubation period, the culture medium has a pH of about 5 and little or no johnin will be obtained
unless the pH is raised, using sodium hydroxide, to about 7.3 before steaming. After thorough mixing, the
cultures are free steamed for 3 hours. The bulk of the killed organisms is removed by coarse filtration and the
filtrate is clarified by further filtration. Protein in the filtrate is precipitated chemically with 40% trichloroacetic acid,
washed and redissolved (alkaline solvent). The product is sterilised by filtration. An antimicrobial preservative
that does not give rise to false-positive reactions, such as phenol (not more than 0.5% [w/v]), may be added.
Glycerol (not more than 10% [w/v]) may be added as a stabiliser. The product is dispensed aseptically into sterile
glass containers, which are then sealed.
pH (not adjusted) 5.65.8

When required, the above medium is solidified by the addition of 1.5% granulated agar (Difco). Sterilised at 121°C for
15 minutes.

Vaccine: Adequate growth of culture and cultural purity need to be checked. Presence of contaminating
organisms may be detected by conventional sterility tests on harvests. Tests for pathogenic mycobacteria are
carried out by injection of moist culture, taken prior to blending with adjuvant and diluted tenfold in saline, into two
guinea-pigs, each receiving 1 ml. These are observed for 8 weeks, killed humanely, and examined for any
abnormal lesions.
Johnin: After final filtration the sterility of each filtrate of the PPD solution is checked.
Sterile filtrates are tested for protein content by a Kjeldahl method (1). The protein content is adjusted to give
between 0.475 and 0.525 mg/ml of protein in the final product. The pH is adjusted to the range 6.5–7.5.
4. Batch control
a) Sterility
Tests for sterility and freedom from contamination may be found in Chapter 1.1.9 Tests for sterility and
freedom from contamination of biological materials. The vaccine organism will not normally grow to a
detectable level in conventional sterility tests.
b) Safety
Vaccine: These tests are normally performed in laboratory animals, although multidose tests in target
animals would also be satisfactory. A typical laboratory animal test would be as follows. Each of two guinea-
pigs is inoculated, subcutaneously, with an acceptable batch of vaccine at a fraction of the cattle dose
previously determined to give a nodule but no overt necrosis at the injection site. Animals are observed for
8 weeks, killed humanely and examined for any abnormal lesions.
Johnin: Two guinea-pigs should each be injected subcutaneously with 0.5 ml of the johnin under test. No
significant local or systemic lesions should be seen within 7 days (1).
Tests on johnin for living mycobacteria may be performed either on the material immediately before it is
dispensed into final containers or on samples taken from final containers themselves. A sample of at least
10 ml should be taken, and this should be injected intraperitoneally or subcutaneously into at least two
guinea-pigs, dividing the volume to be tested equally between the guinea-pigs. It is desirable to take a
larger sample, say 50 ml, and to concentrate any residual mycobacteria by centrifugation or membrane
filtration. The guinea-pigs are observed for at least 42 days and post-mortem examinations are carried out.
Any macroscopic lesions are examined microscopically and culturally.
c) Potency
Vaccine: As protection tests appear to be impractical, a test of sensitising ability may be used. This may
then be related to bacterial content based on weight. A typical test would be as follows: guinea-pigs are
sensitised by intramuscular injection of 0.5 ml of a 100-fold dilution in liquid paraffin of the vaccine under
test. Skin tests are performed 6 weeks after sensitisation using intradermal inoculations of 0.2 ml of at least
three serial dilutions of an M. paratuberculosis antigen, such as johnin PPD, the dilutions being chosen to
give expected skin reactions of from 8 mm to 25 mm diameter. Each guinea-pig receives several dilutions
per flank, their distribution being chosen by a Latin square design. After 24–48 hours, skin reactions are
measured. A reference preparation for tests of this type has not yet been fully established. Avian tuberculin
PPD of known international unitage may be used as a skin test antigen in tests of this type to ensure that
the vaccine is capable of producing adequate sensitisation (corresponding to the vaccination).
Johnin: The potency of johnin is currently determined by chemical assay for protein using a Kjeldahl
method. A PPD content of 0.5 ± 0.025 mg/ml of final product is recommended (1).
The identity of the material should be confirmed by injecting intradermally into guinea-pigs sensitised by
injections of killed M. paratuberculosis (100 mg powder mycobacteria + 25 ml vaseline + 100 mg pumice
stone) 6 weeks previously.
It is possible to perform a potency test using dilutions of johnin in guinea-pigs sensitised with
M. paratuberculosis, similar to such tests for the potency of bovine and avian tuberculin, but a standard
preparation for this type of test has not yet been fully established.
Vaccine: After vaccination at the age of 14–30 days, the vaccination effect is expressed as the reduction in
the rate of excretors among vaccinated animals as compared with nonvaccinated bovines (21).

There is usually good control of clinical disease, but a reduced level of subclinical infection persists. The
favourable results probably reflect a diminishing exposure to infection resulting from a reduction in the
number of heavy excretors in the herd.
e) Stability
Vaccine: The vaccine may be stored at 2–8°C for 9–12 months without loss of potency. It should not be
frozen.
Johnin: Johnin should be protected from light and stored at 2–8°C. Under these conditions it should retain
its potency for at least 5 years.
f) Preservatives
A preservative is normally included for vaccine in multidose containers. For johnin, the phenol used is no
more than 0.5% (w/v). The concentration of the preservative in the final product and its persistence through
shelf life should be checked.
Vaccine: The vaccine causes some side-effects, nodule formation and sensitisation of animals to the
tuberculin test (15). In humans, accidental injection of vaccine has resulted in chronic inflammatory
reactions requiring surgical treatment (21).
a) Safety
See Section C.4.b.
b) Potency
See Section C.4.c.
REFERENCES
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*
* *
NB: There are OIE Reference Laboratories for Paratuberculosis (see Table in Part 3 of this Terrestrial Manual or

CHAPTER 2.1.12.
Q FEVER
SUMMARY
Query (Q) fever is a zoonosis that occurs in most countries. Humans acquire infection from animal
reservoirs, especially from domestic ruminants. Q fever is a highly infectious disease, which is due
to the proliferation of Coxiella burnetii, a small and pleomorphic bacterium measuring 0.3–1.5 µm
long × 0.2–0.4 µm wide. As an obligate intracellular bacterium, C. burnetii can be grown only in
embryonated eggs or cell cultures or, when necessary, in inoculated laboratory animals. It occurs
in two antigenic forms: the pathogenic phase I, found in infected animals or humans, and the
avirulent phase II, obtained by repeated passages in embryonated eggs or in cell cultures.
Because this microorganism is extremely hazardous, handling viable C. burnetii must be done in
facilities that meet the OIE requirements for Containment Group 3 pathogens.
In humans, Q fever occurs in either an acute form (self-limiting febrile episode, pneumonia,
hepatitis) or a severe chronic form (endocarditis) following an early infection that may be passed
unnoticed. The acute form resolves quite quickly after appropriate antibiotic therapy, but the
chronic form requires prolonged antibiotic therapy (for 2 years or more), coupled with serological
monitoring. In some countries, a vaccine is available for professionally exposed population groups.
The signs of Q fever in cattle include abortion, dead or weak offspring, retained placenta, metritis,
and infertility. In small ruminants, Q fever is often associated with sporadic abortions or outbreaks
of abortions followed by recovery without complications. Coxiella burnetii infection persists for
several years, and is probably life long. Sheep, goats and cows are mainly asymptomatic carriers,
but can shed massive numbers of bacteria at parturition, and intermittently in various secretions
and excreta. Domestic animals, such as dogs, cats, rabbits, birds, etc., are also susceptible to
infection and should be considered as possible sources of infection for animals and humans.
Identification of the agent: For laboratory diagnosis, samples can be taken from the placenta,
vaginal discharges, and liver, lung or stomach contents of aborted fetuses, and from milk,
colostrum and faeces.
The bacteria can be visualised in stained tissue smears using a microscope with an oil-immersion
objective lens. Because it is acid resistant, the bacteria can be stained by several methods: Stamp,
modified Ziehl–Neelsen, Gimenez, Giemsa and modified Koster. This finding is presumptive
evidence of Q fever, but coupled with serological tests, clinical findings and no other infectious
abortive agents, it may be sufficient to establish a diagnosis of the disease at the flock or herd
level.
To date, demonstration of the agent by immunohistology using specific antibodies or by

polymerase chain reaction (PCR) has proven to be more specific and sensitive than classical
staining methods. No specific antibodies for immunochemistry are commercially available, but PCR
can be done in suitably equipped laboratories. PCR is considered a useful test for screening large
numbers of samples and various types of samples. Furthermore, samples can be heat inactivated
and therefore ensure the safety of laboratory personnel.
Coxiella burnetii can be isolated by inoculation of specimens into conventional cell cultures or
embryonated chicken yolk sacs or laboratory animals. Inoculation of laboratory animals (guinea-
pig, mouse, hamster) is helpful in cases requiring isolation from tissues contaminated with various
microorganisms or in order to obtain phase I Coxiella antigens.
Serological tests: The diagnosis of Q fever often relies on serology. A number of tests can be
used, particularly the indirect immunofluorescence test, the enzyme-linked immunosorbent assay,

Chapter 2.1.12. — Q fever
and the complement fixation test. Currently, commercially available tests allow the detection of
anti-C. burnetii phase II antibodies. The presence of specific IgG antibodies provides evidence of a
recent C. burnetii infection or a past exposure.
Requirements for vaccines and diagnostic biologicals: Several inactivated vaccines against Q
fever have been developed, but only vaccines containing or prepared from phase I C. burnetii
should be considered protective. Repeated annual vaccination is recommended in heavily infected
areas, particularly of young animals.
An inactivated phase I vaccine is commercially available in Slovakia. A recent study has proved its
efficacy with regard to both abortion and C. burnetii shedding in experimentally vaccinated then
challenged pregnant goats, but information on its safety is lacking.
A. INTRODUCTION
Q fever is widely distributed throughout the world with the exception of New Zealand. Although Q fever is present
in virtually all ‘animal kingdoms’, including arthropods, the disease affects mostly humans, cattle, sheep and
goats (27, 30). The aetiological agent, Coxiella burnetii, is a gram-negative obligate intracellular bacterium,
adapted to thrive within the phagolysosome of the phagocyte. It has been historically classified in the
Rickettsiaceae family; however, phylogenetic investigations, based mainly on 16S rRNA sequence analysis, have
shown that the Coxiella genus is distant from the Rickettsia genus in the alpha subdivision of Proteobacteria (60).
Coxiella burnetii has now been placed in the Coxiellaceae family in the order Legionellales of the gamma
subdivision of Proteobacteria (26). The complete genome sequencing of C. burnetii has been achieved recently
and confirms its systematic position (47). Unlike rickettsiae, C. burnetii produces a small, dense, highly resistant
spore-like form that is highly stable in the environment, a trait that is important for transmission (31). This ability
has been attributed to the existence of C. burnetii developmental cycle variants: large-cell variants (LCV), small-
cell variants (SCV), and small dense cells (SDC) (10, 17, 48). The SDC and SCV represent the forms of the
bacteria likely to survive extracellularly as infectious particles. Another essential characteristic is that C. burnetii
has two antigenic forms: the pathogenic phase I, isolated from infected animals or humans, and the avirulent
phase II, obtained in ovo or in vitro. An LPS (lipopolysaccharide) change occurs during serial passages: phase I
cells, with full-length LPS O-chains, change to intermediate phases with decreasing LPS O-chain lengths and
then to phase II, with truncated LPS. The LPS phase variation is accompanied by a permanent chromosomal
deletion that makes impossible cell reversion from phase II to phase I (56).
Q fever is a zoonosis. In humans, the infection has an acute, chronic and subclinical form (38). The acute forms
commonly include a self-limiting febrile episode, pneumonia, and granulomatous hepatitis. The main clinical
manifestation of chronic Q fever is endocarditis in patients with valvulopathies. In the absence of any appropriate
antibiotic treatment, complications of the chronic form may be severe to fatal (11). Moreover, C. burnetii infection
of pregnant women can provoke placentitis and often leads to premature birth, growth restriction, spontaneous
abortion or fetal death (36). The infection is endemic in many areas leading to sporadic cases or explosive
epidemics. Its incidence is probably greater than reported. The epidemiology of Q fever suggests that the
infection is principally transmitted by inhalation of desiccated aerosol particles, and through contact with infected
animals and their reproductive tissues (29, 30). Ingestion has been often suggested, particularly through the
consumption of dairy products derived from contaminated raw milk, and even possibly following pasteurisation
(16, 38, 53). Q fever is very rarely transmissible from person to person, although exposure during childbirth,
through sexual transmission and blood transfusions, is possible (12, 32). In animals, in addition to respiratory
and digestive routes, vertical transmission and sexual transmission can occur (25, 58). Arthropods, principally
ticks, may be involved in Q fever transmission.
In cows, ewes and goats, Q fever has been associated mostly with late abortion and reproductive disorders such
as premature birth, dead or weak offspring, metritis and infertility (27). Nevertheless, serological responses in a
given species or even the isolation of C. burnetii do not necessarily correlate with expression of the clinical
disease (18, 27, 44). Indeed, the agent may persist in infected animals, be shed intermittently in milk, faeces,
urine, and may be present in blood. The agent can be recovered in very high numbers in birth products
(placenta, amniotic fluid and fetus), and non-pregnant animals are less of a risk. Domestic ruminants are
considered the main reservoirs for C. burnetii, but cats, dogs, rabbits, birds, etc., have also been reported to be
implicated in human disease/infection (23, 29, 30, 51). Thus, infected domestic and wild animals usually shed the
agent with no outward signs of disease, and should be regarded as possible sources of infection for humans.
Abortions in ruminants have usually been investigated to determine if Q fever is present as it may affect the
health of humans or other animals. Diagnosis of Q fever, and other abortive diseases, traditionally has been
made on the basis of microscopy on clinical samples, coupled with positive serological results, is usually
adequate for this purpose (27, 41, 43). The Q fever diagnostic is also required for epidemiological surveys of ‘at
risk’ and suspected flocks in limited areas (following recent outbreaks in humans or animals), or for export

purpose. However, identification of C. burnetii shedders and asymptomatic carriers is not currently practised (4,
5, 7, 21).
Generally, when several animals are seropositive, an appropriate intervention is advised. The measures could be
adapted according to the seroprevalence and epidemiological context. Proper pasteurisation of milk products
must be ensured (53). The amount of agent can be reduced in the environment by regular cleaning and
disinfection of animal facilities, with particular care of parturition areas, using 10% sodium hypochlorite. Pregnant
animals must be kept in separate pens, and placentas and aborted fetuses must be removed quickly and
disposed properly to avoid being ingested by dogs, cats or wildlife. Spreading manure from contaminated farms
in suburban areas and gardens should be avoided. While the efficiency of fermentation by composting or
decontamination with a chemical treatment (such as 0.4% calcium cyanamid or lime) for inactivating C. burnetii
need to be evaluated, these methods are still recommended. In order to acquire and maintain Coxiella-free
livestock, introduction of animals, regrouping of flocks, contacts with wildlife and infestation by ticks should be
minimised. These methods may be effective in controlling disease but exposed animals may remain infected.
Although vaccines for animal Q fever have been developed, there are not commercially available in most
countries.
Finally, it is important to remember that C. burnetii is extremely hazardous to humans, and laboratory infections
are common. Because of its ability to cause incapacitating disease in large groups of people, its low infectious
dose, resistance in the environment, and aerosol route of transmission, C. burnetii is considered a potential
agent of bioterrorism and is classified by the CDC as a group B agent (9). Appropriate precautions must be taken
with this risk group 3 agent. Live culture or contaminated material from infected animals must only be handled in
facilities that meet the requirements for Containment Group 3 pathogens as outlined in Chapter 1.1.2 Biosafety
and biosecurity in the veterinary microbiological laboratory and animal facilities.
Coxiella burnetii can be demonstrated in various ways, depending on the type of sample and the purpose of
diagnosis (11, 41, 43). Samples should be collected from aborted fetuses, placenta and vaginal discharges soon
after abortion or parturition. Milk from the tank, individual milk or colostrum, and faeces samples can also be
taken.
a) Staining
In a case of an abortion suspected of being caused by an infection, smears are prepared on microscope
slides of placental cotyledon (42). Lung, liver and abomasal contents of the aborted fetus or vaginal
discharge may be used in the same manner. These could be stained according to several methods: Stamp,
Gimenez, Macchiavello, Giemsa and modified Koster (14, 35, 41, 42, 44). The first three techniques give
the best results. These methods are close to the modified Ziehl–Neelsen method involving basic fuchsin to
stain bacteria. For example, the Stamp staining method is performed with 0.4% basic fuchsin solution,
followed by rapid decolouration with 0.5% acetic acid solution, and counterstaining with 1% methylene blue
or malachite green solution. The smears are examined microscopically with an oil-immersion objective lens
(×500 or more). The Stamp method is preferred in veterinary laboratories while the Gimenez method is
widespread in human diagnosis. Gimenez is fastest because an acidic solution is not included for
differentiation. Coxiella burnetii are characterised by a very large number of thin, pink-stained coccobacillary
bacteria against a blue or green background. They may sometimes be difficult to detect due to their small
size (0.3–1.5 µm long × 0.2–0.4 µm wide), but this is compensated for by their large numbers; often
inclusions within the host cells appear as red masses against the blue or green background. Attention must
be taken in the interpretation of the results as, microscopically, C. burnetii can be confused with
Chlamydophila abortus or Brucella spp. However, using the same staining procedure, Chlamydophila have
sharper outlines, are round, small and may resemble globules. Brucella spp, are larger (0.6–1.5 µm long ×
0.5–0.7 µm wide), more clearly defined and stain more intensely. Control positive slides of C. burnetii,
Chlamydophila abortus and Brucella must be used for comparison. Diagnosis made on the basis of
microscopy, coupled with positive serological results, is usually adequate for routine purposes (43). When
biological staining is inconclusive, one of the other methods (below) may be used as a confirmatory test.
b) Specific detection methods

Detection of C. burnetii in samples can also be achieved by specific immunodetection (capture enzyme-
linked immunosorbent assay [ELISA], immunohistochemistry), or DNA amplification (8, 11, 55).
Immunohistology may be used with paraffin-embedded tissues or on acetone-fixed smears (37). The
method is an indirect immunofluorescence or immunoperoxidase assay using polyclonal C. burnetii
antibodies, either a well characterised antiserum of human origin or a specific antiserum produced in

laboratory animals (rabbit or guinea-pig). An anti-species (human, rabbit or guinea-pig) anti-IgG conjugate,
labelled with fluorescein isothiocyanate (FITC) or peroxidase, is then used to visualise the bacteria. Control
positive slides of C. burnetii antigen should be available for comparison. No specific antibodies for
immunochemistry are commercially available.
Polymerase chain reaction (PCR) methods have been used successfully to detect C. burnetii DNA in cell
cultures and biological samples. As the number of C. burnetii is likely to be lower in milk, colostrums and
faeces than in abortion material, PCR can be used for analysis of this large diversity of samples. Before
performing the PCR, biological samples can be inactivated by heating at 90°C for 30–60 minutes,
depending of the samples’ nature, their size or their weight. This technique can be performed in suitably
equipped laboratories using primers derived from various targets, such as multicopy insertion sequence
(accession number M80806), the most popular employed (4, 19). The use of these primers for the
amplification of this sequence allows the sensitivity of the test to be increased and this because of the
presence of several copies in the Coxiella genome. The level of detection of the conventional trans-PCR is
related to the analysed samples (for example 1–500 bacteria/ml of milk sample or 1 bacteria/mg of faeces).
The other target genes reported to be used in the PCR for specific C. burnetii identification are: superoxide
dismutase (sodB) gene (accession number M74242); com1 encoding a 27 kDa outer membrane protein
(accession number AB004712); heat shock operon encoding two heat shock proteins (htpA and htpB)
(accession number M20482); isocitrate dehydrogenase (icd) (accession number AF069035); and
macrophage infectivity potentiator protein (cbmip) (accession number U14170). Different primers used in
PCR can be obtained on the web site (http://ifr48.timone.univ-mrs.fr/Fiches/Fievre_Q), regularly updated by
the French CNR.
The real-time PCR provides an additional means of detection and quantification (21, 22, 52). As the
conventional PCR, various target genes are used: IS1111 (accession number M80806); com1 (accession
number AB004712); and isocitrate dehydrogenase (icd). To quantify the bacteria in biological samples
using the real-time PCR, it is recommended to amplify a unique and specific sequence. Indeed, recent data
show that the number of the insertion sequence (IS) varied widely (between 7 and 110) depending of the
isolates (22). Whereas the use of this sequence could increase the sensitivity of the test, it may not be
accurate for quantification.
Ready-to-use kits are commercially available and can detect the bacteria in several samples. However,
there is an urgent need for the development of a molecular method for the assessment of bacterial viability,
especially in milk samples. The development of a multiplex PCR constitutes another technique for
screening all infectious abortive agents.
c) Isolation of the agent

For specific laboratory investigations, it may be necessary to isolate the agent. Where microscopic
examination has revealed large numbers of C. burnetii combined with a low contamination rate with other
bacteria, direct isolation by inoculation of embryonated chicken eggs or cell culture is possible (18). For
example, a portion of placenta is homogenised in phosphate buffered saline (PBS) containing antibiotics
(streptomycin 100–200 µg/ml and penicillin or gentamicin 50–100 µg/ml). After low-speed centrifugation,
dilutions of the supernatant fluid are inoculated into 5-day-old embryonated chicken eggs via the yolk sac.
Eggs are preferably from specific pathogen free (SPF) hens. Embryos that die during the first 5 days after
inoculation are discarded. The yolk sacs are harvested after 10–15 days of incubation. Stained smears of
the yolk sac wall are examined to ensure the absence of bacterial contamination and to determine the
presence of C. burnetii. PCR analysis can be used to confirm the presence of C. burnetii. Further passages
may be required to obtain an isolate in pure culture.
A cell microculture system from a commercially available method used for virus culture, the shell vial cell
culture 1, has been adapted for isolating strictly or facultatively intracellular bacteria, including C. burnetii.
Such a method was described for C. burnetii in 1990 (11, 39). Suspensions of samples are inoculated into
human embryonic lung (HEL) fibroblasts grown on a 1 cm2 cover-slip within a shell vial. Centrifugation for
1 hour at 700 g enhances the attachment and penetration of bacteria into the cells. Three shell vials are
used for the same sample, and by day 3, 10 and 21, the cytopathic effect (CPE) – C. burnetii characteristic
vacuoles in HEL cells – are examined using an inverted microscope. After 10 days, detection of growing
C. burnetii within the cells is achieved directly on the cover-slip inside a shell vial by a direct
immunofluorescence assay with polyclonal anti-C. burnetii antibodies and an appropriate anti-species
conjugated to FITC. Cells of the remaining shell vial are harvested and transferred in a 25 cm2 culture flask.
Incubation can be conducted for 3 months, with a culture medium change once a week. The infection can
be monitored by microscopy of Gimenez-stained cells cyto-centrifuged from the culture supernatant and by
PCR analysis of the culture supernatant. When the CPE observations and Gimenez staining or PCR results
are positive, a passage in a 75 cm2 culture flask is performed. Culture supernatant is then inoculated on
1 Sterilin, Bibby Sterilin Ltd, Stone, Staffordshire ST15 O5A, United Kingdom.

confluent layers of Vero cells or L929 mouse fibroblasts in a 150 cm2 culture flask in order to establish a
C. burnetii isolate. This method was developed for humans but could be adapted for animals (50).
With heavily multi-contaminated samples, such as placentas, vaginal discharges, faeces, or milk, the
inoculation of laboratory animals may be necessary. Biocontainment level 3 requirements are
recommended for holding experimentally infected rodents (see Chapter 1.1.2). Mice and guinea-pigs are
the most appropriate laboratory animals for this purpose (45). Following intraperitoneal inoculation with a
dose of 0.5 ml per animal, body temperature and antibody status are monitored. This method should always
be performed in conjunction with serological tests on other guinea-pigs or mice that have been inoculated
with the same samples. Sera are collected 21 days after inoculation. A positive result confirms a diagnosis
of C. burnetii infection. If pyrexia develops, the animal is killed and the spleen is removed for isolation of the
agent by inoculation into embryonated chicken eggs or in cell cultures. Microscopic examination of
C. burnetii is done using impressions and staining of the collected spleens. Alternatively, PCR may be
performed on spleens systematically collected 7–9 days post-inoculation (8).
Although characterisation of isolates seems necessary for understanding the varying epidemiology of Q
fever in different geographical areas, no discriminatory typing methods are available currently. For this
purpose, efforts should focus on genetic typing methods. High resolution molecular typing methods that are
currently being developed and evaluated offer promise of mapping progression of clones and contact
traceback studies. Studies using multi-spacer typing (15) and variable number tandem repeat typing (2, 54)
have demonstrated high levels of resolution among C. burnetii isolates. Techniques for genotyping
C. burnetti have been described.
Among the various techniques that can be employed, the three most often used are: the indirect
immunofluorescence assay (IFA), the ELISA and the complement fixation (CF) test (11). Three older serological
tests are no longer used in routine diagnosis: the microagglutination technique, the capillary agglutination test
and the indirect haemolysis test. A high-density particle agglutination (HDPA) test has been evaluated (33).
Serological assays are suitable for screening herds, but interpretation at the individual animal level can be
difficult. Indeed, animals may remain seropositive for several years following an acute infection, some animals
may shed C. burnetii and pose a risk for infection prior to the development of antibodies, and some infected
animals seem not to seroconvert (1, 6, 8). Serological cut-off titres used to diagnose Q fever are given below;
interpretation of the results requires at least ten animals (aborted or not). Both serological responses and
bacterial evidence are necessary for establishing the presence of the infection.
a) Indirect immunofluorescence test

In human medicine, the IFA adapted as a microimmunofluorescence technique is the reference method for
the serodiagnosis of Q fever (11, 57). The procedure can be adapted to perform an immunoperoxidase
assay. Some commercial CF test antigens are suitable, but antigens prepared for the diagnosis in humans
are preferred (11). This method of preparation has been demonstrated to yield antigens with the highest
sensitivity for C. burnetii antibody detection. Briefly, both phase I and phase II C. burnetii antigens are used;
phase II antigen is obtained by growing C. burnetii Nine Mile reference strain in cell culture, while phase I
antigen is obtained from the spleens of laboratory animals inoculated with phase II C. burnetii in cell
cultures. A few phase I cells may still be present in the phase II population and can be selected and
propagated within animals. Antigen is diluted, dropped on to the wells of a glass microscope slide, allowed
to dry, and fixed with acetone. The two forms of the infection, acute and chronic, have different serological
profiles: during acute Q fever, IgG antibodies are elevated against phase II only whereas during chronic Q
fever, high levels of IgG antibodies to both phase I and II of the bacteria are observed (57). In addition,
antigen-spot slide wells may be purchased from a supplier providing the phase II form, or the phase I and II
forms of C. burnetii. These can be adapted by replacing the human conjugate by a conjugate adapted to the
animal species.
Twofold dilutions of the serum under test are placed on immunofluorescence slides with wells previously
coated with one or two antigens. If specific antibodies are present, they are fixed by the antigen on the
slide. The complex is then detected by examination with a fluorescence microscope following the addition of
the fluorescent conjugate recognising the species immunoglobulins.
Antigen should only be prepared in facilities that meet the requirements for Containment Group 3
pathogens as outlined in Chapter 1.1.2 of this Terrestrial Manual.
Phase II C. burnetii Nine Mile are grown in confluent layers of Vero or L929 cells in 150 cm2 culture flasks
with minimal essential medium (MEM) supplemented with 2 mM L-glutamine and 4% fetal bovine serum.
The infection is monitored by microscopic examination of Gimenez-stained cells scraped from the bottoms

of the flasks. When a heavy C. burnetii infection is seen, the supernatants of 15 flasks are individually
pelleted by centrifugation (5000 g, 15 minutes) resuspended in 1 ml of PBS with 0.1% formaldehyde and
incubated for 24 hours at 4°C. After pooling, the remaining cells are broken by sonication. Cellular debris is
removed by two successive centrifugation steps (100 g, 10 minutes each). The 15 ml suspension is then
centrifuged through 20 ml of PBS with 25% sucrose (6000 g, 30 minutes, without a break). The resulting
pellet is washed three times in PBS (6000 g, 10 minutes), resuspended in the smallest possible volume of
sterile distilled water, and adjusted to 2 mg/ml by UV spectroscopy. An antibacterial preservative, such as
sodium azide at a final dilution of 0.1% or thiomersal at 0.01%, is added. Antigen prepared in this manner is
frozen at –20°C.
In order to obtain phase I antigen, mice are inoculated with C. burnetii Nine Mile grown in cells (mainly in
phase II). Nine days after infection, the spleens are removed. Each one is ground in 7.5 ml MEM, and
inoculated into three 75 cm2 culture flasks containing L929 or Vero cell monolayers (2.5 ml per flask).
Amplification of phase I C. burnetii is conducted for 4 weeks, with a culture medium change once a week.
The infected cells are then harvested and the bacteria are purified as described above (mainly in phase I).
Antigen production can also be performed by culture of C. burnetii in SPF embryonated eggs. At 5–6 days
of age, the microorganism is inoculated into the yolk sac of the embryonated eggs, which are harvested
after death of the embryo at 12–15 days. Infected yolk sacs have a characteristic straw-yellow colour.
Uninfected yolk sacs are orange in colour and have a viscous consistency. Any embryos that die between
5 and 10 days of incubation are discarded. The strain used for egg inoculation is a 1/100 homogenate of
yolk sac in PBS containing penicillin (500 International Units/ml) and streptomycin (0.5 mg/ml). The yolk
sacs are pooled and homogenised with three parts PBS. The suspension is inactivated with 1.6%
formaldehyde for 24 hours at 37°C. The lipid supernatant fluid is discarded. The suspension is then
centrifuged at moderate speed (a500 g) for 30 minutes. After removal of the supernatant fluid, more PBS is
added and centrifugation is repeated. The final suspension is diluted with PBS. Sodium azide or thiomersal
is added as an antibacterial preservative. The abundance of C. burnetii and the absence of bacterial
contaminants in homogenates of yolk sacs suspended in PBS are verified by microscopic examination of a
smear on a microscope slide, stained by Stamp’s method. In order to obtain phase I antigen, C. burnetii
recovered from spleen material of infected laboratory animals can be propagated, as ground spleen
extracts are subsequently transferred in the yolk sacs, given that the amount of phase I cells is still high
until the sixth yolk passage (EP6).
Titration of antigen with at least three different known sera (with high, moderate and low titres, respectively)
is sufficient to recover the appropriate dilution for further immunofluorescence tests.
• Materials and reagents

Microscope equipped for fluorescence, humidified incubator, washing basin.
Slides suitable for the antigen are necessary. The latter may be either prepared in the laboratory or
purchased from a supplier (see above). The method described is adapted from the BioMérieux kit, and is
given as an example. Ready-to-use slides contain 12 wells per slide, each of 7 mm diameter, coated with
phase II antigen obtained from culture on Vero cells and can be stored at 4°C or –20°C.
Concentrated fluorescent conjugate, to be diluted when required with PBS + 1% Evans blue at the dilution
recommended by the manufacturer.
PBS, buffered glycerine, Evans blue dye 1% solution.
• Test procedure
i) Inactivate the sera under test for 30 minutes at 56°C, then dilute serially from 1/40 to 1/640 in PBS.
ii) Allow the previously antigen-coated slides to warm to room temperature. Do not touch the wells.
iii) Add 20 µl of each serum dilution to the wells. Add negative and positive control sera. To one well, add
20 µl of PBS to serve as antigen control.
iv) Incubate in a humid chamber for 30 minutes at 37°C. Wash the slide twice with PBS for 10 minutes
each. Rinse with distilled water and air dry.
v) Add to the wells, including the controls, 20 µl of the conjugate directed against the appropriate species
(e.g. FITC-labelled rabbit anti-goat or anti-sheep IgG[H+L]), freshly diluted in PBS + Evans blue.
Incubate in a humid chamber for 30 minutes at 37°C. Rinse with distilled water and air-dry. Add a few
drops of buffered glycerine and cover with a cover-slip. Examine under a fluorescence microscope at
magnification ×400 or more.

• Interpretation of the results

A positive reaction will consist of small brilliant points against a dark background. Verify that the conjugate
by itself and the negative control serum give a negative result (absence of small brilliant points).
Nonspecific fluorescence usually takes the form of spots of irregular shape. The positive control must give
the known titre with ± one dilution.
The reaction is considered to be positive if there is obvious immunofluorescence at the 1/160 dilution and
upwards. In human medicine, this method is used to determine antibodies against phases I and II in the
IgG, IgM, and IgA fractions allowing acute and chronic Q fever to be differentiated. Rheumatoid factor
absorbant is used for remove IgG before the determination of IgM and IgA. Screening of the sera is
performed with phase II antigen, and positive sera are tested subsequently for the presence of the different
classes of Ig directed against phases I and II antigens. However, neither phases I and II antibody responses
nor Ig classes responses have been well studied in domestic animals.
b) Complement fixation test

This cold fixation micromethod of the type developed by Kolmer is performed with 96-well U-bottomed
microtitre plates. The test detects complement-fixing antibodies present in the serum. The CF test is
specific but less sensitive than the ELISA or IFA (11, 34). Seroconversion is detected later by the CF test
than by the IFA or ELISA, but CF antibodies can persist for long periods after illness, and the CF test gives
excellent results for routine diagnosis at the flock level for abortive diseases (41, 43). The CF test is still
largely used by many laboratories in many countries. This method often uses antigen in phase II prepared
from a mixture of two strains (Nine Mile and Henzerling) 2 or antigen in phase I and II mixture prepared from
Nine Mile strain 3. In France, this method has been standardised (AFNOR NFU47-006).
The reaction is done in two stages. Antigen and complement-fixing antibodies are first mixed, and sheep
erythrocytes, sensitised by the anti-sheep erythrocyte serum, are added. Fixation of the complement by the
antigen/antibody complex during the first step does not permit lysis of erythrocytes; in contrast, if there are
no complement-fixing antibodies, the complement induces the lysis of the sensitised erythrocytes. Then the
haemolysis rate is inversely proportional to the level of specific antibodies present in the sample serum.
• Reagents
Veronal/calcium/magnesium buffer (VB), pH 7.2.
The haemolytic system: a mixture of equal parts of a 2% suspension of sheep erythrocytes in VB; and
haemolytic serum diluted to a specified titre in VB.
Complement: commercial freeze-dried preparation or fresh guinea-pig serum.
Antigen: use commercial antigens at the titre recommended by the manufacturer if the antigen titration is
performed with this method.
Positive and negative control sera.
• Pretitrations
i) Dilute the sheep erythrocytes to a final concentration of 2% in VB.
ii) Titrate the haemolytic serum on a microplate: 25 µl of complement at a known haemolytic
concentration (e.g. 1/30); 25 µl of increasing dilutions of haemolytic serum + 2% sheep erythrocytes.
Include controls without complement. Incubate for 30 minutes at 37°C. Establish the dilution
equivalent to 2 haemolytic units.
iii) Dilute the antigen as recommended by the manufacturer. The antigen may also be titrated: make
increasing dilutions of antigen (25 µl horizontally) and a positive serum of known titre (25 µl, vertically).
Add 25 µl of the suspension of sensitised erythrocytes and incubate for 30 minutes at 37°C. The
antigen titre is the highest dilution producing a positive reaction with the highest serum dilution. Verify
the absence of anticomplementary activity of the antigen at different dilutions.
iv) Titrate the complement on a microplate: serially dilute the complement or guinea-pig serum in VB, for
example from 1/15 to 1/200. To each well containing 25 µl of this dilution, add 25 µl of antigen and
25 µl of the haemolytic system. Incubate for 30 minutes at 37°C and establish the dilution equivalent to
2 haemolytic units of complement.
2 Dade Behring, Marburg, Germany.

3 Virion, Zürich, Switzerland.

• Test procedure
i) Make twofold dilutions of decomplemented sample sera from 1/10 to 1/320 in six wells and in four
additional wells at dilutions from 1/10 to 1/80 to detect anticomplementary activity (25 µl per well).
ii) Add 25 µl of diluted antigen or 25 µl of VB to control serum wells.
iii) Add 25 µl diluted complement to all wells. Cover the plate with plastic adhesive film and incubate for
18 hours at 4°C.
iv) Remove the plates from the refrigerator, allow them to reach room temperature, and add 25 µl of
freshly prepared haemolytic system. Incubate at 37°C for 30 minutes. Centrifuge the plates at 500 g
for 5 minutes at 4°C. Examine the controls and read the results.

Titres between 1/10 and 1/40 are characteristic of a latent infection. Titres of 1/80 or above in one or more
sera from a group of from five to ten animals reveal an evolutive phase of the infection.
c) Enzyme-linked immunosorbent assay

This technique has a high sensitivity and a good specificity (11, 40, 59). It is easy to perform in laboratories
that have the necessary equipment (a spectrophotometer) and reagents. The ELISA tends to replace the
IFA and CF tests as the test of choice because it is convenient for large-scale screening and, particularly for
veterinary diagnosis, as it is a reliable technique for demonstrating C. burnetii antibody in various animal
species (20, 49). It requires a relatively pure antigen. Antigens prepared for the CF test may be used for
coating the plates. Ready-to-use kits are commercially available and can detect anti-phase II antibodies or
both anti-phase I and II antibodies.
Wells of the microplate are coated with C. burnetii whole-cell inactivated antigen. Diluted serum samples
are added to the wells and react to antigens bound to the solid support. Unbound material is removed by
washing after a suitable incubation period. Conjugate (horseradish-peroxidase-labelled anti-ruminant Ig)
reacts with specific antibodies bound to the antigen. Unreacted conjugate is removed by washing after a
suitable incubation period. Enzyme substrate is added. The rate of conversion of substrate is proportional to
the amount of bound antibodies. The reaction is terminated after a suitable time and the amount of colour
development is measured spectrophotometrically.
• Materials and reagents

Microtitre plates with 96 flat-bottomed wells, freshly coated or previously coated with Q fever antigen;
microplate reader (spectrophotometer; 405 and/or 450 and/or 492 nm filters); 37°C humidified incubator; 8-
and 12-channel pipettes with disposable plastic tips; microplate shaker (optional).
Positive and negative control sera; conjugate (ruminant anti-immunoglobulin labelled with peroxidase);
tenfold concentration of diluent (PBS–Tween); distilled water; substrate or chromogen (TMB
[tetramethylbenzidine], ABTS [2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)] for peroxidase);
hydrogen peroxide.
• Test procedure
i) Dilute the serum samples, including control sera, to the appropriated dilution (usually 1/100) and
distribute 0.1 ml per well in duplicate. Control sera are positive and negative sera provided by the
manufacturer and an internal positive reference serum from the laboratory in order to compare the
titres between different tests.
ii) Cover the plate with a lid and incubate at room temperature for 30–90 minutes. Empty out the contents
and wash three times in washing solution at room temperature.
iii) Add the appropriate dilution of freshly prepared conjugate to the wells (0.1 ml per well).
iv) Cover each plate and incubate as in step ii. Wash again three times.
v) Add 0.1 ml of freshly prepared chromogen substrate solution to each well (for example: TMB in 0.1 M
acetic acid and 30% H2O2 solution [0.2 µl/ml]; or 0.25 mM ABTS in citrate phosphate buffer, pH 5.0,
and 30% H2O2 solution [0.1 µl/ml]).
vi) Shake the plate; incubate according to the manufacturer recommendations, stop the reaction by
adding stopping solution to each well, e.g. 0.05 ml 2 M sulphuric acid for TMB or 10% sodium dodecyl
sulphate for ABTS.
vii) Read the absorbance of each well with the microplate reader at 405 nm (ABTS) or 450 nm (TMB). The
absorbance values will be used to calculate the results.


For commercial kits, interpretations and values are provided with the kit.
For example: calculate the mean absorbance (Ab) of the sample serum and of the positive (Abpos) and
negative (Abneg) control sera, and for each serum, calculate the percentage:
Ab - Abneg
Abpos - Abneg x 100
Interpret the results as follows:

Ab <30% negative serum
Ab 30–40% doubtful serum
Ab >40% positive serum
1. Vaccine
Vaccination is the most logical strategy for preventing Q fever in exposed subjects and livestock. A C. burnetii
vaccine can only be prepared by trained staff only working in adequate conditions of protection (at a minimum in
a biosafety level 3 laboratory). It is recommended to obtain the vaccine from manufacturers capable of
completing and certifying tests for safety, inactivation and sterility.
In some countries, vaccination is practised for occupationally exposed people, such as abattoir workers,
veterinarians and laboratory personnel. A vaccine inactivated by formaldehyde (Q-VAX, CSL Ltd, Australia),
prepared from the Henzerling strain of phase I C. burnetii, received the approval of the Australian authorities in
1989 (28). Phase I vaccines are more effective, but vaccination is contraindicated for individuals who had
seroconverted or had been exposed to C. burnetii prior to immunisation.
Several vaccines have been developed against animal Q fever. Results converge today towards the use of a
phase I vaccine, as the phase II vaccines are 100 times less effective against the colonisation of mouse spleen
than phase I vaccines (13). An inactivated phase I vaccine is commercially available in Slovakia for vaccination
of cattle. A review on Q fever in Slovakia suggests that the decrease in the occurrence of human and animal Q
fever could be the result of the large-scale vaccination of cattle that was carried out there during 10 years,
together with improved veterinary control of domestic animal transport within the country (46).
This vaccine consists of highly purified antigen prepared from Nine Mile strain in the phase I (egg passage 2 to
egg passage 6) and inactivated by formaldehyde. Recently, a French study demonstrated the efficacy of this
vaccine through experimental vaccination and challenge of pregnant goats: the vaccine prevented abortion and
shedding in milk, and decreased considerably the shedding in the vaginal secretions and faeces (3). Ideally,
vaccine efficacy must be demonstrated by tests on all the target species.
In the case of vaccination on already infected animals, there is a lack of information on possible adverse effects
and on the shedding of the Q fever agent. Consequently, some authors believe that it is preferable to select
seronegative herds or animals for immunisation, and to continue vaccination over several years in young animals
(24). To date, no data are available for comparing the cost–benefit of this strategy with a nonselective strategy in
the control of Q fever.
2. Diagnostic biologicals
See Section B.2.a (Antigen preparation).
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*
* *

CHAPTER 2.1.13.
RABIES
SUMMARY
Rabies is a major zoonosis for which diagnostic techniques have been standardised inter-
nationally. As there is no gross pathognomonic lesion for rabies, diagnosis can only be made in the
laboratory. Laboratory techniques are preferably conducted on central nervous system (CNS)
tissue removed from the cranium. A composite of CNS samples should be tested and the brain
stem is the most important component of the sample.
Identification of the agent: Agent identification is preferably done using the fluorescent antibody
test (FAT). A drop of purified immunoglobulin previously conjugated with fluorescein isothiocyanate
is added to an acetone-fixed brain tissue smear, preferably made from several parts of the brain
stem. FAT provides a reliable diagnosis in 98–100% of cases for all serotypes if a potent conjugate
is use. For a large number of samples, as in an epidemiological survey, polymerase chain reaction
(PCR) or immunoenzyme techniques can provide rapid results.
Infected neuronal cells have been demonstrated by histological tests and these procedures will
reveal aggregates of viral material (the Negri bodies) in the cytoplasm of neurones. However, the
sensitivity of histological techniques is much less than that of immunological methods, especially if
there has been some autolysis of the specimen. Consequently, histological techniques can no
longer be recommended.
As a single negative test on fresh material does not rule out the possibility of infection, cell culture
or mouse inoculation tests should be carried out simultaneously. A monolayer culture of
susceptible cells is inoculated with a pool of several CNS tissues, including the brain stem. FAT
carried out after appropriate incubation will demonstrate the presence or absence of viral antigen.
Alternatively, newborn or 3–4-week-old mice may be inoculated intracerebrally with a similar pool
tissues and then kept under observation for 28 days. For any mouse that dies between 5 and
28 days post-inoculation, the cause of death should be confirmed by FAT. Wherever possible,
virus isolation in cell culture should replace mouse inoculation tests.
The identification of the agent can be supplemented in specialised laboratories by identifying any
variant virus strains through the use of monoclonal antibodies, specific nucleic acid probes, or the
polymerase chain reaction followed by DNA sequencing of genomic areas. Such techniques can
distinguish between field and vaccine strains, and possibly identify the geographical origin of the
field strains. These very sensitive tests should be used by well trained personnel in specialised
laboratories.
Serological tests: Virus neutralisation (VN) assays in cell cultures are the prescribed tests for
international trade. Alternatively, use may be made of a test that is known to correlate with these,
notably an enzyme-linked immunosorbent assay using antibody to the G protein or the
neutralisation test in mice. Results are expressed in International Units or equivalent units relative
to an international standard antiserum.
Requirements for vaccines: Rabies vaccines for use in animals contain either live virus
attenuated for the target species (such as Flury low egg passage, Flury high egg passage, Street-
Alabama-Dufferin or Kelev), or virus inactivated by chemical or physical means, or recombinant
vaccines. The virus is cultivated in embryonated egg, or in cell cultures.
Rabies vaccines are usually lyophilised, but inactivated virus vaccines, preferably with an adjuvant,
may be stored in liquid form.
Before newly developed vaccines can be licensed, the duration of immunity resulting from their use
should be determined in vaccinated animals of the target species. Vaccines should confer
protective immunity for at least 1 year.

Chapter 2.1.13. — Rabies
For live virus vaccines, the minimum virus content that will elicit an adequate immune response
must be established.
The potency of inactivated virus vaccines is established and controlled by mouse vaccination
followed by intracerebral challenge using tests formulated by the United States Department of
Agriculture in the United States of America or the European Pharmacopoeia elsewhere. The final
products of both types of vaccine are subjected to tests for innocuity and absence of toxicity.
For live vaccines that are prepared for oral vaccination of wild (or domestic) animals, safety and
efficacy in target animals and safety in nontarget species must be demonstrated.
A. INTRODUCTION
Rabies is caused by a neurotropic virus of the genus Lyssavirus of the family Rhabdoviridae, and is transmissible
to all mammals. As it is transmissible to humans by inoculation or inhalation of infectious virus, all suspected
infected material must be handled under the appropriate safety conditions specified by the World Health
Organisation (WHO) (45).
Seven distinct genetic lineages can be distinguished within the genus Lyssavirus by cross-protection tests and
molecular biological analysis (6, 16, 26), namely the classical rabies virus itself (RABV, genotype 1, serotype 1),
Lagos bat virus (LBV, genotype 2, serotype 2), Mokola virus (MOKV, genotype 3, serotype 3), and Duvenhage
virus (DUVV, genotype 4, serotype 4). The European bat lyssaviruses (EBLV), subdivided into two biotypes
(EBLV1, genotype 5 and EBLV2, genotype 6) and the Australian bat lyssavirus (ABLV, genotype 7), isolated in
Australia (30), are also members of the Lyssavirus genus, but are not yet classified into serotypes. Viruses of
serotypes 2–4, EBLV and ABLV are known as rabies-related viruses. The use of monoclonal antibodies (MAbs)
directed against viral nucleocapsid or glycoprotein antigens, and the sequencing of defined genomic areas has
made possible the definition of numerous subtypes within each serotype. Lyssaviruses cause a clinical disease
indistinguishable from classical rabies. Conserved antigenic sites on the nucleocapsid proteins permit recognition
of all lyssaviruses with modern commercial preparations of anti-rabies antibody conjugates used for diagnostic
tests on brain tissue. There exist two lyssavirus phylogroups with distinct pathogenicity and immunogenicity (5).
For RABV, DUVV, EBLV and ABLV, conserved antigenic sites on the surface glycoproteins allow cross-
neutralisation and cross-protective immunity to be elicited by rabies vaccination. Little or no cross-protection
against infection with MOKV or LBV is elicited by rabies vaccination and most anti-rabies virus antisera do not
neutralise these lyssaviruses. Four new rabies-related viruses (Aravan, Khujand, Irkut, and West Caucasian bat
viruses) have been isolated recently from Eurasian bats, and are described as new putative lyssavirus species.
There is a reduced protection with pre-exposure vaccination and with conventional rabies post-exposure
prophylaxis against all four new bat variants of rabies virus (29).
Humans working with suspect material must be vaccinated against lyssaviruses or other pathogens that may be
present in diagnostic samples. The laboratory must comply with national biocontainment and biosafety
regulations to protect staff from contact with pathogens; it should also comply with the guidelines in Chapter 1.1.2
Biosafety and biosecurity in the veterinary microbiological laboratory and animal facilities.
WHO recommends the preventive immunisation of exposed staff. The immunisation protocol includes three
injections, e.g. at days 0, 7, and 28. The serological evaluation of immunisation is made 1–3 weeks after the last
injection, and checked every 6 months in the case of laboratory workers or every 2 years for other diagnosticians.
Booster vaccination must be given when the titre falls below 0.5 International Units (IU) per ml. In the absence of
serological monitoring, the vaccination regimen should consist of a booster vaccination at 1 year and thereafter
every 1–3 years.
As no clinical sign or gross post-mortem lesion can be considered pathognomonic in domestic or wild animals,
the diagnosis of rabies has to rely on laboratory testing. Serological evidence of infection is rarely useful because
of late seroconversion and the high mortality rate of host species, although such data may be used in some
epidemiological surveys.
Clinical observation may only lead to a suspicion of rabies because signs of the disease are not characteristic
and may vary greatly from one animal to another (43). The only way to perform a reliable diagnosis of rabies is to
identify the virus or some of its specific components using laboratory tests.
As rabies virus is rapidly inactivated, refrigerated diagnostic specimens should be sent to the laboratory by the
fastest means available. Shipment conditions must be considered to be part of the ‘rabies diagnostic chain’.

Several laboratory techniques may be used, and have been detailed and standardised in the fourth edition of the
WHO’s Laboratory Techniques in Rabies (45). The methods vary in their efficiency, specificity and reliability.
They are classically applied to brain tissue, but they can also be applied, though less effectively, to other organs
(e.g. salivary glands). In the brain, rabies virus is particularly abundant in the thalamus, pons and medulla. The
hippocampus (Ammon’s horn), cerebellum and different parts of the cerebrum have been reported to be negative
in 3.9–11.1% of the positive brains. The structure of choice is the thalamus as it was positive in all cases. It is
recommended that a pool of brain tissues that includes the brain stem should be collected and tested (13). To
reach these parts of the brain, it is necessary to remove the entire organ after having opened the skull in a
necropsy room. Under some conditions (e.g. in the field or when sampling for large epidemiological studies), a
simplified method of sampling through the occipital foramen (11), or through the orbital cavity (32), can be used.
Precautions should be taken when handling central nervous system tissues from suspected rabies cases. Gloves
should always be worn and precautions must be taken to prevent aerosols. The use of cutting tools, scissors and
scalpels, should be used with care to prevent injury and contamination.
a) Shipment of samples
During the shipment of suspect material for diagnosis (animal heads, brain or other tissue samples), no risk
of human contamination should arise: brains must be placed in a leak-proof rigid container (animal heads
will be wrapped in absorbent material) as prescribed in the International Air Transport Association (IATA)
Dangerous Goods Regulations must be followed. These regulations are summarised in Chapter 1.1.1
Collection and shipment of diagnostic specimens.
When it is not possible to send refrigerated samples, other preservation techniques may be used. The
choice of the preservative is closely linked to the tests to be used for diagnosis:
i) Formalin inactivates the virus, thus the isolation tests cannot be used and diagnosis depends on using
a modified and less sensitive direct fluorescent antibody test (FAT), immunohistochemistry or
histology (39, 45);
ii) Infectivity at room temperature may be extended for several days if brain material is kept in a mixture
of 50% glycerol in phosphate buffered saline (PBS). Glycerol/PBS slows bacterial action and therefore
protects against the chemical and biological effects of putrefaction. It does not protect against titre
decline due to thermal conditions and therefore, because rabies is thermo-labile, the virus titre will
decline during glycerol/PBS storage. Under normal transport conditions in the tropics, this protection
may only be effective for a matter of several days. Therefore, whenever possible samples in
glycerol/saline should be kept refrigerated. As the virus is not inactivated by glycerol/PBS, all
laboratory tests can be used on these samples.
b) Collection of samples
Usually the brain is collected following the opening of the skull in a necropsy room, and the appropriate
samples are collected. This step may be hazardous if laboratory technicians are not fully trained, or under
field conditions. In such cases, there are two possible methods of collecting some brain samples without
opening the skull:
i) Occipital foramen route for brain sampling

A 5 mm drinking straw (11) or a 2 ml disposable plastic pipette (17) is introduced into the occipital foramen
in the direction of an eye. Samples can be collected from the rachidian bulb, the base of the cerebellum,
hippocampus, cortex, and medulla oblongata. Bovine spongiform encephalopathy (BSE) should be
considered in the differential diagnosis of most cattle that are considered to be ‘rabies suspect’. Sampling of
brain specimens for both diseases can be done using the ‘brain scoop or tool’ developed for BSE tissue
sampling rather than a straw or pipette. The resulting samples are relatively easily recognised as to the
area of brain sampled.
ii) Retro-orbital route for brain sampling

In this technique (32), a trocar is used to make a hole in the posterior wall of the eye socket, and a plastic
pipette is then introduced through this hole. The sampled parts of the brain are the same as in the former
technique, but they are taken in the opposite direction.
c) Routine laboratory tests

Laboratory diagnosis can be performed by using three kinds of procedure.
i) Immunochemical identification of rabies virus antigen

• Fluorescent antibody test
The most widely used test for rabies diagnosis is the FAT, which is recommended by both WHO and
OIE. This test may be used directly on a smear, and can also be used to confirm the presence of
rabies antigen in cell culture or in brain tissue of mice that have been inoculated for diagnosis. The
FAT gives reliable results on fresh specimens within a few hours in more than 95–99% of cases. The

sensitivity of the FAT depends on the specimen (the degree of autolysis and how comprehensively the
brain is sampled, see Section B.1) (1, 9), on the type of lyssavirus and on the proficiency of the
diagnostic staff. Sensitivity may be lower in samples from vaccinated animals due to localisation of
antigen, which is confined to the brainstem. For direct rabies diagnosis, smears prepared from a
composite sample of brain tissue, that includes the brain stem, are fixed in high-grade cold acetone
and then stained with a drop of specific conjugate. Anti-rabies fluorescent conjugates may be
prepared in the laboratory. Those available commercially are either polyclonal conjugates specific to
the entire virus or specific to the rabies nucleocapisid protein, or they may be prepared from a mix of
different MAbs. In the FAT, the specific aggregates of nucleocapsid protein are identified by their
fluorescence. The specificity and sensitivity of these anti-rabies fluorescent conjugates for locally
predominant virus variants should be checked before use.
The FAT may be applied to glycerol-preserved specimens. If the specimen has been preserved in a
formalin solution, the FAT may be used only after the specimen has been treated with a proteolytic
enzyme (7, 8, 38, 39). However, the FAT on formalin-fixed and digested samples is always less
reliable and more cumbersome than when performed on fresh tissue.
• Immunochemical tests
The antibody may be conjugated to an enzyme such as peroxidase instead of fluorescein
isothiocyanate (FITC). This conjugate may be used for direct diagnosis with the same sensitivity as
FAT (27), but attention should be paid to the risk of nonspecific false-positive results. This risk is
considerably reduced by the thorough training of the technicians. It must also be emphasised that this
technique needs one incubation step more than the FAT.
Peroxidase conjugate may be used on sections of formalin-fixed tissue for immunohistochemical tests.
ii) Detection of the replication of rabies virus after inoculation
These tests detect the infectivity of a tissue suspension in cell cultures or in laboratory animals. They should
be used if the FAT gives an uncertain result or when the FAT is negative in the case of known human
exposure.
• Cell culture test

Neuroblastoma cell lines, e.g. CCL-131 in the American Type Culture Collection (ATCC) 1, is used for
routine diagnosis of rabies. The cells are grown in Dulbecco’s modified Eagle’s medium (DMEM) with
5% foetal calf serum (FCS), incubated at 36°C with 5% CO2. Its sensitivity has been compared with
that of baby hamster kidney (BHK-21) cells (34). This cell line is sensitive to street isolates without any
adaptation step, but should be checked for susceptibility to locally predominant virus variants before
use. Presence of rabies virus in the cells is revealed by the FAT. The result of the test is obtained after
at least 18 hours (one replication cycle of virus in the cells); generally incubation continues for
48 hours (10) or in some laboratories up to 4 days.
This test is as sensitive as the mouse inoculation test. Once a cell culture unit exists in the laboratory,
this test should replace the mouse inoculation test as it avoids the use of live animals, is less
expensive and gives more rapid results.
It is often advisable to carry out more than one type of test on each sample, particularly when there
has been human exposure.
• Mouse inoculation test

Five-to-ten mice, 3–4 weeks old (12–14 g), or a litter of 2-day-old newborn mice, are inoculated
intracerebrally. The inoculum is the clarified supernatant of a 20% (w/v) homogenate of brain material
(cortex, Ammon’s horn, cerebellum, medulla oblongata) in an isotonic buffered solution containing
antibiotics. To reduce animal pain, mice should be anaesthetised when inoculated. The young adult
mice are observed daily for 28 days, and every dead mouse is examined for rabies using the FAT. For
faster results in newborn mice, it is possible to check one baby mouse by FAT on days 5, 7, 9 and 11
post-inoculation. Any deaths occurring during the first 4 days are regarded as nonspecific (due to
stress/bacterial infection etc.).
This in-vivo test should be avoided when possible on animal welfare grounds. It is also expensive,
particularly if SPF mice are used, and does not give rapid results (compared with in-vitro inoculation
tests), but when the test is positive, a large amount of virus can be isolated from a single mouse brain
for strain identification purposes. Another advantage of this low-tech test is that it can be easily and
practicably applied in situations where skills and facilities for other tests (e.g. cell culture) are not
available.
1 American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Virginia 20108, United States of America . (USA).

iii) Histological identification of characteristic cell lesions

Negri bodies correspond to the aggregation of viral proteins, but the classical staining techniques detect
only an affinity of these structures for acidophilic stains. Immunohistochemical tests are the only histological
test specific to rabies.
An unfixed tissue smear may be stained by the Seller’s method, diagnosis is then obtained in under 1 hour.
Generally, histological tests, such as Mann’s test, are performed on fixed material after a paraffin-
embedding step, and the result of the test is obtained within 3 days. These techniques have the advantage
that the laboratory equipment needed to perform them is inexpensive and any need to keep specimens cold
after fixation is avoided. Whichever staining method is used, the evidence of infection is provided by
intracytoplasmic acidophilic bodies. These histological methods, especially the Seller’s method, can no
longer be recommended because they have very low sensitivity and should be abandoned.
d) Other identification tests

An enzyme-linked immunosorbent assay (ELISA) that detects rabies antigen is a variation of the
immunochemical test. It is useful for large epidemiological surveys (46). It should only be used after
validation against numerous samples in different laboratories. The specificity and sensitivity of these anti-
rabies enzyme conjugates for locally predominant virus variants should be checked before use. This test
should be used in combination with confirmatory tests by FAT or virus isolation.
The tests above describe methods to accurately diagnose rabies and to isolate and identify the virus.
Typing of the virus can provide useful epidemiological information and should be carried out in specialised
laboratories (such as OIE or WHO Reference Laboratories). These techniques would include the use of
MAbs, nucleic acid probes, or the polymerase chain reaction (PCR), followed by DNA sequencing of
genomic areas for typing the virus (17). This characterisation enables a distinction to be made between
vaccine virus and a field strain of virus, and possibly the geographical origin of the latter.
Serological tests are rarely used in epidemiological surveys, due to late seroconversion and the low percentage
of animals surviving the disease and therefore having post-infection antibodies. The main application of serology
is to determine responses to vaccination, either in domestic animals prior to international travel, or in wildlife
populations following oral immunisation of rabies reservoirs. For follow-up investigations in oral vaccination
campaigns, virus neutralisation (VN) tests in cell culture are preferred. However, if poor quality sera are
submitted, the VN tests in cell culture are sensitive to cytotoxicity, which could lead to false-positive results. For
such samples, the use of an indirect ELISA with rabies glycoprotein-coated plates has been shown to be as
sensitive and specific as the VN test on cells (22).
a) Virus neutralisation test in cell culture: fluorescent antibody virus neutralisation test (a prescribed
test for international trade)
The principle of the fluorescent antibody virus neutralisation (FAVN) test (21) is the neutralisation in vitro of
a constant amount of rabies virus (‘challenge virus standard’ [CVS] strain adapted to cell culture) before
inoculating cells susceptible to rabies virus: BHK-21 C13 cells (ATCC number: CCL-10).
The serum titre is the dilution at which 100% of the virus is neutralised in 50% of the wells. This titre is
expressed in IU/ml by comparing it with the neutralising dilution of the OIE serum of dog origin under the
2
same experimental conditions. The WHO standard for rabies immunoglobulin [human] No. 2, or an internal
control calibrated against the international control may be used. The WHO standard or internal control
should only be used as a control in the test and should not be used to calculate the IU/ml titre of the sera.
This microplate method uses 96-well plates, and is an adaptation of the technique of Smith et al. (36),
modified by Zalan et al. (47) and by Perrin et al. (33). Several publications (18, 21) have shown that the
FAVN test and the rapid fluorescent focus inhibition test (RFFIT) give equivalent results.
• Essential equipment
Humidified incubator at 35°C/37°C with 5% CO2; dry incubator at 37°C; biocontainment cabinet;
fluorescence microscope suitable for FITC fluorescence equipped with ×10 eye-piece and ×10 objective.
The global magnification of the microscope ranges between ×100 and ×125 due to the extra magnification
of some epi-fluorescence systems.
2 National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire
EN6 3QG, United Kingdom (UK).

• Reagents and biologicals

PBS buffer, pH 7.2, without Ca2+ and Mg2+, stored at 4°C;
Trypsin ethylene diamine tetra-acetic acid (EDTA);
High-grade acetone 80% (diluted with deionised water), stored at 4°C;
Dulbecco’s modified Eagle’s medium (DMEM) + 10% heat-inactivated FCS;
FITC anti-rabies conjugate;
Cells: BHK-21 C13 (ATCC CCL-10);
Virus: CVS-11 (ATCC VR 959) strain, which is available from the ATCC or the OIE Reference Laboratory
for Rabies, Nancy, France (see Table given in Part 3 of this Terrestrial Manual). Vials are stored at –80°C;
OIE Standard Serum of dog origin (OIE Reference Laboratory for Rabies, Nancy, France [see Table given
in Part 3 of this Terrestrial Manual] stored at +4°C and diluted to 0.5 IU/ml with sterile deionised or distilled
water according to the titre of the batch).
Naive serum: The pool of negative dog sera is stored at –20°C.
CVS production
i) Cell growth: the BHK-21 C13 cells (ATCC CCL-10) used to produce the CVS virus (ATCC VR 959
CVS-11) are trypsinised during the rapid growth phase, i.e. cells are in the exponential phase of their
kinetic growth. If the confluence of the layer is complete, a new passage should be made. The cells in
the cell suspension should not be aggregated; 2 × 107 cells are used for a 75 cm2 cell culture flask.
Cells are collected within a volume of 20–30 ml in cell culture medium with 10% heat-inactivated FCS.
ii) Infection of cells: the multiplicity of infection (number of infective particles per cell) is adjusted to
between 0.2 and 0.5. The glass bottle containing the virus/cell suspension is incubated for 60 minutes
at 35–37°C. The contents of the bottle are gently stirred every 10–15 minutes.
iii) Virus growth: the virus/cell suspension is then centrifuged at 800-1000 g for 15 minutes and the cell
pellet is resuspended in cell culture medium mixed with 10% heat-inactivated FCS. Virus is harvested
2 days later.
iv) Harvest and storage: the supernatant is centrifuged at 800–1000 g for 15 minutes at 4°C. If several
flasks have been used, the different centrifuged supernatants are mixed and then aliquoted and frozen
at –80°C. The infective titre of the harvest is established at least 3 days after freezing.
Titration of virus in TCID50 (50% tissue culture infective dose)

This titration method uses BHK-21 C13 cells (ATCC CCL-10) in microtitre plates.
Different steps in this procedure may be adapted according to the safety requirements and to the working
practices of the laboratory, but the following should not be changed:
inoculation of a 24-hour cell layer,
tenfold dilutions prepared using 0.9 ml of diluent and 0.1 ml of virus suspension,
six 50 µl replicates per dilution,
incubation for 72 hours,
qualitative reading (i.e. the well is positive or negative),
in every titration session, a vial of a control batch of virus is titrated and this titre is integrated in a
control card to validate the titration process,
calculation according to neoprobit graphic or Spearman–Kärber methods.
i) Cell suspension: the day before titration, a cell suspension containing 105 cells/ml is prepared in cell
culture medium containing 10% heat-inactivated FCS, and is distributed, 200 µl per well, into 96-well
microtitre plates. The plates are then incubated for 24 hours at 35–37°C with 5% CO2.
ii) Dilution of the virus: the serial dilutions are performed in 5 ml tubes using a cell culture medium
without FCS as diluent. Tenfold dilutions from 10–1 to 10–12 are prepared (0.9 ml of diluent with 0.1 ml
of the previous dilution).
iii) Infection of the cells: the medium in the microtitre plates is discarded using an aspiration system. Fifty
µl of each virus dilution is distributed per well. Six replicates are used per dilution. The microtitre plate
is then incubated for 1 hour at 35–37°C with 5% CO2. Then 200 µl of cell culture medium, containing
5% FCS, is added.
iv) Incubation: incubate for 3 days at 35–37°C in 5% CO2.

v) Staining and calculation of titre: The cells are stained using the FAT, as detailed below. Reading is
qualitative, every well that shows specific fluorescence is considered to be positive. The titre
calculation is made using either the neoprobit graphic method (2) or the Spearman–Kärber formula.
vi) The CVS titration must be performed by FAVN test to establish the infective dose in TCID50.
Fig. 1. Proposed use of microplates for the fluorescent antibody virus neutralisation test. Wells to which undiluted
sera must be added are filled with the indicated ‘50 μl’. Wells to which 50 μl of diluted challenge virus standard
must be added are shaded. Dilutions are given in log10.
Plate 1: Controls
H G F E D C B A
Challenge virus 1
standard 2
titration 3
4
OIE 50 µl 50 µl 5 Naive dog
standard serum 50 µl 50 µl 6 serum
(0.5 IU/ml) 50 µl 50 µl 7 (negative)

50 µl 50 µl 8
50 µl 9
Serum or internal 50 µl 10
positive control 50 µl 11
or WHO standard 50 µl 12
serum
log (dilution) 0.48 0.95 1.43 1.91 2.39 2.87 CVS Cells
virus control
control
Plate 2: Sera to be tested
log dilution 0.48 0.95 1.43 1.91 2.39 4.23 0.48 0.95 1.43 1.91 2.39 4.23
1 2 3 4 5 6 7 8 9 10 11 12
A 50 µl 50 µl
Serum 1 B 50 µl 50 µl Serum 3
C 50 µl 50 µl
D 50 µl 50 µl
E 50 µl 50 µl
Serum 2 F 50 µl 50 µl Serum 4
G 50 µl 50 µl
H 50 µl 50 µl
• Test procedure
i) The microplates are used according to the pattern shown in Figure 1. Plate No. 1 is used for the
titration of CVS (rows 1 to 4), and for the controls, standard sera and naive dog serum are used. All
other plates are used for the sera to be tested.
ii) Medium is added to the wells as follows: plate 1, rows 1 to 4 and cells A9 to A12: add 150 µl per well;
in the other plates, rows 6 and 12: add 200 µl per well; all other wells: add 100 µl.
iii) Sera to be tested are heat inactivated for 30 minutes at 56°C. As indicated in Figure 1, 50 µl of each
undiluted serum to be tested is added to four adjacent wells.
iv) Dilutions of sera are conducted in the microplates as follows:

OIE serum, the internal control or WHO standard serum, and the naive dog serum: with a 50–200 µl
multichannel pipette, mix the first dilution wells by sucking in and out at least eight times, transfer 50 µl
from one row to the next one, until the last one is reached. Discard 50 µl from the last row.
If there is a serum to be tested on the control plate, see below for the dilution step.
A minimum of four three-fold dilutions is required.
Sera being tested (all plates): as above, transfer successively 50 µl from one row to the following one
until rows 5 and 11 (dil. 10–2.39). With a 5–50 µl multichannel pipette, transfer 10 µl from rows 5 and 11
to rows 6 and 12, respectively (from dil. 10–2.39 to dil. 10–4.23). Using a multichannel pipette adjusted to
90 µl, mix rows 6 and 12 and discard 180 µl. Then add 70 µl of medium to these rows. This final step
does not lend itself to high throughput testing. To attain or exceed the recommended final dilution
alternative procedures may be used. These may require modifications to the plate layout.
• Addition of challenge virus standard

i) Stock CVS is stored in 1 ml microtubes at –80°C. One tube is thawed rapidly under cold running
water, and placed in melting ice.
ii) One dilution from this tube is prepared in order to obtain 100 TCID50 in 50 µl. Of this dilution, 50 µl is
added to each serum-filled well (see Figure 1). For virus titration, 50 µl is added to wells H1 to H4
(plate 1). Next, transfer 50 µl from row to row (plate 1, lines 1–4). Discard 50 µl from the last row (plate
1, wells A1 to A4). No virus is added to wells A9 to A12 of plate 1 (controls). The range allowed for the
virus dose titre must be between 30 and 300 TCID50/50 µl.
iii) Incubate the microplates at 35°C/37°C in a humid incubator with 5% CO2 for 1 hour.
iv) Addition of cells: trypsinise a subconfluent culture of 3-day-old BHK-21 cells. Resuspend the cells to
obtain a 4 × 105 cells/ml suspension in DMEM supplemented with 10% heat-inactivated FCS. Add
50 µl of the cell suspension to each well.
v) Incubate the microplates for approximately 48 hours at 35–37°C in a humid incubator with 5% CO2.
• Fixation and staining

i) After the 48-hour incubation period, the medium is discarded, and the microplates are rinsed once in
PBS, pH 7.2, and once in 80% acetone. The microplates are then fixed in 80% acetone at room
temperature for 30 minutes, and are dried at room temperature for at least 30 minutes.
ii) Add 50 µl of the FITC anti-rabies conjugate, at the working dilution, to each well, gently rock the
microplates and incubate at 35–37°C for 30 minutes. Discard the fluorescent conjugate and rinse the
microplates twice with PBS. Excess PBS is removed by briefly inverting the microplates on absorbent
paper.
• Reading and interpreting the results

i) The total surface of each well is observed. The reading evaluation is qualitative (plus or minus): no
fluorescent cell – a minus score is recorded for the well; fluorescent cells (one cell or more) – a plus
score is recorded for the well.
ii) Cell and virus controls are read first. For titration of CVS, naïve serum, and OIE standard serum, titres
are calculated according to the Spearman–Kärber method or the neoprobit graphic method (2).
iii) Results of titration of CVS (TCID50), naive serum (D50 [median dose]) and positive standard (D50) are
reported on a control card for each of these three controls. The control results of the current test are
compared with the accumulated control test results from previous tests using the same batch of
control. The test is validated if the values obtained for the three controls in the current test are not
statistically different from the mean (± 2 SD) of all the values obtained in the tests conducted
previously according to this technique.
iv) The result of the test corresponds to the non-neutralised virus after incubation with the reference
serum or with the serum to be tested. These titres are calculated with the neo-probit graphic method
(2) or with the Spearman–Kärber formula (45).The comparison of the measured titre of the tested sera
with that of the OIE positive standard serum of a known neutralising titre allows determination of the
neutralising titre of the tested sera in IU/ml. The conversion to IU/ml can be made by using either the
log D50 value of the day or the mean value of the OIE standard serum.

• Formula to convert the log D50 value in IU/ml titre:
Serum titre (IU/ml) = [(10 (serum log D50 value)) × theoretical titre of OIE serum 0.5 IU/ml]
(10 (log D50 of OIE serum 0.5 IU/ml))
Example of conversion:
• log D50 of the serum = 2.27
• theoretical titre of OIE serum 0.5 IU/ml = 0.5 IU/ml
• log D50 of OIE serum = 1.43
(for the log D50 of OIE, the value of the day or the mean value can be considered)
Serum titre (IU/ml) = [(102.27 × 0.5 = 3.46 IU/ml]
(101.43)
The following parameters have to be strictly respected:
• Rabies virus: only the CVS-11 strain (ATCC number = VR 959 should be used.
• Cells culture: only BHK-21 cells (ATCC number – CCL 10) should be used.
• The FAVN test must be performed only in 96 wells microplate.
• Control charts should be used for rabies virus, naïve serum and OIE standard serum.
• The back titration of the CVS virus, as well as naïve serum and OIE serum, must be present on
control plate.
• A minimum of four three-fold dilutions of sera are required. The reading method is ‘all or nothing’
only.
• Four replicates of each serum should be diluted.
• For the conversion of log D50 in IU/ml, the laboratories should use only the log D50 value of the
OIE standard serum of dog origin.
b) The rapid fluorescent focus inhibition test (RFFIT) for determining rabies virus-neutralising
antibody (a prescribed test for international trade)
Standard procedure (from WHO Laboratory Techniques in Rabies, 1996; [ref. 45])
Preparation of seed virus suspension
i) Trypsinise one 3-day-old 150 ml flask culture of mouse neuroblastoma (MNA) cells. These cells prefer
an acidic medium, supplemented with vitamins (40). A suitable cell line (CCL-131) may be obtained on
request from the ATCC (see footnote 1).
ii) Resuspend 3 × 107 cells in a 50 ml conical centrifuge tube in 2.7 ml of Eagle’s minimal essential
medium supplemented with 10% fetal bovine serum (EMEM-10).
iii) Using standard rabies safety procedures, add 1 × 107 infectious units of CVS-11 rabies virus (ATCC,
VR959) and vortex/mix once. Incubate the cells and virus for 15 minutes at 37°C; vortex/mix the cells
once during this time.
iv) Add 10 ml EMEM-10, vortex/mix, and centrifuge the cells at 500 g for 10 minutes.
v) Discard the supernatant. Resuspend the cells in 30 ml of growth medium and transfer to a 150 ml
flask.
vi) Gently rock the flask to mix the cell suspension, and then prepare three eight-well tissue-culture
chamber slides by pipetting 0.2 ml of the cell suspension into one well of each slide.

vii) Incubate the flask and slides at 37°C in a humidified incubator with 0.5% carbon dioxide (CO2). The
flask should be incubated as a closed culture (tighten the cap).
viii) At 20, 40 and 64 hours after infection, acetone fix and stain one slide using an immunofluorescence
technique (28) to determine the virus infectivity. The supernatant should be harvested 24 hours after
the cells reach 100% infectivity (typically 40 hours after infection).
ix) Transfer the supernatant to a 50 ml centrifuge tube and centrifuge at 4000 g for 10 minutes.
x) Distribute the supernatant into 0.5 ml aliquots and store at –70°C.
Titration of seed virus suspension

i) Thaw one aliquot of the seed virus and prepare serial tenfold dilutions (from 10–1 to 10–8) in EMEM-10.
ii) Distribute 0.1 ml of each virus dilution into one well of an eight-well tissue-culture chamber slide. Add
0.2 ml of MNA cells suspended in EMEM-10 (concentration 5 × 104 cells per 0.2 ml) to each well.
iii) Mix the cells and virus by gently rocking the slide, then incubate at 37°C in a humidified incubator with
0.5% CO2 for 40 hours.
iv) Acetone fix and stain the slide using an immunofluorescence technique. Evidence of virus infection
should be observed at the 10–6 dilution of virus, indicating a virus stock suspension containing at least
1 × 106 infectious units per 0.1 ml. Prepare sufficient seed virus so that frequent serial passage of the
virus is unnecessary.
Preparation of stock virus suspension

i) Infect 3 × 107 MNA cells with 1 × 107 infectious units of the seed virus preparation (see above).
ii) Harvest the supernatant 24 hours after the cells reach 100% infectivity (typically 40 hours after
infection).
iii) Distribute the supernatant into 0.5 ml aliquots and store at –70°C.
Titration of stock virus suspension

i) Thaw one aliquot of the stock virus and use this to prepare serial tenfold dilutions (from 101 to 106)
in EMEM-10.
ii) Distribute 0.1 ml of each virus dilution into one well of an eight-well tissue-culture chamber slide. Add
0.2 ml of MNA cells suspended in EMEM-10 (concentration 1 × 105 cells per 0.2 ml) to each well.
iii) Mix the cells and virus suspension by gently rocking the slide, then incubate at 37°C in a humidified
incubator with 0.5% CO2 for 20 hours.
iv) Acetone fix and stain the slide using an immunofluorescence technique.
Each well of an eight-well tissue-culture chamber slide contains 25–50 distinct microscopic fields when
observed at ×160–200 magnification. One unit of virus for the RFFIT is determined as the dilution at which
50% of the observed microscopic fields contain one or more foci of infected cells (the focus-forming dose,
FFD50). The stock virus suspension should contain at least 1 × 104 FFD50 per 0.1 ml (i.e. the well with cells
infected with the 10–4 dilution of the virus should contain at least one focus of infected cells in 50% of the
observed microscopic fields). A stock virus suspension of this titre can then be diluted to 10–2.3 to obtain a
challenge virus containing 50 FFD50.
Reference sera
A national or international reference serum standard diluted to a potency of 2.0 IU/ml should be included in
each test. The reference serum used at the Centres for Disease Control and Prevention is the first
international standard for rabies immunoglobulin (41), which may be obtained from the NIBSC (see footnote
2). The reference serum should be maintained as frozen aliquots in amounts sufficient for 1 week of tests. A
positive serum control standard diluted to a potency of 0.5 IU/ml and a negative serum control standard with
a potency of <0.1 IU/ml should also be prepared by the laboratory and included in each test.
Test sera
Serum samples should be heated at 56°C for 30 minutes before testing in order to inactivate complement. If
sera are frozen, they should be reheated after thawing. Serial dilutions of test sera may be prepared in an
eight-well tissue-culture chamber slide. Screening dilutions of 1/5 and 1/50 are sufficient for routine
evaluation of vaccination efficacy and may be made as follows:
i) Prepare a 1/2.5 dilution by adding 0.1 ml of inactivated serum and 0.15 ml of EMEM-10 to one of the
slides. Mix by gently rocking the slide.

ii) Transfer 0.05 ml of the 1/2.5 dilution to a second well containing 0.45 ml of EMEM-10. Discard all but
0.1 ml from the well containing the 1/2.5 dilution.
iii) Mix the second well and discard all but 0.1 ml.
iv) Add 0.1 ml of the challenge virus preparation (containing 32–100 FFD50) to all serum dilutions.
v) Mix and incubate at 35°C in a humidified incubator with 0.5% CO2 for 90 minutes.
Addition of cells
i) During the incubation period, trypsinise a stock culture of 3–5-day-old MNA cells.
ii) Resuspend the cells in EMEM-10 to give a final concentration of 1 × 105 cells per 0.2 ml.
iii) Distribute 0.2 ml of the cell suspension into each well of the slide and incubate at 35°C in a humidified
incubator with 0.5% CO2 for a further 20 hours.
Acetone fixation and staining by immunofluorescence

i) After 20 hours, remove the slides from the incubator and pour off the medium into a virucidal solution.
ii) Rinse the slides once in PBS and then fix for 10 minutes at room temperature in cold acetone (–20°C).
iii) Leave the slides to dry for 10 minutes before adding FITC-conjugated anti-rabies serum. The
conjugate may be prepared in EMEM-10 or PBS; there is no need to adsorb the conjugate with tissue
or cells. The working dilution of the conjugate should be determined by titration. The slides should be
stained for 20–30 minutes at 37°C and then rinsed in PBS and distilled water, respectively.
iv) Observe the slides under a fluorescence microscope.
Calculation of virus-neutralising antibody titres

Residual virus is detected using a standard fluorescence microscope. The serum neutralisation end-point
titre is defined as the dilution factor of the highest serum dilution at which 50% of the observed microscopic
fields contain one or more infected cells (i.e. a 97% reduction in the virus inoculum). This value may be
obtained by mathematical interpolation. Alternatively, a 100% neutralisation titre may be determined by
recording the highest serum dilution at which 100% of the challenge inoculum is neutralised and there are
no infected cells in any of the observed fields. For both titration methods, the titre of antibody in the test
serum (in IU/ml) can be obtained by comparison with the titre of the national reference standard included in
each test. It should be noted that it is also valid to perform the RFFIT using BHK-21 cells instead of
neuroblastoma cells. A modified protocol for this has been published (45).
The following parameters have to be strictly adhered to:
• Rabies virus; only the CVS-11 strain (ATCC number VR 959) should be used.
• Cells cultures: only BHK-21 cells (ATCC number CCL10) or MNA cells (ATCC number CCL131)
should be used.
• The test should be performed only on Labteck chamber slides.
• Control charts should be used for rabies virus, naïve serum and OIE standard serum.
• The back titration of the CVS virus, as well as the naïve serum and OIE serum, must be present on
control plate.
• Reading method for the test: each chamber slide should contain 25–50 fields and be observed at
×160–200 magnification.
• A minimum of three-to-five-fold dilutions of sera is required.
• For the conversion of log D50 to IU/ml, only the log D50 value of the OIE standard serum of dog origin
should be used.

c) Virus neutralisation in mice

This method is no longer recommended by either OIE or WHO and should be discontinued.
d) Enzyme-linked immunosorbent assay (a prescribed test for international trade or movement)

Commercial kits are available for indirect ELISA that allow a qualitative detection of rabies antibodies in
individual dog and cat serum samples following vaccination. In accordance with the WHO
recommendations (41), 0.5 IU per ml rabies antibodies is the minimum measurable antibody titre
considered to represent a level of immunity that correlates with the ability to protect against rabies
infection. The ELISA provides a rapid (~ 4 hours) test that does not require handling of live rabies virus, to
determine if vaccinated dogs and cats have sero-converted. The sensitivity and specificity of any kit
used should be determined by comparison with virus neutralisation methods. The ELISA is acceptable
as a Prescribed Test for international movement of dogs or cats provided that a kit is used that
has been validated and adopted on the OIE Register as fit for such purposes (see
http://www.oie.int/vcda/eng/en_VCDA_registre.htm?e1d9)]. Virus neutralisation methods may be used as
confirmatory tests if desired.
ELISA methods are also useful for monitoring of vaccination campaigns in wildlife populations, provided the
kit used has been validated for the wildlife species under study.
C. REQUIREMENTS FOR VACCINES

Rabies vaccines prepared from Pasteur’s original 1885 strain and its derivative strains (Pasteur Virus, Challenge
Virus Standard, Pitman-Moore, etc.), and strains isolated more recently (Flury, Street-Alabama-Dufferin [SAD],
Vnukovo and Kelev), protect against all strains of genotype 1 isolated so far. Conventional rabies virus vaccines
may not provide adequate cross-protection against other lyssaviruses, especially in phylogroup II; there is no
protection provided against Mokola virus (37) and the recently identified West Caucasian Bat Virus (29). Cross
neutralisation using conventional rabies virus vaccines has been demonstrated against two phylogroup I viruses:
EBLV type-1 and EVLV type-2 (20). The principles governing the preparation of inactivated rabies vaccines are
identical whether they are to be used in humans or animals, although an adjuvant may be added to vaccines for
animal use.
Recombinant vaccine (e.g. vaccinia rabies-glycoprotein recombinant) has also proved to be effective (19, 31).
The rabies-glycoprotein recombinant vaccines are not live rabies vaccines. They are prepared by inserting non-
infectious rabies nucleic acid into a vector such as vaccinia or canary pox. Since these do not contain live rabies
virus, animals vaccinated with rabies-glycoprotein recombinant vaccines should not be restricted from entry into
countries that have restrictions on entry of animals vaccinated with live rabies vaccines.
For animals, live and recombinant vaccines are effective by the oral route and can be distributed in baits in order
to immunise wild (or domestic) animals.
Different standards apply to vaccines containing live virus modified by passage in eggs or cell cultures to reduce
its virulence for the target animal, and to vaccines prepared from inactivated virus. Both types of vaccine have
their advantages and disadvantages (6), but they can both be used to immunise animals for periods of between 1
and 3 years. Live attenuated rabies vaccines are not accepted in some countries. They are not to be relied on to
protect previously unvaccinated animals that have been exposed to infection (15). Only in humans has the
efficiency of post-exposure prophylaxis with vaccine alone been proven and even in these cases there is an
additional strong recommendation to administer anti-rabies immunoglobulin.
All handling of the virus during manufacture and testing of vaccines must conform to the strict safety precautions
specified by WHO (43, 45), the OIE (Chapter 1.1.2) and to national guidelines and regulations.
1. Seed management

Any strain belonging to serotype 1, which has been proved to protect against field rabies viruses (currently
found in the country where the vaccine is to be used), is suitable. The strain of virus used should have well-
known biological (e.g. pathogenicity) and antigenic properties (typing by MAbs). If it is to be used as a live
vaccine, the master seed virus must be shown not to cause clinical rabies. At least two animals (preferably
five to six per group) of each of the species for which the vaccine is intended and, so far as possible, any
species that might be in contact with vaccine or vaccinated animals, should be tested. This can be done by

inoculating in or adjacent to a major nerve, a dose equivalent to ten times the intended viral titre in one
dose of the proposed final product. Animals should be observed for at least 90 days for any adverse effect
attributable to the master seed.
A master cell stock of the seed virus should be prepared and kept at or below –70°C. Subculture from this
stock will be used for vaccine production. Virus multiplication is verified by titration during growth of the
seed virus.
Before a vaccine is licensed, evidence of efficacy should be established by the challenge of vaccinated and
control animals of each target species. The challenge should be performed at the end of the period after
vaccination for which the manufacturer claims maintenance of immunity. Antibody kinetics should also be
determined in order to establish the correlation between antibody titre and resistance to challenge.
The efficacy of the produced vaccine is assessed by studies on every target species previously vaccinated
as recommended. Protection at the end of the period of immunity is monitored by a measurement of
specific neutralising antibodies and by challenge with rabies virus. The experimental conditions of this
challenge should mimic the natural conditions of infection. The challenge virus should preferably be
prepared from locally isolated strains. In animals vaccinated with inactivated vaccines, the percentage of
seroconversion and the mean level of antibody allow a good prognosis for survival to challenge (3).
The correlation between potency in the target species and antigenic value as estimated in mice should be
established (see Section C.4.c below).
For the purposes of licensing a vaccine, safety tests should be conducted in the target species. In the case
of live virus vaccines used in oral vaccination campaigns (including recombinant vaccines), safety tests
should also be carried out on those other species that live in the area of vaccination and could become
exposed to the vaccine (6).
Vaccine stability is ascertained by testing batches after prolonged storage, usually 1–2 years. A process of
accelerated ageing, by storage at 37°C for 1 week, is sometimes used. The storage life claimed by the
manufacturer is checked by the national licensing authority. In general, it is 12–18 months for fluid vaccines,
and possibly 24 months for lyophilised vaccines.
Whatever method is adopted, close attention should be paid to the quality of the substrate. Both animals and
eggs should be of SPF origin, and the cell cultures, such as BHK cell lines, should conform to international
standards of sterility and innocuity.
a) In cell cultures
Cultures are infected with cell-culture-adapted strains of rabies virus and incubated at 35–36°C. These may
then be used as live virus vaccines (as in Flury and SAD vaccines), or as inactivated vaccines after the
addition of phenol (Semple vaccine) or some other chemical, such as beta-propiolactone.
Cell culture can also be used to grow the vector viruses (e.g. vaccinia virus) harbouring the gene coding for
the expression of rabies virus glycoprotein (31).
During manufacture, the multiplication of the virus in one of the substrates mentioned above is monitored,
followed by harvesting at the most appropriate time, usually 4–6 days after inoculation of animals, eggs or
cell cultures. The virus harvest is suspended in a buffer solution at a dilution that will provide an optimum
antigenicity of the end-product. If required, the suspension is either inactivated or lyophilised. An adjuvant is
recommended for vaccines prepared from inactivated virus, as well as for other vaccine antigens that may
be incorporated in polyvalent vaccines.
b) In eggs
A modified egg-adapted strain of virus is inoculated into SPF-embryonated chicken eggs, which are then
incubated at 38°C for 5–6 days. The virus is harvested in the form of infective embryo tissues, and is
usually lyophilised and used as a live vaccine. Examples of such vaccines include those that contain the
Flury low egg passage (LEP), or the more desirable high egg passage (HEP) variant strain, which is safer
for some animal species such as the cat.

c) In animals
Nervous tissue vaccines prepared in animals are no longer considered safe or effective, and their use
should be discontinued.
This consists of monitoring virus growth to provide an optimum titre and ensure the absence of undesirable
microbial contamination.
In live virus vaccines, kinetics of virus growth should be established in order to ensure a final titre of virus
correlated to the desired protection in target species.
In inactivated virus vaccines, immunogenic properties of the final product may be evaluated by in-vitro
techniques (e.g. ELISA, agar gel immunodiffusion, antibody-binding tests or infected cell staining). These
evaluations will indicate the best time for harvesting the virus in cell cultures.
4. Batch control
a) Sterility
b) Safety
Safety tests for batches of inactivated virus vaccines are carried out by inoculation of cell culture or
intracerebrally into mice to detect viable virus. A suitable safety test for live rabies vaccines should be
carried out on each lot of vaccine, in the intended host species. At least three, preferably five to six animals
of the intended host species should be given a dose equivalent to ten times the recommended field dose,
by the recommended route of administration. The animals should be observed for 180 days for adverse
reactions attributable to the vaccine (23).
c) Potency/biological activity
The amount of virus present in live attenuated and recombinant vaccines is determined by titration. Once a
correlation has been established between the activity of the vaccine in the target species and virus titres,
virus titrations become reliable indicators of vaccine efficacy. This is carried out using cell cultures or by the
intracerebral inoculation of unweaned mice (in mice it is only possible with a few attenuated viruses).
Recombinant vaccines should be monitored for the expressed rabies protein until assured that expression
stablilty is maintained in the manufacturing process. Titre of the vector can then be used as a reliable
indicator of vaccine efficacy.
For inactivated virus vaccines, correlation between potency in the target species and antigenic value as
estimated in mice provides a reliable indicator of vaccine activity. The potency of the vaccine is established
in the USA by the National Institutes of Health (NIH) test. Elsewhere, the European Pharmacopoeia test is
widely adopted.
Groups of at least ten mice, aged 3–4 weeks, are inoculated with single, decreasing doses of vaccine in
accordance with the European Pharmacopoeia (23), or with two doses, 1-week apart, according to the NIH
test (45). A sufficient number of dilutions of vaccine are compared to estimate the dilution at which 50% of
the mice are protected against intracerebral challenge 14 days later (23, 45).
A WHO international standard vaccine is available (see footnote 2) for calibration of national standards, so
that the results of testing for antigenicity can be expressed in IUs. The test is not valid unless:
i) For both the vaccine to be examined and the standard preparation, the PD50 (50% protective dose)
lies between the largest and smallest doses given to the mice.
ii) The titration of the challenge virus suspension shows that 0.03 ml of the suspension contained
25 mouse intra-cranial LD50 (MIC LD50). The challenge dose should be in the range 12–50 LD50 for a
valid test.
iii) The confidence interval (p = 0.95) for the test should not be less than 25% and not more than 400% of
the estimated potency: statistical analysis should show a significant slope and no significant deviations
from linearity or parallelism of the dose–response lines.

The vaccine passes the test if the estimated potency is not less than 1 IU per dose, in the smallest
prescribed dose.
A simplified test can also be used for the purpose of anticipating which vaccines are likely to be of an
antigenic value ≥1 IU per dose (4). This test used as a screening test is a good way to reduce the number
of mice used in vaccine potency control tests.
Duration of immunity must be established for the product licence in the target species with a defined
vaccination protocol.
d) Stability
The proposed shelf life must be verified by appropriate tests. These experiments include biological and
physico–chemical stability tests, and should be performed on a sufficient number of batches of vaccine
stored under recommended conditions.
The thermostability of live virus vaccines in liquid form is generally poor. For freeze-dried inactivated virus
vaccines, stability is generally granted for 2 years at 4°C.
e) Preservatives
Inactivated virus vaccines may contain preservatives (formalin, merthiolate). The nature and quantity of
these preservatives should comply with national control regulations.
a) Safety
See Section C.4.b.
b) Potency
See Section C.4.c.
6. Oral vaccination
The concept of oral vaccination is unique: as stray or wild animals are out of physical reach, dropping vaccine
baits into their environment is the only way to immunise them. In the 1980s and 1990s, the Veterinary Public
Health Department of WHO organised several meetings of rabies experts to define the requirements for
guaranteeing the safety and efficacy of vaccines both for the target species (red fox, raccoon dog, skunk, dog,
etc.) and nontarget species, namely wild rodents and any other wild and domestic species that might be in
contact with baits or a recently vaccinated animal (42, 44).
Several guidelines have been established for the quality criteria that vaccines have to satisfy before marketing;
the most precise documents are those produced by WHO, the European Pharmacopoeia and the European
Commission (24, 25, 44). Available oral vaccines have been extensively tested by different routes (cerebral,
muscular and oral) in a variety of species: puppies and adults of carnivores, avian species, nonhuman primates,
rodents and immunocompromised mice. Nonhuman primates have been added to this list since the discovery in
1992 that the original SAD Bern strain is highly pathogenic for baboons by the oral route (12).
All vaccines currently used for oral vaccination programmes are either modified live-virus vaccines or live
recombinant vaccines. At the present time, two oral vaccines are recommended by WHO (44): a recombinant
vaccine – VRG vaccine, and a highly attenuated vaccine – SAG2.
The production controls are closely related to the ones used for parenteral vaccines. The major differences
concern three points:
i) Safety of the vaccine for man, target and non target species.
ii) Efficacy of the protection induced by the vaccine.
iii) Monitoring of the impact of oral vaccination campaigns in the field.

a) Safety considerations
For oral vaccination, either attenuated rabies strains or live-recombinant vaccines may be used. The
vaccine should not induce any adverse signs in target and nontarget species. For vaccines used for dog
immunisation, saliva should be checked for the absence of vaccinal virus because of possible contact with
humans.
The attenuated rabies virus-based vaccines must achieve the lowest residual pathogenicity for target and
nontarget species (24); this is of utmost importance in the case of oral vaccination of dogs as dogs are
often in close contact with humans (44).
The recombinant vaccines cannot induce any risk of rabies; the safety controls concern only the possible
residual pathogenicity of the recombined parental virus.
b) Protection induced by the vaccine

The protection induced by the vaccine must be tested not only with the virus itself (to determine the minimal
vaccinating dose) but also with manufactured baits ready to be used in the field. For foxes for instance, the
vaccine should have a minimal titre corresponding to at least ten times the 100% protective dose (obtained
with the same vaccine experimentally by direct oral instillation) (14).
The protection status cannot be then checked by serology only; a virulent challenge with the homologous
street rabies virus is necessary because of the important implication of cell-mediated immunity in response
to oral vaccination (25).
c) Monitoring the impact of oral vaccination

The stability of the vaccine in the field is important. The European Commission stresses the importance of
checking the 100% protective dose after 7 days of exposure at 25°C (24). Each vaccine bait should be
tested for stability with a melting point above 40°C, and the blister or sachet containing the vaccine should
still be covered by the bait casing after 7 days exposure at 40°C (24).
Aerial distribution of baits is the only way to perform an homogenous, rapid and sufficient distribution for
wildlife vaccination. Quality control measures should be used to monitor different key points of baiting;
control of vaccine titre, control of area coverage by air and of baiting density should at least be constantly
monitored. The cross border cooperation between neighbouring countries is also needed to avoid any
unvaccinated area along the border.
For wildlife in Europe, two campaigns are performed yearly: the spring one aims at vaccinating the young
population of the target species, its period should then be fixed according to the biology of the target
species. The autumn campaign concerns both adult and young animals. It is generally admitted that four
campaigns (i.e. 2 years) should be conducted after the last rabies diagnosis.
The impact of vaccination on the host/vector population is monitored in two different ways:
• Directly by measuring the bait uptake by the wild target species. This supposes that a biomarker
(generally tetracycline) is included in the bait casing. The same examination allows the age of animals
to be determined.
• Directly by measuring the serological response of target animals. This serological control is better
done using validated ELISA techniques (22, 35) as they are more robust than seroneutralisation tests
when testing poor quality field specimens.
• Indirectly by measuring the incidence of rabies in the vaccinated area. Typing of field isolates should
be performed (44) either by using MAbs or by sequencing positive samples from areas where the
target species have been vaccinated with attenuated vaccines to possibly distinguish vaccine and field
virus strains.
The first two controls should be performed on specially killed animals to collect good quality samples.
Rabies monitoring is more sensitive when performed in found dead or ill animals.
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*
* *
NB: There are OIE Reference Laboratories for Rabies (see Table in Part 3 of this Terrestrial Manual or consult
the OIE Web site for the most up-to-date list: www.oie.int).

CHAPTER 2.1.14.
RIFT VALLEY FEVER
SUMMARY
Rift Valley fever (RVF) is a peracute or acute zoonotic disease of domestic ruminants in Africa. It is
caused by a single serotype of a mosquito-borne bunyavirus of the genus Phlebovirus. The
disease occurs in climatic conditions favouring the breeding of mosquito vectors and is
characterised by liver damage. The disease is most severe in sheep, goats and cattle, in which it
produces abortions in pregnant animals and a high mortality rate in the newborn. Older
nonpregnant animals, although susceptible to infection, are more resistant to clinical disease.
There is considerable variation in the susceptibility to RVF of animals of different breeds. Those
breeds or strains that are exotic to Africa or are from areas where RVF is not endemic, tend to be
more susceptible. Camels suffer an inapparent infection with RVF, but abortion rates can be as
high as in cattle. Among ruminant game, buffalo also abort during an inapparent RVF infection.
Humans are susceptible to infection through contact with infected material or mosquito bites.
Infection of humans by vectors is a striking feature in countries with a relatively small population of
animal hosts. In such areas, RVF may be recognised first in humans. It has caused serious
disease in laboratory workers and must be handled with high level biosecurity. It is recommended
that laboratory workers be vaccinated.
Identification of the agent: RVF virus consists of a single serotype of a bunyavirus of the genus
Phlebovirus and has morphological and physicochemical properties typical of bunyaviruses.
The virus can be isolated from blood, preferably collected in an anticoagulant, during the febrile
stage of the disease, or from liver, spleen and brain tissues of animals that have died and from the
organs of aborted fetuses. Primary isolations are usually made on cell cultures of various types,
such as African green monkey kidney (Vero) cells, baby hamster kidney cells, chicken embryo
reticulum, or primary cells of sheep or cattle origin. Alternatively, hamsters, adult or suckling mice,
embryonated chicken eggs or 2-day-old lambs may be used for primary virus isolation.
A rapid diagnosis can be achieved by using the supernatant of homogenised samples as antigen
in virus neutralisation (VN) tests; immunofluorescent staining of impression smears of liver, spleen,
brain or infected cell cultures; or by the demonstration of virus in serum, taken during the febrile
stage of the disease, by enzyme immunoassay or immunodiffusion.
The presence of characteristic histopathological lesions in the liver assists in the diagnosis.
Serological tests: Infected animals develop specific antibodies that may become demonstrable by
VN as early as 3 days following infection and after 6–7 days by enzyme-linked immunosorbent
assay, and by haemagglutination inhibition. Serological tests used less often include
immunofluorescence, complement fixation and immunodiffusion.
Requirements for vaccines and diagnostic biologicals: Live virus vaccines and antigens for
use either in countries where RVF is endemic or during outbreaks, should be prepared from
nonpathogenic mouse- or mutagen-attenuated strains of RVF virus grown in cell cultures. The
mutagen-attenuated strain of RVF is not yet at a stage where it can be recommended for use.
In RVF-free countries, vaccines and diagnostic tests should be limited to those using inactivated
virus. Suitable virus strains can be obtained from the OIE Reference Laboratory for RVF (see
Table given in Part 3 of this Terrestrial Manual).

Chapter 2.1.14. — Rift Valley fever
A. INTRODUCTION
RVF virus consists of a single serotype of a bunyavirus of the genus Phlebovirus and has morphological and
physicochemical properties typical of bunyaviruses. The virus is enveloped, spherical and 80–120 nm in
diameter. Short glycoprotein spikes project through a bilayered lipid envelope and the virus is readily inactivated
by lipid solvents and acid conditions below pH 6. The virus has a three-segmented, single-stranded, negative-
sense RNA genome. and consists of the three segments: L (large), M (medium) and S (small), each of which is
contained in a separate nucleocapsid within the virion. The S segment is ambisense RNA, i.e. has bi-directional
coding (12).
Rift Valley fever (RVF) is a peracute or acute, febrile, mosquito-borne, zoonotic disease caused by a virus of the
family Bunyaviridae, genus Phlebovirus. It usually presents in epizootic form over large areas of a country
following heavy rains and flooding, and is characterised by high rates of abortion and neonatal mortality, primarily
in sheep, goats and cattle. The susceptibility of different breeds to RVF varies considerably. Some indigenous
African animals may have only inapparent infections, while exotic or other breeds suffer severe clinical disease
with mortality and abortion. Susceptible, older nonpregnant animals and some other species usually do not show
signs of disease. Camels have been regularly involved in the RVF epidemics in East Africa and Egypt. Clinical
disease is not seen in adult camels, but abortion occurs and some early post-natal deaths have been observed.
Signs of the disease tend to be nonspecific, rendering it difficult to recognise individual cases (8–11, 13, 22, 34)
and during epidemics; however, the occurrence of numerous abortions and mortalities among young animals,
together with disease in humans, is characteristic. RVF has a short incubation period: 12–36 hours in lambs. A
biphasic fever of up to 41°C may develop, and the fever remains high until shortly before death. Affected animals
are listless, disinclined to move or feed, and may show enlarged superficial lymph nodes and evidence of
abdominal pain. Lambs rarely survive longer than 36 hours after the onset of signs of illness. Animals older than
2 weeks may die peracutely, acutely or may develop an inapparent infection. Some animals may regurgitate
ingesta and may show melaena or bloody, foul-smelling diarrhoea and bloodstained mucopurulent nasal
discharge. Icterus may sometimes be observed, particularly in cattle. In addition to these signs, adult cattle may
show lachrymation, salivation and dysgalactia. In pregnant sheep, the mortality and abortion rates vary from 5%
to almost 100% in different outbreaks and between different flocks. The death rate in cattle is usually less than
10%.
The hepatic lesions of RVF are very similar in all species, varying mainly with the age of the infected individual
(9). The most severe lesion occurring in aborted fetuses and newborn lambs is a moderately to greatly enlarged,
soft, friable liver with a yellowish-brown to dark reddish-brown colour with irregular congested patches. Numerous
greyish-white necrotic foci are invariably present in the parenchyma, but may not be clearly discernible. In adult
sheep, the lesions are less severe and pinpoint reddish to greyish-white necrotic foci are distributed throughout
the parenchyma. Haemorrhage and oedema of the wall of the gallbladder are common. Hepatic lesions in lambs
are almost invariably accompanied by numerous small haemorrhages in the mucosa of the abomasum. The
contents of the small intestine and abomasum are dark chocolate-brown as a result of the presence of partially
digested blood. In all animals, the spleen and peripheral lymph nodes are enlarged, oedematous and may have
petechiae.
Microscopically, hepatic necrosis is the most obvious lesion of RVF in both animals and humans. In fetuses and
neonates of cattle and sheep, foci of necrosis consist of dense aggregates of cellular and nuclear debris, some
fibrin and a few inflammatory cells. There is a severe lytic necrosis of most hepatocytes and the normal
architecture of the liver is lost. In about 50% of affected livers, intranuclear inclusion bodies that are eosinophilic
and oval or rod-shaped are found. Mineralisation of necrotic hepatocytes is also seen. In adult animals, hepatic
necrosis is less diffuse and in sheep, icterus is more common than in lambs (32).
In humans, RVF infections are usually inapparent or associated with a moderate to severe, nonfatal, influenza-
like illness (19, 21). A minority of patients may develop ocular lesions, encephalitis, or severe hepatic disease
with haemorrhagic manifestations, which is generally fatal. RVF virus has caused serious human infection in
laboratory workers. Staff should either be vaccinated and work under containment level 3, work under
containment level 4 conditions, or wear respiratory protection. Particular care needs to be exercised when
working with infected animals or when performing post-mortem examinations (see Chapter 1.1.2 Biosafety and
No significant antigenic differences have been demonstrated between RVF isolates and laboratory-passaged
strains from many countries, but differences in pathogenicity have been shown (5, 33).
Infection of humans by mosquito vectors is a striking feature in countries, such as Egypt, with a relatively small
population of animal hosts and a large population of mosquitoes.
RVF usually occurs in epizootics in Africa, which may involve several countries in a region at one and the same
time. These follow the periodic cycles of exceptionally heavy rain, which may occur very rarely in semi-arid zones

(25–35-year cycles), or more frequently (5–15-year cycles) in higher rainfall savannah grasslands. Low level
undetectable RVF activity may take place in inter-epizootic periods. RVF should be suspected when unusually
heavy rains are followed by the occurrence of abortions together with fatal disease marked by necrosis and
haemorrhages in the liver that particularly affect newborn lambs, kids and calves, concurrent with the occurrence
of an influenza-like illness in farm workers and people handling raw meat.
Preventative measures to protect workers from infection should be employed when there are suspicions that
RVF-virus-infected meat and tissue samples are to be handled.
RVF virus may be isolated from serum and blood collected in an anticoagulant during the febrile stage of the
disease, from liver, spleen and brain of animals that have died, or from aborted fetuses. Primary isolation is
usually performed in hamsters, infant or adult mice, or on cell cultures of various types.
a) Culture
Approximately 5 ml of blood collected during the febrile stage of the disease or approximately 5 g of liver,
spleen and brain collected after death should be presented for virus isolation. The samples should be kept
at 0–4°C during transit. If transport to the laboratory is likely to take more than 24 hours, the samples
should be frozen and sent on dry ice.
Approximately 1 g of homogenised tissue is suspended 1/10 in cell culture medium or buffered saline,
pH 7.5, containing sodium penicillin (1000 International Units [IU]/ml), streptomycin sulphate (1 mg/ml),
mycostatin (100 IU/ml), or fungizone (2.5 µg/ml). The suspension is centrifuged at 1000 g for 10 minutes
and the supernatant fluid is injected intracerebrally into 1–5-day-old mice or intraperitoneally into hamsters
or adult mice. Infant mice will either die or be obviously ill by day 2. Adult mice are affected 1–3 days later.
Although mice or hamsters are the laboratory animal of choice, lambs and embryonated chicken eggs may
also be used.
A variety of cell monolayers including African green monkey kidney (Vero), baby hamster kidney (BHK),
chicken embryo reticulum (CER: cells developed by Tsunemasa Motohashi at the Nippon Institute for
Biological Science, Tokyo, Japan; recharacterised as a hamster line) (4) and primary kidney or testis cells
of calves and lambs may be inoculated with 1 ml of clarified sample supernates and incubated at 37°C for
1 hour. It is advisable to also inoculate some cultures with a further 1/100 dilution of the inoculum. This is to
avoid the production of defective particles, which follows the use of very high virus inocula. Some tubes
containing flying cover-slips should also be prepared. The cultures are washed with phosphate buffered
saline at room temperature and covered with medium containing 2% serum free from antibodies against
RVF. The cultures are observed microscopically for 5–6 days. RVF virus induces a cytopathic effect (CPE)
characterised by slight rounding of cells followed by destruction of the whole cell sheet within 12–24 hours.
Specific identification of RVF virus antigen may be made 18–24 hours after infection by immunofluorescent
staining of the cover-slip preparations.
The virus may also be detected by immunofluorescence carried out on impression smears of liver, spleen
and brain. A rapid diagnosis can sometimes be made by demonstrating viral antigen in tissues or in serum
of febrile animals by a complement fixation or agar gel immunodiffusion (AGID) test. A rapid diagnosis can
also be made by detection of viral RNA using a reverse-transcription polymerase chain reaction (RT-PCR).
b) Agar gel immunodiffusion

The AGID test is useful in laboratories without tissue-culture facilities. Approximately 1 gram of tissue,
preferably liver, is homogenised and made up to a 10–20% suspension in borate saline buffer, pH 9.0. The
material is centrifuged at 1000 g and the supernatant is used in the test. Micro-AGIDs are performed on
standard microscope slides covered with 3 ml of 1% agarose in borate saline. Patterns of six peripheral
wells and a central well are prepared and filled with reagents as follows: a positive, preferably hyperimmune
serum in the central well, positive control antigen in wells 1 and 4, test tissues in wells 2 and 5 and negative
tissues in wells 3 and 6. A precipitin line of continuity should be formed between control antigen and
positive serum that extends to include a line between test tissue and serum for a case to be considered
positive.

c) Polymerase chain reaction

A rapid diagnosis can also be made by detection of viral RNA (30) using RT-PCR. The PCR was used,
among other techniques, for antigen detection in two recent RVF virus outbreaks in Africa – one in Kenya in
1998 and a limited outbreak in South Africa in 1999. It may also be used to detect RVF virus in mosquito
pools (18). RT-PCR followed by sequencing of the NS(S) protein-coding region has been used in
phylogenetic analysis to characterise two distinct lineages of RVF virus – one Egyptian and the other sub-
Saharan – making this technique a powerful molecular epidemiological tool (29).
d) Histopathology
Histopathological examination of the liver of affected animals will reveal characteristic cytopathology, and
immunostaining will allow the specific identification of the RVF viral antigen in infected cells. This is an
important diagnostic tool because liver or other tissue may be placed in formol saline in the field for
diagnostic purposes, which facilitates handling and transport in areas remote from the laboratory.
Virus neutralisation (VN) tests including microneutralisation, plaque reduction neutralisation (PRN) and
neutralisation in mice have been used to detect antibodies against RVF virus in the serum of a variety of species.
Neutralisation tests are highly specific and will record the earliest response, but these tests can only be
performed with live virus and are not recommended for use outside endemic areas or in laboratories without
appropriate biosecurity facilities and vaccinated personnel.
Other available tests include enzyme-linked immunosorbent assay (ELISA), haemagglutination inhibition (HI),
AGID, immunofluorescence, radioimmunoassay and complement fixation. In these tests, however, cross-
reactions may occur between RVF virus and other phleboviruses. An advantage of these tests is the fact that
they can be performed with inactivated antigen and can therefore be used in RVF-free countries.
The ELISA is a reliable and sensitive test that may be employed with several species to detect antibodies against
RVF virus. An IgM-capture ELISA allows diagnosis of a recent infection to be made on a single serum sample.
The HI test can be employed with great confidence in nonendemic areas. However, sera from individuals that
have had previous infections with phleboviruses other than RVF may react with RVF antigen to titres as high as
40 and, rarely, to titres of 320 (33). In suspected cases, the OIE Reference Laboratory for RVF (see Table given
in Part 3 of this Terrestrial Manual) can be of assistance in carrying out neutralisation tests for specificity. The HI
antibody titre after vaccination with RVF virus vaccine may be as high as 640 or, rarely, 1280, whereas titres
following natural infections with RVF virus are usually significantly higher.
a) Virus neutralisation (the prescribed test for international trade)

The VN test may be employed to determine the presence of antibodies in naturally infected animals and in
animals vaccinated with RVF vaccine. The test is highly specific and can be used to test serum of any
species. It is generally used to measure vaccine efficacy. Factors other than neutralising antibodies may
play a part in resistance to RVF. The Smithburn neurotropic mouse brain strain of highly attenuated RVF
virus (31), also referred to as modified live virus and adapted to cell culture, is used as antigen. The antigen
is stored at –80°C or 4°C in freeze-dried form. The stock is titrated to determine the dilution that will give
100 TCID50 (50% tissue culture infective dose) in 25 µl under the conditions of the test.
• Test procedure
i) Inactivate the test sera for 30 minutes in a water bath at 56°C.
ii) Add 25 µl of cell culture medium with 5% RVF-negative serum and antibiotics to each well of a 96-well
cell culture plate.
iii) Add 25 µl of test serum to the first well of each row and make twofold dilutions. Titrate each serum in
duplicate from 1/10 to 1/80 for screening purposes or in quadruplet and to higher dilutions for
determination of end-point titres. Include known positive and negative control sera.
iv) Add 25 µl per well of RVF virus antigen (diluted in cell culture medium and calculated to provide
100 TCID50 per well) to each well that contains diluted test serum and to wells in rows containing
negative and positive control serum. In addition, make twofold dilutions of antigen in at least two rows
each containing cell culture medium only.
v) Incubate for 30 minutes at 37°C.
vi) Add 50 µl per well of Vero, CER or any other suitable cell suspension at 3 × 105 cells/ml or at a dilution
known to produce a confluent monolayer within 12 hours.

vii) Incubate the plates in an atmosphere of 3–5% CO2 for 3–5 days.
viii) Using an inverted microscope, the monolayers are examined daily for evidence of CPE. There should
be no CPE in rows containing positive control serum and clear evidence of CPE in rows containing
negative control serum indicating the presence of virus. Determine the results by the Spearman–
Kärber method.

For the serodiagnosis of RVFV a number of ELISAs using different formats have been published and are
commercially available (1, 28). The use of inactivated whole virus or mouse liver antigens has recently been
replaced by recombinant nucleocapsid (N) protein as antigen.
These ELISAs are at present in an indirect format and apart from the very important safety consideration
also have the advantage of antigen stability and the ability to test 40 sera in duplicate per plate instead of
only 20.
An indirect ELISA with pre-coated plates using a nucleocapsid protein (NC) recombinant antigen and
Protein G peroxidase conjugate is described below (17).
Test procedure
Unless otherwise stated, all dilutions are made with 10% (w/v) dried milk buffer and all washes performed
three times with volumes of 250–300 µl/well.
i) Using pre-coated plates add 50 µl of diluted (1/100) serum in duplicate wells

ii) Add control sera at predetermined dilutions in duplicate wells. Incubate for 60 minutes at 37°C. Wash
the plate.
iii) Add Protein G/horseradish peroxidase conjugate at a working dilution to all wells of the plate. Incubate
for 60 minutes at 37°C. Wash the plate
iv) Add 50 µl of ready-to-use TMB Substrate to all wells of the plate. Cover the plate and incubate at room
temperature in the darkness for 20–30 minutes.
v) Add 50 µl of ready-to-use Stop solution to all wells of the plate. Tap plate gently to allow contents to
mix. Wait 5 minutes and read plate using a spectrophotometer equipped with a 450 nm filter.
vi) Suggested plate layout.
1 2 3 4 5 6 7 8 9 10 11 12
A CC CC 1 2 3 4 5 6 7 8 9 10
B CC CC 1 2 3 4 5 6 7 8 9 10
C C++ C++ 11 12 13 14 15 16 17 18 19 20
D C++ C++ 11 12 13 14 15 16 17 18 19 20
E C+ C+ 21 22 23 24 25 26 27 28 29 30
F C+ C+ 21 22 23 24 25 26 27 28 29 30
G C– C– 31 32 33 34 35 36 37 38 39 40
H C– C– 31 32 33 34 35 36 37 38 39 40
CC: Conjugate control; C++: High positive control serum; C+: low positive control serum;
C–: negative control serum; 1–40: test samples.
Newer ELISA formats are being introduced, including formats that are more specific for IgG and IgM (27).
c) Haemagglutination inhibition
The HI test adapted to a microtechnique is based on Clarke & Casals (7). A sucrose/acetone-extracted
hamster liver antigen is used in a 96-well U-bottomed plate test and antigen is diluted so that
4 haemagglutinating units are used in the test. Nonspecific inhibitors of haemagglutinin are removed by
kaolin extraction of sera followed by adsorption with packed goose erythrocytes (RBC) prior to testing.
Doubling dilutions of sera made in borate saline buffer, pH 9, are tested against equal volumes of antigen.

Plates are held overnight at 4°C before the addition of 50 µl of 0.5% RBC to each of the wells. Plates are
read after 30 minutes at room temperature and end-points are recorded as the reciprocal of the highest
serum dilution producing complete inhibition of agglutination.
Positive and negative control sera are incorporated into each test. A test is considered to be valid only if the
control sera give the expected results. Sera with titres below 1/40 are considered to be negative.
HI is an appropriate screening test for surveys although it is not specific. Marked cross-reactions do occur
between the phleboviruses, but homologous titres exceed heterologous titres. Experimentally, African
phleboviruses other than RVF have been shown to be nonpathogenic for ruminants, and antibodies that
they might induce are unlikely to cause confusion in RVF diagnosis (33).
A live vaccine prepared from Smithburn’s attenuated strain of RVF virus has been used for the control of RVF in
nonpregnant cattle and sheep in endemic areas and during outbreaks (6), while inactivated vaccines for use in
pregnant animals and in RVF-free countries are prepared from virulent field strains (2, 3). Inactivated virus
vaccines should be prepared from highly immunogenic strains of RVF virus produced in cell culture. The virus
should be inactivated with formaldehyde and mixed with an adjuvant to enhance immunogenicity. The inactivated
vaccine should be carefully safety tested to ensure that there is no residual live virus.
In humans, an inactivated experimental RVF vaccine has been used for 25 years with considerable success to
protect persons at risk. This vaccine is currently produced on diploid cells. However, the limited availability of the
vaccine precludes its use in the general population.
Two new vaccine candidates produced from human RVF virus isolates are undergoing extensive testing with a
view to replacing existing vaccines.
The first, MV P12, is a mutagen-derived strain of virus passaged in the presence of 5-florouracil with serial
mutagenesis resulting in attenuation for mice. Immunogenicity and pathogenicity have been tested in sheep and
the virus found to be non-abortogenic in pregnant ewes (23). MV P12 was protective in young lambs (14, 20) and
in cattle (24, 25). In further testing in sheep, the vaccine, when used after 28 days of pregnancy, i.e. in the first
trimester, resulted in abortion and severe fetal teratology (16).
The second candidate, Clone 13, a small plaque variant that did not react with two specific monoclonal
antibodies, was found to be avirulent in mice and hamsters and highly immunogenic. Immunogenicity and
pathogenicity have been tested in lambs, sheep, young and adult goats (26). In further trials it was non-
abortogenic in pregnant sheep and gave more than 80% protection from virulent challenge (15). Clone 13
possesses a large deletion in the portion of the sRNA segment coding for the nonstructural proteins, which
should result in a stable vaccine candidate.
In the following description of vaccine production, information is given on live vaccine production adjacent to
information on inactivated vaccine production. It must be stressed that live and inactivated vaccines must never
be produced in the same facility at the same time, because of the risk of contaminating the attenuated live
vaccine with a virulent strain of virus before it is inactivated. Staff handling live RVF virus should be vaccinated
and work at containment level 3 to minimise the risk of self infection.
1. Seed management
a) Characteristics of the seed virus

Live vaccine: The stock antigen is derived from Smithburn’s original neurotropic strain. This strain is not
lethal to adult mice inoculated intraperitoneally and is safe for use in all breeds of cattle, sheep and goats.
However, it may cause fetal abnormalities or abortion in pregnant animals.
Inactivated vaccine: For seed virus, a highly immunogenic strain of RVF virus adapted to growth in cell
culture may be used. It differs from the attenuated strain in that it is lethal to adult mice when injected
intraperitoneally.

Both attenuated and inactivated virus strains are produced on BHK, Vero or CER cell cultures. The viruses
are stored in a lyophilised form in vials containing 1 ml of a cell culture suspension. The virus titre (following
intracerebral inoculation of infant mice) should be at least 106.5 mouse LD50 (50% lethal dose) per ml.
Seed virus must be shown to be free from adventitious agents, safe for use and able to stimulate effective
immunity in species and breeds for which it is intended.
• Tests
The lyophilised seed virus is reconstituted in sterile cell culture medium without antibiotics and tested for
freedom from bacteria and fungi. The contents of a reconstituted vial are inoculated into two tubes of
thioglycollate and two tubes of soybean casein digest medium. The thioglycollate cultures are incubated at
37°C for 7 days and the soybean casein digest medium cultures at 20°C for 14 days. The cultures should
remain negative.
In addition, 5 ml of reconstituted seed virus is mixed with an equal volume of specific RVF antiserum
produced in rabbits. After incubation of the serum/virus mixture at 37°C for 30 minutes, the virus
suspensions are tested before and after neutralisation on cell cultures, as well as in adult and infant mice,
embryonated eggs, and guinea-pigs. The neutralised virus is:
i) Seeded on to six roller tube cultures of primary lamb kidney cells and six roller tube cultures of BHK
cells. The cell cultures are incubated at 37°C and observed daily for 7 days for CPE, after which they
are subjected to the haemadsorption test with guinea-pig RBCs at 4°C and 37°C. There should be no
evidence of CPE or haemadsorption. If cultures degenerate or show suspicious CPE, the material
from these cultures should again be mixed with antiserum and subinoculated into new cell cultures,
which are observed for a further period of 14 days. The presence of specific CPE or haemadsorption
disqualifies the seed virus pool.
ii) Inoculated intraperitoneally (0.2 ml) into groups of at least six adult and six 2–5-day-old mice. The
mice should remain healthy for 14 days. If any mice should die, appropriate tissue should be
emulsified, mixed with antiserum and subinoculated into further groups of mice, which should again be
observed for a further period of 14 days. If there is any evidence of specific mortality, the seed virus
pool is disqualified.
iii) Inoculated into at least ten 8-day-old embryonated chicken eggs by means of the ‘stab’ method
(combination of chorioallantoic membrane and allantoic sac route). The eggs are incubated at 37°C for
8 days and are candled daily. Embryos that die within 24 hours are discarded. However, the test
should be repeated if <70% of the embryos are alive after 24 hours. The cause of embryo mortality
during the subsequent observation period should be determined by setting up appropriate sterility and
HI tests, and by examination of yolk-sac smears. If these tests are negative, subinoculation of embryo
suspensions mixed with antiserum should be set up as before. On day 4 of incubation, at least four
eggs are opened and allantoic fluid is collected. The remaining eggs are opened on day 8 of
incubation. The membranes of both groups are examined for lesions and abnormalities of the
embryos. The allantoic fluids are subjected to the HI test with guinea-pig and chicken RBCs at 4°C
and 37°C. Specific embryo mortality, haemagglutinating activity of the allantoic fluids or any lesions on
the membranes or embryo abnormalities disqualifies the seed virus pool.
iv) Injected intraperitoneally with 1.0 ml of seed virus into each of two guinea-pigs. The guinea-pigs
should remain healthy over an observation period of 14 days.
Failure to pass any test disqualifies the antigen for use as seed virus.
A vial of lyophilised seed virus is reconstituted and diluted 1/100 to 1/1000 with sterile Eagle’s medium for the
attenuated vaccine and 1/1000 for the inactivated vaccine. To prepare a working suspension, the diluted virus is
seeded on to confluent BHK cell cultures in roller bottles and incubated at 37°C. When 70% of cells is affected
(CPE), the medium and cells are harvested and the material is diluted 1/100 to 1/1000, after which 10 ml is again
seeded on to roller bottles with confluent BHK cells and again incubated. As soon as 70% CPE is observed, the
medium and cells are harvested and pooled.
Virus suspensions for both attenuated and inactivated vaccines are titrated intracerebrally in infant mice and
should have a titre of at least 106.5 mouse LD50/ml. Alternatively, a plaque titration on CER cells may be
performed.

Attenuated vaccine is lyophilised immediately after completion of titration and testing for bacteria and fungi.
A stabiliser should be used, such as 5% peptone in 0.3 M phosphate buffer. The volume of inactivated vaccine is
adjusted so that the final vaccine will contain at least 106.5 mouse LD50/ml. The adjusted virus suspension is then
inactivated at 37°C for 24 hours with formaldehyde at a final concentration of 0.2%. After inactivation, an equal
volume of aluminium hydroxide gel is added to the cell suspension. The vaccine should have a final pH of 7–7.5.
Prior to inoculation of cell cultures, seed virus is subjected to tests for bacteria and fungi in thioglycollate and
soybean casein digest medium (see Section C.1.c and Chapter 1.1.9 Tests for sterility and freedom from
contamination of biological materials).
A representative sample from each batch of vaccine is selected and the contents of each are reconstituted with
5 ml sterile distilled water and tested for freedom from bacteria and fungi.
For inactivated vaccines, inactivation must be checked using two passages in the same type of cell culture as
used in the production of the vaccine.
4. Batch control
a) Sterility
Prior to freeze-drying or inactivation, each container of pooled vaccine, and thereafter representative
samples of the batch, are tested for sterility in thioglycollate and soybean casein digest medium (see also
Chapter 1.1.9 of this Terrestrial Manual).
b) Safety
Live vaccine: Final containers of lyophilised attenuated vaccine are selected at random, and each is
reconstituted in distilled water as for vaccination. Four susceptible sheep are injected subcutaneously with
one dose of vaccine. The sheep are observed daily for 14 days and the rectal temperatures are recorded.
The sheep must remain healthy.
Vaccine is also injected intraperitoneally into six adult mice (0.25 ml each), two hamsters and two guinea-
pigs (1 ml each). The animals are observed for a period of 14 days during which they should remain
healthy. Mortality attributed to the vaccine disqualifies the batch.
Inactivated vaccine: In the case of inactivated RVF vaccine, each of four susceptible sheep is injected
subcutaneously with 2.0 ml of vaccine, observed daily for 3 weeks and rectal temperatures are recorded.
The sheep should remain healthy.
In addition, safety is also determined by intracerebral injection of six adult mice and two litters of at least six
infant mice per litter, and by intraperitoneal injection of two guinea-pigs and two hamsters. The mice,
hamsters and guinea-pigs are observed for a period of 14 days. They should remain healthy. Mortality
attributed to the vaccine disqualifies the batch.
c) Potency
Live vaccine: Lyophilised attenuated vaccine from two final containers is reconstituted and titrated
intracerebrally in infant mice. The final vaccine should contain at least 104.4 mouse LD50/dose. Alternatively,
titrations may be done on cell cultures.
Two final containers are kept at 37°C for 1 week, reconstituted and titrated as before. Each should contain
at least 103.4 mouse LD50/dose. Alternatively, titrations may be done on cell cultures.
Inoculated sheep (see Section C.4.b) are bled 2 and 3 weeks after vaccination, and their antibody response
is determined by PRN. A virus neutralising antibody titre of 100 or more is regarded as satisfactory.
Inactivated vaccine: The sheep, injected subcutaneously to determine safety (Section C.4.b), are bled after
3 weeks and their antibody response is determined by VN test. A virus neutralising antibody titre of 100 or
more is regarded as satisfactory.
Both the live attenuated and the inactivated vaccines have had extensive field use. The live vaccine is
considered to induce lifelong immunity against clinical disease, although controversy exists over the
immunogenicity of the Smithburn vaccine. Nevertheless, cattle can be immunised with the live virus vaccine

using this strain. Experience of the field efficacy of inactivated vaccines is limited because they are used in
areas where RVF is not endemic, consequently natural field challenge of the vaccine does not occur.
However, in South Africa, during the outbreak of RVF in 1976–1978, observations by State Veterinarians
supported the efficacy of the vaccine. In more recent epizootics elsewhere, the inactivated vaccine failed to
protect animals against abortion, following two vaccinations. When using the inactivated vaccine, a booster
dose should be given 3–6 months after the initial vaccination and thereafter vaccination should be repeated
yearly (2, 3).
e) Stability
When stored at 4°C, lyophilised attenuated vaccines are stable for at least 4 years, while inactivated
vaccine may be stored for many years. Storage at higher temperatures is not recommended.
f) Preservatives
No preservatives are used.
Although humans can be infected by handling infected material, no case of disease is known to have
occurred in humans infected with attenuated vaccine virus, but seroconversion often occurs. However, the
strains used to prepare inactivated vaccine may cause disease. Therefore, all staff likely to be exposed to
vaccine virus should be vaccinated with the human formalin-inactivated vaccine.
a) Sterility
Representative samples of the final product are collected and tested as in Section C.4.a.
b) Moisture content
The moisture content of the lyophilised attenuated vaccine should not exceed 3%.
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*
* *
NB: There is an OIE Reference Laboratory for Rift Valley fever (see Table in Part 3 of this Terrestrial Manual or

CHAPTER 2.1.15.
RINDERPEST
SUMMARY
Classical rinderpest is an acute, viral disease of domestic cattle, buffaloes and yaks characterised
by high morbidity and mortality rates. Sheep, goats, pigs and wild ungulates may also be affected.
Clinically this form of the disease is characterised by pyrexia, the progressive development of
shallow erosions on the gums, tongue, cheeks and hard palate, together with serous or
mucopurulent ocular and nasal discharges. Alimentary tract involvement is marked by the
development of diarrhoea or dysentery, leading to severe dehydration and depression. Rinderpest
conforming to this description has not been seen since 2001 (Pibor, Southern Sudan). A milder
form of the disease, with the potential to regain classical characteristics, used to occur in
association with endemic situations, latterly in East Africa; it has not been positively diagnosed
since 1997 (Tanzania) and could have died out, in which case wild-type rinderpest virus may no
longer exist. Based on historical virus collections, three genetically distinct lineages of the virus
have been recognised as causal agents of rinderpest disease in Africa and Asia. The Food and
Agriculture Organisation of the United Nations (FAO) launched a Global Rinderpest Eradication
Programme (GREP) in 1992, calling for eradication of the virus by the year 2010. The success of
this programme may be judged by the fact that two of the three rinderpest lineages have now
assuredly been eradicated and the third might well have joined them.
Identification of the agent: Clinical confirmation of classical rinderpest is based on the finding of
individual or small groups of animals showing pyrexia, inappetance, depression, shallow erosions of
the upper and lower lip and gum, erosions or blunting of the cheek papillae, serous or mucopurulent
ocular discharges and/or nasal discharges, diarrhoea, recumbency and possibly death. Laboratory
confirmation is based on demonstrating the presence of the virus, virus-specific RNA or precipitating
antigens in samples from the spleen, lymph nodes, or ocular or nasal secretions of acutely infected
animals. It is particularly important to isolate the virus if a geographical extension or significant animal
health deterioration has occurred. Following the successes of global eradication, rinderpest-free
countries may now confirm the presence of peste des petits ruminants (PPR) in sheep or goats based
on the clinical appearance of affected animals and the presence of precipitating antigens, even
though both the clinical signs and the virus-induced antigens are common to both viruses.
In cases where rinderpest is suspected, post-mortem examinations should pay particular attention
to the abomasum, which may be highly engorged or show a grey discoloration; to the Peyer’s
patches, which may show lymphoid necrosis; and to the development of linear engorgement and
blackening of the crests of the folds of the caecum, colon and rectum. The principal differential
diagnoses are PPR in sheep and goats, and bovine viral diarrhoea/mucosal disease and malignant
catarrhal fever in cattle; differentiation of these diseases requires the use of appropriate laboratory
methods.
Serological tests: The OIE has developed a set of Recommended Standards for Epidemiological
Surveillance for Rinderpest (the ‘OIE Pathway’) that governs the actions of Member Countries wishing
to demonstrate that they have achieved freedom from infection. To this end, a competitive enzyme-
linked immunosorbent assay (ELISA) has been described to determine the presence of rinderpest
antibodies in animals that have been infected with field virus or received rinderpest vaccine. An
indirect ELISA has also been described. Whatever test is used it should be sensitive with respect to
the lineage of virus likely to be present and be highly specific. Neutralising antibody estimations may
be used for the same purpose. Member Countries may wish to seek expert advice from an OIE
Reference Laboratory or the GREP Secretariat with regard to the selection of the test most
appropriate for their purpose.

Chapter 2.1.15. — Rinderpest
Requirements for vaccines and diagnostic biologicals: A live attenuated cell culture rinderpest
vaccine is available. In recent years its use has been considerably curtailed, as the lifelong
immunity it induces may interfere with post-campaign serological assessments aimed at a
Country’s efforts to gain a rinderpest-free accreditation. Given that the inadvertent use of this
vaccine has given rise to confusing serosurveillance results, and that considerable quantities of it
are still available, Member Countries should catalogue and secure all remaining stocks in order to
safeguard the ability to undertake post-campaign serosurveillance.
A. INTRODUCTION
In recent years the Global Rinderpest Eradication Programme (GREP) of the Food and Agriculture Organisation
of the United Nations (FAO) has made enormous progress in organising and documenting the decline of
rinderpest (13). Historically, the virus was widely distributed throughout Europe, Africa, and Asia; recently
however, it has only occurred in Africa and Asia. Gene sequence analysis has shown that all known rinderpest
isolates fall into one of three non-overlapping phylogenetic lineages, and in recent years it has been possible to
describe the virus’ distribution in lineage-specific terms. Thus, the so-called Asian lineage (lineage 3) was only
ever recorded in Afghanistan, India, Iran, Iraq, Kuwait, Oman, Pakistan, Russia, Saudi Arabia, Turkey, Sri Lanka
and Yemen. As a result of concerted and coordinated vaccination and surveillance campaigns, this virus lineage
has failed to resurface since September 2000 (Pakistan). Although evaluations are not yet complete, it is almost
certain that this virus has been successfully eradicated.
Rinderpest virus lineages 1 and 2 have only been recorded from Africa. Lineage 1 appears to have been
distributed from Egypt to southern Sudan and eastwards into Ethiopia and into northern and western Kenya. On
the other hand, lineage 2 has been recorded from both East and West Africa and at one time may have been
distributed in a sub-Saharan belt running across the whole of the continent (12). Now however, as the result of
further coordinated vaccination and surveillance programmes (Pan African Rinderpest Campaign in particular),
neither West nor Central Africa have reported rinderpest since 1988 (Ghana/Burkina Faso). Until recently both
lineages were being reported from eastern Africa, but it is now clear that lineage 1 was eliminated from southern
Sudan in 2001 by intensive vaccination.
Reappearing in 1994, 1996 and 2001 in wildlife, Lineage 2 has been transmitting within the Somali pastoral
ecosystem (9) where its continued presence caused considerable concern (10). In 1994, this virus reappeared in
south-east Kenya where its effects were expressed most dramatically in buffaloes in Tsavo National Park (7)
thereby illustrating its ability to engage in cryptic persistence for a period of at least 30 years, during which time it
is likely to have been transmitted with a low level of virulence among susceptible cattle. Although this virus is now
seen as having evolved to the point where it has been possible for it to escape veterinary attention in remote
areas, its presence did not go unnoticed by the nomadic pastoralists whose cattle it infected. Nevertheless, it is
clear that this virus has not been confirmed within this last reservoir since 2001 and although not yet an
accredited achievement, it is more than likely that sporadic vaccination has broken the transmission chain of
Lineage 2.
Rinderpest is caused by a negative-strand RNA virus of the Morbillivirus genus within the family
Paramyxoviridae. Classic descriptions of rinderpest refer to it as a highly fatal disease of domestic cattle,
buffaloes and yaks. The virus also affects some breeds of pigs and a very large variety of wildlife species within
the order Artiodactyla, although not always in a clinically apparent form; a recent review views sheep and goats
as susceptible but largely epidemiologically unimportant hosts of rinderpest (14).
Although in its final stages of eradication, some strains of rinderpest had evolved into a mild, nonfatal, infectious
disease of cattle, all strains retained two very dangerous attributes. The first was an almost certain ability to
undergo virulence modulations. The second was an ability to infect game animal species and, in buffaloes, eland,
giraffe, lesser kudu and warthog, to cause an acute infection associated with high levels of mortality.
Classical rinderpest has an incubation period of between 1 and 2 weeks, the clinical disease is characterised by
an acute febrile attack within which prodromal and erosive phases can be distinguished. The prodromal period
lasts approximately 3 days, during which affected animals develop a pyrexia of between 40 and 41.5°C together
with partial anorexia, constipation, congestion of visible mucosae, serous ocular and nasal discharges,
depression and drying of the muzzle. However, it is not until the onset of the erosive phase, and the development
of necrotic mouth lesions, that a tentative clinical diagnosis of rinderpest can be made. At the height of fever,
flecks of necrotic epithelium appear on the lower lip and gum and in rapid succession may appear on the upper
gum and dental pad, on the underside of the tongue, on the cheeks and cheek papillae and on the hard palate.
Through the enlargement of existing lesions and the development of new foci, the extent of the oral necrosis can
increase dramatically over the following 2–3 days. Much of the necrotic material works loose giving rise to
shallow, non-haemorrhagic mucosal erosions.

Diarrhoea is another characteristic feature of rinderpest and develops 1–2 days after the onset of mouth lesions.
The diarrhoea is usually copious and watery at first, but later on may contain mucus, blood and shreds of
epithelium and it may be accompanied, in severe cases, by tenesmus. During the erosive phase, necrosis may
be observed in the nares, in the vulva and vagina, and on the preputial sheath. Anorexia develops, the muzzle
dries out completely, the animal is depressed, the breath is fetid and mucopurulent ocular and nasal discharges
develop.
Deaths will occur but the mortality rate will be variable and may be expected to rise as the virus gains
progressive access to large numbers of susceptible animals. Initial mortality rates will probably be in the order of
10–20% and, in the terminal stages of the illness, animals may become recumbent for 24–48 hours prior to
death. Some animals die while showing severe necrotic lesions, high fever and diarrhoea, others after a sharp
fall in body temperature, often to subnormal values. Alternatively, the pyrexia may remit slightly in the middle of
the erosive period and then, 2–3 days later, return rapidly to normal accompanied by a quick resolution of the
mouth lesions, a halt to the diarrhoea and an uncomplicated convalescence.
Typically the carcass of the dead animal is dehydrated, emaciated and soiled. The nose and cheeks bear
evidence of mucopurulent discharges, the eye is sunken and the conjunctiva congested. In the oral cavity, there
is often extensive desquamation of necrotic epithelium, which always appears sharply demarcated from adjacent
areas of healthy mucosa. The lesions frequently extend to the soft palate and may also involve the pharynx and
the upper portion of the oesophagus; the rumen, reticulum and omasum are usually unaffected, although
necrotic plaques are occasionally encountered on the pillars of the rumen. The abomasum, especially the pyloric
region, is severely affected and shows congestion, petaechiation and oedema of the submucosa. Epithelial
necrosis gives the mucous membrane a grey colour. The small intestine is not commonly involved except for
striking changes to the Peyer’s patches where lymphoid necrosis and sloughing leaves the supporting
architecture engorged or blackened. In the large intestine changes involve the ileocaecal valve, the caecal tonsil
and the crests of the longitudinal folds of the caecal, colonic and rectal mucosae. The folds appear highly
engorged in acute deaths or darkly discoloured in long-standing cases; in either event the lesions are referred to
as ‘zebra striping’.
Taking the lineage 2-associated form of rinderpest as an example of the mild form adopted by the virus within
endemic situations, the incubation period is between 1 and 2 weeks and the ensuing clinical disease is little more
than a subacute febrile attack in cattle. The fever is not invariable; it is short-lived (3–4 days) and not very high
(38–40°C). The depression that characterises more acute forms of rinderpest is absent from mildly affected
animals and, as a result they might not lose their appetite, they will probably continue to graze, water and trek as
well as unaffected animals. These animals do not usually develop diarrhoea. On close examination there may be
some slight congestion of the visible mucous membranes and small, focal areas of raised, whitish epithelial
necrosis may be found on the lower gum – sometimes no larger than a pin head – along with a few eroded cheek
papillae. Some animals may escape the development of such erosions, the appearance of which is fleeting.
Other animals may show a slight, serous, ocular or nasal secretion but, in contrast to the more severe forms of
the disease, these do not progress to become mucopurulent.
Even though infections with lineage 2 may pass unnoticed in cattle, the virus is highly infectious for wildlife
species, and among those generally regarded as highly susceptible (buffalo, giraffe, eland, and lesser kudu) it
causes fever, a nasal discharge, typical erosive stomatitis, gastroenteritis, and death. Kock (7) observed that in
addition, buffaloes infected with lineage 2 showed enlarged peripheral lymph nodes, plaque-like keratinised skin
lesions and keratoconjunctivitis. Lesser kudus were similarly affected, but whereas blindness – caused by a
severe keratoconjunctivitis – was common, diarrhoea was unusual. Eland also showed necrosis and erosions of
the buccal mucosa together with dehydration and emaciation. Therefore, under the present circumstances, a
diagnosis of rinderpest in any of these species points to the likelihood of the simultaneous transmission of the
virus, even at a subclinical level, in neighbouring cattle.
In view of the high level of expectation surrounding the Global Rinderpest Eradication Campaign, any recent
outbreak of rinderpest would be of immense epidemiological significance, not only as a threat of pandemic
eruption but as a indicator of a lacuna in global surveillance. Consequently, until a country has reached an
accredited rinderpest infection free status (based on serosurveillance), samples from all outbreaks considered
rinderpest-like on clinical or pathological grounds must be routinely submitted for laboratory examination. For
rinderpest, a variety of suitable laboratory tests is available, but under the circumstances outlined above it is of
paramount importance to isolate the virus, identify its lineage and assess its virulence in experimental cattle (1).
Blood in anticoagulant is the preferred sample wherever possible. On average, the onset of viraemia slightly
precedes the onset of pyrexia, and may continue for 1–2 days after pyrexia begins to wane. Consequently,
animals showing a pyrexia are probably viraemic and therefore the best source of blood with which to attempt

virus isolation. However, as occasional febrile animals may no longer be viraemic, samples from several febrile
animals should be collected for submission. It is important to ensure that there is adequate tissue available for at
least two virus isolation attempts from the initial submission of a suspected outbreak. The other procedures
described should only be attempted if there is extra tissue available.
a) Virus isolation
Rinderpest virus can be cultured from the leukocyte fraction of whole blood that has been collected into
heparin or EDTA (ethylene diamine tetra-acetic acid) at final concentrations of 10 international units (IU)/ml
and 0.5 mg/ml, respectively. Samples should be thoroughly mixed and transferred to the laboratory on ice,
but never frozen. Virus can also be isolated from samples of the spleen, prescapular or mesenteric lymph
nodes of dead animals; these samples may be frozen for transportation.
To isolate the virus from blood, uncoagulated blood is centrifuged at 2500 g for 15 minutes to produce a
buffy coat layer at the boundary between the plasma and erythrocytes. This is removed as cleanly as
possible, mixed in 20 ml physiological saline and recentrifuged in a washing procedure designed to remove
any neutralising antibody present in the plasma. The resulting cell pellet is suspended in cell culture
maintenance medium and 2 ml aliquots are distributed on to established roller tube monolayers of primary
calf kidney, B95a marmoset lymphoblastoid or African green monkey kidney (Vero) cells. The culture
maintenance medium should be decanted and replaced every 2 or 3 days and the monolayer observed
microscopically for the development of cytopathic effects (CPE). These are characterised by refractility, cell
rounding, cell retraction with elongated cytoplasmic bridges (stellate cells) and/or syncytial formation. The
speed with which the CPE develops varies by substrate and probably by strain of virus also. Up to 12 days
should be allowed in primary cells, a week in Vero and 2–4 days in B95a cells. Blind passages may be
attempted before declaring an important sample negative, but a preferable technique would be to inoculate
the cell suspension, and any residue of the original sample, intravenously into a rinderpest-susceptible ox
and attempt to re-isolate the virus from its blood. Isolates of virus can be partially identified by the
demonstration of morbillivirus-specific precipitinogens in infected cell debris, or completely identified by the
demonstration of specific immunofluorescence using a conjugated monoclonal antibody (MAb).
Alternatively, 20% suspensions (w/v) of lymph node or spleen may be used. These should be made by
macerating the solid tissues in serum-free culture maintenance medium using standard grinding or shearing
techniques and inoculating monolayers as before. The release of virus from solid tissue can be achieved in
several ways. Perhaps the easiest is with a pestle and mortar, but this technique requires the use of sterile
sand as an abrasive. Alternatively, tissue may be ground without an abrasive using all-glass grinders, for
example, a Ten Broeck grinder. Shearing techniques are equally applicable using, for example, Silverson or
Waring blenders. Virus-containing suspensions are clarified by low-speed centrifugation. The volume of the
inoculum is not critical; a working volume is between 1 and 2 ml. Commonly used antibiotics are penicillin
and streptomycin in combination, each at a concentration of 100 IU/ml. A similar broad-spectrum cover can
be obtained using neomycin at 50 µl/ml. Fungizone should be included at 2.5 µg/ml.
b) Antigen detection by agar gel immunodiffusion

The agar gel immunodiffusion (AGID) tests may be conducted in Petri dishes or on glass microscope slides
(5). In either instance the surface should be covered with agar to a depth of about 4 mm using a 1%
aqueous solution of any high quality agar or agarose. Wells are usually cut in a hexagonal pattern of six
peripheral wells around a single central well. For slides, wells should be 3 mm in diameter and 2 mm apart.
For Petri dishes, the wells can be increased to 4 mm in diameter and the distance between wells to 3 mm.
The closer the wells are placed from each other, the shorter the reaction time.
Using a small volume pipette, rinderpest hyperimmune rabbit serum should be placed in the central well.
Similarly, control positive antigen, prepared from the macerated lymph nodes of rabbits infected with the
Nakamura III lapinised strain of rinderpest, should be placed in alternate peripheral wells (i.e. one, three and
five). Negative control antigen is placed in well four. Test antigens are obtained as exudates from the cut
surface of spleen or lymph nodes submitted for testing; if no exudate can be obtained a small portion of the
sample should be ground with a minimum of saline. Ocular exudates may be squeezed directly from the swabs
or, alternatively, by compression in a microtip (the cotton wool should be cut off the swab and placed into the
wide end of a plastic 50–250 µl pipette tip; the stem of the swab may then be used to compress the cotton
wool and force a small volume of exudate out of the narrow end of the tip). Test samples are added to wells
two and six. Tests are best developed at 4°C or low ambient temperatures. The reaction area should be
inspected from 2 hours onwards for the appearance of clean, sharp lines of precipitation between the wells
forming a line of identity with the controls. Tests should be discarded after 24 hours if no result has been
obtained. The result is not acceptable unless precipitation reactions are also obtained giving a line of identity
with the control positive antigen preparation.
Although the test is neither highly sensitive nor highly specific, it is robust and adaptable to field conditions. A
positive reaction from a large domestic ruminant should be treated as if it were rinderpest. From a small

ruminant, a positive result should be treated as having been derived from a case of rinderpest or peste des
petits ruminants (PPR) and requiring further differentiation.
c) Histopathology and immunohistochemistry

At post-mortem examination, tissues should be collected and placed in 10% neutral buffered formalin for
histopathology and immunohistochemistry; the base of the tongue, retropharyngeal lymph node and third
eyelid are suitable tissues. Sections stained with haematoxylin and eosin should be examined for the
presence of syncytial cell formation, and cells with intranuclear viral inclusion bodies. The presence of
rinderpest antigens can be demonstrated in the same formalin-fixed tissues by immunoperoxidase staining
following the quenching of endogenous peroxidase activity. If a polyclonal antiserum is used, this test will
fail to differentiate between rinderpest and PPR. However, this problem can be circumvented by using
monoclonal antibodies specific for rinderpest and PPR in duplicate tests (3).
d) Lineage identification using the reverse-transcription polymerase chain reaction

The reverse-transcription polymerase chain reaction (RT-PCR) (6) produces DNA suitable for gene
sequence analysis. Viral RNA can be purified from spleen (not ideal due to its high blood content), lymph
node and tonsil (ideal), peripheral blood lymphocytes (PBLs), or swabs from eyes or mouth lesions
(contingent). Solid tissues (0.5–1.0 g) are minced and homogenised with 4.0 ml denaturing solution, eye
and mouth swabs are treated with 1.0 ml, and purified PBLs (from 5 to 10 ml whole blood) are treated with
0.4 ml according to the published procedure. Solution D (disruption solution): the procedure is that
recommended to minimise the hazard of handling poisonous guanidium thiocyanate. It should be carried
out in a chemical safety hood. The following are the amounts of guanidium thiocyanate for a 250 g bottle,
but the volumes can be adjusted for other quantities. Do not attempt to weigh out the guanidium
thiocyanate, but dissolve it in the manufacturer’s bottle by adding 293 ml sterile distilled water, 17.6 ml
0.75 M sodium citrate, pH 7.0, and 26.4 ml 10% sarcosyl, then heat to 65°C in a water bath to dissolve. This
solution can be kept for several months in the dark at room temperature in a chemical safety cabinet. The
final solution D is made by the addition of 0.36 ml 2-mercaptoethanol to 50 ml of the stock solution. This
solution should not be kept for more than 1 month. In the past few years, RNA extraction spin columns have
become widely used for fast purification of high quality RNA (RNeasy kit, Qiagen) The resulting RNA is
precipitated with 2.5 volumes of ethanol, washed in 70% ethanol, dissolved in sterile water, or TE buffer
(Tris/EDTA, 10 mM, pH 7.5, 1 mM EDTA) and stored at –70°C or –20°C until required. The cDNA synthesis
is carried out using random hexanucleotide primers to enable several different specific primer sets to be
used in the PCR amplification step. Aliquots of the resulting cDNA are amplified using at least three primer
sets that can detect and differentiate between the two morbilliviruses. These primer sets include two
‘universal’ sets based on highly conserved regions in the phosphoprotein and nucleoprotein genes that
should detect all morbilliviruses, and rinderpest virus-specific sets based on sequences in the fusion protein
genes of the virus. The PCR products are analysed on a 1.5% (w/v) agarose gel along with a suitable DNA
marker to identify the specific DNA product. A positive control such as measles or canine distemper virus
RNA, and a negative control using sterile distilled water instead of RNA, must be included in each RT-PCR.
Positive reactions should be confirmed either by using ‘nested’ primer sets based on the F gene sequences
or by sequence analysis of the DNA product. It is important to use more than one set of primers for the PCR
step when testing for the presence of RNA viruses, as their nucleotide sequences can vary significantly and
one change at the 3’-end of the primer sequence may result in failure of the primers to amplify the DNA.
The World Reference Laboratory in the United Kingdom (UK), which is also an OIE Reference Laboratory
for rinderpest, and the OIE Reference Laboratory in France (see Table given in Part 3 of this Terrestrial
Manual), can advise on use of the technique for field sample analysis.
Most recently, a simple Taqman real-time RT-PCR assay for RPV diagnostic has been described.This real-
time RT-PCR assay for rinderpest virus has been validated to be highly sensitive in infected tissue culture
supernatant and clinical samples from experimentally infected cattle. The assay has proved to be able to
detect isolates representative of all known phylogenetic lineages of the virus and clearly differentiate from
PPR virus and other look-alike diseases (foot and mouth disease virus, bovine viral diarrhoea virus, bovine
herpesvirus, vesicular stomatitis virus). The analytical sensitivity of the L10 primer-probe system exceeded
1–100 TCID50 (50% tissue culture infective dose)/ml, depending on the rinderpest virus strain. Comparison
of samples from experimentally infected animals showed that white blood cells and conjunctival swabs are
the sample of choice for epidemiological surveillance of the disease, allowing the preclinical detection of the
disease by 2–4 days. In the event of a rinderpest virus outbreak, this portable, single-tube format, real-time
RT-PCR has the capability of preclinical diagnosis, thus aiding efforts to prevent further transmission of
disease.
e) Differential immunocapture ELISA

Neither clinical observations nor AGID tests can differentiate between rinderpest and PPR; consequently, if
either disease is suspected in sheep or goats in countries where both diseases occur, other tests like the
real-time PCR must be used. Rapid differentiation can be achieved using a differential immunocapture
ELISA test (8). This test employs MAbs directed against the N protein of the two viruses. One MAb, with a

reactivity against both viruses, is used as a capture antibody, while a second biotinylated MAb specific for a
nonoverlapping antigenic N protein site, and specific against either rinderpest or PPR, is used to determine
which N protein has been captured.
High protein-binding ELISA plates (or strips) are coated with 100 µl/well of capture antibody. After three
washes, the wells are loaded with 50 µl of test sample diluted 1/10 in a lysis buffer, 25 µl of the
manufacturer’s recommended dilution of the virus-specific MAb and 25 µl of streptavidin peroxidase at a
final dilution of 1/3000. The wells are then placed on an orbital shaker for 1 hour at 37°C, after which time
they are again washed; following the addition of 100 µl of ortho-phenylenediamine (OPD), the wells are re-
incubated at room temperature for 10 minutes. Reactions are halted by the addition of 100 µl of 1 N
sulphuric acid, and the results, measured at 492 nm with an automated ELISA reader, are expressed as
absorbance values.
f) Chromatographic strip test

While not a definitive diagnostic test, a rapid chromatographic strip test (penside test; ref. 4) has proved a
useful tool for assisting field personnel in investigating suspected outbreaks of rinderpest.
a) The competitive enzyme-linked immunosorbent assay (the prescribed test for international trade)
A competitive ELISA is available for the detection of rinderpest antibodies in the serum of animals of any
species previously exposed to the virus. The test is based on the ability of positive test sera to compete with
a rinderpest anti-H protein MAb for binding to rinderpest antigen. The presence of such antibodies in the
test sample will block binding of the MAb, producing a reduction in the expected colour reaction following
the addition of enzyme-labelled anti-mouse IgG conjugate and a substrate/chromogen solution. As this is a
solid-phase assay, wash steps are required to ensure the removal of unbound reagents.
The rinderpest antigen is prepared from Madin–Darby bovine kidney cell cultures infected with the
attenuated Kabete ‘O’ strain of rinderpest virus. The viral antigen is extracted from the infected cells by
repeated cycles of sonication and centrifugation. The MAb was obtained by fusing the splenocytes of
hyperimmunised mice with the NSO myeloma cell line, and then shown to be rinderpest H protein specific
(2); this MAb has now been designated as C1. Both C1 and standardised rinderpest antigen are directly
available from the OIE Reference Laboratory for Rinderpest in the UK (see Table given in Part 3 of this
Terrestrial Manual). Kits are available commercially.
• Test procedure
i) Reconstitute the freeze dried rinderpest antigen with 1 ml of sterile water and further dilute it to the
manufacturer’s recommended working dilution using 0.01 M phosphate buffered saline (PBS), pH 7.4.
ii) Immediately dispense 50 µl volumes of the diluted antigen into an appropriate number of wells of a
flat-bottomed, high protein-binding ELISA microplate using two wells per test serum. Tap the sides of
the microplate to ensure that the antigen is evenly distributed over the bottom of each well and, having
sealed the plate, incubate it on an orbital shaker for 1 hour at 37°C. Wash the wells three times with
0.002 M PBS, pH 7.4.
iii) Add 40 µl of blocking buffer (0.01 M PBS, 0.1% [v/v] Tween 20 and 0.3% [v/v] normal bovine serum) to
each test well followed by 10 µl volumes of all test sera.
iv) Follow the manufacturer’s recommendations to prepare a working dilution of the MAb in blocking
buffer, and add 50 µl of this to each test well. Seal the plates and re-incubate on an orbital shaker for
1 hour at 37°C.
v) Follow the manufacturer’s recommendations to prepare a working dilution of rabbit anti-mouse
immunoglobulin horseradish peroxidase conjugate in blocking buffer and add 50 µl to each test well.
Seal the plates and re-incubate on an orbital shaker for 1 hour at 37°C.
vi) At the end of this period the plates are washed as before and immediately refilled with 50 µl volumes
of substrate/chromogen mixture (1 part 3% H2O2 to 250 parts OPD), and incubate at room
temperature for 10 minutes without shaking. Then add 50 µl of a stopping solution consisting of 1 M
sulphuric acid.
vii) The test system must include known rinderpest positive and negative serum samples, a MAb control
and a conjugate control.
viii) Measure the resulting absorbance values on an ELISA reader with a 492 nm interference filter and
express the test results as percentage inhibition values compared with the value obtained using the

MAb control. Inhibition values of 50% or more are considered to be positive and values below 50% are
considered to be negative.
Lowering the positive/negative threshold to 40% or less increases the sensitivity of the test, but inevitably
affects specificity by increasing the proportion of false-positive test results encountered. In practise, the
50% value is recommended by GREP at which level sensitivity is at least 70% and specificity exceeds 99%.
The sensitivity needs to be taken into account when designing sampling frames for serosurveillance.
An indirect ELISA method has been developed and might be useful for rinderpest surveillance programmes,
especially in areas in which lineage 2 rinderpest virus could be present (17). However, the performance
characteristics of the test indicate a problem with specificity and therefore its use will require confirmatory
testing.
b) Virus neutralisation
The ‘gold standard’ virus neutralisation (VN) test is performed in roller-tube cultures of primary calf kidney
cells following the method of Plowright & Ferris (11); the test has been validated in experimentally infected
cattle. In the roller tube procedure, sera, that has not been inactivated, are diluted at intervals of 1 in 10 and
then, starting with undiluted serum, mixed with an equal volume of 103.0 TCID50 per ml of the attenuated
Kabete ‘O’ vaccine strain virus. Mixtures are held overnight at 4°C, after which 0.2 ml volumes are
inoculated into each of five roller tubes, immediately followed by 1 ml of dispersed indicator cells suspended
in growth medium at a rate of 2 × 105 cells per ml. Tubes are incubated at 37°C, sloped for the first 3 days,
after which they are replenished with maintenance medium and placed on a roller apparatus. They are
examined regularly for virus-specific cytopathology and positive tubes recorded and discarded; the final
examination takes place on day 10.
For calculating end-points, the virus dose is regarded as satisfactory if the final dilution falls within the range
101.8 to 102.8 TCID50/tube. This test should be used to examine the sera of ELISA reactors during national
serosurveillance programmes designed to demonstrate freedom from infection, or to qualify susceptible
cattle for vaccine testing. Under these circumstances, the presence of any detectable antibody in the 1/2
final serum dilution is considered to indicate previous infection with rinderpest virus. The VN test is the test
of choice for the examination of wildlife serum samples.
A microplate method may be used as a screening test. In this procedure, an initial serum dilution of 1/5 is
further diluted at twofold intervals. Thereafter, 50 µl volumes of serum are incubated with 50 µl volumes of
virus diluted to contain between 101.8 and 102.8 TCID50 (15). Following a 45-minute or an overnight
incubation period, between 1 and 2 × 105 calf kidney, lamb kidney or Vero cells are added as indicators.
Tests are terminated after 6 or 7 days. Such tests may give indications of non-specific neutralisation at high
serum concentrations. There appear to be factors in some normal (with respect to prior rinderpest
exposure) sera that bring about the failure of the virus to penetrate and replicate in indicator cells. In the
tube test, these factors were probably removed during changes in maintenance medium; in the microplate
method, they remain present the whole time. If the most concentrated final serum dilution is limited to 1/10,
the effect disappears.

Many countries have used rinderpest vaccine to reduce the incidence of rinderpest to zero, and then followed the
OIE Pathway in order to have their rinderpest-free status internationally recognised. To obtain this status, the
process of annual rinderpest vaccination has largely been replaced by active and passive clinical and serological
surveillance. Intensive focal vaccination with homologous vaccine (immunosterilisation) was retained for dealing
with emergency campaign management (16).
The live attenuated tissue culture rinderpest vaccine (TCRV) described in previous editions of this Terrestrial
Manual was developed by Plowright by the serial passage of the virulent bovine rinderpest strain Kabete ‘O’
(RBOK) in primary bovine calf kidney cells. Due to the success of the Global Rinderpest Eradication Programme,
it is believed that few vaccine manufacturers continue to make this product, although a number of them may be
storing considerable stocks. However, the description published in the previous edition of the Terrestrial Manual
will be repeated here so that it is available if conditions change.
1. Seed management

Seed lots used in the manufacture of TCRV must produce a cell-culture vaccine that is safe, that confers an
immunity in cattle lasting at least 5 years, that retains its attenuated characteristics during at least five back

passages in cattle, and that lacks the ability to spread by contact. Substrains of RBOK used in the
manufacture of TCRV must be identifiable by written historical records, which must include information on
the origin of the strain and of its subsequent manipulations.
Vaccine seed must be maintained in a seed-lot system between passage levels 90 and 120. Seed-lot virus
must be preserved in a freeze-dried state at a temperature of –20°C or lower. The virus must be cultured in
Vero cells or primary or serially cultivated kidney cells derived from a normal bovine foetus or a very young
calf. Serially cultivated cells may not be more than ten passages removed from the primary cultivation.
Seed lots must be shown to be:
i) Pure: Free from contamination with viruses, bacteria, fungi or mycoplasmas.

ii) Safe: Inducing no abnormal clinical reaction on inoculation into rinderpest-susceptible cattle.
iii) Efficacious: Inducing an immunity to rinderpest in rinderpest-susceptible cattle.
Individual vaccine batches are prepared by infecting cell cultures and, after an appropriate incubation period,
harvesting the overlying media into which large numbers of live virus particles have been released. To facilitate
long-term storage and cold-chain distribution, this fluid is freeze dried in the presence of a cryoprotectant
consisting of 5% lactalbumin hydrolysate and 10% sucrose. Virus may be grown in primary kidney cells from
bovine embryos or calves, or cells derived in a homogeneous manner by up to ten serial subcultures from either
of these sources. In addition, vaccine may be manufactured in approved continuous cell lines provided the cells
are known to be non-infected with bovine viral diarrhoea (BVD) virus and are maintained in a seed lot system;
Vero cells have been used for this purpose. To constitute a batch, infected cultures must have been inoculated
with the same seed virus and incubated and harvested together. Two harvests are permissible from the same set
of cultures and may be pooled to form a bulk suspension. Written records must accompany all stages of vaccine
manufacture.
Cells: Primary cells, serially cultivated primary cells or continuous cell lines must have been derived from normal
looking animals or embryos, and must retain a normal morphology during cultivation. They must be shown to be
free of contamination with adventitious viruses, particularly BVD virus. Whatever cells are committed for vaccine
production, uninfected control cultures must be maintained using the same media and incubation conditions as
the rinderpest-infected cells. They must be subjected to frequent microscopic observations. After harvesting the
vaccine, the control cultures should be washed to remove ox serum and re-incubated for 10 days in media
containing ox serum substitutes. They are again subject to frequent microscopic observations for evidence of
cytopathic change. Simultaneously a sample of the cultures should be examined for the presence of
noncytopathic BVD virus using an immunofluorescence or immunoperoxidase test or RT-PCR. The serum used
in the culture media must come from rinderpest-susceptible animals.
Virus: A virus titration must be undertaken on the seed lot using tenfold virus dilutions in a microplate or roller
tube system and employing ten replicates per dilution. A similar titration must be undertaken on the final bulk.
Virus should be derived from cultures maintained in roller bottles and may not be harvested more than 10 days
after the date that these cultures were infected. The harvest should be clarified by low-speed centrifugation
before mixing with cryoprotectant. Prior to lyophilisation it may be held for not more than 5 days at 4°C but for
considerably longer if frozen at –20°C to –60°C. As adventitious viral contamination may arise during a
manufacturer's manipulations or from the use of contaminated media, rabbit hyperimmune serum should be used
to neutralise the rinderpest content of the bulk suspension, after which the mixture should be used to infect calf
kidney or Vero cells, which are handled as described above. The final bulk must be tested for freedom from
bacteria, fungi and mycoplasmas.
4. Batch control
a) Identity
The contents of one container from each filling lot must be exposed to neutralisation by rabbit rinderpest
antiserum, using a varying virus/constant serum method, and inoculated into bovine kidney cells. The
identity of the product is established if no rinderpest-specific CPE develop.

b) Sterility
Tests for sterility and freedom of contamination of biological materials may be found in Chapter 1.1.9.
c) Safety and efficacy

Using rinderpest susceptible cattle, the contents of five randomly selected vials are pooled and used to
inoculate one ox with a volume equivalent to 100 cattle field doses and one ox with a volume equivalent to
1/10th of a cattle field dose. These animals are maintained in close contact with an uninoculated ox for the
following 3 weeks. During this period the animals are subjected to daily temperature recording and frequent
clinical inspections. At the end of the 3 weeks, the cattle are examined for rinderpest neutralising antibodies
and challenged with a strain of rinderpest capable of inducing a pyrexia. The vaccine is considered safe and
efficacious if it does not induce any abnormal clinical reaction, if both animals receiving vaccine are
protected and if there is no evidence that the vaccine virus has been transmitted. This test is not a potency
test. Each vaccine lot must also be tested for innocuity in small animals.
d) Potency
The close relationship between immunising potency and infectivity allows the latter to be used as the basis
for potency estimations. Three infectivity titrations are undertaken using cells of an approved continuous
line or cells grown from each of three different bovine calf or embryonic kidneys. For the first titration, the
pool of vials used for the safety test may be employed. The second and third estimates are made on further
pools, each of three final containers. The sensitivity of the cells used in each working session must be
measured using a standard laboratory rinderpest virus preparation. The final titre is the geometric mean of
the three estimates, each undertaken using tenfold dilutions and ten observations per dilution.
e) Duration of immunity
It is unnecessary to routinely establish the duration of immunity to TCRV. Reported results indicate that
lifelong immunity can be expected following the successful vaccination of cattle free of all vestiges of
maternal immunity.
f) Stability
TCRV is highly stable when correctly freeze-dried and will keep for long periods at either +4 or –20°C
provided the product is stored under vacuum. Recent evidence indicates that the rate of degradation of
lyophilised TCRV can be altered by the choice of stabiliser and by variations in the drying cycle. The most
advantageous results were associated with the use of a 5% lactalbumin hydrolysate/10% sucrose stabiliser,
a 72–74 hour drying cycle under reduced vacuum (100 milliTorr), initial drying for 16 hours at –30°C, and a
final shelf temperature of 35°C. With high release titres, such vaccine can be used in the field for 30 days
without refrigeration. Following reconstitution in either normal saline or 1M magnesium sulphate, the virus
becomes much more thermolabile. The period for field distribution of reconstituted vaccine should not
exceed its half-life, but as this parameter is temperature dependent and varies between 8 and 24 hours
over a range from 4°C to 37°C, a common sense limit must be applied; this can be determined by National
Control Authorities, but a universal period of 4 hours can be recommended.
g) Preservatives
TCRV contains lactalbumin hydrolysate and sucrose which are added as cryoprotectants; otherwise it
contains no specific chemical preservative.
h) Precautions (hazards)
There are no known hazards associated with the manufacture or field use of TCRV.
REFERENCES
1. ANDERSON J., BARRETT T. & SCOTT G.R (1966). Manual on the Diagnosis of Rinderpest, Second Edition. FAO
Animal Health Manual No.1. Food and Agriculture Organisation of the United Nations (FAO), Rome, Italy,
143 pp.
2. ANDERSON J., MCKAY J.A. & BUTCHER R.N. (1991). The use of monoclonal antibodies in competitive ELISA for
the detection of antibodies to rinderpest and peste des petits ruminants. In: The Seromonitoring of
Rinderpest Throughout Africa. Phase One. Proceedings of Final Research Co-ordination Meeting. Joint
FAO/IAEA (Food and Agriculture Organisation of the United Nations/International Atomic Energy Agency)
Division, Vienna, Austria, 43–53.

3. BROWN C.C. (1997). A review of three pathology-based techniques for retrospective diagnosis of rinderpest,
with comparison to virus isolation. Res. Vet. Sci., 63, 103–106.
4. BRUNING A., BELLAMY K., TALBOT D. & ANDERSON J. (1999). A rapid chromatographic test for the pen-side
diagnosis of rinderpest virus. J. Virol. Methods, 81, 143–154.
5. FOREMAN A.J., ROWE L.W. & TAYLOR W.P. (1983). The detection of rinderpest antigen by agar gel diffusion and
counterimmunoelectrophoresis. Trop. Anim. Health Prod., 15, 83–85.
6. FORSYTH M.A. & BARRETT T. (1995). Evaluation of polymerase chain reaction for the detection and
characterisation of rinderpest and peste des petits ruminants viruses for epidemiological studies. Virus Res.,
39, 151–163.
7. KOCK R.A. (2006). Rinderpest and wildlife. In: Rinderpest and Peste des Petits Ruminants, Virus Plagues of
Large and Small Ruminants, Barrett T., Pastoret P.-P. & Taylor W.P., eds. Academic Press, Oxford, UK,
143–162.
8. LIBEAU G., DIALLO A., COLAS F. & GUERRE L. (1994). Rapid differential diagnosis of rinderpest and peste des
petits ruminants using an immunocapture ELISA. Vet. Rec., 134, 300–304.
9 MARINER J.C. & ROEDER P.L. (2003). Use of participatory epidemiology in studies of the persistence of
lineage 2 rinderpest virus in East Africa. Vet. Rec., 152, 641–647.
10. OAU-IBAR-PACE (ORGANIZATION OF AFRICAN UNITY-INTERAFRICAN BUREAU FOR ANIMAL RESOURCES-PAN-

AFRICAN PROGRAMME FOR THE CONTROL OF EPIZOOTICS) (2002). Report on the Eastern African Regional
Workshop on Mild Rinderpest. Nairobi, Kenya, 17–19 June 2002.
11. PLOWRIGHT W. & FERRIS R.D. (1961). Studies with rinderpest virus in cell culture. III. The stability of cultured
virus and its use in neutralisation tests. Arch. Gesamte Virusforsch., 11, 516–533.
12. ROEDER P.L. & TAYLOR W.P (2002). Rinderpest. Vet. Clin. North Am. Food Anim. Pract., 18, 515–547.
13. ROEDER P.L., TAYLOR W.P. & RWEYEMAMU M.M. (2006). Rinderpest in the twentieth and twenty first
centuries. In: Rinderpest and Peste des Petits Ruminants, Virus Plagues of Large and Small Ruminants,
Barrett T., Pastoret P.-P. & Taylor W.P., eds. Academic Press, Oxford, UK, 105–142.
14. TAYLOR W.P & BARRETT T. (2007). Peste des Petits Ruminants and Rinderpest in Diseases of Sheep, Fourth
Edition, Aitken I.D., ed. Blackwell Publishing Ltd, Oxford, UK.
15 TAYLOR W.P. & ROWE L.W. (1984). A microneutralisation test for the detection of rinderpest virus antibodies.
Rev. Elev. Med. Vet. Pays Trop., 37, 155–159.
16. TAYLOR W.P., ROEDER P.L., RWEYEMAMU M.M., MELEWAS J.N., MAJUVA P., KIMARO R.T., MOLLEL J.N., MTEI B.J.,
W AMBURA P., ANDERSON J., ROSSITER P.B., KOCK R., MELENGEYA T. & VAN DEN ENDE R. (2002). The control of
rinderpest in Tanzania between 1997 and 1998. Trop. Anim. Health Prod., 34, 471–487.
17. YILMA T., AZIZ F., AHMAD S., JONES L., NGOTHO R., W AMWAYI H., BEYENE B., YESUS M., EGZIABHER B., DIOP M.,
SARR J. & VERARDI P. (2003). Inexpensive vaccines and rapid diagnostic kits tailor-made for the global
eradication of rinderpest, and technology transfer to Africa and Asia. Dev. Biol. (Basel), 114, 99–111.
*
* *
NB: There are OIE Reference Laboratories for Rinderpest (see Table in Part 3 of this Terrestrial Manual or

CHAPTER 2.1.16.
TRICHINELLOSIS
SUMMARY
Trichinellosis in humans is caused by eating raw or undercooked meat from Trichinella-infected

domestic animals or game. The adult worms survive less than 2 months in the small intestine of
host species including humans, pigs, rats, bears, walruses, occasionally in horses and many other
flesh-eating mammals, and birds and reptiles. Trichinella larvae occur in the muscles of their hosts
and susceptible individuals become infected by ingestion of tissue containing these larvae.
Identification of the agent: Diagnostic tests for trichinellosis fall into two categories: 1) direct
detection of first-stage larvae encysted or free in striated muscle tissue, and 2) indirect detection of
infection by tests for specific antibodies.
Tissue digestion and tissue compression methods have been used for the direct detection of
Trichinella larvae in tissues. Trichinella larvae usually localise in preferred muscle sites, particularly
in low level infections, and these sites may vary by host species. It is important that preferred sites
be sampled to maximise test sensitivity. For example, in pigs, the diaphragm (crus), tongue,
masseter and abdominal muscles are preferred sites, whereas in horses, the tongue and masseter
harbour the most worms, followed by the diaphragm and neck muscles.
The artificial digestion methods involve the enzymatic digestion of individual or pooled muscle
tissue samples and incorporate mechanical homogenisation or grinding, stirring, and incubation.
This is followed by filtration and sedimentation procedures to recover and concentrate any larvae
that are released from muscle during digestion. Samples processed by these methods are
examined under a stereomicroscope for the presence of larvae. Digestion tests can detect <1 larva
per gram (lpg) of tissue, but at these low levels of infection, uneven distribution of larvae within
tissues is a limiting factor. This is compensated for by testing larger samples per carcass, such as
a minimum of 1–5 g for pigs and 5–10 g for horses and game. Digestion methods are
recommended for the inspection of individual carcass of food animals such as pigs, horses and
game.
The compression method is less sensitive than artificial digestion and is not recommended as a
reliable test for inspection of carcasses. Although now outdated, this method has been used widely
in the past and is mentioned here for completeness. It involves visual inspection of compressed
pieces of muscle tissue for the presence of larvae in situ. This method can be performed with a
stereomicroscope, or a specialised microscope, (trichinoscope) which has an estimated efficiency
of detecting as few as three larvae/g of tissue. It has the disadvantage of requiring considerable
time for the inspection of multiple samples from each carcass. It is also very difficult to detect the
non-encapsulated Trichinella larvae (T. pseudospiralis, T. papuae and T. zimbabwensis).
Compression techniques are only useful for detecting medium to high infections when few animals
require examination and facilities are not available for testing by artificial digestion.
Serological tests: Serological assays are the most common tests used for indirect detection. The
sensitivity and specificity of serological methods are mainly dependent upon the type and quality of
antigen used. Most serological test performance (validation) data are from pigs. False negative
serological results may occur 3 weeks or longer after muscle larvae become infective in pigs with
light or moderate infections. A low rate of false-positive results has also been reported for
serological tests. For the purposes of individual carcass inspection, only direct methods can be
recommended. For surveillance or verification of Trichinella-free herds or regions, serological
methods are acceptable. Pigs harbouring as few as one larva/100 g of tissue have been detected.
The specificity of enzyme-linked immunosorbent assays (ELISA) for Trichinella infection is directly

Chapter 2.1.16. — Trichinellosis
linked to the type and quality of the antigen employed in the test. Secretory antigens collected by
short-term (18–20 hours) maintenance of T. spiralis muscle larvae in vitro and synthetic
carbohydrate antigens currently provide the most specific and economical source, although a low
rate of false-positive results has been obtained in some studies. It is critical that appropriate
positive and negative control sera be used to ensure that ELISAs are performing at a minimum
acceptable level of sensitivity and specificity. The digestion of 100 g or more of tissue is
recommended as a confirmatory test for serologically positive animals.
Requirements for vaccines and diagnostic biologicals: There are no suitable vaccines for
Trichinella infection in food animals. For indirect (serological) detection methods, appropriate
antigens must be used to ensure adequate test specificity and sensitivity. These antigens may be
obtained from the secretory products of muscle larvae maintained in-vitro. There is a critical need
for an international bank of reference sera to provide a common standard for Trichinella serological
assays.
A. INTRODUCTION
Trichinellosis in humans is caused by eating raw or undercooked meat from Trichinella-infected food animals or
game (10). The short-lived adult worms live in the small intestine of host species including humans, pigs, rats,
bears, walruses, horses, many other flesh-eating mammals, and some birds and reptiles. The parasite has a
direct life cycle. Within hours following consumption of infected muscle by a suitable host, first stage muscle
larvae (L1) are released by digestion and burrow into the villi of the small intestine. They develop rapidly into
adults (males up to 1.8 mm long, females up to 3.7 mm long) and survive for less than 2 months. During this
time, copulation takes place and the ovo-viviparous females release new-born larvae (NBL), which migrate via
venules and lymphatics into the general circulation. NBL are distributed throughout the body where they invade
striated muscles, showing predilection for specific muscle groups. For example, in pigs, the tongue usually
contains the highest concentration of larvae, followed by the diaphragm, and in horses, the tongue followed by
masseter muscle. Predilection sites vary by host species, but in general, tongue, masseter and diaphragm are
optimal sites for sampling. Current knowledge on predilection sites is available for several host species (22). In
cases of severe infection most voluntary muscles contain high numbers of larvae. The larvae of most Trichinella
species become encapsulated in collagen in host musculature where they remain infective for years.
Within the genus Trichinella eleven genotypes have been identified, eight of which have been designated
species status (10, 21, 27). Trichinella spiralis (T-1) is distributed in temperate regions world-wide and is
commonly associated with domestic pigs. It is highly infective for domestic and sylvatic swine, mice and rats, but
it can be also be detected in other mammalian carnivores. Trichinella nativa (T-2) occurs in mammalian
carnivores of arctic and sub-arctic regions of North America, Europe and Asia. Trichinella britovi (T-3) is found
predominantly in wild animals, and occasionally in pigs or horses. It occurs in temperate regions of Europe, Asia,
and in Northern and Western Africa. Trichinella pseudospiralis (T-4) is cosmopolitan in distribution and has been
recovered from raptorial birds, wild carnivores and omnivores, including rats and marsupials in Asia, North
America, Europe and Australia. Unlike most other Trichinella genotypes, T-4 is not enclosed within a collagen
capsule in muscle. Trichinella murrelli (T-5) is found in mammalian carnivores of North America. It has low
infectivity for domestic pigs, but poses a risk to humans who eat game meats. Trichinella T-6 is cold-climate-
adapted and appears to be closely associated with T. nativa in northern North America (27). Both T. nativa and
T-6 are highly resistant to freezing. They have limited infectivity for pigs. Trichinella nelsoni (T-7) has been
isolated from mammalian carnivores and sporadically from wild pigs in Eastern Africa. Trichinella T-8 has been
detected in mammalian carnivores in Namibia and South Africa and Trichinella T-9 in mammalian carnivores in
Japan (27). T-8 and T-9 have some intermediate characteristics with T. britovi and T. murrelli, respectively. Like
T. pseudospiralis, T. papuae (T-10) and T. zimbabwensis (T-11) are non-encapsulated muscle parasites.
Trichinella papuae has been reported from wild and domestic pigs, farmed crocodiles and humans in Papua New
Guinea. Trichinella zimbabwensis has been described in farmed and wild crocodiles in Zimbabwe, Ethiopia and
Mozambique and in monitor lizards in Zimbabwe. Experimentally, it shows a high infectivity for a wide spectrum
of mammalian hosts including pigs and rats (27). All species and genotypes of Trichinella cause disease in
humans.
Human trichinellosis can be a debilitating disease and may result in death. The short-lived adult worms in the
intestine can cause transient gastroenteritis, but the most severe signs and symptoms result from the migration
and presence of the larvae in voluntary muscle. The disease is transmitted by eating infected meat that has not
been sufficiently cooked (or otherwise made safe). Prevention of human infection is accomplished by meat
inspection, by processing (cooking, freezing, or curing of meat), and by preventing the exposure of food animals
to infected meat including uncooked food waste, rodents and other wildlife (10, 12, 13,). Game meats should
always be considered a potential source of infection, and should be tested or properly cooked. Trichinella found
in game meats (mainly T. nativa, T-6 and to a lesser degree T. britovi) may be resistant to freezing and therefore
untested, frozen game poses a public health risk.

Testing methods for the detection of Trichinella infection in pigs and other species either: (a) directly demonstrate
the parasite in tissue samples; or (b) indirectly demonstrate the parasite by using immunological methods to
detect specific antibodies to Trichinella spp. in blood, serum or tissue fluid samples.
1. Identification of the agent (the prescribed test for international trade)
The only recommended procedures for the detection of Trichinella larvae in meat are digestion assays. A number
of digestion assays are officially recognised in various countries for trade purposes. The International
Commission on Trichinellosis (ICT) recommends several of these assays, which are documented standards in
the EU, Canada or the USA. However, a number of other official methods not currently used routinely are not
recommended because of their lack of efficiency or reliability. Modern diagnostic assays should meet
internationally accepted standards of quality assurance, which include scientifically derived validation data and a
design that allows routine monitoring and documentation of critical control points. Although there is general
consensus that the digestion assay is the best procedure, a universally accepted digestion test protocol for trade
and food safety purposes is not yet available. The digestion assay recommended here is based on desirable
innovations inherent in some digestion assays that are accepted for international trade purposes.
a) Recommended direct procedure for testing meat

Sensitivity: the sensitivity of direct testing methods depends on the amount of tissue examined and the site
from which the sample was obtained. Direct methods will identify infected pigs, horses or other animals
infected with T. spiralis as early as 17 days after exposure, coincident with the time that muscle larvae
become infective for a new host. Direct methods remain effective as long as muscle larvae remain viable.
To insure the viability of the trichinae, tissue samples must not be kept for long periods of time or frozen
before testing. Current methods for testing by artificial digestion employing a 1-g sample have a sensitivity
of approximately three larvae/g of tissue, and testing of a 5-g sample increases sensitivity to 1 larva/g of
tissue (13, 22). Where large amounts of tissue (up to 100 g) are available for digestion, the sensitivity of this
test is further increased.
Sampling: tests are usually conducted on carcass samples collected post-mortem. Muscle samples are
taken from predilection sites, usually the diaphragm pillars or tongue of pigs, or tongue or masseter
muscles of horses. Sample sizes can vary; individual samples of 100 g may be taken from one animal, or
multiple samples of lesser amounts may be collected from a number of animals to make a 100-g pool. The
size of the samples that make up the pool will determine the sensitivity of the method. The ICT
recommends 5-g samples per pig for testing in endemic areas. For testing horsemeat, a minimum of 5 g per
carcass is required. For horses originating from endemic areas, a 10-g sample is recommended.
• Digestion and detection

i) Determine the volume of digestive solution required for the digestion (2000 ml of the solution for 100 g
of meat, and 1000 ml for 50 g or less).
ii) Digestive solution: Prepare the appropriate volume of 37% HCl/water solution (0.55% v/v 37% HCl) by
combining the HCl with tap water (e.g. 11 ml of 37% HCl with 1989 ml water). Do not add pepsin to the
solution at this time. This solution should be preheated to 45°C before use.
iii) Remove as much fat and fascia as possible from each sample of meat.
iv) Weigh the appropriate amount of trimmed meat from each sample. Cut each sample into 1–2 g pieces
and pool with other samples into a 100-g amount.
v) Place the pooled meat sample into a blender. Add 50–100 ml of the water/HCl solution for a 100 g
sample pool.
vi) Chop the meat in a blender until it is homogeneous (no chunks of meat should be present; the sample
should be the consistency of pureed baby food). This is usually achieved with several 1–3-second
pulses. Add approximately 100 ml of the prepared water/HCl solution and blend until the mixture is
uniformly liquid. This may take 5–10 seconds (additional solution may be needed).
vii) Sprinkle 10 g of pepsin (1:10,000 NF/1:12500 BP/2000 FIP; granular preferred) on to the homogenate,
add about 200 ml of water/HCl solution, and blend for about 5 seconds.
viii) Transfer the homogenised sample to a 3-litre beaker containing a stir bar. Add the remainder of the
2 litres of water/HCl solution by pouring the water/HCl into the blender and rinsing all residual
homogenate into the 3-litre beaker. Rinse any adhering material from the blender lid into the beaker
using 10–20 ml of digestive solution from a squirt bottle.

ix) Place the beaker on a preheated magnetic stirrer hotplate or in an incubation chamber set at 45±2°C.
Cover the beaker with aluminium foil. Activate the stirrer at a sufficiently high speed to create a deep
vortex without splashing. Note: If the digest temperature at the beginning of digestion is not 45±2°C,
the sample should be allowed to warm to this temperature before the timing of the digestion is started.
x) Allow the digestion to proceed for 30 minutes. If the temperature of the digest has fallen below
45±2°C, additional digestion time may be required to complete the digestion. This can be determined
by observing the digestion mixture. If pieces of undigested muscle tissue are present, the digestion
should be continued for an additional 30 minutes or until the pieces are digested. Care should be
taken to ensure that the digestion temperature range is not exceeded. Alternatively, the digestion may
be performed at 37°C for a longer period of time.
xi) Within 5 minutes of removal from the magnetic stirrer hot plate pour the digestion fluid through a 177–
180-µm sieve and into a 2-litre separatory funnel. Rinse the beaker with room temperature tap water
from a squirt bottle and pour this through the sieve into the 2-litre separatory funnel.
xii) Rinse the sieve into the 2-litre separatory funnel by squirting a small volume of room temperature tap
water through the top of the sieve. There should be no undigested pieces of muscle remaining on the
sieve, although small remnants of fat, fascia and other tissues may be present. Allow the fluid in the
separatory funnel to settle undisturbed for 30 minutes.
xiii) Drain 40 ml of digestion fluid from the separatory funnel into a 50 ml conical tube or measuring
cylinder (Pilsner flask) and allow to stand for 10 minutes.
xiv) At the end of 10 minutes use a pipette to remove 30 ml of the upper part of the fluid (supernatant),
leaving the bottom 10 ml in the tube (do not pour off the upper 30 ml, as this will disturb the sediment).
xv) Gently swirl the remaining 10 ml of fluid and quickly transfer it into a gridded Petri dish or larval-
counting basin. Rinse the tube or cylinder into the Petri dish twice using 5 ml of tap water each time.
The layer of fluid in the petri dish should not be more than a few millimetres deep.
xvi) Wait a minimum of 1 minute to allow larvae to settle to the bottom, then use a stereomicroscope at
×10–16 magnification to systematically examine each grid of the Petri dish for the presence of
Trichinella larvae. The detection of any suspect larvae on the systematic examination must be
confirmed by the identification of morphological details at a higher magnification such as ×40. If the
sediment is cloudy or otherwise difficult to examine, it will require further clarification as described
below.
xvii) Digests should be examined soon after they are ready. Under no circumstance should examination of
digests be postponed until the following day.
xviii) If digests are not examined within 30 minutes of their preparation they may require clarification as
described below.
xix) Sample clarification: transfer the contents of the Petri dish into a 50 ml conical tube using a pipette.
Rinse the Petri dish thoroughly with tap water, adding the rinse water to the conical tube. Add
additional tap water to bring the volume to 45 ml. Let the tube settle undisturbed for 10 minutes.
At the end of 10 minutes use a pipette to withdraw the supernatant, leaving the bottom 10 ml (do not
pour off the supernatant, as this will disturb the sediment). Save the removed fluid for disposal or
decontamination after the sample has been read.
Repeat steps xv and xvi.
xx) In the event of a positive or doubtful result, a further sample should be collected from each carcass
making up the pooled sample. These should be tested individually or in successive smaller pools until
the individual infected animals are identified.
Identification of the larvae: first stage larvae, digested free from muscle cells, are approximately 1 mm in
length and 0.03 mm in width. The most distinguishing feature of Trichinella larvae is the stichosome, which
consists of a series of discoid cells lining the oesophagus and occupying the anterior half of the worm's
body. Trichinella larvae may appear coiled (when cold), motile (when warm) or C-shaped (when dead). In
case of doubt, larvae should be viewed at higher magnification and further tissues should be examined. If
the counts are high, appropriate dilutions must first be made.
Quality assurance: laboratories using artificial digestion methods should maintain a suitable quality
assurance system to ensure test sensitivity. Components of a quality assurance system for digestion testing
are described by the ICT (13) and elsewhere (9) and should include regular use of proficiency testing (7, 8).
b) Other tests
• Other direct detection methods
i) The double separatory funnel method: this assay is recommended as an alternative to the commonly
used digestion procedure described above, and is approved by the EU for export use. The method

was designed to operate under strict conditions of quality control, minimise technical error, and has
been extensively validated for use on pork and horse meat (5). It includes a spin-bar digestion
technique and sequential separatory funnels for sedimentation of the larvae. The procedure has fewer
steps, requires less time and seldom needs further clarification steps. An incubation chamber
equipped with transparent glass doors and set at 45°C is used to perform the digestion. The digestion
is conducted in 3 litres of digest fluid on a magnetic stirrer. Following digestion the suspension is
poured into a 4-litre separatory funnel through a 177–180-µm sieve, which is rinsed thoroughly into the
separatory funnel with tap water. The suspension is allowed to settle for 30 minutes and 125 ml is
drained into a 500-ml separatory funnel. The volume is increased to 500 ml by adding 375 ml of tap
water, and the resultant suspension is allowed to settle for an additional 10 minutes. Finally, 22–27 ml
of sediment is drained into a Petri dish and observed for larvae as previously described.
ii) The mechanically assisted pooled sample digestion method/sedimentation technique (Method 4:
84/319/EEC): this method uses a heated Stomacher blender for the digestion phase, and a separatory
funnel for sedimentation of the larvae (3).
iii) Polymerase chain reaction: limited studies have shown that PCR can be used to detect larvae in the
nucleic acid of larvae in the musculature of infected animals. However, this method lacks sensitivity
and is not practical for routine testing of food animals. Identification of the species or genotype of
Trichinella recovered from muscle tissue is useful in understanding the epidemiology of the parasite in
animals, in assessing the relative risk of human exposure and to trace back the infection to the farm of
origin. Specific primers have been developed that allow the identification of single larva collected from
muscle tissues at the species and genotype by PCR (25). Requests for speciation or genotyping of
Trichinella larvae can be made through the OIE Reference Laboratories in Rome, Italy or Saskatoon,
Canada (see Table given in Part 3 of this Terrestrial Manual; and www.iss.it/Trichinella/index/asp).
• Direct detection methods not recommended for meat inspection

i) Trichinoscopy: This method involves the compression of multiple 2 × 10 mm pieces of muscle tissue
between two glass plates (compressorium) until they become translucent, followed by examination
using a microscopic technique (2). Although this method has been in use for many decades, it is
labour intensive and there are good comparative data available indicating that it is not as sensitive as
digestion assays (6). Increasing the sample size to compensate is not practical for testing large
numbers of animals, and the non-encapsulated Trichinella spp. (T. pseudospiralis, T. papuae and
T. zimbabwensis) may occur uncoiled outside of muscle cells, making them difficult to detect using
trichinoscopy. Because of these limitations, trichinoscopy and similar compression methods are not
recommended for the routine examination of carcasses.
ii) Trichomatic 35: this method involves an automated digestion chamber and a membrane filter for the
recovery and examination of larvae. The critical steps of the digestion and recovery process are
difficult to monitor in this system, and test capacity is only 35 g.
• Immunological methods
A variety of immunological assays have been described for the diagnosis of trichinellosis in domestic and
wild animals (17). Methods include immunofluorescence assay (IFA), immuno-electrotransfer blot (IEBT),
western blot, enzyme immunohistochemical assays, and enzyme-linked immunosorbent assays (ELISA).
Except for the ELISA, these tests have not been standardised, and reagents are not available for routine
use. Nevertheless, the ICT has provided a uniform set of recommendations for the development and use of
serological tests for the detection of circulating antibodies (17). The ELISA is the only immunological assay
endorsed by the ICT. It is only approved as a epidemiological surveillance tool to detect anti-Trichinella
antibodies in pigs; it is not reliable for the detection of Trichinella infection in individual animals.
Although other serological tests may have some practical applications, the ELISA is generally acknowledged as
the test of choice based on economy, reliability, adaptability to good quality assurance practices, increasing body
of validation data and good sensitivity and specificity when conducted under appropriate conditions. It is a useful
tool for testing populations and is routinely used for surveillance programmes and disease outbreak
investigations. Nevertheless, for reasons given below, the ELISA is not recommended for the testing of individual
pigs for food safety purposes.
a) Enzyme-linked immunosorbent assay (ELISA)

• Sensitivity and specificity
Infection levels as low as one larva/100 g of tissue are detectable by ELISA in pigs (17). This high level of
sensitivity makes serological testing by ELISA a useful method for detecting ongoing transmission of
Trichinella infection at the farm or for more broadly based surveillance programmes. A disadvantage of

serology for the detection of trichinellosis is the low rate of false-negative results observed in infected
animals. This is primarily due to the lag time of the immune response following the ingestion of infective
larvae. Detectable levels of antibody are not usually present in pigs until 3–5 weeks or more following
exposure (11, 14). For this reason, serological methods are not recommended for individual carcass testing.
Serological responses in pigs persist for a long time after infection with no decline in titre, however, antibody
has been reported to decline in horses within a few months following infection (22). Serological tests may
be of little practical use in horses as antibody titres eventually drop below diagnostic levels despite the
presence of infective larvae in muscle (19, 26). Little is known of antibody responses to Trichinella infection
in game animals, but high quality serum samples should be obtained to reduce the likelihood of false
positive reactions.
• Samples
The use of ELISA to detect the presence of parasite-specific antibodies provides a rapid method that can be
performed on serum, blood or tissue fluid collected before or after slaughter (15). The dilution used is
different for serum than for tissue fluid (23).
• Antigens
The specificity and sensitivity of ELISA is largely dependent on the quality of the antigen used in the test.
Antigens that are specifically secreted from the stichocyte cells of living L1 larvae and bear the TSL-1
carbohydrate epitope are recognised by Trichinella-infected animals. The antigens recognised in worm ES
products consist of a group of structurally related glycoproteins with molecular weights of 45–55 kDa (24). A
synthetic carbohydrate antigen (Tyvelose) has also been used in ELISA. Studies in swine indicate that
Tyvelose may be as good as ES antigen for surveillance testing in pigs however, the sensibility of the
ELISA using this synthetic antigen is lower than that using ES antigens (4, 18). Antigen preparations have
been developed that provide a high degree of specificity for Trichinella infection in pigs (16). The T. spiralis
ES antigens used in the ELISA are conserved in all species and genotypes of Trichinella (24), and therefore
infection may be detected in pigs or other animals harbouring any of the eight species.
• Antigen production
Diagnosis of Trichinella infection by ELISA can be accomplished by using stichosome antigens collected
from the ES products of Trichinella larvae in culture (16). For purposes of standardisation, it is
recommended that T. spiralis be used for antigen production for food animal testing. However, it has been
demonstrated that antigen prepared from any of the Trichinella species can be used for detection of
antibodies in infected animals regardless of the infecting species (20). Parasites to be used for antigen
preparation may be maintained by serial passage in mice or rats.
To prepare antigen for use in the ELISA (16), T. spiralis (T-1) muscle-stage larvae are recovered from
skinned, eviscerated, ground mouse or rat carcasses by digestion in 1% pepsin with 1% HCl for 30 minutes
at 37°C (as described above). These larvae are washed (three times for 20 minutes each) in Dulbecco’s
modified Eagle’s medium (DMEM) with penicillin (500 units/ml) and streptomycin (500 units/ml), and then
placed (at a density of 5000 L1/ml) into DMEM supplemented with HEPES (N-2-hydroxyethylpiperazine, N-
2-ethanesulphonic acid) (10 mM), glutamine (2 mM), pyruvate (1 mM), and penicillin (250 units/ml)/
streptomycin (250 µg/ml) (complete DMEM) at 37°C in 10% CO2 in air. Culture medium is recovered after
18–20 hours, worms are removed by filtration, and the fluid is concentrated under pressure with a 5000 Da
molecular weight retention membrane. ES antigens thus recovered may be stored frozen for short periods
at –20°C or for longer at –70°C; they consist of approximately 25 protein components as determined by
SDS/PAGE (sodium dodecyl sulphate/polyacrylamide gel electrophoresis), many of which bear the
diagnostic TSL-1 carbohydrate antigen epitope.
Antigen purity is critical to the specificity of the ELISA. Steps should be taken to monitor growth of bacteria
either visually, by phase microscopy, or by plating a sample of media. Cultures showing any bacterial
growth should be discarded. Larvae should not be maintained longer than 18 hours; worm deterioration
after this time contributes to leaking of somatic antigens that reduce test specificity. Antigen, produced as
described, should have a 280:260 nm absorbance ratio of >1.0. The antigens obtained from in-vitro
maintenance of Trichinella larvae, should be tested against a panel of known negative and positive sera
before use.
• Test procedure
An example of an ELISA for detecting Trichinella infection in pigs is given below. It is essential that all
reagents used in the assay be standardised for optimal concentration to obtain reliable results. Typical
values are indicated in the example.
i) Coat 96-well microtitre plates with 100 µl/well of T. spiralis ES antigens diluted to 5 µg/ml in coating
buffer (50 mM carbonate/bicarbonate buffer, pH 9.6). Coating is performed for 60 minutes at 37°C or
overnight at 4°C.

ii) Wash antigen-coated wells three times with wash buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl,
5.0% non-fat milk powder and 1.0% Triton X-100. Following each washing, plates are blotted dry.
iii) Dilute pig sera 1/50 or 1/100 in wash buffer. Alternative sources of antibodies that may be used in
place of sera include whole blood or tissue fluids at the dilution of 1/5 or 1/10 (23). Add 100 µl of
diluted sera to antigen-coated wells. A known positive and known negative serum sample should be
used on each plate at the same dilution as the test sera. Incubate at room temperature for 30 minutes.
iv) Wash wells three times as in step ii.
v) Add 100 µl/well of an affinity-purified rabbit anti-swine IgG–peroxidase conjugate at an appropriate
dilution in wash buffer, e.g. a 1/1000 dilution of rabbit anti-swine IgG (0.1 mg/ml) produced by
Kirkegaard and Perry Laboratories, Gaithersburg, Maryland, USA. Following the addition of the second
antibody, incubate the plates for 30 minutes at room temperature.
vi) Wash wells three times as in step ii. Rinse once with distilled water.
vii) Add 100 µl of a suitable peroxidase substrate (e.g. 5’-aminosalicylic acid [0.8 mg/ml] with 0.005%
hydrogen peroxide, pH 5.6–6.0).
viii) After 5–15 minutes, read plates for colour density at 450 nm on an automated microplate reader.
Values obtained in the ELISA four times that of normal serum pool controls are considered to be
positive. Values three times higher than normal are classified as suspect.
Commercial adaptations of the ELISA are available. The manufacturer must validate the kit prior to
licensure and the user should also evaluate the performance of the kit, prior to use, by using selected
negative and positive reference samples.
The test should be conducted within an environment in which internationally accepted standards of quality
management, such as ISO 17025, have been implemented.

There are no vaccines for trichinellosis in food animals or game. There are no biological reagents required for
direct observation of the parasite. For applicable immunological methods, TSL-1 antigens are recommended to
maximise test specificity. These antigens may be obtained as ES products recovered from in-vitro maintenance
of muscle larvae as described above.
REFERENCES
1. BOIREAU P., VALLEE I., ROMAN T., PERRET C., MINGYUAN L., GAMBLE H.R. & GAJADHAR A. (2000). Trichinella in
horses: a low frequency infection with high human risk. Vet. Parasitol., 93, 309–320.
2. EUROPEAN ECONOMIC COMMUNITY (1977). Commission Directive 77/96/EEC. Off. J. European Communities,
26, 67–77.
3. EUROPEAN COMMISSION (2005). Commission Regulation (EC) No. 2075. Off. J. European Union, 338, 60–82.
4. FORBES L,B., APPLEYARD G.D. & GADJADHAR A.A. (2004). Comparison of synthetic tyvelose antigen with
excretory-secretory antigen for the detection of trichinellosis in swine using enzyme-linked immunosorbent
assay. J. Parasitol., 90, 835–840.
5. FORBES L.B. & GAJADHAR A.A. (1999). A validated Trichinella digestion assay and an associated sampling
and quality assurance system for use in testing pork and horse meat. J. Food Prot., 62, 1308–1313.
6. FORBES L.B., PARKER S. & SCANDRETT W.B. (2003). Comparison of a modified digestion assay with
trichinoscopy for the detection of Trichinella larvae in pork. J. Food Prot., 66, 1043–1046
7. FORBES L.B., RAJIC A. & GAJADHAR A.A. (1998). Proficiency samples for quality assurance in Trichinella
digestion tests. J. Food Prot., 61, 1396–1399.
8. FORBES L.B., SCANDRETT W.S. & GAJADHAR A.A. (2005). A program to accredit laboratories for reliable testing
of pork and horsemeat for Trichinella. Vet. Parasitol., 132, 173–177.
9. GAJADHAR A.A. & FORBES L.B. (2001). An internationally recognized quality assurance system for diagnostic
parasitology in animal health and food safety with example data on trichinellosis. Vet. Parasitol., 103, 133–140.

10. GAJADHAR A.A., SCANDRETT W.B. & FORBES L.B. (2006). Overview of food- and water-borne zoonotic
parasites at the farm level. Rev. sci. tech. Off. int. Epiz., 25 (2), 595–606.
11. GAMBLE H.R. (1996). Detection of trichinellosis in pigs by artificial digestion and enzyme immunoassay. J.
Food Prot., 59, 295–298.
12. GAMBLE H.R. (1997). Parasites associated with pork and pork products. Rev. sci. tech. Off. Int. Epiz., 16,
496–506.
13. GAMBLE H.R., BESSONOV A.S., CUPERLOVIC K., GAJADHAR A.A., VAN KNAPEN F., NOECKLER K., SCHENONE H. &
ZHU X. (2000). International Commission on Trichinellosis: Recommendations on methods for the control of
Trichinella in domestic and wild animals intended for human consumption. Vet. Parasitol., 93, 393–408.
14. GAMBLE H.R., GAJADHAR A.A. & SOLOMON M.B. (1996). Methods for the detection of trichinellosis in horses. J.
Food Prot., 59, 420–425.
15. GAMBLE H.R. & PATRASCU I.V. (1996). Whole blood, serum, and tissue fluids in an enzyme immunoassay for
swine trichinellosis. J. Food Prot., 59, 1213–1217.
16. GAMBLE H.R., RAPIC D., MARINCULIC A. & MURRELL K.D. (1988). Evaluation of excretory-secretory antigens for
the serodiagnosis of swine trichinellosis. Vet. Parasitol., 30,131–137.
17. GAMBLE H.R., POZIO E., BRUSCHI F., NÖCKLER K., KAPEL C.M.O. & GAJADHAR A.A (2004). International
Commission on Trichinellosis: Recommendations on the Use of Serological Tests for the Detection of
Trichinella Infection in Animals and Man. Parasite, 11, 3–13.
18. GAMBLE H.R., W ISNEWSKI N., & W ASSOM D. (1997). Detection of trichinellosis in swine by enzyme
immunoassay using a synthetic glycan antigen. Am. J. Vet. Res., 58, 417–421.
19. HILL D.E., FORBES L.B., KRAMER M., GAJADHAR A.A. & GAMBLE H.R. (2007). Larval viability and serological
response in horses with long-term infection of Trichinella spiralis. Vet. Parasit., 146, 107–116.
20. KAPEL C.M.O. & GAMBLE H.R. (2000). Infectivity, persistence, and antibody response to domestic and sylvatic
Trichinella spp. in experimentally infected pigs. Int. J. Parasitol., 30, 215–221.
21. MURRELL K.D., LICHTENFELS J.R., ZARLENGA D.S. & POZIO E. (2000). The systematics of the genus Trichinella
with a key to species. Vet. Parasitol., 93, 293–307.
22. NOCKLER K., POZIO E., VOIGT W.P. & HEIDRICH J. (2000). Detection of Trichinella infection in food animals.
23. NOCKLER K., SERRANO F.J., BOIREAU P., KAPEL C.M. & POZIO E. (2005). Experimental studies in pigs on
Trichinella detection in different diagnostic matrices. Vet. Parasitol. 132, 85–90.
24. ORTEGA-PIERRES M.G., YEPEZ-MULIA L., HOMAN W., GAMBLE H.R., LIM P., TAKAHASHI Y., W ASSON D.L. &
APPLETON J.A. (1996). Workshop on a detailed characterization of Trichinella spiralis antigens: A platform
for future studies on antigens and antibodies to this parasite. Parasite Immunol., 18, 273–284.
25. POZIO E. & LA ROSA G. (2003). PCR-derived methods for the identification of Trichinella parasites from
animal and human samples. Methods Mol. Biol., 216, 299–309.
26. POZIO E., SOFRONIC-MILOSAVLJEVIC L., GOMEZ MORALES M.A., BOIREAU P. & NÖCKLER K. (2002). Evaluation of
ELISA and Western blot analyses using three antigens to detect anti-Trichinella IgG in horses. Vet.
Parasitol., 108, 163–178.
27. POZIO E. & ZARLENGA D.S. (2005). Recent advances on the taxonomy, systematics and epidemiology of
Trichinella. Int. J. Parasitol., 35, 1191–1204.
*
* *
NB: There are OIE Reference Laboratories for Trichinellosis (see Table in Part 3 of this Terrestrial Manual or

CHAPTER 2.1.17.
TRYPANOSOMA EVANSI INFECTIONS

(INCLUDING SURRA)
SUMMARY
Definition of the disease: Trypanosoma evansi causes a disease known as Trypanosomosis 1

(‘surra’). It affects a number of species of domesticated animals in Asia, Africa and Central and
South America. The principal host species affected varies geographically, but buffalo, cattle,
camels and horses are particularly affected, although other animals, including wildlife, are also
susceptible. It is an arthropod-borne disease. Several species of haematophagous flies, including
Tabanus spp. and Musca spp., are implicated in transferring infection from host to host
mechanically vectors. In Brazil, vampire bats have been implicated in transmission.
Description of the disease: The disease in susceptible animals is manifested by pyrexia, directly
associated with parasitaemia, together with a progressive anaemia, loss of condition and lassitude.
Recurrent episodes of fever and parasitaemia occur during the course of the disease. Oedema,
particularly of the lower parts of the body, urticarial plaques and petechial haemorrhages of the
serous membranes are often observed. Abortions have been reported in buffaloes and camels.
There are indications that the disease causes immunodeficiencies.
Identification of the agent: The general clinical signs of T. evansi infection are not sufficiently
pathognomonic for diagnosis. Laboratory methods for detecting the parasite are required.
Examination of the host blood is problematic as trypanosomes can be detected only when there is
a high parasitaemia. Under these circumstances examination of wet blood films, stained blood
smears or lymph node material might reveal the trypanosomes. In other, more chronic cases, such
as the carrier state, the examination of thick blood smears, as well as methods of parasite
concentration and the inoculation of laboratory animals are required.
Serological tests: Infection gives rise to specific antibody responses and a variety of antibody
detection tests have been introduced for laboratory and field use. Some have been partially
validated, but await large-scale evaluation and standardisation. Among those that are used
regularly in the laboratory are immunoenzyme assays, card agglutination tests and latex
agglutination tests. For field use both card agglutination tests (CATT) for T. evansi and latex can
be applied, yet an individual test format (pen side test) is currently unavailable. Assays for
detection of circulating antibodies have high measures of validity. Estimates of predictive values of
different serological tests indicate that enzyme linked immunosorbent assays (ELISA) for detecting
IgG antibodies are more likely to classify correctly uninfected animals, and are more likely to
classify correctly truly infected animals. An IgG ELISA would thus be suitable for verifying that
animals are free from infection, prior to movement or during quarantine. In situations where there is
overt disease, CATTs can be used to target individual animals for treatment with trypanocidal
drugs. For declaring a disease-free status, serial testing – ELISA followed by re-testing of suspect
samples by CATT – is recommended. It must be stressed however, that there are considerable
antigenic similarities among the different species of pathogenic trypanosomes, hence in areas
where tsetse-transmitted trypanosomoses occur cross-reactions may occur with any serological
test employed.
Requirements for vaccines and diagnostic biologicals: No vaccines are available for the
disease.
1 Nomenclature of parasitic diseases: see the note in Chapter. 2.4.18 Trypanosomosis (Tsetse-borne).

Chapter 2.1.17. — Trypanosoma evansi infections (including surra)
A. INTRODUCTION
The clinical signs of surra, the disease caused by Trypansoma evansi, are indicative but are not sufficiently
pathognomonic and diagnosis must be confirmed by laboratory methods. The disease in susceptible animals,
including cattle, buffalo, camels (dromedary and bactrian), horses, pigs, sheep and goats, is manifested by
pyrexia, directly associated with parasitaemia, together with a progressive anaemia, loss of condition and
lassitude. Recurrent episodes of fever and parasitaemia occur during the course of the disease. Oedema,
particularly of the lower parts of the body, urticarial plaques and petechial haemorrhages of the serous
membranes are often observed. Abortions have been reported in buffaloes and camels (8, 17) and there are
indications that the disease causes immunodeficiency (5, 21).
There is considerable variation in the pathogenicity of different strains and the susceptibility of different host
species to disease. Disease may manifest as an acute or chronic condition, and in the latter case may persist for
many months. The disease is often rapidly fatal in camels, buffaloes, horses, cattle, llama and dogs, but mild and
subclinical infections can also occur in these species. Wild animals such as deer and capybara can become
infected. Animals subjected to stress – malnutrition, pregnancy, work – are more susceptible to disease.
Biologically T. evansi is very similar to T. equiperdum, the causative agent of dourine (2), and morphologically
resembles the slender forms of the tsetse-transmitted trypanosomes, T. brucei, T. gambiense and
T. rhodesiense. Molecular characterisation indicates that various strains of T. evansi isolated from Asia, Africa
and South America have a single origin. Molecular characterisation using random amplified polymorphic DNA
techniques and endonuclease fingerprinting showed that isolates of T. evansi and T. equiperdum formed a
closely homogeneous group. One possibility of this finding is that T. equiperdum is not a genuine species per se
and that the clinical outcome of disease is related primarily to the hosts’ immune response. Like all pathogenic
trypanosomes, T. evansi is covered by a dense protein layer consisting of a single protein called the variable
surface glycoprotein. This acts as a major immunogen and elicits the formation of specific antibodies. The
parasites are able to evade the consequences of these immune reactions by switching the variant surface
glycoprotein, the phenomenon known as antigenic variation.
The classical direct parasitological methods for the diagnosis of trypanosomosis, namely examining blood or
lymph node material, are not highly sensitive. In regions where other Trypanozoon spp. occur in addition to
T. evansi, specific identification by microscopy is not possible. Specific DNA probes (19, 24) may enable
identification of trypanosome species by nonradioactive DNA hybridisation. A species-specific polymerase chain
reaction (PCR) based on T. evansi specific antigen (RoTat 1.2 VSG) has been developed, but has not been
validated in the field (3).
• Direct methods
a) Usual field methods

i) Blood sampling
Trypansoma evansi is a parasite of the blood and tissues often inhabiting the deep blood vessels in
cases of low parasitaemia. For this reason, it is recommended that blood for diagnosis be obtained
from both the peripheral and deep blood vessels. However it should be realised that less than 50% of
infected animals may be identified by examination of peripheral blood.
Peripheral blood is obtained by puncturing a small vein in the ear or tail. Deeper samples are taken
from a larger vein by syringe. Cleanse an area of the ear margin or tip of the tail with alcohol and,
when dry, puncture a vein with a suitable instrument. Ensure that instruments are sterilised or
disposable instruments are used between individual animals, so that infection cannot be transmitted
by residual blood.
ii) Wet blood films

Place a small drop of blood on to a clean glass slide and cover with a cover-slip to spread the blood as
a monolayer of cells. Examine by light microscopy (×200) to detect any motile trypanosomes.
iii) Stained thick smears

Place a large drop of blood on the centre of a microscope slide and spread with a toothpick or the
corner of another slide so that an area of approximately 1.0–1.25 cm in diameter is covered. Air-dry for

1 hour or longer, while protecting the slide from insects. Stain the unfixed smear with Giemsa’s Stain
(one drop of commercial Giemsa + 1 ml of phosphate buffered saline [PBS, 2.4 g Na2HPO4.2H2O,
0.54 g NaH2PO4.2H2O, 0.34 g NaCl], pH 7.2), for 25 minutes. After washing, examine the smears by
light microscopy at high magnification (×500–1000). The advantage of the thick smear technique is
that it concentrates the drop of blood into a small area, and thus less time is required to detect the
parasites. The disadvantage is that the trypanosomes may be damaged in the process, and the
method is therefore not suited for species identification in case of mixed infections.
iv) Stained thin smears

Place a drop of blood 20 mm from one end of a clean microscope slide and draw out a thin film in the
usual way. Air-dry briefly and fix in methyl alcohol for 2 minutes and allow to dry. Stain the smears in
Giemsa (one drop Giemsa + 1 ml PBS, pH 7.2) for 25 minutes. Pour off, stain and wash the slide in
tap water and dry. Unfixed smears can be stained by covering them with May–Grünwald stain for
2 minutes, then adding an equal volume of PBS, pH 7.2, and leaving the slides for a further 3 minutes.
Pour off and add diluted Giemsa for 25 minutes. Pour off, wash the slides with tap water, and dry.
Examine at high magnification (×400–1000). This technique permits detailed morphological studies
and identification of the trypanosome species. Rapid staining techniques also exist (Field’s stain, Diff
Quick®).
v) Lymph node biopsies

Samples are usually obtained from the prescapular or precrural (subiliac) lymph nodes. Select a
suitable node by palpation and cleanse the site with alcohol. Puncture the node with a suitable gauge
needle, and draw lymph node material into a syringe attached to the needle. Expel lymph on to a slide,
cover with a cover-slip and examine as for the fresh blood preparations. Fixed thin or thick smears can
also be stored for later examination.
b) Concentration methods
In most hosts T. evansi can induce mild clinical or subclinical carrier state infections with low parasitaemia
in which it is difficult to demonstrate the parasites. In these circumstances, concentration methods become
necessary.
i) Haematocrit centrifugation
Collect blood (70 µl) into at least two heparinised capillary tubes (75 × 1.5 mm). Seal at the dry end by
heat and centrifuge, sealed end down, at 3000 g for 10 minutes. Place the capillary tube between two
pieces of glass (25 × 10 × 1.2 mm) glued to a slide. Place a cover-slip on top at the level of the buffy
coat junction where the trypanosomes will be concentrated. Flood the space around this part of the
tube with water or immersion oil, and examine the buffy coat area under the microscope (×100–200).
This technique can detect around 400 trypanosomes/ml. A simpler alternative is to examine the
centrifuged capillary tube by placing a drop of immersion oil on the tube and ensuring that there is
contact between the objective lens and the immersion oil.
ii) Dark-ground/phase-contrast buffy coat technique

Collect blood into heparinised capillary tubes and centrifuge as above. Scratch the tube with a glass-
cutting diamond and break it 1 mm below the buffy coat layer – the upper part thus contains the top
layer of red blood cells (RBCs), the buffy coat (white blood cells) and some plasma.
Partially expel the contents of this piece on to a slide, cover with a cover-slip and examine under dark-
ground, phase-contrast or ordinary illumination.
As an alternative to the electrically powered haematocrit centrifuge, hand-powered micro-centrifuges
have been used successfully for detection of trypanosomosis in cattle and camels (9, 14).
iii) Haemolysis techniques

Sodium dodecyl sulphate (SDS) can be used as a reagent to haemolyse RBCs to facilitate detection of
motile trypanosomes in parasitised blood samples. As SDS is toxic, contact with skin, inhalation and
ingestion should be avoided. SDS solution can be stored for several months at ambient temperature.
Both the SDS solution and the blood samples should be used at a temperature above 15°C. At lower
temperatures the trypanosomes may be destroyed.
Two general procedures, namely wet blood film clarification and haemolysis centrifugation, can be used 2.
2 All necessary materials and instructions can be obtained from the Institute of Tropical Medicine, Laboratory of Serology,
Nationalestraat 155, B-2000 Antwerp, Belgium.

Wet blood film clarification method

This method uses the partial lysis of RBCs to facilitate the detection of motile trypanosomes. The
method requires an SDS solution: 1% SDS dissolved in Tris/glucose/saline, pH 7.5, inoculating loops
(10 µl), slides and cover-slips (24 × 24 mm), and a drop of fresh meat or heparinised blood. Dissolve
100 mg of SDS in 100 ml of isotonic Tris/NaCl/glucose buffer, pH 7.5 (Trizma base 14.0 g, NaCl 3.8 g,
glucose 10.8 g; dissolve chemicals in 750 ml distilled H2O, add 90–100 ml 1 N HCl and adjust pH to
7.5 then make up to final volume of 1000 ml with distilled H2O). This buffer can be stored in small vials
at ambient temperature for several months. The test does not give a significantly higher sensitivity
than wet film technique. In addition, the SDS can cause problems when focusing the microscope and
the movements of trypanosomes can be severely curtailed owing to the high viscosity of the SDS.
Put approximately 10 µl of blood on a slide. Add 10 µl of SDS solution using a dip inoculation loop and
mix gently. Apply a cover-slip and examine the preparation without delay at low magnification (×100 or
×200).
Haemolysis centrifugation technique

Nearly complete lysis of RBCs is required for this procedure. The materials needed include: SDS
solution (0.1% SDS dissolved in Tris/glucose/saline, pH 7.5), conical centrifuge tubes, ordinary test
tubes, large and fine plastic tapering pipettes with attached bulb, slides, cover-slips (24 × 24 mm or
24 × 32 mm), and heparinised blood.
Using a pipette or syringe, transfer nine volumes (maximum 6.3 ml) of SDS solution into an ordinary
test tube. Aspirate one volume (maximum 0.7 ml) of heparinised blood into a bulb pipette and expel it
just above the surface of the SDS solution; mix quickly and thoroughly. Avoid foam formation, which
may result in destruction of the trypanosomes. Wait for 10 minutes so as to achieve complete
haemolysis.
Pour the haemolysed suspension into a conical centrifuge tube and spin at approximately 500 g for
10 minutes. With a clean bulb pipette, remove as much supernatant as possible without disturbing the
sediment. Using a fine tapering bulb pipette, remove more supernatant, leaving 10–20 µl of
undisturbed sediment at the bottom.
Very carefully collect the entire sediment and put on to a microscope slide. Apply a cover-slip and
examine the preparation without delay at low magnification (×100 or ×200).
iv) Mini-anion exchange centrifugation technique

When a blood sample from animals infected with salivarian trypanosomes is passed through an
appropriate anion-exchange column, the host blood cells, being more negatively charged than
trypanosomes, are adsorbed onto the anion-exchanger, while the trypanosomes are eluted, retaining
viability and infectivity (15). A simplified field method for detection of low parasitaemia has been
developed (26). The sensitivity of this technique can be increased by approximately tenfold by the use
of buffy coat preparations rather than whole blood (25).
Preparation of phosphate buffered saline glucose, pH 8

Na2HPO4 (anhydrous) (13.48 g); NaH2PO4.2H2O (0.78 g); NaCl (4.25 g); distilled water (1 litre).
Solutions of different ionic strength are made by diluting the stock PBS, pH 8, and adding glucose to
maintain a suitable concentration. For blood of mice, domestic and wild ruminants and dog, add four
parts PBS to six parts distilled water and adjust the final glucose concentration to 1%. For blood of
pigs and rabbits, add three parts PBS to seven parts distilled water and adjust the final glucose
concentration to 1.5%. The PBS/glucose solution (PSG) must be sterile.
Equilibration of DEAE-cellulose
Suspend 500 g of DEAE-cellulose (diethylamino-ethylcellulose) in 2 litres of distilled water. Adjust the
pH to 8 with phosphoric acid. Allow to settle for 30 minutes. Discard the supernatant fluid containing
the fine granules. Repeat the procedure three times. Store the equilibrated concentrated suspension
of DEAE-cellulose (slurry) at 4°C or in small aliquots at –20°C.
Packing of equilibrated DEAE-cellulose

Place a 2 ml syringe without the plunger on a test-tube rack complete with needle (20 G × 1.5 inch).
Put a disc of Whatman No. 41 filter paper at the bottom of the syringe and moisten by adding a few
drops of PSG. Pour 2–2.5 ml of the slurry of equilibrated cellulose into the syringe and allow to pack
by elution of the buffer. The height of the sediment should be approximately 3 cm. Wash and
equilibrate the column with 2 ml of PSG without disturbing the surface.

Adsorption of blood eluate of the trypanosomes

Gently place 100–300 µl of heparinised blood above the surface of the cellulose column. Add ten
drops of PSG and discard the first ten drops of the eluate. Progressively add 1.5 ml of PSG and start
collecting the eluate into a finely tapered Pasteur pipette with a sealed end. Put the filled pipette,
protected by a conical plastic pipette tip, in a tube and centrifuge at 525 g (or up to 1000 g) for
10 minutes. Examine the bottom of the pipette under the microscope (×100 or ×200) using a special
mounting device. Alternatively, the eluate could be collected into 50 ml plastic tubes, with conical
bottoms, centrifuged at 1000 g and the sediment examined by dark-ground microscopy.
The cellulose column should remain wet throughout the procedure.
c) Animal inoculation
Laboratory animals may be used to reveal subclinical (nonpatent) infections in domesticated animals.
Trypanosoma evansi has a broad spectrum of infectivity for small rodents, and so rats and mice are often
used. Rodent inoculation is not 100% sensitive (18) but further improvement in its efficacy can be obtained
by the use of buffy coat material. Such a procedure was able to detect as few as 1.25 T. evansi/ ml blood
(25).
Inoculate heparinised blood intraperitoneally into rats (1–2 ml) or mice (0.25–0.5 ml). Inoculate a minimum
of two animals. Bleed animals from the tail three times a week to detect parasitaemia. The incubation period
before appearance of the parasites and their virulence depends on the strain of trypanosomes, their
concentration in the inoculum, and the strain of laboratory animal used. Sensitivity of this in-vivo culture
system may perhaps be increased by use of immunosuppressed laboratory animals. Drugs such as
cyclophosphamide or hydrocortisone acetate, or X-ray irradiation or splenectomy are used for this purpose.
d) Recombinant DNA probes

Specific DNA probes have been used to detect trypanosomes in infected blood or tissue but are not
routinely applied (28, 31). Although molecular methods have a potentially high analytical sensitivity there
have been few convincing studies to critically evaluate the diagnostic sensitivity of these tests as compared
with other techniques, such as serology.
e) Detection of trypanosomal DNA

Detection of minute amounts of trypanosomal DNA using a PCR procedure is a possible means of
identifying animals with active infections, and could have the sensitivity and specificity required (3, 20, 32).
Experimental studies in buffalo (10) showed the diagnostic sensitivity of a PCR was only 78%, which is
similar to mouse inoculation.
• Indirect methods
These methods involve tests that demonstrate the effects of the parasite on its host rather than directly detecting
the parasite itself.
a) Haematology
Anaemia is usually a reliable indicator of trypanosome infection, although it is not in itself pathognomonic.
However, animals with a mild subclinical infection can have parasitaemia without evidence of anaemia (4).
Anaemia can be estimated by measuring the packed cell volume and may be used in surveys of herds at
risk. The technique is identical to that of haematocrit centrifugation. The capillary tube is examined and the
results are expressed as a percentage of packed RBCs to total blood volume.
Historically, many different methods have been used to detect specific humoral antibodies to trypanosomal
antigens but, more recently, there has been a tendency to concentrate on more easily standardised techniques
such as ELISA (6, 7, 12, 22, 23, 24, 27) card agglutination tests (CATT) (1, 20, 24) and latex agglutination tests
(LAT) (11, 16). Extensive evaluation of ELISA and CATT has been carried out in buffaloes in Indonesia and
Vietnam (6, 11, 29). The collection of samples can be simplified by using filter paper blood spots for later use in
the ELISA, while for the CATT whole blood can be substituted for serum (11). Other innovative modifications that
might be developed in the future are the use of a colloidal-dye dipstick test (13) that could enable the tests to be
carried out under field conditions. It is vitally important that serological tests are validated and standardised if
they are to be suitable for correctly identifying infected animals. This means that standard criteria for interpreting
the tests might have to be developed for each animal species as well as each laboratory operating the
procedure.

a) Enzyme-linked immunosorbent assay

The principle of this technique is that specific antibodies to trypanosomes can be detected by enzyme-
linked anti-immunoglobulins using solid-phase polystyrene plates coated with soluble antigen. The enzyme
may be peroxidase, alkaline phosphatase or any other suitable enzyme. The enzyme conjugate binds to the
antigen/antibody complex and then reacts with a suitable substrate to yield a characteristic colour change
either of the substrate itself or of an added indicator (the chromogen).
The antigen for coating the plates is derived from the blood of a heavily parasitaemic rat. The trypanosomes
are separated on a DEAE-cellulose column and washed three times by centrifugation in cold PSG, pH 8
(PBS with 1% glucose). The final pellet is suspended in cold PSG to a concentration of 3–5%, and briefly
ultrasonicated on ice for 30–120 seconds until disintegration of the organisms is complete. This preparation
is centrifuged at 4°C and 40,000 g for 60 minutes. The supernatant is diluted in water so as to obtain a
protein concentration of 1 mg/ml. The reagent thus obtained can be stored in small aliquots at –70°C for
several months. It can also be freeze-dried and stored at –20°C. Various treatments of the antigen
preparations have been applied to improve the accuracy of antibody detection (32).
Test procedure
i) Dilute or reconstitute the frozen or freeze-dried antigen with freshly prepared 0.01 M
carbonate/bicarbonate buffer, pH 9.6. Add 100 µl to each well of a 96-well microtitre plate and
incubate overnight at 4°C or for 1 hour at 37°C.
ii) Remove excess antigen by washing plate with 0.01 M PBS containing 0.05% Tween 20 (PBST). Add
test serum dilutions in PBST (100 µl). Include control negative and positive sera. Dilutions must be
determined empirically, but are usually between 1/100 and 1/1000.
iii) Incubate plates at 37°C for 30 minutes. Eject contents and wash three times with PBST.
iv) Add a specific peroxidase conjugated species-specific anti-globulin (100 µl) appropriately diluted in
PBST (usually between 1/1000 and 1/50,000).
v) Incubate the plates at 37°C for 30 minutes, eject contents and wash three times with PBST.
vi) For peroxidase conjugates a number of substrate/chromogen solutions can be used, consisting of
hydrogen peroxide with a chromogen, such as tetramethylbenzidene (TMB), 2,2’-azino-bis-(3-
ethylbenzothiazoline-6-sulphonic acid) (ABTS) and ortho-diphenylenediamine (OPD). A suitable
substrate/chromogen solution for peroxidase conjugates is 30% hydrogen peroxide (0.167 ml and
35 mg) in citrate buffer (100 ml), pH 6.0. The citrate buffer is made up as follows: Solution A
(36.85 ml): (0.1 M citric acid [21.01 g/litre]); Solution B (65.15 ml): (0.2 M, Na2HPO4 [35.59 g/litre]);
and distilled water (100 ml). Dissolve 10 mg TMB in 1 ml dimethyl sulphoxide and add to 99 ml of the
citrate buffer. A number of these combinations are available commercially in ready-to-use formulations
that remain stable at 4°C for up to 1 year.
vii) Add the substrate chromogen (100 µl) to the plates and incubate at room temperature for 15–
20 minutes.
viii) Stop the reaction by adding 50 µl 1 M sulphuric acid. Read the absorbance of each well at 450 nm for
TMB chromogen. Other chromogens may require the use of a different wavelength. All tests should
include known high and medium positive control sera, a negative control serum, and a buffer control.
A large variety of other test procedures exists. For closely related animal species, cross-reacting reagents
may often be used (e.g. anti-bovine immunoglobulin for buffaloes and the use of monospecific anti-IgG
conjugates is generally recommended. There are a number of methods that can be used to determine a
cut-off point to discriminate between positive and negative results. The simplest method is to base the cut-
off on visual inspection of the test results from known positive and negative populations. These results are
likely to show some overlap. The operator can choose the most appropriate point to modify the false
negative or false positive results depending on the required application of the assay. An alternative is to
base the cut-off on the mean +2 standard deviations (SD) or +3 SD values from a large sample of negative
animals. Finally, if no suitable negative/positive samples are available a cut-off can be based on the
analysis of the data from animals in an endemic situation. If a bimodal distribution separates infected from
uninfected animals, then an appropriate value can be selected. The ELISA is likely to correctly identify
uninfected animals. Equivocal results can be re-tested using CATT. The Institute of Tropical Medicine in
Antwerp developed an ELISA using the VSG from a T. evansi RoTat 1.2 clone. It was shown (30) that this
antigen is a predominant antigen in T. evansi and absent in T.b brucel. This ELISA/RoTat 1.2 was
successfully used in the field in Vietnam (11, 29). Protocols are available at ITM Antwerp for use in equines,
camelidae and water buffaloes.
b) Card agglutination tests

It is well known that certain predominant variable antigen types (VATs) are expressed in common in
different strains of salivarian trypanosomes from different areas. This finding was used as a basis for a test

for the diagnosis of T. evansi, the card agglutination test – CATT/T.evansi – was developed at the
Laboratory of Serology, Institute of Tropical Medicine, Antwerp 3. The test makes use of fixed and stained
trypanosomes of a defined VAT known as RoTat 1.2. Both variable and invariable surface antigens take
part in the agglutination reaction. The CATT is available in kit form from ITM, Antwerp. It consists of
lyophilised antigen, PBS, pH 7.4, plastic-coated cards, spatulas, positive and negative control sera and a
rotator. The lyophilised antigen can be stored at 2–8°C for up to 1 year. Reconstituted antigen can be
stored at 2–8°C for 2 days, but preferably should be used within 8 hours.
For screening, dilute test sera 1/4 or 1/8 in PBS on to circles inscribed on the plastic cards. Add 45 µl of the
prepared antigen suspension onto circles inscribed on the plastic cards. Add 25 µl of each test serum. Mix
and spread the reagents with a spatula and rotate the card for 5 minutes using the rotator provided in the
kit. Compare the pattern of agglutination with the illustrations of different reactions provided in the kit. Blue
granular deposits reveal a positive reaction visible to the naked eye.
c) Latex agglutination tests

A kit is available from ITM, Antwerp. It comprises a lyophilised latex suspension coated with T. evansi
RoTat 1.2 variable antigens, PBS, positive and negative controls, test cards, plastic spatulas and a rotator.
Reconstitute the antigen-coated latex particles using distilled, deionised water. Mix gently. Add 20 µl of latex
suspension onto each black spot on the test cards.
Dilute test sera with PBS (1/2 to 1/4) and add 20 µl to the latex suspension. Include appropriate controls.
Mix the reagents carefully with a plastic spatula.
Rotate test cards at 70 rotations/minutes for 5 minutes and view cards under a good light source at the end
of the incubation. Positive sera will cause agglutination of the latex particles.

No vaccines are available for this disease.
REFERENCES
1. BAJYANA SONGA E. & HAMERS R. (1988). A card agglutination test (CATT) for veterinary use based on an
early VAT RoTat 1–2 of Trypanosoma evansi. Ann. Soc. Belg. Med. Trop., 68, 233–240.
2. CLAES F., AGBO E.C., RADWANSKA M., TE PAS M.F.W., BALTZ T., DE W AAL D.T., GODDEERIS B.M., CLASSEN E. &
BUSCHER P. (2003). How does Trypanosoma equiperdum fit into the Trypanozoon group? A cluster analysis
by RAPD and multiplex-endonuclease genotyping approach. Parasitology, 26, 425–431
3. CLAES F., RADWANSKA M., URAKAWA T., MAJIWA P.A., GODDEERIS B. & BUSCHER P. (2004) Variable surface
glycoprotein RoTat 1.2 PCR as a specific diagnostic tool for the detection of Trypanosoma evansi
infections. Kinetoplastid Biol. Dis., 3, 3.
4. DARGANTES A.P., REID S.A. & COPEMAN D B. (2005). Experimental Trypanosoma evansi infection in the goat. I
Clinical signs and pathology. J. Comp. Pathol., 133, 261–266.
5. DARGANTES A.P., REID S.A. & COPEMAN D.B. (2005). Experimental Trypanosoma evansi infection in the goat.
II Pathology. J. Comp. Pathol., 133, 267–276.
6. DAVISON H.C., THRUSFIELD M.V., MUHARSINI S., HUSEIN A., PARTOUTOMO S., MASAKE R. & LUCKINS A.G. (1999).
Evaluation of antigen- and antibody-detection tests for Trypanosoma evansi infections of buffaloes in
Indonesia. Epidemiol. Infect., 123, 149–155.
7. FRANKE C.R., GREINER M. & MEHLITZ D. (1994). Investigations on naturally occurring Trypanosoma evansi
infections in horses, cattle, dogs and capybaras (Hydrochaeris hydrochaeris) in Pantanal de Pocone (Mato
Grosso, Brazil). Acta Trop., 58, 159–169.
3 CATT/T. evansi kits are available at the Laboratory of Parasite Diagnostic, Institute of Tropical Medicine, Nationalestraat
155, B-2000 Antwerp, Belgium. ([email protected] ; [email protected])

8. GUTIERREZ C., CORBERA J.A., JUSTE M.C., DORESTE F. & MORALES I. (2005). An outbreak of abortions and high
neonatal mortality associated with Trypanosoma evansi infection in dromedary camels in the Canary
Islands. Vet. Parasit., 130, 163–168.
9. HOLMSTEDT H. (1965). Simple microcentrifuge for use in the field. Science, 149, 977–978.
10. HOLLAND W.G., CLAES F., MY L.N., THANH N.G., TAM P.T., VERLOO D., BUSCHER P., GODDEERIS B. &
VERCRUYSSE J. (2001). A comparative evaluation of parasitological tests and a PCR for Trypanosoma
evansi diagnosis in experimentally infected water buffaloes. Vet. Parasit., 97, 23–33.
11. HOLLAND W.G., THANH N.G., MY L.N., MAGNUS E., VERLOO D., BUSCHER P., GODDEERIS B. & VERCRUYSSE J.
(2002). Evaluation of whole fresh blood and dried blood on filter paper discs in serological tests for
Trypanosoma evansi in experimentally infected water buffaloes. Acta Trop., 81, 159–165.
12. INTERNATIONAL ATOMIC ENERGY AGENCY (IAEA) (1993). Improving the diagnosis and control of
trypanosomiasis and other vector-borne diseases of African livestock using immunoassay methods.
International Atomic Energy Agency, TECDOC, 707, 171 pp.
13. KASHIWAZAKI Y., KANITPUN R., SUTEERAPARP P. & BOONCHIT S. (2000). A preliminary comparative study of a
dipstick colloidal dye immunoassay and two antigen-detection ELISAs for diagnosis of Trypanosoma evansi
infection in cattle. Vet. Res. Commun., 92, 533–544.
14. KELLY S. & SCHILLINGER D. (1983). Improved field diagnostic technique for trypanosomiasis by use of a
microcentrifuge. Vet. Rec., 113, 219.
15. LANHAM S.M. & GODFREY D.G. (1970). Isolation of salivarian Trypanosomes from man and other mammals
using DEAE – Cellulose. Exp. Parasitol., 28, 521–528.
16. LEJON V., CLAES F., VERLOO D., MAINA M., URAKAWA T., MAJIWA P.A. & BUSCHER P. (2005). Recombinant RoTat
1.2 variable surface glycoprotein as antigen for diagnosis of Trypanosoma evansi in dromedary camels. Int.
J. Parasitol., 35, 455–460.
17. LOHR K.F., PHOLPARK S., SIRIWAN P., LEESIRIKUL N., SRIKITJAKARN & STAAK C. (1986). Trypanosoma evansi
infection in buffaloes in North-East Thailand. II. Abortions. Trop. Anim. Health Prod., 18, 103–108.
18. MONZON C.M., MANCEBO O.A. & ROUX J.P. (1990). Comparison between 6 parasitological methods for
diagnosis of Trypanosoma evansi in the subtropical area of Argentina. Vet. Parasitol., 36, 141–146.
19. MASIGA D.K. & GIBSON W.C. (1990). Specific probes for Trypanosoma (Trypanozoon) evansi based on
kinetoplast DNA minicircles. Mol. Biochem. Parasitol., 40, 279–284.
20. NJIRU Z.K., CONSTANTINE C.C., NDUNG’U J.M., ROBERTSON I., OKAYE S., THOMPSON R.C. & REID S.M. (2004).
Detection of Trypanosoma evansi in camels using PCR and CATT/T. evansi tests in Kenya. Vet. Parasitol.,
124, 187–199.
21. ONAH D.N., HOPKINS J.A. & LUCKINS A.G. (1998). Increase in CD5 B cells and depression of immune
responses in sheep infected with Trypanosoma evansi. Vet. Immunol. Immunopathol., 63, 209–222.
22. RAE P.F., THRUSFIELD M.V., HIGGINS A., AITKEN C.G.G., JONES T.W. & LUCKINS A.G. (1989). Evaluation of
enzyme immunoassays in the diagnosis of camel (Camelus dromedarius) trypanosomiasis: a preliminary
investigation. Epidemiol. Infect., 102, 297–307.
23. REID S.A. & COPEMAN D.B. (2002). Evaluation of an antibody-ELISA using five crude antigen preparations for
the diagnosis of Trypansoma evansi infection in cattle. Vet. Parasitol., 104, 79–84.
24. REID S.A. & COPEMAN D.B. (2003). The development and validation of an antibody-ELISA to detect
Trypanosoma evansi infection in cattle in Australia and Papua New Guinea. Prev. Vet. Med., 61, 195–208.
25. REID S.A., HUSEIN A. & COPEMAN D.B. (2001). Evaluation and improvement of parasitological tests for
Trypanosoma evansi infection. Vet. Parasitol., 102, 291–297.
26. SACHS R. (1984). Improvements in the miniature anion exchange centrifugation technique for detecting
trypanosomes in domestic pigs. Trans. R. Soc. Trop. Med. Hyg., 78, 561.

27. TUNTASUVAN D., CHOMPOOCHAN T., VONGPAKORN M. & MOHKAEW K. (1996). Detection of Trypanosoma evansi
antibodies in pigs using an enzyme linked immunosorbent assay. J. Thai Vet. Med. Assoc., 47, 45–53.
28. VENTURA R.M., TAKEDA G.F., SILVA R.A., NUNES V.L., BUCK G.A. & TEIXEIRA M.M. (2002). Genetic relatedness
among Trypanosoma evansi stocks by random amplification of polymorphic DNA and evaluation of a
synapomorphic DNA fragment for species-specific diagnosis. Int. J. Parasitol., 32, 53–63.
29. VERLOO D., HOLLAND W., MY L.N., THANH N.G., TAM P.T., GODDEERIS B., VERCRUYSSE J. & BUSCHER P. (2000).
Comparison of serological tests for Trypanosoma evansi natural infections in water buffaloes from north
Vietnam. Vet. Parasitol., 92, 87–96.
30. VERLOO D., MAGNUS E. & BUSCHER P. (2001). General expression of RoTat 1.2 variable antigen type in
Trypanosoma evansi isolates from different origin. Vet. Parasitol., 97, 183–189.
31. VISESHAKUL N. & PANYIM S. (1990). Specific DNA probe for the sensitive detection of Trypanosoma evansi.
Southeast Asian J. Trop. Med. Public Health, 21, 21–27.
32. W UYTS N., CHODESAJJAWATEE N. & PANYIM S. (1994). A simplified and highly sensitive detection of
Trypanosoma evansi by DNA amplification. Southeast Asian J. Trop. Med. Public Health, 25, 266–271.
*
* *
NB: There is an OIE Reference Laboratory for Trypanosoma evansi infections (including surra) (see Table in Part
3 of this Terrestrial Manual or consult the OIE Web site for the most up-to-date list: www.oie.int).

1 CHAPTER 2.1.18.
2 TULAREMIA
3 SUMMARY
4 Tularemia is a zoonosis caused by Francisella tularensis. The causative bacterium is a Gram-

5 negative coccoid rod, 0.2–0.5 µm × 0.7–1.0 µm, non-motile and non-spore-forming organism that is
6 an obligate aerobe with optimal growth at 37°C. It is oxidase-negative, weakly catalase-positive
7 and cysteine is required for growth. It occurs naturally in lagomorphs (rabbits and hares), and in
8 rodents, especially microtine rodents (such as voles, vole rats and muskrats), and beavers. A wide
9 range of other mammals and several species of birds have also been reported to be infected.
10 Among domestic animals, the cat seems to be able to act as a carrier of the bacterium.
11 Two types of F. tularensis are recognised on the basis of cultural characteristics, epidemiology,
12 and virulence in some hosts. Tularemia is largely confined to the Northern Hemisphere and is not
13 normally found in the tropics or the Southern Hemisphere. Francisella tularensis subsp. tularensis
14 (Type A) is associated with lagomorphs in North America. It is transmitted primarily by ticks and
15 biting flies, is highly virulent for humans and domestic rabbits, and most of the isolates ferment
16 glycerol. Francisella tularensis subsp. palaearctica (Type B) occurs mainly in aquatic rodents
17 (beavers, muskrats) in northern North America, and in hares and small rodents in Eurasia. It may
18 be water- or arthropod-borne, is less virulent to humans and rabbits, and does not ferment glycerol.
19 In addition to vector transmission, tularemia may be spread contact with infected animals or
20 environmental fomites by inhalation, or by ingestion of the poorly cooked flesh of infected animals
21 or contaminated water.
22 The disease is characterised by fever, depression and septicaemia. In humans, there may be
23 ulcers or abscesses at the site of inoculation (this is rarely seen in animals), and swelling of the
24 regional lymph nodes. On post-mortem examination, lesions may include caseous necrosis of
25 lymph nodes and multiple greyish-white foci of necrosis in the spleen, liver, bone marrow and
26 lungs. The spleen is usually enlarged.
27 It is important to understand that there is a high risk of direct infection of humans by direct contact
28 with this organism. Special precautions, including the wearing of gloves, masks and eyeshields,
29 are therefore recommended when handling infective materials. The facility should meet the
30 requirements for Containment Group 3 pathogens (see Chapter 1.1.2 Biosafety and Biosecurity in
31 the veterinary microbiological laboratory and animal facilities).
32 Identification of the agent: The bacterium can be demonstrated in impression smears or in fixed
33 specimens of organs, such as liver, spleen, bone marrow, kidney and lung, as well as in blood
34 smears. Immunological methods, such as the fluorescent antibody test (FAT) are the most reliable
35 way to identify the bacterium. With Gram staining, the bacteria appear as very small punctiform
36 Gram-negative rods, often difficult to distinguish as bacteria. They can also be stained with May–
37 Grunwald–Giemsa or phenol thionin.
38 The organism is highly fastidious. For growth it is necessary to use Francis medium, McCoy and
39 Chapin medium, or Modified Thayer-Martin agar. The colonies are small, round and transparent,
40 and do not appear before 48 hours incubation at 37°C. On Francis medium, the colonies may be
41 confluent and have a milky appearance. If transportation is necessary, samples should be
42 inoculated into sterile nutrient broth and stored at 4–10°C for a few hours or at –70°C if transit is
43 likely to be prolonged.
44 In the past mice or guinea-pigs were experimentally inoculated with infected tissue material or with
45 cultures to aid in the diagnosis of tularemia. Animal inoculation has been replaced by polymerase

Chapter 2.1.18. — Tularemia
46 chain reaction (PCR) protocols for the identification of F. tularensis. The FAT demonstrates
47 F. tularensis in pathological specimens.
48 Serological tests: Serological tests are useful diagnostic aids in human infection, but are of
49 limited value in the more susceptible animal species that usually die before developing antibodies.
50 Epidemiological surveys can be conducted in domestic animals in relatively resistant species that
51 survive the infection, such as sheep, cattle, pigs, dogs, cats and wild ungulates, as these species
52 develop antibodies. Also relatively resistant species of rodents and lagomorphs can also be used
53 in epidemiological surveys. Serological surveys can and are conducted in a number of wildlife
54 species, such as moose, that are exposed to F. tularensis.
55 Requirements for vaccines and diagnostic biologicals: A live attenuated vaccine strain is
56 available for vaccination of humans at high risk of exposure to virulent F. tularensis. However, the
57 vaccine is only available for restricted use. The outcome of vaccination can be estimated by the
58 demonstration of specific antibodies and the ability of lymphocytes to proliferate in the presence of
59 F. tularensis antigen.
60 A. INTRODUCTION
61 Tularemia is a zoonosis caused by Francisella tularensis. It occurs naturally in lagomorphs (rabbits and hares)
62 and rodents, especially microtine rodents such as voles, vole rats and muskrats, as well as in beavers. In
63 addition, a wide variety of other mammals, birds, amphibians and invertebrates have been reported to be
64 infected (19, 20). Tularemia occurs endemically in the Northern Hemisphere. The disease can occur as epizootic
65 outbreaks in many countries in North America and Europe, while it occurs only as sporadic cases in some other
66 countries in Europe and Asia. It is rarely reported from the tropics or the Southern Hemisphere. Several epizootic
67 outbreaks have been reported as a result of importation of subclinically infected lagomorphs.
68 The clinically most relevant two types of F. tularensis are recognised on the basis of culture characteristics,
69 epidemiology, and virulence. Francisella tularensis subsp. tularensis (Type A) is mainly associated with
70 lagomorphs in North America. It is primarily transmitted by ticks or biting flies, or by direct contact with infected
71 lagomorphs. It is highly virulent for humans and domestic rabbits, and most isolates ferment glycerol. Francisella
72 tularensis subsp. palaearctica (Type B) occurs mainly in aquatic rodents (beavers, muskrats) and voles in
73 northern North America, and in lagomorphs (hares) and rodents in Eurasia. It is primarily transmitted by direct
74 contact or by mosquitoes, but may be transmitted through inhalation or through infected water or food. It is less
75 virulent for humans and domestic rabbits, and does not ferment glycerol (1, 7, 14, 15, 17, 22).
76 In sensitive animals, clinical signs of severe depression are followed by a fatal septicaemia. The course of the
77 disease is approximately 2–10 days in susceptible species, and animals are usually dead when presented for
78 diagnosis. Most domestic species do not usually manifest signs of tularemia infection, but they do develop
79 specific antibodies to the organism following infection. Outbreaks with high mortality caused by the Type A
80 organism have occurred in sheep (1, 17). Among domestic animals, the cat has been reported to be able to act
81 as a carrier of the bacterium (6) and the disease is occasionally spread from cats to humans (8).
82 At necropsy, animals that have died from acute tularemia are usually in good body condition. There are signs of
83 septicaemia characterised by whitish foci of necrosis randomly distributed in the liver, bone marrow and spleen.
84 In addition, the spleen is usually enlarged. Necrotic foci vary in size, and in some cases may be barely visible to
85 the naked eye. The lungs are usually congested and oedematous, and there may be areas of consolidation and
86 fibrinous pneumonia or pleuritis. Fibrin may be present in the abdominal cavity. Foci of caseous necrosis are
87 often present in one or more lymph node(s). The lymph nodes that are most often affected are those in the
88 abdominal and pleural cavities and lymph nodes draining the extremities. In less sensitive species, the
89 histological picture can resemble that of tuberculosis with chronic granulomas in liver, spleen, lungs and kidneys.
90 There is a high risk of human infection from F. tularensis, as the infective dose is extremely low and infected
91 animals excrete bacteria in urine and faeces. Infection can occur by simple contact. Suitable precautions, such
92 as the wearing of gloves, masks and eyeshields during any manipulation of pathological specimens or cultures,
93 must be taken in order to avoid human infection. The facility should meet at least the requirements for
94 Containment Group 3 pathogens as outlined in Chapter 1.1.2 Biosafety and biosecurity in the veterinary
95 microbiological laboratory and animal facilities. Countries lacking access to such a specialised national or
96 regional laboratory should send specimens to the OIE Reference Laboratory. Experimentally inoculated animals
97 and their excreta are especially hazardous to humans.

98 B. DIAGNOSTIC TECHNIQUES
99 1. Identification of the agent
100 Francisella tularensis can be demonstrated in smear preparations or in histological sections. It can also be
101 identified by culture or animal inoculation. However, F. tularensis may be difficult to isolate from dead animals
102 and carcasses due to overgrowth of other bacteria. As the post-mortem picture is variable, diagnosis is
103 sometimes difficult and immunological or immunohistochemical methods are preferable, although reagents may
104 be difficult to obtain. It can sometimes be recommended, therefore, that fixed specimens be analysed at
105 laboratories equipped with proper reagents or methods, such as the OIE Reference Laboratory (see Part 3 of this
106 Terrestrial Manual).
107 a) Smear preparations

108 Smear preparations are made on microscope slides as impression smears of organs such as the liver,
109 spleen, bone marrow, kidney, lung or blood. The bacteria are abundant in such smears, but may be
110 overlooked because of their very small size (0.2–0.7 µm). The bacteria can be demonstrated by direct or
111 indirect fluorescent antibody staining. This is a safe, rapid and specific diagnostic tool (13, 16, 18).
112 Gram staining of smears reveals a scattering of small, punctiform Gram-negative bacteria near the limit of
113 visibility. The use of oil microscopy increases the visibility of the bacteria. The bacteria may be difficult to
114 distinguish from precipitates of stain.
115 b) Histological sections

116 Bacteria can be demonstrated in sections using immunohistochemical methods, such as the fluorescent
117 antibody test (FAT) (16). The test is normally performed on specimens from liver, spleen or bone marrow,
118 fixed in neutral buffered formalin and paraffin embedded. Slides are treated with rabbit anti-tularemia
119 serum, washed and thereafter treated with sheep fluorescein-isothiocyanate-conjugated anti-rabbit serum.
120 The samples are examined under a fluorescence microscope. Large numbers of bacteria can be seen in
121 necrotic lesions and in the blood.
122 c) Culture
123 Francisella tularensis will not grow on ordinary media, although an occasional strain can sometimes, on
124 initial isolation, grow on blood agar. Incubation is at 37°C in ambient air or in 5% CO2. Heart blood, liver,
125 spleen and bone marrow from moribund animals should be used for culture. It is necessary to use special
126 culture media, such as:
127 i) Francis medium: Peptone agar containing 0.1% cystine (or cysteine) and 1% glucose, to which is
128 added, before solidification, 8–10% defribrinated rabbit, horse or human blood.
129 ii) McCoy and Chapin medium: This consists of 60 g egg yolk and 40 ml normal saline solution, carefully
130 mixed and coagulated by heating to 75°C.
131 iii) Modified Thayer–Martin agar: Glucose cysteine agar (GCA)-medium base supplemented with
132 haemoglobin and Iso VitaleX.
133 Media can be stored for up to 8–10 days at 4°C. Colonies that form on McCoy medium are small,
134 prominent, round and transparent. A more abundant growth is obtained on Francis medium and modified
135 Thayer–Martin agar, with confluent colonies that have a milky appearance and a mucoid consistency. On
136 either medium, colonies do not appear until after 48 hours’ incubation at 37°C.
137 iv) GCA agar with thiamine (BBL): When used with added blood, the medium is commonly referred to as
138 GBCA and can be substituted for the original, noncommercial medium described by Down et al. (5).
139 Suspend 58 g of the dry material in 1 litre of distilled or demineralised water, and mix thoroughly. Heat
140 with frequent agitation and boil for 1 minute. Dispense into tubes and sterilise by autoclaving at 118–
141 121°C for 15 minutes.
142 For larger volumes (up to several litres) of culture medium,autoclave at the same temperatures for
143 30 minutes. Cool to 45–48°C. Aseptically add 25 ml of packed human blood cells or 50 ml of defibrinated
144 rabbit or sheep blood. Mix thoroughly and pour into plates. Incubate at 37°C for 24 hours before use to
145 decrease surface moisture and to test for sterility (5).
146 The following selective medium can be used in addition to the non-selective media: Cystine heart agar
147 (DIFCO) with 5% rabbit blood, and penicillin (100,000 units), Polymyxin B sulphate (100, 000 units), and
148 cycloheximide (0.1 ml of a 1% stock solution) per litre.
149 Differential criteria for the identification of F. tularensis include absence of growth on ordinary media,
150 distinctive cellular morphology, and specific fluorescent antibody and slide agglutination reactions. The

151 bacteria are nonmotile, nonsporulating, bipolar staining, and of uniform appearance in 24-hour cultures, but
152 pleomorphic in older cultures.
153 Francisella tularensis can be identified in stained smears, by agglutination with tularemia hyperimmune
154 antiserum, or by animal inoculation. In areas of North America where both types of F. tularensis may occur,
155 Type A may be distinguished from Type B by the fact that most Type A ferment glycerol.
156 The bacteria can also be identified by hybridisation with probes specific to the 16S rRNA of F. tularensis,
157 F. tularensis Type A, and F. tularensis Type B (10), or by polymerase chain reaction (PCR) with primers
158 targeting specific regions of the 16 rDNA molecules. The PCR will allow identification at the genus, species
159 and subspecies level (11).
160 d) Capillary tube precipitation test on pathological samples

161 Tissues, such as spleen, liver or bone marrow, are ground with sterile sand in three-to-five times their
162 volume of normal saline. The suspension is transferred to a tube and two volumes of ethyl ether are added.
163 After shaking, the mixture is allowed to stand for 4–5 hours at room temperature. It is again shaken and
164 then allowed to stand overnight.
165 The aqueous phase is drawn off and centrifuged at 2000 g for 30 minutes. The supernatant fluid, containing
166 the antigen, is drawn off and distributed into capillary tubes to which tularemia antiserum is added.
167 The tubes are incubated at 37°C for 3 hours, then kept at 4°C overnight. A positive result is the formation of
168 a ring of precipitate.
169 e) Animal inoculation

170 Animal inoculation is extremely hazardous and is only recommended for agent identification in cases when
171 culture is negative and agent identification is needed for epidemiological reasons. It should only be
172 undertaken where proper biosafety facilities and cages are available (see Chapter 1.1.2). PCR techniques
173 should be used for identification of F. tularmsis.
174 Laboratory animals (preferably mice) are inoculated with culture material to confirm the nature of an isolate.
175 Pathological specimens may be inoculated for the direct detection of F. tularensis. Inoculated mice usually
176 die before lesions can form.
177 Intraperitoneal injection is sufficient for passage of pure cultures. All routes of administration in mice, such
178 as subcutaneous, percutaneous, or intravenous, will lead to an infection that is invariably fatal within 2–
179 10 days.
180 f) Molecular techniques

181 The PCR has recently been developed for the diagnosis of F. tularensis and is an excellent method for
182 identifying the agent in wound specimens from humans (12).
183 A real-time PCR has proven to be a highly sensitive and specific assay and is a major diagnostic
184 improvement, as all other methods for the specific identification of F. tularensis subsp. tularensis are very
185 time-consuming (23).
186 A DNA microarray has also been developed that is capable of distinguishing the highly virulent subspecies
187 F. tularensis subsp. tularensis and the moderately virulent subspecies F. tularensis subsp. holarctica (3).
188 2. Serological tests
189 Serology is currently carried out for diagnosis of tularemia in humans, but is of limited value in sensitive animal
190 species, which usually die before specific antibodies can develop. Serology may be employed, either on sera or
191 on lung extracts (18), in epidemiological surveys of animals that are resistant to infection, such as sheep, cattle,
192 pigs, moose, dogs or birds (18, 20). As there is no antigenic difference between Type A and Type B, the less
193 virulent F. tularensis palaearctica could be used as antigen in all serological tests.
194 a) Tube agglutination

195 The most commonly used serological test is the tube agglutination test. The antigen is a culture of
196 F. tularensis on Francis medium. The culture is harvested after 5–6 days. Younger cultures yield a poorer
197 antigen. The colonies are suspended in 96% alcohol, giving a thick suspension that can be stored for 1–
198 7 days at room temperature. The sediment is washed with normal saline and resuspended in an equal

199 volume of normal saline. Crystal violet powder is added to a final concentration of 0.25%. The bacteria are
200 stained by adding crystal violet and incubating at 37°C for at least 24 hours and at most 7 days.
201 After the supernatant fluid has been discarded, the deposit is suspended in normal saline with or without
202 thimerosal (merthiolate) at a final concentration of 1/10,000, or formaldehyde at a final concentration of
203 0.5%. The suspension is calibrated with positive and negative sera, and adjusted by adding normal saline to
204 provide an antigen that when tested on a slide gives readily visible stained agglutination reactions against a
205 clear fluid background.
206 The test is performed in tubes containing a fixed amount of antigen (0.9 ml) and different dilutions of serum
207 commencing with 1/10, 1/20, 1/40, etc. The results are read after 20 minutes of shaking, or after 1 hour in a
208 water bath at 37°C followed by overnight storage at room temperature. The agglutinated sediment is visible
209 to the naked eye or, preferably, by using a hand lens. The positive tubes are those that have a clear
210 supernatant fluid. Possible cross-reactions with Brucella abortus, B. melitensis and Legionella sp. have to
211 be taken into consideration.
212 b) Enzyme-linked immunosorbent assay

213 Another serological test, the enzyme-linked immunosorbent assay (ELISA), also allows an early diagnosis
214 of tularemia (4). This method is now widely used for clinical purposes. Different antigens, whole bacteria as
215 well as subcellular components (9), have been used as recall antigens against immunoglobulines IgA, IgM
216 and IgG; 2 weeks after the onset of tularemia, specific antibodies can be detected in the serum. IgM is
217 sustained for a long period and cannot be used as an indication of a recent infection (2). For routine
218 diagnosis, whole heat-killed (65°C for 30 minutes) bacteria can be used as antigen. Bacteria can be coated
219 to plastic plates, using the usual procedures (4) followed by serial dilutions of serum to be tested. Positive
220 reactions can be visualised by anti-antibodies labelled with enzyme. The test should also be read in a
221 photometer with positive and negative sera as controls.
222 C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS

223 Vaccines have been produced with the aim of protecting humans, but as all vaccine development involves testing
224 in animals, it is clear that some of the vaccines could be used to protect animals. However, in most countries,
225 there is no vaccine licensed for use in animals.
226 Before 1940, attempts to vaccinate against tularemia were performed by use of whole killed bacteria or bacterial
227 extracts. None of these vaccines induced protection against highly virulent strains of F. tularensis. Instead, viable
228 attenuated vaccines were developed. Attenuation was performed by repeated culture of bacteria on various
229 media with or without antiserum. Such live attenuated vaccines have been used in mass vaccinations of people
230 in the former Soviet Union since 1946, either as monocultures or as a mixture of strains.
231 An attenuated live vaccine strain of F. tularensis biovar palaearctica is available and can be used for restricted
232 vaccination of individuals at high risk (21).
233 REFERENCES
234 1. BELL J.F. (1980). Tularaemia. In: CRC Handbook Series in Zoonoses. Section A: Bacterial, Rickettsial, and
235 Mycotic Diseases. Vol. 2, Steel J.H., ed. CRS Press, Boca Raton, Florida, USA, 161–193.
236 2. BEVANGER L., MAELAND J.A. & KVAN A.I. (1994). Comparative analysis of antibodies to Francisella tularensis
237 antigens during the acute phase of tularemia and eight years later. Clin. Diagn. Lab. Immunol., 1, 238–240.
238 3. BROEKHUIJSEN M., LARSSON P., JOHANSSON A., BYSTRÖM M., ERIKSSON U., LARSSON E., PRIOR R.G., SJÖSTEDT
239 A., TITBALL R.W. & FORSMAN M. (2003). Genome-wide DNA microarray analysis of Francisella tularensis
240 strains demonstates exspensive genetic conservation within the species but identifies regions that are
241 unique to the highly virulent F. tularensis subsp. tularenis. J. Clin. Microbiol., 41 (7), 2924–2931.
242 4. CARLSSON H.E., LINDBERG A., LINDBERG G., HEDERSTEDT B., KARLSSON K. & AGELL B.O. (1979). Enzyme-linked
243 immunosorbent assay for immunological diagnosis of human tulreamia. J. Clin. Microbiol., 10, 615–621.
244 5. DOWN C.M., CORIELL L.L., CHAPMAN S.S. & KLAUBER A. (1947). The cultivation of Bacterium tularense in
245 embryonated eggs. J. Bacteriol., 53, 89–100.

246 6. ELIASSON H., LINDBACK J., NOURTI J.P., ARNEBORN M., GIESECKE J. & TEGNELL A. (2002). The 2000 tularemia
247 outbreak: a case–control study of risk factors in disease-endemic and emergent areas, Sweden. Emerg.
248 Infect. Dis., 8, 956–960.
249 7. ELLIS J., OYSTON P.C., GREEN M. & TITBALL R.W. (2002). Tularemia. Clin. Microbiol. Rev., 15 (4), 631–646.
250 8. FELDMAN K.A. (2003). Tularemia. J. Am. Vet. Med. Assoc., 222 (6), 725–730.
251 9. FULOP M.J., W EBBER T., MANCHEE R.J. & KELLY D.C. (1991). Production and characterization of monoclonal
252 antibodies directed against the lipopolysaccharide of Francisella tularensis. J. Clin. Microbiol., 29, 1407–
253 1412.
254 10. FORSMAN M., SANDSTROM G. & JAURIN B. (1990). Identification of Francisella species and discrimination of
255 Type A and Type B strains of F. tularensis by 16S rRNA Analysis. Appl. Environ. Microbiol., 56, 949–955.
256 11. FORSMAN M., SANDSTROM G & SJOSTEDT A. (1995). Analysis of 16S ribosomal DNA sequences of Francisella
257 strains and utilization for determination of the phylogeny of the genus and for identification of strains by
258 PCR. Int. J. Syst. Bacteriol., 44, 38–46.
259 12. JOHANSSON A., BERGLUND L., ERIKSSON U., GÖRANSSON I., W OLLIN R., FORSMAN M., TÄRNVIK A. & SJÖSTEDT A.
260 (2000). Comparative analysis of PCR versus culture for diagnosis of ulceroglandular tularemia. J. Clin.
261 Microbiol., 38 (1), 22–26.
262 13. KARLSSON K.A., DAHLSTRAND S., HANKO E. & SODERLIND O. (1970). Demonstration of Francisella tularensis in
263 sylvan animals with the aid of fluourescent antibodies. Acta Pathol. Microbiol. Immunol. Scand. (B), 78,
264 647–651.
265 14. MARKOWITZ L.E., HYNES N.A., DE LA CRUZ P., CAMPOS E., BARBAREE J.M., PLIKAYTIS B., MOOSIER D. & KAUFMAN
266 A.F. (1985). Tick-borne tularemia. JAMA, 254, 2922–2925.
267 15. MOLLARET H.H. & BOURDIN M. (1972). Le diagnostique biologique de la tularémie humaine. Med. Mal. Infect.,
268 2, 419–422.
269 16. MORNER T. (1981). The use of FA technique of detecting Francisella tularensis in formalin fixed material.
270 Acta Vet. Scand., 22, 296–306.
271 17. MORNER T. & ADDISON E. (2001). Tularemia. In: Infectious Diseases of Wild Mammals, Third Edition,
272 Williams E.S. & Barker I.K., eds. Iowa State University Press, Ames, Iowa, USA, 303-313.
273 18. MORNER T., SANDSTROM G. & MATTSON R. (1988). Comparison of sera and lung extracts for surveys of wild
274 animals for antibodies against Francisella tularensis biovar palaearctica. J. Wildl. Dis., 24, 10–14.
275 19. PEARSON A. (1998) Tularemia. In: Zoonoses, Palmer S.R, Soulsby E.J.L. & Simpson D.I.H., eds. Oxford
276 University Press, New York, USA, 303–312.
277 20. PHAHLER-JUNG K. (1989). Die globale verbreitungen der tularämie. Diss. Justus. Liebig-Univertsität, Giessen,
278 Germany, 1989.
279 21. SANDSTROM G. (1994). The tularemia vaccine. J. Chem. Tech. Biotechn., 59, 315–20.
280 22. SANDSTRÖM G., SJÖSTEDT A., FORSMAN M., PAVLOVICH N.V. & MISHANKIN B.N. (1992). Characterization and
281 classification of strains of Francisella tularensis isolated in the central Asian focus of the Soviet Union and
282 in Japan. J. Clin. Microbiol., 30 (1), 172–175.
283 23. TOMASO H., SCHOLZ H.C., NEUBAUER H., DAHOUK A.L., SEIBOLD E., LANDT O., FORSMAN M.& SPLETTSTOESSER
284 W.D. (2007). Real-time PCR using hybridization probes for the rapid specific identification of Francisella
285 tularenis subspecies tularensis. Mol. Cell. Probes, 21 (1), 12–16.
286 *
287 * *
288 NB: There is an OIE Reference Laboratory for Tularemia (see Table in Part 3 of this Terrestrial Manual or consult
289 the OIE Web site for the most up-to-date list: www.oie.int).

CHAPTER 2.1.19.
VESICULAR STOMATITIS
SUMMARY
Vesicular stomatitis (VS) is a vesicular disease of horses, cattle and pigs caused by
vesiculoviruses of the family Rhabdoviridae. This disease is clinically indistinguishable from foot
and mouth disease (FMD), vesicular exanthema of swine (VES), or swine vesicular disease (SVD)
when horses are not involved. Sheep, goats and many other wild species can be infected. Humans
are also susceptible. The disease is limited to the Americas; however, it was previously described
in France and in South Africa.
Although virus is transmitted directly by the transcutaneous or transmucosal route, it has been
isolated from sandflies and mosquitoes, suggesting that it could be insect-borne. There is,
therefore, seasonal variation in the occurrence of VS: it disappears at the end of the rainy season
in tropical areas, and at the first frosts in temperate zones. There is also some evidence that it
could be a plant virus and that animals are the end of the epidemiological chain. The pathogenesis
of the disease is unclear, and it has been observed that the humoral-specific antibodies do not
always prevent infection with VS serogroup viruses.
Although VS may be suspected when horses are involved as well as pigs and cattle, prompt
differential diagnosis is essential because the clinical signs of VS are indistinguishable from FMD
when cattle and pigs are affected, and from SVD or VES when only pigs are affected.
Identification of the agent: Virus can be readily isolated by the inoculation of several tissue
culture systems, unweaned mice or embryonated chicken eggs. Viral RNA can be detected from
epithelial tissue and vesicular fluid by conventional and real-time reverse transcriptase polymerase
chain reaction (PCR). Viral antigen can be identified by an indirect sandwich enzyme-linked
immunosorbent assay (IS-ELISA) – this is the least expensive and most rapid test. The
complement fixation (CF) test is also a good alternative. The virus neutralisation (VN) test may be
used, but it is elaborate and time-consuming.
Serological tests: Convalescent animals develop serotype-specific antibodies within 4–8 days of
infection that are demonstrated by a liquid-phase blocking ELISA (LP-ELISA), a competitive ELISA
(C-ELISA) and VN. Other described tests are CF, agar gel immunodiffusion and counter
immunoelectrophoresis.
Requirements for vaccines and diagnostic biologicals: Inactivated virus vaccines with
aluminium hydroxide or oil as adjuvants have been tested in the United States of America and in
Colombia, respectively. Both vaccines generated high levels of specific antibodies in the sera of
vaccinated cattle. However, it is not yet clear if serum antibodies would prevent the disease. An
attenuated virus vaccine has been used in the field with unknown efficacy.
A. INTRODUCTION
Vesicular stomatitis (VS) was described in the United States of America (USA) in 1926 (18) and 1927 (7) as a
vesicular disease of horses, and subsequently of cattle and pigs. Vesicles are caused by virus on the tongue,
lips, buccal mucosa, teats and in the coronary band epithelium of cattle, horses, pigs, and many other species of
domestic and wild animals. Natural disease in sheep and goats is rare, although both species can be
experimentally infected. Mixed infections of foot and mouth disease (FMD) and VS viruses have occurred in the
same herds of cattle and can be induced experimentally. Many species of laboratory animals are also
susceptible. The disease is limited to the Americas; however, it was described in France (1915 and 1917) and in
South Africa (1886 and 1897) (11).

Chapter 2.1.19. — Vesicular stomatitis
Influenza-like signs, normally without vesicles, have been observed in humans who are in contact with animals
with VS or who handle infective virus. All manipulations involving virus, including infective materials from animals,
should be undertaken with using proper biosafety procedures.
There are two distinct immunological classes of vesicular stomatitis virus (VSV) that have been recognised: New
Jersey (NJ) and Indiana (IND). Both viruses are members of the genus Vesiculovirus, family Rhabdoviridae and
have been extensively studied at the molecular level. Several other closely related rhabdoviruses have been
isolated from sick animals over the past decades. There are three subtypes of the IND serogroup based on
serological relationships: IND-1 IND-2 and IND-3; they are also known as classical IND virus (VSIV), cocal virus
(COCV), and alagoas virus (VSAV), respectively (8). Strains of the serotype NJ and subtype IND-1 are endemic
in livestock in areas of southern Mexico, Central America, Venezuela, Colombia, Ecuador and Peru, with VSV NJ
causing the vast majority (>80%) of the clinical cases. Sporadic activity of NJ and IND-1 VSV has been reported
in northern Mexico and western United States. IND-2 has only been isolated in Argentina and Brazil and only
from horses (Salto-Argentina/63, Maipú-Argentina/86, Rancharia-Brazil/66, Riberao-Brazil/79) (2, 3). Cattle living
together with the affected horses did not develop antibodies against VSV (2). The IND-3 subtype, (Alagoas-
Brazil/64), has been identified, sporadically only in Brazil and only in horses until 1977. However, in 1977 the
IND-3 serotype (Espinosa-Brazil/77 strain) was first isolated from cattle in Brazil; it has been observed that this
serotype affects cattle to a lesser degree than horses (2, 3). This finding confirms the first descriptions, in 1926
and 1927 (7, 18), of the NJ and IND serotypes in horses, and subsequently in cattle and pigs; this same
predilection has been observed in other VS outbreaks.
The mechanism of transmission of the virus is unclear. The fact that viruses have been isolated from sandflies,
mosquitoes, and other insects tends to substantiate the hypothesis that it could be transmitted by insects (6, 10,
17). There are also hypotheses that the VS virus is a plant virus present in pasture (17) and that animals are the
end of the epidemiological chain and, in special circumstances, the virus could undergo an adaptation process to
infect animals, followed by direct transmission between susceptible animals. During the 1982 epizootic in
western USA, there were a number of cases where there was direct transmission from animal to animal (20).
While VS is not diagnosed in livestock every year in the USA, it is considered to be endemic in feral pigs on
Ossabaw Island, Georgia (5).
The incidence of disease can vary widely among affected herds. Usually 10–15% of the animals show clinical
signs. Clinical cases are mainly seen in adult animals. Cattle and horses under 1 year of age are rarely affected.
Mortality is close to zero in both species. However, high mortality rates in pigs affected by the NJ virus have been
observed. Sick animals recover in about 2 weeks. The most common complications of economic importance are
mastitis and loss of production in dairy herds (16). Both NJ and IND-1 serotypes in the 1995, 1997 and 1998 US
outbreaks primarily caused clinical disease in horses. Although some clinical signs have been observed in cattle,
the primary finding in cattle was seroconversion.
VS cannot be reliably clinically differentiated from the other vesicular diseases, such as foot and mouth disease
(FMD), vesicular exanthema of swine (VES), and swine vesicular disease (SVD) when horses are not involved.
An early laboratory diagnosis of any suspected VS case is therefore a matter of urgency.
The sample collection and technology used for the diagnosis of VS must be in concordance with the
methodology used for the diagnosis of FMD, VES and SVD, in order to facilitate the differential diagnosis of
these vesicular diseases. Note: VS serogroup viruses can be human pathogens and appropriate precautions
should be taken when working with potentially infected tissues or virus (see Chapter 1.1.2 Biosafety and
Vesicle fluid, epithelium covering unruptured vesicles, epithelial flaps of freshly ruptured vesicles, or swabs of the
ruptured vesicles are the best diagnostic samples. These samples can be collected from mouth lesions, as well
as from the feet and any other sites of vesicle development. It is recommended that animals should be sedated
before samples are collected to avoid injury to helpers and for reasons of animal welfare. Samples from all
species should be placed in containers of Tris-buffered tryptose broth with phenol red, pH 7.6. If complement
fixation (CF) is to be carried out for antigen detection, samples from all species can be collected in
glycerol/phosphate buffer, pH 7.2–7.6. (Note: glycerol is toxic to virus and decreases the sensitivity of virus
isolation; it is therefore only recommended for collection of samples for CF test.) Samples should be kept
refrigerated and if they can arrive at the laboratory within 48 hours after collection, they should be sent
refrigerated. If samples are sent frozen with dry ice, precautions should be taken to protect the sample from
contact with any CO2. There are special packaging requirements for shipping samples with dry ice (see Chapter
1.1.1 Collection and shipment of diagnostic specimens, for further information on shipping of diagnostic
samples).
When epithelial tissue is not available from cattle, samples of oesophageal–pharyngeal (OP) fluid can be
collected by means of a probang (sputum) cup. In pigs, throat swabs can be taken for submission to a laboratory

for virus isolation. This material should be sent to the laboratory refrigerated in Tris-buffered tryptose broth. If the
samples will be in shipment for more than 48 hours after collection, they should be sent frozen with dry ice as
described previously. Probang samples for isolation of virus should not be treated with solvents such as
chloroform. Virus can be isolated from oral and nasal specimens up to 7 days post-infection.
When it is not possible to collect samples for identification of the agent, serum samples from recovered animals
can be used for detecting and quantifying specific antibodies. Paired sera from the same animals, collected 1–
2 weeks apart, are preferred for checking the change in antibody titre.
Specific reagents for VS diagnosis are not commercially available and each laboratory must produce its own or
obtain them from a Reference Laboratory. The two OIE Reference Laboratories for vesicular stomatitis (see
Table given in Part 3 of this Terrestrial Manual), and the Institute for Animal Health 1, produce and distribute
diagnostic reagents on request.
For identification of VS serogroup viruses and the differential diagnosis of vesicular diseases, clarified
suspensions of field samples suspected to contain virus should be submitted for immunological testing. For virus
isolation, the same samples are inoculated into appropriate cell cultures. The inoculation of African green
monkey kidney (Vero), baby hamster kidney (BHK-21) and IB-RS-2 cell cultures with the same sample permits
differentiation of the vesicular diseases: VS serogroup viruses cause a cytopathic effect (CPE) in all three cell
lines; FMD virus causes a CPE in BHK-21 and in IB-RS-2, while SVD virus causes a CPE in IB-RS-2 only. Many
other cell lines, as well as most primary cell cultures of animal origin, are susceptible to VS serogroup viruses.
Virus replicates and can be isolated in 8–10-day-old chicken embryos by inoculation into the allantoic sac, in 2–
7-day-old unweaned mice by inoculation using any route, or in 3-week-old mice by intracerebral inoculation. In all
three cases, virus causes death in between 2 and 5 days after inoculation.
The most susceptible route for horses and cattle is intradermalingual administration. Pigs are inoculated in the
coronary band or on the snout. Vesicular lesions may be observed in the epithelial tissues of the mouth, teats
and feet, 2–4 days after inoculation. The presence of secondary vesicles after inoculation of cattle and horses
depends mainly on the VS virus isolate used. The snout is normally affected in pigs.
If a CPE develops in the cultures, the suspension fluids can be used for identification of the agent by different
immunological tests and the cell culture can be stained with VS-specific fluorescent antibody conjugate and viral
antigen detected by enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR). Similar
tests can be performed on homogenate suspensions of the dissected musculo-skeletal tissues of dead mice and
chicken embryos and with suspensions of epithelial samples. The brain tissue from mice is an excellent source of
virus.
Due to the different morphological characteristics of the rhabdovirus (VS serogroup viruses), picornavirus (FMD
virus and SVD virus), calicivirus (VES) and the large number of virus particles present in vesicular fluids and
epithelial tissues, electron microscopy can be a useful diagnostic tool for differentiating the virus family involved.
The preferred immunological methods for the identification of the viral antigens in the laboratory are the ELISA
(2, 9), the CF test (2, 13) and fluorescent antibody staining. The virus neutralisation (VN) test, with known
positive antisera against the VS virus NJ and IND serotypes, may be used in tissue cultures, unweaned mice or
embryonated eggs, but it is more time-consuming.
a) Virus isolation
i) Inoculate cell culture in Leighton tubes and 25 cm2 flasks with the clarified suspension of tissues or
vesicular fluid.
ii) Incubate inoculated cell cultures at 37°C for 1 hour.
iii) Discard inoculum and wash cell cultures three times with cell culture medium and replace with cell
culture medium containing 2.5% fetal bovine serum (FBS).
Iv) Incubate Leighton tube cell cultures at 33–35°C and observe for CPE.
v) After 18–24 hours of incubation, the cover-slip from one Leighton tube culture per specimen
inoculated is stained with New Jersey and Indiana VS virus-specific fluorescent antibody (FA)
conjugate.
1 Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom.

vi) Remaining Leighton tube cultures and 25 cm2 flask cultures are incubated at 35–37°C for 6 more days
and observed daily for CPE.
vii) At 7 days post-inoculation, the remaining Leighton tube cover-slips are stained with FA conjugate.
viii) If CPE is observed and the FA staining is negative, a second passage as is made, as described
above, using the cells from the 25 cm2 flask. Note: First passage cultures with significant CPE may
yield false-negative immunofluorescence results. Serial tenfold dilutions may be prepared and
inoculated to provide distinct plaques of fluorescing cells.
ix) Interpretation of the results: If no fluorescence is observed and no CPE evident in the flask culture, the
sample is negative for virus isolation. If specific fluorescence is observed, the sample is positive for
virus isolation.
x) Alternatively cell culture in flasks can be inoculated with field samples, incubated at 35–37°C for
48 hours and observed daily for CPE. If no CPE is observed after 48 hours, the flask cultures are
frozen and thawed and a sample of the supernatant is inoculated into fresh cell culture. Up to three
passages are made, of 48 hours each. To detect the presence of VSV antigen, clarified supernatants
of each passage are tested by ELSA or CF test.

The indirect sandwich ELISA (IS-ELISA) (2, 9) is currently the diagnostic method of choice for identification
of viral serotypes of VS and other vesicular diseases. Specifically, the ELISA procedure with a set of
polyvalent rabbit/guinea-pig antisera, prepared against virions of the representative strains of the three
subtypes of the IND serotype, identifies all strains of the VS virus IND serotype (2). For detection of VS
virus NJ strains, a monovalent set of rabbit/guinea-pig antisera is suitable (2, 9).
• Test procedure
i) Solid phase: ELISA plates are coated either for 1 hour at 37°C or overnight at 4°C with rabbit antisera
and normal rabbit serum (as described in refs 2 and 4), and optimally diluted in carbonate/bicarbonate
buffer, pH 9.6. Subsequently, the plates are washed once with phosphate buffered saline (PBS) and
blocked for 1 hour at room temperature with 1% ovalbumin in PBS. The plates are used immediately
or are washed three times and stored at –20°C for future use.
ii) Test samples: Antigen suspensions of test samples (10–20% epithelial tissue suspension, musculo-
skeletal tissue of chicken embryo or mice in PBS or undiluted clarified cell culture supernatant fluid)
are deposited in the corresponding wells and the plates are incubated for 1 hour at 37°C on an orbital
shaker.
iii) Detector: Monovalent and polyvalent guinea-pig antisera to VS virus NJ and IND serotypes,
respectively, that are homologous to coated rabbit serum and that have been diluted appropriately in
PBS containing 0.05% Tween 20, 1% ovalbumin, 2% normal rabbit serum, and 2% normal bovine
serum (PBSTB) are added to the corresponding wells and left to react for 1 hour at 37°C on an orbital
shaker.
iv) Conjugate: Peroxidase/rabbit or goat IgG anti-guinea-pig Ig conjugate, diluted in PBSTB, is added and
left to react for 1 hour at 37°C on an orbital shaker.
v) Substrate: H2O2-activated substrate is added and left to react at room temperature for 15 minutes,
followed by the addition of sulphuric acid to stop the reaction. Absorbance values are measured using
an ELISA reader.
Throughout the test, 50 µl reagent volumes are used. The plates are washed five times between each stage
with PBS containing 0.05% Tween 20. Controls for the reagents used are included.
vi) Interpretation of the results: An antiserum giving an absorbance more than 20% greater than the other
antisera, negative serum and controls is considered to be positive for the corresponding virus subtype.
c) Complement fixation test

The ELISA is preferable to the CF test because it is more sensitive and it is not affected by pro- or anti-
complementary factors. When ELISA reagents are not available, however, the CF test may be performed.
The CF test in U-bottomed microtitre plates, using the reagents titrated by CF50% test, is described.
• Test procedure
i) Antisera: Guinea-pig monovalent anti-NJ VS virus and polyvalent anti-IND VS virus, diluted in veronal
buffer (VB) at a dilution containing 2.5 CFU50 (50% complement fixation units) against homologous
virus, are deposited in plate wells. Those antisera are the detectors used in ELISA.

ii) Test samples: The antigen suspension of test samples, prepared as described for IS-ELISA, is added
to the wells with serum.
iii) Complement: 4 CHU50 (50% complement haemolytic units) are added to the serum and antigen. (An
alternative is to use 7.5, 10 and 20 CHU50 with the goal of reaching 4 CHU50 in the test.) The mixture
of antisera, test samples and complement is incubated at 37°C for 30 minutes.
iv) Haemolytic system: A suspension of sheep red blood cells (SRBC) in VB, sensitised with 10 HU50
(50% haemolytic units) of rabbit anti-SRBC serum, is added to the wells. The haemolytic system has
an absorbance of 0.66 read at 545 nm, in the proportion of two volumes of haemolytic system + three
volumes of distilled water. The mixture is incubated for 30 minutes at 37°C. Subsequently, the plates
are centrifuged and the reaction is observed visually.
Volumes of 25 µl for antisera, test samples and complement, and 50 µl of haemolytic system, are required.
Appropriate controls for the antisera, antigens, complement and haemolytic system are included.
It is possible to perform the CF50% test in tubes (2) using reagent volumes eight times greater than those
indicated for the CF in microtitre plates. With the CF50% test, the reaction can be expressed as absorbance
read spectrophotometrically at 545 nm.
v) Interpretation of the results: When controls are as expected, samples with haemolysis <20% for one
antiserum in comparison with the other antiserum and controls are considered to be positive for the
corresponding type.
Field samples that are negative on the ELISA or CF test should be inoculated into cell culture or unweaned
mice. If there is no evidence of viral infection after three passages, the specimen is considered to be
negative for virus.
d) Nucleic acid recognition methods

The PCR can be used to amplify small genomic areas of the VS virus (12, 19). This technique will detect the
presence of virus RNA in tissue and vesicular fluid samples and cell culture, but cannot determine if the
virus is infectious. In general, PCR techniques have not been routinely used for screening diagnostic cases
for viruses causing VS.
For the identification and quantification of specific antibodies in serum, the ELISA and the VN test are preferable.
The CF test may be used for quantification of early antibodies. Antibody can usually be detected between 5 and
8 days post-infection; the length of time antibody persists has not been accurately determined for the three tests
but is thought to be relatively short for the CF and for extended periods for the VN and ELISA (14).
a) Enzyme-linked immunosorbent assay (a prescribed test for international trade)

The liquid-phase blocking ELISA (LP-ELISA) is the method of choice for the detection and quantification of
antibodies to VS serogroup viruses. The use of viral glycoproteins as antigen is recommended because
they are not infectious, allow the detection of neutralising antibodies, and give fewer false-positive results
than the VN (4).
• Test procedure
i) Solid phase: As described above in Section B.1.a for the IS-ELISA.
ii) Liquid phase: Duplicate, twofold dilution series of each test serum, starting at 1/4, are prepared in U-
bottomed microtitre plates. An equal volume of VS virus NJ or IND glycoprotein, in a dilution providing
70% reaction, is added to each well and the plates are incubated for 1 hour at 37°C. 50 µl of these
mixtures is then transferred to the ELISA plates with the solid phase and left to react for 30 minutes at
37°C on an orbital shaker.
iii) Detector, conjugate and substrate: The same reagents and methods are used as those indicated for
the IS-ELISA.
iv) Interpretation of the results: 50% end-point titres are expressed in log10 in reference to the 50%
reduction of negative serum control, according to the Spearmann–Kärber method. Titres of >1.3 (1/20)
are considered to be positive.
• Competitive enzyme-linked immunosorbent assay (a prescribed test for international trade)

A competitive ELISA for detection of antibodies has also been developed. The procedure described here is
based on a procedure described by Afshar et al. (1). It uses vesicular stomatitis NJ and IND-1 recombinant
antigens as described by Katz et al. (15).

• Test procedure
i) Solid phase: Antigens are diluted in carbonate/bicarbonate buffer, pH 9.6, and 50 µl is added to each
well of a 96-well ELISA plate. The plates are incubated overnight at 4°C; coated plates can be frozen
at –70°C for up to 60 days. The plates are thawed, antigen is decanted, and 100 µl of blocking solution
(5% nonfat dry milk powder solution in PBS [for example, 5 g dry milk powder dissolved in 95 ml PBS)
is added. The plates are then incubated at 25°C for 30 minutes and blocking solution is decanted. The
plates are washed three times with PBS/0.05% Tween 20 solution.
ii) Liquid phase: 50 µl of serum diluted 1/8 in 1% nonfat dry milk in PBS is added to each of the duplicate
wells for each sample. A positive and negative control serum for each serotype should be included on
each ELISA plate. The plates are incubated at 37°C for 30 minutes. Without washing, 50 µl of
polyclonal ascites fluid is added to each well and plates are incubated at 37°C for 30 minutes.
iii) Detector: The plates are washed three times, and 50 µl of goat anti-mouse horseradish-peroxidase
conjugate diluted in 1% nonfat dry milk with 10% normal goat serum is added to each well. The plates
are incubated at 37°C for 30 minutes, washed three times, and 50 µl of tetramethyl-benzidine (TMB)
substrate solution is added to each well. The plates are incubated at 25°C for 5–10 minutes and then
50 µl of 0.05 M sulphuric acid is added to each well. The plates are read at 450 nm and the optical
density of the diluent control wells must be > 1.0.
iv) Interpretation of the results: A sample is positive if the absorbance is ≤50% of the absorbance of the
diluent control.
b) Virus neutralisation (a prescribed test for international trade)

The VN test is carried out in tissue culture microtitre plates with flat-bottomed wells using inactivated serum
as test sample, 1000 TCID50 (50% tissue culture infective dose) of VS NJ or IND virus, and Vero M cells, or
preformed monolayer (4) or a suspension IB-RS-2 cells to test for the presence of unneutralised virus.
• Test procedure
i) Virus: VS NJ or IND virus is grown in Vero cell monolayers and stored in liquid nitrogen or frozen at
–70°C.
ii) Test samples: Sera are inactivated at 56°C for 30 minutes before testing. Positive and negative control
standard sera are included in the test.
iii) Virus neutralisation: Sera are diluted in a twofold or four-fold dilution series across the plates, starting
from 1/4 dilution. Two rows of wells are used per serum. The same volume of NJ or IND VS virus
suspension containing about 1000 TCID50/25 µl is added and incubated at 37°C for 60 minutes to
allow neutralisation to take place. Subsequently, 50 µl of the mixtures is deposited on preformed cell
monolayers in microtitre plates or 150 µl of 300,000/ml IB-RS-2 or Vero cell suspension is added to
each well with the serum/virus mixtures. The plates are covered with loosely fitting lids and incubated
for 48–72 hours at 37°C in an atmosphere of 5% CO2 or sealed with pressure-sensitive tape and
incubated in a normal atmosphere. (It has been determined that a virus titre of 1000 TCID50 will
decrease the nonspecific reactions and maintain a high test sensitivity.)
iv) Interpretation of the results: Wells without CPE are considered to be positive. End-point titres of test
serum titres are determined by the Spearmann–Kärber method when the virus titres are between
750 and 1330 TCID50 and when titres of positive and negative standard sera are within twofold of their
mean values as estimated from previous titration. The 100% neutralisation titres of each serum are
expressed at log 10. Sera with values of 1/32 or greater are considered to be positive for antibodies
against VSV. In an alternative protocol, the end-point titre of the test serum is determined when the
virus doses are between 102±0.5/100 µl and when titres of positive and negative standard sera are
within twofold of their mean values as estimated from the previous titration. The 50% neutralisation
titre of each serum is expressed as log 10. Sera with values of 1.3 (1/20) or greater are considered to
be positive for VS antibodies (4).
c) Complement fixation (a prescribed test for international trade)

A detailed description of this test is given in Section B.1.b. This is modified as follows. The CF test may be
used for quantification of early antibodies, mostly IgM. For this purpose, twofold serum dilutions are mixed
with 2 CFU50 of known antigen and with 5% normal bovine or calf sera included in 4 CHU50 of complement.
The mixture is incubated for 3 hours at 37°C or overnight at 4°C. Subsequently, the haemolytic system is
added followed by incubation for 30 minutes at 37°C. The serum titre is the highest dilution in which no
haemolysis is observed. Titres of 1/5 or greater are considered to be positive. This CF has low sensitivity
and is frequently affected by anticomplementary or nonspecific factors.


Attenuated virus vaccines have been tested in the field in the USA, Panama, Guatemala, Peru and Venezuela
(16, 17) with unknown efficacy. Killed vaccines for the Indiana and New Jersey serotypes are manufactured in
Colombia and Venezuela (2002 OIE vaccine survey).
1. Seed management

Identity of the seed and the source of the serum used in growth and passage of the virus should be well
documented, including the source and passage history of the organism.
Virus seed can be grown in cell culture. Selection of a cell type for culture is dependent on the degree of
virus adaptation, growth in medium, and viral yield in the specific culture system. Vaccine products should
be limited to the number of passages from the master seed virus (MSV) that can be demonstrated to be
effective.
c) Validation of culture
The purity of the seed and cells to be used for vaccine production must be demonstrated. The MSV should
be free from adventitious agents, bacteria, or Mycoplasma, using tests known to be sensitive for detection
of these microorganisms. The test aliquot should be representative of a titre adequate for vaccine
production, but not such a high titre that hyperimmune antisera are unable to neutralise seed virus during
purity testing. Seed virus is neutralised with monospecific antiserum or monoclonal antibody against the
seed virus and the virus/antibody mixture is cultured on several types of cell line monolayers. A cell line
highly permissive for bovine viral diarrhoea virus, types 1 and 2, is recommended as one of the cell lines
chosen for evaluation of the MSV. Bovine viral diarrhoea virus is a potential contaminant introduced through
the use of fetal bovine serum in cell culture systems. Cultures are subpassaged at 7-day intervals for a total
of at least 14 days, then tested for adventitious viruses that may have infected the cells or seed during
previous passages.
d) Validation as a vaccine
Vaccine candidates should be shown to be pure, safe, potent, and efficacious.
Virus(es) used in vaccine production should be antigenically relevant to virus(es) circulating in the field. A
vaccination/challenge study in the species for which the vaccine will be used will indicate the degree of
protection afforded by the vaccine. Species used in vaccination/challenge studies should be free of
antibodies against vesicular stomatitis. Vaccination/challenge studies should be conducted using virus
produced by the intended production method, at the maximum viral passage permitted, and using animal
species of the minimum recommended age listed on the label. Initially, lots are formulated to contain
varying amounts of viral antigen. The test lot containing the least amount of antigen that demonstrates
protection becomes the standard against which future production lots are measured. For vaccines
containing more than one virus (for example, New Jersey and Indiana-1), the efficacy of the different
components of these vaccines must each be established independently and then as a combination in case
interference between different viruses exists.
Once the vaccine is shown to be efficacious, and the proposed conditions for production are acceptable to
regulatory authorities, approval may be granted to manufacture vaccine. Generally, large-scale monolayer or
suspension cell systems are operated under strict temperature-controlled, aseptic conditions and defined
production methods, to assure lot-to-lot consistency. When the virus has reached its appropriate titre, as
determined by CPE, fluorescent antibody assay, or other approved technique, the virus is clarified, filtered, and
inactivated. An inactivation kinetics study should be conducted using the approved inactivating agent on a viral
lot with a titre greater than the maximum production titre and grown using the approved production method. This
study should demonstrate that the inactivation method is adequate to assure complete inactivation of virus.
Samples taken at regular timed intervals during inactivation, then inoculated on to a susceptible cell line, should
indicate a linear and complete loss of titre by the end of the inactivation process. Typically, adjuvant is added to
enhance the immune response.

Cell cultures should be checked macroscopically for abnormalities or signs of contamination and discarded if
unsatisfactory. Virus concentration can be assessed using antigenic mass or infectivity assays.
4. Batch control
a) Sterility
During production, tests for bacteria, Mycoplasma, and fungal contamination should be conducted on both
inactivated and live vaccine harvest lots and confirmed on the completed product (see Chapter 1.1.9 Tests
for sterility and freedom from contamination of biological materials).
b) Safety
The completeness of viral inactivation in a killed product can be determined by multiple passes in cell
culture of the post-inactivation, pre-adjuvant production fluids, followed by testing for the presence of virus.
Final product may be evaluated in the host animal using two animals of the minimum age recommended for
use, according to the instructions given on the label; the animals are observed for 21 days. Field safety
studies conducted on vaccinates, in at least three divergent geographical areas, with at least 300 animals
per area, are also recommended. If the vaccine is to be used in horses, swine, cattle, or other ruminants
destined for market and intended for human consumption, a withdrawal time consistent with the adjuvant
used (generally 21 days) should be established by such means as histopathological examination submitted
to the appropriate food safety regulatory authorities.
c) Potency
During production, antigen content is measured to establish that minimum bulk titres have been achieved.
Antigen content is generally measured before inactivation and prior to further processing. Relative potency
can be used to determine antigen content in final product. It is necessary to confirm the sensitivity,
specificity, reproducibility, and ruggedness of such assays.
The duration of immunity and recommended frequency of vaccination of a vaccine should be determined
before a product is approved. Initially, such information is acquired directly using host animal
vaccination/challenge studies. The period of demonstrated protection, as measured by the ability of
vaccinates to withstand challenge in a valid test, can be incorporated into claims found on the vaccine label.
e) Stability
Vaccines should be stored at 4°C ± 2°C, with minimal exposure to light. The shelf life should be determined
by use of the approved potency test (Section C.5.b) over the proposed period of viability.
f) Preservatives
Preservatives should be avoided if possible, and where not possible, should be limited to the lowest
concentration possible.
g) Precautions
Inactivated vesicular stomatitis vaccines probably present no special danger to the user, although
accidental inoculation may result in an adverse reaction due to the adjuvant and secondary components of
the vaccine.
a) Safety
Final container samples of completed product from inactivated vaccines should be tested.
b) Potency
The potency assay established at the time of the minimum antigen protection study should be used to
evaluate new lots for release. The assay needs to be specific and reproducible. It must reliably detect
vaccines that are not sufficiently potent.

REFERENCES
1. AFSHAR A., SHAKARCHI N.H. & DULAC G.C. (1993). Development of a competitive enzyme linked
immunosorbent assay for detection of bovine, equine, ovine and porcine antibodies to vesicular stomatitis
virus. J. Clin. Microbiol., 31, 1860–1865.
2. ALONSO A., MARTINS M., GOMES M.P.D., ALLENDE R. & SONDAHL M.S. (1991). Development and evaluation of
an enzyme-linked immunosorbent assay for detection, typing and subtyping of vesicular stomatitis virus.
J. Vet. Diagn. Invest., 3, 287–292.
3. ALONSO FERNANDEZ A. & SONDAHL M.S. (1985). Antigenic and immunogenic characterisation of various
strains of the Indiana serotype of vesicular stomatitis isolated in Brazil. Bol. Cen. Panam. Fiebre Aftosa, 51,
25–30.
4. ALLENDE R., SEPULVEDA L., MENDES DA SILVA A., MARTINS M., SONDAHL M.S. & ALONSO FERNANDEZ A. (1992).
An enzyme-linked immunosorbent assay for the detection of vesicular stomatitis virus antibodies. Prev. Vet.
Med., 14, 293–301.
5. BORING W. & SMITH D. (1962). Vesicular Stomatitis Virus: A Survey and Analysis of the Literature. Technical
Study No. 43, US Army Biological Laboratories, Fort Detrick, USA.
6. COMER S.A., CORN J.L., STALLKNECHT D.E., LANDGRAF J.G. & NETTLES V.F. (1992). Titers of vesicular
stomatitis virus New Jersey serotype in naturally infected male and female Lutzomyia shannoni (Diptera:
Psychodidae) in Georgia. J. Med. Entomol., 29, 368–370.
7. COTTON W.E. (1927). Vesicular stomatitis. Vet. Med., 22, 169–175.
8. FEDERER K.E., BURROWS R. & BROOKSBY J.B. (1967). Vesicular stomatitis virus – the relation between some
strains of the Indiana serotype. Res. Vet. Sci., 8, 103–117.
9. FERRIS N.P. & DONALDSON A.I. (1988). An enzyme-linked immunosorbent assay for the detection of VSV
antigen. Vet. Microbiol., 18, 243–258.
10. FRANCY D.B., MOORE C.G., SMITH G.C., TAYLOR S.A. & CALISER C.H. (1988). Epizootic vesicular stomatitis in
Colorado, 1982: isolation of virus from insects collected along the northern Colorado Rocky Mountain Front
Range. J. Med. Entomol., 25, 343–347.
11. HANSON R.P. (1952). The natural history of vesicular stomatitis. Bacteriol. Rev., 16, 179–204.
12. HOFNER M.C., CARPENTER W.C., FERRIS N.P., KITCHING R.P. & BOTERO F.A. (1994). A hemi-nested PCR assay
for the detection and identification of vesicular stomatitis virus nucleic acid. J. Virol. Methods, 50, 11–20.
13. HOLE K., CLAVIJO A. & PINEDA L.A. (2006). Detection and serotype-specific differentiation of vesicular
stomatitis virus using a multiplex, real-time, reverse transcription-polymerase chain reaction assay. J. Vet.
Diagn. Invest., 18, 139–146.
13. JENNY E.W., MOTT L.O. & TRAUB E. (1958). Serological studies with the virus of vesicular stomatitis. I. Typing
of vesicular stomatitis by complement fixation. Am. J. Vet. Res., 19, 993–998.
14. KATZ J.B., EERNISSE K.A., LANDGRAF J.G. & SCHMITT B.J. (1997). Comparative performance of four
serodiagnostic procedures for detecting bovine and equine vesicular stomatitis virus antibodies. J. Vet.
Diagn. Invest., 9, 329–331.
15. KATZ J.B., SHAFER A.L. & EERNISSE K.A. (1995). Construction and insect larval expression of recombinant
vesicular stomatitis nucleocapsid protein and its use in competitive ELISA. J. Virol. Methods, 54, 145–157.
16. LAUERMAN L.H., KUNS M.L. & HANSON R.S. (1962). Field trial of live virus vaccination procedure for prevention
of vesicular stomatitis in dairy cattle. I: Preliminary immune response. Proceedings of the 66th Annual
Meeting of the United States Animal Health Association, 365–369.
17. MASON J. (1978). The epidemiology of vesicular stomatitis. Bol. Cen. Panam. Fiebre Aftosa, 29–30, 35–53.
18. OLTSKY P.K., TRAUM J. & SCHOENING H.W. (1926). Comparative studies on vesicular stomatitis and foot and
mouth disease. J. Am. Vet. Med. Assoc., 70, 147–167.

19. RODRIQUEZ L.L., LETCHWORTH G.J., SPIROPOULOU C.F. & NICHOL S.T. (1993). Rapid detection of vesicular
stomatitis virus New Jersey serotype in clinical samples by using polymerase chain reaction. J. Clin.
Microbiol., 31, 2016–2020.
20. SELLERS R.F. & MAAROUF A.R. (1990). Trajectory analysis of winds in vesicular stomatitis in North America.
Epidemiol. Infect., 104, 313–328.
*
**
NB: There are OIE Reference Laboratories for Vesicular stomatitis (see Table in Part 3 of this Terrestrial Manual
or consult the OIE Web site for the most up-to-date list: www.oie.int).

CHAPTER 2.1.20.
WEST NILE FEVER
SUMMARY
West Nile virus (WNV) is a member of the genus Flavivirus in the family Flaviviridae. The arbovirus
is maintained in nature by cycling through birds and mosquitoes; numerous avian and mosquito
species support virus replication. For many avian species, WNV infection causes no overt signs
while other birds, such as American crows (Corvus brachyrhynchos) and Blue Jays (Cyanocitta
cristata), often succumb to fatal systemic illness. Among mammals, clinical disease is primarily
exhibited in horses and humans.
Clinical signs of WNV infection in horses arise from viral-induced encephalitis or

encephalomyelitis. Infections are dependent on mosquito transmission and are seasonal in
temperate climates, peaking in the early autumn in the Northern Hemisphere. Affected horses
frequently demonstrate mild to severe ataxia. Signs can range from slight incoordination to
recumbency. Some horses exhibit weakness, muscle fasciculation, and cranial nerve deficits.
Fever is not a consistently recognised feature of the disease in horses.
Identification of the agent: Bird tissues generally contain higher concentrations of virus than
equine tissues. Brain and spinal cord are the preferred tissues for virus isolation from horses. In
birds, kidney, heart, brain, liver or intestine can yield virus isolates. Cell cultures (using, for
example, rabbit kidney or Vero cells) are used most commonly for virus isolation. WNV is
cytopathic in susceptible culture systems. Viral nucleic acid and viral antigens can be
demonstrated in tissues of infected animals by reverse-transcription polymerase chain reaction
(RT-PCR) and immuno-histochemistry, respectively. The most sensitive method for identifying
WNV in equine tissues is a nested format of the RT-PCR procedure.
Serological tests: Antibody can be identified in equine serum by IgM capture enzyme-linked
immunosorbent assay (IgM capture ELISA), haemagglutination inhibition (HI), IgG ELISA or plaque
reduction neutralisation (PRN). The ELISA and PRN methods are most commonly used for
identifying antibody against WNV in avian serum. In some serological assays, antibody cross-
reactions with related flaviviruses, such as St Louis encephalitis virus Japanese encephalitis virus,
or tick-borne encephalitis (TBE) virus may be encountered.
Requirements for vaccines and diagnostic biologicals: A formalin-inactivated WNV vaccine

derived from tissue culture, WNV live canarypoxvirus vectored vaccine, a WNV DNA vaccine and a
chimeric vaccine are licensed for use in horses.
A. INTRODUCTION
West Nile virus (WNV) is a zoonotic mosquito-transmitted arbovirus belonging to the genus Flavivirus in the
family Flaviviridae (26). The genus Flavivirus also includes Japanese encephalitis virus (see Chapter 2.1.7),
St Louis encephalitis virus, Murray Valley virus, Usutu virus, and Kunjin virus, among others (6). WNV has a wide
geographical range that includes portions of Europe, Asia, Africa, Australia (Kunjin virus) and in North, Central
and South America. Migratory birds are thought to be primarily responsible for virus dispersal, including
reintroduction of WNV from endemic areas into regions that experience sporadic outbreaks (6). WNV is
maintained in a mosquito–bird–mosquito transmission cycle, whereas humans and horses are considered dead
end hosts. Genetic analysis of WN isolates separates strains into two clades. Lineage 1 isolates are found in
northern and central Africa, Israel, Europe, India, Australia (Kunjin virus) and in North and Central America, and
Columbia and Argentina in South America (18). Lineage 2 strains are endemic in central and southern Africa and
Madagascar, with co-circulation of both virus lineages in central Africa (3, 7). There has been a recent report of
linage 2 from Hungary. While recent human and equine outbreaks have been due to lineage 1 viruses, strains
from each lineage have been implicated in human and animal disease.

Chapter 2.1.20. — West Nile fever
WNV was recognised as a human pathogen in Africa during the first half of the 20th century. Although several
WNV fever epidemics were described, encephalitis as a consequence of human WN infection was rarely
encountered prior to 1996, but since then, outbreaks of human West Nile encephalitis have been reported from
Romania, Russia, Israel, North America, France, and Tunisia (4, 11, 13, 15, 33). During the 1960s, West Nile
viral encephalitis of horses was reported from Egypt and France (23, 25). Since 1998, outbreaks of equine WNV
encephalitis have been reported from France, Italy, Canada, United States of America, Israel and Morocco (8,
14, 19, 21). In the Western Hemisphere, the virus range has dramatically expanded from a discrete region along
the East Coast of New York State to include the contiguous States of the United States of America (USA),
Canada, Mexico, the Caribbean islands, Central America, Argentina, Columbia and Venezuela (10, 18, 21, 30).
Other than in the United States and Canada, the introduction of West Nile virus in the Western Hemisphere has
not been characterised by large disease outbreaks or significant mortality in any species, possibly because of
exposure to indigenous flaviviruses circulating in these regions
The incubation period for equine WN encephalitis following mosquito transmission is estimated to be 3–15 days.
A fleeting viraemia of low virus titre precedes clinical onset (5, 25). WN viral encephalitis occurs in only a small
per cent of infected horses; the majority of infected horses do not display clinical signs (21). The disease in
horses is frequently characterised by mild to severe ataxia. Additionally, horses may exhibit weakness, muscle
fasciculation and cranial nerve deficits (8, 21, 22, 27). Fever is an inconsistently recognised feature. Treatment is
supportive and signs may resolve or progress to terminal recumbency. The mortality rate is approximately one in
three clinically affected horses. Differential diagnoses in horses include other arboviral encephalidites (e.g.
eastern, western or Venezuelan equine encephalomyelitis, Japanese encephalitis), equine protozoal myelitis
(Sarcocystis neurona), equine herpesvirus-1, Borna disease and rabies.
Most species of birds can become infected with WNV; the clinical outcome of infection is variable. Chickens and
turkeys, are resistant to disease, Outbreaks of fatal neurologic disease have been reported in zoo birds in the
USA and in domestic geese in Israel and Canada (1, 28, 33). WNV has been associated with sporadic disease in
small numbers of other species, including squirrels, chipmunks, bats, dogs, cats, white-tailed deer, reindeer,
sheep, alpacas, alligator and a harbour seal during intense periods of local viral activity. Most human infections
occur by natural transmission from mosquitoes, but laboratory acquired infections have been reported. In
clinically suspicious cases, diagnostic specimens from all animals, particularly birds, should be handled at
containment level 3 following appropriate laboratory procedures (see Chapter 1.1.2 Biosafety and biosecurity in
the veterinary microbiology laboratory and animal facilities) (24). There has been confirmed transmission of WNV
in humans by blood transfusion, organ transfer and breast milk.
Due to the occurrence of inapparent WNV infections, diagnostic criteria must include a combination of clinical
assessment and laboratory tests.
a) Culture
Specimens for virus isolation include brain and spinal cord from encephalitic horses (21, 22); a variety of
bird tissues including brain, heart or liver may be used with success (28); WHV has been isolated from
kidney but the tissue may be toxic to cell culture. In general, virus isolates are obtained more easily from
avian specimens. Virus may be propagated in susceptible cell cultures, such as rabbit kidney (RK-13) and
African green monkey kidney (Vero) cells, or embryonating chicken eggs. Intracerebral inoculations of
newborn mice are less likely to yield virus isolates from mammalian tissues than cell culture methods. More
than one cell culture passage may be required to observe cytopathic effect (CPE). Confirmation of WNV
isolates is achieved by indirect fluorescent antibody staining of infected cultures or nucleic acid detection
methods (see below).
Immunohistochemical (IHC) staining of formalin-fixed avian tissues is a reliable method for identification of
WNV infection in birds. Brain, heart, kidney, spleen, liver, intestine, and lung are often IHC-positive tissues
in infected birds. The success rate of IHC detection in positive birds is enhanced by the examination of
multiple tissues. The specificity of identification (e.g. flavivirus specific or WNV specific) depends on the
selection of detector antibody. The brain and spinal cord tissues of horses with WN viral encephalitis are
inconsistently positive in IHC tests; approximately 50% of equine encephalitis cases yield false-negative
results. Failure to identify WNV antigen in equine central nervous system does not rule out infection.
c) Nucleic acid recognition methods

Nucleic acid detection by reverse-transcription polymerase chain reaction (RT-PCR) significantly enhances
the identification of WNV-infected tissues, particularly when a nested PCR approach is applied to fresh,

unfixed, equine brain and spinal cord specimens (16). The RT-nested PCR method to detect WNV nucleic
acid encoding a portion of the E gene is described below. This method was developed using a 1999 North
American isolate and has been successful in detecting WNV RNA in animal tissues during recent North
American outbreaks. St Louis encephalitis virus is not detected by this method. Lineage 1 West Nile viruses
from China (People’s Rep. of), France, Egypt, Israel, Italy, Kenya, Mexico and Russia demonstrate a highly
conserved nucleotide sequence in the target region, regardless of species of origin (17). Analysis of
sequence information for the Uganda 1937 Lineage 2 strain (GenBank M12294) in the region targeted by
the PCR primers indicate that amplification of lineage 2 strains of WNV would not be expected. Other
viruses from, the Japanese encephalitis serogroup have not been examined. Non-nested methods,
including real-time PCR, pose less risk of laboratory cross-contamination and may be applied successfully
to avian tissue samples (17). A real-time RT-PCR has been described for the detection of WNV nucleic acid
(29). In order to standardise WNV molecular techniques a proficiency study based on formalin-fixed tissues
was developed, administered and reported (20). Tissues selected for PCR are the same as those selected
for virus isolation attempts.
• Reverse-transcription nested polymerase chain reaction (RT-nPCR) procedure

The RT-nPCR described here includes several procedures: extraction of RNA, reverse transcription to
generate DNA from RNA and first stage PCR, second stage PCR using ‘nested’ primers and, finally,
detection of the appropriately sized amplicon by gel electrophoresis. WNV E protein gene regions of 445 bp
(base pairs) and 248 bp are amplified in the first-stage and nested procedures, respectively. The kits and
reagents described below are provided as examples. Equivalent products may be available from other
sources. Extreme care in handling all materials and inclusion of proper controls are essential to ensure
accurate results. The precautions to be taken have been covered in Chapter 1.1.5 Validation and quality
control of polymerase chain reaction methods used for the diagnosis of infectious diseases. Duplicate
samples of each diagnostic specimen should be processed and tested to increase confidence in test
results. Use appropriate precautions when handling hazardous reagents such as ethidium bromide.
• Extraction of viral RNA

From 50 to 100 mg of tissue, extract total RNA using Trizol® reagent (Life Technologies, Grand Island, NY,
USA) according to the manufacturer’s instructions. Also extract total RNA from WNV control stock virus
containing 10–100 tissue culture infective dose (TCID50) per 100 µl volume.
• Reverse transcription and first stage PCR

First stage primers:
1401: 5’-ACC-AAC-TAC-TGT-GGA-GTC-3’
1845: 5’-TTC-CAT-CTT-CAC-TCT-ACA-CT-3’
i) Suspend extracted RNA samples in 12 µl RNase-free water.
ii) Incubate at 70°C for 10 minutes.
iii) Add 2 µl of each RNA sample to 48 µl of RT-PCR mixture containing a final composition of:
10 mM Tris/HCl, pH 8.3
50 mM KCl
2.0 mM MgCl2
0.8 mM deoxynucleoside triphosphate (dNTP) pool
25 units M-MLV (Moloney murine leukaemia virus) RT
1.25 units RNase inhibitor
1.25 units AmpliTaq GoldTM (Applied Biosystems, Foster City, CA, USA)
37.5 pmol of the first stage primers.
Include ‘no RNA’ controls using 2 µl RNase-free water in place of denatured RNA.
iv) Incubate reaction tubes at 45°C for 45 minutes.
v) Incubate reaction tubes at 95°C for 11 minutes.
vi) PCR amplification through 35 cycles:
Denaturation at 95°C for 30 seconds,
Primer annealing at 55°C for 45 seconds,
Primer extension at 72°C for 60 seconds (for the 35th cycle, primer extension at 72°C for 5 minutes).
vii) Hold samples at 4°C until second stage PCR.
• Second stage (nested) PCR

Second stage primers:
1485: 5’-GCC-TTC-ATA-CAC-ACT-AAA-G-3’
1732: 5’-CCA-ATG-CTA-TCA-CAG-ACT-3’

i) For each sample and control, add 1.5 µl of the first-stage amplification product to 48.5 µl of PCR
mixture with a final composition of:
10 mM Tris/HCl, pH 8.3
50 mM KCl
2.0 mM MgCl2
0.8 mM deoxynucleoside triphosphate (dNTP) pool
1.25 units AmpliTaq GoldTM (Applied Biosystems, Foster City, CA, USA)
37.5 pmol each of the nested primers.
ii) Incubate reactions tubes at 95°C for 11 minutes.
iii) PCR amplification through 35 cycles:
Denaturation at 95°C for 30 seconds,
Primer annealing at 55°C for 45 seconds,
Primer extension at 72°C for 60 seconds (for the 35th cycle, primer extension at 72°C for 5 minutes).
iv) Hold samples at 4°C or –20°C until electrophoresis.
• Analysis of PCR products by gel electrophoresis

i) Prepare a 2.5% NuSieve® 3/1 (FMC Bioproducts, Rockland, Maine, USA) agarose solution in
0.045 mM Tris/borate, pH 8.6, 1.5 mM EDTA (ethylene diamine tetra-acetic acid) (1 × TBE buffer). Boil
the agarose on a hotplate or in a microwave oven until completely dissolved. Cool the agarose to 45–
55°C. Add 5.0 µl ethidium bromide solution (10 mg/ml) per 100 ml warm agarose and pour agarose
gel with comb. Allow to solidify then remove comb.
ii) Add 30 µl ethidium bromide solution (10 mg/ml) per 600 ml 1 × TBE tank buffer. Position gel in
apparatus and fill buffer tanks.
iii) Mix 15 µl of each sample and control with 5 µl gel loading solution (e.g. Sigma product G-2526, St
Louis, MO, USA). Include 100 bp DNA ladder (e.g. Life Technologies, Grand Island, NY, USA product
15268-019, range 100–1500 bp) in at least one gel well. Load samples into agar wells and
electrophorese at 65–75 volts until the gel loading dye has travelled approximately two-thirds the
length of the gel.
iv) Visualise and photograph gel under ultraviolet illumination.
• Interpretation of the test

For the PCR test to be valid, the positive controls must show the appropriate size band (248 bp). The ‘no
RNA’ controls should have no bands. Samples are considered to be positive if there is a band of the same
size as the positive control. Duplicate samples should both show the same reaction. If there is a disparity,
the test should be repeated, starting with extraction from tissue. If further validation is required, the final
nested PCR product can be sequenced and compared with the published sequences of WNV from
GenBank.
Antibody can be identified in equine serum by IgM capture enzyme-linked immunosorbent assay (IgM capture
ELISA), hemagglutination inhibition (HI), IgG ELISA or plaque reduction neutralisation (PRN) (2, 12). The IgM
capture ELISA described below is particularly useful to detect antibodies resulting from recent natural exposure
to WNV. Equine WNV-specific IgM antibodies are usually detectable from 7–10 days post-infection to 1–
2 months post-infection. Most horses with WN encephalitis test positive in the IgM capture ELISA at the time that
clinical signs are first observed. WNV neutralising antibodies are detectable in equine serum by 2 weeks post-
infection and can persist for more than 1 year. The HI and PRN methods are most commonly used for identifying
WNV antibody in avian serum. In some serological assays, antibody cross-reactions with related flaviviruses,
such as St Louis encephalitis virus or Japanese encephalitis virus, will be encountered. The PRN test is the most
specific among WNV serological tests; when needed, serum antibody titres against related flaviviruses can be
tested in parallel. Finally, WN vaccination history must be considered in interpretation of serology results,
particularly in the PRN test and IgG ELISA. IgM capture ELISA may be used to test avian or other species
provided that species-specific capture antibody is available (e.g. anti-chicken IgM). The PRN test is applicable to
any species, including birds.
a) Equine IgM capture ELISA

WNV and negative control antigens for the IgM capture ELISA may be prepared from mouse brain (see
Chapter 2.5.5), tissue culture or recombinant cell lines (9). Commercial sources of WNV testing reagents
are available in North America. Characterised equine control serum, although not an international standard,
can be obtained from the National Veterinary Services Laboratories, Ames, Iowa, USA. Virus and negative
control antigens should be prepared in parallel for use in the ELISA. Antigen preparations must be titrated

with control sera to optimise sensitivity and specificity of the assay. Equine serum samples are tested at a
dilution of 1/400 and equine cerebrospinal fluid samples are tested at a dilution of 1/2 in the assay. To
ensure specificity, each serum sample is tested for reactivity with both virus antigen and control antigen.
• Test procedure
i) Coat flat-bottom 96-well ELISA plates (e.g. Immulon 2HB, Dynex Technologies, Chantilly, VA, USA)
with 100 µl/well anti-equine IgM diluted in 0.5 M carbonate buffer, pH 9.6, according to the
manufacturer’s suggested dilution for use as a capture antibody.
ii) Incubate plates overnight at 4°C in a humid chamber. Coated plates may be stored for several weeks.
iii) Prior to use, wash plates twice with 200–300 µl/well 0.01 M phosphate buffered saline, pH 7.2,
containing 0.05% Tween 20 (PBST).
iv) Block plates by adding 300 µl/well freshly prepared 5% nonfat dry milk in PBST and incubate
60 minutes at room temperature. After incubation, remove blocking solution and wash plates three
times with PBST.
v) Test and control sera are diluted 1/400 (cerebrospinal fluid is diluted 1/2) in PBST and 50 µl/well of
each sample is added to duplicate sets of wells (total of four wells per sample) on the plate. Include
control positive and negative sera prepared in the same manner as samples.
vi) Cover the plates and incubate 75 minutes at 37°C in a humid chamber.
vii) Remove serum and wash plates three times in PBST.
viii) Dilute virus and negative control antigens in PBST and add 50 µl of virus antigen to one set of wells
per test and control sera and add 50 µl normal antigen to the second set of wells per test and control
sera.
ix) Cover the plates and incubate overnight at 4°C in a humid chamber.
x) Remove antigens from the wells and wash the plates three times in PBST.
xi) Dilute horseradish peroxidase conjugated anti-Flavivirus monoclonal antibody 1 in PBST according to
manufacturer’s directions and add 50 µl per well.
xii) Cover the plates and incubate at 37°C for 60 minutes.
xiii) Remove conjugate and wash plates six times in PBST.
xiv) Add 50 µl/well freshly prepared ABTS (2,2’-azino-di-[3-ethyl-benzthiazoline]-6-sulphonic acid)
substrate with hydrogen peroxide (0.1%) and incubate at room temperature for 30 minutes.
xv) Measure absorbance at 405 nm. A test sample is considered to be positive if the absorbance of the
test sample in wells containing virus antigen is at least twice the absorbance of negative control serum
in wells containing virus antigen and at least twice the absorbance of the sample tested in parallel in
wells containing control antigen.
b) Plaque reduction neutralisation (applicable to serum from any species)

The PRN test is performed in Vero cell cultures in either 25 cm2 flasks or 6-well plates. The sera can be
screened at a 1/10 and 1/100 final dilution or may be titrated to establish an endpoint. A description of the
test as performed in 25 cm2 flasks using 100 plaque-forming units (PFU) of virus is as follows.
Prior to testing, serum is heat inactivated at 56°C for 30 minutes and diluted (e.g. 1/5 and 1/50) in media.
Virus (200 pfu per 0.1 ml) working dilution is prepared in media containing 10% guinea-pig complement.
Equal volumes of virus and serum are mixed and incubated at 37°C for 75 minutes before inoculation of
0.1 ml on to confluent cell culture monolayers. The inoculum is adsorbed for 1 hour at 37°C, followed by the
addition of 4.0 ml of primary overlay medium. The primary overlay medium consists of two solutions that are
prepared separately. Solution I contains 2 × Earle’s Basic Salts Solution without phenol red, 4% fetal bovine
serum, 100 µg/ml gentamicin and 0.45% sodium bicarbonate. Solution II consists of 2% Noble agar that is
sterilised and maintained at 47°C. Equal volumes of solutions I and II are adjusted to 47°C and mixed
together just before use. The test is incubated for 72 hours at 37°C. A second 4.0 ml overlay prepared as
above, but also containing 0.003% neutral red is applied to each flask. Following a further overnight
incubation at 37°C, the number of virus plaques per flask is assessed. Endpoint titres are based on 90%
reduction compared with the virus control flasks, which should have about 100 plaques.
Standard microneutralisation or microtitre plaque reduction neutralisation assays may be more suitable
when small volumes of samples are available (32).
1 Available from the Centers for Disease Control and Prevention, Biological Reference Reagents, 1600 Clifton Road NE,
Mail Stop C21, Atlanta, Georgia, 30333, USA.


In February 2003, the United States Department of Agriculture (USDA) issued a license for a formalin-inactivated
WNV vaccine derived from tissue culture for use in horses. This was followed in December 2003, by a USDA
licensed live canarypoxvirus vectored WNV vaccine for use in horses. In 2004, an inactivated human cell line-
derived WNV vaccine developed by Crucell NV (the Netherlands) and Kimron Veterinary Institute (Israel)
obtained a market authorisation in Israel as a veterinary vaccine for geese. In July 2005, the USDA issued the
first fully licensed WNV DNA vaccine for animals in the USA. The vaccine contains genes for two WNV proteins,
and therefore, does not contain any whole WNV, live or killed. In late 2006, a chimeric vaccine, based on a
yellow fever virus vector, was licensed by USDA for use in horses. These vaccines have demonstrated sufficient
efficacy and safety in adequately vaccinated horses. Vaccination may be helpful in preventing neurological signs
associated with WNV infection.
1. Seed management

The isolate of WNV used for vaccine production must be accompanied by documentation describing its
origin and passage history. The isolate must be safe in host animals at the intended age of vaccination and
provide protection after challenge.
The WNV should be propagated in cell lines known to support the growth of WNV. Cell lines should be free
from extraneous viruses, bacteria, fungi, and mycoplasma. Viral propagation should not exceed five
passages from the master seed virus (MSV), unless further passages prove to provide protection in the host
animal.
The MSV should be free from bacteria, fungi and mycoplasma. The MSV must be tested for and be free of
extraneous viruses, including equine herpesvirus, equine adenovirus, equine viral arteritis virus, bovine viral
diarrhoea virus, reovirus, and rabies virus by the fluorescent antibody technique. The MSV must be free
from extraneous virus by CPE and haemadsorption on the Vero cell line and an embryonic equine cell type.
In an immunogenicity trial, the MSV at the highest passage level intended for production must protect
susceptible horses against a virulent challenge strain. A statistically significant number of vaccinated horses
must be protected from viraemia when compared with the controls. Field trial studies should be conducted
to determine the safety of the vaccine.
The susceptible cell line is seeded into suitable vessels. Minimal essential medium, supplemented with fetal
bovine serum (FBS), is used as the medium for production. Incubation is at 37°C.
Cell cultures are inoculated directly with WN working virus stock, which is generally from 1 to 4 passages from
the MSV. Inoculated cultures are incubated for 1–8 days before harvesting the culture medium. During
incubation, the cultures are observed daily for CPE and bacterial contamination.
Killed virus vaccines are chemically inactivated with either formalin or binary ethylenimine and mixed with a
suitable adjuvant.
The DNA vaccine expression cassette is amplified in Escherichia coli using a plamid vector cutting out plasmid
backbone and purified for formulation into a vaccine.
Production lots of WNV must be titrated in tissue culture for standardisation of the product. Low-titred lots may be
concentrated or blended with higher-titred lots to achieve the correct titre.

Production lots of DNA are quantified by analytical methods and characterised before standardisation and
blending at the correct DNA content. The highest level of lipopolysaccharide (LPS) contamination of the DNA
vaccine is 100 EU/dose (EU = endotoxin units).
4. Batch control
Final container samples are tested for purity, safety and potency.
a) Purity
Samples are examined for bacterial and fungal contamination. To test for bacteria, ten vessels, each
containing 120 ml of soybean casein digest medium, are inoculated with 0.2 ml from ten final-container
samples. The ten vessels are incubated at 30–35°C for 14 days and observed for bacterial growth. To test
for fungi, ten vessels, each containing 40 ml of soybean casein digest medium, are inoculated with 0.2 ml
from ten final-container samples. The vessels are incubated at 20–25°C for 14 days and observed for
fungal growth.
b) Safety
Safety tests can be conducted in a combination of guinea-pigs, mice or horses. Field safety studies should
be conducted before the vaccine receives final approval. Generally, two serials should be used, in three
different geographical locations, and a minimum of 600 animals. About one-third of the animals should be at
the minimum age recommended for vaccination (correlated to efficacy). If the final product is a modified-live
vaccine, additional safety testing of the MSV is required to demonstrate a lack of virulence.
c) Potency
Killed virus vaccines may use host animal or laboratory animal vaccination/serology tests or
vaccination/challenge tests to determine potency of the final product. Parallel-line assays using ELISA
antigen-quantifying techniques to compare a standard with the final product are acceptable in determining
the relative potency of a product. The standard should be shown to be protective in the host animal (31).
Live viral products are titred in cell cultures to determine the potency of the final product. The final release
potency titre should include an additional 0.7 log10 for test variability and 0.5 log10 for end-of-dating stability
than the minimum protective dose established in the immunogenicity trial.
DNA vaccines are tested for bioactivity and DNA content using parallel-line direct quantification methods
that compare a standard preparation to the final product.
Duration of immunity studies are conducted before the vaccine receives final approval. The duration should
be for the length of the mosquito season in the infected areas. For animals at higher risk and in infected
areas with year-round mosquito activity, more frequent vaccine boosters may be advised.
e) Stability
All vaccines are initially given 24 months before expiry. Real-time stability studies are conducted to confirm
the appropriateness of all expiration dating.
f) Preservatives
Antibiotics are added during production, generally gentamicin sulphate or neomycin not to exceed 30 µg/ml.
Vaccination is only recommended for horses in WN-positive areas. Vaccinated horses may develop a
serological titre that may interfere with the ability to export the horse.
a) Safety
See Section C.4.b.
b) Potency
See Section C.4.c.

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http://www.aphis.usda.gov/vs/cvb/vsmemos.htm
32. W EINGARTL H.M., DREBOT M.A., HUBALEK Z., HALOUZKA J., ANDONOVA M., DIBERNARDO A., COTTAM-BIRT C.,
LARENCE J. & MARSZAL P. (2003) Comparison of assays for detection of West Nile virus antibodies in chicken
sera. Can. J. Vet. Res., 67, 128–132.
33 ZELLER H.G. & SCHUFFENECKER I. (2004). West Nile virus: An overview of its spread in Europe and the
Mediterranean Basin in contrast to its spread in the Americas. Eur. J. Clin. MIcrobiol. Infect. Dis., 23, 147–
156.
*
* *
NB: There is an OIE Reference Laboratory for West Nile fever (see Table in Part 3 of this Terrestrial Manual or

SECTION 2.2.
APIDAE
INTRODUCTORY NOTE ON BEE DISEASES
Bees are insects that are closely related to ants and wasps. There are many thousands of species
of bee, most of which are not social insects, living solitary lives. The honey bee, Apis species, lives
as a colony, which is a family of social insects. There are many species, subspecies, races and
subraces of honey bees that are adapted to their environment.
Two species are important for bee keeping – the western honey bee Apis mellifera, and the
eastern honey bee A. cerana. The Africanised bee, which is found in South and Central America
and some states of the United States of America, is a cross between two subspecies of the
western honey bee, the European bees and the South African bee. Apis cerana is important in
South and South-East Asia. The colonies are small and docile, but the honey yields are low. In a
suitable climate, the western honey bee, A. mellifera, is sometimes preferred for its greater honey
production.
It is thought that all bees are susceptible to the known diseases of bees, but different races may
have varying susceptibility. For example, A. cerana is less susceptible to varroosis. When
sampling a colony of bees for diagnosis of diseases, live bees must first be killed with diethyl ether
or in a deep freezer (–20°C) overnight. Bees may also be killed by submersion in 70% ethyl
alcohol, e.g., when collected for diagnosis of acariosis (Acarapis). Larval and pupal smears must
be made when testing for brood diseases or a piece of comb containing brood showing visible
signs of disease may be sent to the laboratory.
*
* *

CHAPTER 2.2.1.
ACARAPISOSIS OF HONEY BEES
SUMMARY
Acarapisosis or acariosis or acarine disease is a disease of the adult honey bee Apis mellifera L.
and other Apis species. It is caused by the Tarsonemid mite, known as the tracheal mite, Acarapis
woodi (Rennie). The mite is approximately 150 µm in size, and is an internal parasite of the
respiratory system, living and reproducing mainly in the large prothoracic trachea of the bee.
Sometimes they are also found in the head, thoracic and abdominal air sacs. Mites feed on the
haemolymph of their host.
The pathogenic effects found in infected bees depend on the number of parasites within the
trachea and are attributable both to mechanical injuries and to physiological disorders consequent
to the obstruction of air ducts, lesions in the tracheal walls, and the depletion of haemolymph. As
the parasite population increases, the tracheal walls, normally white and translucent, become
opaque and discoloured with blotchy black areas, probably due to melanin crusts.
The mortality rate may range from moderate to high. Early manifestations of infection normally go
unnoticed, and only when infection is heavy does it become apparent. This is usually in the early
spring. The infection spreads by direct contact. Generally, only newly hatched bees under 10 days
old are susceptible. Reproduction occurs within the tracheae of adult bees, where female mites
may lay 8–20 eggs. There are 2–4 times as many females as males. Development takes 11–
12 days for males and 14–15 days for females.
Identification of the agent: The parasites are demonstrated only by laboratory methods and
under the microscope. The mites need to be observed inside the tracheae or removed from them
to be observed microscopically. Several techniques are available for demonstrating the mites, such
as dissection, grinding and staining.
The thoraces of suspect bees are dissected to expose the trachea. Each trachea is examined
under a dissecting microscope (×18–20), where the mites will be seen through the transparent wall
as small oval bodies.
Alternatively, larger samples of suspect bees can be ground or homogenised in water, followed by
coarse filtration of the suspension, and centrifugation. The deposit is treated with undiluted lactic
acid for 10 minutes. This is then mounted for microscopic examination.
The parasites may be stained by histological techniques so that they can be observed within the
bee trachea. The tracheae are separated out, cleared with 8% potassium hydroxide, and stained
with 1% methylene blue. This is the best method for large numbers of samples.
Serological tests: Serological tests are not available.
Requirements for vaccines and diagnostic biologicals: There are no biological products
available. Menthol crystals or oil patties made with vegetable oil (not animal fat) and white
granulated sugar will keep mite levels under control.
A. INTRODUCTION
Acarapisosis is a disease of the adult honey bee Apis mellifera L. and other Apis species, caused by the
microscopic Tarsonemid mite Acarapis woodi (Rennie). The mite is approximately 150 µm in size and is an

Chapter 2.2.1. — Acarapisosis of honey bees
internal parasite of the respiratory system (Figure 1). These tracheal mites enter, live and reproduce mainly in the
large prothoracic tracheae of all bees, feeding on the haemolymph of their host (Figure 2). Sometimes they are
also found in the head, thoracic and abdominal air sacs (6, 18).
Fig. 1. Acarapis woodi (Rennie). Top: Adult male, Centre: Adult Female, Bottom: Egg.
Fig. 2. Main thoracic tracheae of a honey bee where Acarapis is commonly found;
light infestations are near the spiracle opening.

The pathogenic effects on individual bees depend on the numbers of parasites within the tracheae and are
attributable both to mechanical injuries and to physiological disorders consequent to the obstruction of the air
ducts, lesions in the tracheal walls, and to the depletion of haemolymph. As the parasite population increases,
the tracheal walls, which are normally whitish and translucent, become opaque and discoloured with blotchy
black areas, probably due to melanin crusts (5).
The mortality rate may range from moderate to high. Early signs of infection normally go unnoticed, except for a
slow dwindling in the colony size. Only when infection is heavy does it become apparent. This is generally in the
early spring after the winter clustering period when the mites have bred and multiplied undisturbed into the
longer-living winter bees. This applies mainly to the Northern Hemisphere where there are seasonal variations in
the reproduction of bees.
Infection spreads from one bee to another by direct contact. Generally, only newly hatched bees under 10 days
old, are susceptible. Attempts to rear A. woodi on artificial and synthetic diets have been unsuccessful, while
culturing them on immature stages of the honey bee itself has been only partially successful (7). The life span of
the mites in dead bees is approximately 1 week. Reproduction occurs within the tracheae of adult bees, where
female mites may lay 8–20 eggs. There are 2–4 times as many females as males; development takes 11–
12 days for males and 14–15 days for females.
There are no reliable clinical signs for the diagnosis of acarapisosis as the signs of infection are not specific and
the bees behave in much the same way as do bees affected by other diseases or disorders. They crawl around
in the front of the hive and climb blades of grass, unable to fly. Dysentery may be present.
Acarapisosis can be detected only in the laboratory using microscopic examination or an enzyme-linked
immunosorbent assay (ELISA). There is no reliable method for detection of very low levels of infection. The
number of bees sampled determines the detection threshold of the method. It has been shown that a 1 to 2%
rate of infection can be detected by sampling 50 bees Sequential sampling data are available (4, 17). The best
time to take bee samples is in the early spring or late autumn (Northern hemisphere), when Acarapis populations
are high. Visualisation of mites is easier in older bees, which have more mites. Samples of queens, drones or
workers can be used, but Acarapis prefer drones.
a) Dissection (8)
A sample of 50 bees (see above) is collected at random from the suspected colony. These are mainly bees
crawling and unable to fly, found within about 3 metres of the front of the hive. This is preferable to random
collection from within the colony. The bees may be living, dying, or dead. Live bees must first be killed with
ethyl alcohol or in a deep freezer (–20°C); bees must not have been dead for over 2–3 days unless kept at
4°C for up to 4 weeks or –20°C for several months. They may be preserved indefinitely in a preservative
such as Oudemann solution: glacial acetic acid (80 ml); glycerol (50 ml); 70% ethanol (870 ml).
• Test procedure: direct preparation (15, 18)

i) Remove the abdomen at the thorax of the bees (see Figure 3).
ii) Pick up the thorax with the beginning of the head and examine it under the binocular magnifying glass
at 20–30-fold magnification.
Iii) Remove the pleural sclerite of the first thoracic segment with the first pair of legs, by means of a pair of
tweezers. In the circular opening the main strains of the thoracic tracheae and the branches of the
head tracheae can be seen.
iv) By means of a fine pair of tweezers, remove the thoracic tergite of the first thoracic segment and part
of the second thoracic tergite. After removing the overlying musculature, the two thoracic tracheae are
exposed. Positive diagnosis consists of either the presence of melanisation of one or both tracheae or,
in light infestation, of the presence of oval translucent bodies (eggs etc.) easily seen within the
tracheae.
v) For further microscopis examination (e.g. confirmation of light infestation), remove the tracheae and
put them onto a slide, with a drop of water. Under the microscope at 100-fold magnification the adult
mites as well as their individual stages of development can be recognised.

Fig. 3. Preparation of bees to reveal Acarapis woodi in the first thoracic pair of tracheae.
• Test procedure: maceration (15)

i) Lay and secure bees on their backs or hold with thumb and first finger.
ii) Remove the heads and forelegs using a small forceps and remove the collar surrounding the neck
opening to expose the tracheae (Figure 4). Check the tracheae nearest to the spiracle (as mites enter
through the spiracle) to see light infestations. Heavy infestations are easily visible as shadows or dark
objects in clear to dark brown tracheae. Old and heavy infestations will make the tracheae brown to
black.
iii) Cut through the thorax in front of the middle pair of legs and the base of the forewings with a sharp
razorblade. These thin disks can be further treated to clear muscle tissue.
iv) Macerate either by gentle heating in an 8% solution of potassium hydroxide for approximately
20 minutes or by leaving them to stand overnight without heating.
v) Examine the first pair of tracheae, which are covered by muscle tissue, under a dissecting microscope
at a magnification of ×18–20, or transfer the tracheae to another slide, add glycerin or water and
observe at higher magnification.
vi) Mites are easily seen through the transparent wall as small, oval bodies.
Fig. 3. Left: front view of bee thorax with head removed and collar intact.
Right: Collar removed and tracheae exposed to spiracle openings.
This is the simplest and most reliable technique for the laboratory diagnosis of acarapisosis, allowing the
detection of early infections and enabling the infection rate to be established. Even light infections can be
detected by using a dissecting microscope with this technique. Only in very exceptional instances will it be
necessary to employ higher magnifications in order to make a diagnosis. However, this is a demanding
technique, especially when a large number of acarapisosis diagnoses have to be made. If it is necessary
only to distinguish between heavily infected and lightly or non-infected colonies, dissection can be stopped
at step ii and the colour of the tracheae observed.

b) Grinding (3)
A sample of about 200 bees is collected at random from the suspect colony. The wings and legs of each
bee are removed from the thorax, and the bodies are pooled in a 100 ml container that has been one-
quarter filled with water. This suspension is homogenised three times, each time for several seconds, in a
homogeniser at 10,000 rpm with the addition of more water. The resulting suspension is strained through a
sieve (mesh 0.8 mm) and the sieve is rinsed with water to a final volume of approximately 50 ml. The filtrate
is centrifuged at 1500 g for 5 minutes and the supernatant fluid is discarded. A few drops of undiluted lactic
acid solution are added to the debris of the deposit, which will contain the mites. This is left for 10 minutes
to allow the muscle fibres to dissolve, and is then mounted under a cover-slip for microscopic examination.
This technique is quicker than dissection, but may be less accurate. External mites A. externus, A. vagans
and A. dorsalis, all of which are morphologically similar to A. woodi, are often found on the thorax of healthy
bees and can very easily be mistaken for A. woodi (Table 1). It seems, however, that they do not cause any
serious threat to bees or beekeeping. This method should therefore only be chosen if all that is required is a
rough estimation of the degree of infection in a region. It is not suitable for determining a first outbreak.
Table 1. Differential diagnosis of Acarapis species (15)
Character A. dorsalis A. externus A. woodi
Notch of the coxal plate Deep Short Flat

Space between stigmata 16.7 µm 16.8 µm 13.9 µm
Length of tarsal limb (IV leg pair) 7.6 µm 11.4 µm 7.5 µm
c) Staining (11)
The mites and trachea can be stained specifically, rendering them easily visible by microscopy.
• Test procedure
i) Remove the head and forelegs.
ii) Make a transverse cut through the membranous areas behind the forelegs.
iii) Make a second transverse cut in front of the middle pair of legs at the base of the forewings.
iv) To clear the sections (1–1.5 mm thick), place them in an 8% solution of potassium hydroxide.
v) Stir gently and heat near to boiling point for approximately 10 minutes until the soft internal tissues are
dissolved and cleared, leaving the chitinous tissues intact.
vi) Retrieve sections by filtration and wash with tap water.
vii) Stain and mount the sections.
viii) Examine for mites by low-power microscopy.
Permanent mounts are prepared by the usual histological techniques.
Cationic stains are the most suitable and specific as they stain the mites intensely but the tracheae only
weakly. A solution of 1% aqueous methylene blue is the most suitable, prepared by dissolving the
methylene blue first and then adding sodium chloride to make a 0.85% NaCl solution.
• Test procedure
i) Stain in 1% aqueous methylene blue.
ii) Differentiate sections in distilled water for 2–5 minutes.
iii) Rinse the sections in 70% alcohol.
When kept in 95% ethanol, the mites will retain the stain for 6 hours (1). It is essential with this technique to
macerate the tissues effectively in the potassium hydroxide solution. Using this method, it is possible to
process a large number of samples rapidly and conveniently.

d) Enzyme-linked immunosorbent assay

An ELISA for trachea mites has been developed (9, 13, 14). This test may produce false-positive results,
and is therefore only recommended for survey examinations. Another method is the visualisation of
guanine, a nitrogenous waste product of mites (12).

There are no biological products available. Menthol crystals (50 g for a two story colony) control mites if left in the
colony for 28 days, providing the ambient temperature is at least 18°C. The optimum temperature range for the
vapours to work is 27–29°C. Small cakes made with vegetable shortening (e.g. margarine, not animal fat) and
white granulated sugar will keep mite levels to 10%. The cake (about 100 g in weight) should be placed on the
top bars of the frames in the brood nest in the autumn and early spring (16). Formic acid may be used to treat
infected colonies (10).
Some races of bees, such as Buckfast bees (2) and some hygienic strains, are less susceptible to attack by
Acarapis.
ACKNOWLEDGEMENTS
Illustrations by Diana Sammataro and Wolfgang Ritter are reproduced with their permission.
An FAO publication, Honey bee diseases and pests: a practical guide, W. Ritter & P. Akratanakul (eds).
Agricultural and Food Engineering Technical Report No. 4. FAO, Rome, Italy, 42 pp. ISSN 1814-1137
TC/D/A0849/E, is available free of charge at:
http://www.fao.org/WAICENT/faoINFO/AGRICULT/ags/subjects/en/industFoodAg/pdf/AGST_techrep_4.pdf
REFERENCES
1. BANCROFT J.D. & STEVENS A. (1982). Theory and Practice of Histological Techniques. Churchill Livingstone,
Edinburgh, UK.
2. BROTHER A. (1968). ‘Isle of Wight’ or acarine disease: its historical and practical aspects. Bee World, 49, 6–
18.
3. COLIN M.A., FAUCON J.P., GIANFERT A. & SARRAZIN C. (1979). A new technique for the diagnosis of Acarine
infestation in honey bees. J. Apic. Res., 18, 222–224.
4. FRAZIER M.T., FINLEY J., HARKNESS W. & RAJOTTE E.G. (2000). A sequential sampling scheme for detecting
infestation levels of tracheal mites (Heterostigmata:Tarsonemidae) in honey bee (Hymenoptera: Apidae)
colonies. J. Economic Entomol., 93 (3), 551.
5. GIORDANI G. (1964). Recherches au laboratoire sur Acarapis woodi (Rennie), agent de l’acariose des
abeilles (Apis mellifera L.). Note 3. Bull. Apic., 7, 43–60.
6. GIORDANI G. (1965). Recherches au laboratoire sur Acarapis woodi (Rennie), agent de l’acariose des
abeilles (Apis mellifera L.). Note 4. Bull. Apic., 8, 159–176.
7. GIORDANI G. (1970). Ricerche di laboratorio su Acarapis woodi (Rennie), agente dell’acarosi delle api
mellifiche (Apis mellifera L.) Nota 6. Ann. Acc. Naz. Agric., 90, 69–76.
8. GIORDANI G. (1974). Méthodes de diagnostic des maladies des abeilles adultes. Diagnostic de l’acariose.
Bull. Apic., 17.
9. GRANT G., NELSON D., OLSEN P. & RICE W.A. (1993). The ELISA detection of tracheal mites in whole honey
bee samples. Am. Bee J., 133, 652–655.
10. HOOD W.M. & MCCREADIE J.W. (2001). Field tests of the Varroa Treatment Device using formic acid to
control Varroa destructor and Acarapis woodi. J. Agric. Urban Entomol., 18 (2), 87.

11. PENG Y. & NASR M.E. (1985). Detection of honey bee tracheal mites (Acarapis woodi) by simple staining
techniques. J. Invertebr. Pathol., 46, 325–331.
12. MOZES-KOCH R. & GERSON U. (1997). Guanine visualization, a new method for diagnosing tracheal mite
infestation of honey bees. Apidologie, 28, 3–9.
13. RAGSDALE D. & FURGALA B. (1987). A serological approach to the detection of Acarapis woodi parasitism in
honey bees using an enzyme-linked immunosorbent assay. Apidologie, 18, 1–10.
14. RAGSDALE D. & KJER K.M. (1989). Diagnosis of tracheal mite (Acarapis woodi Rennie) parasitism of honey
bees using a monoclonal based enzyme-linked immunosorbent assay. Am. Bee J., 129, 550–553.
15. RITTER W. (1996). Diagnostik und Bekämpfung der Bienenkrankheiten (Diagnosis and control of bee
diseases). Gustav Fischer Verlag, Jena, Stuttgart, Germany.
16. SAMMATARO D. & NEEDHAM G.R. (1996). Host-seeking behaviour of tracheal mites (Acari: Tarsonemidae) on
honey bees (Hymenoptera: Apidae). Exp. Appl. Acarol., 20, 121–136.
17. TOMASKO M., FINLEY J., HARKNESS W. & RAJOTTE E. (1993). A sequential sampling scheme for detecting the
presence of tracheal mite (Acarapis woodi) infestations in honey bee (Apis mellifera L.) colonies. Penn.
State Agric. Exp. Stn Bull., 871.
18. W ILSON W.T., PETTIS J.S, HENDERSON C.E. & MORSE R.A. (1997). Tracheal mites. In: Honey Bee Pests,
Predators and Diseases, Third Edition. AI Root publishing, Medina, Ohio, USA, pp 255–277.
*
* *
NB: There are OIE Reference Laboratories for Bee diseases (see Table in Part 3 of this Terrestrial Manual or

CHAPTER 2.2.2.
AMERICAN FOULBROOD OF HONEY BEES
SUMMARY
American foulbrood (AFB) affects the larval stage of the honey bee Apis mellifera and other Apis
spp., and occurs throughout the world. Paenibacillus larvae, the causative organism, is a
bacterium that can produce over one billion spores in each infected larva. The spores are
extremely resistant to heat and chemical agents, and can survive for many years in scales (from
diseased dead brood), hive products and equipment. Only the spores are capable of inducing the
disease.
Identification of the agent: Combs of infected colonies have a mottled appearance due to a
mixture of healthy capped brood, uncapped cells containing the remains of diseased larvae, and
empty cells. This is not a characteristic of AFB only. Cell cappings of a diseased larva appear
moist and darkened, becoming concave and possibly punctured as infection progresses. The larval
or pupal colour changes to creamy brown and then to a dark brown with a ropy appearance when
drawn out. In some cases the larval remains are rather watery. The diseased brood eventually
dries out to form characteristic brittle scales that adhere tightly to the lower sides of the cell. The
formation of a pupal tongue is one of the most characteristic but rarely seen signs of the disease
and precedes the formation of the scales.
Diagnosis of AFB is based on identification of the pathogenic agent and the presence of clinical
signs. The analyst can rely on a broad range of samples. However, in practice, the samples of
choice will depend on whether it concerns a suspicious or diseased honey bee colony/apiary, or
analysis in the context of an AFB monitoring/prevention programme. Some of the identification
methods require a previous culturing step, while others can be performed directly on collected
samples. Four solid culture media are recommended: PLA (Paenibacillus larvae agar), MYPGP
agar, BHIT agar and Columbia sheep blood agar. Two polymerase chain reaction (PCR) protocols
are described in this chapter. The first protocol can be used for rapid confirmation of clinical AFB
and for identification of bacterial colonies after a cultivation step. The second protocol is a so-
called nested PCR that also permits direct analysis of spore solutions. The biochemical profiling of
P. larvae is based on the catalase test, the production of acid from carbohydrates and the
hydrolysis of casein. Further, antibody-based techniques and the microscopic identification of the
pathogenic agent are described.
Serological tests: There are no serological tests available.
Requirements for vaccines and diagnostic biologicals: Monoclonal and polyclonal antibodies
produced for the development of diagnostic tests should be sufficiently specific.
A. INTRODUCTION
American foulbrood (AFB) is an infectious disease of the larval stage of the honey bee Apis mellifera and other
Apis spp., and occurs throughout the world where such bees are kept. Paenibacillus larvae, the causative
organism, is a Gram positive bacterium that can produce over one billion spores in each infected larva. The
bacterium is a round-ended, straight and sometimes curved rod, which varies greatly in size (0.5 µm wide by
1.5 to 6 µm long), occurring singly and in chains and filaments; some strains are motile. The sporangia are often
sparse in vitro, and the ellipsoidal, central to subterminal spores, which may swell the sporangia, are often found
free (16). The spores are extremely heat stable and resistant to chemical agents. Only spores are capable of
inducing the disease.

Chapter 2.2.2. — American foulbrood of honey bees
The infection can be transmitted to larvae by nurse bees or by spores remaining at the base of a brood cell.
Although the larval stages of worker bees, drones and queens are susceptible to infection, infected queens and
drone larvae are rarely seen under natural conditions. The susceptibility of larvae to AFB disease decreases with
increasing age (35); larvae cannot be infected later than 53 hours after the egg has hatched. The mean infective
dose (LD50= spore dose at which 50% of the larvae are killed) needed to initiate infection, though very variable,
is 8.49 spores in 24–48 hour-old bee larvae (14). Exchanging combs containing the remains of diseased brood is
the most common way of spreading the disease from colony to colony. In addition, feeding or robbing of spore-
laden honey or bee bread, package bees and the introduction of queens from infected colonies can also spread
the disease. Wax contaminated with the spores of P. larvae, which are used in the production of combs
foundation, can also spread the disease. The early detection of AFB helps to prevent further spread.
Diagnosis of AFB is based on identification of the pathogenic agent only. The analyst can rely on a broad range
of samples. However, in practice, the samples of choice will depend on whether it concerns a suspicious or
diseased honey bee colony/apiary, or analysis in the context of an AFB monitoring/prevention programme. An
initial overview of clinical signs of the disease will be provided in this chapter, followed by identification methods
that require a previous culturing step, or that can be performed directly on collected samples. The techniques
involved are microbiological characterisation, the polymerase chain reaction (PCR), biochemical profiling,
antibody-based techniques and microscopy. The analyst should be aware of differences in sensitivity between
the presented approaches and should select the most appropriate for a given situation.
Fig. 1. Progression of the disease: (a) Point of infection. (b) Larval development to the prepupal stage. (c) Cell
contents reduced and capping is drawn inwards or is punctured. (d) Cell contents become glutinous.
(e) Residual scale tightly adherent to bottom of cell.
a) Epizootology and clinical signs

Spores of P. larvae can survive in bee products (honey, wax, dry larval scales) and in the environment for
3 to 10 years and purified spores can survive even more than 70 years (29).
The clinical signs of AFB are very diverse and depend on the genotype involved, the stage of the disease
and the strength of the bee colony (and possibly its resistance to AFB). Larvae can be killed rapidly at an
early age when they are curled at the base of uncapped brood cells. Adult worker bees will remove these
dead larvae leaving only an empty cell (4). Other larvae will die later on in their development, when they are
in an upright position, filling most of the brood cell. Often the larvae or pupae will die after brood cell
capping.
In severely infected colonies, the combs have a mottled appearance caused by a pattern of healthy capped
brood, uncapped cells containing the remains of diseased larvae, and empty cells. The capping of a cell
that contains a diseased larva appears moist and darkened and becomes concave and punctured as the
infection progresses. Also, the larva or pupa changes colour, first to a creamy and eventually to a dark
brown. The larvae can become glutinous in consistency and can be drawn out as threads when a probe is

inserted into the larval remains and removed from the cell (match-stick test). This is probably the best-
known technique for field diagnosis of the disease, but in some cases the larval remains are rather watery,
resulting in a negative match-stick test. Finally, 1 month or more after the larva becomes ropy, the remains
of the diseased brood dry out to form typical hard, dark scales that are brittle and adhere strongly to the
lower sides of the cell (Figure 1). If death occurs in the pupal stage, the pupal tongue protrudes from the
pupal head, extending to the top of the brood cell or may angle back towards the bottom of the cell. The
protruding tongue is one of the most characteristic signs of the disease, although it is rarely seen (Figure 2).
The tongue may persist also on the dried scale. European foulbrood needs to be taken into consideration
as a differential diagnosis.
Fig. 2. Clinical American foulbrood (a-c) and Gram staining (d): (a) Combs have mottled appearance.
(b) A matchstick draws out the brown, semi-fluid larval remains in a ropy thread. (c) The formation of a pupal
tongue is a very characteristic sign, but rarely seen. (d) Microscopic examination reveals Gram-positive rods,
occurring singly and in chains.
b) Selection of samples
i) Collection of samples from a suspicious or diseased colony/apiary
While maintaining their colonies, beekeepers often find brood combs with signs of disease. In this
case a brood sample can be collected for diagnosis. The brood is sampled by cutting out a piece
comb of about 20 cm2 in size, containing as much of the dead or discoloured brood as possible. An
experienced person can collect infected larval/pupal remains directly from the cells with a sterile swab,
significantly reducing the sample size and facilitating packaging and sample transportation to the
laboratory (see below). When microscopic examination is the method of choice, smears of the
remains of diseased larvae can also be made at the apiary (17). After air-drying they can be forwarded
to the laboratory.
Every bee colony in the vicinity of such a clinical case of AFB should be considered as suspicious and
a broad range of samples should be taken for confirmation. Apart from brood samples, food stores
(honey [27, 34], pollen [12] and royal jelly), adult workers (21) and wax debris (32) can be used to
detect the presence of P. larvae spores. Honey samples can be collected from cells close to the brood
with separate disposable spoons to prevent cross-contamination between samples; however, honey
may have been sitting in the comb for months at the time of sampling. Adult bees can be shaken or
brushed from the combs of the brood chamber or the honey supers into a plastic bag or container. For
the most reliable picture of the actual situation, bees from the brood nest (and not the honey supers)
should be analysed. Wax debris can be collected at the hive bottom all year round.
ii) Samples for AFB monitoring/prevention programmes

To prevent the propagation of diseased brood, honey, adult bee and debris samples can be used to
detect AFB in colonies where no clinical signs are observed. Routine collection of samples from

colonies or from harvested honey can be used as part of an operational or regional AFB detection
programme.
Microscopic examination of smears from larvae with no clinical signs is far less sensitive at detecting
spores in colonies compared with bacteriological or PCR-based methods. In fact, bacteriological and
PCR-based methods will often detect spores in colonies that never develop clinical signs of AFB. High
numbers of spores cultured from honey and bee samples using bacteriological methods, however, can
often predict the presence of clinical AFB signs at colony, apiary and operational levels.
b) Packaging and transportation of samples to the laboratory

Brood comb should be wrapped in a paper bag, paper towel or newspaper and placed in a wooden or
heavy cardboard box for transport. Swabs with larval remains can be put into appropriate test tubes with a
cap. Holders for microscope slides are commercially available. Adult bees can be kept frozen or submerged
in 70% ethanol during transportation, although dried bees are adequate. Food supplies can be put into a
test tube or a suitable pot, or wrapped in a plastic bag together with the spoon. Leaking and cross-
contamination of the samples must be prevented. If possible, fresh material for laboratory tests should be
sent refrigerated.
c) Sample preparation
i) Samples for cultivation
In general, an aqueous solution containing P. larvae spores should be prepared for further analysis.
This spore suspension is heat-shocked at 80°C for 10 minutes or 95–96°C for 3–5 minutes in order to
kill other spore-formingmicroorganisms.
Larval/pupal remains from brood comb are collected with a sterile swab and suspended in 5–10 ml of
sterile water or physiological solution (phosphate buffered saline or 0.9% NaCl) in a test tube.
Honey samples to be examined for spores are heated to 45–50°C and shaken to distribute any spores
that may be present. Dilution with an equal volume (25 ml) of water permits easier handling. The
diluted honey is transferred into 44 mm width dialysis tubing that has been tied at one end. The open
end is tied after filling. The tubes are submerged in running water for 18 hours or in a water bath with
3–4 water changes over the same time period. After dialysis, the contents are centrifuged at 2000 g
for 20 minutes. The supernatant liquid is discarded leaving approximately 1 ml (or less) of residue in
each sample. The residue is then resuspended in 9 ml of water (31).
Honey can also be prepared for cultivation without the dialysis step, however this requires longer
(30 minutes) and faster (3000 g) centrifugation. Likewise, the volume in which the deposit is finally
resuspended can be much smaller (200 µl) in order to improve the sensitivity of the test (6).
Direct plating of diluted honey (27) is widely used, but its sensitivity is inferior to that of the
centrifugation method as only a fraction of the total volume will be plated out. Whatever the method of
choice is, when honey is analysed quantitatively and threshold values are set, the methodology that
was used to establish these values should always be strictly followed.
An aqueous filtrate of pollen can be made by thoroughly dispersing 1 g of pollen in 10 ml final volume
sterile distilled water and filtering it through Whatman No. 1 paper (12).
When adult bees are dispatched in ethanol, the latter should be decanted and replaced by sterile
water or physiological solution before crushing.
Debris and bee wax (1.5 g) should be dissolved in an organic solvent (10 ml): toluene (32), chloroform
(19) or diethyl ether (28). The liquid part (2 ml) is then diluted in physiological solution (6 ml). After
shaking roughly, this suspension can immediately be plated out (no heat-shock) (32). In another
protocol, bee wax is first diluted in water (wax/water 1/10) and heated up to 90°C for 6 minutes. After
cooling down, the organic solvent is added (organic solvent/water 1/9) and the mixture is shaken
carefully. After 2minutes standing time, a deposit of a watery solution containing P. larvae spores
forms (28).
ii) Samples for PCR

Cell/spore suspensions and suspensions containing only spores have to be differentiated, the latter
requiring a more complex DNA extraction step (except for the nested PCR).
If the PCR is aimed at identifying bacterial colonies (= cell/spore suspension) after a cultivation step,
the pre-treatment is as follows: one colony is suspended in 50 µl of distilled water and heated to 95°C
for 15 minutes. Following centrifugation at 5000 g for 5 minutes, 1–5 µl of the supernatant is used as
template DNA in a PCR 50 µl mixture (9).
For rapid confirmation of clinical AFB, the samples should be prepared as follows: the remains of two
diseased honey bee larvae (= cell/spore suspension) are suspended in 1 ml of sterile distilled water

and mixed thoroughly. 100 µl of this suspension is diluted with 900 µl distilled water. This dilution is
vortexed and 100 µl of it is used to extract DNA by heating and centrifugation (see above) (9).
All aqueous solutions resulting from the sampling of honey, adult bees, debris, bee wax, pollen and
royal jelly should be considered as a spore suspension. Here, the extraction of DNA demands another
approach. Indeed, spore suspensions are centrifuged at 6000 g and 4°C for 30 minutes. Next, the
pellet is subjected to microwave treatment for 5 minutes at maximum power to break the spores, and
the released DNA is suspended in 30 µl of 10 mM Tris/HCl, pH 8.0, containing 1 mM EDTA (26).
When spores are to be detected from honey, DNA is serially diluted with sterile distilled water to
eliminate PCR inhibition caused by honey (26). Another DNA extraction method, based on lysozyme
and proteinase K treatment, has been described (3).
Good results can also be obtained by incubating a pelleted spore suspension in MYPGP broth at 37°C
for 2–24 hours. Thereafter, the suspension is centrifuged at 14,500 g for 5 minutes, washed with
sterile distilled water and resuspended in 200 µl of sterile distilled water. This short incubation step
causes spores to germinate, making them sensitive for DNA preparation by heat treatment again (see
above) (20).
When the nested PCR is chosen, the spore solution should only be boiled at 100°C for 10 minutes
and thereafter centrifuged at 14,500 g for 2 minutes. The supernatant can immediately serve as
template DNA sample in the nested PCR reaction (20).
d) Culture
Several media for cultivating P. larvae have been described but best results were obtained with PLA
(Paenibacillus larvae agar) (30), MYPGP agar (the abbreviation refers to its constituents: Mueller-Hinton
broth, yeast extract, potassium phosphate, glucose and pyruvate) (7), BHIT agar (Brain–Heart Infusion
medium supplemented with thiamine) (11) and CSA (Columbia sheep blood agar). The formulations of the
first two media are as follows:
PLA
This selective medium combines three different media to comprise the base, to which is added antibiotics
and egg yolk supplements (30). Equal quantities (100 ml) of sterile, molten Bacillus cereus selective agar
base (Oxoid CM617), trypticase soy agar (Merck 5458) and supplemented nutrient agar (SNA) are
combined and mixed. SNA is composed of (per litre): nutrient agar 23 g, yeast extract 6 g, meat extract 3 g,
NaCl 10 g, Na2HPO4 2 g: final pH is 7.4 ± 0.2. All solid media are sterilised at 121°C/15 minutes. Nalidixic
acid stock solution (18) is prepared by dissolving 0.1 g in 2 ml of 0.1 N NaOH and diluting to 100 ml with
0.01 M phosphate buffer (pH 7.2). Pipemidic acid stock (2) is prepared by dissolving 0.2 g in 2 ml of 0.1 N
NaOH and then diluting to 100 ml with the same phosphate buffer. Both antibiotic solutions are filter
sterilised.
After the three molten media are combined, 3 ml of stock nalidixic acid, 3 ml of stock pipemidic acid, and
30 ml of 50% egg-yolk suspension (13) is added to form the PLA medium. The PLA medium is poured
(20 ml) into sterile Petri dishes and plates are dried before use (45–50°C for 15 minutes).
MYPGP agar
MYPGP agar is composed of (per litre): Mueller-Hinton broth (Oxoid CM0405) 10 g, yeast extract 15 g,
K2PO4 3 g, glucose 2 g, Na-pyruvate 1 g and agar 20 g (7). Addition of nalidixic acid and pipemidic acid is
as above.
If cultivation of P. larvae is hampered by the occurrence of fungi, the addition 16.8 µg/ml medium of
amphotericin B (Sigma) works very well.
A sterile cotton swab is used to transfer a portion of the sample on to the surface of the solid medium. For a
quantitative evaluation, it is recommended to spread a fixed volume of the suspension on the solid agar with
a sterile scraper or pipette rather than using cotton swabs.
Inoculated plates are best incubated at 34–37°C for 2–4 days in an atmosphere of 5–10% CO2 in air,
although aerobic incubation will do as well.
e) Identification
i) Colony morphology
Samples from clinically diseased larvae will result in confluently grown plates after 2–4 days, leading
to a subculturing step in order to isolate colonies.

On PLA, colonies of P. larvae are small, pale green to yellow (= the same colour as the medium), with
a slightly opaque and rough surface; sometimes the centre is raised.
On MYPGP agar, colonies are small, regular, mostly rough, flat or raised and whitish to beige
coloured.
On Columbia sheep blood agar, colonies are small, regular, glossy, butyrous and greyish.
Paenibacillus larvae colonies with orange to red pigmentation have been described (10, 22).
It is advised to run P. larvae reference strains in parallel, for instance LMG 9820 (other designation:
ATCC 9545, DSM 7030) for the non-pigmented variant and DSM 16115 or DSM 16116 for the
pigmented genotype.
A proven positive brood or honey sample can serve as a positive control for the entire examination.
Colony morphology is not conclusive but might serve to select the bacterial colonies for further
identification.
ii) Polymerase chain reaction

PCR reactions are set up as 50 µl mixtures containing 5 µl template DNA (see sample preparation),
50 pmol forward (AFB-F) and reverse primer (AFB-R; primer sequences are given below), 10 nmol of
each deoxynucleoside triphosphate and 1–2.5 U of Taq polymerase, in the appropriate PCR buffer
(provided together with Taq polymerase) containing 2 mM MgCl2 (ref. 9 with modifications). Reducing
the volume of the PCR mixtures to 25 µl is possible. Amplification of a specific DNA fragment occurs in
a thermocycler under to the following PCR conditions: a 95°C (1–15 minutes) step; 30 cycles of 93°C
(1 minute), 55°C (30 seconds), and 72°C (1 minute); and a final cycle of 72°C (5 minutes).
Nested PCR comprises an external and an internal amplification step (20). The external amplification
is performed using primers PleF and PleR (see below). Each 50 µl PCR reaction contains: 10 µl
template DNA (see sample preparation), 1 × PCR buffer (with 1.5 mM MgCl2), 0.5 µM PleF primer,
0.5 µM PleR primer, 0.2 mM of each dNTP, additional 0.75 mM MgCl2, 1.25 U Taq polymerase. A
‘touchdown’ PCR protocol was performed in which annealing is lowered by 0.5 C/cycle, from 69 to
59°C, for a total of 20 cycles with each annealing step lasting 30 seconds. Twenty more cycles are
then performed with the annealing temperature at 59°C for 30 seconds. Denaturation steps are all
executed at 94°C (for 30 seconds) and extensions at 72°C (for 45 seconds). Following this, a final
extension at 72°C for 5 minutes is performed, and then the reaction is cooled at 4°C. Internal
amplification is performed using primers PliF and PliR (see below). Each 50 µl PCR reaction contains
1 µl of the external PCR amplification, 1 × PCR buffer (with 1.5 mM MgCl2), 0.5 µM PliF primer, 0.5 µM
PliR primer, 0.2 mM of each dNTP, additional 1 mM MgCl2, 1.25 U Taq polymerase. Cycling conditions
are: 94°C (30 seconds), 59°C (30 seconds), 72°C (45 seconds) for a total of 30 cycles followed by
5 minutes at 72°C and then the reaction is cooled at 4°C.
The molecular weights of the PCR products are determined by electrophoresis in a 0.8% agarose gel
and staining with ethidium bromide.
Ref. Name Sequence PCR-product Specificity

size level
(9) AFB-F 5’-CTT-GTG-TTT-CTT-TCG-GGA-GAC-GCC-A-3’ 1106 bp species

AFB-R 5’-TCT-TAG-AGT-GCC-CAC-CTC-TGC-G-3’
(20)
PleF 5’-TCG-AGC-GGA-CCT-TGT-GTT-3’ 969 bp
PleR 5’-CTA-TCT-CAA-AAC-CGG-TCA-GAG-3’
PliF 5’-CTT-CGC-ATG-AAG-TCA-TG-3’
572 bp species
PliR 5’-TCA-GTT-ATA-GGC-CAG-AAA-GC-3’
iii) Biochemical tests

Paenibacillus larvae can be also be identified by its biochemical profile. The bacteria are catalase
negative or weak delayed positive, they have a typical carbohydrate acidification profile with acid from
glucose and trehalose, not from arabinose and xylose, and they can hydrolyse casein or milk. Some
strains of P. larvae can change the biochemical signs.
Catalase test
A drop of 3% hydrogen peroxide is placed on an actively growing culture on solid medium. Most
aerobic bacteria break down the peroxide to water and oxygen, producing a bubbly foam, but P. larvae

is negative or weak delayed positive for this reaction (15). When Colombia sheep blood agar is used
for cultivation, the test cannot be done on the solid medium, as the presence of sheep blood will cause
a false-positive reaction. In this case, colonies should be transferred to a clean microscope slide for
the execution of the test. Here the evaluation of the test occurs as above with the naked eye.
Production of acid from carbohydrates (13)

Bacteria are grown in J-broth (per litre: yeast extract 15 g, tryptone 5 g and K2HPO4 3 g) in which 0.5%
of the test substrate, separately sterilised in aqueous solution, is substituted for the glucose. The
carbohydrates used are L (+)-arabinose, D (+)-glucose, D (+)-xylose and D (+)-trehalose. The cultures
are tested at 14 days by aseptically removing one ml or less to a spot plate, mixing the sample with a
drop of 0.04% alcoholic bromocresol purple, and observing the colour of the indicator. Paenibacillus
larvae produces acid aerobically from glucose and trehalose. No acid is produced from arabinose and
xylose (1).
The use of commercial kits, such as API 50 CHB (5), BBL CRYSTAL (8) and Biolog system (22) for
the biochemical characterisation of P. larvae can be taken into consideration.
Hydrolysis of casein (30)

Casein hydrolysis is assayed using milk agar plus thiamine (per litre: agar 20 g, yeast extract 10 g;
sterilised at 121°C/15 minutes). Add to each 70 ml cooled medium 30 ml of UHT (ultra heat treated)
skimmed milk and 1.5 ml filter sterilised 0.1% thiamine solution. Plates are streaked and examined
after 5 days of incubation at 36 ± 1°C. Paenibacillus larvae hydrolyses casein, hence zones of clearing
are observed around bacterial colonies.
iv) Antibody-based techniques

Different antibody-based techniques have been developed for the diagnosis of AFB. Most of them rely
on polyclonal rabbit serum developed against pure cultures of P. larvae. They can be used for
identification of bacterial colonies resulting from a culturing step or for direct examination of suspicious
larval remains.
In an immunodiffusion test the antibodies interact with the bacterial antigen during a double diffusion
process, leaving precipitation marks behind (25). In the fluorescent antibody technique these
antibodies are conjugated with a fluorochrome dye. The resulting fluorescent antibody reacts with a
bacterial smear on a slide. Any excess antiserum is washed off and the smear is examined by
fluorescence microscopy. Paenibacillus larvae stains can be recognised specifically as brightly
fluorescing bacteria on a dark background (24, 33, 36). An enzyme-linked immunosorbent assay using
a monoclonal antibody specific to P. larvae exists (23). A lateral flow device for rapid confirmation of
AFB has been commercialised.
v) Microscopy
Two microscopic techniques are commonly used. Gram staining is often done on smears of bacteria
from isolated bacterial colonies. Paenibacillus larvae is Gram positive. Carbol fuchsin staining is done
on larval smears and can confirm clinical AFB based on spore morphology. These techniques are
outlined below:
Gram staining of bacteria

Flood (cover completely) the entire slide with crystal violet. Let the crystal violet stand for about
60 seconds. When the time has elapsed, wash the slide for 5 seconds with water. The specimen
should appear blue-violet when observed with the naked eye. Now, flood the slide with the iodine
solution. Let it stand for about a minute as well. When the time has expired, rinse the slide with water
for 5 seconds and immediately proceed. At this point, the specimen should still be blue-violet. This
step involves addition of the decolouriser, ethanol. This step is somewhat subjective because using
too much decolouriser could result in a false Gram (–) result. Likewise, not using enough decolouriser
may yield a false Gram (+) result. To be safe, add the ethanol drop-wise until the blue-violet colour is
no longer emitted from the specimen. As in the previous steps, rinse with the water for 5 seconds. The
final step involves applying the counter-stain, safranin. Flood the slide with the dye and let this stand
for about a minute to allow the bacteria to incorporate the saffranin. Gram-positive cells will
incorporate little or no counter-stain and will remain blue-violet in appearance. Gram-negative
bacteria, however, take on a pink colour and are easily distinguishable from the Gram positives.
Again, rinse with water for 5 seconds to remove any excess of dye. Blot the slide gently with bibulous
paper or allow it to air dry before viewing it under the microscope.
Carbol fuchsin staining of larval smears (17)

Heat-fix smears. Flood the slides with 0.2% carbol fuchsin for 30 seconds. Wash off the stain and
allow to air dry or gently blot dry with absorbent material. Examine under the microscope for P. larvae
spores, which are about 1.3 × 0.6 µm, ellipsoidal and thick rimmed.

No serological tests are available.
1. Antibody production
VITA diagnostic kit for the early detection of AFB was developed by the Central Science Laboratory Pocket
Diagnostic (UK).
When monoclonal or polyclonal P. larvae-specific antibodies are produced for the development of a diagnostic
test, no cross-reactivity may occur with closely related bacteria or bacteria that commonly occur in beehives, for
example against Paenibacillus alvei, often found in late phase European foulbrood.
• Acknowledgement
Illustrations by Karl Weiss, extracted from Bienen-Pathologie, 1984, are reproduced with the kind permission of
the author and Ehrenwirth-Verlag, Munich (Germany). Photographs are from the Central Science Laboratory,
York (UK) and the Informatiecentrum voor Bijenteelt, Ghent (Belgium) and published with kind permission of
respectively Ruth Waite and Frans J. Jacobs.
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12. GOCHNAUER T.A. & CORNER J. (1987). Detection and identification of Bacillus larvae in a commercial pollen
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13. GORDON R.E., HAYNES W.C. & PANG C.H.N. (1973). The genus Bacillus. Agriculture Handbook N° 427,
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*
* *

CHAPTER 2.2.3.
EUROPEAN FOULBROOD OF HONEY BEES
SUMMARY
The causal organism of European foulbrood of honey bees is the bacterium Melissococcus
plutonius. The identification of its presence by the observation of signs of disease in the field is
unreliable. The most usual and obvious sign is the death of larvae shortly before they are due to be
sealed in their cells, but this may be for reasons other than European foulbrood. Most infected
colonies display few visible signs, which themselves often quickly abate spontaneously before the
end of each active season. Infection remains enzootic within individual colonies because of
mechanical contamination of the honeycombs by the durable organism. Recurrences of disease
can therefore be expected in subsequent years.
Identification of the agent: Examination, by high-power microscopy, of suitable preparations of
larval remains for the presence of numerous lanceolate cocci is adequate for most practical
purposes, especially when it is done by experienced individuals.
Traditionally the diagnosis of European foulbrood is done by isolating and identifying the causative
organism. This can be differentiated quite readily from all other bacteria associated with bees by its
fastidious cultural requirements.
The isolated bacterium can be identified and differentiated by means of simple tube agglutination
tests. A polymerase chain reaction and a hemi-nested polymerase chain reaction are also
available. The latter permits direct analysis of larvae, adult bees and honey bee products.
Serological tests: No tests for detecting antibodies in bees are available.
available.
A. INTRODUCTION
Bee larvae usually die of European foulbrood 1–2 days before being sealed in their cells, or sometimes shortly
afterwards, and always before transformation to pupae. The disease is caused by Melissococcus plutonius and
occurs mostly during the period when colonies are growing quickly. Most sick larvae become displaced from their
coiled position in the bottom of their cells before they die. Many are quickly detected and removed by nurse bees,
leaving empty cells scattered randomly among the remaining brood. Some infected larvae survive, successfully
pupate and emerge as adults. These surviving larvae are able to defecate and their infected-faeces contribute to
the continued propagation of the disease (2).
Infected larvae that escape detection by adult bees and then die, first become flaccid and turn a light yellow
colour that becomes increasingly brown, and at the same time they dissolve into a semi-liquid mass. They then
become dry and form a dark brown scale that can easily be removed from the cells. Severely affected brood may
have a very stale or sour odour, sometimes acidic, like vinegar, but often there is no smell.
Signs of disease usually disappear spontaneously from infected colonies before the end of the active season, but
are likely to return in subsequent years (4, 10).
a) Microscopy
Freshly dead larvae are best for diagnosis. Before any decomposition occurs, diseased larvae can be
smeared on a microscope slide or pulled apart by pinching the cuticle about the centre of the body with two

Chapter 2.2.3. — European foulbrood of honey bees
pairs of forceps, which are then pulled apart. The mid gut contents are left exposed on the slide, still within
the gelatinous, transparent peritrophic membrane. This is partially or almost completely filled with bacteria,
which are easily seen as opaque chalk-white clumps. The contents of the mid-guts of healthy larvae, which
are less easily dissected, have a golden-brown colour. Apparently healthy larvae may contain a mixture of
bacteria and pollen. The mid-gut of healthy larvae that contain much light-coloured pollen may resemble
those that are filled with bacteria.
a b c
Fig. 1. Bacteria associated with European foulbrood.
(a) Melissococcus plutonius: the cause of European foulbrood occurs singly, in longitudinal chains or in clusters.
Morphologically resembles Enterococcus faecalis, a common secondary invader.
(b) Paenibacillus alvei: vegetative rods 2.0—7.0 × 0.8—1.2 μm with flagella; sporulating with spores lying adjacently.
Both rods and spores are larger than those of Paenibacillus larvae (see American foulbrood).
(c) Bacterium eurydice: slender, square-ended rods in vivo but can form chains of cocci in vitro in certain media.
For a bacteriological investigation, a loopful of a dilute aqueous suspension of the midgut contents is
transferred to a clean microscope slide and mixed with a loopful of 5% aqueous nigrosin. This is spread
over one or two square centimetres, dried gently over a flame, and examined directly by high-power
microscopy. The presence of numerous lanceolate cocci, about 0.5 × 1.0 µm in size, occurring either singly
or in clusters, and arranged end to end in pairs or short chains, is almost certainly diagnostic of European
foulbrood. Some very slender square-ended rod-like bacteria are also usually present (Figure 1). The cocci
are Gram positive and the rods are Gram negative. Similar preparations made from aqueous suspensions
of whole dead or decomposing larvae are likely to present a confusing array of bacteria in which
M. plutonius will be difficult to distinguish.
b) Culture methods
Melissococcus plutonius (type strain NCIMB 702443) is the most abundant bacterium during the early
stages of an infection (5, 6). Melissococcus plutonius can be cultivated on a medium (expressed in g/litre or
ml/litre) comprising: yeast extract or certain peptones, 10 (5); cysteine or cystine, 0.2–2.0; glucose or
fructose, 10; soluble starch, 10; 1 M KH2PO4, 100 at pH 6.6; and agar, 2. The medium is preferably
autoclaved in 100 ml lots in screw-capped bottles at 116°C for 20 minutes and poured into Petri plates
immediately before use. These plates are streaked with dilute aqueous suspensions of diseased larvae, or
ideally, of diseased larval mid-gut. The latter can be prepared beforehand by allowing them to dry on a
slide, which may then be kept, for years if necessary, at 4°C or –20°C. All culture media should be
subjected to quality control and must support the growth of M. plutonius from small inocula. The reference
strain should also be cultured in parallel with the suspect samples to ensure that the tests are working
correctly.
The preparation and storage of dried smears also eliminates most secondary organisms after a few weeks
without affecting the viability of M. plutonius. This organism is isolated most efficiently by inoculating
decimal dilutions of the aqueous suspension into agar that has been maintained molten at 45°C and which
is then poured into plates. The plates must be incubated anaerobically, such as in McIntosh and Fildes jars
in an atmosphere of approximately 5–10% carbon dioxide (CO2) at 35°C. Small white opaque colonies of
M. plutonius usually appear within 4 days. This bacterium is somewhat pleomorphic in vitro, often appearing
in rod-like forms. The final pH of the medium may reach 5.5. Decreasingly fastidious strains become
selected in vitro. Simplified or modified forms of the medium then support multiplication, especially of a
serologically distinct M. plutonius group from Brazil (1) that will multiply on chemically defined media (3).
CO2 remains essential. Inoculated slopes should be sealed when bacterial growth is apparent and may
then be kept at 4°C for up to 6 months. Alternatively, the cultures can be suspended in a medium of 10%
sucrose, 5% yeast extract and 0.1 M KH2PO4, pH 6.6, and then lyophilised.
A number of other bacteria are often associated with and may be confused with M. plutonius. Bacterium
eurydice inhabits the alimentary tract of adult bees and occurs commonly in the gut of healthy larvae in
small numbers. It is more numerous in larvae infected with M. plutonius. The incidence of B. eurydice in
healthy bees is very low in winter and early spring, but it increases in summer. It forms thin square-ended
rods, which can grow either singly or in chains. When grown in certain media, it sometimes resembles

streptococci and has been confused with M. plutonius. However, its cultural characteristics closely resemble
those of Corynebacterium pyogenes (10), and it multiplies poorly in the form of thin rods, under the
conditions necessary for the cultivation of M. plutonius.
Enterococcus (= Streptococcus) faecalis closely resembles M. plutonius morphologically and has often
been confused with it, although they are both culturally and serologically distinct. Unlike M. plutonius, it
does not remain viable for long when dried, or persist as mechanical contamination within bee colonies. It is
probably brought into the hive by foraging adult bees, and is responsible for the sour smell sometimes
encountered with European foulbrood.
Enterococcus faecalis grows well in vitro under the conditions suitable for M. plutonius, but it may be readily
differentiated by its ability to grow aerobically. It forms small transparent colonies within 24 hours and is a
facultative anaerobe. It multiplies on a variety of the more common media with or without carbohydrates or
CO2. The final pH in the presence of glucose is 4.0. Enterococcus faecalis rarely exceeds the number of
M. plutonius in bee larvae, and can usually be diluted out. When it is not diluted out it produces sufficient
acid to prevent the in-vitro multiplication of M. plutonius.
Enterococcus faecalis does not multiply in bee larvae in the absence of M. plutonius, so its presence in
large numbers can be taken as presumptive evidence of European foulbrood.
Paenibacillus (= Bacillus) alvei is generally more common than E. faecalis in bee colonies affected with
European foulbrood, but it is not invariably associated with the disease and so cannot act as a reliable
indicator of it. In bee colonies, it multiplies only in the decomposing remains of larvae, and then its spores
often predominate over all other bacteria, even to their apparent exclusion. Paenibacillus alvei forms very
resistant spores and becomes well established in bee colonies with enzootic European foulbrood. It causes
a characteristic stale odour. Paenibacillus alvei multiplies poorly under the conditions necessary for the in-
vitro growth of M. plutonius. It produces a spreading growth of transparent colonies, some of which are
motile and move in arcs over the surface of the agar. Cultures have the characteristic stale odour that is
associated with European foulbrood when the bacillus is present. Spores are formed rapidly.
c) Immunological methods
For the identification of M. plutonius, antisera can be prepared in rabbits against washed cultures of
M. plutonius either by intravenous injections (7) or by a single intramuscular injection of 1 ml of antigen
suspension mixed with an equal volume of Freund’s incomplete adjuvant.
Assays are made by agglutination tests in tubes containing suspensions of bacteria equivalent to 0.25 mg
dry weight/ml. End-points are noted after tubes have been incubated for 4 hours at 37°C.
A test kit for the identification of antibodies against M. plutonius has recently been developed and is
commercially available. It provides rapid confirmatory on-site diagnosis of European foulbrood infection in
honeybee larvae.
d) Polymerase chain reaction

Polymerase chain reaction (PCR) can be done on suspicious bacterial colonies transferred to and grown in
liquid medium (9). Genomic DNA is prepared according to standard methods (12). The DNA pellet is
resuspended in 50 µl of 1 × TE buffer (10 mM Tris/HCl, pH 7.5; 1 mM EDTA [ethylene diamine tetra-acetic
acid]). Approximately 1–3 µg of genomic DNA is amplified in a 50 µl reaction. The PCR reaction can also be
done with larvae. Each larva is incubated individually in liquid medium overnight at 30°C in an anaerobic jar
containing hydrogen plus 10% CO2. Two millilitres of each sample is then centrifuged at 1000 g for
2 minutes, and the supernatant is centrifuged at 10,000 g for 5 minutes. The resultant pellet is resuspended
in 100 µl of sterile H2O and heated at 95°C for 15 minutes. One microlitre is amplified in a 50 µl PCR
mixture. Besides template DNA this mixture also contains 2 mM MgCl2, 50 pmol of forward (EFB-F) and
reverse primer (EFB-R; primer sequences are given below) per µl, 25 mM (each) deoxynucleoside
triphosphate and 1 U of Taq polymerase. Amplification of a specific DNA fragment occurs in a thermocycler
under to the following PCR conditions: a 95°C (1 minute) step; 30 cycles of 93°C (1 minute), 55°C
(30 seconds), and 72°C (1 minute); and a final cycle of 72°C (5 minutes).
A hemi-nested PCR was first developed by Djordjevic et al. (8) and thereafter improved for sensitive
detection of M. plutonius in honey, pollen, whole larvae and adult bees (11). Here the first 50 µl reaction
mixture contains 5–30 ng genomic DNA, 3 mM MgCl2, 200 µM of each deoxyribonucleotide triphosphate,
100 ng of the primers MP1 and MP2, 5 µl of 10 × PCR buffer (100 mM Tris/HCl, pH 8.3; 15 mM MgCl2;
500 mM KCl) and 1 U of Taq polymerase. Conditions of amplification consist of an initial denaturation cycle
at 95°C for 2 minutes followed by 40 cycles of denaturation (95°C, 30 seconds), primer annealing (61°C,

15 seconds), primer extension (72°C, 1 minute) followed by an additional extension step of 5 minutes at
72°C. The third primer MP3 is used in conjunction with MP1 to amplify a DNA fragment from 1 µl of the
primary PCR product obtained in the previous reaction. PCR conditions for the hemi-nested PCR are
exactly as described above except that the MgCl2 concentration is lowered to 1.5 mM and the annealing
temperature to 56°C.
The molecular weights of the PCR products are determined by electrophoresis in a 1.0–1.5 % agarose gel
and staining with ethidium bromide.
Ref. Name Sequence PCR-product

size
(9) EFB-F 5’-GAA-GAG-GAG-TTA-AAA-GGC-GC-3’ 812 bp

EFB-R 5’-TTA-TCT-CTA-AGG-CGT-TCA-AAG-G-3’
(8, 11) MP1 5’-CTT-TGA-ACG-CCT-TAG-AGA-3’ 486 bp

MP2 5’-ATC-ATC-TGT-CCC-ACC-TTA-3’
MP3 5’-TTA-ACC-TCG-CGG-TCT-TGC-GTC-TCT-C-3’ 276 bp
No tests for detecting antibodies in bees are available.

There are no biological products available.
ACKNOWLEDGMENT
Illustrations by Karl Weiss, extracted from Bienen-Pathologie, 1984. Reproduced with the kind permission of the
author and Ehrenwirth-Verlag, Munich (Germany).
REFERENCES
1. ALLEN M.F. & BALL B.V. (1993). The cultural characteristics and serological relationships of isolates of
Melissococcus pluton. J. Apic. Res., 32, 80–88.
2. BAILEY L. (1960). The epizootiology of European foulbrood of the larval honey bee, Apis mellifera Linnaeus.
J. Insect Pathol., 2, 67–83.
3. BAILEY L. (1984). A strain of Melissococcus pluton cultivable on chemically defined media. FEMS Microbiol.
Lett., 25, 139–141.
4. BAILEY L. & BALL B.V. (1991). Honey Bee Pathology. Academic Press, London, UK, and New York, USA.
5. BAILEY L. & COLLINS M.D. (1982). Taxonomic studies on Streptococcus pluton. J. Appl. Bacteriol., 53, 209–
213.
6. BAILEY L. & COLLINS M.D. (1982). Reclassification of Streptococcus pluton (White) in a new genus
Melissococcus, as Melissococcus pluton nom. rev.; Comb. nov. J. Appl. Bacteriol., 53, 215–217.
7. BAILEY L. & GIBBS A.J. (1962). Cultural characters of Streptococcus pluton and its differentiation from
associated enterococci. J. Gen. Microbiol., 28, 385–391.
8. DJORDJEVIC S.P., NOONE K., SMITH L. & HORNITZKY M.A.Z. (1998). Development of a semi-nested PCR assay
for the specific detection of Melissococcus pluton. J. Apic. Res., 37, 165–174.

9. GOVAN V.A., BROZEL V., ALLSOPP M.H. & DAVISON S. (1998). A PCR detection method for rapid identification
of Melissococcus pluton in honeybee larvae. Appl. Environ. Microbiol., 64, 1983–1985.
10. JONES D. (1975). A numerical taxonomic study of Coryneform and related bacteria. J. Gen. Microbiol., 87,
52–96.
11. MCKEE B.A., DJORDJEVIC S.P., GOODMAN R.D. & HORNITZKY M.A. (2003). The detection of Melissococcus
pluton in honey bees (Apis mellifera) and their products using a hemi-nested PCR. Apidologie, 34, 19–27.
12. W ILSON K. (1990). Preparation of genomic DNA from bacteria. In: Current Protocols in Molecular Biology,
Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Smith J.A., Seidman J.G. & Struhl K., eds. Greene
Publishing Association and Wiley Interscience, New York, USA, 241–245.
*
* *

CHAPTER 2.2.4.
NOSEMOSIS OF HONEY BEES
SUMMARY
To date, two microsporidian parasites have been described from honey bees: Nosema apis
(Zander) and Nosema ceranae (Fries). Nosema apis is a parasite of the European honey bee (Apis
mellifera) and Nosema ceranae of the Asian honey bee (Apis cerana) (11) and the European
honey bees). The latter has recently been detected in several geographically separated
populations of European honey bees in Europe (12), South and North America (14) and Asia (13).
The pathological consequences of Nosema ceranae in Apis mellifera are not well known. In the
following chapter, only Nosema apis is described. Both types are presumably very similar.
Nosema apis is a parasite that invades the epithelial cells of the ventriculus of the adult honey bee.
Infections are acquired by the uptake of spores during feeding or grooming. The disease occurs
throughout the world, but treatment of bees can help to prevent the spread of infection to
unaffected bee colonies.
The parasite invades the posterior region of the ventriculus, giving rise to large numbers of spores
within a short period of time. The parasite is ubiquitous. Nosema levels generally increase when
bees are confined, such as in the autumn and winter in colder climates when the amount of brood
is decreasing and perhaps in the early spring when there is an increase in the brood. The disease
is transmitted among bees via the ingestion of contaminated comb material and water, and by
trophallaxis; honey stores and crushed infected bees may also play a role in disease transmission.
Spores are expelled with the faeces where they may retain their viability for more than 1 year.
Spores may also remain infective after immersion in honey and in the cadavers of infected bees;
however they may lose viability after 3 days when submerged in honey at hive temperature. The
relative importance of faeces, honey and cadavers as reservoirs of infective spores is not fully
understood. However, it seems likely that faecal contamination of wax, especially in combs used
for brood rearing, or other hive interior surfaces, provides sufficient inoculum for N. apis to be
successfully transmitted to the next generation of bees. The spores are inactivated by acetic acid
or by heating to 60°C for 15 minutes. To be effective, these treatments, which inactivate spores on
hive surfaces and combs, can be combined with feeding colonies with the antibiotic fumagillin to
suppress infections in live bees. The EU prohibits the use of antibiotic fumigation (EU 3/01/081).
Identification of the agent: In some acute cases, brown faecal marks are seen on the comb and
the front of the hive, with sick or dead bees in the vicinity of the hive. However, the majority of
colonies show no obvious signs of infection, even when the disease is sufficient to cause
significant losses in honey production and pollination efficiency. During winter, there may be an
increase in bee mortality. In affected bees, the ventriculus, which is normally brown, can be white
and very fragile. Microscopic examinations (×400 magnification) of homogenates of the abdominal
contents of affected bees will reveal the oval spores of Nosema apis, which are approximately 5–
7 × 3–4 µm with a dark edge (Nosema ceranae is slightly smaller). Their internal contents can be
distinguished after staining with Giemsa’s stain. Nosema apis spores have a distinctive
appearance, with a thick unstained wall and a blue-stained featureless interior. The nuclei within
the spores are not visible. This method can help to distinguish N apis from other microbes found in
bees.
The appearance of Nosema apis spores can be confused with yeast cells, fungal spores, fat and
calciferous bodies or cysts of Malpighamoeba mellificae. The latter are similar in size to Nosema
spores, being 6–7 µm in diameter, but are completely spherical instead of oval.
Positive identifications can be made only by observation of typical spores in the ventriculus or
faeces. Very mild infections may not be demonstrable. The extent of infection is determined by

Chapter 2.2.4. — Nosemosis of honey bees
counting the spores on a microscope grid and calculating the average number of spores per area
and estimating from that the number of spores per bee.
Serological tests: There are no applicable serological tests.
available.
A. INTRODUCTION
The microsporidium Nosema apis (Zander) is a protozoan parasite exclusive to the epithelial cells of the
ventriculus of adult bees and the disease occurs throughout the world (16). Infection occurs by the ingestion of
spores in the feed (5, 19), via trophallaxis (19) or perhaps after grooming of the body hairs (6, 10, 19).
The polar tube of the spore is everted and penetrates the peritrophic matrix of the intestine, particularly in the
posterior region of the ventriculus. The sporoplasm passes down the tube and enters the cytoplasm of the
epithelial cells, where it reproduces. Autoinfections can occur at the same time as new infections. After a short
interval, spores develop in large quantities. The parasite is ubiquitous and multiplies at a specific rate throughout
the year. Nosema levels generally increase when bees are confined, such as in the autumn and winter in colder
climates when the amount of brood is decreasing and perhaps in the early spring when there is an increase in
the brood (19, 20). In winter, spores are rarely to be found, or are only found in heavily infected bees.
Any inherent natural defence by a bee colony against a heavy infection with the parasite depends on the colony
size as well as on the prevailing weather conditions during the early part of the autumn of the previous year (18).
If these conditions are unfavourable, the overall life expectancy of the colony is reduced. This may lead to the
premature death of bees during winter or early spring. In a typical case of a colony being depleted because of a
Nosema infection, the queen can be observed surrounded by a few bees, confusedly attending to brood that is
already sealed.
In faecal droppings, spores may retain their viability for more than 1 year (3). Spores may also remain viable for
up to 4 months after immersion in honey (21) and for up to 4.5 years in the cadavers of infected bees (18). The
spores may lose viability after only 3 days when submerged in honey at hive temperature (17). It is likely that
faecal contamination of wax, especially in combs used for brood rearing, or other hive interior surfaces, provides
sufficient inoculum for N. apis to be successfully transmitted to the next generation of bees. The relative
importance of faeces, honey and cadavers as reservoirs of infective spores is not fully understood and it seems
that temperature may have a marked effect on the rates at which spores lose viability, regardless of their medium
(17).
Spores may be killed by heating hive equipment or tools to a temperature of at least 60°C for 15 minutes. Combs
may be sterilised by heating to 49°C for 24 hours (8). Fumes from a solution of at least 60% acetic acid will
inactivate any spores within a few hours, depending on the concentration; higher concentrations are even more
effective and will kill spores within a few minutes (2, 9). Such procedures come under the jurisdiction of national
control authorities with protocols that vary from country to country. Disinfection can be carried out, for example,
by putting acetic acid solution into bowls or on to sponges that can soak up the liquid. Following disinfection after
an outbreak, all combs should be well ventilated for at least 14 days prior to use. Suppression of Nosema
disease can also be achieved by feeding an antibiotic, fumagillin, in sugar syrup to the colony (8). This is
forbidden in many countries and in the EU.
In acute forms of infection, especially in early spring, brown faecal marks may be noted on the comb and the
front of the hive (4). At the entrance to the hive, sick and dead bees may be seen, although other causes, such
as pesticide poisoning and diseases of adult honey bees (such as acarapidosis should be eliminated first if this is
the case. The detection of these infectious diseases requires microscopic examination. During winter, Nosema
apis-infected colonies may become severely depleted of bees or die out altogether. The majority of Nosema
apis-infected colonies will appear normal, with no obvious signs of disease even when the disease is sufficient to
cause significant losses in honey production and pollination efficiency (1, 11). A proper diagnosis can be made
only by microscopic examination of adult bee abdomen or ventriculus. To diagnose a Nosema apis infection, the
posterior pair of abdominal segments is removed with a forceps to reveal the ventriculus, complete with the
malpighian tubules, the small intestine and rectum. The ventriculus is normally brown but, following a Nosema
infection, it can become white and fragile. However, this appearance is given by other causes of intestinal

disturbance, for example feeding on indigestible food stores, such as syrup containing actively growing yeast.
For a reliable diagnosis, a number of bees in a sample should be examined.
a) Microscopy
It is necessary to attempt to distinguish between a Nosema apis infection and an infection caused by
Malpighamoeba mellificae (19). There is quite often an indication of dysentery in a Nosema apis infection. In
an M. mellificae infection, there may be diarrhoea, often of a sulphur-yellow colour and with a distinct odour.
Characteristics of M. mellificae cysts are described later. Secondary mixed infections may occur (17). A
simple, nonquantitative method for detecting Nosema apis infection is as follows: sampled bees should be
obtained from the hive entrance in order to avoid sampling individuals under the age of 8 days, which would
lead to ‘false negatives’ because no spores from the protozoan in question would be determined. At least
60 bees should be collected in order to detect 5% of diseased bees with 95% confidence (10). Before
sending to the laboratory, the bees should be fixed in 4% formol, 70% ethyl alcohol or frozen in a standard
freezer in order to prevent them from decomposing and to improve their reception and organisation in the
laboratory. The abdomens of the bees to be examined are separated and ground up in 2–3 ml of water.
Three drops of the suspension are placed on a slide under a cover-slip and examined microscopically at
×400 magnification, under bright-field or phase-contrast optics. This is a slight simplification of Cantwell’s
original method (7). The spores are about 5–7 µm long and 3–4 µm wide (Nosema ceranae is slightly
smaller than Nosema apis). They are completely oval with a dark edge. Their contents, consisting of
nucleus, sporoplasm and polar tube, cannot be seen. Dyes are usually not necessary.
Nosema spores must be differentiated from yeast cells, fungal spores, fat and calciferous bodies, and from
M. mellificae cysts, which are spherical and approximately 6–7 µm in diameter.
When air-dried, ethanol-fixed smears of infected tissue are stained with Giemsa’s stain (10% in 0.02 M
phosphate buffer) for 45 minutes. Nosema apis spores will have a distinctive appearance, with thick
unstained walls and an indistinct blue interior, without visible nuclei. Insect cells, fungal spores and other
protozoa stained in this way will generally have thinner walls, blue/purple cytoplasm and magenta-coloured
nuclei.
In order to obtain accurate, reliable and meaningful quantification of levels of Nosema infections in honey
bees, a standardised procedure must be used. A suitable protocol is as follows:
A sample of older worker honey bees is taken, from which the abdomens of ten individuals are macerated
in 5 ml of water using a mortar and pestle. When tissue pieces have become quite fine, the suspension is
filtered through two layers of muslin (thin loosely woven cotton fabric) in a funnel leading to a graduated
centrifuge tube. A second 5 ml of water is used to rinse the pestle, swirl around the inside of the mortar and
pour through the subsample in the funnel. Water levels are equalised in the tubes and the suspensions are
centrifuged for 6 minutes at 800 g. The supernatants are decanted and the tubes are refilled to the 10 ml
level. Using disposable pipettes and a rubber bulb, the pellets are resuspended by repeated uptake and
forcible ejection through the pipette tips. When the solution appears to be homogenous, a sample is taken
to fill the calibrated volume under the cover-slip of a haemocytometer (blood cell counting chamber). After a
few minutes the spores will have settled to the bottom of the chamber. Nosema spores appear transparent
but with a very distinct dark edge and are 5–7 µm long and 3–4 µm wide. They are best seen using a
magnification of ×400 and bright-field or phase-contrast optics. The number of spores in each square is
counted. Where a spore lies over the edge of a square, count only those spores that straddle the left and
upper edges of the square, not the right and bottom edges. One Nosema apis spore, observed in the
haemocytometer’s entire central square millimetre grid (25 × 16 = 400 small squares), is equal to an
average of 10.000 spores per bee. If no spores are seen, the result should be designated ‘not detected’, but
that does not mean that the bees are not infected. Regulatory agencies will decide on the level of infection
useful for their purposes.
A laboratory method for the simultaneous detection of Nosema spores and M. mellificae cysts consists of
the individual examination of the colonies using 30–60 bees per colony. A suspension of the abdomens of
dead bees is prepared by grinding with 5–10 ml water; the volume of water depending on the number and
condition of the bees. The suspension must be filtered to remove debris that would interfere with the
examination, first through a 100 µm and then a 40 µm filter. Parts of the malpighian tubules pass through
the 100 µm filter, but are collected on the 40 µm filter. They are placed on a slide or bacterial counting
chamber and examined at ×400 magnification. Only a few tubules are filled with cysts after an M. mellificae
infection. The normal structure of malpighian tubules is not visible in this case. Only cysts inside the
malpighian tubules can be taken as a positive result, because M. mellificae cysts are often confused with
fungal spores and yeast cells.
b) Culture
There are no cultural methods for growing these organisms.

c) Polymerase chain reaction (PCR)

Different methods have been developed to distinguish N. apis from N. ceranae. A multiplex PCR is
described below with which both pathogen types can be clearly identified at the same time (15).
• Sample preparation for PCR

The abdomens of 10–20 adult honey bees from each sample are macerated in 10 ml distilled water (PCR
grade) and the suspension is then filtered and centrifuged at 800 g for 6 minutes. For DNA extraction, spore
germination is induced with 200 µl freshly prepared germination buffer (0.5 M sodium chloride, 0.5 M
sodium hydrogen carbonate, pH to 6.0 with ortophosphoric acid), and the mixture is incubated at 37°C for
15 minutes. The DNA extraction can be easily carried out using routine procedures or commercial kits, such
as High Pure PCR Template Preparation Kit (No. 1796828 Roche Diagnostic).
• Multiplex PCR
With this technique both microsporidians (N. apis and N. ceranae) can be distinguished in just one PCR
because of the use of specific primers with no interference. PCR reactions are performed in 50-µl volumes
containing 5 µl of template DNA, 25 µl of High Fidelity PCR Master Mixture (catalogue no. 12140314001;
Roche Diagnostic), 0.4 µM of each primer, 0.4 mM of each deoxynucleoside triphosphate, 3 mM Cl2Mg,
0.2 mg/ml bovine serum albumin, 0.1% Triton X-100, and 5 µl of N. apis or N. ceranae DNA template. The
parameters for amplification are: an initial PCR activation step of 2 minutes at 94°C, followed by 10 cycles
of 15 seconds at 94°C, 30 seconds at 61.8°C, and 45 seconds at 72°C, and 20 cycles of 15 seconds at
94°C, 30 seconds at 61.8°C, and 50 seconds at 72°C plus a 5-second elongation cycle for each successive
cycle and a final extension step at 72°C for 7 minutes. Negative controls (from DNA extraction) are included
in all PCR experiments.
The molecular weights of PCR products are determined by electrophoresis in a 2% agarose TAE (Tris-
acetate-ethylene diamine tetra-acetic acid) gel in standard TAE buffer, stained with ethidium bromide, and
visualised using UV illumination.
Primers selected for detection of N. ceranae and N. apis in multiplex PCR:
Primer Sequencea PCR product size (bp) Specificity
218MITOC FOR 5’-CGGCGACGATGTGATATGAAA-ATATTAA-3’ 218–219b N. ceranae
218MITOC REV 5’-CCCGGTCATTCTCAAACAAAA-AACCG-3’
321APIS FOR 5’-GGGGGCATGTCTTTGACGTACTATGTA-3’ 321 N. apis
321APIS REV 5’-GGGGGGCGTTTAAAATGTGAAACAACTATG-3’
aCG tails added to primers are underlined.
bThere is a 1-bp difference in the N. ceranae amplicon size depending on the sequences for N. ceranae available in GenBank
(http://www.ncbi.nlm.nih.gov).
There are no serological tests available.

No biological products are available.
REFERENCES
1. ANDERSON D.L. & GIACON H. (1992). Reduced pollen collection by honey bee (Hymenoptera: Apidae)
colonies infected with Nosema apis and sacbrood virus. J. Econ. Entomol., 85, 47–51.
2. BAILEY L. (1957). Comb fumigation for Nosema disease. Am. Bee J., 97, 24–26.

3. BAILEY L. (1962). Bee diseases. In: Report of the Rothamsted Experimental Station for 1961, Harpenden,
UK, 160–161
4. BAILEY L. (1967). Nosema apis and dysentery of the honey bee. J. Apic. Res., 6, 121–125.
5. BAILEY L. (1981). Honey Bee Pathology. Academic Press, London, UK.
6. BULLA (1977). In: Comparative Pathobiology. Vol. 1: Biology of Microsporidia (1976); Vol. 2: Systematics of
the Microsporidia, Lee A. & Cheng T.C., eds. Plenum Press, New York, USA, and London, UK.
7. CANTWELL G.E. (1970). Standard methods for counting nosema spores. Am. Bee J., 110, 222–223.
8. CANTWELL G.E. & SHIMANUKI H. (1970). The use of heat to control Nosema and increase production for the
commercial beekeeper. Am. Bee J., 110, 263.
9. DERUITER A. & VAN DER STEEN J. (1989). Disinfection of combs by means of acetic acid (96%) against
Nosema. Apidologie, 20, 503–506.
10. FRIES I. (1988). Contribution to the study of Nosema disease (Nosema apis Z.) in honey bee (Apis mellifera
L.) colonies. Rapport 166, Sveriges Landbruksuniversitet, Institutionen för husdjurens utfodring och värd,
Uppsala, Sweden.
11. FRIES I., FENG F., DA SILVA A., SLEMENDA S.B. & PIENIAZEK N.J. (1996). Nosema ceranae n.sp. (Microspora,
Nosematidae), morphologycal and molecular characterization of a Microsporidian parasite of the Asian
honey bee Apis cerana (Hymenoptera, Apidae). Eur. J. Protistol., 32, 356–365.
12. HIGES M., MARTÍN R. & MEANA A. (2006). Nosema ceranae, a new microsporidian parasite in honeybees in
Europe. J. Invertebr. Pathol. 92, 93–95.
13. HUANG W.F., JIANG J.H., CHEN Y.W. & W ANG C.H. (2005). Complete rRNA sequence of Nosema ceranae
from honey bee (Apis mellifera). https:/gra103.aca.ntu.edu.tw/gdoc/F90632004a.pdf (Date: 2005-11-25).
14. KLEE J., BESANA A.M., GENERSCH E., GISDER S., NANETTI A., TAM D.Q., CHINH T.X., PUERTA F., RUZ J.M.,
KRYGER P., MESSAGE D., HATJINA F., KORPELA S., FRIES I., PAXTON R.J. (2007). Widespread dispersal of the
microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera. J.
Invertebr. Pathol., 96, 1–10.
15. MARTIN-HERNANDEZ R., MEANA A., PRIETO L., SALVADOR A.M., GARRIDO-BAILON E. & HIGES M. (2007). Outcome
of colonization of Apis mellifera by Nosema ceranae. Appl. Environ. Microbiol., 73, 6331–6338.
16. MATHESON A. (1996). World bee health update 1996. Bee World, 77, 45–51.
17. MORGENTHALER D. (1939). Die ansteckende Frühjahrsschwindsucht (Nosema-Amoeben-Infection) der

Bienen. Erweiterter Sonderdruck aus der Scheizerischen Bienenzeitung Heft 2, 3 und 4.
18. STECHE W. (1985). Revision of ZANDER & BOTTCHER. Nosematose. In: Krankheiten der Biene, Handbuch der
Bienenkunde.
19. W EBSTER T.C. (1993). Nosema apis spore transmission among honey bees. Am. Bee J., 133, 869–870.
20. WEISER J. (1961). Die Mikrosporidien als Parasiten der Insekten. Verlag Paul Pavey, Hamburg and Berlin,
Germany.
21. W HITE G.F. (1919). Nosema Disease. United States Department of Agriculture Bull., No. 780, 54 pp.
*
* *

CHAPTER 2.2.5.
SMALL HIVE BEETLE INFESTATION

(Aethina tumida)
SUMMARY
The small hive beetle Aethina tumida Murray 1867 (Coleoptera: Nitidulidae), is a parasite and
scavenger of honey bee colonies. Adults and larvae feed on honey bee brood, honey and pollen,
causing death of brood, fermenting of honey and comb destruction, often resulting in the full
structural collapse of the nest and absconding of the colony. The small hive beetle can be a
serious problem in honey-extracting facilities where stored comb, honey and wax cappings are all
potential feeding and breeding areas. Development requires 3–12 weeks, depending on
temperature and food availability. The flying adult beetles actively infest colonies.
Identification of the agent: An infestation by Aethina tumida can be recognised either indirectly
via colony-wide damage or directly via eggs, larvae and adults. An early diagnosis can be made
after opening the colony and finding adult beetles on the bottom board or hiding in the combs.
Intra- and extra-colonial acaricides and insecticides presently are used to kill the adult beetles and
larvae while intra- and extra-colonial traps can be used to find the beetles.
Serological tests: No serological tests are applicable.
Requirements for vaccines and diagnostic biologicals: No biological products are available.
A. INTRODUCTION
The small hive beetle, Aethina tumida Murray, Coleoptera: Nitidulidae (17), is native to sub-Saharan Africa (12)
but has been introduced to the United States of America (1996), Egypt (2000) and Australia (2002) (18). It was
introduced in to Canada in 2002 but did not establish; it was reintroduced in 2006 and it has not been determined
if it is established. Aethina tumida can be spread by active flying, migratory beekeepers, or transportation of
infested hive products (13, 18). Larvae and eggs of A. tumida have been identified in cages of imported queens
in Portugal (2004), but all bee hives were immediately destroyed (pers. comm.). Within its native range, it is
usually considered a minor pest, and reproduction appears more successful in weak and stressed colonies or in
recently abandoned nests (18). However, within its new ranges it can cause considerable damage in colonies of
European honey bee subspecies (3, 11, 13, 18).
1. Life cycle
The infesting A. tumida females mate in the colony (more than 1000 adult beetles may occur within a colony [11])
and oviposit several eggs in typical clutches in small cracks, in cells or within capped brood (9, 15, 18). The
larvae hatch after 1–6 days and feed on pollen, honey and bee brood like the adults (15, 18, 22). Adult beetles
also can be fed by worker bees via trophallaxis (8). Larval development takes 8–29 days (depending on food
availability and temperature [8, 15, 18, 22]) following which they reach the wandering phase (15) and leave for
pupation in the soil, mostly in close proximity to the hive (21). Pupation takes 2–12 weeks depending on
temperature and soil moisture (7). Emerging adults leave the soil and can actively fly over long distances
(>10 km) to search for new host colonies, thereby completing the life cycle of A. tumida.
The reasons for the apparent difference in the impact of the small hive beetle with its native range compared with
its new ranges are not well understood (3). They may include quantitative behavioural differences between
African and European honey bee subspecies, different beekeeping techniques and climatic differences (3, 13,
18).
Adult beetles can survive up to 6 months and females can oviposit about 1000 eggs in their life time (15).

Chapter 2.2.5. — Small hive beetle infestation (Aethina tumida)
While damage due to the adults is relatively minor, it nevertheless can cause absconding of the colony (6). If not
prevented by the bees (9, 19), larval growth (several hundreds or thousands of individuals) is usually associated
with fermentation of the honey, causes severe damage to combs and often results in the full structural collapse of
the nest (12). Economic losses can also be associated with beetle infestations in the honey-extracting facility.
Environmental conditions generally associated with extracting facilities, such as high temperature and humidity,
provide optimal condition for beetle development. Cryptic low-level reproduction may also occur either in the
debris or underneath hive inserts without any signs of damage to the beekeeper (22).
The first sign of an infestation by A. tumida is the occurrence of adult beetles (~5 mm length and ~3 mm width,
females slightly longer than males [10]), with a dark brown to black colour (lighter shortly after eclosion) in the
colony (Fig. 1). During inspections, adults avoid sunlight, hide, and can be observed while running for cover into
corners or in a typical fashion over the combs. Adults can be confused with other beetles from the same family,
which can also be associated with colonies (e.g. Cychramus luteus [20]).
Fig. 1. Aethina tumida adult. Photo by N. Ruppert.
a) Beetle eggs, larvae and pupae

Eggs are white and bean-shaped (~2/3 of the size of a honey bee egg) and oviposited in clutches (up to
210) in cracks, on the bottom board, on the combs and underneath sealed brood (9). Larvae are whitish,
often covered with a slimy sticky coating, up to 1.2 cm long (wandering phase) and have three pairs of legs
and dorsal spikes. Larvae can be found mining in combs (15) or in the debris (23). Larval infestations are
typically associated with a rotten smell (e.g. rotting orange). Wandering larvae often leave smear trails
inside and outside the colony. Such wandering larvae and pupae (whitish, ~5 mm long and 3 mm wide) can
be found in small pupation chambers 1–20 cm deep in the soil usually in close proximity to colonies
(<180 cm, [21]).
b) Colony examination
When monitoring honey bee colonies for the presence of A. tumida, an examination of the hive may provide
an early indication of infestation. Adult beetles can be observed hiding inside cells and in the debris. Colony
examinations start by removing the hive roof and placing it upside down on the ground next to the hive.
Remove the hive chamber, i.e. supers and upper brood chamber (in double brood chamber colonies) and
place them on the upturned roof for a few minutes. Place the hive crown board on top. A few minutes later
lift the boxes out of the way and scan for beetles on the inner surface of the upturned roof. Then frames are
screened one by one for the presence of adults, larvae and eggs. During cool weather, adults tend to stay
close to or within the bee cluster. In warmer periods beetles are found more often on the bottom board or
the outer-most frames.
c) Board examination using traps

Less labour intensive diagnosis is feasible using hive inserts. Such inserts allow for the beetles to hide in
the corrugations but prevent bees from entering. They can be placed on the bottom board. To detect the
beetles, place a piece of corrugated cardboard (15 cm × 15 cm), with one surface peeled to expose the
ridges, on the bottom board of the bee hive with the ridged side down. Cover it with wood to fit underneath
the frames on the bottom board. Leave the insert in the colony for ≤ 3 days, remove it and examine for
adults and larvae. Acaricides can be used to kill adults in these inserts. The debris and all areas to which
bees have no access should also be screened carefully.

No serological tests are available for routine laboratory diagnosis.
3. Treatment
In countries with infestations of A. tumida, control has focused on chemical treatments with acaricides and
insecticides (1, 2, 11). Acaricides, non-toxic to bees, are used in traps intra-colonially (11) to kill the adults.
Similarly, insecticides are used as a ground drench to kill wandering larvae and pupae (1, 2). Such treatments
carry the risks of both pest resistance and residues in the hive products (18). Alternative, more sustainable
controls are moderately efficient so far (3, 13, 14, 18) and currently under investigation (3–5, 13, 14, 16).

REFERENCES
1. BAXTER J.R., ELZEN P.J., W ESTERVELT D., CAUSEY D., RANDALL C., EISCHEN F.A. & W ILSON W.T. (1999). Control
of the small hive beetle in package bees. Am. Bee J., 139, 792–793.
2. BAXTER J.R., ELZEN P.J. & W ILSON W.T. (1999). Gardstar 40% EC (Perimithrin) efficacy trials as a ground
drench for the control of small hive beetle around honey bee colonies. Tektran, USDA Agricultural Research
Service, 1 pp.
3. ELLIS J.D. (2004). The Ecology and Control of Small Hive Beetles. PhD dissertation, Rhodes University,
Grahamstown, South Africa.
4. ELLIS J.D., DELAPLANE K.S., HEPBURN H.R. & ELZEN P.J. (2003). Efficacy of modified hive entrances and a
bottom screen device for controlling Aethina tumida (Coleoptera: Nitidulidae) infestations in Apis mellifera
(Hymenoptera: Apidae) colonies. J. Econ. Entomol., 96, 1647–1652.
5. ELLIS J.D., DELAPLANE K.S. & HOOD W.M. (2001). Small hive beetle (Aethina tumida) weight, gross biometry,
and sex proportion at three locations in the southeastern Untied States. Am. Bee J., 142 (7), 520–522.
6. ELLIS J.D., HEPBURN H.R., DELAPLANE K., NEUMANN P. & ELZEN P.J. (2003). The effects of adult small hive
beetles, Aethina tumida (Coleoptera: Nitidulidae), on nests and flight activity of Cape and European honey
bees (Apis mellifera). Apidologie, 34, 399–408.
7. ELLIS J.D., HEPBURN H.R., LUCKMANN B. & ELZEN P.J. (2004). The effects of soil type, moisture, and density
on pupation success of Aethina tumida (Coleoptera: Nitidulidae). Environ. Entomol., 33, 794–798.
8. ELLIS J.D., PIRK C.W.W., HEPBURN H.R., KASTBERGER G. & ELZEN P.J. (2002). Small hive beetles survive in
honeybee prisons by behavioural mimicry. Naturwissenschaften, 89, 326–328.
9. ELLIS J.D., RICHARDS C.S., HEPBURN H.R. & ELZEN P.J. (2003). Oviposition by small hive beetles elicits
hygienic responses from Cape honeybees. Naturwissenschaften, 90, 532–535.
10. ELLIS J.D., RONG I.H., HILL M.P., HEPBURN H.R. & ELZEN P.J. (2004). The susceptibility of small hive beetle
(Aethina tumida Murray) pupae to fungal pathogens. Am. Bee J., 144 (6), 486–488.
11. ELZEN P.J., BAXTER J.R., W ESTERVELT D., RANDALL C., DELAPLANE K.S., CUTTS L. & W ILSON W.T. (1999). Field
control and biology studies of a new pest species, Aethina tumida Murray (Coleoptera, Nitidulidae) attacking
European honey bees in the Western hemisphere. Apidologie, 30, 361–366.
12. HEPBURN H.R. & RADLOFF S.E. (1998). Honeybees of Africa. Springer Verlag, Berlin, Heidelberg, New York.
13. HOOD M.W. (2004). The small hive beetle, Aethina tumida: a review. Bee World, 85, 51–59.

14. HOOD W.M. & MILLER G.A. (2005). Evaluation of an upper Hive entrance for control of small hive beetles
(Coleoptera: Nitidulidae) in colonies of honey bees (Hymenoptera: Apidae). J. Econ. Entomol., 98, 1791–
1795.
15. LUNDIE A.E. (1940). The small hive beetle Aethina tumida, Science Bulletin 220, Dep. Agr. Forestry,
Government Printer, Pretoria, South Africa.
16. MUERRLE T.M., NEUMANN P., DAMES J.F., HEPBURN H.R. & HILL M.P. (2006). Susceptibility of Adult Small Hive
Beetle to Entomopathogenic Fungi. J. Econ, Entomol., 99, 1–6.
17. MURRAY A. (1867). List of Coleoptera received from Old Calabar. Ann. Magazine Nat. Hist., London, 19,
167–179.
18. NEUMANN P. & ELZEN P.J. (2004). The biology of the small hive beetle (Aethina tumida, Coleoptera:
Nitidulidae): Gaps in our knowledge of an invasive species. Apidologie, 35, 229–247.
19. NEUMANN P. & HÄRTEL S. (2004). Removal of small hive beetle (Aethina tumida) eggs and larvae by African
honeybee colonies (Apis mellifera scutellata). Apidologie, 35, 31–36.
20. NEUMANN P. & RITTER W. (2004). A scientific note on the association of Cychramus luteus (Coleoptera:
Nitidulidae) with honeybee (Apis mellifera) colonies. Apidologie, 35, 665–666.
21. PETTIS J. & SHIMANUKI H. (2000). Observations on the small hive beetle, Aethina tumida, Murray, in the
United States. Am. Bee J., 140, 152–155.
22. SCHMOLKE M.D. (1974). A study of Aethina tumida: the small hive beetle, Project Report, University of
Rhodesia, Zimbabwe, pp. 178.
23. SPIEWOK S. & NEUMANN P. (2006). Cryptic low-level reproduction of small hive beetles in honeybee colonies.
J. Apic. Res., 45, 47–48.
FURTHER READING
*
* *

CHAPTER 2.2.6.
TROPILAELAPS INFESTATION OF HONEY BEES

(Tropilaelaps spp.)
SUMMARY
The mites in the genus Tropilaelaps are parasites of honey bee brood. Feeding on bee larvae and
pupae causes brood malformation, death of bees and subsequent colony decline or absconding.
Development requires about 1 week, and the mites are dispersed on bees. There are at least four
species in the genus Tropilaelaps. Each species is closely associated with a particular giant honey
bee in Asia. Two species (Tropilaelaps clareae and Tropilaelaps mercedesae) are damaging pests
of Apis mellifera. The other two species (Tropilaelaps koenigerum and Tropilaelaps thaii) appear to
be harmless to Apis mellifera (1).
Identification of the agent: Molecular and morphological methods are available for identifying
each species (1). An infestation by Tropilaelaps can be recognised either visually on bees or by
examining hive debris. Irregular brood pattern, dead or malformed immatures, bees with malformed
wings that crawl at the hive’s entrance, and especially the presence of fast-running, large, red-
brown, elongated mites on the combs, are diagnostic for the presence of T. clareae. An early
diagnosis can be made after opening brood cells and finding immature and adult mites therein. The
hive (colony) may be treated with various chemicals that cause the mites to drop off combs and
bees. Sticky boards on the bottom of the colony can be used to examine hive debris and mites.
A. INTRODUCTION
The mite species Tropilaelaps clareae, previously assumed to be ubiquitous in Asia, has been found to be two
species. Tropilaelaps clareae occurs in Asia where it is a parasite of the native honey bee Apis dorsata
breviligula. It is also a parasite of the introduced honey bee species A. mellifera in the Philippines and the native
honey bee species A. dorsata binghami on Sulawesi Island in Indonesia. Tropilaelaps mercedesae, which until
now was mistaken for T. clareae, together with T. koenigerum, parasitises the native A. dorsata dorsata in
mainland Asia and Indonesia (except Sulawesi Island). Tropilaelaps mercedesae is also a parasite of the
introduced A. mellifera in these and surrounding regions and, with another species, T. thaii, also parasitises
A. laboriosa in mountainous Himalayan regions (1).
1. Life cycle
The colonising Tropilaelaps female (or females; as many as a dozen may occur within individual a single cells)
places from one to four eggs on mature bee larvae shortly before the brood cell is capped. The drone brood is
preferred by Tropilaelaps and may be almost 100% parasitised (4). The mite progeny, usually one male and
several females feed on and seriously damage the bee brood. Development of the mite requires about 1 week.
The adults, including the foundress female, emerge with the adult bee and search for new hosts.
The short life-cycle, as well as a very brief stay on adult bees, explains why populations of T. clareae increase
faster than those of Varroa mites. When both T. clareae and Varroa destructor infest the same colony, the former
may out-compete the Varroa mite (4, 13). It has been reported that when both mite species are in the same cell,
the reproduction of both mites declines (12).

Chapter 2.2.6. — Tropilaelaps infestation of honey bees (Tropilaelaps spp.)
Phoretic survival on bees is quite short (only 1–2 days) because Tropilaelaps cannot pierce the integument of
adult bees. The phoretic time for Tropilaelaps spp. is important in understanding the life cycle, and recent
research suggests the period can be as long as 5–10 days (15, 16). Gravid female mites will die within 2 days
unless they deposit their eggs (17).
Infestation by Tropilaelaps causes the death of many bee larvae (up to 50%), resulting in an irregular brood
pattern and of which the cadavers that may partially protrude from the cells. Many malformed bees occur, with
distorted abdomens, stubby wings and deformed or missing legs. Some of the affected bees crawl at the hive’s
entrance (2). In addition, perforated cappings are seen, the result of sanitation activities by the worker bees,
which evict the infested bee pupae or young adults. Some infested colonies abscond, carrying the mites to a new
location.
The first sign of an infestation by Tropilaelaps species is often the occurrence of large, red-brown, elongated
mites on the combs or on adult bees (Figs 1 and 2). Tropilaelaps clareae (<1 mm in length), and T. mercedesae
(< 9 mm in length) differ in body size but otherwise are alike. Tropilaelaps koenigerum is slightly smaller, only
about 0.7 mm in length (5). The females also differ in the structure of their ventral anal plate and subapical tooth
of the chelicerae (1). Tropilaelaps can easily be recognised and separated from the Varroa mite using a
×10 magnifying glass. The body of the Varroa mite is wider than it is long and it moves slowly, whereas the body
of Tropilaelaps is elongated, with a heavily sclerotised holoventral or similar shield (Fig. 3), and it is a fast-
running mite.
Fig. 1. Tropilaelaps clarea. Photo by J. Waddell.
Fig. 2. Tropilaelaps on Apis dorsata larvae. Photo by D. Anderson.

Fig. 3. Tropilaelaps offspring on Apis mellifera pupae. Photo by W. Ritter.
a) Mite collection
Methods to collect mites include an ether or sugar roll (13). Collect approximately 100–200 bees in a wide-
mouthed jar with lid. Scrape the bees into the jar or use a modified vacuum to suck them in. Knock the bees
to the bottom of the jar with a sharp blow; there should be about a 1–2 inch (2.54–5.08 cm) layer of bees on
the bottom. Remove the lid and spray a 2-second burst with ether starter fluid. Alternatively, use enough
70% alcohol or soapy water to cover the bees; or add around 25 g (1 oz) powdered sugar (or flour). If using
ether replace the lid and agitate or roll the jar for about 10 seconds; mites should stick to walls. If using
soap or alcohol, agitate and then strain out the bees with a coarse hardware cloth or mesh strainer; mites
will be in the liquid. If using sugar or other powder, put screening material (such as hardware cloth) on top
of the jar and shake the mites on to white paper to count; repeat every 2 minutes. For a more accurate
count, finish with an alcohol or soapy water wash to collect all the mites.
b) Colony and brood examination

When monitoring honey bee colonies for the presence of Tropilaelaps (or Varroa), an examination of both
drone and worker brood may provide an early indication of infestation. Mites can be observed inside capped
bee brood by using a honey scratcher (with fork-like tines) to pull up capped pupae. The mites are clearly
visible. The younger mite stages are whitish and may be almost motionless while feeding on their hosts’
bodies, as their mouthparts and front legs are fixed to the cuticle of the bee host (13). The extent of
parasitisation can be estimated by opening a predetermined number of brood cells; infestation rates are
then calculated as per cent of capped brood containing live mites (3).
c) Sticky board examination

A precise diagnosis can be made using a sticky board covered with a mesh, such as fly screen, that
prevents the bees from removing the dislodged mites. The mesh must be large enough for mites to pass
through. Make a sticky board with poster board, cardboard or other white, stiff paper coated with Vaseline or
other sticky substance (8, 10, 14), or use a sheet of sticky shelf paper. Cut the paper to fit the bottom board
of a hive. Cut a piece of hardware cloth or screen to fit on top of the sticky board. To keep the bees from
cleaning off the board, fold under the outside edges of the screen to raise it off the board, and staple or tape
in place. Leave the board in the colony for up to 3 days, collecting and examining the debris for mites. For
faster mite diagnosis, smoke each colony adding 25 g (1 oz) pipe tobacco in the smoker. Puff the bees 6–
10 times, close up the hive for 10–20 minutes. Pull out the sticky board after 10 minutes and count the
mites. Acaricides are sometimes used to knock mites off bees and will appear on the sticky boards.
No serological tests are available for diagnosis.
3. Treatment
In countries with infestations of Tropilaelaps spp., fluvalinate in slow-release formulations controls Tropilaelaps
(9, 11), as do monthly dustings with sulphur (2) and treatments with formic acid (6). The inability of this mite to
feed on adult bees, or to survive outside sealed brood for more than a few days, such as caging the queen for a
few weeks, is being used as a non-chemical control method (17, 18).

Many of the same acaricides used for Varroa will kill Tropilaelaps. Strips of plastic-impregnated fluvalinate
(Apistan™) will kill mites. Alternatively, tobacco smoke in the smoker will cause mites to drop off bees. Strips of
filter paper, available in some countries are prepared by soaking in an aqueous solution of 15% potassium nitrate
to which two drops of amitraz (usually 12.5%) are added (9). After the paper dries, the strip is ignited and
inserted into the hive. The smoke causes many mites to drop off. Another method is to use plates or pads
soaked with 20 ml of 65% formic acid (very caustic and will burn hands and face). The pads are placed in the
colonies, near the top (7).

REFERENCES
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mites (Acari: Laelapidae): new and re-defined species. Exp. Appl. Acarol., 43, 1–24.
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Apic. Res., 10, 137–142.
3. BURGETT D.M. & KITPRASERT C. (1990). Evaluation of Apistan™ as a control for Tropilaelaps clareae (Acari:
Laelapidae), an Asian honey bee brood mite parasite. Am. Bee J., 130, 51–53.
4. BURGETT M., AKRATANAKUL P. & MORSE R.A. (1983). Tropilaelaps clareae: a parasite of honeybees in south-
east Asia. Bee World, 64, 25–28.
5. DELFINADO-BAKER M. & BAKER E.W. (1982). A new species of Tropilaelaps parasitic on honey bees. Am. Bee
J., 122, 416–417.
6. GARG R., SHARMA O.P. & DOGRA G.S. (1984). Formic acid: an effective acaricide against Tropilaelaps clareae
Delfinado & Baker (Laelapidae: Acarina) and its effect on the brood and longevity of honey bees. Am. Bee
J., 124, 736–738.
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Tropilaelaps clareae with formic acid. Am. Bee J., 129, 739–742.
8. KOENIGER G., KOENIGER N., ANDERSON D.L., LEKPRAYOON C. & TINGEK S. (2002). Mites from debris and
sealed brood cells of Apis dorsata colonies in Sabah, (Borneo) Malaysia, including a new haplotype of
Varroa jacobsoni. Apidologie, 33, 15–24.
9. LUBINEVSKI Y., STERN Y., SLABEZKI Y., LENSKI Y. ,BEN YOSSEF H. & GERSON U. (1988). Control of Varroa
jacobsoni and Tropilaelaps clareae mites using Mavrik under subtropical and tropical climates. Am. Bee J.,
128, 48–52.
10. OSTIGUY N. & SAMMATARO D. (2000). A simplified technique for counting Varroa sticky boards. Apidologie,
31, 707–716.
11. PONGTHEP A. (1990). Bee Mites. FAO Agricultural Services Bulletin 68/4. Food and Agriculture Organization
of the United Nations, Rome, Italy.
12. RATH W., BOECKING O. & DRESCHER W. (1995). The phenomena of simultaneous infestation of Apis meliferea
in Asia with the parasitic mites Varroa jacobsoni OUD, and Tropilaelaps clareae Delfinado and Barker. Am.
Bee J., 135, 125–127.
13. RITTER W. & SCHNEIDER-RITTER U. (1988). Differences in biology and means of controlling Varroa jacobsoni
and Tropilaelaps clareae, two novel parasitic mites of Apis mellifera. In: Africanized Honey Bees and Bee
Mites, Needham G.R., Page R.E. Jr., Delfinado-Baker M.& Bowman C.E., eds. Ellis Horwood, Chichester,
UK, 387–395.
14. SAMMATARO D., GERSON U. & NEEDHAM G.R. (2000). Parasitic mites of honey bees: life history, implication s
and impact. Ann. Rev. Entomol., 45, 519–548.

15. W ILDE J. (2000). How long can Tropilaelaps clareae survive on adult honeybee workers? In: Proceedings of
the Euroconference on Molecular Mechanisms of Disease Tolerance in Honeybees (MOMEDITO), held in
Kralupy near Prague, Czech Republic, 17–19 October 2000. Bee Research Institute, Dol, Czech Republic,
217–221.
16. W ILDE J. (2000). Varroa destructor and Tropilaelaps clareae in Apis meliferea colonies in Nepal. In:
Proceedings of the Euroconference on Molecular Mechanisms of Disease Tolerance in Honeybees
(MOMEDITO), held in Kralupy near Prague, Czech Republic, 17–19 October 2000. Bee Research Institute,
Dol, Czech Republic, 223–238.
17. W OYKE J. (1987). Length of stay of the parasitic mite Tropilaelaps clareae outside sealed honeybee brood
cells as a basis for its effective control. J. Apic. Res., 26, 104–109.
18. W OYKE J. (1993). Practical control method of the parasitic bee mite Tropilaelaps clareae. Am. Bee J., 133,
510–511.
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CHAPTER 2.2.7.
VARROOSIS OF HONEY BEES
SUMMARY
The mite Varroa destructor (formerly Varroa jacobsoni) is a parasite of adult bees and their brood.
It penetrates the intersegmental skin between the abdominal sclera of adult bees to ingest
haemolymph. It can sometimes be found between the head and thorax. The number of parasites
steadily increases with increasing brood activity and the growth of the bee population, especially
late in the season when clinical signs of infestation can first be recognised. The life span of the
mite depends on temperature and humidity but, in practice, it can be said to last from some days to
a few months.
Identification of the agent: The clinical signs of varroosis can only be recognised at a late stage
of infestation, so that diagnosis entails the examination of the hive debris. The debris produced
during the summer is especially useful for diagnosis. The earliest and most precise diagnosis can
be made only after the application of a medication that forces the mites to drop off the bees or kills
them directly. Larger amounts of debris can be examined using a flotation procedure. Bees are
washed in petroleum spirit, alcohol or detergent solution. However, this method is less accurate
due to the unequal distribution of mites and the usually small sample sizes.
A. INTRODUCTION
The Varroa mites are parasites of adult bees and their brood. Four species have been recorded: Varroa
jacobsoni, V. destructor, V. underwoodi and V. rinderi. Until recently Varroa mites that affect Apis mellifera world-
wide were assumed to be V. jacobsoni. However it has been shown that these mites are V. destructor (Figure 1).
Fig. 1. Varroa on pupa and adult bee. Left: pupa with four Varroa female mites.
Right: worker bee with two female mites.
They are responsible for the condition of varroosis or varroatosis (1, 2). The mite inserts itself between the
abdominal sclera in adult bees (10) where it penetrates the intersegmental membranes in order to ingest
haemolymph. Sometimes it can also be found between the head and thorax. For reproduction, the female enters
the cells with the bee brood shortly before the cells are sealed. They prefer drone brood to worker brood. After
the brood cell is sealed, the mite lays after 2 to 3 days the first egg (generally male). Later up to seven eggs
(generally females) are laid in intervals of about 1–2 days. These hatch into nymphs, but only two to three reach
the adult stage (Figs 2 and 3).

Chapter 2.2.7. — Varroosis of honey bees
Fig. 2. Oviposition and development of Varroa in brood cells of worker bee (until about 9th day
unsealed brood, until about 21st day sealed brood).
Fig. 3. Development of Varroa: E = Egg, L = Larva, P = Protonymph, D = Deutonymph, A = Adult (Sex of eggs,
larvae and protonymphs can only be distinguished by examining the chromosomes).

The number of mites usually increases slowly at the beginning of the season. Clinical signs may be seen at any
time during the active season, although usually maximum numbers are reached late in the season (Figure 4),
when the first clinical signs of infestation can be recognised. The course of this parasitism is usually lethal,
except in some areas, such as tropical Latin America (6, 12). The life span of mites on larval or adult bees
depends on temperature and humidity. Under practical conditions, the life span may vary from some days to a
few months.
Fig. 4. Graph of populations of bees and mites over 1 year in a temperate Northern Hemisphere climate:
brood numbers (solid line); mite numbers (broken line).
In heavily infested bee colonies, clinical signs of varroosis can often first be seen in the latter part of the season
when the brood is reduced (12). Heavy infestations are usually reached 3–4 years after the primary invasion, but
can occur within weeks if infested by bees from nearby colonies that are collapsing.
Essentially, the brood is damaged by the parasitic mites. Bees and their offspring that have been infected during
the brood phase by only one parasitic mite show various ill effects, such as a shortened life span, changes in
behaviour and an increased disease susceptibility (8). The parasitism is critical if more than one mite enters the
brood cell for reproduction. Only in the lethal stage immediately before the collapse of the colonies do clinical
signs, such as shrunken wings and shortened abdomen, appear (Figure 5). This is due to an increased
susceptibility to deformed wing and acute paralysis virus, as well as to the infection of wounds and loss of
haemolymph (3, 4). If the brood dies shortly before or after sealing, clinical signs of European foulbrood appear
without the presence of the specific agent Melissococcus pluton. If the brood survives, the emerging bees show
various behavioural changes and their life span is considerably shortened (7, 11).
Fig. 5. Effect of Varroa on bee morphology. Left: normal bee appearance. Right: bee heavily attacked by mites. This
newly emerged bee has a deformed wing and reduced abdominal volume.
The female mite is a dark reddish/brown colour and has a flat, oval-shaped body approximately 1.1 mm ×
1.5 mm. It is the only common parasite of honey bees that can be seen with the naked eye (13).

a) Debris examination
An easy method of diagnosis of varroosis is by the examination of the debris generated by bees
themselves. An insert covered with a screen mesh is placed on the floor of the hive. Unless this insert is
covered with such a gauze, or smeared with grease, the bees will dispose of the mites outside the hive.
The debris produced within a few days in the late season usually contains little other than visible mites (9,
11). The debris collected in winter, however, must be examined in the laboratory. An insert is placed in the
hive as before, but an effective medication is used to cause the mites to fall off the bees, so that after a
given time, a number of mites may be observed on the floor insert. Some countries demand the diagnostic
application of certain medications for proving the absence of mites.
Large amounts of debris can be examined in the laboratory using a flotation procedure (5).
• Test procedure
i) Dry the debris for 24 hours.
ii) Flood the debris with industrial alcohol.
iii) Stir continuously for around 1 minute or, if debris contains wax or propolis particles, stir for 10–
20 minutes.
iv) Identify and observe the mites that float to the surface.
b) Brood examination
For the second method, drone brood is examined, if available, otherwise worker brood is examined.
When a large number of samples are examined, a rough determination of the degree of infection can be
obtained.
• Test procedure
i) Remove the cappings of the brood cells with a knife.
ii) Wash the brood cells directly into a sieve system with warm water from a hand-held shower.
iii) Collect the mites in the lower fine sieve (mesh width 1 mm) while the brood is gathered in the upper
coarse sieve (mesh width 2–3 mm).
iv) Place the contents of the sieve on a bright plate, where the mites can be easily identified and counted.
When a smaller number of samples are being studied, the individual cells are examined using an
appropriate source of light. After removing the cappings and the bee brood, infected cells can be identified
by the presence of small white spots – the faeces of the mite – found on the cell wall. The mites themselves
should be sought for confirmation, by examining the bottom of the cell and the bee brood for attached mites.
c) Bee examination
In a third method, approximately 200–250 bees are removed from unsealed brood combs. Samples should
be taken from both sides of at lest three uncapped brood combs. To determine an apiary’s percentage of
infestation, it is necessary to collect and analyse individual samples from at least 10% of the beehives, and
to determine later the average infestation rate based on these individual results.
• Test procedure
i) Kill the bees in a special container by submersion in alcohol.
ii) Stir the container for 10 minutes.
iii) Separate the bees from the mites by means of a sieve with a mesh size of approximately 2–3 mm.
Under some circumstances, the Varroa mite may be confused with the bee louse, Braula coeca (Figure 6).
The latter is round, not oval, and being an insect, has only three pairs of legs. A number of different species
of mite may be associated with Varroa mites on bees, but these are easily distinguished. In addition, other
parasitic mites, such as those of the Tropilaelaps spp., are known to cause similar damage to bee colonies
as the Varroa mites.
No serological tests are available for routine laboratory diagnosis.

Fig. 6. Diagram of Varroa destructor (formerly Varroa jacobsoni Oudemans) (female).

a) Dorsal aspect ⎫
b) Anterior aspect ⎬ Note the flat shell-like back and four pairs of legs.
c) Ventral aspect ⎭
d) The bee louse (Braula coeca, female). Note the lack of shell-like back and only three pairs of legs.

There are no biological products and vaccines available. Several medicaments and substances like Formic acid,
oxalic acid, lactic acid and thymol can be used to control Varroa mites
(http://www.apis.admin.ch/english/Themes/Varroa.htm). Some hygienic honeybee strains are less susceptible to
Varroa parasites.
• Acknowledgement
Illustrations by Karl Weiss, extracted from Bienen-Pathologie, 1984. Reproduced with the kind permission of the
author and Ehrenwirth-Verlag, Munich (Germany).
REFERENCES
1. ANDERSON D.L. (2000). Variation in the parasitic bee mite Varroa jacobsoni Oud. Apidologie, 31, 281–292.
2. ANDERSON D.L. & TRUEMAN J.W.H. (2000). Varroa jacobsoni (Acari: Varroidae) is more than one species.
Exp. Appl. Acarol., 24, 165–189.
3. BAILEY L. (1981). Honey Bee Pathology. Academic Press, London, UK.
4. BALL B.V. (1985). Acute paralysis virus isolated from honey bee colonies infested with Varroa jacobsoni.
J. Apic. Res., 24, 115–119.
5. BREM S. (1980). Laboruntersuchungen von Wintergemull. In: Diagnose und Therapie der Varroatose.
Apimondia Publishing House, Bucharest, Romania, 116–117.
6. DE JONG D. (1997). Varroa and other parasites of brood. In: Pests, Predators and Diseases of Honey Bees,
Third Edition, Morse R.A., ed. A. I. Root, Medina, Ohio, USA, 231–279.
7. DE JONG D. & DE JONG P.H. (1983). Longevity of Africanized honey bees (Hymenoptera Apidae) infested by
Varroa jacobsoni (Parasitiformes Varroidae). J. Econ. Entomol., 76, 766–768.
8. DE JONG P.H. & GONCALVES L.S. (1982). Weight loss and other damage to developing worker honey bees
from infestation with Varroa jacobsoni. J. Apic. Res., 21, 165–167.

9. FRIES I., CAMAZINE S. & SNEYD J. (1994). Population dynamics of Varroa jacobsoni: a model and a review.
Bee World, 75, 4–28.
10. RITTER W. (1980). Varroatosis: A new disease of honey bee Apis mellifera. Bee World, 6, 141–153.
11. RITTER W. (1996). Diagnostik und Bekämpfung der Bienenkrankheiten (Diagnosis and control of bee
diseases). Gustav Fischer Verlag, Jena, Stuttgart, Germany.
12. RITTER W., LECLERQ E. & KOCH W. (1984). Observations des populations d’abeilles et de Varroa dans les
colonies à différents niveaux d’infestation. Apidologie, 14, 389–400.
13. SHIMANUKI H. & KNOX D.A. (1991). United States Department of Agriculture (USDA) Handbook No. 690. 53p.
*
* *

SECTION 2.3.
AVES
CHAPTER 2.3.1.
AVIAN CHLAMYDIOSIS
SUMMARY
Avian chlamydiosis (AC) is caused by the bacterium Chlamydophila psittaci. AC occurring in

humans and all birds was originally called psittacosis, but later the term ornithosis was introduced
to identify the disease contracted from or occurring in domestic and wildfowl, while the name of the
disease contracted from or occurring in psittacine birds remained psittacosis. These diseases are
similar when contracted by humans. The genus Chlamydia was recently divided into two genera,
Chlamydia and Chlamydophila. All known avian strains are now in the species Chlamydophila
psittaci. Chlamydiosis is still the term used for diseases produced by both genera. The avian
strains include at least six serotypes that correlate with the avian species from which they are
usually isolated. Chlamydiosis as it occurs naturally in mammalian species and not contracted from
avian species is caused by distinctly different strains of the organism.
Depending on the chlamydial serovar and the avian host, chlamydiae cause pericarditis,
conjunctivitis, sinusitis, airsacculitis, pneumonia, lateral nasal adenitis, peritonitis, hepatitis, and
splenitis. Generalised infections result in fever, anorexia, lethargy, diarrhoea, and occasionally
shock and death. Special laboratory handling is recommended because avian chlamydial strains
can cause serious illness and possibly death in humans. The disease in ducks and turkeys is of
particular concern as transmission to humans is common during handling and slaughter of the
birds. The diagnosis of AC requires the isolation and identification of the organism, the
demonstration of chlamydiae in tissues, or the demonstration of a four-fold increase in specific
humoral antibody as well as typical clinical signs.
Identification of the agent: Isolation of chlamydiae requires the inoculation of embryonated eggs
or cell cultures and testing for chlamydiae by cytochemical stains or immunohistochemical
methods. The direct inoculation of samples into cell cultures is preferable as they are as sensitive
for the isolation of most avian strains of chlamydiae as are chicken embryos. The cell cultures are
then stained by direct immunofluorescence or by other appropriate stains at appropriate times to
demonstrate the presence of inclusions.
Histochemical staining of impression smears from the liver, heart, and spleen are commonly made.
The technique gives a rapid diagnosis, but requires some experience.
Enzyme-linked immunosorbent assays (ELISAs) developed for detecting trachomatis antigen in

humans have been used for diagnosing chlamydiae in birds. Many of the earlier tests were
developed using monoclonal or polyclonal antisera against lipopolysaccharide epitopes, some of
which were shared with other Gram-negative bacteria. Their use when screening individual birds is
questionable, as they lack sensitivity and specificity.
Molecular tools (polymerase chain reaction restriction length polymorphism, DNA microarray or
sequencing) and immunohistochemical staining of histological sections are new techniques
showing promise for the future. All of them are rapid and do not require the live agent. The current
PCR tests target the MOMP gene or the ribosomal RNA genes (16S–23S), and some will amplify
all chlamydial strains and allow identification at the level of the chlamydial species. Nested and

Chapter 2.3.1. — Avian chlamydiosis
real-time PCRs can be as sensitive as isolation. There has been an increase in the use of
immunohistochemical staining of histological sections because of the recent development and
availability of automated staining equipment.
Serological tests: The standard serological test for chlamydial antibodies is the complement
fixation (CF) test. The modified direct CF test can be used with most sera. The antigen is a group-
reactive lipopolysaccharide antigen present in all strains. The occurrence of high CF titres in the
majority of individuals in a flock with clinical signs is presumptive evidence of active infection. The
demonstration of a four-fold increase in titre in an individual bird is considered to be diagnostic of a
current infection.
Other serological tests, such as the ELISA, latex agglutination, elementary body agglutination,
micro-immunofluorescence, and the agar gel immunodiffusion tests are available. These tests are
of value in specific cases and may replace the CF test; however, comparisons of reliability and
reproducibility are not yet available.
Requirements for vaccines and diagnostic biologicals: There are no commercial vaccines
available for chlamydiosis control in poultry. Antibiotics are the only current means of control.
Chlamydophila psittaci is susceptible to a number of antibiotics. The drug of choice varies from
country to country.
A. INTRODUCTION
Avian chlamydiosis (AC) is caused by the bacterium Chlamydophila psittaci. The disease in birds was originally
called psittacosis, but the term ornithosis was introduced later to differentiate the disease in domestic and wild
fowl from the disease in psittacine birds. The two syndromes are currently considered to be the same (5). Their
earlier separation was based on the assumption that in humans, ornithosis was a milder disease than psittacosis.
However, it should be noted that the disease in humans contracted from turkeys and ducks is often as severe as
that contracted from psittacine birds.
Chlamydophila psittaci produces a systemic and occasionally fatal disease in birds. The clinical signs vary
greatly in severity and depend on the species and age of the bird and the strain of chlamydia. AC can produce
lethargy, hyperthermia, abnormal excretions, nasal and eye discharges, and reduced egg production. Mortality
rates will vary greatly. In pet birds the most frequent clinical signs are conjunctivitis, anorexia and weight loss,
diarrhoea, yellowish droppings, sinusitis, biliverdinuria, nasal discharge, sneezing, lacrimation and respiratory
distress (24). Many birds, especially older psittacine birds, may show no clinical signs; nevertheless, they may
often shed the agent for extended periods. Necropsy of affected birds will often reveal multifocal hepatic
necrosis, spleen and liver enlargement, fibrinous airsacculitis, pericarditis and peritonitis (5, 39, 40). Histological
lesions are suggestive of infection but are non-pathognomonic unless there are identifiable chlamydiae present.
The family Chlamydiaceae was recently reclassified into two genera and nine species based on sequence
analysis of its 16S and 23S rRNA genes (13). The two new genera, Chlamydia and Chlamydophila, correlate with
the former species Chlamydia trachomatis and C. psittaci. The genus Chlamydia includes C. trachomatis
(human), C. suis (swine), and C. muridarum (mouse, hamster). The genus Chlamydophila includes C. psittaci
(avian), C. felis (cats), C. abortus (sheep, goats, cattle), C. caviae (guinea-pigs), and the former species
C. pecorum (sheep, cattle) and C. pneumonia (human).
The two genera and nine species have merit both molecularly and for classification of host range and clinical
disease. The species show a high degree of correlation with host range, disease syndrome, and virulence, and
thus provide an understanding of the epidemiology of the various species and serovars affecting livestock and
birds. The terms ‘chlamydiosis’ and ‘chlamydia(e)’ are used as generic terms to refer to members of either and
both genera. However, the new scientific names are used when referring to a specific chlamydial species.
The avian strains all belong to the species Chlamydophila psittaci. This species includes six known avian
serovars and two mammalian serovars, M56 from muskrats and WC from cattle (13). M56 and WC were each
isolated from a single outbreak. The six avian serovars are labelled A through F and each shows host specificity.
The hosts that each serovar has been associated with are: A, psittacine birds; B, pigeons; C, ducks and geese;
D, turkeys; E, pigeons and ratites; and F is a single isolate from a psittacine bird. What is not known is how many
of these birds and mammals are the natural hosts of the serovars.
The strains of avian chlamydiae can infect humans and should be handled carefully under conditions of
biocontainment (31). Most infections occur through inhalation of infectious aerosols. Post-mortem examinations
of infected birds and handling of cultures should be done in laminar flow hoods or with proper protective

equipment. Human infection can result from transient exposures. The incubation period is usually 5–14 days;
however, longer incubation periods are known. Human infections vary from inapparent to severe systemic
disease with interstitial pneumonia and encephalitis. The disease is rarely fatal in properly treated patients;
therefore, awareness of the danger and early diagnosis are important. Infected humans typically develop
headache, chills, malaise and myalgia, with or without signs of respiratory involvement. Pulmonary involvement is
common; auscultatory findings, however, may appear to be normal or to underestimate the extent of involvement.
Diagnosis can be difficult and is usually established through testing paired sera for antibodies to chlamydia by
the complement fixation (CF) test. In humans, tetracycline, doxycycline, or azithromycin are usually the drugs of
choice unless contraindicated. The length of treatment will vary with the drug, but should be continued for at least
14 days for tetracycline.
The preferred method for the identification of AC is the isolation and identification of the organism. Because of
the time involved, the need for high quality samples, and the hazard to laboratory personnel, other techniques
are often used. These include histochemical staining of smears of exudate and faeces, and impression smears
of tissues, immunohistochemical staining of cytological and histological preparations, antigen-capture enzyme-
linked immunosorbent assays (ELISA), polymerase chain reaction (PCR), PCR-RFLP (restriction fragment length
polymorphism), DNA microarray based detection and identification systems or sequencing.
a) Collection and treatment of samples

The samples to be collected will depend on the disease signs in evidence. They must be taken aseptically.
Contaminant bacteria may interfere with the isolation of the chlamydiae. Specimens from acute cases
should include inflammatory or fibrinous exudate in or around organs that display lesions, ocular and nasal
exudates, impression smears of liver, whole blood and tissue samples from kidney, lung, pericardium,
spleen, and liver. In cases with diarrhoea, colon contents or excrement should be cultured. In live birds, the
preferred samples are pharyngeal and nasal swabs (2). Intestinal excrement, cloacal swabs, conjunctival
scrapings, and peritoneal exudate can also be taken.
Proper handling of clinical samples is necessary to prevent loss of infectivity of chlamydiae during shipping
and storage. A special medium consisting of sucrose/phosphate/glutamate (SPG) was developed for
rickettsiae and has proven to be satisfactory for transport of chlamydial field samples. The medium as
recommended for chlamydiae (32) consists of SPG buffer: sucrose (74.6 g/litre); KH2PO4 (0.512 g/litre);
K2HPO4 (1.237 g/litre); and L-glutamic acid (0.721 g/litre), which can be sterilised by autoclaving or filtering.
Added to this are fetal calf serum (10%), vancomycin, kanamycin, and streptomycin (200–500 µg/ml),
amphotericin B and gentamicin (50 µg/ml). The addition of antibiotics reduces the effect of contamination,
even when samples are shipped at ambient temperatures. This medium can also be used as a laboratory
diluent and for freezing of chlamydiae.
Contaminated samples must be pre-treated before being used to inoculate animals or cell cultures. There
are three basic methods: treatment with antibiotics (7, 8), treatment with antibiotics together with low-speed
centrifugation (4, 5), and treatment with antibiotics with filtration (4, 7, 8). A number of antibiotics that do not
inhibit chlamydia can be used. Samples are homogenised in phosphate buffered saline (PBS), pH 7.2,
containing a maximum of the following: streptomycin (1 mg/ml), vancomycin (1 mg/ml), and kanamycin
(1 mg/ml). Gentamicin (200 µg/ml) can be used. Amphotericin B (50 µg/ml) can be added to control yeast
and fungal growth. Other antibiotic solutions are often used. Penicillin, tetracycline and chloramphenicol
should be avoided as these inhibit the growth of chlamydiae.
When contamination is light, samples should be homogenised in an antibiotic solution prior to inoculation
into chicken embryos or tissue cultures. Samples are often left to stand in the antibiotic solution for 24 hours
at 5°C before inoculation. Heavily contaminated samples, such as faecal samples, should be homogenised
in antibiotics and then centrifuged at 500 g for 20 minutes. The surface layer and the bottom layer are
discarded. The supernatant fluid is collected and recentrifuged. The final supernatant fluid is used for
inoculation. Samples should be passed through a filter of 450–800 µm average pore size if contamination
persists.
b) Isolation in cell culture

Cell cultures are the most convenient method for the isolation of C. psittaci. Cell lines are satisfactory, the
more common ones being buffalo green monkey (BGM), McCoy, HeLa, African green monkey kidney
(Vero), and L cells (38). The cells are grown as monolayers using standard tissue culture media containing
5–10% fetal calf serum and antibiotics that are not inhibitory to chlamydia (as described previously).

When selecting cell culture equipment, it is important to remember that:
i) Chlamydiae can be identified by direct or indirect immunofluorescence or some other appropriate

staining technique;
ii) The inoculum is usually centrifuged on to the monolayer to enhance its infectivity;
iii) The sample may need to be blind passaged at 4–5 days to increase sensitivity of isolation;
iv) The sample will need to be examined from two to three times during any one passage; and
v) Chlamydia can be infectious to humans.
Small flat-bottomed vials, such as 1-dram (3.7 ml, 15 × 45 mm) shell vials or bottles containing cover-slips
that are 12 mm in diameter, will meet these requirements (7, 8). A number of vials, often four to six, are
inoculated with each sample to permit fixing and staining at various intervals, and to permit repassaging of
apparently negative samples 6 days after inoculation. When testing multiple samples, 96-well multiwell
dishes can also be used as they have a labour-saving advantage. However, it should be noted that cross-
contamination between samples can be a problem.
Chlamydiae can be isolated from cells that are replicating normally, but the use of nonreplicating cells is
preferable as these may provide increased nutrients for the growth of chlamydiae. Suppressed cells can
also be observed for longer periods. Host cell division can be suppressed either by irradiation or, more
commonly, by cytotoxic chemicals. The latter include 5-iodo-2-deoxyuridine, cyto-cholasin B, cycloheximide,
and emetine hydrochloride (27). Cycloheximide is the most commonly used and can be added to the
medium at the rate of 0.5–2.0 µg/ml at the time of inoculation of the monolayer (4, 5, 7, 8). Emetine is
removed after treatment and replaced by medium. The monolayer is first treated for 5 minutes with emetine
(0.5 µg/ml), after which the emetine is removed and replaced with culture medium; the monolayer is then
ready for use. The growth of most chlamydial strains will be enhanced by the treatment of the monolayer by
one of these drugs; the treatment will have little or no effect on the growth of other strains.
Attachment of chlamydia to cells is increased by centrifuging the inoculum on to the monolayer at 500–
1500 g for 30–90 minutes at 37°C. The inoculum is removed and replaced with tissue culture medium
containing a cell-division inhibitor, and then incubated at 37–39°C. Cultures must be examined for
chlamydiae at regular intervals using an appropriate staining method. This is usually done on day 2 or 3, as
well as on day 4 or 5. Cultures that appear to be negative at the fifth day are harvested and repassaged.
When repassaging chlamydiae, cells and culture media should be passaged without using freeze–thawing
to disrupt cells, as this will destroy the chlamydiae.
Before staining the cultures, the medium is first removed, the cultures are washed with PBS and fixed with
acetone for 2–10 minutes. The fixation time will depend on the tissue culture vessel used. As acetone will
soften most plastics, the use of a mixture of 50% acetone and 50% methyl alcohol may be preferable. A
number of staining methods can be employed to demonstrate chlamydial inclusions. The preferred method
is direct immunofluorescence (4, 7, 25). A chlamydial fluorescein-conjugated antiserum is applied to the
infected cells and incubated in a humid chamber for 30 minutes at 37°C. The cover-slips are then washed
three times with PBS, air-dried, mounted, and examined. Chlamydial inclusions fluoresce a green colour.
Commercial conjugate preparations using monoclonal antibodies (MAbs) are available and are highly
specific. Conjugates may also be prepared from polyclonal sera, but it is important to obtain specific, high-
titred antisera. Polyclonal antisera can be prepared in rabbits, guinea-pigs, sheep or goats. Sheep and
goats are excellent sources because of the volume and high titres that are readily obtained following
infection. Conjugates are then prepared using standard techniques (4, 5, 7).
Chlamydial inclusions can also be demonstrated by indirect fluorescent antibody and immunoperoxidase
techniques (4, 6, 25). Direct staining can be done with Gimenez, Giemsa, Ziehl–Neelsen, or Macchiavello’s
stains. Except for immunofluorescence, all these stains have the advantage that standard light microscopes
can be used.
c) Isolation in eggs
Chicken embryos are still used for the primary isolation of chlamydiae. The standard procedure is to inject
up to 0.5 ml of inoculum into the yolk sac of a specific pathogen free 6–7-day-old embryo (4, 5). The eggs
are then incubated in a humid atmosphere at 39°C, rather than at 37°C, as multiplication of chlamydia is
greatly increased at the higher temperature. Replication of the organism usually causes the death of the
embryo within 3–10 days. If no deaths occur, two additional blind passages are usually made before
designating any sample as negative. Chlamydial infections will give rise to a typical vascular congestion of
the yolk sac membranes. These are harvested and homogenised as a 20% (w/v) suspension in SPG buffer,
and can be frozen to preserve the strain, or inoculated into eggs or on to cell cultures.
The organism can be identified by preparing an antigen from an infected yolk sac and testing it by direct
staining of smears using appropriate stains or by using the antigen in a serological test. Cell culture
monolayers can be inoculated with the yolk sac suspension and examined by direct immunofluorescence

48–72 hours later for the presence of chlamydial inclusions. Typical inclusions are intracytoplasmic round or
hat-shaped bodies. With some virulent strains, the inclusions rapidly break up and the chlamydial antigen is
dispersed throughout the cytoplasm.
d) Differentiating among species/strains

All avian isolates are in the Chlamydophila psittaci group, as discussed earlier (13). The avian strains can
be differentiated from other chlamydiae by PCR-RFLP of either the MOMP gene or the 16S–23S rDNA
operon (12). A DNA microarray technology was recently developed in order to differentiate chlamydiae
strains (29). A provisional C. psittaci determination can be made using the source of the isolate and
serovar-specific MAbs.
The avian strains of C. psittaci contain at least six serotypes determined by serovar-specific MAbs (1, 3, 6).
The syndromes caused by the various strains are quite specific; the natural host range of a particular strain
may also be fairly specific. Serotypes are labelled A through F. The hosts from which they are mainly
isolated are: serotype A, psittacine birds; serotype B, pigeons; serotype C, ducks; serotype D, turkeys;
serotype E, pigeons and ratites; and one isolate of serotype F from a psittacine bird.
Avian strains could be differentiated by molecular tools like PCR-RFLP (3, 30, 37). Serotyping and PCR-
RFLP have been compared (33, 35) and sometimes incongruent results have been observed. A new
genotype, named E/B, was recently identified; sequencing clearly demonstrated that new genotypes cannot
always be discovered by PCR-RFLAP or serotyping.
e) Histochemical staining
Giemsa, Gimenez, Ziehl–Neelsen and Macchiavello’s stains are commonly used to detect chlamydiae in
impression smears of liver and spleen. The following modified Giminez technique is used by several
laboratories (4):
Modified Gimenez technique or (Pierce-van der Kamp) stain

Reagents:
Solution 1: Distilled H2O (450.0 ml) and phenol (5.0 ml) added to basic fuchsin (2.5 g) and 95%
ethanol (50.0 ml). Incubate at 37°C for 48 hours. Filter and store in the dark at room temperature.
Solution 2: Na2HPO4 (11.65 g); Na2HPO4.H2O (2.47 g); distilled H2O, pH 7.5 (to 1.0 litre).
Solution 3: Solution 1 (20.0 ml); and solution 2 (25.0 ml). Let stand for 10 minutes, filter and use.
Solution 4: 0.5% citric acid.
Solution 5: Fast green (0.2 g); distilled H2O (100.0 ml); and glacial acetic acid (0.2 ml).
Solution 6: Solution 5 (20.0 ml); and distilled H2O (50.0 ml).
Procedure for smears is as follows:

i) Fix in methanol for 5 minutes.
ii) Stain in Solution 3 for 10 minutes and rinse in tap water.
iii) Counterstain in Solution 6 for 2 minutes.
iv) Rinse in tap water and air-dry.
Procedure for paraffin sections is as follows:

i) Deparaffinise and hydrate with distilled H2O.
ii) Stain in Solution 3 for 10 minutes and rinse in tap water.
iii) Dip in Solution 4 until no more red runs out of the section. Rinse in tap water.
iv) Counterstain in Solution 6 for 20 dips.
v) Dip in two changes of 95% alcohol, for five dips each. Dehydrate, clear, and mount.
Chlamydiae will appear red against a green background.
f) Immunohistochemical staining
Immunohistochemical staining can be used to detect chlamydiae in cytological and histological
preparations. The technique is more sensitive than histochemical staining, but some experience is
necessary as cross-reactions with some bacteria and fungi require that morphology must be considered.

Most widely used immunohistochemical staining procedures can be adapted to give satisfactory results.
The selection of the primary antibody is very important. Both polyclonal and monoclonal antibodies have
been used. Because formalin affects chlamydial antigens, it is recommended that polyclonal antibodies be
made to purified formalin-inactivated chlamydiae. The chlamydial strain used is not important, as the
antibodies will be mainly to the group-reactive antigens. MAbs should also be selected for reactions to
formalin-fixed chlamydia. A pool of group-reactive MAbs can be used.
g) Enzyme-linked immunosorbent assays

The ELISA is a relatively new technique that has been extensively promoted as kits for use in the diagnosis
of human chlamydiosis. These test kits detect the lipopolysaccharide (LPS) antigen (group reactive) and will
detect all species of chlamydiae. A number of these kits have been tested for use in detecting chlamydia in
birds (41), but none of the kits has been licensed for detection of C. psittaci. One problem with some of
these tests is that the chlamydial LPS shares some epitopes with other Gram-negative bacteria, and these
epitopes can cross-react, resulting in a high number of false-positive results. This problem has been
reduced or eliminated in more recently developed kits by careful selection of the MAbs used. These kits,
however, still lack sensitivity because a few hundred organisms are still needed to give a positive reaction.
Most diagnosticians believe that a diagnosis of AC can be made when a strong positive ELISA reaction is
obtained from birds with signs of psittacosis. Because of the number of false-positive results, a positive in
an individual bird without signs of disease is not considered to be significant, but indicates the need for
further testing using different methods.
h) Polymerase chain reaction and PCR-based systems

PCR techniques are replacing isolation for the detection of chlamydiae in animals. The sensitivity and
specificity can equal or exceed isolation and the sample can be inactivated prior to testing. Current PCR
tests for detection of C. psittaci target the MOMP gene or the 16S–23S rRNA gene (14–16, 22, 23, 28, 34,
36). The sensitivity and specificity varies on sample preparation and the PCR test. Reagents designed to
stabilise the DNA should be considered when a delay in processing the sample is anticipated (11). DNA
samples can be prepared using inexpensive reagents or using commercially available kits (4). Sensitivity is
increased by targeting a relatively short DNA segment, using a nested procedure or using real-time PCR
techniques. The nested procedure can equal isolation in sensitivity and specificity. However, the risk of
contamination is increased if extreme care is not taken when manipulating the reactions (see Chapter 1.1.5
Validation and quality control of polymerase chain reaction methods used for the diagnosis of infectious
diseases). Some excellent nested procedures are available (23, 28, 36). The real-time PCR requires a
labelled probe and special equipment, which increases costs. The sensitivity can approach that of the
nested system and the contamination problems and labour are reduced as it uses one reaction in a closed
system (14–16). Targeting the 16S–23S gene also increases sensitivity as multiple copies are usually
present in the organism; however, cross-reactions with other bacteria can be a problem. Sequencing of
PCR products will allow comparison with the sequences of reference avian chlamydia isolates and the
sequence can be used in phylogenic analysis for classification and epidemiological purposes.
DNA microarray technology was recently developed in order to detect chlamydiae (29). The assay has also
proved suitable for unambiguous species identification of Chlamydia in cell cultures and has demonstrated
a potential for direct detection of these bacteria from clinical tissues.
a) Modified direct complement fixation test for Chlamydia

The following is a widely used modified direct CF test for the detection of antibody. The reagents are
relatively easy to prepare and standardise. There are other CF tests; each has advantages. The modified
direct CF test is performed in 96-well round-bottom multiwell dishes. Incubation steps are usually done by
floating the plates in a 37°C water bath. The chlamydial antigen can be prepared from either infected yolk
sacs or cell culture preparations. The modified direct CF test differs from the direct CF test in that normal,
unheated chicken serum from chickens without chlamydial antibody is added to the complement dilution.
The normal serum increases the sensitivity of the CF procedure so that it can be used to test sera from
avian species whose antibodies do not normally fix guinea-pig complement.
Test procedure
i) Dilution of sera
Figure 1 gives a suggested pattern for performing the test in round-bottom, 96-well multiwell dishes.
All sera must be heat-inactivated at 60°C for 30 minutes prior to use. The sera are diluted in Veronal
(barbiturate) buffer saline (VBS) as shown in Figure 1. The dilutions are made in the multiwell dish by
adding 100 µl of VBS to each well of rows A and E, and then adding 25 µl of the undiluted sera,
positive serum, or negative serum to each of three wells. This gives a starting dilution of 1/5. Then,
25 µl of VBS is added to each well in row B through to D and row F through to H. Twofold dilutions are

made, using a 25 µl micropipette, from row A through to D and row E through to H. Appropriate
volumes are discarded from the starting and finishing rows to give 25 µl per well. Diluters are rinsed
twice in distilled H2O and once in VBS between each serum.
Serum #1 Serum #2 Serum #3 Serum #4

Antigens: +Ag VBS -Ag +Ag VBS -Ag +Ag VBS -Ag +Ag VBS -Ag
1 2 3 4 5 6 7 8 9 10 11 12
A
1/5
/
1/10 B
1/20 C
1/40 D
1/5 E
1/10 F
1/20 G
1/40 H
Serum #5 Serum #6 + Control – Control
Fig. 1. Suggested test pattern for the modified direct complement fixation test when using 96-well dishes.
ii) Addition of antigen

To each well in columns 1, 4, 7, and 10, add 25 µl of positive chlamydial antigen. In columns 2, 5, 8,
and 11, add 25 µl of VBS (anticomplementary control wells), and in columns 3, 6, 9, and 12, add 25 µl
of negative antigen (normal yolk sac or cell culture prepared the same as the chlamydial antigen). The
chlamydial antigens are stored undiluted at 4°C and diluted to proper concentration in VBS prior to
use.
iii) Addition of complement

Complement (C’) is stored at –70°C and should be thawed and diluted in VBS prior to the addition of
the antigen. Fresh chicken serum is added before diluting the C’ to give a 5% concentration in the
complement. Dilutions of C’ are made as in previous tests or from titrations. C’ should be allowed to
stand in an ice bath to stabilise for 15 minutes. The diluted C’ should be stored at 4°C following
stabilisation and should be used within 2 hours: 50 µl of the C’ is added to each well immediately
following the addition of the antigens. The plates are incubated uncovered in a 37°C water bath for
2 hours.
iv) Addition of sheep red blood cells

Mix 4% standardised sheep red blood cells (SRBCs) with an equal volume of VBS. To this add an
equal volume of haemolysin diluted in VBS. The final dilution is incubated in a 37°C water bath for
15 minutes to sensitise the SRBCs. To each well add 50 µl of sensitised SRBCs. The plates are then
incubated for 1 hour in a 37°C water bath. The plates can be centrifuged at 400 g for 5 minutes before
reading or they can be refrigerated at 4°C overnight prior to reading.
v) Interpretation of the results

The wells are often scored 1+, 2+, 3+, or 4+ corresponding to reduction of haemolysis of 25, 50, 75, or
100%. A positive reaction is 2+ or higher, which is equivalent to 50% or less lysis of the SRBCs. This
indicates that the C’ was fixed by antibody prior to the addition of the SRBCs. Negative wells are
indicated by the complete lysis of the cells: the C’ remains unbound and reacts with the SRBCs and
the haemolysin to produce lysis of the SRBCs.
Invalid tests occur when the serum is anticomplementary and a positive reaction occurs in the dilution
with VBS as the antigen. Nonspecific serum reactions give positive reactions in both the positive and
negative wells.

Reagents
i) Antigen preparation
The simplest methods start with the growth of chlamydiae in cell culture. The two methods described
below produce antigens that can be used in the micro-CF test. The procedures are quite similar: both
include the growth of chlamydiae in cell culture, the inactivation of the chlamydiae, partial purification
of the antigen, mechanical disruption, and dilution into the appropriate buffer. The method selected will
depend on the equipment available.
The first procedure (17, 19) starts with the chlamydiae and cell culture debris harvested when
cytopathic effects are noted. The culture is inactivated by the addition of phenol to a final
concentration of 1.0%, incubated for 24 hours at 37°C, and concentrated by centrifugation at 10,000 g
for 1 hour. The sediment is reconstituted to 10% of the original volume using VBS, pH 7.2, containing
1.0% phenol and 1.0% glycerol.
The sediment is then homogenised in an omnimixer at top speed for three 1-minute periods while
cooled in ice water. The homogenate is centrifuged for 15 minutes at 100 g to remove debris. Some
procedures suggest heating the antigen for 30 minutes in a boiling water bath at this time. The
supernatant is saved and diluted to the desired concentration.
In the second procedure for the production of antigen for the CF test (9, 10), antigen is prepared from
L cells infected with a psittacine strain. The cell culture medium is discarded, and the cells are heated
for 40 minutes at 56°C. The cells are lysed in distilled water, the chlamydiae are disrupted by
ultrasonication, and then made isotonic in VBS. The antigen is tested against a standard sheep
convalescent serum and used at 2 units in the micro-CF test.
There are a number of procedures for preparing the antigen from infected yolk sacs, some of which
are quite elaborate. However, with the following procedure it is relatively easy to prepare a crude
infected yolk sac antigen that works well in the modified direct CF test. An egg-adapted strain of
chlamydia is used to inoculate 6–7-day-old embryonated chicken eggs via the yolk sac. The yolk sacs
are harvested from embryos that die between 3 and 7 days post-inoculation. The yolk-sac harvest is
diluted 1/3 in PBS, Tris buffer, or cell culture medium, and then autoclaved for 20 minutes. The
suspension is cooled and then homogenised thoroughly. The use of a high-speed tissue homogeniser
for 3–5 minutes is recommended. After homogenisation, phenol is added to make a final concentration
of 0.5% phenol (prepare a 5% phenol stock solution and add 1 ml for every 9 ml of antigen). The
antigen preparation is prepared, held for 3 days, and then the supernatant is used after centrifugation
for 20 minutes at 1000 g. The antigen can be stored for long periods of time at 4°C.
ii) Preparation of sensitised SRBCs

Defibrinated SRBCs are preserved by mixing in an equal volume of Alsever’s solution. These can be
stored at 4°C for up to 4 weeks. Wash 25 ml of the stock SRBCs with 25 ml of VBS. Centrifuge at
400 g for 10 minutes. Aspirate off the VBS and resuspend in 50 ml of VBS. Repeat the wash a total of
three times. Following the final wash, dilute the SRBCs at a ratio of 2.2 ml of packed SRBCs to 98 ml
of VBS. The SRBCs can then be standardised by optical density: mix 1 ml of the diluted, washed
SRBCs with 14 ml distilled H2O, determine the absorbance using a spectrophotometer, and
standardise to 0.25 at a wave length of 550 mm. The reading obtained can be used in the following
formula to determine the dilution needed:
(absorbanc e reading x current volume)

Final volume of SRBCs =
0.25 desired absorbance
The SRBCs are sensitised by rapidly adding an equal volume of VBS containing the appropriate
dilution of haemolysin (dilution determined by titration). Incubate at 37°C for 15 minutes prior to use.
iii) Veronal buffer saline

VBS is prepared as a 5 × stock solution and diluted 1/5 with distilled H2O prior to use. The following
formula makes 4 litres. To distilled water add sodium barbital (7.5 g); barbital H2O (dissolve in boiling
H2O) (11.5 g); MgSO4.7 H2O (4.056 g); NaCl (170.0 g); and CaCl2 (0.078 g). Add distilled H2O to
make to 4 litres.
iv) Complement titration

Complement (C’) is unstable and will deteriorate if improperly handled. Normally it should be kept
frozen at –70°C in aliquots that are used at one time to eliminate refreezing. To obtain the desired
working concentration (2 units per test well) first add 5% normal chicken serum for the modification to

enhance sensitivity as described earlier. Then estimate a starting point based on previous lots. A good
starting point is a dilution of 1/30 after the chicken serum has been added. Set up a series of tubes
with various amounts of complement in VBS. The VBS should contain the antigen to be used in the
reaction and take into account any anticomplementary properties of the antigen. A common method is
to dilute 0.10 ml C’ + 0.90 ml VBS; 0.12 ml complement + 0.88 ml VBS, etc. through 0.25 ml C’ +
0.75 ml VBS. Incubate the tubes for 2 hours in a 37°C water bath. Add 0.5 ml of sensitised SRBCs to
each tube. Incubate for 1 additional hour in the 37°C water bath. The highest dilution giving complete
haemolysis equals 1 unit. Twice that amount equals 2 units. The following formula can be used to
obtain 2 units/0.05 ml:
x = (di) (v)/2dh
where:
x = reciprocal of C’ dilution desired to yield 2 units C’/well

di = reciprocal of C’ initial dilution used in titration (1/30)
v = volume of diluted C’ to be added
dh = twice the volume of C’ giving complete haemolysis in titration
v) Titration of haemolysin
Haemolysin can be obtained from commercial sources. It must be standardised by titration. The
following procedure is recommended:
Prepare a 1/100 dilution of the stock haemolysin in VBS. From this, prepare 1/300, 1/400, and
1/500 dilutions in tubes. From each of these dilutions, make 0.5 ml of twofold dilutions in VBS for a
block titration.
To determine haemolysin concentration, add the following to 0.5 ml of each dilution: 0.5 ml of C’ at
1/30 dilution, 0.5 ml of unsensitised SRBCs at 0.25 optical density, and 1.5 ml of VBS. Incubate for
1 hour at 37°C, and then centrifuge at 400 g for 5 minutes. One unit of the haemolysin is the dilution
that gives complete lysis of the SRBCs. The haemolysin solution is prepared in VBS at the dilution
containing 2 units of haemolysin. This is then added to an equal volume of SRBCs at the proper
concentration.
vi) Titration of antigen and positive control serum

In order to standardise the CF test, it is also necessary to have titres of both the antigen and the
positive control serum. If the titre is known for either the positive serum or antigen, the titre of the other
component can be determined by performing the CF test using dilutions of the component being titred.
If titres of both the positive serum and antigen are unknown, a block titration (chequerboard) can be
used to determine the limiting dilutions of both the antigen and the antibody where haemolysis starts.
It is very critical to obtain these titres accurately.
For both the antigen and the positive control serum, 4 units are used. A unit is the highest dilution that
will give a positive test. That is, if a dilution of 1/160 gives a positive test, then a 1/40 dilution has
4 units and is used for the test.
Complement-fixing antibodies usually appear within 7–10 days of infection. For a positive diagnosis, a
four-fold rise in CF antibody titre is required. A presumptive diagnosis by serological tests on a flock
can only be made if typical clinical signs are present and a majority of the birds have antibody titres of
>1/64.
b) Other tests
Other serological tests have been developed, but their specificity has not yet been sufficiently evaluated.
The ELISA for group-specific chlamydial antibodies is more rapid and sensitive than the CF test; it can be
automated. Evaluations of ELISAs for the detection of antibodies to both C. trachomatis and C. psittaci
indicate that the tests are highly sensitive but lack specificity. New tests are being developed that use
peptides or recombinant antigens which may correct the specificity problem.
Other tests include the agar gel immunodiffusion test (26), the latex agglutination (LA) test, the elementary
body agglutination (EBA) test (18, 21) and the micro-immunofluorescence test (MIFT). Immunodiffusion is
less sensitive than the CF test. The LA test will detect antibodies to C. psittaci, and is easy and rapid to
perform (20). Latex beads are coated with purified chlamydial antigen, mixed thoroughly with the test serum
on a glass plate, and rotated for 2 minutes to enhance agglutination. The test is read against a dark
background. Sera giving positive reactions should be retested with uncoated beads to eliminate possible
nonspecific agglutination. The LA and direct CF tests correlate in 72.5% of tests with paired sera. The LA

test has a sensitivity of 39.1% and a specificity of 98.8% relative to the direct CF test (20). The test detects
both IgM and IgG, but it is best at detecting IgM. It has been suggested for use in detecting recent or active
infections. The EBA test detects only IgM, and it is indicative of a current infection. The MIFT is rapid and
easy to perform; however, fluorescence-conjugated anti-species sera are not always available.

There are no commercial vaccines available for chlamydiosis in poultry. Attempts to produce a vaccine have met
with limited success, and most have been based on bacterins produced by formalin inactivation of concentrated
suspensions of chlamydiae. There is evidence that immunity involves cell-mediated immune responses (30, 31),
but vaccine manufacture has not been directed towards reactions of this type.
Antibiotics are the only current means of control. Chlamydophila psittaci is susceptible to a number of antibiotics:
the drug of choice varies from country to country. Chlortetracycline, doxycycline, and other tetracyclines are the
most commonly used. Treatment needs to be maintained for extended periods of time. For pet birds, 45 days is
often recommended (31, 39).
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detection of Chlamydia psittaci in cloacal and conjunctival specimens from turkeys. J. Clin. Microbiol., 32,
1470–1474.
*
* *
NB: There is an OIE Reference Laboratory for avian chlamydiosis (see Table in Part 3 of this Terrestrial Manual

CHAPTER 2.3.2.
AVIAN INFECTIOUS BRONCHITIS
SUMMARY
Avian infectious bronchitis (IB) is caused by coronavirus infectious bronchitis virus (IBV). The virus
causes infections mainly in chickens and is a significant pathogen of commercial meat and egg
type birds. IB is an acute, contagious disease characterised primarily by respiratory signs in
growing chickens. In hens, decreased egg production and quality are often observed. Some strains
of the virus are nephropathogenic and produce interstitial nephritis and mortality. The severity of
IBV-induced respiratory disease is enhanced by the presence of bacterial pathogens leading to
chronic complicated airsacculitis. Diagnosis of IB requires virus isolation or demonstration of viral
nucleic acid from diseased flocks. Demonstration of an ascending serum antibody response may
also be useful. The widespread use of live and inactivated vaccines may complicate both the
interpretation of virus isolation and serology findings. The occurrence of antigenic variant strains
may overcome immunity induced by vaccination.
Diagnosis requires laboratory testing. Virus isolation and identification is preferred. Reverse-
transcription polymerase chain reaction (RT-PCR) techniques are commonly used to identify the
IBV genotype. Haemagglutination inhibition (HI) tests to determine serotype and enzyme-linked
immunosorbent assays (ELISA) for general monitoring are often used for sero-diagnosis.
Supplementary tests include electron microscopy, the use of monoclonal antibodies, virus
neutralisation (VN), immunohistochemical or immunofluorescence tests, and immunisation-
challenge trials in chickens.
Identification of the agent: For the common respiratory form, IBV is most successfully isolated
from tracheal mucosa and lung several days to one week following infection. For other forms of IB,
kidney, oviduct, the caecal tonsils of the intestinal tract or proventriculus tissues are better sources
of virus depending on the pathogenesis of the disease.
Specific pathogen free chicken embryos or tracheal organ cultures (TOCs) from embryos may be
used for virus isolation. Following inoculation of the allantoic cavity, IBV produces embryo stunting,
curling, clubbing of the down, or urate deposits in the mesonephros of the kidney, usually within
three serial passages. Isolation in TOCs has the advantage that IBV produces stasis of the
tracheal cilia on initial inoculation. RT-PCR is increasingly being used to identify the spike (S)
glycoprotein genotype of IBV field strains. Genotyping using primers specific for the S1 subunit of
the S gene or sequencing of the same gene generally provides similar but not always identical
findings to HI or VN serotyping. Alternatively, VN or HI tests using specific antiserum may be used
to identify the serotype.
Serological tests: Commercial ELISA kits may be used for monitoring serum antibody responses.
The antigens used in the kits are broadly cross-reactive among serotypes and allow for general
serological monitoring of vaccinal responses and field challenges. The HI test is used for
identifying serotype-specific responses to vaccination and field challenges especially in young
growing chickens. Because of multiple infections and vaccinations, the sera of breeders and layers
contain cross-reactive antibodies and the results of HI testing cannot be used with a high degree of
confidence.
Requirements for vaccines and diagnostic biologicals: Both live attenuated and oil emulsion
inactivated vaccines are available. Live vaccines, attenuated by serial passage in chicken embryos
or by thermal heat treatment, confer better local immunity of the respiratory tract than inactivated
vaccines. The use of live vaccines carries a risk of residual pathogenicity associated with vaccine

Chapter 2.3.2. — Avian infectious bronchitis
back-passage in flocks. However, proper mass application will generally result in safe application
of live vaccines.
Inactivated vaccines are injected and a single inoculation does not confer protection unless
preceded by one or more live IBV priming vaccinations. Both types of vaccines are available in
combination with Newcastle disease vaccine; in some countries inactivated multivalent vaccines
are available that include Newcastle disease, infectious bursal disease, reovirus and egg-drop
syndrome 76 viral antigens.
A. INTRODUCTION
Avian infectious bronchitis (IB) was first described in the United States of America (USA) in the 1930s as an
acute respiratory disease mainly of young chickens. A viral aetiology was established, and the agent was termed
avian infectious bronchitis virus (IBV). The virus is a member of the genus Coronavirus, family Coronaviridae, in
the order Nidovirales. IBV and other avian coronaviruses of turkeys and pheasants are classified as group 3
coronaviruses, with mammalian coronaviruses comprising groups 1, 2 and 4 (group 4 being the more recently
identified severe acute respiratory syndrome (SARS) coronavirus) (6). Coronaviruses have a nonsegmented,
positive-sense, single-stranded RNA genome.
IB affects chickens of all ages, which, apart from pheasants (10) are the only species reported to be naturally
affected. The disease is transmitted by the air-borne route, direct chicken to-chicken contact and indirectly
through mechanical spread (contaminated poultry equipment or egg-packing materials, manure used as fertiliser,
farm visits, etc.). IB occurs world-wide and assumes a variety of clinical forms, the principal one being respiratory
disease that develops after infection of the respiratory tract tissues following inhalation or ingestion. Infection of
the oviduct can lead to permanent damage in immature birds and, in hens, can lead to cessation of egg-laying or
production of thin-walled and misshapen shells with loss of shell pigmentation. IB can be nephropathogenic
causing acute nephritis, urolithiasis and mortality (11). After apparent recovery, chronic nephritis can produce
death at a later time. IBV has also been reported to produce disease of the proventriculus (49). Vaccine and field
strains of IBV may persist in the caecal tonsils of the intestinal tract and be excreted in faeces for weeks or
longer in clinically normal chickens (2). For an in-depth review of IB, refer to Cavanagh & Naqi (11). A detailed
discussion of IBV antigen, genome and antibody detection assays prepared by De Wit (24) is also available.
There have been no reports of human infection with IBV.
Confirmation of diagnosis is based on virus isolation, often assisted by serology. Extensive use is made of live
and inactivated vaccinations, which may complicate diagnosis by serological methods as antibodies to
vaccination and field infections can not always be distinguished. Persistence of live vaccines may also confuse
attempts at recovering the causative field strain.
a) Sampling
Samples appropriate to the form of IB observed must be obtained as soon as signs of clinical disease are
evident. Samples must be placed in cold transport media and be frozen as soon as possible. The cold chain
from bird to laboratory should be maintained. For acute respiratory disease, swabs from the upper
respiratory tract of live birds or tracheal and lung tissues from diseased birds should be harvested, placed in
transport medium containing penicillin (10,000 International Units [IU]/ml) and streptomycin (10 mg/ml) and
kept on ice and then frozen. For birds with nephritis or egg-production problems, samples from the kidneys
or oviduct, respectively, should be collected in addition to respiratory specimens. In some cases, IBV
identification by reverse-transcription polymerase chain reaction (RT-PCR) may be desirable without virus
isolation. In this case, swabbings from the respiratory tract or cloaca may also be submitted alone, without
being placed in liquid transport media (8). In situations where IB-induced nephritis is suspected, kidney
samples should also be selected from fresh carcases for histopathological examination as well as virus
isolation. Blood samples from acutely affected birds as well as convalescent chickens should be submitted
for serological testing. A high rate of virus recovery has been reported from the caecal tonsil or faeces (2).
However, isolates from the intestinal tract may have no relevance to the latest infection or clinical disease.
IBV isolation may be facilitated using sentinel specific pathogen free (SPF) chickens placed at one or more
times in contact with commercial poultry (25).

b) Culture
Suspensions of tissues (10–20% w/v) are prepared in sterile phosphate buffered saline (PBS) or nutrient
broth for egg inoculation, or in tissue culture medium for tracheal organ culture (TOC) inoculation (17). The
suspensions are clarified by low-speed centrifugation and filtration through bacteriological filters (0.2 µ)
before inoculation of eggs or TOCs.
Embryonated chicken eggs and TOCs are used for primary isolation of IBV. Cell cultures are not
recommended for primary isolation as it is often necessary to adapt IBV isolates to growth in chicken
embryos before cytopathic effect (CPE) is produced in chick embryo kidney cells.
Embryonated eggs used for virus isolation should originate preferably from SPF chickens or from breeder
sources that have been neither infected nor vaccinated with IBV. Most commonly, 0.1–0.2 ml of sample
supernatant is inoculated into the allantoic cavity of 9–11-day-old embryos. Eggs are candled daily for
7 days with mortality within the first 24 hours being considered nonspecific. The initial inoculation usually
has limited macroscopic effects on the embryo unless the strain is derived from a vaccine and is already
egg adapted. Normally, the allantoic fluids of all eggs are pooled after harvesting 3 days after infection; this
pool is diluted 1/5 or 1/10 in antibiotic broth and further passaged into another set of eggs for up to a total of
three to four passages. Typically, a field strain will induce observable embryonic changes consisting of
stunted and curled embryos with feather dystrophy (clubbing) and urate deposits in the mesonephros on the
second to fourth passage. Embryo mortality in later passages may occur as the strain becomes more egg
adapted. Other viruses, notably adenoviruses that are common to the respiratory tract, also produce
embryo lesions indistinguishable from IBV. The IBV-laden allantoic fluid should not agglutinate red blood
cells and isolation of IBV must be confirmed by serotyping or genotyping. Infective allantoic fluids are kept
at –20°C or below for short-term storage, –60°C for long-term storage or at 4°C after lyophilisation.
TOCs prepared from 20-day-old embryos can be used to isolate IBV directly from field material (17). An
automatic tissue-chopper is desirable for the large-scale production of suitable transverse sections or rings
of the trachea for this technique (21). The rings are about 0.5–1.0 mm thick, and are maintained in a
medium consisting of Eagle’s N-2-hydroxyethylpiperazine N’-2-ethanesulphonic acid (HEPES) in roller
drums (15 rev/hour) at 37°C. Infection of tracheal organ cultures usually produces ciliostasis within 24–
48 hours. Ciliostasis may be produced by other viruses and suspect IBV cases must be confirmed by
serotyping or genotyping methods.
c) Methods for identification

The initial tests performed on IBV isolates are directed at eliminating other viruses from diagnostic
consideration. Chorioallantoic membranes from infected eggs are collected, homogenised, and tested for
avian adenovirus group 1 by immunodiffusion or PCR. Group 1 avian adenovirus infections of commercial
chickens are common, and the virus often produces stunted embryos indistinguishable from IBV-infected
embryos. Furthermore, harvested allantoic fluids do not hemagglutinate (HA) chick red blood cells. Genetic
based tests (RT-PCR or RT-PCR-RFLP [restriction fragment length polymorphism]) are used commonly to
identify an isolate as IBV. Other techniques may be used, for example cells present in the allantoic fluid of
infected eggs may be tested for IBV antigen using fluorescent antibody tests (12) and direct negative-
contrast electron microscopy will reveal particles with typical coronavirus morphology in allantoic fluid or
TOC fluid concentrates. The presence of IBV in infective allantoic fluid may be detected by RT-PCR
amplification and use of a DNA probe in a dot-hybridisation assay (32). Direct immunofluorescence staining
of infected TOCs for the rapid detection of the presence of IBV has been described (3). Immuno-
histochemistry, with a group-specific monoclonal antibody (MAb), can be used to identify IBV in infected
chorioallantoic membranes (43).
d) Serotype identification
Antigenic variation among IBV strains is common (11, 16, 23, 28, 31), but at present there is no agreed
definitive classification system. Nevertheless, antigenic relationships and differences among strains are
important, as vaccines based on one particular serotype may show little or no protection against viruses of
a different antigenic group. As a result of the regular emergence of antigenic variants, the viruses, and
hence the disease situation and vaccines used, may be quite different in different geographical locations.
Ongoing assessment of the viruses present in the field is necessary to produce vaccines that will be
efficacious in the face of antigenic variants that arise. Serotyping of IBV isolates and strains has been done
using haemagglutination inhibition (HI) (1, 36) and virus neutralisation (VN) tests in chick embryos (23),
TOCs (22) and cell cultures (29). Neutralisation of fluorescent foci has also been applied to strain
differentiation (19).
MAbs, usually employed in enzyme-linked immunosorbent assays (ELISA), have proven useful in grouping
and differentiating strains of IBV (30, 38). The limitations of MAb analysis for IB serotype definition are the
lack of availability of MAbs or hybridomas and the need to produce new MAbs with appropriate specificity to
keep pace with the ever-growing number of emerging IB-variant serotypes (34).

e) Genotype identification
RT-PCR genotyping methods have largely replaced HI and VN serotyping for determining the identity of a
field strain. The molecular basis of antigenic variation has been investigated, usually by nucleotide
sequencing of the gene coding for the spike (S) protein or, more specifically, nucleotide sequencing of the
gene coding for the S1 subunit of the S protein (5, 40) where most of the epitopes to which neutralising
antibodies bind are found (39). An exact correlation with HI or VN results has not been seen, in that while
different serotypes generally have large differences (20–50%) in the deduced amino acid sequences of the
S1 subunit (40), other viruses that are clearly distinguishable in neutralisation tests show only 2–3%
differences in amino acid sequences (5). However, there is, in general, good agreement between data
represented by the S1 sequence and the VN serotype, and it may eventually be possible to select vaccine
strains on the basis of sequence data.
The primary advantages of genotyping methods are a rapid turnaround time, and the ability to detect a
variety of genotypes, depending on the tests used. RFLP RT-PCR differentiates IBV serotypes based on
unique electrophoresis banding patterns of restriction enzyme-digested fragments of S1 following
amplification of the gene by RT-PCR (33, 41). The RFLP RT-PCR procedure may be used in conjunction
with a biotin-labelled DNA probe to first detect IBV in egg fluids harvested following the inoculation of eggs
with clinical samples (32). The RFLP RT-PCR test can identify all known serotypes of IBV as well as variant
viruses.
S1 genotype-specific RT PCR may be used to identify specific IBV serotypes (35). S1 gene primers specific
for serotypes Massachusetts (Mass), Connecticut, Arkansas, and JMK are used in conjunction with a
universal primer set that amplifies all IBV serotypes. Primers for the DE/072/92 and California serotypes
have also been developed. Other variant serotypes may be determined to be IBV using the general primers,
but the specific serotype cannot be identified. Infections caused by multiple IBV serotypes may be
identified.
Nucleotide sequencing of a diagnostically relevant fragment of the S1 gene is the most useful technique for
the differentiation of IBV strains and has become the genotyping method of choice in many laboratories.
Nucleotide sequencing has also produced evidence that recombination between IB strains occurs often (7,
50). RT-PCR product cycle sequencing of the hypervariable amino terminus region of S1 may be used
diagnostically to identify previously recognised field isolates and variants (37). Comparison and analysis of
sequences of unknown field isolates and variants with reference strains for establishing potential
relatedness are significant advantages of sequencing.
Recently, it has been shown that coronaviruses isolated from turkeys and pheasants are genetically similar
to IBV, having approximately 90% nucleotide identity in the highly conserved region II of the 3’ untranslated
region (UTR) of the IBV genome (9, 10). The potential role of these coronaviruses in IBV infections has not
been determined.
The major uses of RT-PCR tests are virus identification and its application in the understanding of
epidemiological investigations during IBV outbreaks. The RT-PCR tests, as they now exist however, do not
provide information on viral pathogenicity.
RT-PCR test procedure

i) Extraction of viral RNA
Any RNA extraction method can be used. There are many protocols available in journals, books and
on the web. However, for extracting high quality RNA from allantoic fluid, the Qiagen Viral RNA Mini Kit
(www.qiagen.com) is recommended. The Qiagen RNeasy Mini Kit is recommended for extracted IBV
RNA from tissue or swabbings. All extracted RNA should be stored between –20°C and –80°C until
tested. It is advised that for long-term storage, RNA be kept at –80°C.
ii) Custom oligos
Custom oligos can be purchased through any commercial supplier. Operon (www.operon.com) has
been making quality custom oligos for years. The target gene for IBV characterisation is the S1
subunit of the spike glycoprotein gene. A commonly used primer pair for amplification of genotypically
diverse IBV strains is oligo S15’ mod (forward): 5’-TGA-AAA-CTG-AAC-AAA-AGA-3’ and CK2
(reverse): 5’-CNG-TRT-TRT-AYT-GRC-A-3’ (26). The oligo S15’mod/CK2 amplicon is approximately
700 bp in length beginning from the start of the S1 gene spanning two hypervariable regions used for
genotyping.
iii) Reverse-transcription polymerase chain reaction
Many one and two-step RT-PCR kits are commercially available from manufacturers claiming superior
enzyme sensitivity and fidelity. The recommended RT-PCR kit is the basic, two-step, RNA PCR kit
from Applied Biosystems (http://www.appliedbiosystems.com). Reverse transcription is performed

according to the manufacturer’s instructions. RT priming is accomplished with the use of random
hexamers (supplied with the kit) or with the reverse PCR primer, in this case CK2 (35). One cycle of
RT is performed with the following parameters: 25°C for 10 minutes, 42°C for 25 minutes, 95°C for
5 minutes, hold at 4°C. The full RT reaction volume is added to the PCR sample master mix. PCR is
performed using the following parameters: 95°C for 2 minutes, 45 cycles of 95°C for 30 seconds, 52°C
for 30 seconds, 68°C for 30 seconds, final extension of 68°C for 12 minutes, hold at 4°C. Samples are
concentrated in a desiccator overnight or by the use of a vacuum centrifuge. Dried samples are
resuspended in 12 µl of DEPC-treated water and 6 µl of loading buffer prior to electrophoresis on a
1.8% agarose gel containing ethidium bromide. Gels are visualised with a UV light box. Bands are
compared to a commercially available 100 bp ladder and an IBV positive control.
iv) S1 gene sequencing
Bands visualised in the agarose gel that are of similar size to the positive control are excised
from the gel. The PCR product is separated from the agarose gel using the Qiagen Gel Extraction
kit (www.qiagen.com) or any other commercial gel extraction kit. Purified PCR products are run
on a second 1.8% agarose-ethidium bromide gel to determine the quantity of product present.
Approximately 20 µl (10 ng/µl) of PCR product is required for sequencing. Sequencing can be
performed at the University of Delaware Sequencing & Genotyping Center, Newark, DE
(http://www.udel.edu/dnasequence) or another university or commercial sequencing facility. Sequence
chromatagrams are edited using the DNAStar analysis software or on-line freeware 4peaks
(http://www.mekentosj.com/4peaks/)
or chromas lite(http://www.technelysium.com.au/chromas_lite.html).
Edited sequences of IBV isolates are characterised using BLASTn for nucleotide or BLASTp for
protein analysis (http://www.ncbi.nlm.nih.gov/BLAST/).
A number of tests have been described. Those considered here include VN (23), agar gel immunodiffusion
(AGID) (48), HI (1) and ELISA (42). Each test has advantages and disadvantages in terms of practicality,
specificity, sensitivity and cost. In general, for routine serological testing, the VN tests are too expensive and
impractical, and AGID tests lack sensitivity. ELISA and HI tests are most suitable for routine serology. ELISAs
are useful for general monitoring of IBV exposure and can detect antibody responses to all serotypes. HI when
used on sera from young growing chickens such as pullets and broilers can give information on the serotype-
specific antibody status of a flock. Regular monitoring of sera from flocks for IB antibody titres may help to
indicate the level of vaccine or field challenge responses. Because chicken sera from older birds contain
antibodies that are highly cross-reactive against antigenically unrelated strains, serodiagnosis of suspected
disease outbreaks of IB cannot be used with a high degree of confidence.
In VN tests, all sera should first be heated to 56°C for 30 minutes. Virus is mixed with serum and incubated
for 30–60 minutes at 37°C or room temperature. Chicken embryos are most often employed, but antibodies
can be measured using TOC or cell culture systems. Two methods have been used to estimate neutralising
antibodies. One employs a constant serum concentration reacted with varying dilutions of virus (the alpha
method) and the other employs a constant amount of virus and varying dilutions of serum (the beta
method).
In the alpha method, tenfold dilutions of egg-adapted virus are reacted with a fixed dilution (usually 1/5) of
antiserum, and the mixtures are inoculated into groups of from five to ten eggs. The virus alone is titrated in
parallel. End-points are calculated by the Kärber or the Reed and Muench methods. The results are
expressed as a neutralisation index (NI) that represents the log10 difference in the titres of the virus alone
and that of the virus/antiserum mixtures. The NI values may reach 4.5–7.0 in the case of homologous
virus/serum mixtures; values of <1.5 are not specific, but a heterologous virus will give a value as low as
1.5.
The beta method is the more widely used neutralisation test for antibody assay with chicken embryos. Two-
or four-fold dilutions of antiserum are reacted in equal volumes with a dilution of virus, usually fixed at
100 or 200 EID50 (median embryo-infective doses) per 0.05 ml and 0.1 ml of each mixture inoculated into
the allantoic cavity of each of from five to ten embryonated eggs. A control titration of the virus is performed
simultaneously to confirm that the fixed virus dilution in the virus/serum mixtures was between 101.5 and
102.5 EID50. End-points of the serum titres are determined by the Kärber or Reed and Muench method as
before, but here are expressed as reciprocals of log2 dilutions. This fixed-virus/varying-serum method is
also employed for neutralisation tests in tracheal organ cultures using five tubes per serum dilution, as is
conventional with other viruses (22). The results are calculated according to Reed and Muench, and the
virus titres are expressed as median ciliostatic doses per unit volume (log10 CD50). Serum titres are again
expressed as log2 dilution reciprocals. This test is more sensitive than others, but technical logistics hamper
its more widespread adoption.

b) Haemagglutination inhibition
A standard protocol for a HI test for IBV has been described (1), and the test procedure detailed below is
based on that standard. Strains and isolates of IBV will agglutinate chicken red blood cells (RBCs) after
neuraminidase treatment (44, 45). The strain selected to produce antigen may be varied, depending on the
requirements of diagnosis. The antigen for the HI test is prepared from IBV-laden allantoic fluids.
For HA and HI tests, procedures are carried out at 4°C.
Haemagglutination test
i) Dispense 0.025 ml of PBS, pH 7.0–7.4, into each well of a plastic U or V-bottom microtitre plate.
ii) Place 0.025 ml of virus antigen in the first well. For accurate determination of the HA content, this
should be done from a close range of an initial series of dilutions, i.e. 1/3, 1/4, 1/5, 1/6, etc.
iii) Make twofold dilutions of 0.025 ml volumes of the virus antigen across the plate.
iv) Dispense a further 0.025 ml of PBS into each well.
v) Dispense 0.025 ml of 1% (v/v) chicken RBCs to each well.
vi) Mix by tapping the plate gently and allow the RBCs to settle for about 40 minutes at 4°C, when control
RBCs should be settled to a distinct button.
vii) HA is determined by tilting the plate and observing the presence or absence of tear-shaped streaming
of the RBCs. The titration should be read to the highest dilution giving complete HA in which there is
no streaming; this is 100% HA and represents 1 HA unit (HAU) and can be calculated accurately from
the initial range of dilutions.
Haemagglutination-inhibition test
The HI test is used in the diagnosis and routine flock monitoring of vaccine responses.
i) Dispense 0.025 ml of PBS into each well of a plastic U or V-bottom microtitre plate.
ii) Place 0.025 ml of serum into the first well of the plate.
iii) Make twofold dilutions of 0.025 ml volumes of the serum across the plate.
iv) Add 4 HAU of virus antigen in 0.025 ml to each well and leave for 30 minutes.
v) Add 0.025 ml of 1% (v/v) chicken RBCs to each well and, after gentle mixing, allow the RBCs to settle
for about 40 minutes when control RBCs should be settled to a distinct button.
vi) The HI titre is the highest dilution of serum causing complete inhibition of 4 HAU of antigen. The
agglutination is assessed more exactly by tilting the plates. Only those wells in which the RBCs
‘stream’ at the same rate as the control wells (containing 0.025 ml RBC and 0.05 ml PBS only) should
be considered to show inhibition.
vii) The validity of results should be assessed against a negative control serum, which should not give a
titre >22, and a positive control serum, for which the titre should be within one dilution of the known
titre.
viii) Sera are usually regarded as positive if they have a titre of 24 or more. However, it should be noted
that even in SPF flocks, a very small proportion of birds may show a nonspecific titre of 24, but usually
in birds over 1 year of age.

The ELISA technique is a sensitive serological method and gives earlier reactions and higher antibody titres
than other tests (42). It lacks type or strain specificity, but is valuable for monitoring vaccination responses
under field conditions. Commercial kits for ELISAs are available – these are based on several different
strategies for the detection of IBV antibodies. Usually, such tests have been evaluated and validated by the
manufacturer, and it is therefore important that the instructions specified for their use be followed carefully.
The ELISA is widely used to identify IBV-infected flocks (broilers) based on high antibody titres. If IB
reoccurs in the next flock on the farm, virus isolation attempts are performed and the virus is genotyped by
RFLP or S1 sequencing.

d) Agar gel immunodiffusion

AGID can be used in diagnosis (48). The antigen is prepared from a homogenate of the chorioallantoic
membranes of infected chicken embryos. The Beaudette embryo-lethal strain is often employed to produce
antigen. The test lacks sensitivity and is liable to yield inconsistent results as the presence and duration of
precipitating antibodies may vary with individual birds.

All live and inactivated commercial vaccines must be licensed. Strains used in live virus vaccines generally
require attenuation. At present, many countries only permit live vaccines of the Massachusetts type, such as the
H 120. Some countries may also have licensed vaccines to other live strains such as Connecticut, Arkansas, or
Delaware 072 (USA) or the 4/91 (United Kingdom). Live vaccines may be given as aerosols, in the drinking
water, or by the intraocular route (eyedrop).
The efficacy of inactivated vaccines depends heavily on proper priming with a live vaccine(s). Inactivated
vaccines must be administered to birds individually, by intramuscular or subcutaneous injection. Variant strains
may be used to prepare inactivated autogenous vaccines for controlling IB in layers and breeders.
Live vaccines confer better local immunity in the respiratory tract and also may protect against a wider antigenic
spectrum of field strains (18). However, vaccination with live vaccines may not protect layer flocks against variant
serotype challenge especially common on farms with flocks of multiple ages where production drops as early as
40 weeks of age are not uncommon (27). Live vaccines carry a risk of residual pathogenicity associated with
vaccine back-passage in flocks. However, proper mass application techniques (e.g. spray or drinking water) can
achieve uniform distribution of the vaccine in the flock and avoid backpassage. Furthermore, the use of vaccines
at manufacturer’s recommended dosages will also help avoid backpassage reversion that may be caused by
fractional dose application.
There are prospects for genetically engineered vaccines (4), and in-ovo vaccination (47).
production. The guidelines given here and in Chapter 1.1.8 are intended to be general in nature. National and
international standards that apply in the country in which IB vaccines are manufactured must be complied with.
The licensing authority should provide information and guidance on requirements. These are now often
presented in general terms, as applying to all vaccines – avian and mammalian, live and inactivated, or viral and
bacterial vaccines. There may also be specific requirements applying to IB vaccines, live and inactivated. As
examples, references are given to the European and USA regulations (13–15, 46).
The list of extraneous agents that must be shown to be absent continues to grow. Manufacturers must be familiar
with those that currently apply in their country. Recent additions are avian nephritis virus and avian pneumovirus.
For IB vaccines, important differences among countries may arise regarding the challenge virus to be used for
potency tests, and its validation. Traditionally, the virulent M-41 (Mass 41) strain of the Massachusetts type has
been used for challenge tests of both live and inactivated vaccines. Although this type is still common, it is often
not the only or the dominant type in many countries and it may be advisable to prepare vaccines from other
types. It is logical for challenges to be made by the same type as present in the vaccine. Establishing criteria for
validating the challenge virus may be more difficult for non-Massachusetts types, because of their lower virulence
in general. Inactivated vaccines are usually expected to protect against drops in egg production. The traditional
M-41 challenge, as described in this chapter, should cause a drop of at least 67% in the unvaccinated controls,
but when using other types much lower drops may be regarded as satisfactory, depending on published evidence
of the effects of these strains in the field. There is also a tendency to relax the criteria for Massachusetts type
challenges, and the European Pharmacopoeia now defines a satisfactory drop for Massachusetts types to be at
least 35%, and for non-Massachusetts types to be at least 15%, provided that the drop is ‘commensurate with
the documented evidence’ (15).
1. Seed management

The seed-lot (master seed) system should be employed for whatever type of vaccine is produced. Each
virus must be designated as to strain and origin and must be free from contamination with other strains of
IBV and extraneous agents. Separate storage facilities should be provided between the strains of virus
intended for vaccines or for challenge.

For live virus vaccines, many countries permit only strains of the Massachusetts type. Some countries allow
other strains, usually on the basis that those strains are already present in their national flocks. The
antigenic type incorporated in both live and inactivated vaccines requires justification if there is doubt as to
its existence in a country.
All seed viruses are grown in the allantoic sac of developing chicken embryos or in suitable cell cultures.
The eggs should be from an SPF flock.
• Purity
Every seed lot must be free from bacterial, fungal, mycoplasmal and viral contamination.
For the detection of extraneous viruses, the seed is first treated with a high-titred monospecific antiserum
prepared against the strain under examination or against one of identical type. This mixture is cultured in a
variety of ways, designed to confirm the absence of any viruses considered from past experience to be
potential contaminants. The antiserum must not contain antibodies to adenovirus, avian encephalomyelitis
virus, avian rotavirus, chicken anaemia virus, fowlpox virus, infectious laryngotracheitis virus, influenza A
virus, Newcastle disease virus, infectious bursal disease virus, leukosis virus, reovirus, Marek’s disease
virus, turkey herpesvirus, adeno-associated virus, egg-drop syndrome 76 (EDS76) virus, avian nephritis
virus, avian pneumovirus or reticulo-endotheliosis virus. The inoculum given to each unit of the culture
system used should contain a quantity of the neutralised IBV component under test that had an initial
infectivity of at least ten times the minimum field dose. These systems include:
1. SPF chicken embryos, incubated for 9–11 days, inoculated via both allantoic sac and chorioallantoic
membrane (two passages);
2. Chicken embryo fibroblast cultures, for leukosis virus subgroups A and B. The COFAL test (test for
avian leukosis using complement fixation) or double-antibody sandwich ELISA for group-specific
leukosis antigen is performed on cell extracts harvested at 14 days. An immunofluorescence test for
reticulo-endotheliosis virus is done on cover-slip cultures after two passages.
3. SPF chicken kidney cultures that are examined for CPEs, cell inclusions and haemadsorbing agents
passaged at intervals of no fewer than 5 days for up to 20 days’ total incubation.
4. SPF chickens of minimum vaccination age inoculated intramuscularly with 100 field doses, and on to
the conjunctiva with ten field doses; this is repeated 3 weeks later when the chickens are also
inoculated both into the foot pad and intranasally with ten field doses. Observations are made for
6 weeks overall, and serum is collected for tests for avian encephalomyelitis, infectious bursal
disease, Marek’s disease, Newcastle disease and Salmonella pullorum infection.
• Potency
Vaccines intended to protect against loss of egg production should be tested for duration of antibody
response. Mean HI titres should be >6 log2 up to at least 60 weeks of age. Serological tests should be done
at intervals frequent enough to show that titres have not been boosted by extraneous IBV infection.
Vaccines intended for protection of broiler chickens or rearing chickens against the respiratory form of the
disease should be similarly tested for duration of antibody responses; in the case of broilers this would be
up to the normal age for slaughtering, and in the case of pullets up to the age when a booster vaccination
would be administered (often at 16–18 weeks of age).
• Safety
Tests on seed virus should include a test for any potential ability to revert to virulence. Live and inactivated
vaccine seed must be tested for safety as in Section C.4.b.
• Efficacy
To demonstrate efficacy, a trial vaccine must be made from the master seed and the working seed at five
passages from the master seed and subjected to tests that demonstrate their protective effect.
For live vaccines, a minimum of ten SPF chickens aged 3–4 weeks are vaccinated intranasally or by
eyedrop with the recommended dose. Ten unvaccinated control birds from the same age and source are
retained separately. All birds of both groups are challenge inoculated intranasally or by eyedrop 3–4 weeks

later, each with 103.0–103.5 EID50 of the virulent Massachusetts M-41 strain. A swab of the trachea is taken
from each bird 4–5 days after challenge and placed in 3 ml of antibiotic broth. Each fluid is tested for IBV by
the inoculation (0.2 ml) of five embryonated eggs after 9–11 days of incubation. An alternative test to that of
taking swabs is to kill birds at 4–6 days after challenge and examine microscopically the tracheal rings for
ciliary activity (20). Failure to resist challenge is indicated by an extensive loss of ciliary motility. The live
vaccine is suitable for use if at least 90% of the challenge vaccinated birds show no evidence of IBV in their
trachea, while 90% or more of the control birds should have evidence of the presence of the virus.
To assess an inactivated vaccine intended to protect laying birds, 30 or more SPF chickens are vaccinated
as recommended at the earliest permitted age. If a primary vaccination with live vaccine is first undertaken,
an additional group of birds is given only the primary vaccination. In both cases, these primary vaccinations
should be done at no later than 3 weeks of age. The inactivated vaccine is given 4–6 weeks after the live
priming vaccination. A further group of 30 control birds are left unvaccinated. All groups are housed
separately until 4 weeks before peak egg production, and then are housed together. Individual egg
production is monitored and once it is regular, all birds are challenged, egg production being recorded for a
further 4 weeks. The challenge should be sufficient to ensure loss of production during the 3 weeks after
challenge. The loss in the control group should be at least 67%; the group that received primary live virus
vaccine followed by inactivated vaccine should remain at the previous level, and the group given only a
primary vaccination should show an intermediate drop in production. Sera are collected from all birds at
vaccination, 4 weeks later, and at challenge; there should be no response in the control birds.
To assess an inactivated vaccine intended to protect birds against respiratory disease, 20 SPF chickens
aged 4 weeks are vaccinated as recommended. An additional 20 control birds of the same age and origin
are housed with this first group. Antibody responses are determined 4 weeks later; there should be no
response in the control birds. All birds are then challenged with 103 CID50 (50% chick infective dose) of
virulent virus, killed 4–7 days later, and tracheal sections are examined for ciliary motility. At least 80% of
the unvaccinated controls should display complete ciliostasis, whereas the tracheal cilia of a similar
percentage of the vaccinated birds should remain unaffected.
Both live and inactivated vaccines containing Newcastle disease, infectious bursal disease, reovirus and
EDS76 viruses are available in some countries. The efficacy of the different components of these vaccines
must each be established independently and then as a combination in case interference between different
antigens exists.
All virus strains destined for live vaccines are cultured in the allantoic sac of SPF chicken embryos or in suitable
cell cultures. For inactivated vaccines, hens’ eggs from healthy non-SPF flocks may be used. The pooled fluid is
clarified and then titrated for infectivity. For live vaccines this fluid is lyophilised in vials, and for inactivated
vaccines it is blended with high-grade mineral oil to form an emulsion to which a preservative is added.
The required antigen content is based on initial test batches of vaccine of proven efficacy in laboratory and field
trials. Infectivity titrations are done in chicken embryos.
Live vaccine should contain not less than 103.5 EID50 per dose per bird until the expiry date indicated, and not
less than 102.5 EID50 per dose per bird after incubation at 37°C for 7 days at the time of issue. For inactivated
vaccine, the inactivating agent and inactivation procedure must be shown under manufacture to be effective on
both IBV and potential contaminants. With the use of beta-propiolactone or formalin, any live leukosis viruses
and Salmonella species must be eliminated; and with other inactivating agents, the complete range of potential
contaminants must be rendered ineffective. Before inactivation procedures, it is important to ensure homogeneity
of suspensions, and a test of inactivation should be conducted on each batch of both bulk harvest after
inactivation and the final product.
Tests of inactivation should be appropriate to the vaccine concerned and should consist of two passages in cell
cultures, embryos or chickens, using inoculations of 0.2 ml and ten replicates per passage.
4. Batch control
a) Sterility
Every batch of live vaccine should be tested for the absence of extraneous agents as for the seed virus
(see Chapter 1.1.9 Tests for sterility and freedom from contamination of biological materials).

b) Safety
• For live vaccines
Use no fewer than ten chickens from an SPF flock that are of the minimum age stated on the label for
vaccination. Administer by eyedrop to each chicken ten doses of the vaccine reconstituted so as to obtain a
concentration suitable for the test. Observe the chickens for 21 days. For vaccines intended for chickens
that are 2 weeks old or more, use the chickens inoculated in the ‘test for extraneous agents using chickens’
(see Section C.1.c.4). If during the period of observation, more than two chickens die from causes not
attributable to the vaccine, repeat the test. The vaccine complies with the test if no chicken shows serious
clinical signs, in particular respiratory signs, and no chicken dies from causes attributable to the vaccine.
• For inactivated vaccines

Inject a double dose of vaccine by the recommended route into each of ten 14–28-day-old chickens from an
SPF flock. Observe the chickens for 21 days. Ascertain that no abnormal local or systemic reaction occurs.
c) Potency
The potency test is developed from the results of efficacy tests on the master seed virus. Live vaccines are
tested for potency by titration of infectivity, and inactivated vaccines by measuring antibody production. The
potency test for a batch of inactivated vaccine consists of vaccinating 20 SPF chickens, 4 weeks of age,
and showing that their mean HI titre 4 weeks later is not less than 6 log2.
Vaccine must be shown to have the required potency to achieve the claimed duration of immunity at the
end of the claimed shelf life.
e) Stability
At least three batches should be tested for stability and must give satisfactory results for 3 months beyond
the claimed shelf life.
The stability of a live vaccine must be measured by maintenance of an adequate infectivity titre.
The stability of an inactivated vaccine is measured at intervals by batch potency tests. The concentration of
preservative and persistence through the shelf life should be assessed. There should be no physical
change in the vaccine and it should regain its former emulsion state after one quick shake.
f) Preservatives
There are maximum level requirements for the use of antibiotics, preservatives and residual inactivating
agents.
IBV itself is not known to present any danger to staff employed in vaccine manufacture or testing.
Extraneous agents may be harmful, however, and the initial stages of handling a new seed virus should be
carried out in a safety cabinet. It is a wise precaution with all vaccine production to take steps to minimise
exposure of staff to aerosols of foreign proteins. Persons allergic to egg materials must never be employed
in this work.
a) Safety
A safety test must be carried out on each batch of final product, as in Section C.4.b.
b) Potency
A potency test must be carried out on each batch of final product, as in Section C.4.c, at manufacture and
at the end of the stated shelf life.
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*
* *

CHAPTER 2.3.3.
AVIAN INFECTIOUS LARYNGOTRACHEITIS
SUMMARY
Avian infectious laryngotracheitis (ILT) is a respiratory disease caused by Herpesviridae

alphaherpesvirinae gallid herpesvirus 1. It is principally a disease of chickens, although it can also
affect pheasants, partridges and peafowl. The clinical signs and pathological reactions may vary
from extremely severe, with some birds dying due to asphyxiation, to very mild, indistinguishable
from other mild respiratory diseases of chickens. The principal lesion is tracheitis. In infected birds
the virus can become latent and re-excreted at a later date without clinical signs.
Laboratory diagnosis depends on isolation of the virus, demonstration of the presence of the virus
or viral antigens, and detection of specific antibodies in the serum. Histopathological examination of
the trachea for characteristic intranuclear inclusions may be of value.
Identification of the agent: Virus isolation may be done by inoculation of suspected material on to
the dropped chorioallantoic membrane of embryonated eggs, or into avian embryonic cell cultures.
These methods are time-consuming but sensitive. Rapid methods include direct electron
microscopy on tracheal exudate, immunofluorescence on tracheal exudate or frozen sections, agar
gel immunodiffusion (AGID) to detect viral antigens in tracheal samples or infected egg material,
and an enzyme-linked immunosorbent assay (ELISA) to demonstrate viral antigen in mucosal
scrapings. Polymerase chain reaction (PCR) methodology has been shown to be more sensitive
than virus isolation for the examination of clinical material and is now widely used. Virus
characterisation and differentiation of vaccine and wild-type viruses are possible using PCR
followed by restriction fragment length polymorphism.
Serological tests: Antibodies to ILT virus (ILTV) can be detected by virus neutralisation (VN) tests
conducted in eggs or in cell cultures, or by AGID reactions, indirect immunofluorescence, or ELISA.
The latter is preferred for screening flocks.
Requirements for vaccines and diagnostic biologicals: Vaccines against ILT are usually
prepared from attenuated live virus. Those available at present afford some degree of protection,
but are not completely satisfactory. There have been recent promising studies on the efficacy of a
genetically engineered vaccine.
A. INTRODUCTION
Avian infectious laryngotracheitis (ILT) is a respiratory disease of chickens caused by an alphaherpesvirus. It can
also affect pheasants, partridges and peafowl. In the virulent form, the history, clinical signs and very severe
tracheal lesions are highly characteristic of the disease, but the mild form may be indistinguishable from other
mild respiratory diseases. Laboratory diagnosis depends on the demonstration of the presence of the virus or viral
antigens or products (9, 24, 29) or specific antibodies in the serum (1, 20).
Clinically, the disease may appear in three forms, namely peracute, subacute, and chronic or mild. In the peracute
form, onset of disease is sudden with a rapid spread. The morbidity is high and mortality may exceed 50%. Some
birds may die in good body condition before the appearance of signs, which are characteristic and comprise
difficulty in breathing with extension of the neck and gasping in an attempt to inhale. There is also gurgling,
rattling and coughing when birds try to expel obstructions in the trachea. Clots of blood may be coughed up and
can be found on the floor and walls of the house. Post-mortem changes are confined to the upper respiratory tract
and are also characteristic, consisting of haemorrhagic tracheitis with blood clots, mucoid rhinitis, and blood-
stained mucus along the length of the trachea.

Chapter 2.3.3. — Avian infectious laryngotracheitis
In the subacute form, the onset of illness is slower and respiratory signs may extend over some days before
deaths are seen. The morbidity is high but the mortality is lower than in the peracute form, between 10% and
30%. Post-mortem findings are less severe and consist of mucoid exudate with or without blood in the trachea.
Yellow caseous diphtheritic membranes may be found adherent to the larynx and upper tracheal mucosa.
Chronic or mild ILT may be seen among survivors of either of the above forms of the disease, although some
outbreaks themselves may be entirely mild. Incidence of chronic ILT within a flock may be only 1–2%, with most
affected birds dying of suffocation. Signs include spasms of coughing and gasping, with nasal and oral discharge
and reduced egg production. Infection is acquired via the upper respiratory tract and transmission occurs most
readily from acutely infected birds, but clinically inapparent infection can persist for long periods with intermittent
re-excretion of the virus, and these recovered carrier birds are also a potential means of transmission of the
disease (12). On post-mortem examination, diptheritic and caseous necrotic plaques and plugs are found in the
trachea, larynx and mouth. Outbreaks of mild ILT may affect large numbers of birds simultaneously, in which case
gross lesions may consist only of conjunctivitis, sinusitis and mucoid tracheitis. Given that transmission of ILT
takes place by close contact, transmission is slower in cage houses than where birds are loose-housed, and the
path of infection through a cage house may be apparent. Recent work has confirmed considerable variation
among ILTV strains in their tropism for trachea or conjunctiva and those with affinity for the latter site can severely
affect weight gain (18).
The virus may be isolated in chick embryo liver (19), chicken embryo kidney (6) or in chicken kidney (26) cell
cultures. Of these, monolayers of chicken embryo liver cells have been found to be the most sensitive (11). The
virus can also be grown on the dropped chorioallantoic membrane (CAM) of 10–12-day-old specific pathogen free
embryonated chicken eggs (14).
The causative herpesvirus may be demonstrated directly in tracheal exudate by electron microscopy (26). Viral
antigens may be detected by immunofluorescence (4, 28), agar gel immunodiffusion (AGID) (15), or enzyme-
linked immunosorbent assay (ELISA), using tracheal mucosal scrapings (30). Histopathological examination of
the trachea for typical herpesvirus intranuclear inclusions may also be helpful (3, 23). Methods of detecting ILT
virus (ILTV) using polymerase chain reaction (PCR) have been described and PCR has been reported to be
generally more sensitive than virus isolation (2, 16, 19, 29).
a) Virus isolation
When samples are taken from live birds for virus isolation, tracheal swabs are superior to oropharyngeal or
conjunctival swabs. These are placed in transport medium containing antibiotics. When selecting material for
virus isolation from chronic outbreaks, it is more productive to cull a bird in the early stages of the infection,
rather than to attempt to isolate virus from a bird that has died of asphyxiation after a long illness. The quality
of sample is further improved if the bird is killed by barbiturate or other injection rather than by cervical
dislocation. The whole head and neck from dead birds may be submitted, or only the trachea and larynx
after their removal with minimal contamination. Tracheas should be transported in antibiotic broth for virus
isolation, but wrapped in moist tissue paper if destined for electron microscopy. Any prolonged storage of
infected tissues should be at –70°C or below to minimise loss of virus titre. Repeated freezing and thawing
must be avoided as this reduces virus infectivity.
Exudate and epithelial cells are scraped from the tracheas, diluted approximately 1/5 in nutrient broth
containing penicillin and streptomycin, and agitated vigorously. The resulting suspension is centrifuged at
low speed to remove debris, and 0.1 ml of the supernatant fluid is inoculated on to the dropped CAM of at
least three embryonated chicken eggs of 10–12 days’ incubation. The eggs are sealed with paraffin wax and
incubated at 37°C for up to 7 days. They are candled daily and the CAMs of dead embryos or of those
surviving for 7 days are examined for typical pocks. Alternatively, at least two confluent chick embryo liver or
chicken embryo kidney cell monolayers, with their medium removed, are inoculated and allowed to adsorb
for 1–2 hours. Cultures are overlaid with fresh medium, incubated for up to 7 days and examined daily under
the microscope for evidence of a typical syncytial cell cytopathic effect (CPE).
In each instance, up to three passages of material may be necessary before a specimen is considered to be
negative. A virus isolate can be confirmed as ILTV by a neutralisation test in eggs or cell culture using
hyperimmune antiserum to ILTV. Alternatively, virus particles may be identified rapidly in cell culture fluid or
in pocks on CAMs by electron microscopy, viral antigens by immunofluorescence in acetone-fixed ILT-virus-
infected cell cultures or in frozen sections of CAM and viral nucleic acid by PCR.

b) Electron microscopy
To demonstrate the presence of virus by electron microscopy, tracheal exudate or epithelial scrapings from
the trachea are smeared on to a microscope slide and mixed with a few drops of distilled water. One drop of
suspension is placed on a carbon and formvar-coated grid and left for 2 minutes, after which excess
moisture is removed using filter paper. One drop of 4% phosphotungstic acid, pH 6.4, is added and the
excess removed after a further 3 minutes. The grid is allowed to dry thoroughly and examined using the
electron microscope at a magnification of ×30–45,000 for typical herpesvirus particles, measuring 100 nm
diameter with icosahedral symmetry.
c) Immunofluorescence
In immunofluorescence tests for viral antigens, epithelial cell scrapings from the trachea are smeared on to a
glass slide. Alternatively, 5 µm thick cryostat sections of trachea, snap-frozen in liquid nitrogen may be used.
The preparations are fixed in acetone at room temperature for 10 minutes. These can be stained directly by
applying chicken anti-ILTV immunoglobulin labelled with fluorescein isothiocyanate (FITC) for 1 hour,
followed by rinsing for 15 minutes in a bath of phosphate buffered saline (PBS), pH 7.2, agitated with a
magnetic stirrer. Otherwise, they can be stained indirectly by applying an appropriate dilution of chicken anti-
ILT serum for 1 hour. The slide is rinsed thoroughly with PBS for 15 minutes as above, and an FITC-labelled
anti-chicken immunoglobulin is applied for 30 minutes. After a final rinse, cover-slips are applied over non-
fade mountant. The preparations are examined for specific intranuclear fluorescence in the epithelial cells
using a microscope with epifluorescent ultraviolet illumination. Suitable controls include the use of known
uninfected specimens and, for the indirect method, the application of nonimmune chicken serum. Particular
care should be taken in the reading of indirect immunofluorescence preparations, as endogenous chicken
IgG in the trachea may cause unwanted attachment of FITC-labelled anti-chicken IgG.
d) Agar gel immunodiffusion

ILT viral antigens may be demonstrated by AGID tests on tracheal exudate, infected CAMs and infected cell
culture material using hyperimmune ILTV antiserum. The gel is made with Noble agar (1.5%) containing
sodium chloride (8%) and sodium azide (0.02%) – as preservative – in distilled water. The ingredients are
autoclaved at 15 lb/sq. inch (2.4 bar) for 15 minutes; 5 ml of the molten agar is poured into a 5 cm diameter
Petri dish. When the agar has set, a pattern of wells is punched in the agar, consisting of a central well and
six surrounding wells. The wells are usually 8 mm in diameter and 4 mm apart. The hyperimmune serum is
pipetted into the central well, while the surrounding wells are filled with suspect virus samples under test, but
with at least one well containing positive viral antigen. Dishes are incubated in a humid atmosphere at room
temperature or at 37°C, and examined 24–48 hours later by oblique illumination for lines of precipitation
(reactions of identity). Tests should include uninfected material as negative antigen and known negative
antiserum as controls. For economy of materials, the test can be done on a microscale – the agar being
poured in a thin layer on to a microscope slide and holes punched of 4 mm diameter and 2 mm apart.
e) Enzyme-linked immunosorbent assay

When the monoclonal antibody (MAb) ELISA is used for detecting viral antigens (19), tracheal exudate is
mixed with an equal volume of PBS containing 1% (v/v) of a detergent, such as Nonidet P40 (BDH
Chemicals, Poole, United Kingdom), then vortexed for 30 seconds and centrifuged at 10 g for 1 minute. The
supernatant fluid is dropped in 50 µl volumes in wells of microtitre plates previously coated with rabbit IgG
against ILTV, diluted 1/200 in 0.05 M carbonate/bicarbonate buffer, pH 9.0, and incubated for 1 hour. Next,
50 µl of MAb against major glycoproteins of ILTV, diluted 1/50 in PBS, is added to each well, followed by
50 µl of a 1/1000 dilution of affinity-purified goat anti-mouse IgG conjugated to horseradish peroxidase. The
substrate, 5-aminosalicylic acid (6.5 mM), is added to the wells in 100 µl volumes. After 30 minutes, the
plates are read at 450 nm on a spectrophotometer and the absorbance reading for each well is corrected by
subtracting the reading obtained for wells containing diluting buffer instead of tracheal exudate. The
positive/negative cut-off point is taken as the mean absorbance value for several negative (i.e. tracheal
material without ILTV) samples plus 3 standard deviations.
f) Histopathology
Tracheas for histopathological examination should be placed in formol saline immediately after removal from
the birds and embedded in paraffin wax. Eyelids and lung are sometimes examined. Intranuclear inclusions
may be seen in the epithelial cells of the trachea in longitudinal sections after staining by haematoxylin and
eosin. They are the classical Cowdry type A inclusions of herpesviruses, but they may be present for only 3–
5 days after infection. In severe cases where most infected cells have detached from the tracheal lining,
inclusions may be seen in intact cells among the cellular debris in the lumen of the trachea. Longitudinal
rather than transverse sections of trachea permit examination of the whole length of the organ.
g) Molecular methods
Several molecular methods for identifying ILTV DNA in clinical samples have been reported, but the PCR
has proved he most useful. Dot-blot hybridisation assays and cloned virus DNA fragments were shown to be
highly sensitive for detecting virus when isolation and ELISA were negative (16, 17). Humberd et al. (13),

using a nested PCR, showed that ILTV DNA could be detected in formalin-fixed, paraffin-embedded tissues
independently of the presence of syncytial cells, intranuclear inclusions or both.
PCR has been found to be more sensitive than virus isolation for clinical samples, especially when other
contaminant viruses such as adenoviruses are present (29). Alexander & Nagy (2) found that during the
middle to the end of the infection phase, PCR and virus isolation were similar in sensitivity, but PCR was
superior in the recovery phase.
A problem with the PCR for ILTV was that initially it was not able to differentiate between field and vaccine
strains. However, the combination of PCR with restriction fragment length polymorphism (RFLP) analysis of
single and multiple viral genes and genome regions has enabled the characterisation of different strains
within a country or region (5). Several reports have shown that while some field strains are closely related to
and likely to be derived from vaccines viruses, others are true ‘wild types’ (21). Genes commonly examined
by different international authors include ICP4, TK (thymidine kinase), glycoprotein G (gG), glycoprotein E
(gE) and UL47. Oldoni & Garcia (22) used 36 restriction enzymes, while others have used as few as four.
i) PCR protocol
In a typical PCR protocol for ILTV, viral DNA is extracted from clinical samples (swabs, tissues pieces),
chorioallantoic membrane plaques, cell culture supernatants or vaccines using DNA extraction kits. Primers
used can be obtained from previously published work or designed using ILTV sequences on the Genbank
international database. Amplifications are made using Taq DNA polymerase. Typical amplification reactions
use an initial denaturing step of 94°C for 1 minute followed by 35 amplification cycles of 94°C for 1 minute
with annealing temperatures ranging from 54–60°C for 30 seconds. Extension may be performed at 68°C,
with extension times varying according to the size of the target region amplified and a final extension at 68°C
for 7 minutes. The PCR products are separated by electrophoresis in 1% agarose gels, stained with
ethidium bromide and exposed to UV light for visualisation.
ii) Real-time PCR

Recently a real-time PCR assay had been described for ILTV (7). This has the advantage that, including
amplification and melt-curve analysis, it can be conducted in less than 2 hours. It therefore provides a very
rapid method of ILT diagnosis in comparison with traditional virus isolation, or even the standard PCR
followed by gel electrophoresis.
iii) Restriction fragment length polymorphism (RFLP)

A range of restriction endonucleases (RE) have been described for RFLP analysis of ILTV PCR products
and several genes have been targeted for digestion. They include ICP4, TK (thymidine kinase), UL 15, UL47
glycoprotein G and ORF-BTK genes. Amplification products are digested separately with 10U RE for
3 hours. Digestion fragments are separated in 15% polyacrylamide gels. Fragments are observed after DNA
silver staining and analysed under a light box. Pattern differences are recorded for each enzyme and results
can be developed into dendrograms. The combination of PCR and RFLP has enabled field strains of ILTV to
be distinguished from vaccine strains (7, 10, 21, 26).
Antibodies to ILTV in chicken serum can be detected by virus neutralisation (VN), AGID, indirect
immunofluorescence tests and ELISA (1).
VN tests may be conducted on the dropped CAMs of embryonating chicken eggs that have been incubated
for 9–11 days, where antibody specifically neutralises pock formation due to ILTV. Alternatively, the tests
can be performed in cell cultures, where antibody specifically neutralises the ILTV thus preventing CPE.
Doubling dilutions of serum are added to equal volumes of a constant concentration of virus. This
concentration may either be 100 median egg infectious doses (EID50) for egg inoculations, or 100 median
tissue culture infectious doses (TCID50) for the inoculation of cultures. The mixtures are incubated at 37°C
for 1 hour to allow any neutralisation to occur.
When the test is performed in eggs, the virus/serum mixtures are inoculated on to the dropped CAMs, using
at least five eggs per dilution. Eggs are sealed and incubated at 37°C for 6–7 days. The end-point is
recorded as the highest dilution of the serum where no pocks are present on the CAMs. When the tests are
done in cell cultures, serum dilutions are prepared in 96-well microculture plates and virus is then added.
After the period allowed for neutralisation, freshly prepared chicken embryo liver or kidney cells are added to
each well. The plates are incubated at 37°C in an atmosphere of 5% CO2 and examined daily for CPE; 50%
end-points are read after approximately 4 days when the virus control titre indicates that 30–300 TCID50 of

virus have been used in the test. For the cell culture method of testing, virus neutralisation at 1/8 (initial
dilution) or greater is considered positive.

For AGID tests, antigen is prepared from virus-infected CAMs or infected cell cultures. For the former, at
least 104 TCID50 of ILTV is inoculated into the allantoic cavity of a batch of 10-day-old embryonating specific
pathogen free (SPF) chicken eggs. The CAMs are harvested after 4 days’ incubation, and those with large
pocks are homogenised and sonicated in a small amount of PBS, pH 7.1. Alternatively, heavily infected
monolayers of chicken embryo liver or kidney, or chicken kidney cells are incubated at 37°C until the CPE is
maximal. Any remaining attached cells are scraped from the culture vessel into the medium. Total culture
harvests may be concentrated up to 100-fold by dialysis against polyethylene glycol (PEG 20,000 or PEG
30,000). For the test, the agar is prepared as described previously for antigen detection, but this time the
CAM or cell culture antigen is placed in the central well with test sera in the surrounding wells. Known
positive and negative antisera are incorporated in the test, which is read after 24–48 hours’ incubation at
room temperature or at 37°C. AGID tests are simple, economical to perform, and useful for flock screening,
although they are less sensitive than the other methods.
c) Indirect fluorescent antibody test

For indirect fluorescent antibody tests, the antigen consists of ILT-virus-infected cell culture monolayers
grown on teflon-coated multispot slides. When CPE is beginning to develop, the cultures are fixed in acetone
for 10 minutes. Dilutions of test sera prepared in PBS are applied to each spot culture and the slides are
incubated at 37°C for 1 hour. The slides are washed in PBS as described previously, drained and treated
with an appropriate dilution of a commercial FITC-labelled rabbit anti-chicken IgG. After incubation at 37°C
for 1 hour, the slides are rewashed and cover-slips are applied over a non-fade mountant. They are
examined by epifluorescence with ultraviolet illumination, and end-point titres are read as the highest serum
dilutions giving specific fluorescent staining. This test is more sensitive than AGID, but the interpretation of
results may be subjective.
d) Enzyme-linked immunosorbent assay

The antigen for ELISA is obtained by sonication of heavily infected cell cultures at the time of maximum
CPE, which is then absorbed on to the wells of microtitre plates. A negative antigen is provided by
uninfected cell culture material treated in the same way. The test consists essentially of the addition of
0.1 ml of 1/10 dilutions of test sera to duplicate wells coated with positive or negative antigen. After
incubation at 37°C for 2 hours, the plates are washed four times and a 1/4000 dilution of a rabbit anti-
chicken IgG conjugated with peroxidase is added. After incubation at 37°C for 1 hour, the plates are washed
again four times. Finally a substrate consisting of 5-aminosalicylic acid is added to each well followed by
hydrogen peroxide to a final concentration of 0.0005%, and the absorbance of the fluid in each well is read
at 450 nm on a spectrophotometer. The result for each serum is expressed as the difference between the
mean absorbance produced with the positive and negative antigens. The positive/negative cut-off point is
taken as the mean absorbance value for numerous negative sera plus 3 standard deviations. The test is
very sensitive and possibly the best available for surveillance purposes. Antibody responses as measured
by ELISA are detectable 7–10 days after infection and peak at about 2 weeks. The response to ILT vaccines
may be variable and testing is not worthwhile before 14 days post-vaccination. Several laryngotracheitis
antibody ELISA kits are available commercially.

ILT is usually controlled with live vaccines, although inactivated vaccines have also been used for safety reasons.
There has been some recent work with genetically engineered vaccines and the results of the initial studies look
promising (8, 27). The live virus seed is a suitably attenuated or naturally avirulent strain of ILTV. Vaccines may
be administered by eye-drop, spray or in the drinking water. If administered by spray and a small droplet size is
produced and inhaled, clinical disese may be precipitated. Young chickens may require vaccinating in endemic
areas, but show more severe reactions to the vaccine. Repeated doses may be required to afford good
protection. The level of virulence of the vaccine virus is critical. Strains of low virulence may not be effective, and
those of higher virulence may cause severe disease. The spray route of administration requires care over droplet
size and uniformity of application. It can be more effective with low virulence strains, but may be more dangerous
with high virulence strains. At present, the available vaccines attempt to make a compromise between lack of
efficacy and poor safety. Because of persistence of virulent vaccine virus on a site, it may be difficult to
discontinue vaccination once it has been started. Subclinical mixed infections of vaccine and field virus, in
vaccinated birds, can cause severe disease in unvaccinated in-contacts.
supplemented by national and regional requirements (e.g. ref. 25).

1. Seed management

The master seed virus (MSV) is selected and can be propagated in SPF chicken embryos or tissue cultures
derived from such embryos. The MSV is tested in chicken embryos or chickens for the following: 1) purity,
2) Mycoplasma spp., 3) Salmonella spp., 4) avian leukosis virus, 5) haemagglutinating viruses, 6) virus
identity, and 7) extraneous pathogens. Additionally, initial tests are performed to demonstrate the safety and
efficacy of the chosen master seed. The safety test on the MSV must include tests to show lack of reversion
to virulence on serial passage and also safety in birds. Evidence of shed and spread is also required. The
MSV is stored in aliquots at –70°C. The MSV should not cause mortality or a severe respiratory reaction in
chickens following ocular instillation, although pheasants are more susceptible. Administration by spray is
convenient but may cause quite severe respiratory disease in some flocks.
In large-scale vaccine production, the virus is propagated in SPF chicken embryos or tissue culture derived
from such embryos, up to the fifth passage from the MSV. The acceptable passage level is supported
experimentally by the passage level used to prepare the experimental product used in the efficacy study.
A test must be carried out to establish the efficacy of the vaccine in birds of the minimum age for which the
product is destined and also for each avian species. This is repeated in further batches of chickens for each
of the recommended routes of administration and/or age of bird. Three weeks later (or 10–14 days in the
USA), the birds, together with ten controls of the same age and source, are challenged intratracheally or in
the orbital sinus with a strain of ILTV of known high virulence. To be satisfactory, only 5% of the vaccinated
birds should die or show severe signs of ILT. No more than four should show mild signs of ILT. At least eight
of the controls should die or show severe signs of ILT.
The vaccine is made by inoculation of the production seed virus into 9–11-day-old chicken embryos or tissue
culture prepared from chicken embryos derived from SPF flocks. Eggs are inoculated through a hole in the shell,
on to the dropped CAM. They are sealed and incubated at 37°C for 4–6 days. All eggs are candled before harvest
and only those with living embryos are used. To harvest the virus, the eggs are chilled, then cleansed and opened
aseptically. The CAMs and fluids are pooled in sterile, cooled containers. The CAMs should show the thick grey
plaques typical of ILTV growth. Tissue culture-derived product would be prepared from virus-bearing cell culture
fluids, which would also be subsequently pooled and tested.
The infected tissue or tissue culture homogenate may be tested for purity, potency, and virus content, mixed with
a stabiliser (usually beef peptone and sucrose) and then lyophilised and stored at 4°C.
4. Batch control
a) Sterility
b) Safety
Using the recommended route of administration, each batch of vaccine is tested in ten SPF chickens, or ten
birds of other target species, using ten doses per bird. The birds are observed for at least 21 days for
adverse effects attributable to the vaccine.
c) Potency
Once the in-vivo efficacy of the vaccine has been established, the batch potency may be determined by
measuring the virus content. Serial dilutions of the vaccine are inoculated on to the dropped CAM of 9–
11-day-old SPF chicken embryos, using at least seven eggs per dilution, in a volume of 100 µl. The eggs are
incubated for 5 days and the virus titre is calculated by observing characteristic lesions on the CAMs. The
virus content should be at or above a release value and above and expiration titre during dating of the
product. Both the release and expiration titres are based on the minimum protective dose described above.

The results of vaccination will depend on many factors, including dose schedule and route of administration.
Some degree of protection should be given, over a period of several months.
e) Stability
Stability is tested by taking samples of correctly stored vaccine at intervals and measuring virus content.
Tests should be carried out on at least six batches of the vaccine or until a statistically valid number of
serials have been evaluated and be continued for 3 months after the claimed shelf-life.
f) Preservatives
Preservatives may not be required, but some antibiotics may be added to the tissue harvest or at serial
assembly during manufacture. For products licensed in the USA, any antibiotics added are listed on the
label.
Care should be taken over diluting and administering the vaccine, and over the proper disposal of unused
vaccine.
a) Safety
In the USA, 25 susceptible chickens are injected intratracheally and observed for 14 days. Deaths are
counted as failures. Four or fewer failures are allowed for satisfactory serials. In the European Union, tests
of virus content are carried out. The virus titre shall normally be no higher than one-tenth of the dose at
which the vaccine has been shown to be safe.
b) Potency
The test of virus content (see above) may also be used as a measure of potency. It must be no lower than
the agreed minimum release titre. Each serial or subserial shall have a virus titre of 107 greater than the
minimum protective dose, but not less than 1025 EID50 (or TCID50 for tissue culture prepared product)/dose.
c) Tests of final product

The lack of chicken pathogens should be confirmed in embryos or chickens. It should also be confirmed by
testing for Mycoplasma spp., Salmonella spp., avian leukosis virus, and haemagglutinating viruses.
REFERENCES
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2. ALEXANDER H.S. & NAGY E. (1997). Polymerase chain reaction to detect infectious laryngotracheitis virus in
conjunctival swabs from experimentally infected chickens. Avian Dis., 41, 646–653.
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84.
4. BRAUNE M.O. & GENTRY R.F. (1985). Standardization of the fluorescent antibody technique for the detection
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5. CHANG P.C., LEE Y.L., SHIEN J.H. & SHIEH H.K. (1997). Rapid differentiation of vaccine strains and field
isolates of infectious laryngotracheitis virus by restriction fragment length polymorphism of PCR products.
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6. CHANG P.W., YATES V.J., DARDIRI A.H. & FRY D.E. (1960). Some observations of the propagation of infectious
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length. Avian Pathol., 35, 173–179.

8. DAVISON S., GINGERICH, E.N., CASAVANT S. & ECKROADE R.J. (2006). Evaluation of the efficacy of a live
fowpox-vectored infectious laryngotracheitis/avian encephalomyelitis vaccine against ILT viral challenge.
Avian Dis., 50, 50–54.
9. GUY J.S. & BAGUST T.J. (2003) Laryngotracheitis. In: Diseases of Poultry, Eleventh Edition, Saif Y.M., Barnes
H.J., Glisson J.R., Fadly A.M., McDougald L.R. & Swayne D., eds. Iowa State University Press, USA, 124–
134.
10. HAN M.G. & SIM S.J. (2001). Analysis of Korean strains of infectious laryngotracheitis virus by nucleotide
sequences and restriction fragment length polymorphism. Vet. Microbiol., 83, 321–331.
11. HUGHES C.S. & JONES R.C. (1988). Comparison of methods of isolation of infectious laryngotracheitis from
field material. Avian Pathol., 17, 295–303.
12. HUGHES C.S., JONES R.C., GASKELL R.M., JORDAN F.T.W. & BRADBURY J.M. (1987). Demonstration in live
chickens of the carrier state in infectious laryngotracheitis virus. Res. Vet. Sci., 42, 407–410.
13. HUMBERD J., GARCIA M., RIBLET S.M., RESURECCION R.S. & BROWN T.P. (2002). Detection of infectious
laryngotracheitis virus in formalin-fixed, paraffin-embedded tissues by nested polymerase chain reaction.
Avian Dis., 46, 64–74.
14. JORDAN F.T.W. (1964). Diagnosis of infectious laryngotracheitis by chick embryo inoculation. J. Comp.
Pathol., 74, 119–128.
15. JORDAN F.T.W. & CHUBB R.C. (1962). The agar gel diffusion technique in the diagnosis of infectious
laryngotracheitis (ILT) and its differentiation from fowl pox. Res. Vet. Sci., 3, 245–255.
16. KEAM L., YORK J.J., SHEPPARD M. & FAHEY K.J. (1991). Detection of infectious laryngotracheitis virus in
chickens using a non-radioactive DNA probe. Avian Dis., 35, 257–262.
17. KEY D.W., GOUGH B.C., DERBYSHIRE J.B. & NAGY E. (1994). Development and evaluation of a non-isotypically
labeled DNA probe for the diagnosis of infectious laryngotracheitis. Avian Dis., 38, 467–474.
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of field isolates of infectious laryngotracheitis virus from Ontario. Avian Pathol., 35, 286–292.
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regions. Avian Pathol., 36, 167–176.
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25. UNITED STATES DEPARTMENT OF AGRICULTURE (2000). Code of Federal Regulations, Part 9, Section 113.328,
Fowl Laryngotracheitis Vaccine. US Government Printing Office. Washington DC, USA.
26. VAN KAMMEN A. & SPADBROW P.B. (1976). Rapid diagnosis of some avian virus diseases. Avian Dis., 20,
748–751.
27. VEITS J., LUSCHOW D., KINDERMANN K., W ERNER O., TEIFKE J. P., METTENLEITER TC. & FUCHS W. (2003).
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plague. J. Gen. Virol., 84, 3343–3352.

28. W ILKS C.R. & KOGAN V.G. (1979). An immunofluorescence diagnostic test for avian infectious
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29. W ILLIAMS R.A., SAVAGE C.E. & JONES R.C. (1994). A comparison of direct electron microscopy, virus isolation
and a DNA amplification method for the detection of avian infectious laryngotracheitis virus in field material.
30. YORK J.J. & FAHEY K.J. (1988). Diagnosis of infectious laryngotracheitis using a monoclonal antibody ELISA.
*
* *

1 CHAPTER 2.3.4.
2 AVIAN INFLUENZA
3 SUMMARY
4 Avian influenza (AI) is caused by specified viruses that are members of the family Orthomyxoviridae
5 and placed in the genus influenzavirus A. There are three influenza genera – A, B and C; only
6 influenza A viruses are known to infect birds. Diagnosis is by isolation or detection and
7 characterisation of the virus. This is because infections in birds can give rise to a wide variety of
8 clinical signs that may vary according to the host, strain of virus, the host’s immune status,
9 presence of any secondary exacerbating organisms and environmental conditions.
10 Identification of the agent: Suspensions in antibiotic solution of oropharyngeal and cloacal swabs
11 (or faeces) taken from live birds, or of faeces and pooled samples of organs from dead birds, are
12 inoculated into the allantoic cavity of 9– to 11-day-old embryonated fowl eggs. The eggs are
13 incubated at 35–37°C for 4–7 days. The allantoic fluid of any eggs containing dead or dying
14 embryos during the incubation and all eggs at the end of the incubation period are tested for the
15 presence of haemagglutinating activity. The presence of influenza A virus can be confirmed by an
16 immunodiffusion test between concentrated virus and an antiserum to the nucleocapsid and/or
17 matrix antigens, both of which are common to all influenza A viruses. Isolation in embryos has
18 recently been replaced, under certain circumstances, by reverse-transcription polymerase chain
19 reaction (RT-PCR).
20 For subtyping the virus, the laboratory must have monospecific antisera prepared against the
21 isolated antigens of each of the 16 haemagglutinin (H1–H16) and 9 neuraminidase (N1–N9)
22 subtypes of influenza A viruses that can be used in immunodiffusion tests. Alternatively, the newly
23 isolated virus may be examined by haemagglutination and neuraminidase inhibition tests against a
24 battery of polyclonal antisera to a wide range of strains covering all the subtypes.
25 As the term highly pathogenic avian influenza and the historical term ‘fowl plague’ refer to infection
26 with virulent strains of influenza A virus, it is necessary to assess the virulence of an isolate for
27 domestic poultry. Any highly pathogenic avian influenza isolate is classified as notifiable avian
28 influenza (NAI) virus. Although all virulent strains isolated to date have been either of the H5 or H7
29 subtype, most H5 or H7 isolates have been of low virulence. Due to the risk of a low virulent H5 or
30 H7 becoming virulent by mutation in poultry hosts, all H5 and H7 viruses have also been classified
31 as NAI viruses. The methods used for the determination of strain virulence for birds have evolved
32 over recent years with a greater understanding of the molecular basis of pathogenicity, but still
33 primarily involve the inoculation of a minimum of eight susceptible 4–8-week-old chickens with
34 infectious virus; strains are considered to be highly pathogenic if they cause more than 75%
35 mortality within 10 days or have an intravenous pathogenicity index (IVPI) of greater than 1.2.
36 Characterisation of suspected virulent strains of the virus should be conducted in a virus-secure
37 laboratory. All virulent AI isolates are identified as highly pathogenic notifiable avian influenza
38 (HPNAI) viruses. Regardless of their virulence for chickens, H5 or H7 viruses with an HA0 cleavage
39 site amino acid sequence similar to any of those that have been observed in virulent viruses are
40 considered HPNAI viruses. H5 and H7 isolates that are not pathogenic for chickens and do not
41 have an HA0 cleavage site amino acid sequence similar to any of those that have been observed in
42 HPNAI viruses are identified as low pathogenicity notifiable avian influenza (LPNAI) viruses and
43 non-H5 or non-H7 AI isolates that are not highly pathogenic for chickens are identified as low
44 pathogenicity avian influenza (LPAI) viruses.
45 Serological tests: As all influenza A viruses have antigenically similar nucleocapsid and matrix
46 antigens, agar gel immunodiffusion tests are used to detect antibodies to these antigens.
47 Concentrated virus preparations containing either or both type of antigens are used in such tests.
48 Not all species of birds develop demonstrable precipitating antibodies. Haemagglutination inhibition

Chapter 2.3.4. — Avian influenza
49 tests have also been employed in routine diagnostic serology, but it is possible that this technique
50 may miss some particular infections because the haemagglutinin is subtype specific. Enzyme-
51 linked immunosorbent assays have been used to detect antibodies to influenza A type-specific
52 antigens.
53 Requirements for vaccines and diagnostic biologicals: Historically, in most countries, vaccines
54 specifically designed to contain or prevent HPNAI were banned or discouraged by government
55 agencies because they may interfere with stamping-out control policies. During the 1990s the
56 prophylactic use of inactivated oil-emulsion vaccines was employed in Mexico and Pakistan to
57 control widespread outbreaks of NAI, and a recombinant fowl poxvirus vaccine expressing the
58 homologous HA gene was also used in Mexico, El Salvador and Guatemala. During the 1999–2001
59 outbreak of LPNAI in Italy, an inactivated vaccine was used with the same haemagglutinin type as
60 the field virus, but with a different neuraminidase. This allowed the differentiation of vaccinated
61 birds from birds infected with the field virus and ultimately resulted in eradication of the field virus.
62 Prophylactic use of H5 and H7 vaccines has been practised in parts of Italy aimed at preventing
63 LPNAI infections and several countries in SE Asia have used prophylactic vaccination as an aid in
64 controlling HPNAI H5N1 virus infections. HPNAI viruses should not be used as the seed virus for
65 production of vaccine.
66 If HPNAI is used in challenge studies, the facility should meet the OIE requirements for
67 Containment Group 4 pathogens.
68 A. INTRODUCTION
69 Notifiable avian influenza (NAI) is caused by infection with viruses of the family Orthomyxoviridae placed in the
70 genus influenzavirus A. Influenza A viruses are the only orthomyxoviruses known to affect birds. Many species of
71 birds have been shown to be susceptible to infection with influenza A viruses; aquatic birds form a major reservoir
72 of these viruses, but the overwhelming majority of isolates have been of low pathogenicity for chickens and
73 turkeys,. Influenza A viruses have antigenically related nucleocapsid and matrix proteins, but are classified into
74 subtypes on the basis of their haemagglutinin (H) and neuraminidase (N) antigens (80). At present, 16 H subtypes
75 (H1–H16) and 9 N subtypes (N1–N9) are recognised. To date, the highly virulent influenza A viruses that produce
76 acute clinical disease in chickens, turkeys and other birds of economic importance have been associated only
77 with the H5 and H7 subtypes. Most viruses of H5 and H7 subtype isolated from birds, have been of low virulence
78 for poultry (2). Due to the risk of a H5 or H7 virus of low virulence becoming virulent by mutation, all H5 and H7
79 viruses have been identified as notifiable avian influenza (NAI) viruses (81).
80 Depending on the species, age and type of bird, specific characteristics of the viral strain involved, and on
81 environmental factors, the highly pathogenic disease, in fully susceptible birds, may vary from one of sudden
82 death with little or no overt clinical signs to a more characteristic disease with variable clinical presentations
83 including respiratory signs, such as ocular and nasal discharges, coughing, snicking and dyspnoea, swelling of
84 the sinuses and/or head, apathy, reduced vocalisation, marked reduction in feed and water intake, cyanosis of the
85 unfeathered skin, wattles and comb, incoordination and nervous signs and diarrhoea. In laying birds additional
86 clinical features include a marked drop in egg production usually accompanied by an increase in numbers of poor
87 quality eggs. Typically, high morbidity is accompanied by high and rapidly escalating unexplained mortality.
88 However, none of these signs can be considered pathognomonic. In addition, low pathogenicity avian influenza
89 (LPAI) viruses, which normally cause only a mild or no clinical disease, may in certain circumstances produce a
90 spectrum of clinical signs the severity of which may approach that of highly pathogenic avian influenza,
91 particularly if exacerbating infections are present. Confirmatory diagnosis of the disease, therefore, depends on
92 the isolation of the causal virus and the demonstration that it fulfils one of the defined criteria described in section
93 B.2. In some specific circumstances this may be achieved by detection of the virus in the infected host; especially
94 using molecular techniques that allow the determination of virus virulence. Testing sera from suspect birds using
95 antibody detection methods may supplement diagnosis, but these methods are not suitable for a detailed
96 identification. Diagnosis for official control purposes is established on the basis of agreed official criteria for
97 pathogenicity according to in vivo tests or to molecular determinants (i.e. the presence of multiple basic amino
98 acids at the cleavage site of the haemagglutinin precursor protein HA0) and haemagglutinin typing. These
99 definitions evolve as scientific knowledge of the disease increases.
100 HPNAI and NAI are subject to official control and the virus has a high risk of spread from the laboratory;
101 consequently, a risk assessment should be carried out to determine the level of biosecurity needed for laboratory
102 diagnosis and chicken inoculation; characterisation of the virus should be conducted at biocontainment level 3 (at
103 least). The facility should meet the requirements for the appropriate Containment Group as determined by the risk
104 assessment and as outlined in Chapter 1.1.2 Biosafety and biosecurity in the veterinary microbiology laboratory
105 and animal facilities. Countries lacking access to such a specialised national or regional laboratory should send
106 specimens to an OIE Reference Laboratory.

107 B. DIAGNOSTIC TECHNIQUES
108 1. Identification of the agent (the prescribed test for international trade)
109 Samples taken from dead birds should include intestinal contents (faeces) or cloacal swabs and oropharyngeal
110 swabs. Samples from trachea, lungs, air sacs, intestine, spleen, kidney, brain, liver and heart should also be
111 collected and processed either separately or as a pool.
112 Samples from live birds should include both oropharyngeal and cloacal swabs. To avoid harming them, swabbing
113 of small delicate birds should be done with the use of especially small swabs that are usually commercially
114 available and intended for use in humans. Where these are not available, the collection of fresh faeces may serve
115 as an alternative.
116 The samples should be placed in isotonic phosphate buffered saline (PBS), pH 7.0–7.4 with antibiotics or a
117 solution containing protein and antibiotics. The antibiotics can be varied according to local conditions, but could
118 be, for example, penicillin (2000 units/ml), streptomycin (2 mg/ml), gentamycin (50 µg/ml) and mycostatin
119 (1000 units/ml) for tissues and oropharyngeal swabs, but at five-fold higher concentrations for faeces and cloacal
120 swabs. It is important to readjust the pH of the solution to pH 7.0–7.4 following the addition of the antibiotics. It is
121 recommended that a solution for transport of the swabs should contain protein to stabilise the virus (e.g. brain–
122 heart infusion, cattle serum up to 5% [v/v] or bovine albumen – 0.5% [w/v]). Faeces and finely minced tissues
123 should be prepared as 10–20% (w/v) suspensions in the antibiotic solution. Suspensions should be processed as
124 soon as possible after incubation for 1–2 hours at room temperature. When immediate processing is
125 impracticable, samples may be stored at 4°C for up to 4 days. For prolonged storage, diagnostic samples and
126 isolates should be kept at –80°C. Repeated freezing and thawing should be avoided.
127 The preferred method of growing avian influenza A viruses is by the inoculation of embryonated specific pathogen
128 free (SPF) fowl eggs, or specific antibody negative (SAN) eggs. The supernatant fluids of faeces or tissue
129 suspensions obtained through clarification by centrifugation at 1000 g are inoculated into the allantoic sac of at
130 least five embryonated SPF or SAN fowl eggs of 9–11 days’ incubation. The eggs are incubated at 35–37°C for
131 4–7 days. Eggs containing dead or dying embryos as they arise, and all eggs remaining at the end of the
132 incubation period, should first be chilled to 4°C and the allantoic fluids should then be tested for
133 haemagglutination (HA) activity (see Section B.3.b). Detection of HA activity, in bacteria-free amnio-allantoic
134 fluids, indicates a high probability of the presence of an influenza A virus or of an avian paramyxovirus. Fluids that
135 give a negative reaction should be passaged into at least one further batch of eggs.
136 The presence of influenza A virus can be confirmed in agar gel immunodiffusion (AGID) tests by demonstrating
137 the presence of the nucleocapsid or matrix antigens, both of which are common to all influenza A viruses (see
138 Section B.3.a). The antigens may be prepared by concentrating the virus from infective allantoic fluid or extracting
139 the infected chorioallantoic membranes; these are tested against known positive antisera. Virus may be
140 concentrated from infective allantoic fluid by ultracentrifugation, or by precipitation under acid conditions. The
141 latter method consists of the addition of 1.0 M HCl to infective allantoic fluid until it is approximately pH 4.0. The
142 mixture is placed in an ice bath for 1 hour and then clarified by centrifugation at 1000 g at 4°C. The supernatant
143 fluid is discarded. The virus concentrates are resuspended in glycin/sarcosyl buffer: this consists of 1% (w/v)
144 sodium lauroyl sarcosinate buffered to pH 9.0 with 0.5 M glycine. These concentrates contain both nucleocapsid
145 and matrix polypeptides.
146 Preparations of nucleocapsid-rich antigen can also be obtained from chorioallantoic membranes for use in the
147 AGID test (7). This method involves removal of the chorioallantoic membranes from infected eggs that have
148 allantoic fluids with HA activity. The membranes are then homogenised or ground to a paste. This is subjected to
149 three freeze–thaw cycles, followed by centrifugation at 1000 g for 10 minutes. The pellet is discarded and the
150 supernatant is used as an antigen following treatment with 0.1% formalin.
151 Use of the AGID test to demonstrate nucleocapsid or matrix antigens is a satisfactory way to indicate the
152 presence of avian influenza virus in amnioallantoic fluid, but various enzyme-linked immunosorbent assays
153 (ELISAs) are also available. There is a sensitive and specific ELISA that demonstrates nucleoprotein of type A
154 influenza virus using a monoclonal antibody against type A influenza nucleoprotein (47, 49, 64). This is available
155 as a commercial kit.
156 Any HA activity of sterile fluids harvested from the inoculated eggs is most likely to be due to an influenza A virus
157 or to an avian paramyxovirus (a few strains of avian reovirus will do this, or nonsterile fluid could contain HA of
158 bacterial origin). There are currently nine recognised serotypes of avian paramyxoviruses. Most laboratories will
159 have antiserum specific for Newcastle disease virus (avian paramyxovirus type 1), and in view of its widespread
160 occurrence and almost universal use as a live vaccine in poultry, it is best to evaluate its presence by
161 haemagglutination inhibition (HI) tests (see Chapter 2.3.14 Newcastle disease).

162 Alternatively, the presence of influenza virus can be confirmed by the use of reverse-transcription polymerase
163 chain reaction (RT-PCR) using nucleoprotein-specific or matrix-specific conserved primers (3, 53). Also, the
164 presence of subtype H5 or H7 influenza virus can be confirmed by using H5- or H7-specific primers (21, 46, 53,
165 79).
166 The method recommended for definitive antigenic subtyping of influenza A viruses by the World Health
167 Organization (WHO) Expert Committee (80) involves the use of highly specific antisera, prepared in an animal
168 giving minimum nonspecific reactions (e.g. goat), directed against the H and N subtypes (45). An alternative
169 technique is the use of polyclonal antisera raised against a battery of intact influenza viruses. Subtype
170 identification by this technique is beyond the scope of most diagnostic laboratories not specialising in influenza
171 viruses. Assistance is available from the OIE Reference Laboratories (see Table given in Part 3 of this Terrestrial
172 Manual).
173 2. Assessment of pathogenicity
174 The term highly pathogenic avian influenza relates to the assessment of virulence in chickens and implies the
175 involvement of virulent strains of virus. It is used to describe a disease of fully susceptible chickens with clinical
176 signs such as ocular and nasal discharges, coughing, snicking and dyspnoea, swelling of the sinuses and/or
177 head, apathy, reduced vocalisation, marked reduction in feed and water intake, cyanosis of the unfeathered skin,
178 wattles and comb, incoordination and nervous signs and diarrhoea. In laying birds additional clinical features
179 include a marked drop in egg production usually accompanied by an increase in numbers of poor quality eggs.
180 Typically, high morbidity is accompanied by high and rapidly escalating unexplained mortality. However, none of
181 these signs can be considered pathognomonic and high mortality may occur in their absence. In addition, low
182 pathogenicity avian influenza (LPAI) viruses that normally cause only a mild or no clinical disease, may cause a
183 much more severe disease if exacerbating infections or adverse environmental factors are present and, in certain
184 circumstances, the spectrum of clinical signs may mimic highly pathogenic avian influenza. At the First
185 International Symposium on Avian Influenza held in 1981 (5), it was resolved to abandon the term ‘fowl plague’
186 and to define highly pathogenic avian influenza strains on the basis of their ability to produce not less than 75%
187 mortality within 8 days in at least eight susceptible 4–8-week-old chickens inoculated by the intramuscular,
188 intravenous or caudal air sac route. However, this definition proved unsatisfactory when applied to the viruses
189 responsible for the widespread outbreaks in chickens occurring in 1983 in Pennsylvania and the surrounding
190 states of the United States of America (USA). The problem was mainly caused by the presence of a virus of
191 demonstrable low pathogenicity in laboratory tests, but which was shown to be fully pathogenic following a single
192 point mutation. Further consideration of a definition to include such ‘potentially pathogenic’ viruses was
193 undertaken by several international groups.
194 The eventual recommendations made were based on the finding that while there have been numerous isolations
195 of strains of H5 and H7 subtypes of low pathogenicity, all the highly pathogenic avian influenza strains isolated to
196 date have possessed either the H5 or H7 haemagglutinin. Further information concerning the pathogenicity or
197 potential pathogenicity of H5 and H7 subtypes may be obtained by sequencing the genome, as pathogenicity is
198 associated with the presence of multiple basic amino acids (arginine or lysine) at the cleavage site of the
199 haemagglutinin. For example, most H7 subtype viruses of low virulence have had the amino acid motif at the HA0
200 cleavage site of either -PEIPKGR*GLF- or -PENPKGR*GLF-, whereas examples of amino acids motifs for highly
201 pathogenic avian influenza H7 viruses are: -PEIPKKKKR*GLF-, -PETPKRKRKR*GLF-, -PEIPKKREKR*GLF-,
202 -PETPKRRRR*GLF-. Amino acid sequencing of the cleavage sites of H5 and H7 subtype influenza isolates of low
203 virulence for birds should identify viruses that, like the Pennsylvania virus, have the capacity, following simple
204 mutation, to become highly pathogenic for poultry. In 1992, the OIE adopted criteria for classifying an avian
205 influenza virus as highly pathogenic based on pathogenicity in chickens, growth in cell culture and the amino acid
206 sequence for the connected peptide (41). The European Union adopted similar criteria in 1992 (16).
207 The following criteria, which are a modification of the previous OIE procedure, have been adopted by the OIE for
208 classifying an avian influenza virus as HPNAI:
209 a) One of the two following methods to determine pathogenicity in chickens is used. A HPNAI virus is:
210 i) any influenza virus that is lethal 1 for six, seven or eight of eight 4– to 8-week-old susceptible chickens
211 within 10 days following intravenous inoculation with 0.2 ml of a 1/10 dilution of a bacteria-free, infective
212 allantoic fluid
213 or
214 ii) any virus that has an intravenous pathogenicity index (IVPI) greater than 1.2. The following is the IVPI
215 procedure:
1 When birds are too sick to eat or drink, they should be killed humanely.

216 Fresh infective allantoic fluid with a HA titre >1/16 (>24 or >log2 4 when expressed as the reciprocal)
217 is diluted 1/10 in sterile isotonic saline.
218 0.1 ml of the diluted virus is injected intravenously into each of ten 6-week-old SPF or SAN chickens.
219 Birds are examined at 24-hour intervals for 10 days. At each observation, each bird is scored 0 if
220 normal, 1 if sick, 2 if severely sick, 3 if dead. (The judgement of sick and severely sick birds is a
221 subjective clinical assessment. Normally, ‘sick’ birds would show one of the following signs and
222 ‘severely sick’ more than one of the following signs: respiratory involvement, depression, diarrhoea,
223 cyanosis of the exposed skin or wattles, oedema of the face and/or head, nervous signs. Dead
224 individuals must be scored as 3 at each of the remaining daily observations after death 2.)
225 The intravenous pathogenicity index (IVPI) is the mean score per bird per observation over the 10-
226 day period. An index of 3.00 means that all birds died within 24 hours, and an index of 0.00 means
227 that no bird showed any clinical sign during the 10-day observation period.
228 b) For all H5 and H7 viruses of low pathogenicity in chickens, the amino acid sequence of the connecting
229 peptide of the haemagglutinin must be determined. If the sequence is similar to that observed for other
230 highly pathogenic AI isolates, the isolate being tested will be considered to be highly pathogenic.
231 The OIE has the following classification system to identify viruses for which disease reporting and control
232 measures should be taken (81):
233 a) All AI isolates that meet the above criteria are identified as highly pathogenic notifiable avian influenza
234 (HPNAI).
235 b) H5 and H7 isolates that are not virulent for chickens and do not have an HA0 cleavage site amino acid
236 sequence similar to any of those that have been observed in HPNAI viruses are identified as low
237 pathogenicity notifiable avian influenza (LPNAI).
238 c) Non-H5 or non-H7 AI isolates that are not virulent for chickens are identified as low pathogenicity avian
239 influenza (LPAI).
240 A variety of strategies and techniques have been used successfully to sequence the nucleotides at that portion of
241 the HA gene coding for the cleavage site region of the haemagglutinin of H5 and H7 subtypes of avian influenza,
242 enabling the amino acids there to be deduced. The most commonly used method has been RT-PCR using
243 oligonucleotide primers complementing areas of the gene either side of the cleavage site coding region, followed
244 by cycle sequencing (78). Various stages in the procedure can be facilitated using commercially available kits and
245 automatic sequencers.
246 Now the presence of multiple basic amino acids at the HA0 cleavage site is well-established as an accurate
247 indicator of virulence or potential virulence for H5 and H7 influenza viruses, it appears inevitable that
248 determination of the cleavage site by sequencing or other methods will become the method of choice for initial
249 assessment of the virulence of these viruses and incorporated into agreed definitions. This will have the
250 advantage of reducing the number of in vivo tests, although at present the inoculation of birds is still required to
251 confirm a negative result as the possibility of virus cultures containing mixed populations of viruses of high and
252 low virulence cannot be ruled out.
253 Although all the truly highly pathogenic AI viruses isolated to date have been of H5 or H7 subtypes, at least two
254 isolates, both of H10 subtype (H10N4 and H10N5), have been reported that would have fulfilled both the OIE and
255 EU definitions for highly pathogenic AI viruses (76) as they killed 7/10 and 8/10 chickens with IVPI values >1.2
256 when the birds were inoculated intravenously. These viruses did not induce death or signs of disease when
257 inoculated intranasally and did not have multiple basic amino acids at their haemagglutinin cleavage sites. It
258 appears that some H10 AI viruses are nephrotropic and birds that die have high titre virus in their kidneys
259 indicating a renal pathogenic mechanism (50). Conversely, four viruses have been described that have HA0
260 cleavage sites containing multiple basic amino acids, but which show low virulence (IVPI <1.2) when inoculated
261 into 6-week-old chickens intravenously (33). Other anomalies are the Chile 2002 (57) and the Canada 2004 (42)
262 H7N3 HPAI viruses, which show distinct and unusual cleavage site amino acid sequences of
263 PEKPKTCSPLSRCRETR*GLF and PENPKQAYRKRMTR*GLF, respectively. These viruses appear to have
264 arisen as a result of a recombination event between the HA gene and nucleoprotein gene and matrix gene,
265 respectively, resulting in an insertion at the HA0 cleavage site of 11 amino acids for the Chile virus and 7 amino
266 acids for the Canadian virus. They are both extremely virulent when inoculated into 6-week-old chickens
267 intravenously.
2 When birds are too sick to eat or drink, they should be killed humanely and scored as dead at the next observation.

268 3. Serological tests
269 a) Agar gel immunodiffusion

270 All influenza A viruses have antigenically similar nucleocapsid and antigenically similar matrix antigens. This
271 fact enables the presence or absence of antibodies to any influenza A virus to be detected by AGID tests.
272 Concentrated virus preparations, as described above, contain both matrix and nucleocapsid antigens; the
273 matrix antigen diffuses more rapidly than the nucleocapsid antigen. AGID tests have been widely and
274 routinely used to detect specific antibodies in chicken and turkey flocks as an indication of infection. These
275 have generally employed nucleocapsid-enriched preparations made from the chorioallantoic membranes of
276 embryonated fowl eggs (7) that have been infected at 10 days of age, homogenised, freeze–thawed three
277 times, and centrifuged at 1000 g. The supernatant fluids are inactivated by the addition of 0.1% formalin or
278 1% betapropiolactone, recentrifuged and used as antigen. Not all avian species may produce precipitating
279 antibodies following infection with influenza viruses.
280 Tests are usually carried out using gels of 1% (w/v) agarose or purified agar and 8% (w/v) NaCl in 0.1 M
281 phosphate buffer, pH 7.2, poured to a thickness of 2–3 mm in Petri dishes or on microscope slides. Using a
282 template and cutter, wells of approximately 5 mm in diameter, and 2–5 mm apart, are cut in the agar. A
283 pattern of wells must place each suspect serum adjacent to a known positive serum and antigen. This will
284 make a continuous line of identity between the known positive, the suspect serum and the nucleocapsid
285 antigen. Approximately 50 µl of each reagent should be added to each well.
286 Precipitin lines can be detected after approximately 24–48 hours, but this may be dependent on the
287 concentrations of the antibody and the antigen. These lines are best observed against a dark background
288 that is illuminated from behind. A specific, positive result is recorded when the precipitin line between the
289 known positive control wells is continuous with the line between the antigen and the test well. Crossed lines
290 are interpreted to be due to the test serum lacking identity with the antibodies in the positive control well.
291 b) Haemagglutination and haemagglutination inhibition tests

292 Variations in the procedures for HA and HI tests are practised in different laboratories. The following
293 recommended examples apply in the use of V-bottomed microwell plastic plates in which the final volume for
294 both types of test is 0.075 ml. The reagents required for these tests are isotonic PBS (0.01 M), pH 7.0–7.2,
295 and red blood cells (RBCs) taken from a minimum of three SPF or SAN chickens and pooled in an equal
296 volume of Alsever’s solution. Cells should be washed three times in PBS before use as a 1% (packed cell
297 v/v) suspension. Positive and negative control antigens and antisera should be run with each test, as
298 appropriate.
299 • Haemagglutination test

300 i) Dispense 0.025 ml of PBS into each well of a plastic V-bottomed microtitre plate.
301 ii) Place 0.025 ml of virus suspension (i.e. infective allantoic fluid) in the first well. For accurate
302 determination of the HA content, this should be done from a close range of an initial series of dilutions,
303 i.e. 1/3, 1/4, 1/5, 1/6, etc.
304 iii) Make twofold dilutions of 0.025 ml volumes of the virus suspension across the plate.
305 iv) Dispense a further 0.025 ml of PBS to each well.
306 v) Dispense 0.025 ml of 1% (v/v) chicken RBCs to each well.
307 vi) Mix by tapping the plate gently and then allow the RBCs to settle for about 40 minutes at room
308 temperature, i.e. about 20°C, or for 60 minutes at 4°C if ambient temperatures are high, by which time
309 control RBCs should be settled to a distinct button.
310 vii) HA is determined by tilting the plate and observing the presence or absence of tear-shaped streaming
311 of the RBCs. The titration should be read to the highest dilution giving complete HA (no streaming); this
312 represents 1 HA unit (HAU) and can be calculated accurately from the initial range of dilutions.
313 • Haemagglutination inhibition test

314 i) Dispense 0.025 ml of PBS into each well of a plastic V-bottomed microtitre plate.
315 ii) Place 0.025 ml of serum into the first well of the plate.
316 iii) Make twofold dilutions of 0.025 ml volumes of the serum across the plate.
317 iv) Add 4 HAU of virus/antigen in 0.025 ml to each well and leave for a minimum of 30 minutes at room
318 temperature (i.e. about 20°C) or 60 minutes at 4°C.

319 v) Add 0.025 ml of 1% (v/v) chicken RBCs to each well and after gentle mixing, allow the RBCs to settle
320 for about 40 minutes at room temperature, i.e. about 20°C, or for 60 minutes at 4°C if ambient
321 temperatures are high, by which time control RBCs should be settled to a distinct button.
322 vi) The HI titre is the highest dilution of serum causing complete inhibition of 4 HAU of antigen. The
323 agglutination is assessed by tilting the plates. Only those wells in which the RBCs stream at the same
324 rate as the control wells (containing 0.025 ml RBCs and 0.05 ml PBS only) should be considered to
325 show inhibition.
326 vii) The validity of results should be assessed against a negative control serum, which should not give a
327 titre >1/4 (>22 or >log2 2 when expressed as the reciprocal), and a positive control serum for which the
328 titre should be within one dilution of the known titre.
329 HI titres may be regarded as being positive if there is inhibition at a serum dilution of 1/16 (24 or log2 4 when
330 expressed as the reciprocal) or more against 4 HAU of antigen. Some laboratories prefer to use 8 HAU in HI
331 tests. While this is permissible, it affects the interpretation of results so that a positive titre is 1/8 (23 or
332 log2 3) or more. The meaning of a minimum positive titre should not be misinterpreted; it does not imply, for
333 example, that immunised birds with that titre will be protected against challenge or that birds with lower titres
334 will be susceptible to challenge.
335 Chicken sera rarely give nonspecific positive reactions in this test and any pretreatment of the sera is
336 unnecessary. Sera from species other than chickens may sometimes cause agglutination of chicken RBCs,
337 so this property should first be determined and then removed by adsorption of the serum with chicken RBCs.
338 This is done by adding 0.025 ml of packed chicken RBCs to each 0.5 ml of antisera, shaking gently and
339 leaving for at least 30 minutes; the RBCs are then pelleted by centrifugation at 800 g for 2–5 minutes and
340 the adsorbed sera are decanted. Alternatively, RBCs of the avian species under investigation could be used.
341 The neuraminidase-inhibition test has been used to identify the AI neuraminidase type of isolates and to
342 characterise the antibody in infected birds. The procedure requires specialised expertise and reagents;
343 consequently this testing is usually done in an OIE Reference Laboratory. The DIVA (differentiating infected
344 from vaccinated animals) strategy used in Italy also relies on a serological test to detect specific anti-N
345 antibodies; the test procedure has been described (12).
346 Commercial ELISA kits that detect antibody against the nucleocapsid protein are available. Kits with an
347 indirect and competitive format have been developed and are now being used to detect of AIV-specific
348 antibodies. The kits should be validated for specific species being tested. Several different test and antigen
349 preparation methods are used. Such tests have usually been evaluated and validated by the manufacturer,
350 and it is therefore important that the instructions specified for their use be followed carefully.
351 4. Antigen capture and molecular techniques
352 At present the conventional virus isolation and characterisation techniques for the diagnosis of AI remain the
353 methods of choice, for at least the initial diagnosis of AI infections. However, conventional methods tend to be
354 costly, labour intensive and slow. There have been enormous developments and improvements in molecular and
355 other diagnostic techniques, many of these have been applied to the diagnosis of AI infections.
356 a) Antigen detection

357 There are several commercially available antigen-capture kits that can detect the presence of influenza A
358 viruses in poultry (49). Most of the kits are enzyme immunoassays and use a monoclonal antibody against
359 the nucleoprotein; they should be able to detect any influenza A virus. The main advantage of these tests is
360 that they can demonstrate the presence of AI within 15 minutes. The disadvantages are that they may lack
361 sensitivity, they may not have been validated for different species of birds, subtype identification is not
362 achieved and the kits are expensive. The tests should only be interpreted on a flock basis and not as an
363 individual bird test. Oropharyngeal or tracheal samples from clinically affected or dead birds provide the best
364 sensitivity. Nevertheless, the lack of sensitivity is a major drawback to the use of available antigen detection
365 tests. Chua et al. (14) evaluated five detection tests and showed overall sensitivities from 36.3% to 51.4%;
366 these authors pointed out that in terms of sensitivity using cloacal and tracheal swabs, the tests performed
367 less well with samples from waterfowl or wild birds than they did with samples from chickens.
368 b) Direct RNA detection

369 Although, as demonstrated by the current definitions of HPNAI, molecular techniques have been used in the
370 diagnosis of AI for some time, recently there have been developments in their application for detection and
371 characterisation of AI virus directly from clinical specimens from infected birds. It is imperative that when
372 using highly sensitive molecular detection methods that allow rapid direct detection of viral RNA for
373 confirmatory laboratory diagnosis of avian influenza infections, stringent protocols are in place to prevent the

374 risk of cross-contamination between clinical samples. In addition, RT-PCR test methodologies should be
375 validated to the OIE standard (see Chapter 1.1.4 Principles of validation of diagnostic assays for infectious
376 diseases) using clinical material to demonstrate the tests as being ‘fit for purpose’ for application in a field
377 diagnostic setting, which may include the use of internal test standards. For example, PCR amplification of a
378 ‘house-keeping gene’ that aids the normalisation of results by providing information on this target gene that
379 equates quantitatively to the presence of clinical sample present on the swab. The control reactions enable
380 greater confidence in the integrity of the molecular reactions, clinical samples and results.
381 RT-PCR techniques on clinical specimens can with the correctly defined primers, result in rapid detection
382 and subtype (at least of H5 and H7) identification, plus a cDNA product that can be used for nucleotide
383 sequencing (37, 54, 55). The real application of direct RT-PCR tests may be on rapidly identifying
384 subsequent outbreaks once the primary infected premises has been detected and the virus characterised.
385 This technique was used with success during the 2003 highly pathogenic AI outbreaks in The Netherlands.
386 Ring trials conducted recently in the European Union identified H5 and H7 conventional RT-PCR protocols
387 that were sufficiently sensitive to amplify directly from swabs obtained from HPAI-infected poultry (51).
388 Modifications on the use of RT-PCR have been applied to reduce the time for both identification of virus
389 subtype and sequencing. For example Spackman et al. (53) used a ‘real time’ single-step RT-PCR
390 primer/fluorogenic hydrolysis probe system to allow detection of AI viruses and determination of subtype H5
391 or H7. The authors concluded that the test performed well relative to virus isolation and offered a cheaper
392 and much more rapid alternative with diagnosis on clinical samples in less than 3 hours. The test provides
393 high sensitivity and specificity similar to virus isolation from tracheal and oropharyngeal swabs of chickens
394 and turkeys, but may lack sensitivity for detection of influenza A virus in faecal swabs, faeces and tissues in
395 some bird species, because of the presence of PCR inhibitors resulting in false negative result (18).
396 Incorporation of a positive internal control into the test will verify a proper test run.
397 Real-time RT-PCR, usually based around the hydrolysis probe or ‘TaqMan’ method for generation of the
398 target-specific fluorescence signal, has become the method of choice in many laboratories for at least partial
399 diagnosis directly from clinical specimens. The method offers rapid results, with sensitivity and specificity
400 comparable to virus isolation, and these are ideal qualities for AI outbreak management, where the speed
401 with which an unequivocal diagnosis can be obtained is crucial for decision making by the relevant
402 Veterinary Authority. In addition, RT-PCR systems can be designed to operate in a 96-well format and
403 combined with high-throughput robotic RNA extraction from specimens (1).
404 The approach to diagnosis using real-time RT-PCR adopted in most laboratories has been based on initial
405 generic detection of AIV in clinical specimens, primarily by initially targeting the matrix (M) gene, which is
406 highly conserved for all type A influenza viruses, followed by specific real-time RT-PCR testing for H5 and
407 H7 subtype viruses. For subtype identification, primers used in TaqMan real-time RT-PCRs are targeted at
408 the HA2 region as this is relatively well conserved within the haemagglutinin genes of the H5 and H7
409 subtypes, and has served as the target region for H5 and H7. Spackman et al. (53) demonstrated specific
410 detection of these subtypes but cautioned that their H5 and H7 primer/probe sequences had been designed
411 for the detection of North American H5 and H7 isolates and might not be suitable for all H5 and H7 isolates.
412 This proved to be the case. Slomka et al. (52) described modification of the H5 oligonucleotide sequences
413 used by Spackman et al. (53) to enable the detection of this Asian lineage HPAI H5N1 AI virus and other
414 Eurasian H5 AI viruses that have been isolated within the past decade in both poultry and wild birds. This
415 validated Eurasian H5 real-time RT-PCR has proved valuable in the investigation of many H5N1 HPAI
416 clinical specimens submitted to International Reference Laboratories from Europe, Africa and Asia since
417 autumn 2005 (52).
418 One of the problems with rapidly emerging new tests is that methods and protocols may be developed and
419 reported without the test being properly validated. This has been addressed for some of the real-time RT-
420 PCR protocols (52, 56). In the European Union, National Reference Laboratories have collaborated to define
421 and validate protocols that can be recommended for use within the European Union (51, 52).
422 Real-time RT-PCR protocols have been described that amplify regions across the cleavage site of the HA0
423 gene. This may result in useful tests for specific viruses. For example, Hoffman et al. (27) have described a
424 real-time RT-PCR test specific to the Asian HPAI H5N1 Quinghai clade 2.2 viruses that represents a rapid
425 means of determining the pathotype for this subgroup of H5N1 HPAI viruses without sequencing.
426 Modifications on the straightforward RT-PCR method of detection of viral RNA have been designed to
427 reduce the effect of inhibitory substances in the sample taken, the possibility of contaminating nucleic acids
428 and the time taken to produce a result. For example, nucleic acid sequence-based amplification (NASBA)
429 with electrochemiluminescent detection (NASBA/ECL) is a continuous isothermal reaction in which
430 specialised thermocycling equipment is not required. NASBA assays have been developed for the detection
431 of AI virus subtypes H7 and H5 in clinical samples within 6 hours (15, 29). The loop-mediated isothermal
432 amplification (LAMP) system for H5 detection appeared to show high sensitivity and reliable specificity (28).

433 It seems highly likely that within a very short time molecular-based technology will have developed
434 sufficiently to allow rapid ‘flock-side’ tests for the detection of the presence of AI virus, specific subtype and
435 virulence markers. The extent to which such tests are employed in the diagnosis of AI will depend very much
436 on the agreement on and adoption of definitions of statutory infections for control and trade purposes.
437 C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS

438 It is important that vaccination alone is not considered the solution to the control of NAI or LPAI subtypes if
439 eradication is the desired result. Without the application of monitoring systems, strict biosecurity and depopulation
440 in the face of infection, there is the possibility that these viruses could become endemic in vaccinated poultry
441 populations. Long-term circulation of the virus in a vaccinated population may result in both antigenic and genetic
442 changes in the virus and this has been reported to have occurred in Mexico (31).
443 Experimental work has shown, for both NAI and LPAI that vaccination protects against clinical signs and mortality,
444 reduces virus shedding and increases resistance to infection, protects from diverse field viruses within the same
445 hemagglutinin subtype, protects from low and high challenge exposure, and reduces excretion and thus contact
446 transmission of challenge virus (13, 19, 59, 65). However, the virus is still able to infect and replicate in clinically
447 healthy vaccinated birds. Most of the work evaluating vaccines has been done in chickens and turkeys and some
448 care must be taken in extrapolating the results obtained to other species. For example, in an experimental system
449 using HPAI H7N7 as a challenge virus it was shown for chickens and ringed teal ducks, Callonetta leucophrys,
450 that vaccination sufficiently reduced excretion and increased the infective dose that transmission between birds
451 was dramatically reduced, but for golden pheasants, Chrysolophus pictus, while giving clinical protection
452 vaccination had no effect on excretion of challenge virus and no influence on transmission (69, 70). In some
453 countries, vaccines designed to contain or prevent NAI are specifically banned or discouraged by government
454 agencies because it has been considered that they may interfere with stamping-out control policies. However,
455 most AI control regulations reserve the right to use vaccines in emergencies.
456 Live conventional influenza vaccines against any subtype are not recommended.
457 Conventional vaccines
458 Conventionally, vaccines that have been used against NAI or LPAI have been prepared from infective allantoic
459 fluid inactivated by beta-propiolactone or formalin and emulsified with mineral oil.
460 The existence of a large number of virus subtypes, together with the known variation of different strains within a
461 subtype, pose serious problems when selecting strains to produce influenza vaccines, especially for LPAI. In
462 addition, some isolates do not grow to a sufficiently high titre to produce adequately potent vaccines without costly
463 prior concentration. While some vaccination strategies have been to produce autogenous vaccines, i.e. prepared
464 from isolates specifically involved in an epizootic, others have been to use vaccines prepared from viruses
465 possessing the same haemagglutinin subtype that yield high concentrations of antigen. For example, in the USA,
466 some standardisation of the latter has been carried out in that the Center for Veterinary Biologics have
467 propagated and hold influenza viruses of several subtypes for use as seed virus in the preparation of inactivated
468 vaccines (6).
469 Since the 1970s in the USA, there has been some use of inactivated vaccines produced under special licence on
470 a commercial basis (25, 35, 43). These vaccines have been used primarily in turkeys against viruses that are not
471 highly pathogenic, but which may cause serious problems, especially in exacerbating circumstances. Significant
472 quantities of vaccine have been used (26, 35). In recent years in the US, most of the special license inactivated
473 vaccine has used in breeder turkeys to protect against H1 and H3 swine influenza viruses (58). Conventional
474 vaccination against the prevailing strain of LPAI has also been used in Italy for a number of years (17).
475 Vaccination against H9N2 infections has been used in Pakistan (39), Iran (72) and the People’s Republic of China
476 (32) and several countries in the Middle East.
477 Inactivated vaccine was prepared from the LPNAI virus of H7N3 subtype responsible for a series of outbreaks in
478 turkeys in Utah in 1995 and used, with other measures, to bring the outbreaks under control (26). Similarly in
479 Connecticut in 2003 vaccination of recovered hens and replacement pullets with a H7N2 or H7N3 vaccine was
480 implemented following an outbreak of LPNAI caused by a H7N2 virus (61).
481 Vaccination against HPNAI of H5N2 subtype was used in Mexico following outbreaks in 1994–1995 (21, 22, 31),
482 and against H7N3 subtype in Pakistan (38) following outbreaks in 1995. In Mexico, the HPNAI virus appears to
483 have been eradicated, but LPNAI virus of H5N2 has continued to circulate, while in Pakistan highly pathogenic AI
484 viruses genetically close to the original highly pathogenic AI virus were still being isolated in 2001 (66) and 2004.
485 Following the outbreaks of HPNAI caused by H5N1 virus in Hong Kong in 2002 (48) a vaccination policy was
486 adopted there using an H5N2 vaccine. In 2004 the widespread outbreaks of highly pathogenic AI H5N1 in some

487 countries of South-East Asia resulted in emergency and prophylactic vaccination being used in the People’s
488 Republic of China, Indonesia and Vietnam. Inactivated H7N7 AI vaccine was used in North Korea during 2005 to
489 control a HPAI outbreak. Prophylactic vaccination has also been used in limited areas in Italy to aid the control of
490 H5 and H7 LPNAI viruses. Similar preventive vaccination has been allowed in outdoor poultry and in zoo birds in
491 several European Union countries in recent years.
492 Recombinant vaccines
493 Recombinant vaccines for AI viruses have been produced by inserting the gene coding for the influenza virus
494 haemagglutinin into a live virus vector and using this recombinant virus to immunise poultry against AI (60).
495 Recombinant live vector vaccines have several advantages: [1] they are live vaccines able to induce both humoral
496 and cellular immunity, [2] they can be administered to young birds and induce an early protection, e.g. the fowl
497 poxvirus can be administered at 1 day of age, is compatible with the Marek’s disease vaccine, and provides
498 significant protection 1 week later, [3] they enable differentiation between infected and vaccinated birds, since, for
499 example, they do not induce the production of antibodies against the nucleoprotein or matrix antigens that are
500 common to all AI viruses. Therefore, only field-infected birds will exhibit antibodies in the AGID test or ELISA tests
501 directed towards the detection of influenza group A (nucleoprotein and/or matrix) antibodies. However, these
502 vaccines have limitations in that they will replicate poorly and induce only partial protective immunity in birds that
503 have had field exposure to or vaccination with the vector virus, i.e. fowl poxvirus or infectious laryngotracheitis
504 viruses for currently available recombinant vaccines (34, 62). If used in day-old or young birds the effect of
505 maternal antibodies to the vector virus on vaccine efficacy may vary with the vector type. In the case of fowl
506 poxvirus recombinant vaccine, it has been reported that effective immunisation was achieved when given to 1-
507 day-old chicks with varying levels of maternal immunity (4). However, when very high levels of maternal
508 antibodies are anticipated due to previous infection or vaccination, the efficacy of the fowlpox vector vaccine in
509 such day-old chicks should be confirmed. In addition, because the vectors are live viruses that may have a
510 restricted host range (for example infectious laryngotracheitis virus does not replicate in turkeys) the use of these
511 vaccines must be restricted to species in which efficacy has been demonstrated.
512 The use of recombinant vaccines is restricted to countries in which they are licensed and are legally available.
513 The recombinant fowlpox-AI-H5 vaccine has been licensed in El Salvador, Guatemala, Mexico, China and the
514 USA (59, 82). Recombinant fowl poxvirus vaccines containing H5 HA have been prepared and evaluated in field
515 trials (8, 23, 44, 63), but the only field experience with this vaccine has been in Mexico, El Salvador, Guatemala
516 and China where it has been used in the vaccination campaign against the H5N2 LPAI and H5N1 HPAI viruses.
517 Between 1995 and 2006, Mexico used more than 1.788 billion doses of inactivated H5N2 vaccine in their H5N2
518 control programme (73, 74). In addition, Mexico, Guatemala and El Salvador have used over 1.6 billion doses of
519 the recombinant fowlpox-AI-H5 vaccine for control of H5N2 LPNAI from 1997 to 2005 and China used 606 million
520 doses in 2005 (82).
521 Newcastle disease virus can also be used as a vector for expressing influenza HA genes (40). A recombinant
522 Newcastle disease vaccine virus (clone 30) containing and expressing an H5 HA gene was shown to protect
523 chickens against challenge with either virulent Newcastle disease virus or an HPAI H5N2 virus (71). A similar
524 recombinant virus based on Newcastle disease virus vaccine strain La Sota and expressing the Asian lineage H5
525 HA gene was produced in China (24) and reported to be efficacious in protection studies with either virus. This
526 latter virus has been licensed in China and used widely as one of the four H5 vaccines allowed under the
527 compulsory vaccination policy currently in place that resulted in the vaccination of 8.2 billion birds between
528 January and September 2006 (36). As with other recombinant vaccines it seems doubtful that this vaccine will be
529 appropriate for use in older birds that are well-immunised against Newcastle disease and it is not clear how much
530 the efficacy will be affected by the presence of maternal immunity to either the vector or the AI HA in young
531 chicks. A baculovirus-expression system has been used to produce recombinant H5 and H7 antigens for
532 incorporation into vaccines (75). DNA encoding H5 haemagglutinin has been evaluated as a potential vaccine in
533 poultry (30).
534 Detection of infection in vaccinated flocks and vaccinated birds
535 A strategy that allows differentiation of infected from vaccinated animals (DIVA), has been put forward as a
536 possible solution for the eventual eradication of NAI without involving mass culling of birds and the consequent
537 economic damage that would do, especially in developing countries (20). This strategy has the benefits of
538 vaccination (less virus in the environment), but the ability to identify infected flocks would still allow the
539 implementation of other control measures, including stamping out. DIVA strategies use two broad detection
540 strategies within the vaccinated population: 1) detection of influenza A virus, or 2) detection of antibodies against
541 influenza A virus infection. At the flock level, a simple method is to regularly monitor sentinel birds left
542 unvaccinated in each vaccinated flock, but this approach does have some management problems, particularly in
543 identifying the sentinels in large flocks. As an alternative or adjunct system, testing for field exposure may be
544 performed on the vaccinated birds either by detection of field virus or antibodies against the virus. In detection of
545 the field virus, oropharyngeal or cloacal swabs from normal daily mortality or sick birds can be tested, individually

546 or as pools, by molecular methods, such as real-time RT-PCR or antigen-capture enzyme immunoassay of the
547 vaccinated populations.
548 In order to use serological DIVA, vaccination systems that enable the detection of field exposure in vaccinated
549 populations should be used. Several systems have been developed in recent years. These include the use of a
550 vaccine containing a virus of the same haemagglutinin (H) subtype but a different neuraminidase (N) from the
551 field virus. Antibodies to the N of the field virus act as natural markers of infection. This system has been used in
552 Italy following the re-emergence of a LPNAI H7N1 virus in 2000. In order to supplement direct control measures,
553 a ‘DIVA’ strategy was implemented using a vaccine containing H7N3 to combat an H7N1 field infection.
554 Vaccinated and field exposed birds were differentiated using a serological test to detect specific anti-N antibodies
555 (10, 11). The same strategy was used to control LPNAI caused by H7N3 in Italy in 2002–2003 (9), in this case
556 with an H7N1 vaccine. In both cases vaccination with stamping out using this DIVA strategy resulted in
557 eradication of the field virus. Problems with this system would arise if a field virus emerges that has a different N
558 antigen to the existing field virus or if subtypes with different N antigens are already circulating in the field.
559 Alternatively the use of vaccines that contain only HA, e.g. recombinant vaccines, allows classical AGID and NP-
560 or matrix-based ELISAs to be used to detect infection in vaccinated birds. For inactivated vaccines, a test that
561 detects antibodies to the nonstructural virus protein has been described (67).This system is yet to be validated in
562 the field.
563 Production of conventional vaccines
564 The information below is based primarily on the experiences in the USA and the guidance and policy for licensing
565 avian influenza vaccines in that country (68). The basic principles for producing vaccines, particularly inactivated
566 vaccines, are common to several viruses e.g. Newcastle disease (Chapter 2.3.14).
567 Guidelines for the production of veterinary vaccines are given in Chapter 1.1.8 Principles of veterinary vaccine
568 production. The guidelines given here and in Chapter 1.1.8 are intended to be general in nature and may be
569 supplemented by national and regional requirements.
570 The vaccine production facility should operate under the appropriate biosecurity procedures and practices. If
571 HPNAI virus is used challenge studies, that part of the facility where this work is done should meet the
572 requirements for Containment Group 4 pathogens as outlined in Chapter 1.1.2.
573 1. Seed management
574 a) Characteristics of the seed

575 For any subtype, only well characterised influenza A virus of proven low pathogenicity, preferably obtained
576 from an international or national repository, should be used to establish a master seed for inactivated
577 vaccines. HPAI viruses should not be used as seed virus for AI vaccine.
578 b) Method of culture

579 A master seed is established, and from this, a working seed. The master seed and working seed are
580 produced in SPF or SAN embryonated eggs. The establishment of a master culture may only involve
581 producing a large volume of infective allantoic fluid (minimum 100 ml), which can be stored as lyophilised
582 aliquots (0.5 ml).
583 c) Validation as a vaccine

584 The master seed should be checked after preparation for sterility, safety, potency and absence of specified
585 extraneous agents.
586 2. Method of manufacture
587 For vaccine production, a working seed, from which batches of vaccine are produced, is first established in SPF
588 or SAN embryonated eggs by expansion of an aliquot of master seed to a sufficient volume to allow vaccine
589 production for 12–18 months. It is best to store the working seed in liquid form at below –60°C as lyophilised virus
590 does not always multiply to high titre on subsequent first passage.
591 The inactivated influenza vaccines prepared from conventional virus are produced in embryonated fowl eggs. The
592 method of production is basically that of propagating the virus aseptically; all procedures are performed under
593 sterile conditions.

594 It is usual to dilute the working seed in sterile isotonic buffer (e.g. PBS, pH 7.2), so that about 103–104 EID50 (50%
595 egg-infective dose) in 0.1 ml are inoculated into each allantoic cavity of 9 to 11-day-old embryonated SPF or SAN
596 fowl eggs. These are then incubated at 37°C. Eggs containing embryos that die within 24 hours should be
597 discarded. The incubation time will depend on the virus strain being used and will be predetermined to ensure
598 maximum yield with the minimum number of embryo deaths.
599 The infected eggs should be chilled at 4°C before being harvested. The tops of the eggs are removed and the
600 allantoic fluids collected by suction. The inclusion of any yolk material and albumin should be avoided. All fluids
601 should be stored immediately at 4°C and tested for bacterial contamination.
602 In the manufacture of inactivated vaccines, the harvested allantoic fluid is treated with either formaldehyde (a
603 typical final concentration is 1/1000) or beta-propiolactone (a typical final concentration is 1/1000–1/4000). The
604 time required must be sufficient to ensure freedom from live virus. Most inactivated vaccines are formulated with
605 non-concentrated inactivated allantoic fluid (active ingredient). However, active ingredients may be concentrated
606 for easier storage of antigen. The active ingredient is usually emulsified with mineral or vegetable oil. The exact
607 formulations are generally commercial secrets.
608 3. In-process control
609 For inactivated vaccines, the completeness of the inactivation process should be tested in embryonated eggs,
610 taking at least 10 aliquots of 0.2 ml from each batch and passaging each aliquot at least two times through SPF or
611 SAN embryos.
612 4. Batch control
613 Most countries have published specifications for the control of production and testing of vaccines, which include
614 the definition of the obligatory tests on vaccines during and after manufacture.
615 a) Sterility
616 Tests for sterility and freedom from contamination of biological materials may be found in Chapter 1.1.9.
617 b) Safety
618 For inactivated vaccines, a double dose is administered by the recommended route to ten 3-week-old birds,
619 and these are observed for 2 weeks for absence of clinical signs of disease or local lesions.
620 c) Potency
621 Potency of avian influenza vaccine is generally evaluated by testing the ability of the vaccine to induce a
622 significant HI titre in SPF or SAN birds. Conventional potency testing involving the use of three diluted doses
623 and challenge with virulent virus (e.g. chapter 2.3.14) may also be used for vaccines prepared to give
624 protection against HPNAI or LPNAI subtypes. For inactivated vaccines to other subtypes where virulent
625 viruses are not available, potency tests may rely on the measurement of immune response or challenge and
626 assessment of morbidity and quantitative reduction in challenge virus replication in respiratory
627 (oropharyngeal or tracheal) and intestinal (cloaca) tracts. Assessment of haemagglutinin antigen content
628 (77) could allow in vitro extrapolation to potency for subsequent vaccine batches.
629 d) Stability
630 When stored under the recommended conditions, the final vaccine product should maintain its potency for at
631 least 1 year. Inactivated vaccines must not be frozen.
632 e) Preservatives
633 A preservative may be used for vaccine in multidose containers.
634 f) Precautions (hazards)

635 Care must be taken to avoid self-injection with oil emulsion vaccines.
636 5. Tests on the final product
637 a) Safety
638 See Section C.4.b. above

639 b) Potency
640 See Section C.4.c. above.
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853 217.
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855 pathogenicity for chickens at different sites in chickens and ducks following intranasal inoculation. Avian
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862 Control of Avian Influenza. Presented 22 March 2007, Verona, Italy.
863 *
864 * *
865 NB: There are OIE Reference Laboratories for Avian influenza (see Table in Part 3 of this Terrestrial Manual or
866 consult the OIE Web site for the most up-to-date list: www.oie.int).

CHAPTER 2.3.5.
AVIAN MYCOPLASMOSIS
(Mycoplasma gallisepticum, M. synoviae)
SUMMARY
Definition of the disease: Avian mycoplasmosis is caused by several pathogenic mycoplasmas of

which Mycoplasma gallisepticum (MG) and M. synoviae (MS) are the most important; they are the
only ones listed by the OIE.
Description of the disease: MG causes chronic respiratory disease of domestic poultry, especially
in the presence of management stresses and/or other respiratory pathogens. Disease is
characterised by coryza, conjunctivitis, sneezing, and by sinusitis, particularly in turkeys and game
birds. It can result in loss of production and downgrading of meat-type birds, and loss of egg
production. MS may cause respiratory disease, synovitis, or may result in a silent infection. MG and
MS strains vary in infectivity and virulence, and infections may sometimes be unapparent.
Identification of the agent: MG and MS can be identified by immunological methods after isolation
in mycoplasma media or by detection of their DNA in field samples or cultures.
Samples for isolation can be swabs of organs or tissues, exudates, diluted tissue homogenates,
aspirates from the infraorbital sinuses or joint cavities, or material from egg yolk or embryos.
Clinical signs and lesions will influence the sample selection. Broth and agar are used for isolation,
but it is normally necessary to obtain mycoplasma colonies on agar before attempting identification.
Basic biochemical tests can be helpful in preliminary classification of isolates but final identification
is by immunological tests, the most satisfactory being fluorescent antibody and immunoperoxidase
tests.
DNA detection methods based on the polymerase chain reaction are used in specialised
laboratories. Once validated, they can be used on swab material or cultures.
Serological tests: Several serological tests are used to detect MG or MS antibodies, but due to
variations in specificity and sensitivity, they are recommended for flock screening rather than for
testing individuals.
The most commonly used are the rapid serum agglutination (RSA) test, the enzyme-linked
immunosorbent assay (ELISA) and the haemagglutination inhibition (HI) test. In the RSA test, sera
are mixed with commercially produced stained antigen and sera that react within 2 minutes are
heated at 56°C for 30 minutes and retested. Sera that still react, especially when diluted, are
considered positive and are tested by either ELISA or HI for confirmation. Several commercial MG
and MS antibody ELISA kits are available.
Requirements for vaccines and diagnostic biologicals: Although the preferred method of
control is maintenance of MG- and MS-free flocks, both live and inactivated vaccines are used in
chickens. Vaccination should be considered only on multi-age sites where infection is inevitable.
The normal use is to prevent egg-production losses in commercial layers, although vaccines may
also be used to reduce egg transmission in breeding stock or to aid MG eradication on multi-age
sites. It is important to vaccinate before field challenge occurs.
Available live vaccines for MG are produced from the F strain, and, more recently, strains ts-11 and
6/85, which are apathogenic strains with improved safety characteristics. Administration of the
F strain by the intranasal or eyedrop route is preferred, but aerosol or drinking water administration
may be used. The eyedrop method is recommended for ts-11, and a fine spray for 6/85. Pullets are
generally vaccinated between 12 and 16 weeks of age. One dose is sufficient and vaccinated birds

Chapter 2.3.5. — Avian mycoplasmosis (Mycoplasma gallisepticum, M. synoviae)
remain permanent carriers. Long-term use of the F strain on multi-age sites results in displacement
of field strains. The ts-11 strain has been successfully used to eradicate F strain in multi-age
commercial layers. A live MS vaccine has been produced from the MS-H strain and should be
administered by eyedrop.
Bacterins consist of a concentrated suspension of MG organisms in an oil emulsion. They are

administered parenterally to pullets at 12–16 weeks of age, usually subcutaneously in the neck.
Two doses are desirable. Bacterins are effective in preventing egg-production losses and
respiratory disease, but they do not prevent infection with wild-type MG. A similar bacterin has been
licensed in the United States of America for MS, but it is not widely used.
A. INTRODUCTION
Mycoplasma gallisepticum (MG) and M. synoviae (MS) belong to the class Mollicutes, order Mycoplasmatales,
family Mycoplasmataceae. It should be noted, however, that M. meleagridis and M. iowae can also cause disease
in poultry, but MG and MS are considered to be the most important of the pathogenic mycoplasmas, and both
occur world-wide.
MG infection is particularly important in chickens and turkeys as a cause of respiratory disease and decreased
meat and egg production (3, 24). It can also cause upper respiratory disease in game birds. More recently MG
has been recognised in North America in house finches as a cause of conjunctivitis (27). In poultry the infection is
spread vertically through infected eggs and horizontally by close contact; the MG nucleic acid has been identified
in environmental samples (30). Other methods of spread are less well documented.
The clinical signs of MG in infected poultry can vary from subclinical to obvious respiratory signs including coryza,
conjunctivitis, coughing and sneezing. Nasal exudate, rales and breathing through the partially open beak may
occur. Unilateral or bilateral sinusitis may also be a feature, particularly in turkeys and game birds and the
infraorbital sinuses may become so swollen that the eyelids are closed. Conjunctivitis, with frothy ocular exudate
is also a common feature in turkeys and game birds, and sometimes in chickens. In turkeys there is often soiling
of the wing feathers as the result of attempts to remove exudate from the eyes. Infected finches may reveal ocular
and nasal discharge and swollen eyelids in addition to the conjunctivitis.
Mycoplasma gallisepticum may be associated with acute respiratory disease in chickens and turkeys, especially
in young birds, with the turkey being more susceptible. The severity of the disease is greatly affected by the
degree of secondary infection with viruses such as Newcastle disease and infectious bronchitis, and/or bacteria
such as Escherichia coli. In turkeys there is synergism with avian pneumovirus infection. A more chronic form of
the disease may occur and can cause reduced egg production in breeders and layers.
Lesions of the respiratory tract take the form initially of excess mucous exudate followed by catarrhal and caseous
exudate, which may form amorphous masses in the air sacs. In turkeys and game birds the swollen infraorbital
sinuses contain mucoid to caseous exudate.
MG or MS disease in chickens may superficially resemble respiratory disease caused by other pathogens such as
mild strains of Newcastle disease (Chapter 2.3.14) and avian infectious bronchitis (Chapter 2.3.2). These may be
present in mixed infection with MG or MS. Infections with Haemophilus paragallinarum (now Avibacterium
paragallinarum), and Pasteurella multocida, should also be ruled out. MG in turkeys may be confused with avian
pneumovirus infections and the presence of sinusitis may also suggest infection with Pasteurella multocida,
Chlamydia (Chapter 2.3.1) or MS. Infectious synovitis caused by MS should be differentiated from
Staphylococcus aureus infection and from infectious tenosynovitis caused by reovirus.
Chickens with infectious synovitis may exhibit pale combs, lameness and retarded growth. Swellings may occur
around joints. Greenish droppings containing large amounts of urates are commonly seen. Joints may contain a
viscous, creamy to grey exudate in the joint and along tendon sheaths, along with hepatosplenomegaly and
mottled, swollen kidneys (17). Respiratory signs and lesions are similar to those observed with MG, except that
they are generally milder, and, as with MG, there is a synergistic effect with other respiratory agents (19). MS
strains exhibit significant variability with respect to their virulence and tissue tropism (18, 22, 26).
The presence of MG or MS can be confirmed by isolating the organism in a cell-free medium or by detecting its
DNA directly in infected tissues or swab samples. Serological tests are also widely used for diagnosis. When

results are equivocal the birds are usually resampled, although chicken embryos or chickens may be inoculated
with suspect material.
Culture
Samples are taken from live birds, fresh carcasses or the carcasses of birds that have been frozen when
fresh. From live birds, swabs may be taken from the choanal cleft, oropharynx, oesophagus, trachea, eyes,
cloaca and phallus. In the case of dead birds, samples may be taken from the nasal cavity, infraorbital sinus,
trachea, or air sacs. Exudates can be aspirated from the infraorbital sinuses and joint cavities.
Samples may also be collected from dead-in-shell embryos or chickens or poults that have broken the shell
but failed to hatch. Samples can be taken from the inner surface of the vitelline membrane, and from the
oropharynx and air sacs of the embryo.
All samples should be examined as soon as possible after collection. If transportation is necessary, small
pieces of tissue should be placed in mycoplasma broth, or swabs should be vigorously agitated in 1–2 ml of
mycoplasma broth and then discarded. Alternatively, the swabs can be dipped in mycoplasma broth before
the specimens are taken (36) and then replaced in the swab holders for transportation. An ice pack or some
other means of chilling should be included as MG and MS die rapidly at room temperature. Serial dilutions of
specimens in mycoplasma broth may be of value because the presence of specific antibodies or antibiotics
or inhibitory substances in tissues may inhibit mycoplasma growth unless they are diluted out.
Several suitable culture media have been formulated (10) and those suitable for isolation of avian
mycoplasmas can be purchased from Mycoplasma Experience, Reigate, Surrey, United Kingdom.
Mycoplasma media generally contain a protein digest and a meat-infusion base supplemented with serum or
a serum fraction, yeast factors, glucose and bacterial inhibitors. It is important that each new batch of
medium be tested with recently isolated MG cultures of low in-vitro passage because some components,
especially the yeast extract and the serum may vary in their ability to support growth.
The medium developed by Frey et al. is widely used in the United States of America (USA) and other
countries for isolation of MG and MS (2, 11). Nicotinamide adenine dinucleotide (NAD) is a growth
requirement for the primary isolation of MS, but it may be omitted from the medium for the cultivation of MG.
The following broth and agar media are also satisfactory:
Part A: Pleuropneumonia-like organism (PPLO) broth base without crystal violet (Difco) (14.7 g);
distilled or deionised water (700 ml).
Part B: Pig serum (heated at 56°C for 1 hour) (150 ml): 25% (w/v) fresh yeast extract (100 ml); 10%
(w/v) glucose solution (10 ml); 5% (w/v) thallous acetate (10 ml); 200,000 International Units (IU)/ml
penicillin G (5 ml); and 0.1% (w/v) phenol red solution (20 ml). Thallous acetate can be toxic to humans
and the precautions for its use should be followed. The pH is adjusted to 7.8. Pig serum may be
replaced by horse serum, but it is important to ascertain that it supports the growth of MG.
Part A is autoclaved at 121°C, at 1 atmospheric pressure for 15 minutes and, after cooling, is added to Part
B, which has previously been sterilised by filtration.
For the corresponding solid medium, 10 g of purified agar, known to support the growth of MG, is added to
part A above. The mixture is autoclaved as before and kept in a water bath at 56°C. The constituents of part
B, omitting the phenol red, are mixed separately and then incubated at 56°C. Parts A and B are mixed
carefully to avoid the production of air bubbles, and are dispensed into 50 mm dishes using 7–9 ml/dish.
Excess surface moisture can be removed by a short incubation at 37°C. Plates are stored in an airtight
container at approximately 4°C for up to 2 weeks.
Fresh yeast extract is available commercially, although it is preferable to prepare it ‘in-house’ by taking
active dry baker’s yeast (250 g) and suspending it in distilled water (1 litre). This is heated to boiling point,
cooled and then centrifuged for 20 minutes at 3000 g. The supernatant fluid is decanted and adjusted to
pH 8.0 with 0.1 M NaOH. This is clarified by centrifugation or by filtration, and then sterilised by filtration. The
extract is stored at –20°C. Reagent grade glucose (10 g) is dissolved in distilled or deionised water (100 ml)
and adjusted to pH 7.8–8.0 with 0.1 M NaOH. It is sterilised by filtration and stored at 4°C. Reagent grade
thallous acetate is dissolved (5 g) in distilled or deionised water (100 ml), filter-sterilised and stored at
–20°C. Penicillin solution (106 IU benzyl penicillin in 5 ml distilled water) is stored at 4°C, and has a shelf life
of 1 week. For isolation from heavily contaminated samples, penicillin concentration can be increased to

2000 units/ml or ampicillin, 0.5–1.0 mg/ml, maybe used instead. Phenol red (0.1 g) is ground in 0.1 M NaOH
(2.8 ml), and then made up to 100 ml in sterile distilled water and autoclaved at 115°C at 1 atmosphere for
30 minutes. It is stored at 4°C. (Note: Thallous acetate is highly toxic and care should be taken, especially
when preparing the stock solution.)
Specimens are inoculated on to mycoplasma agar and into broth. Solid medium may help detection of slow-
growing mycoplasma colonies, which can be overgrown by saprophytes in broth. It may be necessary to
make serial dilutions up to 10–3 for successful isolation. Inoculated plates are incubated at 37°C in sealed
containers. Increased humidity and CO2 tension in the atmosphere have been reported to enhance growth;
these conditions may be obtained by the inclusion of damp paper or cotton wool, and by flushing the
container with 5–10% CO2 in nitrogen, by placing a lighted candle in the container, or by using a CO2
incubator or suitable gas-generating system.
The caps of liquid medium containers should be tightly sealed before incubation at 37°C to avoid spurious
changes in pH. For the first few days, the plates are examined daily for colonies with a stereoscopic
microscope; after that they are examined less frequently. Cultures from field material should not be
discarded as negative for at least 20 days.
Broth medium should be examined daily for acidity, indicated by a change from red to orange or yellow in
the indicator. Any observable growth is subcultured on to solid medium immediately. Even if no colour
change occurs, subculture on to solid medium should be made after 7–10 days or earlier as the presence of
an arginine-hydrolysing (alkali-producing) mycoplasma species may mask the acid colour change produced
by MG.
Mycoplasma colonies on solid medium can usually be recognised, although they may not have the typical
‘fried egg’ appearance. Bacterial colonies may appear on the first passage, but they are often more
pigmented and fail to passage on mycoplasma media.
Biochemical reactions (e.g. fermentation of glucose and failure to hydrolyse arginine) can assist in
identification, but they are not specific for MG or MS and necessitate purification of the culture by cloning.
Immunological and DNA detection methods can be used to identify mycoplasma isolates. They include the
indirect fluorescent antibody (IFA) and immunoperoxidase (IP) tests, both of which are simple, sensitive,
specific and rapid to perform; growth inhibition (GI); and metabolism inhibition (MI). Purified (cloned) cultures
are required for the GI and MI tests, but not for the IFA or IP test. IFA and IP can detect the presence of
more than one species of mycoplasma, as the colonies specific for the antiserum will react while the others
will not. However, M. imitans, a mycoplasma species that is serologically related to MG and that presents
the same biochemical properties has been isolated from ducks, geese and sometimes from other
nondomestic bird species in some countries. It may be distinguished from MG by use of a PCR-RFLP
(polymerase chain reaction/restriction fragment length polymorphism), as described by Kempf (16).
Alternatively, colonies of the isolate can be examined by immunofluorescence using serial dilutions of
antisera to MG and M. imitans in parallel. The homologous antiserum should have a considerably higher
titre.
DNA detection methods for identifying MG or MS directly in tissues or for identifying laboratory isolates are
discussed below and are usually based on the PCR.
In certain circumstances where results of the above methods are not conclusive, inoculation of chick
embryos or bioassays in live chicks may be appropriate. However these techniques are time-consuming and
costly and tend to have been replaced by PCR technology, although they remain a useful research tool. The
specimens required for inoculation of chicken embryos are the same as those used for the inoculation of
artificial media. They are prepared in broth from which thallous acetate is omitted, incubated for 30–
60 minutes at 37°C, and then a 0.05–0.1 ml aliquot is inoculated into the yolk sac of several 6–8-day-old
chicken embryos derived from mycoplasma-free flocks. The eggs are candled daily and embryos that die
within 24 hours of inoculation are discarded. Any further dead embryos are kept refrigerated until cultured
and those surviving after 5 days are placed at 4°C for 4 hours to kill them and to reduce haemorrhages on
opening. The yolk is subcultured into broth and on to agar. Yolk lipid tends to obscure colonies so it is
essential to streak the yolk thinly or, preferably, to dilute it first in mycoplasma broth.
Bioassays may be performed by the inoculation of a homogenate of suspect material into at least four 8–
16 week-old susceptible mycoplasma-free chickens. Diagnosis is confirmed by the recovery of the
mycoplasma from these birds, demonstration of its DNA and/or the demonstration of specific antibodies (28).
Immunological methods
Immunofluorescence and IP procedures for diagnosis are generally applied to suspect laboratory isolates
rather than directly to infected exudates or tissues. This is because the organisms are too small to recognise

conclusively under the light microscope and because the corresponding negative and positive control
exudate/tissue is unlikely to be readily available.
a) Indirect fluorescent antibody test

The recommended technique for the IFA test (31) requires an agar culture of the unknown isolate, consisting
of numerous small discrete colonies, a known MG or MS culture as a positive control, and a culture of a
another mycoplasma species, such as M. gallinaceum or M. gallinarum as a negative control. Also required
are polyclonal rabbit anti-MG or MS serum, a normal rabbit serum and an anti-rabbit immunoglobulin
fluorochrome-conjugated serum. Sera may be prepared in species other than rabbits, but monoclonal
antibodies (MAbs) should not be used because MG or MS demonstrates variable expression of its surface
epitopes and an MAB may fail to recognise the organism. Suitable working dilutions in sterile phosphate
buffered saline (PBS; 0.01 M, pH 7.2) of the anti-MG or MS serum and the conjugate are first determined by
cross-titration, and are selected for use at two-to-four-fold dilutions less than the actual end-points. These
are applied to the colonies of mycoplasmas to be identified that have been previously grown on agar plates
as indicated below.
Test procedure
i) From colony-bearing agar plates, cut blocks of about 1.0 × 0.5 cm and place them on to labelled
microscope slides with the colonies uppermost.
ii) To make subsequent orientation possible, cut off the lower right hand corner of the blocks. One block
with the unknown isolate, a block with the known MG culture, a block with the known MS culture and a
block with a different but known mycoplasma culture are placed on one slide. A block of the unknown
isolate is placed on another slide.
iii) Add a drop of suitably diluted MG (or MS) antiserum to the surface of each block of the first slide and
add normal rabbit serum to the single block on the second slide.
iv) Incubate all blocks for 30 minutes at room temperature in a humid atmosphere.
v) Place each block in a labelled tube containing PBS, pH 7.2 and wash for 10 minutes in a rotary mixer,
then similarly rewash, and finally return the blocks to the original microscope slides.
vi) Blot excess moisture from the sides of the blocks. Add one drop of the diluted conjugate to each block,
and incubate and wash as before.
vii) Return the blocks to their original slides, and examine the colonies by incident light using fluorescence
microscopy.
Interpretation of the results is subjective and requires some expertise; comparisons with the controls are
essential, and they must give the correct reactions.
Some laboratories use fluorescein-conjugated antiserum in a direct fluorescent antibody test (DFA). A
technique that is widely used for DFA is one in which the reagents are applied successively within stainless
steel cylinders placed on the original mycoplasma agar plate (32). Although this is quick and easy to
perform, the results obtained are less specific than using the indirect method, which is therefore preferred.
b) Indirect immunoperoxidase test

This involves a similar principle to the IFA test except that the binding of specific antibodies to colonies in
situ is detected by adding an anti-rabbit immunoglobulin that has been conjugated to the enzyme
peroxidase. A positive reaction is then developed by adding an appropriate substrate which, on oxidation,
produces coloured colonies. An immunobinding procedure can also be used in which the test colonies are
blotted on to nitrocellulose (21) and then reacted in a similar manner. As with IFA, polyclonal sera should be
used for serotyping isolates by IP. The advantage of the IP test over immunofluorescence is that the IP test
does not require an expensive fluorescence microscope.
c) Growth inhibition test

In the GI test, the growth of mycoplasmas is inhibited by specific antiserum, enabling species to be
identified. It is relatively insensitive and sera must be high-titred, monospecific and prepared in mammalian
hosts as poultry sera do not always inhibit mycoplasma growth efficiently. The organism under test must be
in pure culture (cloned) and several dilutions should be tested; a concentration of 104 colony-forming units
(CFU/ml) is optimal. The rate of growth of the organism may influence growth inhibition, and it is helpful to
retard growth initially by incubating at 27°C for 24 hours, followed by incubation at 37°C thereafter. Details of
the test and its interpretation are published elsewhere (6).

Nucleic acid detection methods
An alternative to conventional culture and identification is the use of specific DNA detection methods. MG or MS
may be detected by hybridisation with DNA probes, but now it is much more common to use the PCR to amplify
specific portions of DNA in the test material. At least one commercial MG DNA test kit uses a PCR directly on
material extracted from swabs. One commercial company produces a kit to detect MG field strains and one that
identifies the vaccine F strain. Several ‘in-house’ PCR-based tests have also been published for MG including a
multiplex PCR, which is designed to detect all four avian mycoplasma pathogens (34), but which has not been
validated with clinical samples. Several methods are cited by Kempf (16) and, in addition, a manual published by
Lauerman (23) contains a validated PCR assay for MG, MS, and other avian mycoplasmas based on unique
sequences contained in the 16S rRNA gene. This method for MG is presented below. In the USA, a PCR based
on the mgc2 gene of MG (12) or the vlhA gene of MS (14) is becoming more widely used, because preliminary
strain identification can be made by sequencing of the PCR product; it must be remembered that unrelated strains
may occasionally share the same sequence.
a) DNA isolation
DNA is extracted from swab samples (three–five may be pooled) suspended in 1 ml of PCR-grade PBS in a
1.5 ml snap-cap Eppendorf tube. The suspension is centrifuged for 30 minutes at 14,000 g at 4°C. The
supernatant is carefully removed with a Pasteur pipette and the pellet is suspended in 25 µl PCR-grade
water. The tube and the contents are boiled for 10 minutes and then placed on ice for 10 minutes before
centrifugation at 14,000 g for 5 minutes. The DNA is in the supernatant.
b) Primers
The MG primers consist of the following sequences.
MG-14F: 5’-GAG-CTA-ATC-TGT-AAA-GTT-GGT-C-3’
MG-13R: 5’-GCT-TCC-TTG-CGG-TTA-GCA-AC-3’
For MS, the following primers are used:
MS–F: 5’-GAG-AAG-CAA-AAT-AGT-GAT-ATC-A-3’
MS–R: 5’-CAG-TCG-TCT-CCG-AAG-TTA-ACA-A-3’
c) Polymerase chain reaction

The reaction mixture should be prepared in a separate clean area using a set of dedicated pipetters. For one
50 µl PCR reaction the mixture is as follows:
H2O Ultra-pure 35.75 µl

10 × PCR Buffer 5.00 µl
dNTP(l0 mM) 1.00 µl
F Primer (20 pmole/µl) 0.50 µl
R Primer (20 pmole/µl) 0.50 µl
Taq (5 U/µl) 0.25 µl
MgCl2 (50 mM) 2.00 µl
A 45 µl volume of the reaction mixture is dispensed into each PCR tube. The reaction mixture should be
overlaid with a few drops of light weight mineral oil unless the thermocyler is equipped with a heated lid. The
tubes are then taken to another clean area where the appropriate DNA sample (5 µl) is added to each tube.
Positive and negative controls should be used in each run.
The tubes are then placed in a thermal cycler for the following cycles: 40 cycles: 94°C for 30 seconds, 55°C
for 30 seconds, 72°C for 60 seconds, 1 cycle (final extension): 72°C for 5 minutes and soak at 4°C.
d) Electrophoresis
PCR products are detected by conventional 2% agarose gel electrophoresis, incorporating appropriate size
markers, followed by examination under UV light. The PCR product for MG is 185 bp. Visualisation of the
PCR products should be carried out in a separate laboratory area, well separated from all other steps in the
PCR procedure.
PCR tests still tend to be carried out by specialist laboratories and should probably be regarded as useful
adjuncts to the present diagnostic methods once their validity is firmly established. Great care needs to be
taken to avoid contamination of samples with MG or MS DNA from nearby post-mortem rooms, culture

laboratories or from positive amplificates from previous PCR runs (see Chapter 1.1.5 Validation and quality
control of polymerase chain reaction methods used for the diagnosis of infectious diseases, for appropriate
safeguards). However one commercial kit referred to above is now licensed by the United States
Department of Agriculture (USDA) as a diagnostic method and approved for use in the National Poultry
Improvement Plan (NPIP). It should be noted that PCR tests are not validated for testing day-old birds for
accurate detection of infection.
Molecular methods are also available for differentiation of MG and MS strains (16), but their use tends to be
restricted at present to specialist laboratories. A rapid and accurate method for DNA fingerprinting uses arbitrary
primed PCR or random amplified polymorphic DNA (RAPD). This technique uses short, arbitrary PCR primers,
which generate reproducible patterns in agarose gels (8). This method is rapid and simple, and has proven to be
very useful for rapid identification of strains of MG for epidemiological studies. However, there may be problems
with reproducibility, so strains to be compared must be run on the same gel. Also, interpretation of banding
patterns which appear to be similar can be difficult.
Gene-targeted sequencing (GTS), using PCR primers for the mgc2, gapA, pvpA, and MGA_0309 genes of MG
can be used to provide an accurate and reproducible method of typing of strains, which will allow rapid global
comparisons between laboratories (9). Preliminary strain identification with diagnostic PCR primers for the mgc2
gene of MG or the vlhA gene of MS by sequencing of the PCR product allows for preliminary strain identification
without the need for prior isolation of the organism (14, 15). However, unrelated strains sometimes have identical
sequences using these primers, and further characterisation may be necessary.
The serological tests in common use may lack specificity and/or sensitivity; their use is strongly recommended for
monitoring flocks rather than for testing individual birds. Diagnosticians wishing to use such tests are advised to
establish the test sensitivity and specificity (Chapter 1.1.4 Principles of validation of diagnostic assays for
infectious diseases) under their own laboratory conditions. It should also be noted that these tests have not been
validated for use with sera from day-old birds or from game birds (4).
The most commonly used tests are RSA, ELISA and HI although several others have been described such as
radioimmunoassay, microimmunofluorescence and IP assay. The number of sera to be tested within a flock
depends on the level of detection and the confidence limits required. Minimal requirements may be laid down for
international trade and the frequency of testing may also be stipulated as, for example, in the European
Communities Council Directive 90/539/EEC. Minimal requirements and approved tests are also set out for
members of the NPIP of the USA.
Poultry companies using ELISA technology for screening large numbers of sera for virus antibodies may find this
type of assay convenient also for mycoplasma testing. The ELISA technology will not be described in detail here
because several MG kits are available commercially. Instead, the details of the HI test are provided as the
reagents needed for this test are not widely available commercially.
a) Rapid serum agglutination test

Sera are collected from a sample of the flock and, if not tested immediately, are stored at 4°C and not
frozen. The test should be carried out at room temperature (20–25°C) within 72 hours of serum collection
and the reagents should also be at room temperature. Prior centrifugation will reduce nonspecific reactions.
The RSA antigens are available commercially, but they may vary in specificity and sensitivity from different
manufacturers and from batch to batch. They must be stored according to the manufacturer’s instructions.
Suitable RSA-stained antigens may also be prepared ‘in-house’ using culture methods as described in
Section B.1.; these are then stained with crystal violet dye. Quality control standards for mycoplasma
antigens for serological tests are described below.
Test procedure (1)

i) Drop one volume (approximately 0.02 ml) of serum on to a clean white tile or glass plate followed by
one volume of stained MG or MS antigen. Do not allow the serum to dry out before addition of the
antigen. It is important to shake the antigen bottle vigorously and frequently during use to keep the
correct amount of antigen in suspension.
ii) Use a stirring rod to spread the mixture over a circular area of approximately 1.5 cm diameter. Rock
the tile or plate for 2 minutes. Agglutination is indicated by flocculation of the antigen within 2 minutes.
iii) Include known positive and negative controls in the test.
iv) Retest serial dilutions of any sera that agglutinate after heating at 56°C for 30 minutes. If they still react
strongly, they are considered to be positive on dilution (1/4 or more).

In the USA, MG and MS positive reference antisera can be obtained from the USDA National Veterinary
Services Laboratories (NVSL), and in Europe from AFSSA Ploufragan 1, France. MG and MS and control
sera produced in chickens or in turkeys and with a range of titres can be purchased. Sets of antisera can be
purchased also from the University of Georgia Department’s of Avian Medicine, subject to availability.
There are no international standards for interpreting these tests, but a high proportion of positive sera in a
flock (10% or more) indicates MG infection, especially if confirmed by HI test or ELISA. For further
confirmation, the flock should be retested within a month. Inconclusive results make it necessary to attempt
to isolate the organism or to demonstrate the presence of its DNA. Doubtful results for MG or MS should be
investigated by performing tests with MS antigen (and vice versa) as infection with these organisms
sometimes causes cross-reactions.
Tests can be conducted on yolk as well as sera although the yolk must first be diluted or extracted.
b) Haemagglutination inhibition test

MG and MS are capable of haemagglutinating avian red blood cells (RBCs), and specific antibodies in sera
cause inhibition. A strain should be selected that grows well and haemagglutinates reliably. The HI test
requires a satisfactory haemagglutinating MG and MS antigen, washed fresh chicken or turkey RBCs, as
appropriate, and the test sera. The antigen can be either a fresh broth culture or a concentrated washed
suspension of the mycoplasma cells in PBS. It may be difficult to sustain a supply of high-titred broth culture
antigen; however, the use of concentrated antigen (usually containing 25–50% glycerol and stored at
–70°C), increases the likelihood of nonspecific reactions. In the USA, MG and MS haemagglutination (HA)
antigen can be purchased from the NVSL.
The HI test follows well-known procedures (1). The HA titre of the antigen is first determined in doubling
dilutions, the HA unit being defined as the least amount of antigen giving complete HA in the test system
employed. The HI test should be performed using 4 HA units by the following method or a method having
equivalent sensitivity as determined by tests with known positive sera.
All HA titrations and HI tests are best performed in multiwell plastic plates with V-shaped wells and using
constant volumes of 50 µl. A positive and a negative control serum are incorporated into each test. One row
of eight wells is required for each serum under test.
• Test procedure
i) Add 50 µl of PBS to the first well in each row.
ii) Add 8 HA units of antigen in 50 µl volumes to the second well in each row and add 50 µl of 4 HA units
of antigen to each of wells 3 to 8.
iii) Add 50 µl of a previously-prepared 1/5 dilution of the serum under test to the first well, mix, and transfer
50 µl to the second well, and so on, and discard 50 µl from the last well. The first well is the serum
control well.
iv) Six wells are required for the antigen control. Add 50 µl of PBS to wells 2 to 6, inclusive, and add 50 µl
of the 8 HA unit antigen to wells 1 and 2. Mix the contents of well 2 and transfer 50 µl to well 3, mix and
repeat up to well 6, and discard 50 µl.
v) Two wells are required for the RBC control. Add 50 µl of PBS to each of these.
vi) Add 50 µl of a 0.5% suspension of RBCs (chicken cells for chicken serum and turkey for turkey serum)
to all wells.
vii) Shake the plate lightly to ensure thorough mixing of the well contents, and read after standing for
approximately 50 minutes at room temperature or when the antigen titration is reading 4 HA units. For
reading, the plate should be tilted and only those wells in which the RBCs ‘stream’ at the same time as
those in the RBC control wells should be considered to be inhibited. The serum control should show a
clear button of RBCs and the positive and negative controls should react as expected. The HI titre is
the highest serum dilution exhibiting complete inhibition of HA.
Sera giving nonspecific HA must be adsorbed to remove all nonspecific haemagglutinins so that a clear
button is obtained in the control well without HA antigen. The adsorption is carried out by incubating 1 ml of
the serum dilution with 6–8 drops of packed washed chicken or turkey RBCs. The cells are removed after
incubation at 37°C for 10 minutes, and the supernatant is tested for haemagglutinating activity.
1 Agence française de sécurité sanitaire des aliments (AFFSA) Ploufragan, Mycoplasmology Bacteriology Unit, 22440
Ploufragan, France.

There is no official definition of positive and negative results for international trade but the NPIP of the USA
states that titres of 1/80 or above are considered to be positive and titres of 1/40 are strongly suspicious.

Several commercial MG and MS antibody ELISA kits are marketed. The sensitivity is determined to some
extent by the manufacturer’s recommendations for the cut-off levels for positive and suspicious reactions.
Sensitivity may sometimes be ‘damped down’, to avoid the well-known cross-reaction between MG and MS.
One ELISA uses an MAb that recognises an epitope on a 56 kDa polypeptide of MG (7). In this system,
ELISA plates are coated with whole cell MG antigen and the sera under test are added as in the
conventional indirect ELISA, but the reaction is assessed by the extent of blocking that occurs when the
conjugated MAb is added. A similar ELISA has also been marketed for MS. One advantage is that the
system can be used for sera from any avian species without adaptation.
• Quality control of Mycoplasma gallisepticum and M. synoviae antigens

i) Mycoplasma gallisepticum antigens
Antigens are usually prepared from the S6 strain or the A5969 strain of MG. Antigens prepared from
other strains may also be used when necessary.
MG antigen for the RSA test: The methods of quality control described below apply solely to
suspensions of MG stained with a suitable dye and containing preservative and intended for use in the
rapid plate agglutination test with serum. Such antigens are available commercially.
On microscopic examination, the antigen should appear as a homogeneous suspension without
floccules or precipitates and the suspending liquid should be free from residual dye. It must be free
from contamination with bacteria and fungi. The pH must be between 6.5 and 7. It must be stored at
5±3°C and be warmed to room temperature before use.
The sensitivity and specificity of the antigen is determined with respect to its reaction with known
positive sera of high and low titre and known negative sera. A positive reaction is recognised by the
formation of coloured floccules and the clearing of the suspending medium. The criteria described
above continue to apply until the expiry date declared by the manufacturer.
MG antigen for the HI test: The test is preferably performed with live, actively growing cultures. The
antigen must be free from contamination with bacteria and fungi.
MG antigen for the ELISA: It may be difficult to prepare satisfactory antigen for use in the indirect
ELISA without considerable prior experimentation and confirmation of sensitivity and specificity. Use of
a reliable commercial kit is probably the best approach for most diagnostic laboratories. Some kits are
now USDA-licensed and approved for use in the NPIP in the USA.
ii) Mycoplasma synoviae antigens

Antigens prepared from the WVU 1853 strain or other suitable strains should be used.
Mycoplasma synoviae antigen for the RSA test: the specifications apply as for MG antigen for the RSA
test.
Mycoplasma synoviae antigen for the HI test: the same specifications apply as for MG antigen for the
HI test.
iii) Additional comments

Sera giving nonspecific reactions to the RSA test do not usually give a positive reaction in the HI test
using live HA antigen. Positive RSA reactions can be confirmed by the HI test with sera taken after the
first 2–3 weeks of infection (the time taken for HI antibodies to develop). However, the HI test tends to
be strain specific (20) and therefore may lack sensitivity. ELISA may be a useful alternative.
Samples of serum should not be frozen before use in RSA tests. They should be free from haemolysis
and contamination to avoid nonspecific reactions. The use of inactivated vaccines for other diseases
may result in nonspecific reactions. Samples should be tested as soon as possible (within 72 hours)
because mycoplasma antibodies may deteriorate on storage. Sera may be inactivated in a water bath
at 56°C for 30 minutes.

The preferred method of control is to maintain MG- and MS-free flocks. Vaccination should be considered only in
situations where field exposure is inevitable, such as on multi-age sites. Potential exposure of neighbouring
poultry flocks should also be carefully considered.

Two types of vaccines are available for the control of MG. These are mild to avirulent MG strains used as live
vaccines, or inactivated oil-emulsion bacterins. The subject of MG vaccination has been reviewed by Whithear
(35). Although there is antigenic variability among MG strains, it is thought that vaccination with a single strain is
sufficient.
Live vaccines: methods of use
The use of live vaccines is equivalent to ‘controlled exposure’. The objective is to infect the flock with a mild,
immunogenic MG strain at an age when little or no significant damage occurs. Such exposure results in
resistance to challenge later in life, such as on multi-age commercial sites. Successfully vaccinated birds are
resistant to respiratory disease, airsacculitis, and egg production drops caused by MG. Vaccination also results in
reduced levels of egg transmission in breeders.
The F strain of MG has been the most commonly used vaccine strain (5). It is a naturally occurring strain of mild
to moderate virulence for chickens, but it is virulent for turkeys. It ordinarily spreads slowly from bird to bird. When
administered to healthy chickens via the upper respiratory tract, little or no respiratory reaction is observed.
However, when administered by aerosol or in the presence of other respiratory disease agents, such as
Newcastle disease or infectious bronchitis virus, respiratory signs and airsacculitis may result. Vaccinated
chickens are permanent carriers, so a single dose is adequate. Use of F strain vaccine in each replacement flock
on a multi-age site will eventually result in displacement of the field strain with the vaccine strain. Strains ts-11
and 6/85 are avirulent and spread to unvaccinated birds does not occur or occurs very poorly when birds are in
very close contact (25).
Commercial pullets are usually vaccinated between 12 and 16 weeks of age, but vaccination of younger or older
birds is permissible. It is essential that vaccination occurs before the flock is naturally infected. Vaccination in
cases of probable early field exposure can be carried out in birds as young as 2–4 weeks of age. For the F strain,
intranasal or eyedrop administration is preferred. Administration in the drinking water may result in some birds
being missed unless the procedure is carried out properly. Aerosol administration should also be done carefully,
so that all birds are exposed. A respiratory reaction should be expected at approximately 5–7 days after
vaccination if aerosol administration is used. Vaccinated flocks should be tested with the agglutination test
approximately 3–4 weeks post-vaccination to be sure that all birds were properly exposed. It is desirable that
birds be vaccinated at an age when there is no reaction to other respiratory vaccines. Strain ts-11 should be
administered by eyedrop, and 6/85 is given as a fine spray. Vaccination with ts-11 results in a low but distinctive
serological response by serum plate agglutination, HI, and ELISA, but vaccination with 6/85 does not ordinarily
result in a serological response. No post-vaccination reaction should be observed with 6/85 or ts-11. Flocks
vaccinated with F strain or ts-11 are culture positive for the life of the flock, but 6/85 may be difficult to recover
later than 4–6 weeks after vaccination.
Commercial live vaccines should be used within 1–2 hours after reconstitution. Lyophilised vaccine should be
stored at 4°C. Some manufacturers supply the vaccine frozen. Such vaccine should be stored in liquid nitrogen,
dry ice, or at –70°C or colder. Live MG vaccine is not stable for long periods at ordinary freezer temperatures.
Storage for more than a few days at –20°C should be avoided.
Strains 6/85 and ts-11 are inherently safer than F strain, although the level of protection may be somewhat less,
and may be useful as the primary vaccine strain on a multi-age site or as a ‘second generation vaccine’ on sites
previously using F strain vaccine. They may also be preferred in situations where inadvertent exposure of
neighbouring poultry flocks is of concern. F strain displaces wild-type MG more efficiently than either ts-11 or
6/85, but ts-11 has been used to eradicate F strain MG from a multi-age commercial egg-production site (33).
Multi-age sites where strain 6/85 is consistently used often test MG-negative, suggesting that it has displaced the
wild-type strain.
Live vaccines have also been used in some countries in broiler breeder pullets. In Australia, ts-11 live vaccine is
being extensively used in broiler breeder pullets as well as in commercial layers. F strain vaccine has been used
in broiler breeder pullets raised under multi-age conditions in some Latin American countries for several years;
more recently there has been limited use of strains ts-11 and 6/85. There has been limited use of the 6/85 strain
as a vaccine for commercial turkeys in the USA, but no good data on its effectiveness are available. Generally,
vaccination of turkeys with live vaccines is not recommended and vaccination of broilers with either live or
inactivated vaccines has not been successful. None of the vaccines has been validated for use in game birds.

A live vaccine for MS is available in several countries for use in broiler breeder and layer chickens. It is produced
from a temperature-sensitive mutant, MS-H (29). Its characteristics and method of use are similar to those for the
MG vaccine, ts-11.
Inactivated vaccines: method of use
MG bacterins are prepared from a concentrated suspension of whole cells that is emulsified into an oil adjuvant. A
high antigen content is essential.
Bacterins are ordinarily used in commercial pullets to provide protection against egg-production drops that occur
after MG exposure on multi-age layer sites (13). They may also be used to reduce the level of egg transmission in
breeder pullets. Use of bacterins in broilers is limited by the fact that birds vaccinated before 1–2 weeks of age
are not protected. Although bacterins may provide protection against respiratory signs, airsacculitis, and egg-
production losses, vaccinated flocks are readily infected. The duration of immunity is not known, but most flocks
are exposed within 1–2 months after vaccination.
Administration is by the intramuscular or subcutaneous route, usually with a dose of 0.5 ml per bird. There is a
risk that a persistent reaction at the site of vaccination will require trimming of carcasses of spent fowl vaccinated
by the intramuscular route, so subcutaneous administration in the upper dorsal part of the neck is the most
commonly used route. Two doses are preferred, but cost and labour considerations may dictate the use of a
single dose, usually between 16 and 18 weeks of age for commercial pullets. A multidose syringe may be used.
All equipment should be cleaned and sterilised between flocks, and vaccination crews should exercise proper
methods of biosecurity when travelling between flocks. Vaccine should be stored at 2–8°C up to the time of use. It
should not be frozen or exposed to strong light.
A similar bacterin for MS is also licensed in the USA, but it has received limited use.
1. Seed management

Live vaccine
The vaccine strain should be immunogenic, must readily colonise the upper respiratory tract, and cause
minimal damage to the respiratory system. A strong antibody response does not necessarily correlate with
immunity.
The seed culture should be free from all extraneous agents. The culture should be cloned to ensure purity. If
desired, restriction endonuclease patterns of the mycoplasmal DNA on agarose gels can be run to be sure
of the identity and purity of the strain.
The seed culture should be stable with no tendency to revert to virulence. This can be confirmed with ten
back passages in susceptible chickens. Contact chickens can be introduced at weekly intervals. If
necessary, tracheal swabs can be taken from infected chickens and can then be inserted into the trachea of
contact chickens. Transmission of the organism should be proven. The resulting isolate can then be used to
challenge susceptible chickens.
Killed vaccine
For killed vaccines the most important characteristics are high yield and good antigenicity. It is assumed, but
not proven, that virulent strains are desirable. The seed culture should be free from all extraneous
organisms.
The seed culture may be propagated in a medium similar to that described above (Section B.1) For live
vaccines, the broth culture is lyophilised or frozen at –70°C or colder. For bacterins the culture must be
concentrated and resuspended in a small volume of saline or PBS before the emulsion is prepared.
Data on efficacy should be obtained before bulk manufacture of vaccine begins. Chickens should be
vaccinated by the same route that will be used in the field. Vaccinated birds should be challenged, and
protection should be determined against respiratory signs, nasal discharge, and/or airsacculitis. Ideally,
protection against egg-production losses should be evaluated, but such challenge trials are expensive and
cumbersome.

Efficacy test: Groups of 20 specific pathogen free (SPF) chickens or at least mycoplasma-free chickens,
2 weeks of age or older, are vaccinated by eyedrop or other route of administration with one field dose of live
vaccine, or subcutaneously or intramuscularly with one dose (usually 0.5 ml) of bacterin. A similar group of
unvaccinated chickens is maintained separately as controls. All chickens should be challenged with a 24-
hour broth culture of a virulent strain of MG, 2–3 weeks post-vaccination. A simple challenge method is
inoculation of 0.1 ml of the challenge culture into the posterior thoracic air sac. All birds are necropsied 7–
10 days post-challenge, and air sac lesions are scored. Alternative methods are to challenge by inoculating
0.1 ml into the infraorbital sinus and examining the birds for nasal discharge from 7 to 14 days post-
challenge or to challenge by aerosol and measure the thickness of the tracheal mucosa on microscopic
sections at four to six equidistant predetermined points (35).
The vaccine must be manufactured in suitable clean and secure accommodation, well separated from diagnostic
facilities or commercial poultry. Special care must be taken to avoid MG contamination of other products
manufactured in the same facility.
Production of vaccine should be on a seed-lot system, using a suitable MG strain of known origin, passage
history, and purity. The growth medium is similar to that given above. The serum used in the growth medium
should be inactivated at 56°C for 1 hour to prevent contamination with any mycoplasmal organism that may be
present, and filter sterilised. A source of SPF serum is desirable.
Broth medium is inoculated, with a rapidly growing inoculum, at a rate of approximately 5% (v/v). Incubation is at
37°C. Production can be in batches using large flasks or in a fermenter. In batch cultures, harvest is
approximately 24 hours after inoculation. Live vaccines are preserved by lyophilisation or by freezing at –70°C, in
liquid nitrogen, or on dry ice.
For bacterin production, the antigen must be concentrated, usually by centrifugation, ultrafiltration, or other
suitable method. Bacterins are made as water-in-oil emulsions, typically 80% mineral oil, 20% aqueous, with
suitable emulsifying agents.
Antigen content: At harvest, the titre should be from 108 to 109 CFU/ml. The antigen concentration of bacterins is
difficult to standardise but may be based on packed cell volume, which is typically 1% (v/v) packed cells in the
final product.
Inactivation of killed vaccines: Inactivation is frequently done with either beta-propiolactone or formaldehyde. The
inactivating agent and the inactivation procedure must be shown under the conditions of vaccine manufacture to
inactivate the vaccine organism and potential contaminants.
Prior to inactivation, care should be taken to ensure a homogeneous suspension free from particles that may not
be penetrated by the inactivating agent. A test for inactivation should be carried out by culture in mycoplasma
broth on each batch of both the bulk harvest after inactivation and the final product. No evidence of growth of
mycoplasma should be observed.
Sterility of killed vaccines: Oil used in the vaccine must be sterilised by heating at 160°C for 1 hour, or by filtration,
and the procedure must be shown to be effective. Tests appropriate to oil-emulsion vaccines are carried out on
each batch of final vaccine as described, for example, in the British Pharmacopoeia (Veterinary) 1985.
4. Batch control
a) Sterility
b) Safety
Live vaccine safety test
The birds vaccinated in the efficacy test given above can be used to evaluate the safety of the vaccine.
Killed vaccine safety test

Birds vaccinated in the efficacy test described above may be observed for adverse local or systemic effects.

c) Potency
Potency tests for both live and killed vaccine can be conducted by the procedures given above for the
efficacy test. The titre of live vaccines should be sufficient to induce infection by the route recommended;
105 CFU/dose is sufficient for eyedrop administration of live F strain vaccine. The recommended dose of ts-
11 is ≥ 107.7 colour changing units (CCU)/dose and for 6/85 a dose of 107–108 CFU was effective in
challenge trials. For MS-H, doses of ≥ 4.8 × 105 were shown to be effective.
d) Duration of immunity (killed vaccine)

Because flocks are generally exposed within 1–2 months after vaccination, duration of immunity is not a
primary consideration. After field challenge, resistance is considered to be permanent.
e) Stability
Evidence should be provided on three batches of vaccine to show that the vaccine passes the batch potency
test at 3 months beyond the requested shelf life.
f) Preservatives
A preservative is normally required for vaccine in multidose containers. The concentration of the
preservative in the final vaccine and its persistency throughout the shelf life should be checked.
A suitable preservative that has already been established for such purposes should be used. Mycoplasmas
are susceptible to many antibacterials except for penicillins; such antibiotics should not be included as
preservatives.
Oil-emulsion vaccines cause serious injury to the vaccinator if accidentally injected into the hand or other
tissues. In the event of such an accident, the person should go at once to a hospital, taking the vaccine
package with him or her. Each vaccine bottle and package should be clearly marked with a warning of the
serious consequences of accidental self-injection. Such wounds should be treated by the casualty doctor as
a ‘grease gun injury’.
Personnel vaccinating birds with live virus vaccines by aerosol spray should wear protective clothes and
masks.
a) Safety
See Section C.4.b.
b) Potency
See Section C.4.c.
REFERENCES
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comparison of macro and micro methods. Vet. Rec., 95, 120–123.
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and Auxiliary Provisions. APHIS Publication 91-55-063. APHIS, USDA, Riverdale, Maryland, USA, 97–100.
3. BRADBURY J.M. (2001). Avian mycoplasmas. In: Poultry Diseases, Fifth Edition, Jordan F., Pattison M.,
Alexander D. & Faragher T., eds. W.B. Saunders, London, UK, 178–193.
4. BRADBURY J.M. (2005). Workshop of European Mycoplasma Specialists. World Poult. Sci. J., 61, 355–357.
5. CARPENTER T.E., MALLINSON E.T., MILLER K.F., GENTRY R.F. & SCHWARTZ L.D. (1981). Vaccination with F-
strain Mycoplasma gallisepticum to reduce production losses in layer chickens. Avian Dis., 25, 404–409.

6. CLYDE W.A., JR. (1983). Growth inhibition tests. In: Methods in Mycoplasmology, Vol. 1, Razin S. & Tully
J.G., eds. Academic Press, New York, USA, and London, UK, 405–410.
7. CZIFRA G., SUNDQUIST B., TUBOLY T. & STIPKOVITS L. (1993). Evaluation of a monoclonal blocking enzyme-
linked immunosorbent assay for the detection of Mycoplasma gallisepticum-specific antibodies. Avian Dis.,
37, 680–688.
8. FAN H.H., KLEVEN S.H. & JACKWOOD M.W. (1995). Application of polymerase chain reaction with arbitrary
primers to strain identification of Mycoplasma gallisepticum. Avian Dis., 39, 729–735.
9. FERGUSON N.M., HEPP D., SUN S., IKUTA N., LEVISOHN S., KLEVEN S.H. & GARCÍA M. (2005). Use of molecular
diversity of Mycoplasma gallisepticum by gene-targeted sequencing (GTS) and random amplified
polymorphic DNA (RAPD) analysis for epidemiological studies Microbiol., 151, 1883–1893.
10. FREUNDT E.A. (1983). Culture media for classic mycoplasmas. In: The Mycoplasmas, Vol. 1, Razin S. & Tully
J.G., eds. Academic Press, New York, USA and London, UK, 127–135.
11. FREY M.L., HANSON R.P. & ANDERSON D.P. (1968). A medium for the isolation of avian Mycoplasmas. Am. J.
Vet. Res., 29, 2163–2171.
12. GARCÍA M., IKUTA N., LEVISOHN S. & KLEVEN S.H. (2005). Evaluation and comparison of various PCR methods
for detection of Mycoplasma gallisepticum infection in chickens. Avian Dis., 49, 125–132.
13. HILDEBRAND D.G., PAGE D.E. & BERG J.R. (1983). Mycoplasma gallisepticum (MG) — laboratory and field
studies evaluating the safety and efficacy of an inactivated MG bacterin. Avian Dis., 27, 792–802.
14. HONG Y., GARCÍA M., LEITING L., BENCINA D., DUFOUR-ZAVALA L., ZAVALA G. & KLEVEN S.H. (2004). Specific
detection and typing of Mycoplasma synoviae strains in poultry with PCR and DNA sequence analysis
targeting the hemagglutinin encoding gene vlhA. Avian Dis., 48, 606–616.
15. HONG Y., GARCIA M., LEVISOHN S., SAVELKOUL P., LEITING V., LYSNYANSKY I., LEY D.H. & KLEVEN S.H. (2005).
Differentiation of Mycoplasma gallisepticum strains using amplified fragment length polymorphism and other
DNA-based typing methods. Avian Dis., 49, 43–49.
16. KEMPF I. (1998). DNA amplification methods for diagnosis and epidemiological investigations of avian
mycoplasmosis. Avian Pathol., 27, 7–14.
17. KLEVEN S.H. (2003). Mycoplasma synoviae infection. In: Diseases of Poultry, Saif Y.M., Barnes H.J., Glisson
J.R., Fadly A.M., McDougald L.R. & Swayne D.E., eds. Iowa State University Press, Ames, Iowa, USA, 756–
766.
18. KLEVEN S.H., FLETCHER O.J. & DAVIS R.B. (1973). Variation of pathogenicity of isolates of Mycoplasma
synoviae with respect to development of airsacculitis and synovitis in broilers. Am. J. Vet. Res., 163, 1196–
1196.
19. KLEVEN S.H., KING D.D. & ANDERSON D.P. (1972). Airsacculitis in broilers from Mycoplasma synoviae: effect
on air-sac lesions of vaccinating with infectious bronchitis and Newcastle virus. Avian Dis., 16, 915–924.
20. KLEVEN S.H., MORROW C.J. & WHITHEAR K.G. (1988). Comparison of Mycoplasma gallisepticum strains by
hemagglutination-inhibition and restriction endonuclease analysis. Avian Dis., 32, 731–741.
21. KOTANI H. & MCGARRITY G.J. (1985). Rapid and simple identification of Mycoplasmas by immunobinding.
J. Immunol. Methods, 85, 257–267.
22. LANDMAN W.J.M. & FEBERWEE A. (2004). Aerosol-induced Mycoplasma synoviae arthritis: the synergistic
effect of infectious bronchitis virus infection. Avian Pathol., 33, 591–598.
23. LAUERMAN L.H. (1998). Mycoplasma PCR Assays. In: Nucleic Amplification Assays for Diagnosis of Animal
Diseases, Lauerman L.H., ed. American Association of Veterinary Laboratory Diagnosticians, Auburn, AL,
USA, 41–52.
24. LEY D.H. (2003). Mycoplasma gallisepticum infection. In: Diseases of Poultry, Saif Y.M., Barnes H.J.,
Glisson J.R., Fadly A.M., McDougald L.R. & Swayne D.E., eds. Iowa State University Press, Ames, Iowa,
USA, 722–744.

25. LEY D.H., MCLAREN J.M., MILES A.M., BARNES H.J., MILLER S.H. & FRANZ G. (1997). Transmissibility of live
Mycoplasma gallisepticum vaccine strains ts-11 and 6/85 from vaccinated layer pullets to sentinel poultry.
Avian Dis., 41, 187–194.
26. LOCKABY S.B., HOERR F.J., LAUERMAN L.H., SMITH B.F., SAMOYLOV A.M., TOIVIO-KINNUCAN M.A. & KLEVEN S.H.
(1999). Factors associated with virulence of Mycoplasma synoviae. Avian Dis., 43, 251–261.
27. LUTTRELL M.P., FISCHER J.R., STALLKNECHT D.E. & KLEVEN S.H. (1996). Field investigation of Mycoplasma
gallisepticum infections in house finches (Carpodacus mexicanus) from Maryland and Georgia. Avian Dis.,
40, 335–341.
28. MALLINSON E.T., ECKROADE R.J. & KLEVEN S.H. (1981). In vivo bioassay and supplemental serologic
techniques for the detection of Mycoplasma in suspect breeding chickens. Avian Dis., 25, 1077–1082.
29. MARKHAM J.F., MORROW C.J. & W HITHEAR K.G. (1998). Efficacy of a temperature-sensitive Mycoplasma
synoviae live vaccine. Avian Dis., 42, 671–676.
30. MAROIS C., DUFOUR-GESBERT F. & KEMPF I. (2002). Polymerase chain reaction for detection of Mycoplasma
gallisepticum in environmental samples. Avian Pathol., 31, 163–168.
31. ROSENDAL S. & BLACK F.T. (1972). Direct and indirect immunoflurescence of unfixed and fixed mycoplasma
colonies. Acta Pathol. Microbiol. Scand. [B], 80, 615–622.
32. TALKINGTON F.D. & KLEVEN S.H. (1983). A classification of laboratory strains of avian Mycoplasma serotypes
by direct immunofluorescence. Avian Dis., 27, 422–429.
33. TURNER K.S. & KLEVEN S.H. (1998). Eradication of live F strain Mycoplasma gallisepticum vaccine using live
ts-11 on a multiage commercial layer farm. Avian Dis., 42, 404–407.
34. WANG H., FADL A.A. & KHAN M.I. (1997). Multiplex PCR for avian pathogenic mycoplasmas. Molec. Cell.
Probes, 11, 211–216.
35. WHITHEAR K.G. (1996). Control of avian mycoplasmoses by vaccination. Rev. sci. tech. Off. int. Epiz., 15,
1527–1553.
36. ZAIN M.Z. & BRADBURY J.M. (1996). Optimising the conditions for isolation of Mycoplasma gallisepticum
collected on applicator swabs. Vet. Microbiol., 49, 45–57.
*
* *
NB: There is an OIE Reference Laboratory for Avian mycoplasmosis (Mycoplasma gallisepticum and
M. synoviae) (see Table in Part 3 of this Terrestrial Manual or consult the OIE Web site for the most up-to-date
list: www.oie.int).

CHAPTER 2.3.6.
AVIAN TUBERCULOSIS
SUMMARY
Avian tuberculosis is an important disease which affects companion, captive exotic, wild and
domestic birds. The disease is most often caused by Mycobacterium avium (serotypes 1, 2 and 3)
and M. genavense. The most significant cause of poultry disease is M. avium.
Clinical signs of the disease vary depending on the organs involved. The classical presentation is
characterised by chronic and progressive wasting and weakness. Diarrhoea is common. Some
birds may show respiratory signs and occasionally sudden death occurs. Some birds may develop
granulomatous ocular lesions.
Mycobacterium tuberculosis is less commonly the cause of infection in birds, often as a result of
transmission from pet bird owners, and clinical signs differ from those caused by the more
commonly occurring species of mycobacteria.
Mycobacterium avium complex and M. intracellulare can also infect an extensive range of different
animal species such as swine, cattle, deer, sheep, goats, horses, cats, dogs, and exotic species.
Mycobacterium genavense has also been reported in a dog and an immunocmpromised cat.
Disease onset in birds is normally more rapid with M. genavense than with M. avium.
In humans, M. avium and M. genavense are capable of inducing a progressive disease that is
refractory to treatment, mostly in immunocompromised hosts. All manipulations involving the
handling of open live cultures or of material from infected birds must be carried out with adequate
biohazard containment.
Diagnosis of tuberculosis in birds depends on the demonstration of Mycobacterium spp. in the dead
bird, or the detection of an immune response, cellular or humoral, in the live bird.
Identification of the agent: Where clinical signs of tuberculosis are seen in the flock, or typical
lesions of tuberculosis are present in birds at necropsy, the demonstration of acid-fast bacilli in
smears or sections made from affected organs is sufficient for a positive diagnosis. If acid-fast
bacilli are not found, but typical signs or lesions are present in the birds, culture of the organism
must be attempted. Any acid-fast organism isolated should be identified by biochemical, nucleic-
acid-based tests, serological or chromatographical (e.g. high performance liquid chromatography
[HPLC]) criteria.
Tuberculin test and serological tests: These tests are normally used to determine the prevalence
of disease in a flock, or to detect infected birds. When used to detect the presence of tuberculosis
in a flock, they should be supported by the necropsy of any birds that give positive reactions.
In domestic fowl, the tuberculin test in the wattle has been the test of choice. This test is less useful
in other species of bird. A better test, especially for waterfowl, is the whole blood stained-antigen
agglutination test (Rozanska). It is more reliable and has the advantage that it will give a result
within a few minutes, while the bird is still being held. The tests are not reliable in caged birds.
Requirements for vaccines and diagnostic biologicals: No vaccines are available for use in
birds. An antigen preparation stained with 1% malachite green is available for the whole blood
agglutination test. Avian tuberculin purified protein derivative is the standard preparation for use in
the tuberculin test of domestic poultry.

Chapter 2.3.6. — Avian tuberculosis
A. INTRODUCTION
Several mycobacterial species can be involved in the aetiology of avian tuberculosis. The disease is most
commonly produced by infection with Mycobacterium avium complex (serotypes 1, 2 and 3) and M. genavense
(25). Other species, such as M. intracellulare, M. scrofulaceum, M. fortuitum, M. tuberculosis and M. bovis are
less common causes of avian tuberculosis (25). Mycobacterium avium complex and M. intracellulare are capable
of infecting an extensive range of different animal species such as swine, cattle, deer, sheep, goats, horses, cats,
dogs, and exotic species (26, 27).
Mycobacterium avium complex consists of three subspecies: M. avium subsp. avium, M. avium subsp.
sylvaticum, and M. avium subsp. paratuberculosis (28). The latter is the causal agent of Johne’s disease, or
paratuberculosis, in ruminants and other mammalian species (see Chapter 2.1.11 Paratuberculosis). Although
successful experimental infections with M. a. paratuberculosis in poultry have been reported (11), there is no
evidence that this organism is involved in the aetiology avian tuberculosis.
Most M. a. avium isolates from birds have a repetitive sequence IS901 in their genome and produce a
characteristic three-band pattern in IS1245 restriction fragment length polymorphism (RFLP) (20). There is
convincing evidence that the presence of IS901 correlates with pathogenicity in birds (6, 15). This repetitive
sequence is also present in M. a. sylvaticum that is capable to produce tuberculosis in birds. IS901 has only been
detected in M. avium strains with serotypes 1, 2 and 3 (15, 20) that are apparently more pathogenic to birds than
other serotypes (25). On the basis of genetic and phenotypic differences it has recently been proposed to divide
M. a. avium into two subspecies: M. a. hominissuis for human and porcine isolates and M. a. avium for bird-type
isolates (13). The isolates designated as M. a. hominissuis had polymorphic multiband IS1245 patterns and were
able to grow at 24 and 45°C. In comparison, the avian isolates designated as M. a. avium had the conserved
three-band pattern in IS1245 RFLP and were unable to grow at 24 and 45°C (13). It is worth noting that the typical
features of bird-isolates, the three-band pattern in IS1245 RFLP and presence of IS901, have also been found in
cervine and bovine isolates of M. a. avium (14).
Tuberculosis in birds is most prevalent in chickens and in wild birds raised in captivity. Turkeys are quite
susceptible, but duck and geese are comparatively resistant. The practices of allowing poultry to roam at large on
the farm (free range) and of keeping the breeders for several years are conducive to the spread of tuberculosis
among them. Infected individuals and contaminated environment (water and soil) are the main source of infection
(25). Mycobacteria can survive for several months in the environment (25).
In most cases, infected birds show no clinical signs, but they may eventually become lethargic and emaciated.
Many affected birds show diarrhoea and comb and wattles may regress and become pale. Affected birds are
usually older than one year. Some show respiratory signs and sudden death may occur, dyspnoea is less
common, and granulomatous ocular lesions (16) and skin lesions have been reported. Under intensive husbandry
conditions, sudden death may occur, often associated with severe lesions in the liver; such lesions are easily
observed at post-mortem examination (25).
The primary lesions of tuberculosis in birds are nearly always in the intestinal tract. Such lesions take the form of
deep ulcers filled with caseous material containing many organisms, and these are discharged into the lumen and
appear in the faeces. Before the intestinal tract is opened, the ulcerated areas appear as tumour-like masses
attached to the gut wall, but when the intestine is opened, the true nature of the mass becomes evident. Typical
caseous lesions are nearly always found in the liver and spleen, and these organs usually are greatly enlarged
because of the formation of new tuberculous tissue. The lungs and the other tissues are ordinarily free from
lesions even in advanced cases.
In most species of affected bird, tuberculous-like lesions are mostly found in the intestinal tract, liver and spleen.
Lesions in other organs are less common. Exceptions include pigeons, waterfowl, and some finches, in which the
disease begins primarily in the respiratory tract.
It is essential to bear in mind that M. avium, M. intracellulare and M. genavense are capable of giving rise to a
progressive disease in humans that is refractory to treatment, especially in immunocompromised individuals (25).
All manipulations involving the handling of open live cultures or of material from infected birds must be performed
with adequate biohazard containment (see Chapter 1.1.2 Biosafety and biosecurity in the veterinary microbiology
laboratory and animal facilities).
If there is a characteristic history of tuberculosis in the flock and typical lesions are found in birds at post-mortem,
the detection of acid-fast bacilli in smears or sections from affected organs, stained by the Ziehl–Neelsen method,

is normally sufficient to establish the diagnosis. Occasionally a case will occur, presumably as a result of large
infecting doses giving rise to acute overwhelming disease, in which affected organs, most obviously the liver,
have a ‘morocco leather’ appearance with fine greyish or yellowish mottling. In such cases acid-fast organisms
may not be found, but careful inspection will reveal parallel bundles of brownish refractile bacilli. Prolongation of
the hot carbol fuchsin stage of Ziehl–Neelsen staining to 10 minutes will usually reveal that these are indeed acid-
fast bacilli, with unusually high resistance to penetration of the stain. Recently, DNA probes and polymerase chain
reaction (PCR) techniques have been used to identify the agent. Traditionally, M. avium is separated from
common nonchromogenic slow-growing organisms by their ability to grow at 42°C (M. a. avium) and biochemical
tests such as the Tween hydrolysis test, pyrazinamidase, growth on thiophen-2-carboxylic acid hydrazide (TCH)-
containing media and tellurite reduction. Mycobacterium genavense is particularly fastidious and has special
requirements for growth and identification.
• Culture
If there is a characteristic flock history and suggestive lesions are found at necropsy, but no acid-fast bacilli are
seen in smears or sections, an attempt must be made to isolate the causative organism from the necropsy
material. Liver or spleen is usually the best organ to use, but if the carcass is decomposed, bone marrow may
prove more satisfactory as it could be less contaminated. As with culture of M. bovis, non-sterile specimens need
to be processed with detergent, alkali or acid to eliminate rapidly growing microorganisms before culture (see
Chapter 2.4.7 Bovine tuberculosis). Mycobacterium avium grows best on media such as Lowenstein–Jensen,
Herrold’s medium, Middlebrook 7H10 and 7H11 or Coletsos, with 1% sodium pyruvate added. It may occasionally
be necessary to incorporate mycobactin, as used for the isolation of M. paratuberculosis and M. silvaticum.
Growth may be confined to the edge of the water of condensation. Cultures should be incubated for at least
8 weeks. Typically M. avium produces ‘smooth’ colonies, within 2–4 weeks; rough variants do occur. Shorter
incubation times can be achieved using the liquid culture BACTEC system.
For M. genavense, use of the BACTEC system with no additives but with pH 6.0 and a lowered oxygen tension is
recommended (18, 19). The optimal solid medium is Middlebrook 7H11 medium acidified to pH 6 and
supplemented with blood and charcoal (17).
Typing of mycobacteria to the species and subspecies level requires a specialised laboratory. Conventional
biochemical tests for species identification are lengthy and fail to distinguish between M. avium and
M. intracellulare. Thus, a miscellaneous group of mycobacteria that includes both species is usually classified
under the denomination of M. avium complex (MAC). Seroagglutination, which is based on sugar residue
specificity of surface glycopeptidolipids, allows classification of MAC organisms into 28 serovars. More
sophisticated typing methods directed at cell-wall-specific targets are currently available, such as enzyme-linked
immunosorbent assays with monoclonal antibodies to major serovars, and high performance liquid
chromatography (HPLC). Serovars 1 to 6, 8 to 11 and 21 are currently ascribed to M. avium, and serovars 7, 12
to 20 and 25 to M. intracellulare. However, no consensus was achieved on other serovars, and some isolates
cannot be typed (9). Tuberculosis in birds is usually caused by M. avium types 1, 2, or 3. If one of these is found,
it may be assumed to be the cause of the disease. If the isolate is not one of these, further identification tests
must be carried out. However, it should be borne in mind that superficial tuberculosis lesions in caged birds,
especially psittacines, may be caused by M. tuberculosis. Hence, if rough colonies of mycobacteria are isolated
from such birds, they should be tested for growth at 42°C. If the isolate will not grow at 42°C, M. tuberculosis
should be suspected.
• Nucleic acid recognition methods
Specific and reliable genetic tests for speciation are currently available (22). Commercial nucleic acid
hybridisation probes have become a ‘gold standard’ for distinction between M. avium and M. intracellulare
cultures. M. genavense can also be distinguished with these tests. A further probe that covers the whole MAC
was also developed, as genuine MAC strains have been described that fail to react with specific M. avium and
M. intracellular probes (23). These tests use a chemiluminescent-labelled, single-stranded DNA probe that is
complementary to the ribosomal RNA of the target organism. The labelled DNA–RNA hybrids are measured in a
luminometer. Various in-house molecular methods have been reported for the identification of mycobacterial
cultures, including MAC. A multiplex PCR method for differentiating M. avium from M. intracellulare and
M. tuberculosis complex has some advantages (4). 16S rRNA sequencing (10) or PCR amplification followed by
either hybridisation with species-specific probes or restriction enzyme analysis (5, 24, 29) may also be used. Even
though some of these methods would theoretically detect the agent directly in tissue samples, none of them has
been validated for this use. Therefore, molecular identification of MAC is currently performed on organisms
previously isolated by culture. Sequencing of hsp65 to distinguish between subsets of M. avium has also been
found useful (30).
Regarding intraspecies genotyping, pulsed-field gel electrophoresis of large DNA restriction fragments has proved
to be highly sensitive (12). Also, a number of DNA mobile elements have been identified that may be exploited for

this purpose. Insertion sequence IS1245, which is virtually M. avium specific, was shown to be the most
discriminative for the analysis of strain relatedness (2, 7). A standardised method consisting of IS1245 restriction
fragment length polymorphism (RFLP) analysis was recently proposed (31). Bird infection was found to be caused
by a particular subset of M. avium strains that are characterised by specific, highly conserved RFLP patterns with
IS1245 and IS901, in addition to serovars 1, 2 or 3 (20).
Recently O’Grady et al. performed RFLP investigation using probes derived from IS901, IS1245 and IS1311 to
study the molecular epidemiology of M. avium and M. intracellulare infection, in particular to gain an
understanding of the sources of infection in humans (14).
If specialised typing facilities are not available, the likelihood that the organism isolated is the cause of the
disease may be established by pathogenicity tests. It is preferred that these be carried out on the species of bird
being investigated, but failing that, domestic fowl or Japanese quail may be used. Young adult birds are best. An
inoculum is prepared by putting a small square of aluminium foil and some glass beads in a screw-capped
container, which is then sterilised and weighed. A loopful of culture is then placed on the foil and the whole is
reweighed. Finally, sufficient sterile normal saline solution is added to suspend the culture at 0.1 mg/ml. Birds are
then inoculated intravenously with 1 ml of the suspension. If the organism is virulent, the bird will die in 5–6 weeks
or, by that time, the bird will have extensive lesions filled with acid-fast bacilli.
2. Immunological methods
Tests used for export depend on the importing requirement of individual countries. In the main, the tuberculin test
or the haemagglutination (stained antigen) test are most frequently used for export testing of poultry.
a) Tuberculin test
The most widely used test in domestic fowl, and the only test for which an international standard for the
reagent exists, is the tuberculin test. The tuberculin is the standard avian purified protein derivative (PPD).
Birds are tested by intradermal inoculation in the wattle with 0.05 ml or 0.1 ml of tuberculin (containing
approximately 2000 International Units [IU]), using a very fine needle of approximately 10 mm × 0.5 mm.
The test is read after 48 hours and a positive reaction is any swelling at the site, from a small firm nodule
approximately 5 mm in diameter to gross oedema extending into the other wattle and down the neck. With
practice, even very small wattles on immature birds can be inoculated successfully. However, in immature
birds the comb may be used, although results are not so reliable. Tuberculin testing of the wattle in turkeys
is much less reliable than in the domestic fowl. Inoculation in the wing web has been recommended as being
more efficient, but this is still not as good as for domestic fowl. Other birds may also be tested in the wing
web, but results are not generally satisfactory. The bare ornamental skin areas on Muscovy ducks and some
species of pheasant can be used, but reliability is doubtful and interpretation difficult. Testing in the foot web
of waterfowl has also been described; the test is not very sensitive and is often complicated by infections of
the inoculation site.
In pheasants, the tuberculin test can be performed in either of two ways. In the first, 0.05 ml or 0.1 ml of
tuberculin is injected into the skin of the lower eyelid. A positive result is indicated by marked swelling at the
site of injection after 48 hours. Alternatively, 0.25 ml of tuberculin is injected into the thoracic muscles and
the birds are observed for 6–10 hours. Infected birds will show signs of depression and keep aside from the
flock, and there may be cases of sudden death. No clinical signs will be provoked in uninfected birds.
b) Stained antigen test

• Preparation of the antigen
An antigen stained with 1% malachite green is used for the rapid whole blood plate agglutination test (21).
The strain used for preparation of the stained antigen must be smooth and not autoagglutinate in saline
suspension. It must conform to the characteristics of the M. avium species.
A strain that will detect infection with any serotype is recommended to be used instead of the specific
serotype that is most likely to be encountered (in Europe serotype 2 for domestic fowl, serotype 1 for
waterfowl). It may be preferable to use a strain that is highly specific for the serotype it detects. The
specificity of strains can be determined only by testing them as antigens, although in general a serotype 2
antigen will always detect serotype 3 infection and vice versa. Serotype 1 strains appear to detect more
often a wide spectrum of infection, and will often also detect infections with mycobactin-dependent
mycobacteria or M. silvaticum. There is no reason not to use a culture containing more than one strain of
M. avium, provided that it shows the desired properties of sensitivity and specificity. Consistency of results
between batches will be easier with the use of pure cultures.

The organism should be grown in a suitable liquid medium, such as Middlebrook 7H9 containing 1% sodium
pyruvate for better growth. Good growth should be obtained in approximately 7 days. The liquid culture is
used as seed for bulk antigen preparation.
Antigen for agglutination tests is best grown on solid medium, such as Löwenstein–Jensen or 7H11,
containing 1% sodium pyruvate instead of glycerol, using Roux flasks or large bottles. The use of solid
medium maximises the chance of detecting any contamination, and antigens grown in some liquid media are
not agglutinated by specific antibody. Liquid seed culture should be diluted (on the basis of experience) to
give discrete colonies on the solid medium. This will usually give the best yield, and again increases the
chance of detecting contamination. About 10 ml of inoculum will usually be enough to allow it to wash over
the whole surface, and provide sufficient moisture to keep the air in the bottle near 100% humidity.
The bottles are incubated at 37°C, and good growth should be obtained in 14–21 days with most strains.
The antigen is harvested by the addition of sterile glass beads and twice the volume of sterile normal saline
(containing 0.3% formalin) as was used to inoculate the bottle. The bottle is then shaken gently to wash off
all the growth and the washing is collected into a sterile bottle and reincubated at 37°C for 7 days. The killed
bacilli are then washed twice in sterile normal saline with 0.2% formalin by centrifugation and resuspension.
This sequence is safer than the original method in which the washing was carried out before the incubation
that kills the organisms. Finally the organisms are again centrifuged and resuspended in sterile normal
saline containing 0.2% formalin and 0.4% sodium citrate, to a concentration of about 1010 bacteria per ml.
This corresponds to a concentration ten times that which matches tube No. 4 on McFarland’s scale.
Cultures for antigen should be inspected for contamination daily for the first 5 days of incubation. The
suspension made from the culture washings is also re-examined microscopically (for likely contaminants
such as yeasts), and rechecked by culture to ensure that the formalin has killed the mycobacteria.
• Validation of the antigen

Cultures should be checked by Gram staining for the presence of organisms other than mycobacteria.
One or more batches for agglutinating antigen must be tested for efficacy in naturally or artificially infected
tuberculous birds by comparison with a standard preparation of known potency. The potency relative to that
of the standard preparation must not differ significantly from that declared on the label. Each bottle of
antigen must be tested with normal chicken serum (to detect autoagglutination) and M. avium positive
chicken serum of low and high antibody content. This should be done, where possible, alongside a previous
batch of stained antigen. Those bottles that give satisfactory agglutination reactions with the antisera can
now be pooled and the antigen stained. This is done by the addition of 3 ml of 1% malachite green solution
per 100 ml of suspension. If possible, the stained antigen should now be checked using whole blood just as
the unstained antigen was tested with serum. The agglutinating antigen should keep for at least 6 months in
the refrigerator at 4°C, and much longer if frozen at –20°C or below. If a batch has not been used for a long
time it should be rechecked, especially for autoagglutination.
The only safety test needed is the culture test of the unwashed antigen after 7 days of incubation, to ensure
that all the bacilli are dead.
• Test procedure
The stained-antigen agglutination test has been used with good results, especially in both domestic and
ornamental waterfowl. A drop (0.05–0.1 ml) of the antigen is mixed with the same volume of fresh whole
blood, obtained by venipuncture, on a white porcelain or enamel tile. The mixture is rocked for 2 minutes and
examined for agglutination. The agglutination may be coarse, in which case it is obvious, or quite fine, in
which case it may be most clearly seen as an accumulation of the malachite-green-stained antigen around
the edge of the drop, leaving the centre a normal blood-red colour. This test is especially useful for
screening large flocks for immediate culling, and therefore has advantages over the tuberculin test for the
control of the disease, even in domestic fowl. It has also been claimed that in domestic fowl it is more
reliable than the tuberculin test.
Note on limitation of use
Neither the tuberculin test with avian tuberculin nor the stained-antigen agglutination test is likely to be of any
value in cases of M. tuberculosis infection in caged birds.

No vaccines are available.

Avian tuberculin is a preparation made from the heat-treated products of growth of M. avium. It is used by
intradermal injection to reveal delayed hypersensitivity as a means of identifying birds infected with or sensitised
to the same species of tubercle bacillus.
1. Seed management

Strains of M. avium used to prepare seed cultures should be identified as to species by appropriate tests.
They should be shown to be free from contaminating organisms and to be capable of yielding a product of
satisfactory quality. The strains recommended by the European Union (EU), for example, are D4ER and
TB56. Reference may also be made to the World Health Organization (32).
The seed material is kept as a stock of freeze-dried cultures. If the cultures have been grown on solid media,
it will be necessary to adapt the organism to grow as a floating culture. This is most easily accomplished by
incorporating a piece of potato in the flasks of liquid medium (e.g. Watson Reid’s medium). When the culture
has been adapted to liquid medium, it can be maintained by passage at 2–4-week intervals (1, 8).
The production culture substrate must be shown to be capable of producing a product that conforms to the
standards of the European Pharmacopoeia or other international standards (3). It must be free from
ingredients known to cause toxic or allergic reactions.
c) Validation
The strains of M. avium used as seed cultures must be shown to be free from contaminating organisms.
Seed lots must be shown to be efficacious in producing tuberculin with sufficient potency. The necessary
tests are described in Section C.4 below.
Avian tuberculin may be made by the following three methods:
a) Old tuberculin
The organism is cultivated in glycerol broth medium, killed by heating in flowing steam, and filtered to
remove cells. The filtrate is concentrated by heat and sterilised by filtration.
b) Heat-concentrated synthetic-medium tuberculin

As for old tuberculin but the glycerol broth medium is replaced by a synthetic medium (modified Dorset-
Henley’s synthetic medium).
c) Purified protein derivative

As for heat-concentrated synthetic-medium (HCSM) tuberculin but, instead of being concentrated by heat,
the protein in the filtrate is precipitated chemically (ammonium sulphate or trichloroacetic acid [TCA] are
used), washed and resuspended. PPD tuberculin is recommended as it gives fewer false-positive reactions
and can be standardised more precisely. An antimicrobial preservative that does not give rise to false-
positive reactions, such as phenol (not more than 0.5% [w/v]), may be added. Mercurial derivatives should
not be used. Glycerol (not more than 10% [w/v]) or glucose (2.2% [w/v]) may be added as a stabiliser. The
product is dispensed aseptically into sterile neutral glass containers, which are then sealed to prevent
contamination. The product may be freeze-dried.
The production flasks, inoculated from suitable seed cultures, are incubated for the appropriate time period. Any
flasks showing contamination or grossly abnormal growth should be discarded after autoclaving. As incubation
proceeds, the surface growth of many cultures becomes moist and may sink into the medium or to the bottom of
the flask. In PPD tuberculins, the pH of the dissolved precipitate (the so-called concentrated tuberculin) should be

pH 6.6–6.7. The protein level of the PPD concentrate is determined by the Kjeldahl method. Total nitrogen and
trichloroacetic acid precipitable nitrogen are usually compared.
4. Batch control
a) Sterility
Sterility testing is generally performed according to the European Pharmacopoeia or other guidelines (see
also Chapter 1.1.9 Tests for sterility and freedom from contamination of biological materials).
b) Safety
Tuberculin PPD can be examined for freedom from living mycobacteria using the culture method described
previously. This culture method, which does not require use of animals, is used in many laboratories and its
use is encouraged over the use of animals for this purpose. The following is the previously described
method, using experimental animals, to evaluate safety of PPD. Two guinea-pigs, each weighing not less
than 250 g and that have not been treated previously with any material that will interfere with the test, are
injected subcutaneously with 0.5 ml of the tuberculin under test. No abnormal effects should occur within
7 days.
c) Residual infectivity
Tests on tuberculin for living mycobacteria may be performed either on the tuberculin immediately before it is
dispensed into final containers or on samples taken from the final containers themselves. A sample of at
least 10 ml must be taken and this must be injected intraperitoneally or subcutaneously into at least two
guinea-pigs, dividing the volume to be tested equally between the guinea-pigs. It is desirable to take a larger
sample, 50 ml, and to concentrate any residual mycobacteria by centrifugation or membrane filtration. The
guinea-pigs are observed for at least 42 days, and are examined macroscopically at post-mortem. Any
lesions found are examined microscopically and by culture.
Each filled container must be inspected before it is labelled, and any showing abnormalities must be
discarded.
d) Sensitising effect
To test the sensitising effect, three guinea-pigs that have not previously been treated with any material that
could interfere with the test are each injected intradermally on each of three occasions with the equivalent of
500 IU of the preparation under test in a 0.1 ml volume. Each guinea-pig, together with each of three control
guinea-pigs that have not been injected previously, is injected intradermally 15–21 days after the third
injection with the same dose of the same tuberculin. The reactions of the two groups of guinea-pigs should
not be significantly different when measured 24–28 hours later.
e) Potency
The potency of avian tuberculin is determined in guinea-pigs sensitised with M. avium, by comparison with a
standard preparation calibrated in IU.
Use no fewer than nine albino guinea-pigs, each weighing 400–600 g. Sensitise the guinea-pigs by
administering to each, by deep intramuscular injection, a suitable dose of inactivated or live M. avium. The
test is performed between 4 and 6 weeks later as follows: Shave the guinea-pigs’ flanks so as to provide
space for three-to-four injections on each side. Prepare at least three dilutions of the tuberculin under test
and at least three dilutions of the standard preparation in isotonic buffer solution containing 0.0005% (w/v)
polysorbate 80 (Tween 80). Choose the dilutions so that the reactions produced have diameters of not less
than 8 mm and not more than 25 mm. Allocate the dilutions to the injection sites randomly according to a
Latin square design. The dilutions are injected intradermally in volumes of 0.1 or 0.2 ml.
Between 24 and 28 hours, the diameters of the reactions are measured and the results are calculated using
standard statistical methods, taking the diameters to be directly proportional to the logarithms of the
concentrations of the tuberculins. The estimated potency must be not less than 75% and not more than
133% of the potency stated on the label. The test is not valid unless the fiducial limits of error (p = 0.95) are
not less than 50% and not more than 200% of the estimated potency. If the batch fails a potency test, the
test may be repeated one or more times provided that the final estimate of potency and of fiducial limits is
based on the combined results of all the tests.
It is recommended that avian tuberculin should contain the equivalent of at least 25,000 IU/ml, giving a dose
for practical use of 2500 IU/0.1 ml.

f) Specificity
One or more batches of tuberculin may be tested for specificity together with a standard preparation of
bovine tuberculin by comparing the reactions produced in guinea-pigs sensitised with M. bovis using a
procedure similar to that described in Section C.4.d. In guinea-pigs sensitised with M. bovis, the potency of
the preparation of avian tuberculin must be shown to be not more than 10% of the potency of the standard
preparation of bovine tuberculin used in the potency test.
g) Stability
During storage, liquid avian tuberculin should be protected from the light and held at a temperature of 5°C
(±3°C). Freeze-dried preparations may be stored at higher temperatures (but not exceeding 25°C) protected
from the light. During use, periods of exposure to higher temperatures or to direct sunlight should be kept at
a minimum.
Provided the tuberculins are stored at a temperature of between 2°C and 8°C and protected from light, they
may be used up to the end of the following periods subsequent to the last satisfactory potency test: Liquid
PPD tuberculins: 2 years; lyophilised PPD tuberculins: 8 years; HCSM tuberculins diluted: 2 years.
h) Preservatives
Antimicrobial preservatives or other substances that may be added to a tuberculin, must have been shown
not to impair the safety and effectiveness of the product. The maximum permitted concentrations for phenol
is 0.5% (w/v) and for glycerol it is 10% (v/v). The pH should be between 6.5 and 7.5.
i) Precautions (hazards)
Experience both in humans and animals led to the observation that appropriately diluted tuberculin injected
intradermally results in a localised reaction at the injection site without generalised manifestations. Even in
very sensitive persons, severe, generalised reactions are extremely rare and limited.
a) Safety
A test for the absence of toxic or irritant properties must be carried out according to the specifications of the
European Pharmacopoeia (see also Section C.4.b).
b) Potency
The potency of tuberculins must be estimated by biological methods. These methods must be used for
HCSM and PPD tuberculins; they are based on the comparison of the tuberculins to be tested with standard
tuberculins (see also Section C.4.d).
REFERENCES
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2. BONO M., JEMMI T., BERNASCONI C., BURKI D., TELENTI A. & BODMER T. (1995). Genotypic characterisation of
Mycobacterium avium strains recovered from animals and their comparison to human strains. Appl. Environ.
Microbiol., 61, 371–373.
3. COUNCIL OF EUROPE (2000). Purified protein derivative (avian). In: European Pharmacopoeia, Fourth Edition.
Editions of the Council of Europe, Strasbourg, France, 1694.
4. COUSINS D., FRANCIS B. & DAWSON D. (1996). Multiplex PCR provides a low-cost alternative to DNA probe
methods for rapid identification of Mycobacterium avium and Mycobacterium intracellulare. J. Clin.
Microbiol., 34, 2331–2333.
5. DEVALLOIS A., PICARDEAU M., GOH K.K., SOLA C., VINCENT V. & RASTOGI N. (1996). Comparative evaluation of
PCR and commercial DNA probes for detection and identification to species level on Mycobacterium avium
and Mycobacterium intracellulare. J. Clin. Microbiol., 34, 2756–2759.

6. DVORSKA L., BULL T.J., BARTOS M., MATLOVA L., SVASTOVA P., W ESTON R.T., KINTR J., PARMOVA I., VAN
SOOLINGEN D. & PAVLIK I. (2003). A standardised restriction fragment length polymorphism (RFLP) method for
typing Mycobacterium avium isolates links IS901 with virulence for birds. J. Microbiol. Methods, 55, 11–27.
7. GUERRERO C., BERNASCONI C., BURKI D., BODMER T. & TELENTI A. (1995). A novel insertion element from
Mycobacterium avium, IS1245, is a specific target for analysis of strain relatedness. J. Clin. Microbiol., 33,
304–307.
8. HAAGSMA J. & ANGUS R.D. (1995). Tuberculin production. In: Mycobacterium bovis Infections in Humans and
Animals, Steele J.H. & Thoen C.O., eds. Iowa State University Press, Ames, USA, 73–84.
9. INDERLIED C.B., KEMPER C.A. & BERMUDEZ L.E.M. (1993). The Mycobacterium avium complex. Clin. Microbiol.
Rev., 6, 266–310.
10. KIRSCHNER P., MEIER P.A. & BOTTGER E.C. (1993). Genotypic identification and detection of mycobacteria. In:
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Society for Microbiology, Washington DC, USA, 173–190.
11. LARSEN A.B. & MOON H.W. (1972). Experimental Mycobacterium paratuberculosis infection in chickens. Am.
J. Vet. Res., 33 (6), 1231–1235.
12. MAZUREK G.H., HARTMAN S., ZHANG Y., BROWN B.A., HECTOR J.S.R., MURPHY D. & W ALLACE R.J. (1993). Large
DNA restriction fragment polymorphism in the Mycobacterium avium, M. intracellulare complex: a potential
epidemiological tool. J. Clin. Microbiol., 31, 390–394.
13. MIJS W., DE HAAS P., ROSSAU R., VAN DER LAAN T., RIGOUTS L., PORTAELS F. & VAN SOOLINGEN D. (2002).
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Evol. Microbiol., 52, 1505–1518.
14. O’GRADY D., FLYNN O., COSTELLO E., QUIGLEY F., GOGARTY A., MCGUIRK J., O’ROURKE J. & GIBBONS N. (2000).
Restriction fragment length polymorphism analysis of Mycobacterium avium isolates from animal and human
sources. Int. J. Tuberc. Lung Dis., 4, 278–281.
15. PAVLIK I., SVASTOVA P., BARTL J., DVORSKA L. & RYCHLIK I. (2000). Relationship between IS901 in the
Mycobacterium avium complex strains isolated from birds, animals, humans, and the environment and
virulence for poultry. Clin. Diagn. Lab. Immunol., 7, 212–217.
16. POCKNELL A.M., MILLER B.J., NEUFELD J.L. & GRAHN B.H. (1996). Conjunctival mycobacteriosis in two emus
(Dromaius novaehollandiae). Vet. Pathol., 33 (3), 346–348.
17. REALINI L., DE RIDDER K., HIRSCHEL B. & PORTAELS F. (1999). Blood and charcoal added to acidified agar
media promote the growth of Mycobacterium genavense. Diagn. Microbiol. Infect. Dis., 34, 45–50.
18. REALINI L., DE RIDDER K., PALOMINO J., HIRSCHEL B. & PORTAELS F. (1998). Microaerophilic conditions promote
growth of Mycobacterium genavense. J. Clin. Microbiol., 36, 2565–2570.
19. REALINI L., VAN DER STUYFT P., DE RIDDER K., HIRSCHEL B. & PORTAELS F. (1997). Inhibitory effects of
polyoxyethylene stearate, PANTA, and neutral pH on growth of Mycobacterium genavense in BACTEC
primary cultures. J. Clin. Microbiol., 35, 2791–2794.
20. RITACCO V., KREMER K., VAN DER LAAN T., PIJNENBURG J.E.M., DE HAAS P.E.W. & VAN SOOLINGEN D. (1998).
Use of IS901 and IS1245 in RFLP typing of Mycobacterium avium complex: relatedness among serovar
reference strains, human and animal isolates. Int. J. Tuberculosis Lung Dis., 2, 242–251.
21. ROZANSKA M. (1965). Preparation of antigen for whole blood rapid agglutination test and its specificity for
diagnosis of avian tuberculosis. Bull. Vet. Inst. Pulawy, 9 (1), 20–25.
22. SAITO H., TOMIOKA H., SATO K., TASAKA H. & DAWSON D.J. (1990). Identification of various serovar strains of
Mycobacterium avium complex by using DNA probes specific for Mycobacterium avium and Mycobacterium
intracellulare. J. Clin. Microbiol., 28, 1694–1697.
23. SOINI H., EEROLA E. & VILJANEN M.K. (1996). Genetic diversity among Mycobacterium avium complex Accu-
Probe-positive isolates. J. Clin. Microbiol., 34, 55–57.

24. TELENTI A., MARCHESI F., BALZ M., BALLY F., BOTTGER E.C. & BODMER T. (1993). Rapid identification of
mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J. Clin.
Microbiol., 31, 175–178.
25. TELL L.A., W OODS L. & CROMIE R.L. (2001). Tuberculosis in birds. Rev. sci. tech. Off. int. Epiz., 20, 180–203.
26. THOREL M.F., HUCHZERMEYER H. & MICHEL A.L. (2001). Mycobacterium avium and M. intracellulare infection in
mammals. Rev. sci. tech. Off. int. Epiz., 20, 204–218.
27. THOREL M.F., HUCHZERMEYER H., W EISS R. & FONTAINE J.J. (1997). Mycobacterium avium infections in
animals. Literature review. Vet. Res., 28, 439–447.
28. THOREL M.F., KRICHEVSKY M. & LEVY-FREBAULT V.V. (1990). Numerical taxonomy of mycobactin-dependent
mycobacteria, emended description of Mycobacterium avium, and description of Mycobacterium avium
subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium
avium subsp. silvaticum subsp. nov. Int. J. Syst. Bacteriol., 40 (3), 254–260.
29. TRUEBA F., FABRE M. & SAINT-BLANCARD P. (2004). Rapid identification of Mycobacterium genavense with a
new commercially available molecular test, INNO-LiPA MYCOBACTERIA v2. J. Clin. Microbiol., 42 (9),
4403–4404.
30. TURENNE C.Y., SEMRET M., COUSINS D.V., COLLINS D.M. & BEHR M.A. (2006). Sequencing of hsp65
distinguishes among subsets of the Mycobacterium avium complex. J. Clin. Microbiol., 44 (2), 433–440.
31. VAN SOOLINGEN D., BAUER J., RITACCO V., CARDOSO LEAO S., PAVLI I., VINCENT V., RASTOGI N., GORI A., BODMER
T., GARZELLI C. & GARCIA M.J. (1998). IS1245 restriction fragment length polymorphism typing of
Mycobacterium avium isolates: proposal for standardization. J. Clin. Microbiol., 36, 3051–3054.
32. WORLD HEALTH ORGANIZATION (WHO) (1987). Requirements for Biological Substances No. 16, Annex 1:
Requirement for Tuberculins. Technical Report Series No. 745, WHO, Geneva, Switzerland, 31–59.
*
* *
NB: There is an OIE Reference Laboratory for Avian tuberculosis Table in Part 3 of this Terrestrial Manual or
consult the OIE Web site for the most up-to-date list: www.oie.int.

CHAPTER 2.3.7.
DUCK VIRUS ENTERITIS
SUMMARY
Duck virus enteritis (DVE) or duck plague is an acute contagious infection of ducks, geese and
swans (order Anseriformes) caused by a herpesvirus. Diagnosis is based on a combination of
assessing the clinical signs, gross pathology and histopathology supported by identification of the
virus by either isolation or polymerase chain reaction.
Identification of the agent: The virus may be isolated from the liver, spleen and kidneys of birds
dying from this infection. Virus may be recovered by infecting susceptible ducklings, in which the
disease can be reproduced; by inoculating embryonated Muscovy duck eggs on the chorioallantoic
membrane; or by inoculating cell cultures of duck embryo or Muscovy duck embryo origin. The
identity of the virus can be confirmed by neutralisation tests using specific antiserum to inhibit
pathological changes in the duck embryos or the cytopathological effects in the cell cultures, or by
direct or indirect immunofluorescence tests on infected cell cultures. Alternatively the DVE DNA
may be detected by the polymerase chain reaction from the oesophagus, liver and spleen of DVE-
infected birds as well as from Muscovy duck embryos or cells used for virus isolation.
Serological tests: Immunological tests have little value in the diagnosis of acute DVE infection.
Serum neutralisation tests in ovo and in vitro have been used to monitor exposure to DVE in
wildfowl.
Requirements for biological products: A live attenuated virus vaccine is available to control
DVE in birds over 2 weeks of age. Ducks are vaccinated subcutaneously or intramuscularly for
active immunity. Vaccine virus is not thought to spread from vaccinated to unvaccinated stock. An
inactivated vaccine has been reported to be efficacious in laboratory tests, but has not been
developed or licensed for large-scale use.
A. INTRODUCTION
Duck virus enteritis (DVE) is an acute, sometimes chronic, contagious virus infection that occurs naturally only in
ducks, geese and swans, all members of the family Anatidae of the order Anseriformes. The aetiological agent, a
herpesvirus, is a member of the alphaherpesvirinae subfamily of the Herpesviridae. DVE may also be referred to
as duck plague, anatid herpes, eendenpest, entenpest and peste du canard. The infection has not been reported
in other avian species, mammals or humans.
In domestic ducks and ducklings, DVE has been reported in birds ranging from 7 days of age to mature
breeders. In susceptible flocks the first signs are often sudden, high and persistent mortality with a significant
drop in egg production. In chronically infected partially immune flocks only occasional deaths occur. Recovered
birds may be carriers and may shed the virus in the faeces or on the surface of eggs over a period of years (18,
21). Recently, DVE limited solely to Muscovy ducks has been observed in the USA (2, 5) .
Clinical signs and gross pathology associated with a DVE outbreak vary with the species, age and sex of the
affected birds, and the virulence of the virus. In breeder ducks the range of signs include eye watering and
pasted eye-lids associated with photophobia, polydypsia, loss of appetite, ataxia, watery diarrhoea and nasal
discharge. Birds often have ruffled feathers and soiled vents. Sick birds may maintain an upright stance by using
their wings for support, but their overall appearance is one of weakness and depression. In ducklings 2–7 weeks
of age, losses may be lower than in older birds and the signs associated with DVE infection include dehydration,
loss of weight, a blue colouration of the beaks and blood-stained vents.
At necropsy, there is little evidence of emaciation in adult ducks that have died. In mature males, prolapse of the
penis may occur. In mature females, haemorrhages may be observed in ovarian follicles. The gross lesions are
characterised by vascular damage, with tissue haemorrhages and free blood in the body cavities, eruptions, or

Chapter 2.3.7. — Duck virus enteritis
annular haemorrhages and diphtheroid lesions of the mucosal surfaces of the digestive tract, lesions of the
lymphoid organs and retrograde changes of the parenchymatous organs. Collectively, these lesions are
pathognomonic for DVE. The pathology and histopathology of DVE in white Pekin ducks has been reviewed (19).
Microscopic lesions are characterised by vascular damage and its consequences in visceral organs. Eosinophilic
intranuclear inclusions and cytoplasmic inclusions in epithelial cells of the digestive tract are typically present.

Primary isolation of the virus is best achieved from samples of liver, spleen or kidney tissue, which have been
homogenised in buffered saline containing antibiotics and clarified by low-speed centrifugation (1800 g). Isolation
may be attempted by inoculating such homogenates into cell cultures, ducklings or duck embryos.
a) Cell cultures
Cell culture is reported as the method of choice for isolation of DVE virus, but may not always be
successful. If attempted, isolations may be made in primary duck embryo fibroblasts (DEF) (23) or,
preferably primary Muscovy duck embryo fibroblasts (MDEF) (9, 14,). Muscovy duck embryo liver (MDEL)
cells are thought to be even more sensitive (R.E. Gough, pers. comm.). Cell monolayers grown in Eagle’s
minimal essential medium (MEM) containing 10% fetal calf serum (FCS), 2 mM glutamine, and 0.17%
sodium bicarbonate and gentamicin are washed with serum-free MEM and then inoculated with the clarified
sample homogenate suspected to contain DVE virus. After incubation for 1 hour at 37°C to allow for virus
adsorption, the cultures are maintained on MEM containing 2% FCS, 2 mM glutamine, and 0.17% sodium
bicarbonate and gentamicin, and incubated in an atmosphere containing 5% CO2. The cytopathic effect
(CPE) is characterised by the appearance of rounded clumped cells that enlarge and become necrotic 2–
4 days later. Cultures should be stained with a fluorescent antibody conjugate using a direct or indirect
method to identify the virus (see Section B.1.d). Cells can also be fixed and then stained with haematoxylin
and eosin to show syncytial formation, intranuclear inclusions and marked cytoplasmic granulation. It has
been reported (11) that the isolation of DVE in MDEF cells is favoured by incubation at temperatures
between 39.5°C and 41.5°C. However, an elevated temperature does not appear to be essential for
isolation, which is often carried out at 37°C. More than one passage in cell culture may be necessary to
isolate the virus. This virus isolation method in cell cultures may be modified to a plaque assay by
overlaying the cell monolayer with maintenance medium containing 1% agarose. As the virus can be cell
associated, sequential passaging should be carried out by trypsinising potentially infected cells and
replanting them, as well as inoculating fresh cells with infected culture supernatant from the previous
passage.
b) Ducklings
When inoculated intramuscularly, 1-day-old susceptible ducklings die within 3–12 days; uninoculated
ducklings, housed separately, should be maintained as controls at the same time. Muscovy ducklings
(Cairina moschata) are more susceptible than white Pekin ducklings. Both macroscopic and microscopic
lesions typical of DVE should be seen on post-mortem examination. The diagnosis may be confirmed either
by vaccinating ducklings against DVE and challenging them subsequently with the virus isolate or by
immunofluorescence. However, virulent strains of the virus exist, against which the vaccine may be
ineffective (13). In the author’s experience of natural infections occurring in Muscovy ducks, this method of
virus isolation has proved more sensitive than cell culture methods.
c) Duck embryos
Primary virus isolations can be made by inoculation on to the chorioallantoic membrane (CAM) of 9–14 day
embryonated Muscovy duck eggs. The embryos may die, showing characteristic extensive haemorrhages
4–10 days after inoculation. Two to four serial blind passages of the homogenised CAMs may be necessary
before isolation can be effected. This method is not as sensitive as that using susceptible day-old ducklings.
Embryonated chicken eggs are not very susceptible to infection with field strains of DVE. The virus can
nevertheless be adapted to chicken embryos by serial passages. Pekin duck embryos vary in their
susceptibility to strains of DVE virus.
d) Immunological methods
Serological tests used to confirm the identity of newly isolated virus include neutralisation assays performed
in either embryonated eggs or cell cultures. A plaque assay for DVE in duck embryo cell cultures has been
described (4). In the author’s laboratory a microtitre assay using primary MDEF or MDEL cells is used.
Provided a hyperimmune antiserum of sufficiently high titre is used, a fluorescent antibody test (direct or
indirect) for DVE in DEF, MDEF or MDEL cells is the next most sensitive assay after isolation in 1–9-day old

ducklings (8). A reverse passive haemagglutination test for DVE has been described (6), but it is reported to
be less sensitive than immunofluorescence and plaque assays. An avidin–biotin–peroxidase method of
immunoperoxidase staining to detect DVE antigen in formalin-fixed, paraffin-embedded sections of liver and
spleen from experimentally infected birds has been described (12). The identity of the virus may also be
confirmed by negative stain electron microscopy, but this alone is not positive confirmation that the
herpesvirus observed is DVE virus. Immunoelectron microscopy has been developed recently using DVE
hyperimmune serum (15).
e) Nucleic acid recognition methods

Recently, detection of DVE virus by polymerase chain reaction (PCR) has been reported (10, 11, 16, 17).
Primers have been identified that are able to amplify DNA from DVE virus present in various tissues,
including oesophagus, liver and spleen, from an original outbreak and after passage from Muscovy duck
embryos. The following is an example protocol for PCR methods for detection of DVE virus; other protocols
exist.
• PCR method 1
This DNA extraction procedure can be used on disrupted cell suspensions from DVE-infected tissue culture,
10% ground tissue suspensions, or cloacal swab material in transport medium. This method is used to
prepare duck plague DNA for the known positive PCR controls.
• Extraction of viral DNA

Note: All product transfers in steps i to v are performed in a biological safety cabinet.
i) For a 10% ground tissue suspension, add 400 µl to a 1.5 ml microfuge tube and microfuge at 16,000 g
for 5 minutes. Transfer the supernatant to a new tube and go to step ii.
ii) For tissue culture suspensions and cloacal swab material, add 400 µl of the sample, or supernatant
from step i above, to a 1.5 ml tube and microfuge at 16,000–20,000 g for 45 minutes to pellet the
virus.
iii) Discard the supernatant and resuspend the pellet with 200 µl of Tris/ethylene diamine tetra-acetic acid
(EDTA) buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA).
iv) Add 10 µl of a 5 µg/µl proteinase K solution to give a final concentration of 0.2 µg/µl, mix thoroughly,
and incubate at 56°C for 1 hour.
v) Add 25 µl of 10% sodium dodecyl sulfate (SDS) solution to give a final SDS concentration of 1%, mix
thoroughly, and incubate at 37°C for 1 hour.
vi) Add 15 µl of 5 M NaCl to give a final concentration of 0.3 M and mix thoroughly.
vii) Add 300 µl of fresh phenol buffered with Tris/HCl, pH 8.0, to the tube, and mix by inverting 50 times.
viii) Microfuge the tube at 16,000 g for 5 minutes and transfer the top aqueous phase (sample) to a new
tube.
ix) Repeat the phenol extraction steps vii and viii once more.
x) Add 500 µl of ether to the tube, mix thoroughly, and microfuge at 16,000 g for 1 minute.
xi) Discard the top aqueous phase (ether) and repeat the ether extraction step (step x) once more.
xii) Heat the tube with the lid open at 56°C for about 15 minutes or until the smell of ether is gone.
xiii) Split the tube contents in two and add 2.25 times the sample volume of 100% ethanol to each tube,
mix the tube contents by inverting the tube several times, and leave at room temperature (22°C) for
30 minutes.
xiv) Microfuge the tube at 16,000 g for 45 minutes and discard the supernatant.
xv) Add 200 µl of 70% ethanol to gently wash the pellet and then microfuge at 16,000 g for 15 minutes.
xvi) Discard the supernatant and dry the pellet at 56°C for 30–45 minutes with the tube lid open.
xvii) Resuspend the DNA in 30 µl distilled water that is RNAase and DNAase free.
xviii) Store the sample tube at 4°C until tested (few days) or at –20°C for long-term storage.
1 Provided by Dr W.R. Hansen, US Geological Survey, Biological Resources Division, National Wildlife Health Center,
6006, Schroeder Road, Madison, WI 53711, USA. This procedure uses the following commercial items: GeneAmp PCR
Reagent Kits containing dNTPs, 10× amplification buffer for hot start PCR, Taq DNA polymerase, Lambda PCR control
reagents, and Ampliwax beads (Applied Biosystems), and a 100 base pair molecular size ladder (Invitrogen)

• Polymerase chain reaction

Lower reaction mixtures for the DVE PCR and the lambda control are prepared in advance in a biosafety
cabinet using the kit manufacturer’s recommended methods for a hot start PCR. The lower reaction mixture
is dispensed into tubes, sealed with Ampliwax at 80°C, as recommended by the manufacturer, and stored
at 4°C for 1–2 months.
PCR primers for DVE DNA-directed DNA polymerase gene

Primer 1 sequence: 5’-GAA-GGC-GGG-TAT-GTA-ATG-TA-3’ (forward)
Primer 2 sequence: 5’-CAA-GGC-TCT-ATT-CGG-TAA-TG-3’ (reverse)
i) The upper reaction mixture is prepared according to the kit manufacturer’s recommendations the day
of the test, and distributed to each sample tube including DVE and lambda control tubes.
ii) Add 10 µl of DNA suspension from the stored sample tubes to the PCR lower reaction tubes with
corresponding labels.
iii) Place known DVE DNA diluted to 1 pg/10 µl into one control tube and 10 µl of distilled water into the
no DNA control tube. Add 10 µl of lambda DNA supplied in the kit and 10 µl of water to the
corresponding lambda control tubes.
iv) Place all the tubes in a thermal cycler that is programmed as follows:
One cycle: Hold 94°C for 2 minutes
Hold 37°C for 1 minute
Hold 72°C for 3 minutes
35 cycles: Hold 94°C for 1 minute
Hold 55°C for 1 minute
Hold 72°C for 2 minutes
One cycle: Hold 72°C for 7 minutes
Hold 4°C until stored
PCR tubes are stored at 4°C until the samples are examined for amplification products.
• Electrophoretic analysis of PCR products

i) A fresh 1 × TAE buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.3) is prepared from a 10× stock for
agarose preparation and for use in the electrophoresis chamber.
ii) A 1% agarose solution is prepared in TAE buffer, heated to dissolve the agarose, and, when cool,
poured into a gel former with a comb.
iii) The solidified gel is placed into the electrophoresis chamber and TAE running buffer is added.
iv) PCR test samples, including the DVE and lambda controls, are mixed 1/10 with 1 µl of loading buffer
(0.25% [w/v] bromophenol blue, 0.25% [w/v] xylene cyanol, 0.01 M Tris/HCl, pH 8.0, and 50% [v/v]
glycerol) and 10 µl of each is added to individual wells of the gel. The 100 bp molecular size markers
are added to each side of the gel.
v) Run the gel for 1 hour at 120 volts and then stain in a 1% ethidium bromide solution for 20 minutes.
Destain the gel for 45 minutes in deionised water and view the gel on a UV-illuminated light box.
Photograph the gel to record results.

A 500 bp amplification band in the lambda control sample indicates the PCR ran successfully. A 446 bp
band in the DVE known DNA control indicates the DVE primers are working. A 446 bp band in the unknown
test sample indicates DVE viral DNA was present. No amplification products will be present in the DVE or
lambda no DNA controls. If bands appear in these negative control products, cross-contamination occurred
during the test set-up and the test must be repeated.
f) Strain variation
Although strains of DVE differ considerably in virulence, there is little reported evidence of serological variation.
Serological tests have little value in the diagnosis of acute DVE infections, but assays based on serum
neutralisation in embryonated eggs and cell cultures have been used to monitor antibodies following exposure to

DVE in wildfowl. The humoral response to natural infection with DVE virus is often low and antibodies may be
short-lived (7); it is assumed that cell-mediated immunity also plays a role in the infection (18). However,
detection of neutralising antibodies to DVE virus in serum is possible. Virus neutralisation (VN) (22) assays using
a constant-serum/varying-virus method may be performed in chicken or duck embryos by using embryo-adapted
virus, or in cell cultures. Neutralisation indices (NI) (28) between 0 and 1.5 were detected in domestic and wild
waterfowl that had not been exposed to DVE; a NI of 1.75 or greater was considered to be evidence of prior
exposure to DVE virus (3). Alternatively, sera may be screened using a constant-virus/varying-serum method. In
the author’s laboratory a microtitre neutralisation assay using primary MDEF or DEF is used. Serial twofold
dilutions of each serum sample (heat-inactivated at 56°C) are prepared in 50 µl of serum-free MEM in microtitre
plates. Approximately 102.0 TCID50 (50% tissue culture infective dose) of DVE virus in 50 µl of MEM is added to
each well and the mixtures are allowed to react at 37°C for 1 hour. A suspension of primary MDEF or DEF in
MEM supplemented with 2 mM L-glutamine, 0.17% sodium bicarbonate and 10% FCS, are adjusted to contain
3 × 105 cells per ml. Cells are next added to the plates at 100 µl per well and the plates are then incubated for up
to 96 hours at 37°C in a humidified 5% CO2 atmosphere. Following incubation, cells are observed daily by light
microscopy and finally fixed with 10% formol-buffered saline and stained with 1% crystal violet. The plates are
read macroscopically. The titre for virus neutralising activity is expressed as the reciprocal of the highest dilution
of serum at which a monolayer grew, i.e. there is no evidence of CPE and therefore complete virus neutralisation
has occurred. A titre of less than 3 log2 is usually considered to be negative. A VN titre of 8 or greater is
considered to be significant and is evidence of exposure to DVE virus (7). VN antibody may also be detected
using cell cultures by mixing sera at a single dilution, e.g. 1/10, with 100–200 TCID50 virus and then testing
inoculated cell cultures for non-neutralised virus by immunofluorescence. Although this method is not
quantitative, it can be useful for screening large numbers of sera. These latter methods, using constant-
virus/varying-serum, are much more economical on sera than the NI methods.

A live attenuated virus vaccine can be used to control DVE in birds over 2 weeks of age (18, 19). Fattening or
breeding ducks may be vaccinated subcutaneously or intramuscularly to produce an active immunity. The
vaccine virus is not thought to spread by contact from vaccinated to unvaccinated ducks, as the unvaccinated
birds remain susceptible to infection.
An inactivated vaccine has been reported to be as efficacious as modified live vaccine (20). This vaccine has
been tested only under laboratory conditions; it has not been tested on a large scale and is not licensed.
1. Seed management

DVE vaccine can be prepared from a strain of the virus that has been attenuated by serial passage in
embryonated chicken eggs. In the USA the vaccine strain seed was originally imported from Holland and
has been serially passaged 41–46 times.
The seed virus should be prepared in 8–11-day-old specific pathogen free (SPF) embryonated chicken
eggs by inoculating on to the CAM followed by incubation at 37°C. The seed may be stored at –70°C or
lower in the form of a homogenate of the embryo CAM in buffered saline.
The seed virus should be shown to be free from extraneous viruses pathogenic to ducks, chickens and
turkeys. It should also be free from bacterial, fungal and mycoplasmal contaminants.
The identity of the virus should be confirmed by a VN test conducted with specific antiserum using the
constant-serum/varying-virus method. This test should be performed in embryonated chicken eggs. The
antiserum should reduce the virus titre by at least 101.75 ELD50 (50% embryo lethal dose).
The immunogenicity of the vaccine can be assessed in DVE-susceptible ducks or ducklings by inoculating
the recommended vaccine dose intramuscularly and challenging intramuscularly 21 days later with virulent
DVE virus. The vaccinated birds should survive challenge while unvaccinated control birds should die. This
test should be carried out on the master seed but need not be done routinely on each vaccine batch

produced. For release of subsequent batches, the titre of the virus should be a sufficient indication of
vaccine potency.
Once frozen at –70°C or lower the vaccine stores well for at least 1 year with little loss in titre. Once issued,
the vaccine should not be refrozen, it should be kept at 4°C and used as soon as possible.
The vaccine is produced in 8–11-day-old SPF embryonated chicken eggs inoculated on to the CAM and
incubated at 37°C. Most embryo deaths occur between 48 and 96 hours after inoculation. The embryos, their
CAMs and chorioallantoic fluids are harvested, pooled and homogenised in buffered saline and clarified by low-
speed centrifugation (1800 g). The preparation is diluted as appropriate, and a stabiliser is incorporated. It is
then dispensed into vials and preferably frozen rapidly to –70°C or lower.
Eggs that have been inoculated should be candled 24 hours later to identify any embryos that have died from
nonspecific causes.
4. Batch control
a) Sterility
b) Safety
A group of 1-day-old ducklings susceptible to DVE should be inoculated subcutaneously or intramuscularly
with the vaccine at 10 times the recommended dose, and observed for 7–14 days for any signs of adverse
reactions.
The final product should be free from contamination by bacteria, fungi, and mycoplasma as well as
extraneous viruses potentially pathogenic to poultry.
c) Potency
The virus titre of the vaccine should be determined in 9–11-day-old embryonated chicken eggs inoculated
on to the CAM and incubated at 37°C. The vaccine should contain a minimum of 103.0 ELD50 per dose at
time of use.
Immunity in vaccinated ducks should last throughout a breeding season. Annual re-vaccination is
recommended (19).
e) Stability
When stored at –70°C or lower the vaccine is stable for at least 1 year. Potency testing should be repeated
after this time on an aliquot of vaccine to determine whether virus titre has been maintained. Once thawed
the vaccine should not be refrozen, it should be maintained at 4°C in a refrigerator but for no longer than
1 week. Lyophilised vaccine should be stored at 4–8°C and used before the stated expiry date.
f) Preservatives
No preservatives are added to the vaccine.
None.
5. Tests on final product
The vaccine is issued as a vial of frozen concentrated vaccine virus together with a bottle of sterile diluent
(phosphate buffered saline), on which standard sterility checks have been made (see Section C.4.a).

a) Safety
No additional testing is performed after the batch testing.
b) Potency
No additional testing is performed after the batch testing.
REFERENCES
1. BURGESS E.C. & YUILL T.M. (1981). Increased cell culture incubation temperatures for duck plague virus
isolation. Avian Dis., 25, 222–224.
2. CAMPAGNOLO E.R., BANERJEE M., PANIGRAHY B. & JONES R.L. (2001). An outbreak of duck viral enteritis (duck
plagues) in domestic Muscovy ducks (Cairina moschata domesticus) in Illinois. Avian Dis., 45, 522–528.
3. DARDIRI A.H. & HESS W.R. (1967). The incidence of neutralizing antibodies to duck plague virus in serums
from domestic ducks and wild waterfowl in the United States of America. Proceedings of the Annual
Meeting of the United States Animal Health Association, 225–237.
4. DARDIRI A.H. & HESS W.R. (1968). A plaque assay for duck plague virus. Can. J. Comp. Med., 32, 505–510.
5. DAVISON S., CONVERSE K.A., HAMIR A.N. & ECKORAGE R.J. (1993). Duck viral enteritis in domestic Muscovy
ducks in Pennsylvania. Avian Dis., 37, 1142–1146.
6. DENG M.Y., BURGESS E.C. & YUILL T.M. (1984). Detection of duck plague virus by reverse passive
haemagglutination test. Avian Dis., 28, 616–628.
7. DOCHERTY D.E. & FRANSON C.J. (1992). Duck Virus Enteritis. In: Veterinary Diagnostic Virology, Castro A.E.
& Heuschele W.P., eds. Mosby Year Book, St Louis, Missouri, USA, 25–28.
8. ERICKSON G.A., PROCTOR S.J., PEARSON J.E. & GUSTAFSON G.A. (1974). Diagnosis of duck virus enteritis
(duck plague). 17th Annual Proceedings of American Association of Veterinary Laboratory Diagnosticians.
Roanoake, Virginia, USA, 85–89.
9. GOUGH R.E. & ALEXANDER D.J. (1990). Duck virus enteritis in Great Britain, 1980 to 1989. Vet. Rec., 126,
595–597.
10. HANSEN W.R., BROWN S.E., NASHOLD S.W. & KNUDSON D.L. (1999). Identification of duck plague virus by
polymerase chain reaction. Avian Dis., 43, 106–115.
11. HANSEN W.R., NASHOLD S.W., DOCHERTY D., E., BROWN S.E. & KNUDSON D.L. (2000). Diagnosis of duck
plague in waterfowl by polymerase chain reaction. Avian Dis., 44, 266–274.
12. ISLAM M.R., NESSA J. & HALDER K.M. (1993). Detection of duck plague virus antigen in tissues by
immunoperoxidase staining. Avian Pathol., 22, 389–393.
13. KISARY S. & ZSAK L. (1983). Comparative studies on duck viral enteritis (DVE) virus strains in geese. Avian
Pathol., 12, 395–408.
14. KOCAN R.M. (1976). Duck plague virus replication in Muscovy duck fibroblasts. Avian Dis., 20, 574–580.
15. PEARSON G.L. & CASSIDY D.R. (1997). Perspectives on the diagnosis, epizootiology and control of the 1973
duck plague epizootic in wild waterfowl at Lake Andes, South Dakota. J. Wildl. Dis., 33, 681–705.
16. PLUMMER P.J., ALEFANTIS T., KAPLAN S., O’CONNELL P., SHAWKY S. & SCHAT K.A. (1998). Detection of duck
enteritis virus by polymerase reaction. Avian Dis., 40, 554–564.
17. PRITCHARD L.I., MORRISSY C., VAN PHUC K., DANIELS P.W. & W ESTBURY H.A. (1999). Development of a
polymerase chain reaction to detect Vietnamese isolates of duck virus enteritis. Vet. Microbiol., 68, 149–
156.

18. RICHTER J.H.M. & HORZINEK M.C. (1993). Duck Plague. In: Virus Infections of Birds, McFerran J.B. & McNulty
M.S., eds. Elsevier Science Publishers B.V., Amsterdam, the Netherlands, 77–90.
19. RICHTER, J. H. M. & HORZINEK, M. C. (1993). DUCK PLAGUE. IN J. B. MCFERRAN & M. S. MCNULTY (EDS.), VIRUS
INFECTIONS OF BIRDS, EDN PP. 77-90). AMSTERDAM, THE NETHERLANDS: ELSEVIER SCIENCE PUBLISHERS B.V.
20. SHAWKY S.A. & SANDHU T.S. (1997). Inactivated vaccine for protection against duck virus enteritis. Avian
Dis., 41, 461–468.
21. SHAWKY S. & SCHAT K.A. (2002). Latency sites and reactivation of duck enteritis virus. Avian Dis., 46, 308–
313.
22. THAYER G.S. & BEARD C.W. (1998). Serologic procedures. In: A Laboratory Manual for the Isolation and
Identification of Avian Pathogens, Fourth Edition, Swayne D.E., Glisson J.R., Jackwood M.W., Pearson J.E.
& Reed W.M., eds. American Association of Avian Pathologists, Kennett Square, Pennsylvania, USA, 255–
266.
23. W OLF K., BURKE C.N. & QUIMBY M.C. (1976). Duck viral enteritis: a comparison of replications by CCL-141
and primary cultures of duck embryo fibroblasts. Avian Dis., 20, 447–454.
*
* *

CHAPTER 2.3.8.
DUCK VIRUS HEPATITIS
SUMMARY
Hepatitis in ducks can be caused by at least three different viruses. The more common and
internationally widespread is duck hepatitis virus (DHV) type I, now designated as an unclassified
picornavirus, which causes a highly lethal, acute, contagious infection in ducklings under 6 weeks
of age and, frequently, under 3 weeks of age. It does not occur in older birds. This infection is often
referred to simply as duck virus hepatitis.
DHV type II has been reported in the United Kingdom only. It occurred in ducklings from 10 days to
6 weeks of age, and caused pathological changes similar to those of DHV type I. From electron
microscopy and molecular studies it is considered to be an astrovirus.
DHV type III has been reported only in the United States of America. It causes similar liver lesions
in young ducklings, but is less virulent than DHV type I. It is believed to be a picornavirus,
serologically unrelated to type I virus.
Diagnosis of hepatitis in ducklings is based on the characteristic disease pattern in the flock, gross
pathological changes, the recovery of virus from dead ducklings, and the reproduction of the
disease in susceptible ducklings.
Identification of the agent: It is not possible to distinguish among DHV types I, II and III on the
basis of clinical findings and pathology, but distinctions can be made from the responses of
ducklings, embryonated eggs and cell cultures to the isolated viruses. Alternatively DHV type I RNA
may be detected by a one-step reverse-transcriptase polymerase chain reaction from duckling liver
and also from allantoic fluid and embryo liver from inoculated duck eggs.
Serological tests: Serological tests have little value in the diagnosis of the acute infections caused
by DHV types I, II and III.
Serum neutralisation tests in ovo have been used with all three viruses and in-vitro tests have been
developed for DHV type 1. These tests have been used for virus identification, assay of immune
responses to vaccination and epidemiological surveys.
Requirements for vaccines and diagnostic biologicals: DHV type I infections can be controlled
by the use of live attenuated virus vaccines and an inactivated virus vaccine. They are administered
to breeder ducks to confer passive immunity to ducklings. Live attenuated virus vaccines may also
actively immunise DHV type-I-susceptible day-old ducklings.
Ducklings susceptible to DHV type I may also be passively protected with a chicken egg yolk
antibody preparation.
DHV type III infections can be controlled by the use of a live attenuated virus vaccine given to
breeder ducks to confer passive immunity to ducklings.
A. INTRODUCTION
Duck hepatitis is caused by at least three different viruses, namely duck hepatitis virus (DHV) types I, II and III.
The most common is DHV type I, which is designated as an unclassified picornavirus and may require to be
assigned to a new genus within the Picornaviridae (4, 10, 15). DHV type II is an astrovirus, and DHV type III is
considered to be a picornavirus serologically unrelated to DHV type I.

Chapter 2.3.8. — Duck virus hepatitis
A new serotype of DHV named N-DHV that can cause high mortality with characteristic liver lesions has been
reported (16). This virus, recovered from both mule ducklings and goslings, is antigenically unrelated to DHV type
I, and although phylogenetically distinct, it is still closely related to DHV type I.
Until the present, DHV type I has only been associated with causing disease in mallard and Pekin ducklings but it
has now been reported to cause pancreatitis and encephalitis in Muscovy ducks (6).
These viruses, which cause acute infections, should not be confused with duck hepatitis B virus, a hepadnavirus
classified in the same group as mammalian hepatitis B virus. The significance of this infection for the duck is not
fully understood.
a) DHV type I
DHV type I causes a highly contagious infection of ducks. It is of no known public health significance. The
disease is an acute, rapidly spreading, often fatal virus infection of young ducklings. It usually affects
ducklings under 6 weeks of age and often much younger. The clinical disease is characterised by lethargy
and ataxia. Ducklings lose their balance, fall on their sides and kick spasmodically prior to death. At death
the head is usually drawn back in the opisthotonos position. The whole disease sequence is rapid and can
take as little as 1–2 hours. Practically all mortality in a flock will occur within 3–4 days, with most deaths on
the second day. Gross pathological changes appear chiefly in the liver, which is enlarged and displays
distinct punctate and ecchymotic haemorrhages. Spleen enlargement and swelling of the kidneys with some
congestion of renal blood vessels may also be apparent. Microscopic changes in the liver are characterised
by extensive hepatocyte necrosis and bile duct hyperplasia, together with varying degrees of inflammatory
cell response and haemorrhage.
The clinical and pathological observations are highly indicative of DHV type I infection. The virus can readily
be recovered from liver tissue by homogenisation as a 20% (w/v) suspension in buffered saline. The
suspension is clarified, and can then be treated further (if desired) with 5% chloroform (v/v) for 10–
15 minutes at ambient temperature. DHV type I is resistant to this treatment.
The presence of DHV type I is usually confirmed by one or more of the following procedures:
i) By subcutaneous or intramuscular inoculation of the isolate into ducklings between 1 and 7 days of age
that are susceptible to DHV type I. The characteristic clinical disease should follow, with deaths
occurring within 18–48 hours of inoculation, often in under 24 hours. The ducklings should show the
gross pathology attributable to DHV type I. Virus should be re-isolated from the livers.
ii) By inoculation of serial dilutions of the liver homogenate into the allantoic sac of embryonated duck
eggs (10–14 days) or chicken eggs (8–10 days). Duck embryos die between 24 and 72 hours later,
whereas chicken embryos are more variable and erratic in their response and usually take 5–8 days to
die. Gross pathological changes in the embryos include stunting and subcutaneous haemorrhages
over the whole body, with oedema particularly of the abdominal and hind limb regions. The embryo
livers may be red and yellowish, swollen and may show some necrotic foci. In embryos that take longer
to die, the greenish colour of the allantois is more pronounced, and both the liver lesions and stunting
become more evident.
iii) By inoculation of primary cultures of duck embryo liver (DEL) cells, which are particularly sensitive (17).
Dilutions of the liver homogenate containing DHV type I cause a cytopathic effect (CPE), which is
characterised by cell rounding and necrosis. When overlaid with a maintenance medium containing 1%
agarose (w/v), the CPE gives rise to plaques approximately 1 mm in diameter.
• Immunological tests
Such tests have not been used extensively for the routine identification of DHV type I infection. Various virus
neutralisation (VN) assays have been described, which may assume greater significance if DHV types II and
III infections become more widespread. The tests that have been described (2, 17–19) include:
i) Passive subcutaneous immunisation of 1–7-day-old ducklings susceptible to DHV type I with 1–2 ml
specific hyperimmune serum or specific egg yolk antibody. These ducklings are then challenged
intramuscularly or subcutaneously, 24 hours later with at least 103.0 LD50 (50% lethal dose) of the virus

isolate. A control group of uninoculated ducklings is similarly challenged. Identification of infection is

based on 80–100% survival in the passively immune ducklings and 80–100% mortality in the controls.
ii) 1–7-day old DHV type-I-susceptible and DHV type I maternally immune ducklings are challenged
intramuscularly or subcutaneously with at least 103.0 LD50 of the virus isolate. Identification is based on
80–100% losses in the susceptible ducklings and 80–100% survival in the maternally immune
ducklings.
iii) Serial tenfold dilutions of the virus isolate are mixed with equal volumes of DHV type-I-specific
hyperimmune serum diluted between 1/5 and 1/10. The mixtures are allowed to react at room
temperature for 1 hour and are then inoculated (0.2 ml) subcutaneously into susceptible ducklings, also
via the allantoic cavity (0.2 ml) of embryonated duck eggs and on to primary DEL cell monolayer
cultures. Controls in each case consist of the virus isolate mixed with control serum.
There is little evidence for antigenic variation among DHV type I isolates. However, a variant, DHV type Ia,
isolated in the United States of America (USA) only partially reacts with the classical type I virus in cross
serum neutralisation tests (12, 20). Other variants have been reported from India and Egypt, but nothing
further is known about them. Recent reports of disease in Muscovy ducks from France (6) and a N-DHV
from Chinese Taipei (16) have raised new questions about duck virus hepatitis.
• Nucleic acid recognition methods

Although some recent publications have revealed the molecular structure of DHV type I (4, 10, 15) only one
has reported a one-step reverse-transcriptase polymerase chain reaction (RT-PCR) to detect DHV type I (9).
This is the method outlined below.

This method has been extracted from (9). It is based on primers specific to amplify a region of the 3D gene.
• Detection of DHV-I from duck and chicken embryo organs

Supernatants prepared from duckling livers infected with DHV type I are collected and filtered (0.2 µm). The
allantoic cavities of each of five 11-day-old duck and 9-day-old chicken embryonated eggs are inoculated
with 0.2 ml viral supernatant. The allantoic fluid and liver samples are collected from embryos inoculated
with two reference strains and each liver sample is ground in a tissue grinder and phosphate buffered saline
is added to make 10% suspensions. Liver sample suspensions and allantoic fluid are centrifuged at 2000 g
for 30 minutes, the supernatants are treated with the Viral Gene-spin™ viral DNA/RNA extraction kit and the
nucleic acids are used for one-step RT-PCR.
• Nucleic acid extraction

Extractions of viral RNA are performed using the Viral Gene-spin™ viral DNA/RNA extraction kit (iNtRON
Biotechnology, Seongnam, Korea). In brief, a total of 150 µl of the sample for extraction is mixed with 250 µl
lysis buffer. For the RT-PCR sensitivity tests, 50 µl of diethylpyrocarbonate (DEPC)-treated distilled water is
added to 100 µl of tenfold virus dilution of the samples before mixing with the lysis buffer. A 350-µl aliquot of
binding buffer is added to the mixture and triturated; the total 750 µl is placed into a minispin column, which
is spun at room temperature for 1 minute at 13,000 rpm in a microcentrifuge. The flow-though is discarded
and two cycles of washing–spinning–flow-through-removal are performed using washing buffers A and B
(500 µl each), followed by a final spin for 1 minute to dry the membrane. The column is transferred to a new
1.5-ml collection tube and RNA is eluted by addition of 40 µl elution buffer and centrifugation for 1 minute at
13,000 rpm.
After measuring RNA concentrations using the NanoDrop ND-1000 (NanoDrop, Wilmington, DE), the
samples are stored at −20°C.
• One-step RT-PCR
The one-step RT-PCR is conducted using the Maxime RT-PCR PreMix kit (iNtRON Biotechnology). The
20-µl reaction mixtures contain 1 U of OptiScript reverse transcriptase, 2.5 mM dNTPs, 2.5 U i-StarTaq DNA
polymerase, and RT-PCR buffer (50 mM Tris/HCl and 75 mM KCl). In addition, the following components
are included in the reaction: 4 µl (50 ng) RNA or DNA template, 1 µl (10 pmol/µl) of each specific primer
(DHV-1 ComF and DHV-1 ComR), and DEPC-treated dH2O to a total reaction volume of 20 µl.
A T-gradient thermal cycler (Biometra, Gottingen, Germany) is used for one-step RT-PCR. Reverse
transcription is performed at 45°C for 30 minutes, after which the enzyme is inactivated at 94°C for
5 minutes. PCR amplification is conducted using an initial denaturation for 20 seconds at 94°C; followed by

40 cycles of annealing for 30 seconds at 52°C, extension for 30 seconds at 72°C, and denaturation for
20 seconds at 94°C; and a final extension for 5 minutes at 72°C. Reactions are stored at 4°C.
• Detection of one-step RT-PCR products

PCR products (10 µl) are separated by electrophoresis (100 V) in horizontal 1.5% agarose gels (iNtRON
Biotechnology) and Tris-acetate buffer (40 mM Tris-acetate, 1 mM ethylenediamine tetra-acetic acid). Gels
are stained with ethidium bromide (0.5 ug/ml), visualised under ultraviolet light, and photographed.
• Interpretation of results
A DNA fragment of 467 bp is amplified by one-step RT-PCR using RNA extracted from the livers of
ducklings infected with reference DHV type I strains. Negative control RNA is obtained from an uninfected
duckling liver and does not amplify under the same conditions.
b) DHV type II
DHV type II infection of ducks has only been reported from the United Kingdom (1, 5). It is an acute, fatal
infection of ducklings producing clinical and pathological signs similar to DHV type I. Affected birds may
show signs of polydypsia and usually die within 1–2 hours of appearing sick.
Gross pathological changes include multiple haemorrhages, both punctate and confluent bands in the liver,
swollen pale kidneys with congested blood vessels, and enlarged spleens. The alimentary tract is often
empty although the small intestine may contain mucus, and haemorrhagic areas are occasionally seen.
Petechial haemorrhages are also occasionally seen on the heart. Histologically, changes in the liver are
similar to those seen in DHV type I infections; the extent of bile duct hyperplasia may be greater than with
DHV type I, but this is relative. DHV type II has astrovirus-like morphology and virions are 28–30 nm in
diameter. It is classified in the family Astroviridae as duck astrovirus I (DAstV-I) (5, 11).
The virus may be recovered in 20% (w/v) homogenised liver suspensions in buffered saline. This can be
used to inoculate:
i) Susceptible ducklings, in which the response can be variable. A mortality rate of up to 20% may occur
within a period of 2–4 days. The gross pathology is similar to that observed in field cases (5). This is in
contrast to the findings with DHV type I infection, which is more virulent and rapid in its effect.
ii) Embryonated chicken or duck eggs, either via the amniotic cavity or yolk sac. These may respond,
erratically, after four passages, but no deaths may be seen during earlier passages. Embryos take 6–
10 days to show evidence of infection; when this occurs there is stunting with green necrotic livers.
• Immunological tests
Immunological tests have not been employed routinely as the serological response to infection of both
ducklings and duck embryos is poor. However, a neutralisation assay has been applied (5) for virus
identification by inoculating chicken embryos via the amniotic cavity with constant-serum/varying-virus
mixtures.
Cross protection tests have been performed in 2–4-day old ducklings (5); these are inoculated with antisera
to types I or II, then challenged 3 days later with the virus isolate. This technique could distinguish DHV type
II from types I and III.
c) DHV type III

DHV type III has been reported in the USA only. Losses of up to 20% occur in ducklings immune to DHV
type I (7, 13). DHV type III causes an acute infection of young ducklings with clinical signs similar to those
seen in type I infections.
The gross pathology is also similar to type I infection. The liver surface is pale and mottled with many red
bands and some petechial haemorrhages. The spleen is paler, but not noticeably enlarged, and the kidneys
may show patchy congestion.
The virus can be recovered from homogenised liver suspensions and is resistant to treatment with 5%
chloroform. The virus can be isolated by:
i) Inoculating the isolate intramuscularly into susceptible ducklings. The mortality rate may reach
20% with 60% morbidity. No deaths occur in the first 24 hours and all losses ensue between day 2 and
day 4 after inoculation. Intravenous inoculation is more effective; type III infection is less virulent than
type I.

ii) Inoculating the isolate on to the chorioallantoic membrane (CAM) of 10-day-old embryonated duck
eggs. The response is erratic, but some embryo mortality always occurs within 7–10 days. The
membranes assume a dry crusty appearance, beneath which they are oedematous. The embryos may
be stunted and oedematous with skin haemorrhages. The liver, kidneys and spleen are enlarged.
Attempts to cultivate the virus in hens’ eggs have not been successful.
Attempts to induce a CPE with the virus in tissue cultures have not been successful, but the virus has been
detected by direct immunofluorescence in experimentally infected DEL and duck embryo kidney (DEK) cell
monolayer cultures (7).
These do not apply to diagnosis as the clinical disease is too acute.
All three DHV types have been used in virus neutralisation tests in ovo, but their success depends on the
expression of the virus in the assay system used; with type II and III viruses this can be a problem. In-vitro tests
have been developed for DHV type I; these include a plaque reduction assay and a microtitre assay (17, 18). The
plaque reduction assay may be performed using either primary DEK or DEL cells. Primary cell culture monolayers
are prepared in Eagle’s minimal essential medium (MEM) containing 5–10% fetal calf serum (FCS), 2 mM
glutamine, 0.17% sodium bicarbonate and gentamicin. Trypsinised cells are seeded into 5 cm diameter Petri
dishes, then incubated at 37°C in a 5% CO2 atmosphere. Monolayers should be nearly confluent at 24–48 hours
post-seeding. The monolayers are washed twice with serum-free MEM or Hank’s balanced salt solution to remove
all traces of FCS before infecting with DHV type I. Equal volumes of DHV type I suspended in serum-free MEM,
adjusted to 200 plaque-forming units (PFU) per 0.1 ml, are mixed with equal volumes of serially diluted duck sera
(twofold dilutions in MEM). The serum samples should be heat inactivated at 56°C for 30 minutes before testing.
The virus/serum mixtures are incubated at 37°C for 1 hour, then 0.1-ml aliquots are added to the confluent cell
monolayers, three dishes per dilution. The plates are left for 30 minutes at room temperature (20–22°C), then
overlaid with agarose maintenance medium (MEM containing 2% chicken serum and 0.1–0.2% FCS to which
agarose had been added to a final concentration of 1% [w/w]). The plates are then placed at 37°C in a 5% CO2
atmosphere. The number of plaques produced is recorded after 48 hours’ incubation. Plaques may be observed
using an oblique light source, or alternatively monolayers may be fixed with 10% formol-buffered saline and
stained with 1% crystal violet. Serum antibody titres are expressed as the reciprocal of the highest serum dilution
that reduces the plaque count by 50%.
A microtitre neutralisation assay may be performed using primary DEK cells. Serial twofold dilutions of each
serum sample (heat-inactivated) are prepared in 50 µl of serum-free Eagle’s basal medium (BME) in microtitre
plates. Approximately 102.0 TCID50 (50% tissue culture infective dose) of DHV type I in 50 µl of BME is added to
each well and the mixtures are allowed to react at 37°C for 1 hour. Primary DEK cells are suspended in BME
supplemented with 10% tryptose phosphate broth, 2 mM L-glutamine, 0.17% sodium bicarbonate and 2–4%
chicken serum, and are adjusted to contain 3 × 105 cells/ml. Cells are next added to the plates at 100 µl per well
and the plates are then incubated for up to 96 hours at 37°C in a humidified 5% CO2 atmosphere. Following
incubation, cells are fixed with 10% formol-buffered saline and stained with 1% crystal violet. The plates are read
macroscopically. The titre for virus neutralising activity is expressed as the reciprocal of the highest dilution of
serum at which a monolayer grew, i.e., there is no evidence of CPE and therefore complete virus neutralisation
has occurred. A titre of less than 4 log2 is considered to be negative.
These neutralisation tests have been used to assay humoral immune responses to vaccination and for
epidemiological surveys, as well as for virus identification.

DHV type I can be controlled by the use of a live attenuated virus vaccine. This is given to breeder ducks so that
immunity is transferred via the yolk to newly hatched birds. Live vaccine virus can also be used to actively
immunise newly hatched DHV type-I-susceptible ducklings (3). An inactivated DHV type I vaccine is also effective
when administered to breeder ducks that have been primed with live vaccine or previously field exposed to live
DHV type I; progeny from these breeders have maternal immunity (18). Ducks may also be passively protected by
inoculation of antibodies in chicken egg-yolk.
An attenuated live virus DHV type II vaccine has been used to protect ducklings only under experimental
conditions (5).
DHV type III infections have been controlled by the use of attenuated live virus vaccines given to breeder ducks,
so that the immunity is transferred via the yolk sac to the hatching ducklings.

1. Seed management

The type I virus vaccine seed used most commonly in Europe is derived from an isolate passaged in
embryonated chicken eggs 53–55 times, that in the USA for live and inactivated vaccines has been
passaged 84–89 times.
The type II virus vaccine seed originated from an isolate attenuated by 25 serial passages in embryonated
chicken eggs (1), and has been employed only experimentally under field conditions (R.E. Gough, personal
communication).
The type III vaccine seed has been attenuated by 30 serial passages in embryonated duck eggs inoculated
via the CAM.
The seed viruses of types I and II are handled similarly. They should be prepared in 9–10-day-old specific
pathogen free (SPF) embryonated chicken eggs inoculated via the allantoic route and incubated at 37°C.
They can be stored as embryo homogenates in buffered saline at –70°C or below for several years.
The type III seed virus is prepared in 10-day-old SPF duck embryos, inoculated on to the CAM, and
incubated for 6–10 days at 37°C. It may be stored as a homogenate of CAM and embryos at –70°C or
below.
All seed viruses should be shown to be free from extraneous viruses that are pathogenic for ducks, chickens
or turkeys. The seeds should be free from all microbiological and fungal contamination.
In the case of newly hatched ducklings, attenuated live DHV type I replicates rapidly and results in an
immunity within 48–72 hours of vaccination. This immunity persists throughout the susceptible period of life
(3). However in ducklings protected by vaccination of their parents, the level of maternally derived immunity
decreases over the first 2 weeks of life, but such ducklings can be actively re-immunised with attenuated
virus given subcutaneously or orally at about 7–10 days of age (8, 14). Alternatively, the immunity can be
enhanced by the administration of specific hyperimmune serum or of egg yolk antibody prepared from eggs
laid by chickens actively hyperimmunised against DHV type I.
Breeder ducks primed with live DHV type I and then given, intramuscularly, a single dose of inactivated type
I vaccine produced maternally immune progeny through a complete laying cycle (18).
DHV types I and II viruses are treated similarly. The vaccine is produced in 9–10-day-old SPF embryonated
chicken eggs inoculated via the allantoic route, and incubated at 37°C. Most embryo deaths occur within 2–3 days
in the case of DHV type I, but with type II, the deaths do not occur until 6–10 days after inoculation, although they
are harvested at 3–5 days for maximum virus yield. The embryo harvests are homogenised in buffered saline and
clarified by low-speed centrifugation. The preparation is diluted as appropriate and dispensed into vials, which are
preferably frozen rapidly at –70°C or below. Subsequently, they may be stored satisfactorily between –20°C and
–40°C. DHV type I attenuated vaccine is also available as a lyophilised preparation that may be stored at 28°C.
The reconstituted vaccine may be used with or without the incorporation of aluminium hydroxide in the diluent.
In the case of inactivated DHV type I vaccine, the embryo harvests are homogenised and clarified by low-speed
centrifugation and then further purified by treatment with chloroform (final concentration 10% [v/v]). This
preparation is then inactivated with freshly prepared binary ethylenimine (BEI). The inactivated virus is then
blended with an adjuvant such as LES-STM 1; 0.2 % (v/v) formalin is added as a preservative (18).
1 A preparation of Salmonella typhimurium (STM), a B cell mitogen, in a lipid emulsion system (LES). Available from Ribi
Immunochem Research, Hamilton, Montana 59840, USA.

The type III vaccine is prepared in 10-day-old SPF duck eggs inoculated via the CAM with attenuated DHV type III
and incubated at 37°C. Most embryo deaths occur between 6 and 10 days. Eggs containing dying embryos,
together with their CAMs, are harvested and homogenised in buffered saline and clarified by low-speed
centrifugation. The preparation is diluted as appropriate and dispensed into vials, which are preferably frozen
rapidly at –70°C or below.
• Egg yolk antibody

Virulent DHV type I prepared from duckling livers or attenuated virus may be used to hyperimmunise SPF
chickens for egg-yolk antibody production. Eggs are collected from the hyperimmunised birds and stored at
4°C until time of production. The yolks are separated, pooled and blended with an antifoaming agent. The
mixture is diluted with buffered saline containing no more than 0.2% (v/v) formalin as a preservative. The
dispensed product is stored at 4°C and has a shelf life of 1 year. Tests are carried out for sterility in the
usual way for the absence of contaminants.
Any embryo deaths within the first 24 hours of inoculation should be discarded as nonspecific deaths.
The identity of the virus type should be confirmed by a VN test conducted with specific antiserum by a constant-
serum/varying-virus method. In the case of types I and II viruses, the tests are performed in embryonated chicken
eggs; with type III virus the tests are done in embryonated duck eggs. The antiserum should reduce the titre of the
respective virus by at least 102.0 ELD50 (50% embryo lethal dose).
4. Batch control
a) Sterility
Tests for sterility and freedom from contamination of biological materials may be found in the Chapter 1.1.9.
b) Safety
A group of 1–3-day-old ducklings susceptible to the type of virus concerned, should be inoculated
subcutaneously or intramuscularly (in the case of types I and II), or subcutaneously (in the case of type III),
with the attenuated vaccine at ten times the recommended dose, and kept under observation for between 10
and 21 days for any adverse reactions. Attenuated live vaccines should be stable and not revert to virulence
on back passages in susceptible ducklings.
A safety test on the inactivated DHV type I vaccine is performed by inoculating the recommended dose
(0.5 ml) intramuscularly into a group of day-old ducklings; no adverse effects should be observed during the
period of testing.
Safety tests on yolk antibody are done by inoculating 1 ml subcutaneously into each of a group of ducklings,
which are then kept under observation for 3 days for signs of adverse effects.
c) Potency
For DHV types I and II viruses, the virus titre of the vaccine should be determined in 9–10-day-old
embryonated chicken eggs inoculated into the allantoic cavity and incubated at 37°C. The immunogenicity,
of the vaccine for ducklings susceptible to type I or II virus can be assessed by inoculating subcutaneously a
minimum of 103.3 ELD50 per duckling of the vaccine virus and challenging subcutaneously 72 hours later
with 103.0 LD50 per duckling of virulent DHV virus type I or II (3). At least 80% of the vaccinated birds should
survive and, in the case of type I, at least 80% of the controls should die; in the case of type II, a 20%
mortality in the controls is more realistic.
The immunogenicity of the inactivated vaccine is considered to be satisfactory if a four-fold or greater

increase in neutralising antibody titre can be demonstrated following administration to ducklings that have
been previously primed with live attenuated DHV type I.
For type III virus, the titre of the vaccine should be determined in 10-day-old embryonated duck eggs
inoculated on to the CAM. Immunogenicity tests in ducklings have proved difficult because of the variable
pathogenicity of the challenge virus for ducklings.

Potency tests on yolk antibody are done by determining the neutralising index (NI) for the product in
embryonated hens’ eggs using the constant-yolk/varying-virus method. A minimum NI of 103.0 is considered
to be satisfactory. The efficacy of the product is determined by inoculating a group of susceptible ducklings
with the recommended dose of egg yolk antibody. A second group is left untreated. After 24 hours each
group is challenged with virulent DHV type I virus. The product is adjudged efficacious if at least 80% of the
treated ducklings survive and at least 80% of the controls die.
Breeder ducks given live attenuated DHV type I vaccine two or three times at 12, 8 and 4 weeks before
coming in to lay, and breeder ducks given live attenuated DHV type III vaccine twice at 12 and 4 weeks
before coming in to lay should produce passively immune progeny throughout a breeding season. However,
it is usually recommended to revaccinate every 3 months with DHV type I vaccine and every 6 months with
DHV type III vaccine after the onset of lay. DVH type I attenuated vaccine can also be supplied as a
lyophilised preparation that is blended with a diluent containing aluminium hydroxide, just before
administration. This is given at 7 weeks of age with a second dose 2 weeks before onset of lay. This should
provide maternally immune progeny throughout a complete laying cycle. No information on the use of DHV
type II vaccine in breeder ducks is available.
Live attenuated DHV type I or type II vaccine given subcutaneously or intramuscularly to 1-day-old ducklings
protects against the disease for the duration of their susceptibility. No information is available on the use of
DHV type III vaccine to actively immunise 1-day-old ducklings.
Breeder ducks primed with live DHV type I and then given a single dose of inactivated DHV type I vaccine
intramuscularly, should produce maternally immune progeny through a complete laying cycle (18).
Egg-yolk antibody offers passive immunisation in the face of an outbreak. The duration of its efficacy is
short-lived.
e) Stability
Aqueous preparations of live attenuated DHV type I, II and III vaccines when stored frozen at –70°C or lower
should remain stable for at least 1 year. Once thawed these vaccines should be held at 4°C and used within
1 week. Live lyophilised vaccines may be stored at 28°C and should retain their potency for at least 1 year.
The inactivated DHV type I vaccine is blended with adjuvant and can be stored at 4°C for at least 20 months
without loss of immunogenicity.
Egg-yolk antibody can be stored for up to 1 year at 4°C.
f) Preservatives
No preservatives are added to the live attenuated DHV type I, II and III vaccines.
Formalin (up to 0.2% [v/v]) is added to the DHV type I inactivated vaccine, and to the egg-yolk antibody
preparation.
The inactivated DHV type I vaccine should be shaken well to ensure that it is completely blended before use.
The live attenuated DHV type I and III vaccines are issued as vials of lyophilised or frozen concentrated vaccine
virus together with bottles of sterile diluent, on which standard sterility checks have been made (see Section
C.4.a). The DHV type II live attenuated vaccine has only been made experimentally.
a) Safety
No additional testing is performed after the batch testing on any of the products.
b) Potency
No additional testing is performed after the batch testing on any of the products.

REFERENCES
1. ASPLIN F.D. (1965). Duck hepatitis. Vaccination against two serological types. Vet. Rec., 77, 1529–1530.
2. CHALMERS W.S.K. & W OOLCOCK P.R. (1984). The effect of animal sera on duck hepatitis virus. Avian Pathol.,
13, 727–732.
3. CRIGHTON G.W. & W OOLCOCK P.R. (1978). Active immunisation of ducklings against duck virus hepatitis. Vet.
Rec., 102, 358–361.
4. DING C. ZHANG D. (2007). Molecular analysis of duck hepatitis virus type 1. Virology, 361, 9–17.
5. GOUGH R.E., BORLAND E.D., KEYMER I.F. & STUART J.C. (1985). An outbreak of duck hepatitis type II in
commercial ducks. Avian Pathol., 14, 227–236.
6. GUÉRIN J.-L., ALBARIC O., NOUTARY V. & BOISSIEU C. (2007). A duck hepatitis virus type I is agent of
pancreatitis and encephalitis in Muscovy duckling. In: Proceedings of the Annual Meeting of the American
Association of Avian Pathologists, 14–18 July 2007, Washington, DC, USA.
7. HAIDER S.A. & CALNEK B.W. (1979). In-vitro isolation, propagation and characterisation of duck hepatitis virus
type III. Avian Dis., 23, 715–729.
8. HANSON L.E. & TRIPATHY D.N. (1976). Oral immunisation of ducklings with an attenuated hepatitis virus. Dev.
Biol. Stand., 33, 357–363.
9. KIM M.C., KWON Y.K., JOH S.J., KWON J.H., KIM J.H. & KIM S.J. (2007). Development of one-step reverse
transcriptase-polymerase chain reaction to detect duck hepatitis virus type 1. Avian Dis., 51, 540–545.
10. KIM M.C., KWON Y.K., JOH S.J., LINDBERG A.M., KWON J.H., KIM J.H. & KIM S.J. (2006). Molecular analysis of
duck hepatitis virus type 1 reveals a novel lineage close to the genus Parechovirus in the family
Picornaviridae. J. Gen. Virol., 87, 3307–3316.
11. KOCI M.D. & SCHULTZ-CHERRY S. (2002). Avian astroviruses. Avian Pathol., 31, 213–227.
12. SANDHU T.S., CALNEK B.W. & ZEMAN L. (1992). Pathologic and serologic characterisation of a variant of duck
hepatitis type I virus. Avian Dis., 36, 932–936.
13. TOTH T.E. (1969). Studies of an agent causing mortality among ducklings immune to duck virus hepatitis.
Avian Dis., 13, 834–846.
14. TRIPATHY D.N. & HANSON L.E. (1986). Impact of oral immunisation against duck viral hepatitis in passively
immune ducklings. Prev. Vet. Med., 4, 355–360.
15. TSENG C.H., KNOWLES N.J. & TSAI H.J. (2007). Molecular analysis of duck hepatitis virus type 1 indicates that
it should be assigned to a new genus. Virus Res., 123, 190–203.
16. TSENG C.H. & TSAI H.J. (2007). Molecular characterization of a new serotype of duck hepatitis virus. Virus
Res., 126, 19–31.
17. WOOLCOCK P.R. (1986). An assay for duck hepatitis virus type I in duck embryo liver cells and a comparison
with other assays. Avian Pathol., 15, 75–82.
18. W OOLCOCK P.R. (1991). Duck hepatitis virus type I: Studies with inactivated vaccines in breeder ducks.
19. WOOLCOCK P.R. (1998). Duck virus hepatitis. In: A Laboratory Manual for the Isolation and Identification of
Avian Pathogens, Fourth Edition, Swayne D.E., Glisson J.R., Jackwood M.W., Pearson J.E. & Reed W.M.,
eds. American Association of Avian Pathologists, Kennett Square, Pennsylvania, USA, 200–204.
20. WOOLCOCK P.R. (2003). Duck hepatitis. In: S.Y.M., Diseases of Poultry, Eleventh Edition, Saif Y.M., Barnes
H.J., Glisson J.R., Fadly A.M., McDougald L.R.& Swayne D.E., eds. Iowa State Press, Iowa, USA, 343–354.
*
* *

CHAPTER 2.3.9.
FOWL CHOLERA
SUMMARY
Fowl cholera (avian pasteurellosis) is a commonly occurring avian disease that can affect all
types of birds and is distributed world-wide. Fowl cholera outbreaks often manifest as acute
fatal septicaemia. Diagnosis depends on isolation and identification of the causative bacterium,
Pasteurella multocida. Presumptive diagnosis may be based on the occurrence of typical signs
and lesions and/or on the microscopic demonstration of myriad bacteria in blood smears, or
impression smears of tissues such as liver or spleen. Mild or chronic forms of the disease also
occur where the disease is endemic, with localised infection primarily of the respiratory and
skeletal systems.
Identification of the agent: Pasteurella multocida is readily isolated, often in pure culture, from
visceral organs such as lung, liver and spleen, bone marrow, gonads or heart blood of birds
that succumb to the acute bacteraemic form of the disease, or from the caseous exudate
characteristic of chronic fowl cholera lesions. It is a facultative anaerobic bacterium that grows
best at 37°C. Primary isolation is usually accomplished using media such as dextrose starch
agar, blood agar, and trypticase–soy agar. Isolation may be improved by the addition of 5%
heat-inactivated serum. Colonies range from 1 to 3 mm in diameter after 18–24 hours of
incubation and are discrete, circular, convex, translucent, and butyraceous. The cells are
coccobacillary or short rod-shaped, 0.2–0.4 × 0.6–2.5 µm in size, stain Gram negative, and
generally occur singly or in pairs. Bipolar staining is evident with Wright or Giemsa stains.
Identification of P. multocida is based on the results of biochemical tests, which include

carbohydrate fermentation, enzyme production, and selected metabolite production.
Serological characterisation of strains of P. multocida includes capsular serogrouping and
somatic serotyping. DNA fingerprinting can differentiate among P. multocida having the same
capsular serogroup and somatic serotype. These characterisations require a specialised
laboratory with appropriate diagnostic reagents.
Serological tests: Serological tests are rarely used for diagnosis of fowl cholera. The ease of
obtaining a definitive diagnosis through isolation and identification of the causative organism
generally precludes the need for serodiagnosis.
Requirements for vaccines and diagnostic biologicals: The P. multocida vaccines in

general use are bacterins, containing aluminium hydroxide or oil as adjuvant, prepared from
multiple serotypes. Two doses of the killed vaccine are typically required. Live culture vaccines
tend to impart greater protective immunity, but are used less frequently because of potential
post-vaccinal sequelae such as pneumonitis and arthritis. Multivalent vaccines typically
incorporate somatic serotypes 1, 3, and 4 as they among the more commonly isolated avian
serotypes. Safety and potency testing of bacterins usually use the host animal. Final containers
of live cultures are tested for potency by bacterial counts.
A. INTRODUCTION
Fowl cholera is a contagious bacterial disease of domesticated and wild avian species caused by infection
with Pasteurella multocida. It typically occurs as a fulminating disease with massive bacteraemia and high
morbidity and mortality. Chronic infections also occur with clinical signs and lesions related to localised
infections. The pulmonary system and tissues associated with the musculoskeletal system are often the seats
of chronic infection. Common synonyms for fowl cholera are avian pasteurellosis and avian haemorrhagic

Chapter 2.3.9. — Fowl cholera
septicaemia. Fowl cholera is not considered to have zoonotic potential as avian isolates are generally
nonpathogenic in mammals exposed by the oral or subcutaneous routes. Other bacterial diseases, including
salmonellosis, colibacillosis, and listeriosis in chickens, and pseudotuberculosis, erysipelas, and chlamydiosis
in turkeys, may present with clinical signs and lesions similar to fowl cholera. Differentiation is based on
isolation and identification as P. multocida is readily cultured from cases of fowl cholera.
Fowl cholera (avian pasteurellosis) is a commonly occurring avian disease that can affect all types of birds
and is often fatal (3, 7). In the peracute form, fowl cholera is one of the most virulent and infectious diseases
of poultry. Diagnosis depends on identification of the causative bacterium, P. multocida, following isolation
from birds with signs and lesions consistent with this disease. Presumptive diagnosis may be based on the
observance of typical signs and lesions and/or on the microscopic demonstration of bacteria showing bipolar
staining in smears of tissues, such as blood, liver, or spleen. Mild forms of the disease may occur.
All avian species are susceptible to P. multocida, although turkeys may be the most severely affected. Often
the first sign of disease is dead birds. Other signs include: fever, anorexia, depression, mucus discharge from
the mouth, diarrhoea, ruffled feathers, drop in egg production coupled with smaller eggs, increased
respiratory rate, and cyanosis at the time of death. Lesions that are often observed include: congested organs
with serosal haemorrhages, enlarged liver and spleen, multiple small necrotic areas in the liver and/or spleen,
pneumonia, and mild ascites and pericardial oedema. Birds that survive the acute septicaemic stage or those
infected with organisms of low virulence may develop chronic fowl cholera, characterised by localised
infections. These infections often involve joints, foot pads, tendon sheaths, sternal bursa, conjunctivae,
wattles, pharynx, lungs, air sacs, middle ears, bone marrow, and meninges. Lesions resulting from these
infections are usually characterised by bacterial colonisation with necrosis, fibrinosuppurative exudate, and
degrees of fibroplasia.
Diagnosis depends on isolation and identification of the causative organism.

Pasteurella multocida is a facultative anaerobic bacterium that grows best at 35–37°C. Primary isolation is
usually accomplished using media such as blood agar, trypticase–soy agar or dextrose starch agar, and
isolation may be improved by supplementing these media with 5% heat-inactivated serum. Maintenance
media usually do not require supplemental serum. Colonies range from 1 to 3 mm in diameter after 18–
24 hours of incubation. They usually are discrete, circular, convex, translucent, and butyraceous. Capsulated
organisms usually produce larger colonies than those of noncapsulated organisms. Watery mucoid colonies,
often observed with mammalian respiratory tract isolates, are very rare with avian isolates. The cells are
coccobacillary or short rod-shaped, usually 0.2–0.4 × 0.6–2.5 µm in size, stain Gram negative, and generally
occur singly or in pairs. Recently isolated organisms or those found in tissue smears show bipolar staining
with Wright or Giemsa stains or methylene blue, and are usually encapsulated.
Isolation of the organism from visceral organs, such as liver, bone marrow, spleen, or heart blood of birds that
succumb to the acute form of the disease, and from exudative lesions of birds with the chronic form of the
disease, is generally easily accomplished. Isolation from those chronically affected birds that have no
evidence of disease other than emaciation and lethargy is often difficult. In this condition or when host
decomposition has occurred, bone marrow is the tissue of choice for isolation attempts. The surface of the
tissue to be cultured is seared with a hot spatula and a specimen is obtained by inserting a sterile cotton
swab, wire or plastic loop through the heat-sterilised surface. The specimen is inoculated directly on to agar
medium or into tryptose or another broth medium, incubated for a few hours, transferred to agar medium, and
incubated again.
Identification is based primarily on the results of biochemical tests. Carbohydrate fermentation reactions are
essential. Those carbohydrates that are fermented include: glucose, mannose, galactose, fructose, and
sucrose. Those not fermented include: rhamnose, cellobiose, raffinose, inulin, erythritol, adonitol, m-inositol,
and salicin. Mannitol is usually fermented. Arabinose, maltose, lactose, and dextrin are usually not fermented.
Variable reactions occur with xylose, trehalose, glycerol, and sorbitol. Pasteurella multocida does not cause
haemolysis, is not motile and only rarely grows on MacConkey agar. It produces catalase, oxidase, and
ornithine decarboxylase, but does not produce urease, lysine decarboxylase, beta-galactosidase, or arginine
dihydrolase. Phosphatase production is variable. Nitrate is reduced; indole and hydrogen sulphide are
produced, and methyl red and Voges–Proskauer tests are negative. Detection of hydrogen sulphide
production may require lead acetate-laden paper strips suspended above a modified H2S liquid medium (8).
Commercial biochemical test kits are available.
Differentiation of P. multocida from other avian Pasteurella spp. and Riemerella (Pasteurella) anatipestifer
can usually be accomplished using the tests and results indicated in Table 1. Laboratory experience has

shown that P. multocida is most easily identified by its colony morphology and appearance in Gram stains.
Positive reactions to indole and ornithine decarboxylase are the most useful biochemical indications.
Table 1. Tests used to differentiate Pasteurella multocida from other avian

Pasteurella species and Riemerella anatipestifer
Test* Pasteurella Riemerella
multocida gallinarum anatipestifer
Haemolysis on blood agar * v
Growth on MacConkey’s agar

Indole production
Gelatin liquefaction u
Catalase production
Urease production v
Glucose fermentation
Lactose fermentation u
Sucrose fermentation
Maltose fermentation u
Ornithine decarboxylase
*Test reaction results: = no reaction; = reaction; v = variable reactions; u = usually no reaction; u usually a reaction.
Antigenic characterisation of P. multocida is accomplished by capsular serogrouping and somatic serotyping.

Capsular serogroups are determined by a passive haemagglutination test (1, 2). Capsular serogroups,
determined by a passive haemaglutination test, are A, B, D, E, and F. All but serogroup E have been isolated
from avian hosts. A nonserological disk diffusion test that uses specific mucopolysaccharidases to
differentiate serogroups A, D, and F has been developed (6).
Somatic serotypes are usually determined by an agar gel immunodiffusion (AGID) test (4, 5). Serotypes 1
through 16 have been reported; all 16 serotypes have been isolated from avian hosts (8). The most effective
characterisation involves determination of both serogroup and serotype. These determinations require a
specialised laboratory with appropriate diagnostic reagents. To determine the serotype, the laboratory
prepares the unknown bacterial culture as antigen for the AGID test and then must test it against all
16 serotype-specific antisera. Antigens present in a single isolate may react with multiple serotype-specific
antisera resulting in bi- or trinomial serotypes, as illustrated by the 3, 4 and 3, 4, 12 strains (8).
Somatic typing procedure using the gel diffusion precipitin test

i) Inoculate a dextrose starch agar (DSA) plate (20 × 150 mm containing 70 ml of medium or two
15 × 100 mm plates containing 20 ml of medium per plate) with cells from a pure culture of
Pasteurella multocida by using a sterile cotton swab. Swab the entire surface of the plate(s).
Incubate the plate(s) in a 37°C incubator for 18–24 hours. This procedure is used to produce
antigen for positive control purposes or to prepare antigen from diagnostic cultures.
ii) Harvest the cells from the plate(s) using 2.5 ml of 0.85% saline with 0.6% formaldehyde and a
sterile hockey stick. Place the cells in a tube using a sterile pipette.
iii) Autoclave the cells at 100°C for 1 hour.
iv) Centrifuge the cell suspension mixture at 13,300 g for 20 minutes.
v) Remove the supernatant and place in a sterile tube.
vi) Prepare the agar gel for use in the gel diffusion precipitin test (GDPT) by placing 17.0 g of NaCl,
1.8 g of agar noble, and 200 ml of distilled water into a 500 ml flask. Microwave the contents of the
flask with the cap loose for 2.5 minutes. Swirl the contents of the flask and microwave again for
2.5 minutes. Allow the agar to cool slightly for 10–15 minutes. Do not prepare less than 200 ml of
agar in a microwave. Dehydration during the microwave process can increase the agar
concentration and negatively impact or inhibit diffusion.

vii) Place 5 ml of melted agar onto the surface of a 75 × 25 mm plain glass microscope slide. It is
important that the slides are level prior to dispensing the agar. Allow the agar to cool
(approximately 30 minutes) completely.
viii) Wells are cut in the agar bed. The wells are 3 mm in diameter and 3 mm apart from edge-to-edge.
Frequently an Ouchterlony template is used to create two or three replicates of wells per slide.
Each replicate has a centre well and is surrounded by four wells located at 90° angles (from
centre).
ix) Reference antiserum is always placed in the centre well (of a replicate). Antigen from a diagnostic
or reference culture is placed in one of the surrounding wells within a replicate. Each well is filled to
capacity.
x) The slides are incubated within a moist chamber in a 37°C incubator for 48 hours. Precipitin lines
of a reaction can be best observed with subdued lighting from underneath the slide. When present,
reactions should occur between the centre and surrounding well(s) as an arc of precipitin.
Sometimes these reactions are close to the edge of a well. Slides should be carefully examined.
Diagnostic cultures can react to more than one reference somatic antiserum.
xi) Positive controls should be used. Reference antiserum should be tested against reference antigen
each time the test is performed.
DNA fingerprinting of P. multocida by restriction endonuclease analysis (REA) has proved valuable in
epidemiological investigations fowl cholera in poultry flocks. Isolates of P. multocida having both capsular
serogroup and somatic serotype in common may be distinguished by REA. Ethidium-bromide-stained
agarose gels are analysed following electrophoresis of DNA digested with either Hhal or Hpall endonuclease
(10).
Serological tests for the presence of specific antibodies are not used for diagnosis of fowl cholera. The ease
of obtaining a definitive diagnosis by isolation and identification of the causative organism precludes the need
for serodiagnosis. Serological tests, such as agglutination, AGID, and passive haemagglutination, have been
used experimentally to demonstrate antibody against P. multocida in serum from avian hosts; none were
highly sensitive. Determinations of antibody titres using enzyme-linked immunosorbent assays have been
used with varying degrees of success in attempts to monitor seroconversion in vaccinated poultry, but not for
diagnosis.

Fowl cholera may be caused by any of 16 Heddleston serotypes of P. multocida, although certain serotypes
appear to be more often associated with disease. The P. multocida vaccines in general use are bacterins,
containing aluminium hydroxide or oil adjuvant, prepared from inactivated cells of serotypes selected on the
basis of epidemiological information. Commercial bacterins are usually composed of serotypes 1, 3, and 4.
Vaccination plays a significant role in the control of this disease. Live vaccines containing modified
P. multocida are not generally used except in North America.
Guidelines for the production of veterinary vaccines are given in Chapter 1.1.8 Principles of veterinary
vaccine production. The guidelines given here and in Chapter 1.1.8 are intended to be general in nature and
may be supplemented by national and regional requirements.
Bacterin is normally administered by intramuscular injection in the leg or breast muscles, or subcutaneously
at the back of the neck. Two doses are typically administered at 2–4-week intervals. As with most killed
vaccines, full immunity cannot be expected until approximately 2 weeks after the second dose of a primary
vaccination course. Live vaccines are typically administered in the drinking water. Vaccination of diseased
birds or those in poor nutritional status should be avoided as a satisfactory immune response may not be
generated in such circumstances.
The general method for production of P. multocida bacterins is presented here. Production cultures of each
bacterial isolate to be included in the final product are prepared. The cultures are typically started in small
vessels and subpassaged into progressively larger volumes of media until the desired production volume is
achieved. Each production culture is inactivated by formalin or other acceptable means. All of the component
cultures are mixed, and usually blended, with an adjuvant prior to filling sterile final containers.

The following section is based on the requirements for P. multocida bacterins and vaccines as found in Title
9, United States Code of Federal Regulations. Other countries may have slightly different requirements.
2. Master seed management

All strains of P. multocida to be incorporated into a bacterin or vaccine must be well characterised, of
known serotype, pure, safe and immunogenic. The culture(s) that is evaluated and characterised is
designated by lot number and called a master seed. All cultures used in the production of licensed
bacterins or vaccines must be derived from an approved master seed(s) and must be within an
accepted number of passages from the master seed lot.
b) Validation as a vaccine
i) Efficacy
Products prepared from candidate master seeds must be shown to be effective against challenge
infection. Efficacy must be demonstrated in each animal species (chickens, turkeys, ducks,
psittacines) and by each route of administration for which the product will be recommended, and
protection must be demonstrated against each challenge serotype for which protection is claimed.
The lot of product used to demonstrate efficacy must be produced from the highest allowable
passage of master seed.
For live avian Pasteurella vaccines, 20 vaccinates and 10 controls are used in each efficacy trial.
Birds are challenged not less than 14 days after vaccination and are observed for 10 days after
challenge. A satisfactory test requires that at least eight of the controls die and at least 16 of the
vaccinates survive.
The arithmetic mean count of colony-forming units in the lot of product that is used to demonstrate
efficacy is used as the minimum standard (immunogenicity standard) for all subsequent production
lots of vaccine.
Efficacy of bacterins must be demonstrated similarly prior to licensure. However, no
immunogenicity standards are derived from the lot that was used to demonstrate initial efficacy;
each production lot is satisfactorily tested in a vaccination-challenge trial prior to release for sale
and distribution.
ii) Safety
The safety of master seeds used in the production of live vaccines must be evaluated prior to
licensing. Safety must be tested in each animal species (chickens, turkeys, ducks, psittacines) for
which the product is recommended. Each of 10 birds is given an equivalent of 10 vaccine doses
and observed for 10 days. At least 8 of 10 birds must show no unfavourable reactions attributable
to the master seed. Additionally, the master seeds must be tested for reversion to virulence and
evaluated for excretion from the host and transmission to other target species.
The safety of each production lot is tested by methods described in Section C.4.c.
The purity of the cultures is determined at each stage of production prior to inactivation. This may be
achieved by microscopic examination (e.g. phase–contrast microscopy, Gram strain) and/or by culture. Killed
cultures are tested for completeness of inactivation. Analytical assays to determine the levels of
formaldehyde or other preservatives are done on bulk vaccine and must be within specified limits. During
manufacturing, production parameters must be tightly controlled to ensure that all serials (batches) are
produced in the same manner as that used to produce the serials used for immunogenicity studies.
4. Batch control
a) Sterility
Sterility tests are done on filled vaccine. Each lot must pass sterility requirements, for example those
detailed in the 9 CFR Part 113.26 or 113.27 (9). (See also Chapter 1.1.9 Tests for sterility and freedom
from contamination of biological materials).
b) Safety
Safety testing is conducted on each bulk or filled vaccine lot. Live vaccines are tested according to the
method described in C.1.c.ii, except that only one representative animal species is required. Bacterins

are administered according to label recommendations, and the birds are observed for 14 days; at least
18 of 20 birds must show no unfavourable reactions attributable to the bacterin.
c) Potency
Each production lot of bacterin or live vaccine must be tested for potency by a test that is related to, and
considered predictive of, efficacy. Potency tests are performed on the product in its final form.
Bacterins are tested for potency in a vaccination-challenge trial. Separate groups of birds
(20 vaccinates, 10 controls) must be challenged with each of the serotypes of P. multocida for which
protection is claimed. Bacterins are administered according to the dose and route recommended on the
label. Two doses are administered 3 weeks apart, and all birds are challenged 2 weeks after the second
dose. The birds are observed for 14 days after challenge. For a satisfactory test, at least 14 of
20 vaccinates must survive and at least 8 of 10 controls must die.
The potency of live vaccine lots is determined by a bacterial count performed on reconstituted
lyophilised product in its final container. The mean bacterial count of any vaccine lot at the time of
preparation must be sufficiently high to ensure that at any time prior to product expiration, the count is at
least twice the immunogenicity standard. (The European Pharmacopoeia requires a count that is at least
equal to the immunogenicity standard.)
d) Stability
The acceptability of the shelf life of a vaccine is confirmed by testing the product for potency at the end
of the approved shelf life. At least three lots of vaccine are tested and must pass established potency
requirements. Vaccines are stored at 2–7°C and protected from freezing. Partly used packs should be
discarded at the end of a day’s operations.
e) Preservatives
Any preservatives must be added within specified limits. Preservatives are generally added to vaccines
to limit the growth of any contaminants introduced when the rubber cap is pierced with a needle. Ideally,
multidose vaccination equipment should be used whereby the vaccine pack is entered only once with a
sterile needle.
Vaccines prepared with aluminium-based adjuvants may cause temporary nodules at the site of
injection. Operator self-injection poses no immediate problems, but medical advice should be sought as
there is a risk of infection via a contaminated needle.
Vaccines prepared with oil based adjuvants may cause more severe reactions at the site of injection,
which may manifest as large nodules. Care should be taken to administer these vaccines correctly.
Operator self injection requires immediate medical attention, involving prompt incision and irrigation of
the site.
5. Tests on final product
a) Safety
See Section C.4.b.
b) Potency
See Section C.4.c.
REFERENCES
1. CARTER G.R. (1955). Studies on Pasteurella multocida. I. A hemagglutination test for the identification of
serological types. Am. J. Vet. Res., 16, 481–484.
2. CARTER G.R. (1972). Improved hemagglutination test for identifying type A strains of Pasteurella
multocida. Appl. Microbiol, 24, 162–163.

3. DERIEUX W.T. (1978). Response of young chickens and turkeys to virulent and avirulent Pasteurella
multocida administered by various routes. Avian Dis., 22, 131–39.
4. HEDDLESTON K.L. (1962). Studies on pasteurellosis. V. Two immunogenic types of Pasteurella multocida
associated with fowl cholera. Avian Dis., 6, 315–321.
5. HEDDLESTON K.L., GALLAGHER J.E. & REBERS P.A. (1972). Fowl cholera: Gel diffusion precipitin test for
serotyping Pasteurella multocida from avian species. Avian Dis., 16, 925–936.
6. RIMLER R.B. (1994). Presumptive identification of Pasteurella multocida serogroups A, D, and F by

capsule depolymerisation with mucopolysaccharidases. Vet. Rec., 134, 191–192.
7. RIMLER R.B. & GLISSON J.R. (1997). Fowl cholera. In: Diseases of Poultry, Tenth Edition, Calnek B.W.,
Barnes H.J., Beard C.W., McDougald L.R. & Saif Y.M., eds. Iowa State University Press, Ames, Iowa,
USA. 143–159.
8. RIMLER R.B., SANDHU T.S. & GLISSON J.R. (1998). Pasteurellosis, Infectious Serositis, and
Pseudotuberculosis. In: A Laboratory Manual for the Isolation and Identification of Avian Pathogens,
Fourth Edition, Swayne D.E., Glisson J.R., Jackwood, M.W., Pearson, J.E. & Reed W.M., eds. American
Association of Avian Pathologists, Kennett Square, Pennsylvania, USA, 17–28.
9. UNITED STATES DEPARTMENT OF AGRICULTURE (USDA) (2001). Code of Federal Regulations, Title 9,
Animals and Animal Products. Office of the Federal Register, National Archives and Records
Administration. US Government Printing Office, Washington D.C., USA.
10. W ILSON M.A., RIMLER R.B. & HOFFMAN L.J. (1992). Comparison of DNA fingerprints and somatic serotypes
of serogroups B and E Pasteurella multocida isolates. J. Clin. Microbiol., 30, 1518–1524.
*
* *

CHAPTER 2.3.10.
FOWLPOX
SUMMARY
Fowlpox is a disease of chickens and turkeys caused by a DNA virus of the genus Avipoxvirus of
the family Poxviridae. Its distribution is world-wide. It is slow-spreading and characterised by the
formation of proliferative lesions and scabs on the skin, and diphtheritic lesions in the upper parts of
the digestive and respiratory tracts. In the case of the cutaneous form, the mortality rate is usually
low and affected birds are more likely to recover than those with the diphtheritic form. In the
diphtheritic form, proliferative lesions involving the nasal passages, larynx or trachea can result in
respiratory distress and death from suffocation.
Fowlpox causes a transient drop in egg production and a reduced growth rate in young birds.
Identification of the agent: Fowlpox should be suspected where skin eruptions occur on exposed
areas. Histological examination of cutaneous or diphtheritic lesions reveals epithelial hyperplasia
with intracytoplasmic inclusions in affected cells. Elementary bodies may be detected in smears
from lesions by the use of the Gimenez method. Electron microscopy of lesions will detect virus
particles with the characteristic poxvirus morphology by negative staining or in ultrathin sections of
the lesion.
The diphtheritic form of fowlpox involving the trachea must be differentiated from infectious
laryngotracheitis, which is caused by a herpesvirus and is characterised by the presence of
intranuclear inclusion bodies.
Virus isolation is done by inoculation on to chorioallantoic membranes of 9–12-day-old developing

chicken embryos or avian cell cultures.
Serological tests: Immune responses to fowlpox virus may be demonstrated by the use of virus
neutralisation, agar gel immunodiffusion, immunofluorescence, or passive hemagglutination tests,
enzyme-linked immunosorbent assay and by immunoblotting.
Requirements for vaccines and diagnostic biologicals: Modified live fowlpox or pigeon pox
virus vaccines of chicken embryo or avian cell culture origin are available commercially. The use of
vaccines is indicated in areas where the disease is endemic, or on premises where infection has
been diagnosed.
A. INTRODUCTION
The morphology of the fowlpox virus is like that of other viruses of the poxviridae family. The mature virus
(elementary body) is brick shaped and measures about 330 × 280 × 200 nm. The outer coat is composed of
random arrangements of surface tubules. The virion consists of an electron-dense centrally located biconcave
core or nucleoid with two lateral bodies in each concavity and surrounded by an envelope. The 288 kbp fowlpox
virus genome encodes for over 250 genes.
Fowlpox has a world-wide distribution and is caused by a DNA virus of the genus Avipoxvirus of the family
Poxviridae (17, 21). Its incidence is variable in different areas because of differences in climate, management and
hygiene or the practice of regular vaccination. It can cause drops in egg production, or retarded growth in younger
birds.
Fowlpox is a slow-spreading virus disease of chickens and turkeys, characterised in the cutaneous form (dry pox)
by the development of proliferative lesions, ranging from small nodules to spherical wart-like masses on the skin
of the comb, wattle and other unfeathered areas. In the diphtheritic form (wet pox), slightly elevated white opaque

Chapter 2.3.10. — Fowlpox
nodules develop on the mucous membranes. They rapidly increase in size to become a yellowish diphtheritic
membrane. Lesions occur on the mucous membranes of the mouth, oesophagus, larynx or trachea. The mortality
rate is higher in the diphtheritic form than in the cutaneous form, sometimes nearing 50% particularly in young
birds. Integration of reticuloendotheliosis virus (REV) sequences has been observed in the genome of fowlpox
virus (11, 13). It is interesting that this insertion event occurred over 50 years ago (7). While most field strains
contain REV provirus, vaccine strains have only remnants of long terminal repeats (13). Virulence is enhanced by
the presence of REV provirus in the genome of field strains of fowlpox virus. Complete sequence of the genome
of a vaccine-like strain of fowlpox virus has been determined (1). The functions of the majority of the genes are
not known at this time. It is however, interesting that the virus tends to persist in the poultry environment for
extended periods of time where other viruses may not survive. In this regard the presence of photolyase gene and
A-type inclusion body gene in the virus genome appear to protect the virus from environmental insults (15, 16).
Antigenic cross-reactivity is observed among avianpox viruses and it appears that many genes are conserved.
Limited studies on antigenic, genetic and biologic comparison of fowlpox virus with other avianpox viruses
especially those that infect the wild birds are available. Recently, complete sequence of canarypox virus genome
has become available.
Fowlpox virus multiplies in the cytoplasm of epithelial cells with the formation of large intracytoplasmic inclusion
bodies (Bollinger bodies) that contain smaller elementary bodies (Borrel bodies). The inclusions can be
demonstrated in sections of cutaneous and diphtheritic lesions by the use of haematoxylin and eosin (H&E),
acridine orange or Giemsa stains (19). The elementary bodies can be detected in smears from lesions, for
example by the Gimenez method (18), which is described below. Electron microscopy can be used to
demonstrate viral particles of typical poxvirus morphology by negative staining or in ultrathin sections of infected
tissues (3).
a) A smear technique for fowlpox

i) Place a drop of distilled water and the lesion (cutaneous or diphtheritic) on a clean slide. Prepare a thin
smear by pressing the lesion with another clean slide and rotating the upper slide several times.
ii) Air dry and gently fix the smear over a flame.
iii) Stain the smear for 5–10 minutes with freshly prepared primary stain (8 ml stock solution 1 of basic
fuchsin mixed with 10 ml of phosphate buffer 2, pH 7.5, and filtered through Whatman filter paper
No. 1).
iv) Wash thoroughly with tap water.
v) Counterstain with malachite green (0.8% in distilled water) for 30–60 seconds.
vi) Wash the smear with tap water and then dry.
vii) Examine the smear under oil immersion. The elementary bodies appear red and are approximately
0.2–0.3 µm in size.
b) Virus isolation
Fowlpox virus can be isolated by the inoculation of suspected material into embryonated chicken eggs.
Approximately 0.1 ml of tissue suspension of skin or diphtheritic lesions, with the appropriate concentration
of antibiotics, is inoculated on to the chorioallantoic membranes (CAMs) of 9–12-day-old developing chicken
embryos. These are incubated at 37°C for 5–7 days, and then examined for focal white pock lesions or
generalised thickening of the CAMs. Histopathological examination of the CAM lesions will reveal
eosinophilic intracytoplasmic inclusion bodies following staining with H&E (19, 22).
Primary chicken embryo fibroblasts, chicken embryo kidney cells, chicken embryo dermis cells, or the
permanent quail cell line QT-35, can also be used to propagate fowlpox virus (6, 10). The adaptation of virus
strains to cell cultures is an important requirement for plaque formation, as not all strains will form plaques
initially.
1 Stock solution: A solution of basic fuchsin (5 g) in 95% ethanol (100 ml) is slowly added to a second solution of crystalline
phenol (10 g) in distilled water (900 ml). This stock solution, kept in a tightly screw-capped glass bottle, is incubated for
48 hours at 37°C, and then stored at room temperature.
2 Phosphate buffer, pH 7.5: NaH2PO4H2O (2.47 g) and Na2HPO4 (11.65 g) are added to distilled water (1000 ml) and
stored at 4°C.

c) Molecular methods
Restriction fragment length polymorphism (RFLP) analysis can be used for comparison of field isolates and
vaccine strains of fowlpox virus (6, 10). However, this procedure is not used in routine diagnosis.
Cloned genomic fragments of fowlpox virus can be used effectively as nucleic acid probes for diagnosis of
fowlpox. Viral DNA isolated from lesions can be detected by hybridisation either with radioactively or
nonradioactively labelled genomic probes. This method is especially useful for differentiation of fowlpox from
infectious laryngotracheitis when tracheal lesions are present (5).
Genomic DNA sequences of various sizes can be amplified by the polymerase chain reaction (PCR) using
specific primers (4, 8). This technique is useful when there is only an extremely small amount of viral DNA in
the sample.
Although both cell-mediated immunity (CMI) and humoral immunity play an important role in poxvirus infections,
routine use of the CMI test is not convenient. Therefore, serological tests, such as virus neutralisation (VN), agar
gel immunodiffusion (AGID), passive haemagglutination and fluorescent antibody tests as well as the enzyme-
linked immunosorbent assay (ELISA), are used to measure specific humoral antibody responses. Evidence of
successful immunisation with vaccine can be determined by examining a flock 7–10 days after vaccination for
‘takes’. A take consists of a swelling of the skin or a scab at the site where the vaccine was applied and its
presence is evidence of successful immunisation.
After virus/serum interaction, the residual virus activity may be assayed in embryonating chicken eggs or in
cell cultures (9). This technically demanding test may not be convenient for routine diagnosis. Only some
selected strains of the virus have plaque-forming ability in chicken embryo cells. Neutralising antibodies
develop within 1–2 weeks of infection.

Precipitating antibodies can be detected by reacting test sera against viral antigens. The antigen can be
derived by sonication and homogenisation of infected skin or CAM lesions as well as by treatment of
infected cell cultures as described in Section B.2.f below. The lysed suspension is centrifuged and the
supernatant is used as antigen. Gel-diffusion medium is prepared with 1% agar, 8% sodium chloride and
0.01% thiomersol. The viral antigen is placed in the central well and the test sera are placed in the peripheral
wells. It is important to include a positive and negative control serum. The plates are incubated at room
temperature. Precipitation lines develop in 24–48 hours after incubation of the antigen with antibody to
homologous or closely related strains. The test is less sensitive than the ELISA (2) or the passive
haemagglutination test (23).
c) Passive haemagglutination
Tanned sheep or horse red blood cells are sensitised with a partially purified fowlpox viral antigen (20). The
antigen is prepared from infected CAMs or cells as described in Section B.2.f below. Passive
haemagglutination is more sensitive than AGID. The test will give cross-reactions among avian pox viruses.
d) Fluorescent antibody tests

Direct or indirect immunofluorescence tests will reveal specific intracytoplasmic fluorescence in infected
cells. The latter test is commonly used and involves two steps: the antibody against fowlpox virus is reacted
with the antigen in the infected cells, followed by a secondary fluorescein-isothiocyanate-labelled antibody
against chicken gamma globulin (e.g. goat anti-chicken). Such labelled antibodies are available
commercially. In this regard, formalin-fixed tissue sections can be used effectively for fluorescent antibody
tests.
e) Immunoperoxidase
Specific staining of cytoplasmic inclusions is achieved when horseradish-peroxidase-conjugated specific
polyclonal antibody against fowlpoxvirus is reacted with the hydrated sections of fowlpox-infected fixed
tissues (CAM and skin) or cell culture. Similar results are obtained when either polyclonal or monoclonal
antibodies are used in an indirect test. An advantage of the technique is that the sections can be examined
with the light microscope and can be stored for an extended period without loss of colour (19).

f) Enzyme-linked immunosorbent assay

ELISAs have been developed to detect humoral antibodies to fowlpox virus. They are capable of detecting
antibody 7–10 days after infection (2), but commercial kits for this test are not available.
Fowlpox virus antigens are prepared either from infected QT-35 cell monolayers or CAM lesions. Infected
QT cells are pelleted (700 g for 10 minutes at 4°C), washed with isotonic buffer (10 mM Tris, pH 8.0,
150 mM NaCl, 5 mM ethylene diamine tetra-acetic acid [EDTA]) followed by lysis in hypotonic buffer (10 mM
Tris, pH 8.0, 10 mM KCl, 5 mM EDTA) containing 0.1% Triton X-100 and 0.025% beta-mercaptoethanol.
Nuclei and cellular debris are removed by low-speed centrifugation (500 g for 5 minutes at 4°C) and the
resulting supernatant is used as a source of fowlpox virus antigens for ELISA or immunoblotting. To isolate
viral antigen from CAM lesions, initial grinding of the lesions with subsequent detergent treatment as
described earlier would be required. Virus propagated in chicken embryo fibroblasts and chicken embryo
dermis cells has also been used for antigen. The antigen preparation is as described for QT cells.
Wells of microtitre plates are coated with 1 µg of soluble fowlpox virus antigen in 100 µl of coating buffer
(15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and incubated overnight at 4°C (2, 19). Each well is then rinsed
once with wash solution (0.29 M NaCl, 0.05% Tween 20) and then blocked with phosphate buffered saline
(PBS, pH 7.4) containing 3% bovine serum albumin (BSA) for 1 hour at 37°C. After one wash, serial
dilutions of the test sera in PBS containing 1% BSA are added to the wells. After rocking for 2 hours at 37°C,
the wells are washed three times prior to the addition of 100 µl/well horseradish-peroxidase-conjugated goat
anti-chicken IgG (H+L) antibodies 3 at a recommended dilution in PBS. After 2 hours’ incubation at 37°C and
three subsequent washes, 100 µl of the peroxidase substrate TMB3 is added to each well. Reactions are
terminated by the addition of 1 M phosphoric acid and absorbance at 450 nm is recorded using an ELISA
plate reader 4.
g) Immunoblotting
Antigenic variations r between strains of fowlpox virus can be evaluated by means of immunoblotting or
Western Blotting. In this method, viral antigens separated by SDS-PAGE (sodium dodecyl sulphate-
polyacrylamide gel electrophoresis) are reacted either with polyclonal or monoclonal antibodies against
fowlpox virus (6, 12, 14). This method is not convenient for routine diagnosis.
Early studies indicated the feasibility of protecting chickens from fowlpox by the use of pigeon pox or fowlpox
viruses (21, 23). Vaccination is indicated in areas where fowlpox is endemic or on premises where infection has
been diagnosed previously. Live fowl and pigeon pox virus vaccines, and also fowlpox vectored vaccines that
protect against pox, are available commercially. These vaccines are derived from chicken embryos or avian cell
cultures.
Passively acquired immunity should be taken into consideration during vaccination of progeny from flocks that
have either had a recent natural infection or been recently vaccinated. As passive immunity (for 2–3 weeks) may
interfere with vaccine virus multiplication, such progeny should be vaccinated only after the decline of passively
acquired antibody. Fowlpox vaccine is applied by a wing web stab method.
1. Seed management

A master seed virus (MSV) must be established and used according to a seed-lot system. A record must be
kept of its origin, passage history and characteristics. Viruses used may be either fowlpox or pigeon pox
viruses. The MSV must be propagated in suitable premises with materials that meet approved standards,
and must be tested for freedom from contamination as well as for identity and purity.
3 Kirkegaard and Perry Laboratories, Gaithersberg, Maryland, United States of America.

4 Dynatech, Chantilly, Virginia, United States of America.

The MSV may be propagated in specific pathogen free (SPF) chicken embryos, using the CAMs, or in avian
cell cultures, such as primary chicken embryo fibroblasts, chicken embryo kidney or chicken embryo dermis.
i) Purity
The MSV may be neutralised with a specific hyperimmune serum before testing for purity. Because of
difficulty in neutralising avian pox virus, it is acceptable to centrifuge the MSV at 1000 g for 20 minutes,
followed by filtration through a 0.2 µm filter. The neutralised or filtered MSV is then used in tests to
demonstrate freedom from extraneous agents. These tests should be done in embryonating eggs or
avian cell cultures, to demonstrate absence of extraneous virus replication, and in SPF chickens, to
demonstrate freedom from antibodies to extraneous agents.
ii) Safety
Vaccines should be prepared only from virus that is a stable attenuated strain or a naturally occurring
isolate of low virulence.
The vaccine must be shown to be safe by the recommended route of administration, which is wing web
stab, in all ages of susceptible birds. A suitable test is to take ten SPF chickens and inoculate each by
piercing the wing web with a needle dipped in the vaccine. The birds are observed for 7–10 days for
evidence of ‘takes’ and for the absence of adverse effects attributable to the vaccine. A ‘take’ consists
of swelling of the skin or a scab at the site where the vaccine was applied and is evidence of
successful vaccination. The safety test should be repeated after at least six serial passages of the virus
in SPF chickens to show that there has been no reversion to virulence.
iii) Efficacy
Data should be obtained using the highest passage level (fifth passage from the master seed) and the
lowest titre of virus to be used in the final product: 20 SPF chickens of the minimum age indicated for
vaccination should receive one dose of vaccine by the recommended method. The chickens, together
with 20 unvaccinated chickens of the same age and source, should be challenged 3 weeks later by
scarification with a virulent strain of fowlpox virus. The birds should be observed for 3 weeks. Ninety
per cent of the control birds should develop lesions due to the challenge virus and at least 90% of the
vaccinated birds should remain free from such lesions.
Vaccine is manufactured on a seed-lot system from the validated MSV. This must be done in approved premises
designed to avoid the risk of contamination. All media and cell cultures must be tested to ensure freedom from
contamination.
During the process of validation as a vaccine, the efficacy data must be compared to the virus content of the
vaccine. A suitable potency can thus be established. The vaccine should be filled into final containers to ensure
that each container has sufficient virus to achieve the specified potency.
4. Batch control
a) Sterility
Tests for sterility and freedom from contamination of biological materials may be found in the Chapter 1.1.9.
b) Safety
The safety test described in Section C.1.c.ii above, except the requirement for six passages in SPF
chickens, should be used on each batch of vaccine.
c) Potency
Virus content tests should be carried out using each of at least three containers. The dilutions should span
0–100% infection range, using five-fold dilution steps and at least seven replicates per dilution. Tests should

be done in parallel with a standard vaccine, if available. Each lot of vaccine should be titrated in the diluent
provided for its use. The virus titre should not normally be higher than 1/10 of the dose at which the vaccine
has been shown to be safe and must not be lower than the release titre determined in the test for efficacy. A
suitable potency for an attenuated live fowlpox vaccine is likely to be in the region of 105 EID50 (50% embryo
infective dose) per ml.
The efficacy test given in Section C.1.c.iii may be used to determine the duration of immunity (approximately
6–12 months) by testing at intervals after vaccination, using separate groups of birds for each test.
e) Stability
Evidence of stability must be presented to justify the shelf life. This should be based on virus titrations
carried out at intervals until 3 months beyond the requested shelf life on at least six batches of vaccine kept
under recommended storage conditions.
f) Preservatives
Preservatives are not used in live vaccines.
It is usually recommended not to vaccinate birds that are in lay. Avoid human contact with the live vaccine.
Standard fowlpox vaccine is not to be used in pigeons, though they can be vaccinated with pigeon pox
vaccine. In many countries, pigeon pox vaccine has been superseded by attenuated live fowlpox vaccine
designed for use in day-old chicks. These products have been safely used in pigeons in the absence of an
available pigeon pox vaccine.
a) Safety
The safety test described in Section C.1.c.ii above is used on each batch.
b) Potency
The potency test described in Section C.4.c above is used on each batch.
ACKNOWLEDGMENT
This work was supported by funds from Illinois Agricultural Experiment Station Regional Project.
REFERENCES
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Virol., 74, 3815–3831.
2. BUSCAGLIA C., BANKOWSKI R.A. & MIERS L. (1985). Cell-culture virus neutralization test and enzyme-linked
immunosorbent assay for evaluation of immunity in chickens against fowlpox. Avian Dis., 29, 672–680.
3. DOANE F.W. & ANDERSON N. (1987). Electron Microscopy in Diagnostic Virology: A Practical Guide and Atlas.
Cambridge University Press, Cambridge, UK.
4. FALLAVENA L.C., CANAL C.W., SALLE C.T., MORAES H.L., ROCHA S.L. & PEREIRA DA SILVA A.B. (2002). Presence
of avipoxvirus DNA in avian dermal squamous cell carcinoma. Avian Pathol., 31, 241–246.
5. FATUNMBI O.O., REED W.M., SCHWARTZ D.I. & TRIPATHY D.N. (1995). Dual infection of chickens with pox and
infectious laryngotracheitis (ILT) confirmed with specific pox and ILT DNA dot-blot hybridization assays.
Avian Dis., 39, 925–930.

6. GHILDYAL N., SCHNITZLEIN W.M. & TRIPATHY D.N. (1989). Genetic and antigenic differences between fowlpox
and quailpox viruses. Arch. Virol., 106, 85–92.
7. KIM T.J. & TRIPATHY D.N. (2001). Reticuloendotheliosis virus integration in the fowlpoxvirus genome: not a
recent event. Avian Dis., 45, 663–669.
8. LEE L.H. & LEE K.H. (1997). Application of the polymerase chain reaction for the diagnosis of fowlpoxvirus
infection. J. Virol. Methods, 63, 113–119.
9. MORITA C. (1973). Studies on fowlpox viruses. II. Plaque neutralization test. Avian Dis., 17, 93–98.
10. SCHNITZLEIN W.M., GHILDYAL N. & TRIPATHY D.N. (1988). Genomic and antigenic characterisation of
avipoxviruses. Virus Res., 10, 65–76.
11 SINGH P., KIM T.-J. & TRIPATHY D.N. (2000). Re-emerging fowlpox: evaluation of isolates from vaccinated
flocks. Avian Pathol., 29, 449–455
12 SINGH P., KIM T.-J. & TRIPATHY D.N. (2003). Identification and characterisation of fowlpox virus strains using
monoclonal antibodies. J. Vet. Diagn. Invest., 15, 50–54.
16.13 SINGH P., SCHNITZLEIN W.M. & TRIPATHY D.N. (2003). Reticuloendotheliosis virus sequences within the
genomes of field strains of fowlpox virus display variability. J. Virol., 77, 5855–5862.
14 SINGH P. & TRIPATHY D.N. (2000). Characterization of monoclonal antibodies against fowlpoxvirus. Avian Dis.,
44, 365–371.
15 SRINIVASAN V., SCHNITZLEIN W.M. & TRIPATHY D.N. (2001). Fowlpox virus encodes a novel DNA repair
enzyme, CPD-photolyase, that restores infectivity of UV light-damaged virus. J. Virol., 75,1681–1688.
16 SRINIVASAN V. & TRIPATHY D.N. (2005). The DNA repair enzyme, CPD-photolyase restores the infectivity of
UV-damaged fowlpox virus isolated from infected scabs of chickens. Vet. Microbiol., 108, 215–223.
17 TRIPATHY D.N. (1993). Avipoxviruses. In: Virus Infections of Vertebrates Virus Infections of Birds, Vol. 4,
McFerran J.B. & McNulty M.S., eds. Elsevier Science Publishers, Amsterdam, the Netherlands, 5–15.
18 TRIPATHY D.N. & HANSON, L.E. (1976). A smear technique for staining elementary bodies of fowlpox. Avian
Dis., 20, 609–610
19 TRIPATHY D.N., HANSON L.E. & KILLINGER A.H. (1973). Immunoperoxidase technique for detection of fowlpox
antigen. Avian Dis., 17, 274–278.
20 TRIPATHY D.N., HANSON L.E. & MYERS W.L. (1970). Passive hemagglutination test with fowlpox virus. Avian
Dis., 14, 29–38.
21 TRIPATHY D.N. & REED W.M. (2003) Pox. In: Diseases of Poultry, 11th Edition, Saif, Y.M., Barnes, H.J.
Glisson, J.R. Fadly, A.M., McDougald L.R. & Swayne, D.E., eds. Iowa State University Press, Ames, Iowa,
USA, 253-269.
22. TRIPATHY D.N. & REED W.M. (1998). Pox. In: A Laboratory Manual for the Isolation and Identification of Avian
Pathogens, Fourth Edition, Swayne D.E., Glisson J.R., Jackwood M W., Pearson J.E., & Reed W.M., eds.
American Association of Avian Pathologists, University of Pennsylvania, New Bolton Center, Kennett
Square, PA 19348-1692, USA, 137–140.
23. W INTERFIELD R.W. & HITCHNER S.B. (1965). The response of chickens to vaccination with different
concentrations of pigeon pox and fowlpox viruses. Avian Dis., 9, 237–241.
*
* *

CHAPTER 2.3.11.
FOWL TYPHOID AND PULLORUM DISEASE
SUMMARY
Definition of the disease: Pullorum disease of chickens is caused by Salmonella enterica

subspecies enterica serovar Gallinarum biovar Pullorum (Salmonella Pullorum) 1. At this time the
serovar is referred to as Gallinarum in some parts of the world and Pullorum in others; in this
chapter the serovar will be referred to as Gallinarum or Pullorum according to the biovar under
discussion. In its acute form, Pullorum disease is almost exclusively a septicaemic disease of
young chickens. However, the organism may also be associated with disease in turkey poults and
may be carried subclinically or lead to reduced egg production and hatchability plus a range of
atypical signs in older birds. Ovarian transmission is a major route by which the organism can
spread. Game birds and ‘backyard’ poultry flocks may act as reservoirs of infection, and wild birds
may act as vectors for the organism and as such are important in the epidemiology of the disease.
Fowl typhoid in chickens and turkeys is caused by S. Gallinarum biovar Gallinarum and is more
often observed in the later growing period and in mature stock. Disease is often characterised by
rapid spread with high morbidity and acute or subacute mortality. Red mites may be involved in the
transmission of disease and persistence in poultry houses
Salmonellosis caused by Salmonella bongori or other subspecies of Salmonella enterica is

covered in chapter 2.9.9 of this Terrestrial Manual.
Description of disease: Clinical signs in chicks and poults include anorexia, diarrhoea,
dehydration, weakness and death. In mature birds Pullorum disease is less severe but decreased
egg production, poor hatchability and some increased mortality may occur. Fowl typhoid is a more
acute septicaemic condition which mainly affects mature birds and may be particularly severe in
commercial laying flocks.
Identification of the agent: Samples should not be taken from birds or eggs that have recently
been treated with antimicrobial drugs. Swabs or aseptically collected samples from infected
tissues, or intestinal and cloacal contents should be used for diagnostic testing. Other materials
that may be sampled include eggs, embryos, faecal droppings and hatcher debris, especially fluff,
dust and broken eggshells and chick box linings. Samples of tissues such as caecal tonsils and
spleen from infected birds are preferable to faecal and environmental samples. Tissue samples
should be inoculated into nonselective and selective enrichment broth and into selective agar
medium, such as brilliant green agar, as soon as possible after collection. In case of delay,
samples should be stored at 4°C. Typical colonies can be identified by serological and biochemical
tests.
Serological tests: These are satisfactory for establishing the presence and estimating the
prevalence of infection within a flock. The test used in the field is the rapid whole blood plate
agglutination test. This test is unreliable in turkeys and ducks as many uninfected birds may give
positive reactions. In the laboratory a serum agglutination test is used, either as a rapid plate test
or as a tube test. These can be applied as macro- or microagglutination tests though the latter may
be more likely to give false-positive results with turkey sera. Any positive reactors should be
confirmed as being infected by culture at post-mortem examination. Enzyme-linked immunosorbent
assays have been reported but no commercial test is available.
The use of vaccines to control S. Enteritidis infections in chickens may cause problems in the
interpretation of serological results.
1 See the note in Chapter 2.9.9 Salmonellosis for the principles followed concerning the nomenclature of Salmonella.

Chapter 2.3.11. — Fowl typhoid and Pullorum disease
Requirements for vaccines and diagnostic biologicals: Live and inactivated vaccines are
available for fowl typhoid in some countries. The most commonly used vaccine is a live vaccine
derived from the stable rough strain of S. Gallinarum known as ‘9R’.
A. INTRODUCTION
Fowl typhoid and pullorum disease, caused by Salmonella enterica subspecies enterica serovars Gallinarum
biovars Gallinarum and Pullorum, respectively, are widely distributed throughout the world but they have been
eradicated from commercial poultry in many developed countries of Western Europe, the United States of
America (USA), Canada, Australia and Japan. In the United States and the United Kingdom the serovar is
referred to as Pullorum (8); in this chapter the terms serovar Gallinarum or Pullorum will be used. However,
S. Gallinarum has recently recurred in some European countries (18). Salmonella Pullorum remains in wild and
game birds. Salmonella Gallinarum and S. Pullorum are host adapted to avian species and are considered to
pose a minimal zoonotic risk (19), although the genome is continually evolving, which could theoretically widen
the host range in future (12).
Salmonellosis caused by Salmonella bongori or other subspecies of Salmonella enterica is covered in chapter
2.9.9 of this Terrestrial Manual.
Clinical signs are typical of a septicaemic condition in poultry and include increased mortality and poor quality in
chicks hatched from infected eggs. Older birds show signs of anaemia, depression, laboured breathing and
diarrhoea causing adherence of faeces to the vent. The highest mortality occurs in birds of 2–3 weeks of age. In
older birds disease may be mild or inapparent. In breeding flocks reduced egg production and hatchability may
be the only signs, and trans-ovarian infection resulting in infection of the egg and hatched chicks or poults is one
of the most important transmission routes for the disease.
Post-mortem signs of pullorum disease in newly hatched chicks are those of peritonitis with generalised
congestion of tissues and an inflamed unabsorbed yolk sac. Longer standing infections commonly lead to
typhlitis with development of necrotic caecal casts and small necrotic foci in the liver, lungs and other viscera.
Small lesions in the liver and spleen of Pullorum-infected birds may show a ‘white spot’ appearance that is not
seen with Gallinarum; however, this lesion is not pathognomic. These Salmonella are very poor at colonisation
and survival in the gastrointestinal tract is often indicative of later stages of clinical disease. Adult birds may
develop misshapen or shrunken ovaries with follicles attached by pedunculated fibrous stalks. Variant strains of
S. Pullorum do not normally cause clinical disease or may result in mild, nonspecific signs but may lead to
seroconversion.
In fowl typhoid, as well as generalised signs of septicaemia, the liver is usually enlarged, dark and friable with a
distinctive coppery bronze sheen that may only develop after exposure to air. The bone marrow is also often dark
brown. Although clinical signs and post-mortem findings of pullorum disease and fowl typhoid may be highly
suggestive of the conditions, they are not sufficiently distinct from other causes of septicaemia to be
pathognomic. It is therefore necessary to confirm disease by isolation of the organisms. Serological tests can be
used to establish the presence of the disease in a flock.
In its acute form, pullorum disease is almost exclusively a disease of young chickens, and the agent can be
recovered from almost all organs, tissues and faeces. In older birds that have become carriers, S. Pullorum is
most commonly recovered from the ova and oviduct; and it is recovered only occasionally from other organs and
tissues, including the alimentary tract. In the acute phase of fowl typhoid the organism is also widely distributed,
but in carrier birds, the organism is found most often in the liver, spleen and reproductive tract, and occasionally
in the caeca.
• Culture
Salmonella Pullorum and S. Gallinarum belong to the Kauffmann–White scheme serogroup D, along with
S. Enteritidis, which is thought to be closely related. The organisms are Gram negative nonsporogenic rods 1.0–
2.5 µm in length and 0.3–1.5 µm in width. They are considered to be non-motile under normal conditions but
inducement of flagellar proteins and motility has been shown in some strains of S. Pullorum when grown in
special media (9).
For optimal recovery of the organisms, the birds being sampled should not have been treated with antimicrobial
drugs for approximately 2–3 weeks previously.

Samples may be obtained from live birds, preferably after identifying highly sero-positive birds. Fresh or freshly
chilled carcasses, egg materials, fresh faeces, or any contaminated materials from housing, incubators or
transport boxes may also be taken. Swabs may be taken from the cloaca of live birds. Samples from visibly
abnormal tissues are preferable to faecal and environmental samples. Aseptic samples can be taken from the
spleen, liver, gall-bladder, kidneys, lungs, heart, ova, testes, alimentary tract or joint lesions. The surface is
seared with a hot spatula and a sample is obtained by inserting a sterile cotton swab or sterile loop through the
heat-sterilised surface. The demonstration of infection in serological reactor birds that are apparently normal
may, in some cases, require the culture of large volumes of homogenised tissues as well as direct swabbing.
Tissue pools may be made from tissues collected from a number of birds.
When floor litter or faecal material is sampled, it should be remembered that S. Pullorum and S. Gallinarum are
more difficult to isolate from faecal and environmental samples than other salmonellae and it is always preferable
to culture sick or recently deceased birds . Environmental samples may include sock or boot swabs, floor faeces,
moist and dry litter and swabs from open drinkers. Red mites associated with poultry which are infected with
S. Gallinarum often contain the organism after feeding and can be cultured. These samples should be cultured
by direct inoculation of a selective enrichment broth such as selenite cysteine, followed by plating on selective
media such as brilliant green agar (17).
Both S. Pullorum and S. Gallinarum grow well on nonselective media, but selective and enrichment media have
been described that contain substances to inhibit the growth of extraneous organisms. Salmonella Pullorum may
grow slowly and produce very small colonies on selective media so incubation of plates for 48 hours is
recommended. The efficiency of recovery of Salmonella varies according to circumstances, and experience in
the use of a medium is an important but unquantifiable factor. Some complex media may have an inhibitory effect
on these organisms, so that it is advisable to use both nonselective and selective media for isolation from
tissues. Both solid media and broths can be employed. As the toxic properties of selective media may vary, it is
preferable to monitor these by comparing growth of control cultures on both types of medium. The inhibitory
media should grow at least 75% of the colonies of the corresponding non-inhibitory medium (3, 4, 7, 13).
All the media mentioned below are examples of commonly used media, but there are many others found to be
equally satisfactory.
Non-inhibitory media include nutrient agar and blood agar, on which colonies are seen to be smooth, translucent,
slightly raised, and about 1–2 mm in diameter. Salmonella Gallinarum grows more rapidly than S. Pullorum and
produces larger colonies with a distinctive smell resembling that of seminal fluid on most media. Broths include
buffered peptone water and nutrient and meat infusion broths or universal pre-enrichment broth.
• Selective media include:

MacConkey agar: the agar is inhibitory to non-enteric organisms, it differentiates lactose fermenters (pink
colonies) from nonlactose fermenters (colourless colonies). NaCl is omitted to limit the spread of Proteus
colonies. Salmonella colonies are smooth and colourless. Salmonella Pullorum produces smaller colonies
than other salmonellae. MacConkey is the agar of choice for direct plating from tissues.
Xylose lysine deoxycholate agar: the agar is inhibitory to non-enteric organisms. Salmonella Pullorum
grows sparsely as small red translucent colonies. S. Gallinarum colonies are small, dome-shaped, and may
have a central black spot due to H2S production, but this reaction may be delayed or variable.
Brilliant green agar (BGA): the agar is inhibitory to coliforms and most Proteus strains; useful for
distinguishing enteric organism colonies. Salmonellae form low, convex, pale red, translucent colonies of 1–
3 mm in diameter, similar to Citrobacter. Proteus forms pin-point colonies, Pseudomonas aeruginosa
appears as small red colonies, and lactose fermenters are green. Salmonella Pullorum produces smaller
more pale colonies than other salmonellae. BGA is the agar of choice following enrichment.
Brilliant green sulphapyridine agar: the agar is inhibitory to coliforms and Proteus strains. The
sulphapyridine is added to stabilise selectivity in the presence of egg materials. Salmonella Pullorum
produces small colonies.
Salmonella Pullorum and Gallinarum grow poorly and do not produce typical colonies on newer
chromogenic agars such as Rambach agar.
• Liquid enrichment and selective media include:

Selenite F broth: inhibitory to coliforms but not Proteus, improved by addition of brilliant green. Loss of
activity after 24 hours. Selenite cysteine broth is more stable. Although selenite broths are considered to be
preferable for isolation of S. Pullorum and S. Gallinarum from faeces, if there are difficulties with issues of
toxicity or shelf life in particular laboratories the other enrichment broths mentioned below may be used.
Most of these other broths are however designed to be used following a nonselective enrichment stage and

S. Gallinarum and S. Pullorum are readily overgrown by competitor organisms in nonselective faecal culture
resulting in false-negative tests. Direct selective enrichment is therefore recommended for faeces and
intestinal or environmental samples. Nonselective enrichment may give better results for tissues obtained
by aseptic post-mortem where there should be no competing organisms (16).
Tetrathionate/brilliant green broth: inhibitory to coliforms and Proteus, but may also inhibit some strains of
S. Pullorum/Gallinarum.
Rappaport–Vassiliadis soya (RVS) peptone broth: for selective enrichment following pre-enrichment, use
1 part inoculum to 100 parts medium. Salmonella Pullorum and Gallinarum are more likely to be overgrown
by other organisms during pre-enrichment of faeces or intestinal contents than salmonellae that are not
host-adapted so direct enrichment with RVS may also be attempted.
• Recovery of salmonellae
The methods for recovering S. Pullorum and S. Gallinarum vary according to the origin of the samples. Although
their isolation from cloacal swabs and faeces may be unrewarding, examination of tissues taken at post-mortem
is usually more successful. The methods are as follows:
Cloacal swabs and fresh faeces from live birds: swabs dipped in nutrient broth are suitable, small swabs being
used for young chickens. The swabs are streaked on selective media, and placed in enrichment broth. The
plates and the broth are incubated at 37°C. At this temperature, Proteus and Pseudomonas organisms tend to be
inhibited relative to Salmonella. Higher temperatures may be used with some broths, e.g. 41.5°C for Rappaport–
Vassiliadis (RV). Subcultures are made on to selective media after 24 and 48 hours.
Gall-bladder contents: swabs of gall-bladder contents are streaked on to nonselective and selective agars and
placed in inhibitory and non-inhibitory broths, followed by incubation at 37°C and subculture on to selective agar
after 24 and 48 hours.
Organs and tissues: swabs or segments of tissues are taken in an aseptic manner from individual tissues and
lesions and cultured on to nonselective and selective media, and into similar nonselective and selective broths.
These are incubated at 37°C and subcultured on to selective agar after 24 and 48 hours. Intestinal material in
selective broths may also be incubated at 40°C; S. Gallinarum grows well but there may be some inhibition of
S. Pullorum at this temperature.
Carrier birds: larger amounts of material may be required to identify the carrier birds. The ovary and oviduct are
the tissues of choice for S. Pullorum, and the liver and gall-bladder as well as ovary and oviduct should be tested
for S. Gallinarum. In practice it is usually best to pool samples from a variety of tissues including the spleen.
Tissues are homogenised in a small volume of broth and directly plated out. Approximately 10 ml of homogenate
is also added to 100 ml of nonselective enrichment broth (e.g. buffered peptone water) and selective enrichment
broth (e.g. selenite cysteine broth or brilliant green broth), and incubated at 37°C. These broths are subcultured
on to nonselective and selective agar after 24 hours.
Alimentary canal, including the caecal tonsils and intestinal contents: after grinding or homogenisation in a small
volume of broth, 10 ml of the homogenate is incubated in 100 ml of selective enrichment broth at 37°C. In
general better isolation is achieved with selenite cysteine broth.
Eggshells: the broken eggshells are placed in a tenfold volume of enrichment broth (e.g. selenite cysteine broth).
The broth is incubated at 37°C and subcultured on to selective agar after 24 and 48 hours.
Egg contents: Aseptically taken contents of fresh eggs are homogenised and mixed with 200 ml of buffered
peptone water or nutrient broth, incubated at 37°C, and subcultured on to nonselective and selective agar after
24 and 48 hours. Incubated eggs, whether infertile or containing small embryos, can be similarly treated.
Embryos: Homogenised viscera and swabs from the yolk sacs of well developed embryos may be streaked on to
nonselective and selective agar, one swab being placed in 10 ml of both nonselective and enrichment broth (e.g.
selenite cysteine broth or brilliant green broth). Incubation is carried out at 37°C, and subcultures are made on to
nonselective and selective agars after 24 and 48 hours.
Environmental samples: These include hatcher fluff, debris and macerated egg/chick waste samples and chick
box liners or floor faecal or litter samples; 25 g is mixed with 225 ml of enrichment broth (e.g. selenite cysteine
broth, brilliant green broth), incubated at 37°C, and subcultured on to selective agar after 24 and 48 hours.
Polymerase chain reaction (PCR) based tests may also be used, but have not been fully validated internationally
(15).

Typical S. Gallinarum colonies on nonselective media are round, translucent, glistening, domed, smooth, and 1–
2 mm in diameter after 24–48 hours’ incubation. Salmonella Pullorum colonies are slightly smaller and
translucent. On selective media their appearance varies with the medium, but suspect colonies can be
investigated serologically by testing for ‘O’9 somatic antigens, observing for motility and testing biochemically.
After incubation for 20–24 hours, the plates should be examined carefully for the presence of typical S. Pullorum
and S. Gallinarum colonies. If growth is slight after 24 hours’ incubation, the plates should be reincubated for a
further 24 hours and examined again. For biochemical and serological confirmation from each plate, five typical
or suspect colonies should be chosen for further examination. If there are fewer than five typical or suspect
colonies, all of them should be taken for further examination. Selected colonies should be streaked on to the
surface of nutrient agar, in a manner that allows the growth of separate colonies. For biochemical confirmation,
only pure cultures taken from nonselective media should be used. The following media should be streaked using
an inoculating loop: triple sugar iron (TSI) agar; lysine iron agar (or l-lysine decarboxylation medium); urea agar
according to Christensen; tryptone/tryptophan medium for indole reaction; glucose with an inverted Durham tube
for acid and gas production; dulcitol, maltose, ornithine decarboxylation medium and semisolid agar, for motility.
The reactions shown in Table 1 occur.
Identification kits are commercially available, for example Analytical Profile Index (API) system for
Enterobacteriaceae. However, care must be taken when using API because S. Pullorum may be misidentified as
Hafnia spp. Molecular tests using ribotyping techniques and PCR have been developed in research laboratories
(10), but are not generally available for confirmation of S. Gallinarum and S. Pullorum.
For serological confirmation as to serogroup, colonies from nonselective media (nutrient or blood agar) are used.
The first stage is elimination of autoagglutinable strains. For this, material taken from a single colony of pure
culture is transferred to a glass slide and mixed with a drop of sterile saline. The slide is rocked gently or the drop
stirred with a loop for 30–60 seconds and observed for agglutination against a dark background, preferably with
the aid of a magnifying glass or dissecting microscope. If the bacteria have clumped into more or less distinctive
units, the strain is considered to be autoagglutinable and must not be submitted to the following tests. If the
bacterial sample is recognised as non-autoagglutinable, it is tested with a polyvalent ‘O’ (A–G) antiserum. For
this purpose, the material from a single colony is dispersed in the drop of polyvalent ‘O’ antiserum on the glass
slide to obtain a homogenous and turbid suspension. After gently rocking for 30–60 seconds, the reaction is
observed against a dark background for agglutination. Alternatively the slide agglutination test may be carried out
with smaller volumes of suspension under a dissecting microscope. In this case a portion of the colony to be
checked is added to a loopful of saline on the microscope slide to produce a light suspension to check for
autoagglutination (‘rough strains’). If no agglutination takes place, one or two loops of antisera are added, the
drop stirred with a loop and observed for agglutination. Salmonella Pullorum and S. Gallinarum should
agglutinate with polyvalent ‘O’ antisera but not with polyvalent flagella (poly ‘H’ phase 1 and phase 2) antisera. If
the reaction is positive, the single colony is tested further in the same manner using group-specific sera for
S. Pullorum and S. Gallinarum serotypes (‘O’9 antiserum). After serogrouping, isolates may be sent to a
reference laboratory for serotyping.
Table 1. Biochemical investigation of Salmonella Pullorum and S. Gallinarum
Salmonella Pullorum Salmonella Gallinarum

TSI glucose (acid formation)
TSI glucose (gas formation) v –
TSI lactose – –
TSI saccharose – –
TSI hydrogen sulphide v v
Gas from glucose (medium with Durham tube) –
Urea hydrolysis – –
Lysine decarboxylation
Ornithine decarboxylation –
Maltose fermentation – or late
Dulcitol –
Motility – –
= 90% or more positive reaction within 1 or 2 days; – = No reaction (90% or more); v = Variable reactions.
It is also possible to confirm and differentiate S.Gallinarum by specific PCR (18).

• Test procedure for culture of visceral, faecal, intestinal and environmental samples for S. Pullorum
and S. Gallinarum
i) Where possible, begin laboratory procedures on the same day as samples are collected.
ii) Homogenise the material as much as possible by manual mixing, gentle macerating or stomaching
with a small volume of sterile saline if the material is dry.
iii) Stir the mixture with a small rectal swab or loop and streak thickly on to one-quarter of a brilliant green
agar plate. (Swabs from uncontaminated tissues sampled in an aseptic manner can also be streaked
on to blood agar.)
iv) From this deposit of material on the plate, streak the rest of the plate to obtain individual colonies.
v) Add 5–25 g of the homogenised sample to freshly made selenite cysteine broth (see note on liquid
enrichment and selective media above) to make a 1:10 sample to broth ratio. Shake or stir to disperse
the sample in the broth.
vi) Incubate the brilliant green agar plates and selenite cysteine broth at 37°C for 24 hours.
vii) Examine the plate after 24 hours’ culture. Carry out agglutination tests on up to five suspect colonies
with polyvalent ‘O’ (A–G) antisera and polyvalent H (phase 1 and phase 2) antisera. If agglutination is
unclear subculture suspect colonies on to nutrient agar or blood agar and repeat tests after 24 hours’
incubation of those media.
viii) If poly ‘O’ is positive then check with ‘O’9 antiserum. If ‘O’9 is positive and poly ‘H’ is negative, this is
indicative of the possible presence of S. Pullorum or S. Gallinarum.
ix) If there are no positive colonies on the brilliant green agar plate, streak out a 10 µl loop of incubated
selenite cysteine broth onto brilliant green agar as in step iv above.
xi) Incubate the brilliant green agar plates at 37°C for 24 hours and re-incubate the previous (negative)
brilliant green agar plates and the selenite cysteine broths for a further 24 hours.
xi) Repeat examination of plates as in step vii above.
xii) If plates are still negative, replate from selenite cysteine broth and incubate brilliant green agar plate,
that was inoculated in step ix, for a further 24 hours and examine as in step vii above.
xiii) Confirm S. Pullorum and S. Gallinarum using biochemical tests as shown in Table 1. Isolates can be
sent to a Salmonella reference laboratory for confirmation of serotype and for phage typing of
S. Pullorum.
• Molecular epidemiology
Standard molecular ‘fingerprinting’ techniques used for Salmonella, such as plasmid profile analysis, pulsed
field gel electrophoresis or ribotyping can be used for investigating outbreaks of S. Pullorum or
S. Gallinarum. It is often necessary to use combinations of such methods to obtain maximum
discrimination.
Serological tests are best applied as a flock test as results for individual birds will vary according to the stage of
infection. It is therefore necessary to take sufficient individual samples to determine infection in the flock. The
number of samples will depend on the expected prevalence and level of confidence desired (see Chapter 1.1.1
Collection and shipment of diagnostic specimens). If the test is to be used for detecting individual infected birds
for culling, it should be repeated at least twice and preferably until the whole flock has given at least two negative
tests.
The tests that are most readily applied include rapid whole blood agglutination, rapid serum agglutination (RST),
tube agglutination and micro-agglutination (21). Other invasive Salmonella such as S. Enteritidis and
S. Typhimurium may give false-positive results in serological tests for S. Pullorum.
Both S. Pullorum and S. Gallinarum possess ‘O’ antigens 9 and 12 and may also posess O antigen 1. However,
in the case of S. Pullorum, there is a variation in the ratio of 121, 122 and 123; the standard strain contains more
123 than 122, while the reverse is true of the variant form. Intermediate forms also exist. (There appears to be no
such form variation in the case of S. Gallinarum.) As this variation occurs, it is necessary to use a polyvalent
antigen in immunodiagnostic tests. The same antigen is used to detect both S. Pullorum and S. Gallinarum, but
detection of the latter may be relatively poor (17).
a) Rapid whole blood agglutination test

The rapid whole blood agglutination test can be used under field conditions for detecting both S. Pullorum
and S. Gallinarum, and the reactors can be identified immediately. However, it is not reliable in turkeys as
the test results in a significant proportion of false-positive results. Chickens can be tested at any age,

although some authorities specify a minimum age of 4 months (21, 22) and positive results from chicks less
than 4 weeks of age may be due to maternal antibodies.
• Preparation of stained antigen for the rapid whole blood or rapid serum agglutination test
Incubate one standard form strain of S. Pullorum (antigenic structure 9, 121, 123) and one variant form
(antigenic structure 9, 121, 122) at 37°C and harvest separately until final mixing for the complete antigen.
Sow strains on to separate agar slopes, incubate at 37°C for 24 hours, emulsify growth with sterile normal
saline and spread an inoculum over an agar plate to produce easily selected discrete colonies. For this the
plates are incubated for 48 hours, a number of colonies are marked out and each is tested for agglutination
on a slide with 1/500 acriflavine in saline. Smooth-phase colonies do not produce agglutination. Pick off
typical colonies that do not produce any agglutination, seed on to agar slopes, and incubate for 24 hours.
Emulsify the growth in saline and evenly distribute 2 ml over the surface of the medium (200 ml) in a Roux
or similar flask. Incubate the flasks for 60 hours.
For harvesting the bacterial growth, flood the surface of each flask with enough sterile buffered formol
saline, pH 6.5 (8.5 g/litre sodium chloride, 10 ml/litre neutral formalin, 4 ml/litre 0.5 M sodium phosphate:
made up to 1 litre with distilled water, pH adjusted to 6.5 using 1 M orthophosphoric acid or 1 M sodium
hydroxide), to give dense cell suspensions (about 10 ml per flask). Add 12–15 sterile glass beads of 3–
5 mm diameter and rock the flasks until all the growth is in even suspension; leave in a vertical position for
at least 15 minutes. Check the morphology and purity of the suspensions by preparing and examining
Gram-stained films. Bulk the suspension from each flask containing the same strains. To each 100 ml of
suspension, add 200 ml of absolute alcohol. Shake the mixture and allow to stand for 36 hours or until
precipitation is complete. Check the agglutinability of the standard and variant precipitate by first
centrifuging a sample to separate the alcohol, which is removed, dilute with normal saline and test with a
known positive and negative serum. If satisfactory, remove the clear supernatant alcohol (centrifugation at
2000 g for 10 minutes may be helpful in precipitation), and add sufficient phosphate buffered saline (PBS)
containing 10% (v/v) glycerol to standardise the density to 75 × No. 1 Wellcome opacity tube (or 50 × tube
No. 1.0 on the McFarland scale). Add equal volumes of standard and variant strains, and add 1% (v/v) of
3% (w/v) alcoholic crystal violet solution to the final mixture, and allow to stand for 48 hours at room
temperature. Store in a tightly closed container at 0–4°C for up to 6 months. To assess safety, carry out a
culture test on blood agar for nonviability of the unwashed antigen before standardisation. Each bottle of
antigen must be tested after alcoholic precipitation and before standardisation against standard titre
antisera for S. Pullorum and S. Gallinarum, and against a negative serum. If possible, also test with known
positive and negative serum and blood from positive and negative chickens.
Stained antigen products for the whole blood plate agglutination test are available commercially, and
although there seems to be some slight differences in their sensitivity (5), it is unlikely that poultry flocks
infected with the different variants of S. Pullorum would be missed.
• Test procedure
i) Use a clean white tile marked into squares of about 3 × 3 cm. If a tile with 3 × 4 squares is used, up to
12 blood samples can be tested at the same time.
ii) Place 1 drop (about 0.02 ml) of crystal-violet-stained antigen in the centre of each square.
iii) Obtain a sample of fresh whole blood. This is conveniently done by making a stab of a wing vein using
a needle with a triangular point.
iv) Place an equal size drop of fresh whole blood next to a drop of antigen.
v) Mix the drops of antigen and blood using a fine glass rod, which is wiped clean between samples.
vi) Use a gentle rocking motion to keep the drops agitated for up to 2 minutes. Several tests may be
carried out simultaneously on the same tile, but the drops should not be allowed to dry out during this
time. In very warm conditions, larger drops may be required to avoid drying out.
vii) A positive reaction is indicated by easily visible clumping of the antigen within 2 minutes.
viii) A negative reaction is indicated by absence of clumping of the antigen within 2 minutes.
ix) Include known positive and negative control sera on each testing occasion, using them in the same
way as the blood.
x) On completion of a set of tests, the tile is washed and dried, ready for further use.
In the absence of positive reactions, any doubtful reactions can only be interpreted in the light of the
previous Salmonella testing history of the flock. Where there are positive reactors, any doubtful reactor
should be regarded as positive. Also, recently infected birds may not show a typical positive reaction until
they are retested after 3–4 weeks.

b) Rapid serum agglutination test

The RST is performed in the same manner, except that serum is substituted for whole blood. For export test
purposes an initial screening of sera by RST followed by confirmation of positives by the tube agglutination
test is the optimal approach. Ideally serum samples tested by any method should be tested within 72 hours
of collection as nonspecific reactions may increase in older samples. Fresh samples can be frozen for later
testing if a delay is unavoidable.
c) Tube agglutination test

Fresh serum from chickens, turkeys or other birds is used at an initial dilution of 1/25, obtained by mixing
0.04 ml of serum with 1.0 ml of antigen 2. Positive and negative control sera are included in each test. The
antigen is prepared from unstained S. Pullorum or S. Gallinarum cultures diluted to a concentration of No. 1
on the McFarland scale (as described above). The mixture is incubated at 37 or 50°C for 18–24 hours
before reading. A positive reaction consists of a granular white deposit with a clear supernatant fluid; a
negative reaction shows uniform turbidity. Samples positive at a dilution of 1/25 are retested at a higher
range of dilutions and a titre of 1/50 is usually considered to be positive, although this figure seems to vary
in the literature. In many cases a single dilution of 1/50 is used but this may fail to detect some flock
infections if only small numbers of samples are taken.
d) Micro-agglutination test
This resembles the tube agglutination test, but requires much smaller volumes of reagents. The test is
performed in microtest plates. Sera are first diluted by adding 10 µl of serum to 90 µl of normal saline, and
then adding 100 µl of previously standardised stained microtest antigen to give a final dilution of 1/20. By
titrating the serum in doubling dilutions and adding an equal volume of standardised stained antigen, an
end-point (titre) can be obtained. The plates are sealed and incubated at 37°C for 18–24 or 48 hours. A
positive reaction consists of a fine diffuse precipitation, whereas a negative reaction shows a button-like
precipitate. Titres of 1/40 are usually considered to be positive but this test is more liable to produce false-
positive results with turkey sera.
Other serological tests include micro-antiglobulin (Coombs), immunodiffusion, haemagglutination and enzyme-
linked immunosorbent assay (ELISA).
ELISA techniques have been described for detecting antibodies to S. Pullorum and S. Gallinarum (14). The
indirect ELISA using lipopolysaccharide antigen is likely to be the most sensitive and specific serological flock
test for Salmonella, including S. Gallinarum and S. Pullorum. It is relatively easy to perform with serum or yolk,
and can be used for quantifying the titre of antibody (1, 2, 22). No commercial ELISA kits for S. Pullorum and
S. Gallinarum are currently available.

Although both live and inactivated vaccines have been prepared for use against S. Gallinarum, the vaccine most
widely used is made from the rough 9R strain (6). It has only been employed in chickens. The number of viable
organisms per dose is important; these organisms can survive in vaccinated birds for many months and may be
transmitted through the egg (and perhaps from bird to bird). Vaccination may reduce flock losses, but will not
prevent infection with field strains. In addition, vaccination with 9R may sometimes precipitate high mortality in
infected birds (20), and may stimulate the production of transient antibodies. It is usual to vaccinate at 8 weeks
and again at 16 weeks of age. Antimicrobials should be avoided before and after vaccination.
Currently available vaccines, however, have only a minor role to play in the control of fowl typhoid as they offer
short-lived protection against clinical disease and limited or variable protection against infection. Autogenous
vaccines can also be used to control clinical disease. Control can best be achieved by biosecurity, hygiene, good
management, monitoring and removal of infected flocks. Commercially available 9R vaccines are commonly
used for reduction of S. Enteritidis in laying flocks (11).
1. Seed management

Live fowl typhoid vaccine is a suspension of suitably attenuated living organisms of a rough strain of
S. Gallinarum, e.g. 9R. The organisms in the vaccine give the biochemical reactions characteristic of
S. Gallinarum. Colonies of a 24-hour culture prepared from the vaccine on nutrient agar plates are rough
2 For preparation of small volumes of somatic antigens see Chapter 2.9.9 Salmonellosis.

when examined by the acriflavine slide test. The culture should not contain the somatic antigens
characteristic of the smooth forms of S. Gallinarum.
Salmonella Gallinarum is grown on or in a suitable medium, such as nutrient agar or broth, for 24 hours at
37°C.
There is no satisfactory method of assessing the protection afforded by the vaccine in the field. However,
experience has shown that the vaccine can provide some benefit in some situations where control cannot
be achieved by hygiene and management alone. The potency test described below may be used to provide
evidence of efficacy.
The vaccine may be prepared by inoculation of a suitable medium, such as nutrient broth, with a fresh culture of
S. Gallinarum (9R) and incubation at 37°C for 24 hours, with or without aeration. The organisms are harvested by
sedimentation or centrifugation.
Alternatively the organisms may be grown on and harvested from a solid medium, such as nutrient agar. In either
case, the suspension is diluted in PBS solution, pH 7.0, and may be freeze-dried. The dose used per bird is
between 5 × 106 and 5 × 107 organisms.
The culture used for inoculation of the production cultures and the harvested cells are examined microscopically
using Gram staining to check for purity.
4. Batch control
a) Sterility
b) Safety
Six healthy, susceptible (preferably specific pathogen free [SPF]) chickens, 8–16 weeks of age, are each
injected subcutaneously with ten doses of vaccine, and are observed for at least 7 days; no local or
systemic reaction should develop.
c) Potency
Fifteen healthy chickens, 8–16 weeks of age, of the Light Sussex or Rhode Island Red breeds, or crosses
of these, and taken from a stock that is free from S. Pullorum infection, are each injected subcutaneously
with a quantity of vaccine corresponding to one field dose, i.e. 5 × 107 viable organisms. After an interval of
21–28 days, the vaccinated chickens and an equal number of similar unvaccinated chickens are deprived of
food for approximately 18 hours. The chickens are then challenged by oral administration of 1 ml of a broth
suspension containing 5 × 107 organisms of a virulent strain of S. Gallinarum mixed with 300 mg of a
powder consisting of chalk (40%), light kaolin (43%) and magnesium trisilicate (17%). All the chickens are
observed for 14–21 days. The vaccine passes the test if at the end of this period the number of surviving
vaccinated chickens that show no macroscopic lesions of fowl typhoid at post-mortem exceeds by eight or
more the number of similarly defined control chickens.
The vaccine should provide protection throughout the laying period, and this can be measured by potency
(efficacy) tests at stages during lay. A booster dose during lay may be required, but should not be used
during lay in flocks providing eggs for human consumption.
e) Stability
The shelf life of the vaccine can be measured by conducting potency tests at periods after manufacture.
These should be done on at least six samples. Potency should remain satisfactory for at least 1 year.

f) Preservatives
No preservatives are used.
The organism is not known to be pathogenic to humans, and there are no special risks associated with the
manufacture of either the vaccine or the antigen. However, the vaccine may establish a persistent infection
in carrier birds and can precipitate disease in already infected chickens.
a) Safety
The safety test described in Section C.4.b should be used on a representative sample from each batch of
final product.
b) Potency
The potency test described in Section C.4.c should be used on a representative sample from each batch of
final product.
REFERENCES
1. BARROW P.A. (1992). ELISAs and the serological analysis of salmonella in poultry: a review. Epidemiol.
Infect., 109, 361–369.
2. BARROW P.A. (1994). Serological diagnosis of Salmonella serotype enteritidis infections in poultry by ELISA
and other tests. Int. J. Food Microbiol., 21, 55–68.
3. ELLIS E.M., W ILLIAMS J.E., MALLINSON E.T., SNOEYENBOS G.H. & MARTIN W.J. (1976). Culture Methods for the
Detection of Animal Salmonellosis and Arizonosis. Iowa State University Press, Ames, Iowa, USA.
4. FRICKER C.R. (1987). The isolation of salmonellas and campylobacters. J. Appl. Bacteriol., 63, 99–116.
5. GAST R.K. (1997). Detecting infections of chickens with recent Salmonella Pullorum isolates using standard
serological methods. Poult. Sci., 76, 17–23.
6. HARBOURNE J.F., W ILLIAMS B.M., PARKER W.H. & FINCHAM I.H. (1963). The prevention of fowl typhoid in the
field using a freeze-dried 9R vaccine. Vet. Rec., 75, 858–861.
7. HARVEY R.W.S. & PRICE T.H. (1975). Isolation of Salmonellas. Public Health Laboratory Service, London,
UK.
8. HITCHNER S.B. (2004). History of biological control of poultry diseases in the U.S.A. Avian Dis., 48, 1–8.
9. HOLT P.S. & CHAUBAL L.H. (1997). Detection of motility and putative synthesis of flagellar proteins in
Salmonella pullorum cultures. J. Clin. Microbiol., 35, 1016–1020.
10. ITOH Y., HIROSE K., MIYAKE M., KHAN A.Q., HASHIMOTO Y. & EZAKI T. (1997). Amplification of rfb and flic gene
by polymerase chain reaction for identification and detection of Salmonella serovar enteritidis, dublin and
gallinarum-pullorum. Microbiol. Immunol., 41, 791–794.
11. LEE, Y.J., MO I.P. & KANG M.S. (2005). Safety and efficacy of Salmonella gallinarum 9R vaccine in young
laying chickens. Avian Pathol., 34, 362–366.
12. LIU G.-R., RAHN, A., LIU W.-Q., SANDERSON K.E., JOHNSTON R.N. & LIU S.-L. (2002). The evolving genome of
Salmonella enterica serovar Pullorum. J. Bacteriol., 184, 2626–2633.
13. MALLINSON E.T. & SNOEYENBOS G.H. (1989). Salmonellosis. In: Isolation and Identification of Avian
Pathogens, Third Edition, Purchase H.G. et al., eds. American Association of Avian Pathologists, Kendall
Hunt Publishing, Iowa, USA, 3–11.

14. OLIVEIRA G.H. DE, BERCHIERI JUNIOR A., MONTASSIER H.J. & FERNANDES A.C. (2004). Assessment of serological
response of chickens to Salmonella Gallinarum and Salmonella Pullorum by Elisa. Rev. Bras. Cienc. Avic.,
6, 111–115.
15. OLIVEIRA S.D., RODENBUSCH C.R., CE M.C., ROCHA S.L.S. & CANAL, C.W. (2003). Evaluation of selective and
non-selective enrichment PCR procedures for Salmonella detection. Lett. Appl. Microbiol., 36, 217–221.
16. PARMAR D. & DAVIES R.H. (2007). Fowl typhoid in a small backyard laying flock. Vet. Rec., 160, 348.
17. PROUX K., HUMBERT F., JOUY E., HOUDAYER C., LALANDE F., OGER A. & SALVAT G. (2002). Improvements
required for the detection of Salmonella Pullorum and Gallinarum. Can. J. Vet. Res., 66, 151–157.
18. SHAH D.H., PARK J.-H., CHO M.-R., KIM M.-C. & CHAE J.-S. (2005). Allele-specific PCR method based on rfbS
sequence for distinguishing Salmonella Gallinarum from Salmonella pullorum: serotype-specific rfbS
sequence polymorphism. J. Microbiol. Methods, 60, 169–177.
19. SHIVAPRASAD H.L. (2000). Fowl typhoid and pullorum disease. Rev. sci. tech. Off. int. Epiz. 19, 405–424.
20. SILVA E.N., SNOEYENBOS G.H., W EINACK O.M. & SMYSER C.F. (1981). Studies on the use of 9R strain
Salmonella Gallinarum as a vaccine in chickens. Avian Dis., 25, 38–52.
21. UNITED STATES DEPARTMENT OF AGRICULTURE (1996). Auxiliary Provisions on National Poultry Improvement
Plan. Code of Federal Regulations, Title 9, Part 147, 717–727.
22. W RAY C. & W RAY A. (EDS) (2000). Salmonella in Domestic Animals. CAB International, Wallingford, Oxon,
UK, 407–427.
*
* *

CHAPTER 2.3.12.
INFECTIOUS BURSAL DISEASE

(Gumboro disease)
SUMMARY
Infectious bursal disease (IBD) is caused by a virus that is a member of the genus Avibirnavirus of
the family Birnaviridae. Although turkeys, ducks, guinea fowl and ostriches may be infected, clinical
disease occurs solely in chickens. Only young birds are clinically affected. Severe acute disease of
3–6-week-old birds is associated with high mortality, but a less acute or subclinical disease is
common in 0–3-week-old birds. This can cause secondary problems due to the effect of the virus
on the bursa of Fabricius. IBD virus (IBDV) causes lymphoid depletion of the bursa, and if this
occurs in the first 2 weeks of life, significant depression of the humoral antibody response may
result. Two serotypes of IBDV are recognised. These are designated serotypes 1 and 2. Both
serotypes can be differentiated by cross-neutralisation assays. Clinical disease has been
associated with only serotype 1 and all commercial vaccines are prepared against this serotype.
Antigenic variants of IBD serotype 1 have been described and these may require special vaccines
for maximum protection. Very virulent strains of classical serotype 1 are now common and are
causing serious disease in many countries.
Clinical disease due to infection with the IBDV, also known as Gumboro disease, can usually be
diagnosed by a combination of characteristic signs and post-mortem lesions. Laboratory
confirmation of disease, or detection of subclinical infection, can be carried out by demonstration of
a humoral immune response in unvaccinated chickens or by detecting the presence of viral antigen
or viral genome in tissues. In the absence of such tests, histological examination of bursae may be
helpful.
Identification of the agent: Isolation of IBDV is not usually carried out as a routine diagnostic
procedure. Specific antibody-negative chickens may be used for this purpose, as may cell cultures
or embryonating eggs from specific antibody-negative sources. However, some difficulty may be
experienced if using the latter two systems as the virus does not readily adapt to them. If
successful, the identity of the virus can be confirmed by the virus neutralisation (VN) test.
The agar gel immunodiffusion (AGID) test can be used to detect viral antigen in the bursa of
Fabricius. A portion of the bursa is removed, homogenised, and used as antigen in a test against
known positive antiserum. This is particularly useful in the early stages of the infection, before the
development of an antibody response. An immunofluorescence test using IBDV-specific chicken
antiserum can also be used to detect antigen in bursal tissue. Antigen-capture enzyme-linked
immunosorbent assays (ELISAs) based on plates coated with IBDV-specific antibodies have also
been described for the demonstration of IBDV antigens in bursal homogenates. The reverse-
transcription polymerase chain reaction (RT-PCR) with specific primers may be used to detect viral
genomic RNA in the bursa of Fabricius.
Strain characterisation: IBDV strains can be further identified by testing their pathogenicity in
specific antibody-negative chickens, by investigating their antigenic reactivity in cross VN assays
or in tests based on monoclonal antibodies, by determining the nucleotide sequence of RT-PCR
amplification products derived from IBDV genome, or by studying the number and size of the
restriction fragments obtained following digestion of such RT-PCR products with restriction
endonucleases. Several protocols have been described for each of these different approaches.
Tests should be performed by specialised laboratories and should include a panel of reference
strains as controls. Although the molecular basis for antigenic variation is now better understood,
no validated virulence marker has been described yet.

Chapter 2.3.12. — Infectious bursal disease (Gumboro disease)
Serological tests: An AGID, VN or ELISA may be carried out on serum samples. The infection
usually spreads rapidly within a flock of birds. Because of this, only a small percentage of the flock
needs to be tested to detect the presence of antibodies. If positive reactions are found in
unvaccinated birds then the whole flock must be regarded as infected.
Requirements for vaccines and diagnostic biologicals: Both live attenuated and inactivated
(killed) vaccines are available to control the disease. A live recombinant vaccine expressing the
VP2 antigen of IBDV has also been licensed recently. It is important that live vaccines be stable,
with no tendency to revert to virulence on passage. To be effective, the inactivated vaccines need
to have a high antigen content.
Live vaccines are used to produce an active immunity in young chickens. A complementary
approach to this is to provide chickens with passive protection by vaccinating the parents using a
combination of live and killed vaccines. Effective vaccination of breeding stock is therefore of great
importance.
Live vaccines: Attenuated strains of IBD viruses are used. These are referred to as either mild,
intermediate, or ‘intermediate plus’ (‘hot’) vaccines. The mild vaccines cause limited bursal
damage, while the intermediate and intermediate plus vaccines cause some lymphocytic depletion
in the bursa of Fabricius. Usually none of the vaccine types causes immunosuppression when
used in birds over 14 days old that have been hatched from IBD immune parents.
Mild vaccines are rarely used in broilers, but are used widely to prime broiler parents prior to
inoculation with inactivated vaccine. Intermediate and ‘hot’ vaccines are more capable of
overcoming low levels of maternally derived antibodies (MDA). Live vaccines are usually
administered by spray or in drinking water. In the absence of MDA, mild vaccines are given at 1-
day old. When MDA are present at 1 day of age, vaccination should be delayed until MDA in most
of the flock has waned. The best schedule can be determined by serological testing of the birds to
detect the time at which MDA has fallen to a low level. More recently, vaccines have been
developed that can be administered in ovo at 18 days of incubation.
Killed vaccines: These are usually used to stimulate high and uniform levels of antibody in parent
chickens so that the progeny will have high and uniform levels of MDA. The killed vaccines may
occasionally be used in young valuable birds with MDA. The killed vaccines are manufactured in
oil emulsion adjuvant and given by injection. They must be used in birds already sensitised by
primary exposure, either to live vaccine or to field virus. This can be checked serologically. High
levels of MDA can be obtained in breeder birds by giving, for example, live vaccine at
approximately 8 weeks of age, followed by inactivated vaccine at approximately 18 weeks of age.
A. INTRODUCTION
Infectious bursal disease (IBD) is caused by a virus that is a member of the genus Avibirnavirus of the family
Birnaviridae. Although turkeys, ducks, guinea fowl and ostriches may be infected, clinical disease occurs solely
in chickens. Only young birds are clinically affected. Severe acute disease of 3–6-week-old birds is associated
with high mortality, but a less acute or subclinical disease is common in 0–3-week-old birds. This can cause
secondary problems due to the effect of the virus on the bursa of Fabricius. IBD virus (IBDV) causes lymphoid
depletion of the bursa, and if this occurs in the first 2 weeks of life, significant depression of the humoral antibody
response may result. Two distinct serotypes of infectious bursal disease virus (IBDV) are known to exist.
Serotype 1 virus causes clinical disease in chickens younger than 10 weeks. Older chickens usually show no
clinical signs. Antibodies are sometimes found in other avian species, but no signs of infection are seen.
Serotype 2 antibodies are very widespread in turkeys and are sometimes found in chickens and ducks. There are
no reports of clinical disease caused by infection with Serotype 2 virus (20).
Isolation and identification of the agent provide the most certain diagnosis of IBD, but are not usually attempted
for routine diagnostic purposes as the virus may prove difficult to isolate (25). In practice, laboratory diagnosis of
IBD depends on detection of specific antibodies to the virus, or on detection of the virus in tissues, using
immunological or molecular methods.

Clinical IBD has clearly characteristic signs and post-mortem lesions. A flock will show very high morbidity with
severe depression in most birds lasting for 5–7 days. Mortality rises sharply for 2 days then declines rapidly over
the next 2–3 days. Usually between 5% and 10% of birds die, but mortality can reach 30–40%. The main clinical
signs are watery diarrhoea, ruffled feathers, reluctance to move, anorexia, trembling and prostration. Post-
mortem lesions include dehydration of the muscles with numerous ecchymotic haemorrhages, enlargement and
discoloration of the kidneys, with urates in the tubules. The bursa of Fabricius shows the main diagnostic lesions.
In birds that die at the peak of the disease outbreak, the bursa is enlarged and turgid with a pale yellow
discoloration. Intrafollicular haemorrhages may be present and, in some cases, the bursa may be completely
haemorrhagic giving the appearance of a black cherry. Peribursal straw-coloured oedema will be present in many
bursae. Confirmation of clinical disease or detection of subclinical disease is best done by using immunological
methods as the IBDV is difficult to isolate. For virus isolation, the methods described below should be followed.
Differentiation between serotypes 1 and 2 or between serotype 1 subtypes or pathotypes should be undertaken
by a specialised laboratory (e.g. the OIE Reference Laboratories for infectious bursal disease [see Table given in
Part 3 of this Terrestrial Manual]).
a) Sample preparation
Remove the bursae of Fabricius aseptically from approximately five affected chickens in the early stages of
the disease. Chop the bursae using two scalpels, add a small amount of peptone broth containing penicillin
and streptomycin (1000 µg/ml each), and homogenise in a tissue blender. Centrifuge the homogenate at
3000 g for 10 minutes. Harvest the supernatant fluid for use in the investigations described below. Filtration
through a 0.22 µ filter may prove necessary to further control bacterial contamination, although this may
cause a reduction in virus titre.
b) Identification by the agar gel immunodiffusion test

A protocol for the AGID test is described in Section B.2.a. For detection of antigen in the bursa of Fabricius
by AGID, the bursae should be removed aseptically from about ten chickens at the acute stage of infection.
The bursae are minced using two scalpels in scissor movement, then small pieces are placed in the wells of
the AGID plate against known positive serum. Freeze–thaw cycles of the minced tissue may improve the
release of IBDV antigens from the infected bursal tissue, and the freeze–thaw exudate may be used to fill
the wells.
c) Identification by immunofluorescence
Sections of bursa are prepared using a microtome cryostat, dried at room temperature and then fixed in
cold acetone. Fluorescent-labelled IBDV-specific antisera are applied to the sections, which are then
incubated at 37°C for 1 hour in a humid atmosphere. At the end of the incubation period, they are washed
for 30 minutes using phosphate buffered saline (PBS), pH 7.2, then rinsed in distilled water. The sections
are mounted using buffered glycerol, pH 7.6, and examined by UV microscopy for IBDV-specific
fluorescence (27).
d) Identification by antigen-capture enzyme-linked immunosorbent assay (AC-ELISA)

Different protocols have been described for the detection of serotype 1 IBDV using an antigen-capture
enzyme-linked immunosorbent assay (AC-ELISA) (12, 19, 36). Briefly, ELISA plates are coated with IBDV-
specific antibodies. Depending on the chosen AC-ELISA protocol, the capture antibody may be a mouse
anti-IBDV monoclonal antibody (MAb), or a mix of such MAbs, or a chicken post-infectious anti-IBDV
polyclonal serum. It has been suggested that AC-ELISAs using polyclonal antibodies may have a higher
sensitivity. Samples of bursal homogenates (see above) diluted 1/10 to 1/25 (w/v) in a suitable dilution
buffer are incubated in the coated wells. Unbound antigens are discarded at the end of the incubation
period by washing with a suitable washing buffer (e.g. PBS, pH 7.2 + 0.2% Tween 20). The captured
antigens are then revealed, as in an indirect ELISA, with a detection antibody (which must have been
developed from a different animal species than the capture antibody), followed by an enzyme conjugate that
binds to the detection antibody only (in some protocols the detection antibody may be directly conjugated to
the enzyme), followed by the enzyme substrate. Finally, optical densities, which parallel the amount of
captured IBDV antigens, are read with an ELISA reader.
The AC-ELISA is based on the use of samples possibly containing live virus and should be performed only
in suitable containment facilities such as a class II safety cabinet. All liquid (washing buffers) and solid
wastes should be considered to be contaminated by IBDV and decontaminated accordingly before disposal.
Critical steps in the implementation or assessment of AC-ELISA are i) the need to perform extensive
washings between each step of the reaction to keep background reactions low, ii) the requirement for
known positive and negative samples to be included in each assay as controls, and iii) the need for both the

capture and detection antibodies to positively react with all serotype 1 IBDV strains (i.e. neither capture nor
detection should critically depend on IBDV antigenic variation that occurs among serotype 1 strains).
e) Identification by molecular techniques

Molecular virological techniques have been developed that allow IBDV to be identified more quickly than by
virus isolation (8, 16, 43). The most frequently used molecular method is the detection of IBDV genome by
the reverse-transcription polymerase chain reaction (RT-PCR) (24, 43). This method can detect the genome
of IBDV, which is unable to grow in cell culture, because it is not necessary to grow the virus before
amplification.
RT-PCR is performed in three steps: extraction of nucleic acids from the studied sample, reverse
transcription (RT) of IBDV RNA into cDNA, and amplification of the resulting cDNA by PCR. The two latter
steps require that the user selects oligonucleotidic primers that are short sequences complementary to the
virus-specific nucleotidic sequence. Different areas of the genome will be amplified depending on the
location from which the primers have been selected. The example below allows the amplification of the
middle third of the gene encoding the outer capsid protein VP2 (9, 10).
• Extraction of nucleic acids

Single-stranded RNA is extremely susceptible to degradation by RNases. IBDV double-stranded RNA
(dsRNA) genome resists degradation by RNases. However, infected cells also contain IBDV-derived
positive-sense single-stranded RNA species that can be used as a template at the RT step and may
contribute to improving the sensitivity of the assay. It is thus important that RNA extraction be performed
using gloves and RNase-free reagents and labware.
IBDV RNA can be extracted from infected tissues using some kits available from commercial suppliers of
molecular biology reagents. Alternatively, IBDV RNA can be extracted by adding 1% (weight/volume final
concentration) sodium dodecyl sulphate and 1 mg/ml proteinase K to 700 µl of virus suspension (e.g. bursal
homogenate). Incubate for 60 minutes at 37°C. Nucleic acids are obtained using a standard protocol for
phenol/chloroform extraction (caution: phenol is toxic and should be handled and disposed accordingly).
Nucleic acids are harvested from the final aqueous phase by ethanol precipitation and are resuspended in
RNase-free distilled water or a suitable buffer. Water-diluted RNA should be kept frozen at a temperature
below –20°C until use.
• Reverse transcription
A variety of reverse transcriptases are commercially available. Follow the supplier’s instructions to prepare
the RT reaction mix. Use the ‘lower’ PCR primer (complementary to the positive strand of IBDV genome,
see below) for reverse transcription, as this allows the synthesis of cDNA both from the positive strand of
IBDV dsRNA genome and from IBDV-derived positive-sense single-stranded RNAs previously contained in
infected cells. Alternatively, random primers (hexanucleotides) can be used to prime cDNA synthesis.
The IBDV RNA matrix must be denaturated before transfer to the RT reaction mix. Add one part (by volume)
molecular biology grade dimethylsulphoxide to four parts the unfrozen solution of IBDV RNA. Heat for
3 minutes at 92°C and chill on ice; an alternative method is to heat for 5 minutes and immediately incubate
the mixture in liquid nitrogen. Transfer the relevant volume of denaturated matrix to the reaction mix.
Incubate according to the instructions of the enzyme supplier.
The cDNA solution obtained after the RT step should be kept frozen at a temperature below –20°C.
Delaying the PCR step for several weeks after the cDNA synthesis may cause false-negative PCR results.

A variety of DNA polymerases suitable for PCR are commercially available. Follow the manufacturer’s
instructions to prepare the PCR reaction mix. Protocols for the amplification and molecular typing of IBDV
have been reviewed recently (44). As an example, the U3/L3 and +290/–861 pairs of PCR primers shown
below can be suggested and have been found useful for amplifying the middle third of the VP2 gene in
segment A of serotype 1 IBDV strains (9, 10), and a region at the 5’ extremity of IBDV segment B (21),
respectively. Both regions have been shown to be suitable for molecular epidemiology studies (22).
Although a significant number of IBDV strains have two nucleotide changes at position 35 (G–A) and 38 (T–
C) of the U3 primer (including isolates from Japan [OKYM], Hong Kong [HK46], UK [UK661], Nigeria [N4]), it
has been shown that the U3-L3 primer pair successfully amplifies some of these viruses that exhibit both
mutations. This is probably because the 3’ extremity of U3 is highly conserved. However, as with most PCR
assays, IBDV strains may exist with nucleotide changes at the annealing positions of the primers, thus
requiring the use of other primers for optimised RT-PCR detection.
The combination of segment A- and segment B-targeted RT-PCR protocols enhances the probability that, if
present, serotype 1 IBDV will indeed be detected; it also allows a thorough genetic characterisation of the
IBDV strains detected.

Nucleotide sequence of the U3 and L3 IBDV-specific PCR primers (specific for Segment A, VP2 gene):
Upper U3: 5’-TGT-AAA-ACG-ACG-GCC-AGT-GCA-TGC-GGT-ATG-TGA-GGC-TTG-GTG-AC-3’
Lower L3: 5’-CAG-GAA-ACA-GCT-ATG-ACC-GAA-TTC-GAT-CCT-GTT-GCC-ACT-CTT-TC-3’
Nucleotide sequence of the +226 and –793 IBDV-specific PCR primers (specific for Segment B, VP1 gene):
Upper +290: 5’-TGT-AAA-ACG-ACG-GCC-AGT-GAA-TTC-AGA-TTC-TGC-AGC-CAC-GGT-CTC-T-3’
Lower -861: 5’-CAG-GAA-ACA-GCT-ATG-ACC-CTG-CAG-TTG-ATG-ACT-TGA-GGT-TGA-TTT-TG-3’
The U3 and L3 primers are both 44 nucleotides long, whereas primers +290 and –861 are 46 and
47 nucleotides long, respectively. The four primers include an IBDV-specific 3’ extremity (in italics in the
sequence shown above) corresponding to nucleotide positions 657–676 and 1193–1212 of IBDV segment
A in primers U3 and L3, respectively (numbering as in segment A of strain P2, Acc No X84034), and to
nucleotide positions 290–311 and 861–883 of IBDV segment B in primers +290 and –861, respectively
(numbering as in segment B of strain D6948, Acc No AF240687). The IBDV-specific extremity is coupled to
a non-IBDV 5’ extremity (bold type in the sequence above) corresponding to the M13 and RM13 universal
primers in the upper and lower primers, respectively. The M13 and RM13 universal primers are commonly
used as primers in DNA sequencing reactions, so that purified PCR products resulting from amplification
with the U3/L3 and +290/–861 primer pairs can be easily sequenced in both directions. Finally, restriction
sites (underlined in the above sequence) are included for the following restriction endonucleases: SphI (in
primer U3), EcoRI (in primers L3 and +290), and Pst I (in primer –861). These restriction sites are
positioned so that the PCR products resulting from amplification with the U3/L3 or +290/–861 pairs can be
cloned if required. The U3/L3 pair generates a 604 base pair (bp) product, 516 bp of which are specific of
the amplified IBDV sequence and encompass the region encoding the hyper-variable region of the VP2
protein. The +290/–861 pair generates a 642 bp product, 549 bp of which are specific of the amplified IBDV
sequence. Both products are derived from genomic regions that are suitable for phylogenetic analysis (9,
10, 21, 22).
Perform an initial denaturation step as recommended by the DNA polymerase supplier, followed by
35 cycles, each including one denaturation, one annealing and one elongation step. In such cycles,
denaturation at 95°C for 30 seconds and annealing at 64°C for 45 seconds may be used with both the
U3/L3 and +290/–861 primer pairs (the annealing temperature should be adapted if other primers are
used). The parameters for the elongation step should be set according to the supplier’s recommendations.
Revelation may be performed by electrophoresis with the PCR products and DNA molecular weight markers
in a 1% agarose gel stained with ethidium bromide (caution: ethidium bromide is toxic and carcinogenic. It
should be handled and disposed accordingly).
Three PCR reactions should be performed for each cDNA sample (pure, 10- and 100-fold diluted cDNA) to
avoid false-negative results due to PCR inhibition in mixes containing high amounts of the cDNA
preparation.
Each PCR should include negative and positive control reactions. Protocols that include an internal control
to test for the presence of PCR inhibitors have been developed (35).
Delaying the PCR for several weeks after the RT step may cause false negative PCR results.
f) Isolation of virus in cell culture

Inoculate 0.5 ml of sample into each of four freshly confluent chicken embryo fibroblast (CEF) cultures (from
a specific pathogen free [SPF] source) in 25 cm2 flasks. Adsorb at 37°C for 30–60 minutes, wash twice with
Earle’s balanced salt solution and add maintenance medium to each flask. Incubate the cultures at 37°C,
observing daily for evidence of cytopathic effect (CPE). This is characterised by small round refractive cells.
If no CPE is observed after 6 days, discard the medium, then freeze and thaw the cultures and inoculate the
resulting lysate into fresh cultures. This procedure may need to be repeated at least three times. If CPE is
observed, the virus should be tested against IBDV antiserum in a tissue culture virus neutralisation (VN)
test (see below). The more pathogenic IBDV strains usually cannot be adapted to grow in CEF unless the
virus has first been submitted to extensive serial passage in embryos (see below).
g) Isolation of virus in embryos

Inoculate 0.2 ml of sample into the yolk sac of five 6–8-day-old specific antibody negative (SAN) chicken
embryos and on to the chorioallantoic membrane (1) of five 9–11-day-old SAN chicken embryos. SAN
embryos are derived from flocks shown to be serologically negative to IBDV. Candle daily and discard dead
embryos up to 48 hours post-inoculation. Embryos that die after this time are examined for lesions.
Serotype 1 IBD produces dwarfing of the embryo, subcutaneous oedema, congestion and subcutaneous or
intracranial haemorrhages. The liver is usually swollen, with patchy congestion producing a mottled effect.

In later deaths, the liver may be swollen and greenish, with areas of necrosis. The spleen is enlarged and
the kidneys are swollen and congested, with a mottled effect. If lesions are observed, the virus should then
be tested against a monospecific anti-IBDV serum in an embryo-revealed virus neutralisation assay.
Serotype 1 IBDV usually causes death in at least some of the embryos on primary isolation.
Serotype 2 IBDV does not induce subcutaneous oedema or haemorrhages in the infected embryos, but
embryos are of a smaller size with a pale yellowish discolouration.
For the preparation of embryo-propagated stock virus or for subsequent passaging, embryos with lesions or
embryos suspected to be infected, respectively, are harvested aseptically. Their head and limbs are
discarded and the main body is minced as described in Section B.1.a for the preparation a virus
suspension.
h) Isolation of virus in chickens

This method has been used in the past but is no longer recommended due to animal welfare concerns. Five
susceptible and five IBD-immune chickens (3–7 weeks of age) are inoculated by the eye-drop route with
0.05 ml of sample. Kill the chickens 72–80 hours after inoculation, and examine their bursae of Fabricius.
The bursae of chickens infected with virulent serotype 1 IBDV appear yellowish (sometimes haemorrhagic)
and turgid, with prominent striations. Peribursal oedema is sometimes present, and plugs of caseous
material are occasionally found. The plicae are petechiated.
The presence of lesions in the bursae of susceptible chickens along with the absence of lesions in immune
chickens is diagnostic of IBD. The bursae from both groups may be used as antigen in an agar gel
immunodiffusion (AGID) test against known positive IBD antiserum (see Section B.1.b).
The extent of bursal damage may vary considerably with the pathogenicity of the studied IBDV strain.
However, as the samples submitted for virus isolation may vary in virus content, the extent of bursal
damage observed in susceptible chickens at the isolation stage gives a limited indication on strain
pathogenicity.
The bursae of chickens infected with serotype 2 IBDV do not exhibit any gross lesions.
i) Strain differentiation
IBDV strains can be further identified by testing their pathogenicity in SAN chickens, by investigating their
antigenic reactivity in cross VN tests or using MAbs, by determining the nucleotide sequence of RT-PCR
amplification products derived from IBDV genome, or by studying the number and size of the restriction
fragments obtained following digestion of such RT-PCR products with restriction endonucleases. Several
protocols have been described for each of these approaches. Tests should be performed by specialised
laboratories and should include a panel of reference strains as controls. Although the molecular basis for
antigenic variation is now better understood, no validated virulence marker has been described yet.
• Pathogenicity testing
Studies to compare the pathogenicity of IBDV strains must be carried out in secure biocontainment facilities
to avoid the dissemination of the studied virus (see Chapter 1.1.2 Biosafety and biosecurity in the veterinary
microbiological laboratory and animal facilities). SAN birds with a known microbial status (ideally SPF
chickens) must be used to avoid interference by contaminating agents.
The main variables when comparing the results of pathogenicity trials are the breed, age and immune
status of the challenged chickens, the dose and route of inoculation of the challenge virus, and the possible
presence of contaminating agents in the inoculum. Light layer breeds have been reported to be more
susceptible than heavy broilers (42). Differences in susceptibility may also occur between different SPF
chicken lines. The highest susceptibility to acute IBD occurs in chickens between 3 and 6 weeks of age
(25). (The influence of the immune status is described in Section C.) A high dose of challenge virus such as
that recommended in Section C.1.c is necessary so that all inoculated chickens become infected at once
without requiring bird-to-bird transmission of the inoculated virus. Finally, the presence in the inoculum of
contaminating agents, such as adenovirus or chicken infectious anaemia virus, may modify the severity of
IBD and signs observed after challenge (32).
The terms ‘variant’, ‘classical’ and ‘very virulent’ have been used to describe IBDV strains that exhibit a
different pathogenicity. Based on the signs and lesions observed in two lines of White Leghorn SPF chickens
during acute experimental IBD following a 105 50% embryo infective dose (EID50) challenge, North American
‘Variant’ IBDVs induce little if any clinical signs and no mortality but marked bursal lesions, ‘Classical’ IBDVs
induce approximately 10–50% mortality with typical signs and lesions whereas ‘very virulent’ IBDVs induce
approximately 50–100% mortality with typical signs and lesions (Eterradossi et al., personal observation).

• Antigenicity testing
Antigenic relatedness among IBDV strains may be assayed in cross VN tests, which correlate best with
cross protection. Such tests have to be performed in SAN embryonated eggs when the studied viruses do
not grow in CEF (e.g. very virulent IBDV [vvIBDV]). Differences in cross VN results among serotype 1 IBDV
strains have led to the definition of serotype 1 ‘subtypes’, some of which include the antigenically ‘variant’
North American IBDV isolates (15).
Another approach to the study of genetic relatedness is the use of mouse MAbs that bind to IBDV
neutralising epitopes. Several panels of MAbs exist world-wide (12, 13, 37). Some of the MAbs have been
included in commercially available kits, but no unified MAb panel as yet been proposed. All neutralising
epitopes of IBDV characterised to date have been mapped into a major immunogenic domain in the middle
third (amino acid positions 200 to 340) of the VP2 outer capsid protein (10, 33, 40). This region is termed
‘VP2 variable domain’ because most amino acid changes observed among IBDV strains are clustered in it.
Within vVP2, four amino acid stretches are of critical importance to antigenicity and are referred to as vVP2
hydrophilic peaks. These are amino acid positions 210 to 225 (major peak A), 249 to 252 (minor peak 1),
281 to 292 (minor peak 2) and 313 to 324 (major peak B) (2, 41). Both North American ‘variants’ and ‘very
virulent’ IBDV exhibit in these areas amino acid changes that correlate with epitope variation (9, 40). To
date, no antigenic marker has been shown to correlate strictly with IBDV pathogenicity.
• Molecular identification
Most efforts at molecular identification have focused on the characterisation of the larger segment of IBDV
(segment A) and especially of the vVP2 encoding region. Several protocols have been published on
characterisation using restriction endonucleases of RT-PCR products. These approaches are known as RT-
PCR/RE or RT-PCR-RFLP (restriction fragment length polymorphism) (17, 24, 46). The usefulness of the
information they provide depends on the identification of enzymes that cut in restriction sites that are
phenotypically relevant. Some sites involved in antigenicity have already been identified (see above),
however, restriction sites reliably related to virulence still need to be defined and validated. Nucleotide
sequencing of RT-PCR products, although more expensive than restriction analysis, provides an approach
to assessing more precisely the genetic relatedness among IBDV strains. Markers have been demonstrated
experimentally, using a reverse genetics approach, for cell culture-adapted strains, which exhibit amino acid
pairs 279 N–284 T (23) or 253 H–284 T (28). In most very virulent viruses, four typical amino acids are
present (222 A, 256 I, 294 I and 299 S) (3, 9, 24). However, it is not yet known whether these amino acids
play a role in virulence or if they are merely an indication of the clonal origin of most vvIBDV isolates.
Several recent studies indicate that although VP2 is an important virulence determinant, it may not be the
only one (4). It has been reported that segment A and B of IBDV mostly co-evolve (i.e. most significant
IBDV clusters, such as vvIBDV-related strains, may be identified by analysis of both genome segments),
however some potentially reassortant viruses have been identified (21).
a) Agar gel immunodiffusion test

The AGID test is the most useful of the serological tests for the detection of specific antibodies in serum, or
for detecting viral antigen or antibodies in bursal tissue.
Blood samples should be taken early in the course of the disease, and repeat samples should be taken 3 weeks
later. Because the virus spreads rapidly, only a small proportion of the flock needs to be sampled. Usually 20 blood
samples are enough. For detection of antigen in the bursa of Fabricius, the bursae should be removed aseptically
from about ten chickens at the acute stage of infection. The bursae are minced using two scalpels in a scissor
movement, then small pieces are placed in the wells of the AGID plate against known positive serum. Freeze–
thaw cycles of the minced tissue may improve the release of IBDV antigens from the infected bursal tissue.
• Preparation of positive control antigen

Inoculate 3–5-week-old susceptible chickens, by eye-drop, with a clarified 10% (w/v) bursal homogenate
known to contain viable IBDV 1. Kill the birds 3 days post-inoculation, and harvest the bursae aseptically.
Discard haemorrhagic bursae and pool the remainder, weigh and add an equivalent volume of cold distilled
water (or of a suitable buffer such as PBS or tryptose phosphate broth) and an equivalent volume of
undiluted methylene chloride. (Caution: methylene chloride is toxic and possibly carcinogenic. It should be
handled and disposed accordingly. A possible alternative to avoid health hazards caused by methylene
chloride is to use trichlorotrifluoroethane). Thoroughly homogenise the mixture in a tissue blender and
centrifuge at 2000 g for 30 minutes. Harvest the supernatant fluid and dispense into aliquots for storage at
–40°C. The antigen contains live virus and should be handled only in suitable containment facilities such as
1 A suitable classical strain of IBDV (serotype 1, classical pathotype) is strain 52/70, otainable from one of the OIE
Reference Laboratories (see Table given in Part 3 of this Terrestrial Manual).

a class II safety cabinet. If required, the antigen can be inactivated prior to dispensing: add 0.3% (v/v) ß-
propiolactone to the harvested supernatant, then further incubate at 37°C for 2 hours. It is important that
incubation takes place on an orbital shaker or a mechanical rocker, so that any inner part of the vial that
has been in contact with live virus indeed gets into contact with ß-propiolactone. Dispense and store as
above. Check the efficacy of the inactivation process by attempting IBDV isolation from the inactivated
antigen, with three serial passages on SAN embryonated eggs (see Section B.1.g).
• Preparation of positive control antiserum

Inoculate 4–5-week-old susceptible chickens, by eye-drop, with 0.05 ml of a clarified 10% (w/v) bursal
homogenate known to contain viable IBDV1. Exsanguinate 28 days post-inoculation. Pool and store serum
in aliquots at –20°C.
• Preparation of agar
Dissolve sodium chloride (80 g) and phenol (5 g) in distilled water (1 litre) (caution: phenol is toxic and
should be handled and disposed of accordingly). Add agar (12.5 g) and steam until the agar has dissolved.
To avoid the health and environmental hazards caused by the use of phenol, another suitable recipe for the
preparation of agar is as follows: sodium chloride (80 g), kalium dihydrogenophosphate (0.45 g), sodium
hydrogenophosphate dihydrate (1.19 g), agar (10 g) and distilled water to a final volume of 1 litre (final
pH 7.1 at 20–25°C). This second recipe can be homogenised by heating up to 90°C under agitation. While
the mixture is still very hot, filter it through a pad of cellulose wadding covered with a few layers of muslin
and dispense the medium in 20 ml volumes into glass bottles. The medium without phenol can further be
sterilised by autoclaving at (at most) 115°C for 15 minutes. Store the bottles at 4°C until required for use.
• Test procedure
i) Prepare plates from 24 hours to 7 days before use. Dissolve the agar by placing in a steamer or
boiling water bath. Take care to prevent water entering the bottles.
ii) Pour the contents of one bottle into each of the required number of 9 cm plastic Petri dishes laid on a
level surface. (Some laboratories prefer to pour the gel on 25 × 75 mm glass slides, 3 mm deep.)
iii) Cover the plates and allow the agar to set, and then store the plates at 4°C. Poured plates may be
stored for up to 7 days at 4°C. (If the plates are to be used the same day that they are poured, dry
them by placing them opened but inverted at 37°C for from 30 minutes to 1 hour.)
Notes:
1. The linear pattern of wells is preferred although a hexagonal pattern may be used. Each test serum or bursa should
be placed adjacent to a positive control antibody (AB) or antigen (AG), respectively.
2. Wells, 3 mm deep, 6 mm in diameter, and 3 mm apart (or wells of any other size previously shown to be effective),
are used.
iv) Cut three vertical rows of wells 6 mm in diameter and 3 mm apart, using a template and tubular cutter.
v) Remove the agar from the wells by aspiration or remove using a pen and nib, taking care not to
damage the walls of the wells.
vi) Using a pipette, dispense 50 µl of the test sera into the wells as shown in Figure 1.

Or, for the detection of IBDV antigens in bursae:

Dispense small pieces of finely minced test bursae by means of curved fine-pointed forceps into the
wells, as shown in Figure 2, to just fill the wells. Alternatively, the freeze–thaw exudate of minced
tissues can be used to fill the wells.
vii) Dispense 50 µl of the positive and negative control reagents into the relevant wells.
viii) Incubate the plates at between 22°C and 37°C for up to 48 hours in a humid chamber to avoid drying
the agar.
ix) Examine the plates against a dark background with an oblique light source after 24 and 48 hours.
• Quantitative agar gel immunodiffusion tests

The AGID test can also be used to measure antibody levels by using dilutions of serum in the test wells and
taking the titre as the highest dilution to produce a precipitin line (5). This can be very useful for measuring
maternal or vaccinal antibodies and for deciding on the best time for vaccination; however, this AGID
quantitative determination has now been largely replaced by the ELISA.
b) Virus neutralisation tests

VN tests are carried out in cell culture. The test is more laborious and expensive than the AGID test, but is
more sensitive for detecting antibody. This sensitivity is not required for routine diagnostic purposes, but
may be useful for evaluating vaccine responses or for differentiating between IBDV 1 and 2 serotypes.
First, 0.05 ml of virus diluted in tissue culture medium to contain 100 TCID50 (50% tissue culture infective
doses) per 0.05 ml is placed in each well of a tissue-culture grade microtitre plate (Spearman–Kärber [1] or
the Reed & Muench [30]). The test sera are heat-inactivated at 56°C for 30 minutes. Serial doubling
dilutions of the sera are made in the diluted virus. After 30 minutes at room temperature, 0.2 ml of SPF
chicken embryo fibroblast cell suspension, with a cell density allowing confluent layers to be obtained after
24 hours of incubation, is dispensed into each well. Plates are sealed and incubated at 37°C for 4–5 days,
after which the monolayers are observed microscopically for typical CPE. The end-point (serum titre) is
expressed as the reciprocal of the highest serum dilution that did not show CPE. To reduce test-to-test and
operator-to-operator variation, a standard reference antiserum may be included with each batch of tests 2
and the titre of the virus suspension must be reassessed in each new experiment using a sufficient number
of repeats (wells) per virus dilution.

ELISAs are in use for the detection of antibodies to IBD. Coating the plates requires a purified, or at least
semipurified, preparation of virus, necessitating special skills and techniques. Methods for preparation of
reagents and application of the assay were described by Marquardt et al. in 1980 (26). Commercial kits are
available.
The test sera are diluted according to the established protocol or kit instructions and each is dispensed into
the requisite number of wells. After incubation under the appropriate conditions, the sera are discarded from
the plates, and the wells are washed thoroughly. Anti-chicken immunoglobulins conjugated to an enzyme
are dispensed into the wells, and the plates are again incubated as appropriate. The plates are emptied and
rewashed before substrate containing a chromogen that gives a colour change in the presence of the
enzyme used is added to the plate. After a final incubation step, the substrate/chromogen reaction is
stopped by addition of a suitable stopping solution and the colour reactions are quantified by measuring the
optical density of each well. The Sample to Positive (S/P) ratio for each test sample is calculated.
d) Interpretation of results
The AGID test is surprisingly sensitive, though not as sensitive as the VN test; the latter will often give a titre
when the AGID test is negative. Positive reactions indicate infection in unvaccinated birds without maternal
antibodies. As a guide, a positive AGID reaction in a vaccinated bird or young bird with maternal antibody
indicates a protective level of antibody. ELISA gives more rapid results than VN or AGID and is less costly
in terms of labour, although the reagents are more expensive. VN and AGID titres correlate well, but as VN
is more sensitive, AGID titres are proportionally lower. Correlation between ELISA and VN and between
ELISA and AGID is more variable depending on the source of the ELISA reagents. When testing for the
decay of maternally derived antibodies (MDA), it is not uncommon to find residual VN antibodies at an age
when ELISA results are already negative. A formula has been devised that allows ELISA titres to be used to
calculate the optimal age for vaccination (18), which will vary depending on the vaccine used. Nonspecific
positive reactions may occur with most ELISAs because they are usually designed for monitoring vaccine
2 A suitable reference antiserum may be obtained from the OIE Reference Laboratories (see Table given in Part 3 of this
Terrestrial Manual).

responses, in which case sensitivity is regarded as more important than specificity. This should be taken
into account when the ELISA is used for diagnosis. In commercial chicken flocks, the possibility that a
serotype 1 ELISA antigen also detects antibodies induced by a natural infection with serotype 2 IBDV
cannot be ruled out, however this possible cross reactivity has not yet been demonstrated to interfere with
serological monitoring programmes of IBD based on the ELISA.

Two types of vaccine are mostly available for the control of IBD. These are live attenuated vaccines, or
inactivated oil-emulsion adjuvanted vaccines (39). A live recombinant vaccine expressing IBDV antigens has also
been licensed (7).
To date, IBD vaccines have been made with serotype 1 IBDV only, although a serotype 2 virus has been
detected in poultry. The serotype 2 virus has not been associated with disease, but its presence will stimulate
antibodies. Serotype 2 antibodies do not confer protection against serotype 1 infection, neither do they interfere
with the response to type 1 vaccine. There have been numerous descriptions of antigenic variants of serotype 1
virus (31). Cross-protection studies have shown that inactivated vaccines prepared from ‘classical’ serotype 1
virus require a high antigenic content to provide good protection against some of these variants. IBD vaccines
that contain both classical and variant IBD serotype 1 viruses are now licensed. vvIBDV strains with limited
antigenic changes as compared with ‘classical’ serotype 1 viruses have emerged since 1986. Active
immunisation with a ‘classical’ serotype 1 virus or vaccine provides a good protection against the vvIBDVs (16),
however the latter viruses are less susceptible to neutralisation by maternally derived antibodies than ‘classical’
pathogenic viruses (42).
• Live vaccines: methods of use

Live IBD vaccines are produced from fully or partially attenuated strains of virus, known as ‘mild’, ‘intermediate’,
or ‘intermediate plus’ (‘hot’), respectively.
Mild or intermediate vaccines are used in parent chickens to produce a primary response prior to vaccination
near to point-of-lay using inactivated vaccine. They are susceptible to the effect of MDA so should be
administered only after all MDA has waned. Application is by means of intramuscular injection, spray or in the
drinking water, usually at 8 weeks of age (34).
Intermediate or intermediate plus vaccines are used to protect broiler chickens and commercial layer
replacements. Some of these vaccines are also used in young parent chickens if there is a high risk of natural
infection with virulent IBD. Although intermediate vaccines are susceptible to the presence of MDA, they are
sometimes administered at 1-day old, as a coarse spray, to protect any chickens in the flock that may have no or
only minimal levels of MDA. This also establishes a reservoir of vaccine virus within the flock that allows lateral
transmission to other chickens when their MDA decay. Second and third applications are usually administered,
especially when there is a high risk of exposure to virulent forms of the disease or when the vaccinated chicks
exhibit uneven MDA levels. The timing of additional applications will depend on the antibody titres of the parent
birds at the time the eggs were laid. As a guide, the second dose is usually given at 10–14 days of age when
about 10% of the flock is susceptible to IBD, and the third dose 7–10 days later. The route of administration is by
means of spray or in the drinking water. Intramuscular injection or eye-drop is used rarely. If the vaccine is given
in the drinking water, clean water with a neutral pH must be used that is free from smell or taste of chlorine or
metals. Skimmed milk powder may be added at a rate of 2 g per litre. Care must be taken to ensure that all birds
receive their dose of vaccine. To this end, all water should be removed (cut off) for 2–3 hours before the
medicated water is made available and care must be taken that no residual water remains in the water adduction
pipes or in the drinkers. It is possible to divide the medicated water into two parts, giving the second part
30 minutes after the first.
Recently, technology has been developed to deliver live vaccine into eggs during the incubation period. Live
vaccine virus is blended with IBD antibody and the complex is injected in ovo at 18 days of incubation. The eggs
go on to hatch and the vaccine virus is released when the chicks are about 7 days of age. In this way, the
problem of maternally derived IBD antibody is overcome and the chicks are effectively immunised (14).
A live recombinant vaccine that uses a viral vector (herpes virus of turkeys) to express the VP2 antigen of IBDV in
chickens has been licensed recently in Europe. There is limited information available on the use of this vaccine.
Live IBD vaccines are generally regarded as compatible with other avian vaccines. However, it is possible that
IBD vaccines that cause bursal damage could interfere with the response to other vaccines. Only healthy birds

should be vaccinated. The vials of lyophilised vaccine should be kept at temperatures between 2°C and 8°C up
to the time of use.
• Inactivated vaccines: method of use

Inactivated IBD vaccines are mostly used to produce high, long-lasting and uniform levels of antibodies in
breeding hens that have previously been primed by live vaccine or by natural exposure to field virus during
rearing (6). The usual programme is to administer the live vaccine at about 8 weeks of age. This is followed by
the inactivated vaccine at 16–20 weeks of age. Occasionally, inactivated vaccines may be used in programmes
combining inactivated and live vaccines, in young valuable birds with high MDA levels reared in areas with high
risk of exposure to virulent IBDV. The inactivated vaccine is manufactured as a water-in-oil emulsion, and has to
be injected into each bird. The preferred routes are intramuscular into the leg muscle, avoiding proximity to joints,
tendons or major blood vessels or the subcutaneous route. A multidose syringe may be used. All equipment
should be cleaned and sterilised between flocks, and vaccination teams should exercise strict hygiene when
going from one flock to another. Vaccine should be stored at between 4°C and 8°C. It should not be frozen or
exposed to bright light or high temperature.
Only healthy birds, known to be sensitised by previous exposure to IBDV, should be vaccinated. Used in this way
the vaccine should produce such a good antibody response that chickens hatched from those parents will have
passive protection against IBD for up to about 30 days of age (45). This covers the period of greatest
susceptibility to the disease and prevents bursal damage at the time when this could cause immunosuppression.
It has been shown that bursal damage occurring after about 15 days of age has little effect on
immunocompetence as by that time the immunocompetent cells have migrated into the peripheral lymphoid
tissues. However, if there is a threat of exposure to infection with very virulent IBDV, live vaccines should be
applied as described above. The precise level and duration of immunity conferred by inactivated IBD vaccines
will depend mainly on the concentration of antigen present per dose. The manufacturing objective should be to
obtain a high antigen concentration and hence a highly potent vaccine.
1. Seed management

• Live vaccine
The seed virus must be shown to be free from extraneous viruses, bacteria, mycoplasma and fungi,
particularly avian pathogens. This includes freedom from contamination with other strains of IBDV. For
vaccine strains that claim to be attenuated and nonimmunosuppressive, the seed virus must be shown to
be stable, with no tendency to revert to virulence. This can be confirmed by carrying out at least five
consecutive chicken-to-chicken passages at 3–4-day intervals using bursal suspension as inoculum in SPF
chickens of the minimum age recommended for vaccination. It must be shown that the virus was
transmitted. A histological comparison is then made to show that there is no difference between bursae
from birds inoculated with the initial and the final passage material. Bursal scoring (29) and imaging
techniques have been developed.
Test for immunosuppression: An important characteristic is that the virus should not produce such damage
to the bursa of Fabricius that it causes immunosuppression in susceptible birds. Live vaccines of the
‘intermediate’ or ‘intermediate plus’ type may be licensed even though they may be capable of causing
immunosuppression. A possible protocol for the experimental assessment of immunosuppression is the
following: The vaccine is administered by injection or eye-drop, one field dose per bird, to each of 20 SPF
chickens, at 1-day old. A further group of birds of the same age and source are housed separately as
controls. At 2 weeks of age, each bird in both groups is given one field dose of live Newcastle disease
vaccine by eye-drop. Alternatively, the IBDV vaccine may be administered at the minimum age
recommended for vaccination, and the Newcastle disease vaccine at the time when bursal lesions induced
by the IBDV vaccine are maximal. The haemagglutination inhibition (HI) response of each bird to Newcastle
disease vaccine is measured 2 weeks after the administration of the Newcastle Disease vaccine, and the
protection is measured against challenge with 105.0 to 106.5 ELD50 (50% embryo lethal doses) Herts 33/56
strain (or similar) of Newcastle disease virus. The IBD vaccine fails the test if the HI response and
protection afforded by Newcastle disease vaccine is significantly less (<0.01) in the group given IBD
vaccine than in the control group. In countries where Newcastle disease virus is exotic, an alternative is to
use sheep erythrocytes or Brucella abortus-killed antigen as the test antigen, measuring the response using
the haemagglutination or serum agglutination test, respectively. However, another live vaccine is a
preferable test system because it also evaluates cell-mediated immunity.
• Killed vaccine
For killed vaccines, the most important characteristics are high yield and good antigenicity. Both virulent
and attenuated strains have been used. The seed virus must be shown to be free from extraneous viruses,
bacteria, mycoplasma and fungi, particularly avian pathogens (38).

Seed virus may be propagated in various culture systems, such as SPF chicken embryo fibroblasts, or
chicken embryos. In some cases, propagation in the bursa may be used. The bulk is distributed in aliquots
and freeze-dried in sealed containers. There have been claims that bursal origin vaccines are better
immunogens than tissue culture vaccines. In controlled studies, it was concluded that both vaccines, when
containing similar antigenic mass, elicited similar immune responses.
Data on efficacy should be obtained before bulk manufacture of vaccine begins. The vaccine should be
administered to birds in the way in which it will be used in the field. Live vaccine can be given to young birds
and the response measured serologically and by resistance to experimental challenge. In the case of killed
vaccines, a test must be carried out in older birds that go on to lay, using the recommended vaccination
schedule, so that their progeny can be challenged to determine resistance due to MDA at the beginning and
end of lay.
• Live vaccine
Efficacy test: Administer one vaccine dose of the minimum recommended titre to each of 20 SPF chickens
of the minimum age of vaccination. Inoculate separate groups for each of the recommended routes of
application. Leave 20 chickens from the same hatch as uninoculated controls. After 14 days, challenge
each of the chickens by eye-drop with approximately 100 CID50 (50% chicken infective dose) of a virulent
strain of IBDV as recommended by one of the OIE Reference Laboratories for IBD (see Table given in Part
3 of this Terrestrial Manual). Observe the chickens daily for 10 days. Register the number of birds that die
or exhibit IBD signs. Perform a histological examination of the bursa in chickens that survive at day 10. The
vaccine fails the test unless at least 90% of the vaccinated chickens survive without showing either clinical
signs or severe lesions in the bursae of Fabricius at the end of the observation period. If more than half the
controls do not show IBD signs, or one or more control chicken does not exhibit severe lesions of the bursa
of Fabricius, or control or inoculated birds die from causes not attributable to the test, the test is invalid.
Lesions are considered to be severe if at least 90% of follicles show greater than 75% depletion of
lymphocytes. Providing results are satisfactory, this test need be carried out on only one batch of all those
batches prepared from the same seed lot.
• Killed vaccine
Efficacy test: At least 20 unprimed SPF birds are given one dose of vaccine at the recommended age (near
to point-of-lay) at least one of the recommended routes; an alternative recommended procedure is to test
one dose of vaccine in the recommended routes listed on the label, using 20 unprimed SPF birds for each
route. The antibody response is measured between 4 and 6 weeks after vaccination by serum neutralisation
with reference to a standard antiserum 3.
Eggs are collected for hatching 5–7 weeks after vaccination, and 25 progeny chickens are then challenged
at 3 weeks of age by eye-drop with approximately 100 CID50 of a recognised virulent strain of IBDV. Ten
control chickens of the same breed but from unvaccinated parents are also challenged. Protection is
assessed 3–4 days after challenge by removing the bursa of Fabricius from each bird; each bursa is then
subjected to histological examination or tested for the presence of IBD antigen by the agar gel precipitin
test. Not more than three of the chickens from vaccinated parents should show evidence of IBD infection,
whereas all those from unvaccinated parents should be affected.
These procedures should be repeated towards the end of the period of lay when the vaccinated birds are at
least 60 weeks of age, but, on this occasion challenge of the progeny should be undertaken when they are
15 days old.
The efficacy test should be repeated on primed birds vaccinated by the recommended schedule. The final
dose of killed vaccine is given at the earliest recommended age. Chickens hatched from fertile eggs
collected at the beginning and the end of lay are tested for protection against challenge as described
above.
These tests need to be performed once only using a typical batch of vaccine.
The vaccine must be manufactured in suitable clean and secure accommodation, well separated from diagnostic
facilities or commercial poultry.
3 See footnote 2

Production of the vaccine should be on a seed-lot system using a suitable strain of virus of known origin and
passage history. SPF eggs must be used for all materials employed in propagation and testing of the vaccine.
Live vaccines are made by growth in eggs or cell cultures. Inactivated IBD vaccines may be made using virulent
virus grown in the bursae of young birds, or using attenuated, laboratory-adapted strains of IBDV grown in cell
culture or embryonated eggs. A high virus concentration is required. These vaccines are made as water-in-oil
emulsions. A typical formulation is to use 80% mineral oil to 20% suspension of bursal material in water, with
suitable emulsifying agents.
Antigen content: Having grown the virus to high concentration, its titre should be assayed by use of cell cultures,
embryos or chickens as appropriate to the strain of virus being used. The antigen content required to produce
satisfactory batches of vaccine should be based on determinations made on test vaccine that has been shown to
be effective in laboratory and field trials.
Inactivation of killed vaccines: This is often done with either ß-propiolactone or formalin. The inactivating agent
and the inactivation procedure must be shown under the conditions of vaccine manufacture to inactivate the
vaccine virus and any potential contaminants, e.g. bacteria, that may arise from the starting materials.
Prior to inactivation, care should be taken to ensure a homogeneous suspension free from particles that may not
be penetrated by the inactivating agent. A test for inactivation of the vaccine should be carried out on each batch
of both the bulk harvest after inactivation and the final product. An alternative approach is to test inactivation of
the final or bulk harvest, but not both. The test selected should be appropriate to the vaccine virus being used
and should consist of at least two passages in susceptible cell cultures, embryos or chickens, with ten replicates
per passage. No evidence of the presence of any live virus or microorganism should be observed.
Sterility of killed vaccines: Oil used in the vaccine must be sterilised by heating at 160°C for 1 hour, or by
filtration, and the procedure must be shown to be effective. Tests appropriate to oil-emulsion vaccines are carried
out on each batch of final vaccine as described, for example, in the European Pharmacopoeia.
4. Batch control
a) Sterility
b) Safety
• Live vaccine safety test
Ten field doses of vaccine are administered by eye-drop to each of 15 SPF chickens of the minimum age
recommended for vaccination and not older than 2 weeks. The chickens are observed for 21 days. If more
than two chickens die due to causes not related to the vaccine, the test must be repeated. The vaccine fails
the test if any chickens die or show signs of disease attributable to the vaccine. This test is performed on
each batch of final vaccine.
• Killed vaccine safety test

Ten SPF birds, 14–28 days of age, are inoculated by the recommended routes with the recommended dose
or twice the field dose. The birds are observed for 3 weeks. No abnormal local or systemic reaction should
develop. The test is performed on each batch of final vaccine.
c) Potency
• Live vaccine potency test
A potency test (virus titration) in eggs or cell cultures must be carried out on each serial (batch) of vaccine
produced. In addition, the method described in Section C.1.c ‘Live vaccine (efficacy test)’ must be used on
one batch representative of all the batches prepared from the same seed lot.
• Killed vaccine potency test

Ten SPF chickens, approximately 4 weeks of age, are each vaccinated with one dose of vaccine given by
the recommended route. An additional ten control birds of the same source and age are housed together
with the vaccinates. The antibody response of each bird is determined 4–6 weeks after vaccination in a VN
test with reference to a standard antiserum. The mean antibody level of the vaccinated birds should not be

significantly less than the level recorded in the test for protection. No antibody should be detected in the
control birds. This test must be carried out on each batch of final vaccine.
d) Stability
Evidence should be provided on three batches of vaccine to show that the vaccine passes the batch
potency test at the requested shelf life or as an alternative at 3 months beyond
e) Preservatives
A preservative is normally required for vaccine in multidose containers. The concentration of the
preservative in the final vaccine and its persistence throughout shelf life should be checked. A suitable
preservative already established for such purposes should be used.
Oil-emulsion vaccines cause serious injury to the vaccinator if accidentally injected into the hand or other
tissues. In the event of such an accident the person should go at once to a hospital, taking the vaccine
package with them. Each vaccine bottle and package should be clearly marked with a warning of the
serious consequences of accidental self-injury. Such wounds should be treated by the casualty doctor as a
‘grease gun injury’.
a) Safety
See Section C.4.b.
b) Potency
See Section C.4.c.
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RECENT REVIEWS
A. ETERRADOSSI N. (2001). Major advances in infectious bursal disease virus (IBDV) research since the first
nd
International IBDV/CIAV Symposium (Rauischolzhausen, Germany, 1994). Proceedings of the 2
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Redmann U., eds. Rauischholzhauzen, Germany, 16–20 June 2001, 6–23.
B. VAN DEN BERG T.P. (2000). Acute infectious bursal disease in poultry: a review. Avian Pathol., 29, 175–194.
*
* *
NB: There are OIE Reference Laboratories for Infectious bursal disease (Gumboro disease) (see Table in Part 3
of this Terrestrial Manual or consult the OIE Web site for the most up-to-date list: www.oie.int).

CHAPTER 2.3.13.
MAREK’S DISEASE
SUMMARY
Marek’s disease (MD) is a lymphomatous and neuropathic disease of domestic fowl caused by an
alphaherpesvirus.
Diagnosis is made on clinical signs and gross or microscopic lesions. Chickens may become
persistently infected with MD virus (MDV) without developing clinical disease. Infection by MDV is
detected by virus isolation and the demonstration of viral antigen or antibodies.
MD is prevented by vaccination with monovalent or multivalent live virus vaccines of various types.
The vaccine is injected in ovo or at hatch.
In chickens, MD occurs at 3–4 weeks of age or older and is most common between 12 and
30 weeks of age. Clinical signs observed are paralysis of the legs and wings, with enlargement of
peripheral nerves, but nerve involvement is sometimes not seen, especially in adult birds.
Depending on the strain of MDV, lymphomatosis can occur, especially in the ovary, liver, spleen,
kidneys, lungs, heart, proventriculus and skin. As opposed to the uniform cell population that
comprises the tumours caused by lymphoid leukosis, the nerve infiltration and lymphomas caused
by MDV consist of lymphoid cells of various types. Tumours that resemble those produced by MDV
are induced by the avian retrovirus, reticulo-endothelial virus (REV). Differentiation of MD from
lymphoid leukosis is important. Lesions caused by reticuloendotheliosis virus can also be confused
with those of MD.
Identification of the agent: Under field conditions, most chickens become infected with MDV
during the first few weeks of life and then carry the infection throughout their lives, often without
developing overt disease. The infection is usually detected by inoculating live buffy coat cells on to
monolayer cultures of chicken kidney cells or duck embryo fibroblasts, in which characteristic viral
plaques develop within a few days. Two serotypes of MDV are recognised – 1 and 2 – and a third
serotype is represented by the related herpesvirus of turkeys (HVT). Serotype 1 includes the
virulent strains and serotype 2 the naturally avirulent strains. MD viral antigen can be detected in
the feather tips of infected birds using a radial precipitin test.
Serological tests: Antibodies to MDV develop within 1–2 weeks of infection and are commonly
recognised by the agar gel immunodiffusion test, the indirect fluorescent antibody test, and
sometimes by other serological tests such as enzyme-linked immunosorbent assay.
Requirements for vaccines and diagnostic biologicals: MD is prevented by vaccinating

chickens in ovo or at 1 day of age. Live viral vaccines are used. HVT, in either a cell-free
(lyophilised) form, or a cell-associated (‘wet’) form, is most commonly used. Attenuated variants of
serotype 1 strains of MDV are the most commonly used vaccine type; also serotype 2 strains may
also be used, particularly in bivalent vaccines, together with HVT (serotype 3). Serotype 1 and 2
vaccines are only available in the cell-associated form. Bivalent vaccines consisting of serotypes 1
and 3 or trivalent vaccines consisting of serotypes 1, 2, and 3 are also used. The bivalent and
trivalent vaccines have been introduced to combat the very virulent strains of MDV that are not well
controlled by the usual monovalent vaccines.
Vaccination greatly reduces clinical disease, but not persistent infection by MDV. The vaccine
viruses are also carried throughout the life of the fowl and are continued to be shed, which results
in the ubiquitous presence of MDV.

Chapter 2.3.13. — Marek’s disease
A. INTRODUCTION
Marek’s disease (MD) (14, 25, 33) is a disease of domestic fowl (chickens) caused by a herpesvirus. Birds get
infected by inhalation of infected dust from the poultry houses, and following a complex life cycle, the virus is
shed from the feather follicle of infected birds (4). MD occurs at 3–4 weeks of age or older and is most common
between 12 and 30 weeks of age. MD is associated with several distinct pathological syndromes, of which the
lymphoproliferative syndromes are the most frequent and are of most practical significance. In the classical form
of the disease, characterised mainly by the involvement of nerves, mortality rarely exceeds 10–15% and can
occur over a few weeks or many months. In the acute form, in which there is usually lymphoma formation in the
viscera, a disease incidence of 10–30% in the flock is not uncommon and outbreaks involving up to 70% can
occur. Mortality may increase rapidly over a few weeks and then cease, or can continue at a steady or slowly
falling rate for several months. Currently, the acute form of the disease with extensive visceral lymphomas is
most prevalent. In its classical form, the most common clinical sign of MD is partial or complete paralysis of the
legs and wings. In the acute form, birds are often severely depressed and some may die without showing signs
of clinical disease. Non-neoplastic disease involving brain pathology with vasogenic oedema resulting in transient
paralysis is increasingly recognised with MD induced by the more virulent strains.
In the classical form, the characteristic finding is enlargement of one or more peripheral nerves. Those most
commonly affected and easily seen at post-mortem are the brachial and sciatic plexuses, celiac plexus,
abdominal vagus and intercostal nerves. Affected nerves are often two or three times their normal thickness, the
normal cross-striated and glistening appearance is absent, and the nerve may appear greyish or yellowish, and
sometimes oedematous. Lymphomas are sometimes present in the classical form of MD, most frequently as
small, soft, grey tumours in the ovary, and sometimes also in the lungs, kidneys, heart, liver and other tissues.
‘Grey eye’ caused by an iridocyclitis that renders the bird unable to accommodate the iris in response to light and
causes a distorted pupil is common in older (16–18 week) birds, and may be the only presenting sign.
In the acute form, the typical finding is widespread, diffuse lymphomatous involvement of the liver, gonads,
spleen, kidneys, lungs, proventriculus and heart. Sometimes lymphomas also arise in the skin around the feather
follicles and in the skeletal muscles. Affected birds usually have enlarged peripheral nerves, as in the classical
form. In younger birds, liver enlargement is usually moderate in extent, but in adult birds the liver may be greatly
enlarged and the gross appearance identical to that seen in lymphoid leukosis, from which the disease must be
differentiated. Nerve lesions are often absent in adult birds with MD.
In both the classical and acute forms of MD, the disease starts as a proliferation of lymphoid cells, which is
progressive in some cases and regressive in others. The peripheral nerves may be affected by proliferative,
inflammatory or minor infiltrative changes, which are termed type A, B, and C lesions, respectively. The A-type
lesions consist of infiltration by proliferating lymphoblasts, large, medium and small lymphocytes, and macrophages,
and appear to be neoplastic in nature. The B-type lesion is characterised by interneuritic oedema, infiltration by
mainly small lymphocytes and plasma cells, and Schwann cell proliferation, and appears to be inflammatory. The C-
type lesion consists of a light scattering of mainly small lymphocytes, and is often seen in birds that show no gross
lesions or clinical signs. It is thought to be a regressive, inflammatory lesion. Demyelination frequently occurs in
nerves affected by the A- and B-type lesions, and is responsible for the clinical paralysis.
Lymphomas in the visceral organs and other tissues are similar cytologically to the lymphoproliferations in the A-
type lesions in nerves. Usually the lymphoid cells are of mixed types, often with a preponderance of small and
medium lymphocytes, but sometimes, particularly in acute MD in adult birds, large lymphocytes and
lymphoblasts may predominate.
The heterogeneous population of lymphoid cells in MD lymphomas, as seen in haematoxylin-and-eosin-stained

sections, or in impression smears of lymphomas stained by May–Grünwald–Giemsa, is an important feature in
differentiating the disease from lymphoid leukosis, in which the lymphomatous infiltrations are composed of
uniform lymphoblasts. Another important difference is that, in lymphoid leukosis, gross lymphomas occur in the
bursa of Fabricius, and the tumour has an intrafollicular origin and pattern of proliferation. In MD, although the
bursa is sometimes involved in the lymphoproliferation, the tumour is less apparent, diffuse and interfollicular in
location. Peripheral nerve lesions are not a feature of lymphoid leukosis as they are in MD. The greatest difficulty
comes in distinguishing between lymphoid leukosis and forms of MD sometimes seen in adult birds in which the
tumour is lymphoblastic with marked liver enlargement and absence of nerve lesions. If post-mortems are
conducted on several affected birds, a diagnosis can usually be made based on gross lesions and
histopathology. However there are other specialised techniques described. The expression of a Meq biochemical
marker has been used to differentiate between MD tumours, latent MDV infections and retrovirus-induced
tumours (3). The procedure may require specialised reagents and equipment and it may not be possible to carry
out these tests in laboratories without these facilities. Other techniques, such as detection by
immunofluorescence of activated T cell antigens present on the surface of MD tumour cells (MD tumour-
associated surface antigen or MATSA), or of B-cell antigens or IgM on the tumour cells of lymphoid leukosis can
give a presumptive diagnosis, but these are not specific to MD tumour cells.
Nerve lesions and lymphomatous proliferations induced by certain strains of reticuloendotheliosis virus are
similar, both grossly and microscopically, to those present in MD. Although reticuloendotheliosis virus is not

common in chicken flocks, it should be borne in mind as a possible cause of lymphoid tumours; its recognition
depends on virological and serological tests on the flock. Reticuloendotheliosis virus can also cause neoplastic
disease in turkeys, ducks, quail and other species. Another retrovirus also causes lymphoproliferative disease in
turkeys. Although chicken flocks may be seropositive for reticuloendotheliosis virus, neoplastic disease is rare.
The main features in the differential diagnosis of MD, lymphoid leukosis and reticuloendotheliosis are shown in
Table 1. Peripheral neuropathy is a syndrome that can easily be confused with the neurological lesions caused
by MD virus (MDV). This is not very common but its incidence may be increasing in some European flocks (3).
There are no recognised health risks to humans working with MDV or the related herpesvirus of turkeys (HVT).
Table 1. Features useful in differentiating Marek's disease, lymphoid leukosis and reticuloendotheliosis
Feature Marek’s disease Lymphoid leukosis Reticuloendotheliosis*
Age Any age. Usually 6 weeks or older Not under 16 weeks Not under 16 weeks
Signs Frequently paralysis Non-specific Non-specific
Incidence Frequently above 5% in Rarely above 5% Rare

unvaccinated flocks. Rare in
vaccinated flocks
Macroscopic lesions
Neural involvement Frequent Absent Infrequent
Bursa of Fabricius Diffuse enlargement or atrophy Nodular tumours Nodular tumours
Tumours in skin, muscle May be present Usually absent Absent

and proventriculus, ‘grey
eye’
Microscopic lesions
Neural involvement Yes No Infrequent
Liver tumours Often perivascular Focal or diffuse Focal
Spleen Diffuse Often focal Focal or diffuse
Bursa of Fabricius Interfollicular tumour and/or Intrafollicular tumour Intrafollicular tumour

atrophy of follicles
Central nervous system Yes No No
Lymphoid proliferation in Yes No No

skin and feather follicles
Cytology of tumours Pleomorphic lymphoid cells, Lymphoblasts Lymphoblasts

including lymphoblasts, small,
medium and large lymphocytes
and reticulum cells. Rarely can be
only lymphoblasts
Category of neoplastic T cell B cell B cell

lymphoid cell
*Reticuleondotheliosis virus may cause several different syndromes. The bursal lymphoma syndrome is most likely to occur in
the field and is described here.
Infection by MDV in a flock may be detected by isolating the virus from the tissues of infected chickens. The
ubiquitous nature of MDV must be remembered and the diagnosis of MD should be based on a combination of

MDV isolation or detection of the genome by PCR and clinical disease. Commonly used sources are buffy coat
cells from heparinised blood samples, or suspensions of lymphoma cells or spleen cells. When these samples
are collected in the field, it is suggested that they be transported to the laboratory under chilled conditions. As
MDV is highly cell associated, it is essential that these cell suspensions contain viable cells. The cell
suspensions are inoculated into monolayer cultures of chicken kidney cells or duck embryo fibroblasts (chicken
embryo fibroblasts are less sensitive for primary virus isolation). Serotype 2 and 3 viruses (see Section C.1.a) are
more easily isolated in chicken embryo fibroblasts than in chicken kidney cells. Usually a 0.2 ml suspension
containing from 106 to 107 live cells is inoculated into duplicate monolayers grown in plastic cell culture dishes
(60 mm in diameter). Inoculated and uninoculated control cultures are incubated at 38.5°C in a humid incubator
containing 5% CO2. Alternatively, closed culture vessels may be used. Culture medium is replaced at 2-day
intervals. Areas of cytopathic effects, termed plaques, appear within 3–5 days and can be enumerated at about
7–10 days.
Another, less commonly used source of MDV for diagnostic purposes is feather tips, from which cell-free MDV
can be extracted. Tips about 5 mm long, or minced tracts of skin containing feather tips, are suspended in an
SPGA/EDTA (sucrose, phosphate, glutamate and albumin/ethylenediamine tetra-acetic acid) buffer for extraction
and titration of cell-free MDV (9). The buffer is made as follows: 0.2180 M sucrose (7.462 g); 0.0038 M
monopotassium phosphate (0.052 g); 0.0072 M dipotassium phosphate (0.125 g); 0.0049 M L-monosodium
glutamate (0.083 g); 1.0% bovine albumin powder (1.000 g); 0.2% EDTA (0.200 g); and distilled water (100 ml).
The buffer is sterilised by filtration and should be at approximately pH 6.5.
This suspension is sonicated and then filtered through a 0.45 µm membrane filter for inoculation on to 24-hour-
old drained chicken kidney cell monolayers. After absorption for 40 minutes, the medium is added, and cultures
are incubated as above for 7–10 days.
Using these methods, MDV of serotypes 1 and 2 may be isolated, together with the HVT (serotype 3), if it is
present as a result of vaccination. With experience, plaques caused by the different virus serotypes can be
differentiated fairly accurately on the basis of time of appearance, rate of development, and plaque morphology.
HVT plaques appear earlier and are larger than serotype 1 plaques, whereas serotype 2 plaques appear later
and are smaller than serotype 1 plaques.
MDV and HVT plaques may be identified as such using specific fluorescent antibodies raised in chickens.
Monoclonal antibodies may be used to differentiate serotypes (17).

Genomes of all three serotypes have been sequenced (2, 18). Polymerase chain reaction (PCR) tests have
been developed for the diagnosis MD; and a real time RT-PCR has been described (1, 5, 16). In addition
differentiation of some oncogenic and non-oncogenic strains of serotype 1 MDV, and of MDV vaccine
strains of serotypes 2 and 3 (6, 7, 15, 27, 34) have been described. PCR may also be used to quantitate
virus load in tissues (5, 7, 8, 24) or differentially detect MDV and HVT in the blood or feather tips (5, 13).
The presence of antibodies to MDV in non-vaccinated chickens from about 4 weeks of age is an indication of
infection. Before that age, such antibodies may represent maternal transmission of antibody via the yolk and are
not evidence of active infection.
Viruses, antigens and antisera are usually available from OIE Reference Laboratories for Marek’s Disease (see
Table in Part 3 of this Terrestrial Manual), but international standard reagents have not yet been produced.
a) Agar gel immunodiffusion

There is no prescribed test for trade, but the agar gel immunodiffusion (AGID) test is employed most
commonly to detect antibody. The test is conducted using glass slides coated with 1% agar in phosphate
buffered saline containing 8% sodium chloride. Adjacent wells are filled with antigen or serum and these are
incubated in a humid atmosphere at 37°C for 24 hours for diffusion to take place; positive sera show
reactions of identity with known positive serum and antigen. The antigen used in this test is either disrupted
MDV-infected tissue culture cells or an extract of feather tips, or skin containing feather tracts obtained from
MDV-infected chickens. The cell culture antigen is prepared by propagating MDV in chicken kidney cells or
chicken embryo fibroblast cells. When cytopathic effect is confluent, the cells are detached from the culture
vessel and suspended in culture medium or phosphate buffered saline without tryptose phosphate broth
(presence of tryptose phosphate broth may produce non-specific precipitin lines) at a concentration of about
1 × 107 cells/ml. This suspension is then freeze–thawed three times and used as antigen.

Test procedure
i) Make a 1% solution of Difco Bactoagar in 8% sodium chloride by standing the mixture in a boiling
water bath.
ii) Either a microscope slide or a Petri dish can be used and the agar is poured to a thickness of 2–3 mm.
iii) Cut holes in the agar using a template with a centre well and 6 wells spaced at equal distance around
the centre well. The diameter of wells should be approximately 5.3 mm, and the wells should be about
2.4 mm apart. A template with cutters is commercially available.
iv) The antigen is placed in the centre well and the standard antiserum is placed in alternate exterior
wells. Serum samples to be tested are placed in the remaining three wells so that .a continuous line of
identity is formed between an unknown sample that is positive and the known positive control sera.
v) Incubate the slide for 24 hours at 37°C in a humid container and read the results over a lamp in a
darkened room.
A variation of the AGID test may be used to detect MDV antigen in feather tips as an indication of infection
by MDV. Glass slides are prepared with a coating of 0.7% agarose (e.g. A37) in 8% sodium chloride,
containing MDV antiserum. Tips of small feathers are taken from the birds to be examined and are inserted
vertically into the agar, and the slides are maintained as described above. The development of radial zones
of precipitation around the feather tips denotes the presence in the feather of MDV antigen and hence of
infection in the bird.
b) Other tests
Other tests for MDV antibody include the direct and indirect fluorescent antibody test. These demonstrate
the ability of a test serum to stain MDV plaques in cell cultures (28, 29). These tests are group specific and
more sensitive than the AGID test. A virus neutralisation test for the ability of a serum to neutralise the
plaque-forming property of cell-free MDV can also be employed. However, this test is more suitable for
research purposes than for routine diagnostic use. Enzyme-linked immunosorbent assays (ELISA) for
detecting MDV antibodies are available (10, 25, 35). To prepare antigen for the ELISA, wells of a 96-well
microtitre plate are coated with MDV-infected cells.

Control of MD is essentially achieved by the widespread use of live attenuated vaccines (21). Commercial
biological products used in the control of MD are the cell-associated or cell-free (lyophilised) live virus or
HVT, respectively (see below). Although genetically engineered recombinant vaccines have been
developed (23), they are currently not in commercial use. Marek’s disease vaccines are injected in ovo at
the 17th or 18th day of embryonation (26) or subcutaneously at hatch. The requirements for producing
vaccines are outlined below, and in Chapter 1.1.8 Principles of veterinary vaccine production, but other
sources should be consulted for further information on the procedures (11, 12, 19, 20, 22, 30). Protocols
are given in the British Pharmacopoeia Monograph 589, and the US Code of Federal Regulations, Volume
9, part 113 (31). The guidelines in this Terrestrial Manual are intended to be general in nature and maybe
1. Seed management

Viruses of the MDV group are classified under three serotypes – 1, 2, and 3 – on the basis of their antigenic
relatedness.
Serotype 1: This includes all the pathogenic strains of the virus, ranging from strains that are very virulent
plus (e.g. 648A), very virulent (e.g. Md/5, Md/11, Ala-8, RB-1B), virulent (e.g. HPRS-16, JM GA), mildly
virulent (e.g. HPRS-B14, Conn A) and finally to weakly virulent (e.g. CU-2, CVI-988). These strains may be
attenuated by passage in tissue culture, with loss of pathogenic properties but retention of immunogenicity,
to provide strains that have been used as vaccines. Those that have been used commercially include
attenuated HPRS-16 and CVI-988 (Rispens) strains. Attenuated variants of the very virulent stains have
been used in experimental vaccines to protect against the variant form of acute MD caused by the very
virulent stains. Md11/75C/R2/23 is one such strain (32) licensed for use in the United States of America.
Serotype 1 vaccines are prepared in a cell-associated (‘wet’) form that must be stored in liquid nitrogen.
Serotype 2: This includes naturally avirulent strains of MDV (e.g. SB-1, HPRS-24, 301B/1, HN-1), and
several of these have been shown to provide protection against virulent strains. The SB-1 and 301B/1
strains have been developed commercially and used, particularly with HVT, in bivalent vaccines for
protection against the very virulent strains. Serotype 2 vaccines exist only in the cell-associated form.

Serotype 3: This contains the strains of naturally avirulent HVT (e.g. FC126, PB1), which are widely used as
a monovalent vaccine, and also in combination with serotype 1 and 2 strains in bivalent or trivalent vaccines
against the very virulent strains of MDV. HVT may be prepared in a cell-free form as a freeze-dried
(lyophilised) vaccine or in a cell-associated (‘wet’) form.
The substrates used for commercial vaccine production are primary chicken embryo fibroblasts (CEF)
derived from specific pathogen free (SPF) flocks or duck embryo fibroblasts. CEF from SPF flocks are
preferred to duck cells because more is known about chicken-embryo-transmitted pathogens and methods
for their detection.
Methods for testing SPF flocks for freedom from infection are available (20, 30). SPF chicken flocks should
be free from avian adenoviruses, including egg-drop syndrome 76 virus, avian encephalomyelitis virus,
avian leukosis virus (subgroups A, B and J), avian nephritis virus, avian reoviruses, avian rotaviruses,
chicken anaemia virus, fowl pox virus, infectious bronchitis virus, infectious bursal disease virus, infectious
laryngotracheitis virus, influenza type A virus, MDV, Mycoplasma gallisepticum, Mycoplasma synoviae,
Newcastle disease virus, reticuloendotheliosis virus, Salmonella spp., and turkey rhinotracheitis virus.
SPF duck flocks should be free from avian adenoviruses, avian reoviruses, Chlamydia, duck virus enteritis,
duck virus hepatitis types I and II, influenza type A virus, Newcastle disease virus, Pasteurella (now
Riemerella) anatipestifer, reticuloendoetheliosis virus, and Salmonella infections.
Freedom from other infections may also be required as they become recognised.
The master seed virus should be shown to be non-pathogenic for chickens by inoculating ten times the field
dose into 1-day-old SPF chickens of a strain susceptible to MD, to ensure that it does not cause gross
lesions or significant microscopic lesions of MD by 120 days of age. It should be noted that some vaccine
strains of MDV and HVT may produce minor and transient microscopic nerve lesions.
No increase in virulence should occur during six serial passages of the vaccine strain in 1-day-old SPF MD-
susceptible chickens. Ten times the field dose of vaccine is inoculated initially and then passaged by
inoculation of heparinised blood at 5–7-day intervals, and tests for viraemia are run to check that virus is
transferred at each passage. The birds receiving the final passage are kept for 120 days and should be free
from MD lesions. However, some strains such as Rispens, may cause some mild MD lesions. The important
observation is that the virulence should not change. This is a difficult test because the genetic resistance of
the chickens fundamentally affects the apparent virulence of the virus, so does the type of inoculum. After
successful completion of laboratory safety tests, the safety of the strain should be confirmed in extensive
field trials.
Seed virus must be free from the agents listed for SPF flocks and from other contaminants that may be
acquired in the laboratory. A vaccine strain derived from turkeys must also be free from lymphoproliferative
disease virus and haemorrhagic enteritis virus.
The ability of the master seed virus – and derived virus at the limit of the passage range used to produce
vaccinal virus (usually not more than five tissue culture passages) – to protect against MD must be
determined. Standardised protection tests are published. They involve vaccination of MD-susceptible SPF
chickens at 1 day of age and challenge with sufficient virulent MDV 8 days later to cause at least a 70%
incidence of MD in unvaccinated chickens. Two types of tests are used. In the protection index test, a single
field dose (1000 PFU) (plaque-forming units) of vaccine is given and the incidence of MD in vaccinated
birds is compared with that in unvaccinated birds. Protective indices should be greater than 80, i.e.
vaccinated birds should show at least 80% reduction in the incidence of gross MD, compared with
unvaccinated controls.
A PD50 (50% protective dose) test is also used, involving the inoculation of five four-fold serial dilutions of
vaccine virus selected to provide protection above and below the 50% level, followed by challenge 8 days
later to determine the PD50 value. The assays are conducted using a standard reference vaccine for
comparison. The PD50 may be as low as 4 PFU, but higher values can be obtained depending on the
vaccine strain, whether cell-free or cell-associated and the presence or absence of maternal antibodies in
the test chickens. On the basis of the PD50 test, it has been suggested that the minimum vaccine field dose
should be the greater of two values: 103 PFU or 100 PD50.
Extensive field trials of a new vaccine strain in the presence of field challenge should be conducted, using
different breeds of birds of varying MDV maternal antibody status, to ensure efficacy and persistence of
immunity. Experience suggests that vaccinal immunity, once acquired, is lifelong.

Substrate cells are seeded into flat-bottomed vessels for stationary incubation, or into cylindrical vessels for
rolled incubation. Media commonly used are Eagle’s minimal essential medium, or 199 medium, buffered with
sodium bicarbonate and supplemented with 5% calf serum. Incubation is at 38–39°C for 48 hours.
For cell-associated vaccine, cultures are infected with production HVT or MDV seed-virus stock, in cell-
associated form, which is usually two passages beyond the master seed stock. Cultures are incubated for
48 hours then the infected cells are harvested by treating the washed cell sheet with an EDTA/trypsin solution to
allow the cells to begin to detach. The flasks are then returned to the incubator (38.5°C) to allow complete
detachment. The cells are subjected to low-speed centrifugation, and then resuspended in the freezing mixture
consisting of cell growth medium containing 7.5–15% dimethylsulphoxide (DMSO), and held at 4°C or dispensed
immediately into the final vaccine containers, usually glass ampoules, which are flame sealed and frozen in liquid
nitrogen.
Cell-free lyophilised vaccine may be prepared from HVT, but not from MDV strains. For the production of this
form of vaccine, HVT-infected cultures are incubated for 72 hours, infected cells are detached from the vessel as
described above, or scraped from the walls of the vessel. The cells are suspended in a small volume of growth
medium, centrifuged, and resuspended in a buffered stabiliser solution containing 8% sucrose, but free from
protein to prevent frothing. The cell suspension is sonicated to release virus, the cell debris is removed, the
suspension is diluted with a complete stabiliser – such as SPGA – filled into the final containers, and lyophilised.
The dilution rate for both cell-associated and cell-free vaccines is based on previous experience, as is the
number of doses required per container, because the virus content of the harvested material cannot be assayed
prior to filling the final containers. The virus content of the finished product can subsequently be added to the
label.
For optimal results in preparing cell-associated vaccine, a slow rate of freezing (1–5°C per minute) and rapid
thawing are essential. The infectivity titre of the infected cells, and hence the number of doses per ampoule, are
determined after filling the ampoules. Similarly for cell-free vaccine, the virus content of the final suspension, and
hence the number of doses per container, is determined after filling.
4. Batch control
a) Identity
Using immunofluorescence assay (IFA) with monospecific serum, checks should be carried out to show that
the product is of the same specificity as the seed virus. This is best done using monoclonal antibodies.
b) Safety and sterility

Extensive testing is required of the materials used to produce the vaccine, and of the final product.
Substrate cells should come from an SPF flock, in particular, free from vertically transmitted agents.
Substances of animal origin used in the preparation of vaccines such as serum, trypsin, and bovine serum
albumin, must be free from extraneous agents.
Batches of the final vaccine produced should be tested for freedom from contaminating bacteria, fungi,
mycoplasma and the viruses listed for SPF flocks; tests for purity of the diluent should also be conducted.
Suitable tests for the detection of extraneous agents at all stages of vaccine production are recommended
by several official bodies (20, 22, 30) and in Chapter 1.1.9 Tests for sterility and freedom from
contamination of biological materials.
Ten doses of vaccine or a quantity of diluent equivalent to two doses of vaccine should be inoculated into
separate groups of ten 1-day-old SPF chickens. No adverse reactions should occur during a 21-day
observation period.
c) Potency
The standard dose of each type of vaccine is 1000 PFU per chicken or egg. Virus content assays are
conducted on batches of vaccine to ensure that the correct dose per bird will be achieved.
A test for duration of immunity is carried out on the seed virus only. Such immunity is apparently lifelong.

e) Stability
Tests for stability are carried out on six representative batches of vaccine to show that titre is maintained
during the stated shelf life of the vaccine. These tests should be conducted under the conditions of storage
of the vaccine. The lyophilised product should have a shelf life of 12 months when stored at 2–8°C.
Manufacturers may double the virus content of the vaccine to compensate for some loss of titre during
storage. Appropriate diluting fluids are provided for use with cell-associated and freeze-dried vaccines. The
stability of reconstituted vaccine over a 2-hour period should be tested.
f) Preservatives
Preservatives are not included in the vaccine or diluent.
With cell-associated vaccine, care is necessary to avoid injury from ampoules that may explode when they
are removed from liquid nitrogen. Eye protection must be worn. During use, reconstituted vaccine must be
kept cool and cell-associated vaccine should be agitated to keep cells in suspension.
a) Safety
See Section C.4.b.
b) Potency
See Section C.4.c.
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(MDVs) by the polymerase chain reaction amplification of the tandem direct repeats within the MDV
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30. THORNTON D.H. (1985). Quality control and standardisation of vaccines. In: Marek’s Disease, Payne L.N. ed.
Martinus Nijhoff, Boston, USA, 267–291.
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199. US Government Printing Office, Washington D.C., USA.
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Springer-Verlag, Berlin, Germany, 58–90.
33. W ITTER R.L. & SCHAT K.A. (2003). Marek’s disease. In: Diseases of Poultry, Eleventh Edition, Saif Y.M. et al.,
eds. Iowa State University Press, Ames Iowa, USA, 407–465.
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DNA amplification. Avian Dis., 36, 637–645.
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*
* *
NB: There are OIE Reference Laboratories for Marek’s disease (see Table in Part 3 of this Terrestrial Manual or

CHAPTER 2.3.14.
NEWCASTLE DISEASE
SUMMARY
Newcastle disease (ND) is caused by specified viruses of the avian paramyxovirus type I (APMV-
1) serotype of the genus Avulavirus belonging to the family Paramyxoviridae. There are nine
serotypes of avian paramyxoviruses designated APMV-I to APMV-9.
NDV has been shown to be able to infect over 200 species of birds, but the severity of disease
produced varies with both host and strain of virus. The less pathogenic strains may induce severe
disease when exacerbated by the presence of other organisms or by adverse environmental
conditions. The preferred method of diagnosis is virus isolation and subsequent characterisation.
Identification of the agent: Suspensions in an antibiotic solution prepared from tracheal and
cloacal swabs (or faeces) obtained from live birds, or of faeces and pooled organ samples taken
from dead birds, are inoculated into the allantoic cavity of 9–11-day-old embryonating fowl eggs.
The eggs are incubated at 37°C for 4–7 days. The allantoic fluid of any egg containing dead or
dying embryos, as they arise, and all eggs at the end of the incubation period are tested for
haemagglutinating activity.
Any haemagglutinating agents should be tested for specific inhibition with a monospecific
antiserum to NDV. NDV (APMV-1) may show some antigenic cross-relationship with some of the
other avian paramyxovirus serotypes, particularly APMV-3 and APMV-7.
The pathogenicity of any newly isolated virus can be assessed by determining the intracerebral
pathogenicity index. The pathogenicity of isolates can also be evaluated using molecular
techniques, i.e. reverse-transcription polymerase chain reaction and sequencing. NDV is subject to
official control in most countries and the virus has a high risk of spread from the laboratory;
consequently, appropriate laboratory biosafety and biosecurity must be maintained; a risk
assessment should be carried out to determine the level needed.
Serological tests: The haemagglutination inhibition test is used most widely in NDV serology, its
usefulness in diagnosis depends on the vaccinal immune status of the birds to be tested and on
prevailing disease conditions.
Requirements for vaccines and diagnostic biologicals: Live viruses of low virulence
(lentogenic) or of moderate virulence (mesogenic) are used for the vaccination of poultry
depending on the disease situation. Inactivated vaccines are also used.
Live vaccines may be administered to poultry by various routes. They are usually produced by
harvesting the infective allantoic/amniotic fluids from inoculated embryonating fowl eggs; some are
prepared from infective cell cultures. The final product should be derived from the expansion of
master and working seeds.
Inactivated vaccines are given intramuscularly or subcutaneously. They are usually produced by
the addition of formaldehyde to infective virus preparations, or by treatment with beta-
propiolactone. Most inactivated vaccines are prepared for use by emulsification with a mineral or
vegetable oil.
If pathogenic forms of NDV are used in the production of vaccine or in challenge studies, the
facility should meet the OIE requirements for Containment Group 4 pathogens.

Chapter 2.3.14. — Newcastle disease
A. INTRODUCTION
Newcastle disease (ND) is caused by specified viruses of the avian paramyxovirus type I (APMV-I) serotype of
the genus Avulavirus belonging to the subfamily Paramyxovirinae, family Paramyxoviridae. The paramyxoviruses
isolated from avian species have been classified by serological testing into nine serotypes designated APMV-1 to
APMV-9; ND virus (NDV) has been designated APMV-1 (6).
Since its recognition in 1926, ND is regarded as being endemic in many countries. Prophylactic vaccination is
practised in all but a few of the countries that produce poultry on a commercial scale.
One of the most characteristic properties of different strains of NDV has been their great variation in
pathogenicity for chickens. Strains of NDV have been grouped into five pathotypes on the basis of the clinical
signs seen in infected chickens (15). These are:
1. Viscerotropic velogenic: a highly pathogenic form in which haemorrhagic intestinal lesions are frequently
seen;
2. Neurotropic velogenic: a form that presents with high mortality, usually following respiratory and nervous
signs;
3. Mesogenic: a form that presents with respiratory signs, occasional nervous signs, but low mortality;
4. Lentogenic or respiratory: a form that presents with mild or subclinical respiratory infection;
5. Asymptomatic enteric: a form that usually consists of a subclinical enteric infection.
Pathotype groupings are rarely clear-cut (7) and even in infections of specific pathogen free (SPF) birds,
considerable overlapping may be seen. In addition, exacerbation of the clinical signs induced by the milder
strains may occur when infections by other organisms are superimposed or when adverse environmental
conditions are present.
As signs of clinical disease in chickens vary widely and diagnosis may be complicated further by the different
responses to infection by different hosts, clinical signs alone do not present a reliable basis for diagnosis of ND.
However, the characteristic signs and lesions associated with the virulent pathotypes will give rise to strong
suspicion of the disease.
NDV is a human pathogen. Reported infections have been non-life threatening and usually not debilitating for
more than a day or two (18). The most frequently reported and best substantiated clinical signs in human
infections have been eye infections, usually consisting of unilateral or bilateral reddening, excessive
lachrymation, oedema of the eyelids, conjunctivitis and sub-conjunctival haemorrhage. Although the effect on the
eye may be quite severe, infections are usually transient and the cornea is not affected. Reports of other clinical
symptoms in humans infected with NDV are less well substantiated, but suggest a more generalised infection
may sometimes occur resulting in chills, headaches and fever, with or without conjunctivitis. There is evidence
that both vaccinal and virulent (for poultry) strains of NDV may infect and cause clinical signs in humans. There is
no evidence of human-to-human spread.
ND, as defined in Section B.1.f of this chapter, is subject to official control in most countries and the virus has a
high risk of spread from the laboratory; consequently, a risk assessment should be carried out to determine the
level of biosafety and biosecurity needed for the diagnosis and characterisation of the virus. The facility should
meet the requirements for the appropriate Containment Group as determined by the risk assessment and as
outlined in Chapter 1.1.2 Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities.
Countries lacking access to such a specialised national or regional laboratory should send specimens to an OIE
Reference Laboratory.
a) Samples for virus isolation

When investigations of ND are the result of severe disease and high mortality in poultry flocks, it is usual to
attempt virus isolation from recently dead birds or moribund birds that have been killed humanely. Samples
from dead birds should consist of oro-nasal swabs, as well as samples collected from lung, kidneys,
intestine (including contents), spleen, brain, liver and heart tissues. These may be collected separately or
as a pool, although intestinal samples are usually processed separately from other samples.

Samples from live birds should include both tracheal and cloacal swabs, the latter should be visibly coated
with faecal material. Small delicate birds may be harmed by swabbing, but the collection of fresh faeces
may serve as an adequate alternative.
Where opportunities for obtaining samples are limited, it is important that cloacal swabs (or faeces) and
tracheal swabs (or tracheal tissue) be examined as well as organs or tissues that are grossly affected or
associated with the clinical disease. Samples should be taken in the early stages of the disease.
The samples should be placed in isotonic phosphate buffered saline (PBS), pH 7.0–7.4, containing
antibiotics. Protein-based media, e.g. brain–heart infusion (BHI) or tris-buffered tryptose broth (TBTB), have
also been used and may give added stability to the virus, especially during shipping. The antibiotics can be
varied according to local conditions, but could be, for example, penicillin (2000 units/ml); streptomycin
(2 mg/ml); gentamycin (50 µg/ml); and mycostatin (1000 units/ml) for tissues and tracheal swabs, but at
five-fold higher concentrations for faeces and cloacal swabs. It is important to readjust the solution to
pH 7.0–7.4 following the addition of the antibiotics. If control of Chlamydophila is desired, 0.05–0.1 mg/ml
oxytetracycline should be included. Faeces and finely minced tissues should be prepared as 10–20% (w/v)
suspensions in the antibiotic solution. Suspensions should be processed as soon as possible after
incubation for 1–2 hours at room temperature. When immediate processing is impracticable, samples may
be stored at 4°C for up to 4 days.
b) Virus culture
The supernatant fluids of faeces or tissue suspensions obtained through clarification by centrifugation at
1000 g for about 10 minutes at a temperature not exceeding 25°C are inoculated in 0.2 ml volumes into the
allantoic cavity of each of at least five embryonated SPF fowl eggs of 9–11 days’ incubation. After
inoculation, these are incubated at 35–37°C for 4–7 days. Eggs containing dead or dying embryos as they
arise, and all eggs remaining at the end of the incubation period, should first be chilled to 4°C and the
allantoic fluids tested for haemagglutination (HA) activity. Fluids that give a negative reaction should be
passaged into at least one further batch of eggs.
c) Virus identification
HA activity detected in bacteriologically sterile fluids harvested from inoculated eggs may be due to the
presence of any of the 16 haemagglutinin subtypes of influenza A viruses or of the eight other
paramyxovirus serotypes. (Nonsterile fluid could contain bacterial HA.) NDV can be confirmed by the use of
specific antiserum in a haemagglutination inhibition (HI) test. Usually chicken antiserum that has been
prepared against one of the strains of NDV is used.
Cross-reactions in HI tests between NDV and some of the other APMVs, especially APMV-3 and APMV-
7 serotype viruses may cause some problems that can be resolved by the use of suitable antigen and
antiserum controls.
d) Pathogenicity index
The extreme variation in virulence of different NDV isolates and the widespread use of live vaccines means
that the identification of an isolate as NDV from birds showing clinical signs does not confirm a diagnosis of
ND, so that an assessment of the virulence of the isolate is also required (see Section B.1.f below
‘Definition of Newcastle disease’). In the past such tests as the mean death time in eggs, the intravenous
pathogenicity test and variations of these tests have been used (27), but by international agreement, a
definitive assessment of virus virulence is based on the intracerebral pathogenicity test. The current OIE
definition (Section B.1.f below) also recognises the advances made in understanding the molecular basis of
pathogenicity and allows confirmation of virus virulence, but not lack of virulence, by in-vitro tests that
determine the amino acid sequence at the F0 protein cleavage site.
• Intracerebral pathogenicity index

i) Fresh infective allantoic fluid with a HA titre >24 (>1/16) is diluted 1/10 in sterile isotonic saline with no
additives, such as antibiotics.
ii) 0.05 ml of the diluted virus is injected intracerebrally into each of ten chicks hatched from eggs from
an SPF flock. These chicks must be over 24-hours and under 40-hours old at the time of inoculation.
iii) The birds are examined every 24 hours for 8 days.
iv) At each observation, the birds are scored: 0 if normal, 1 if sick, and 2 if dead. (Birds that are alive but
unable to eat or drink should be killed humanely and scored as dead at the next observation. Dead
individuals must be scored as 2 at each of the remaining daily observations after death.)
v) The intracerebral pathogenicity index (ICPI) is the mean score per bird per observation over the 8-day
period.

The most virulent viruses will give indices that approach the maximum score of 2.0, whereas lentogenic and
asymptomatic enteric strains will give values close to 0.0.
e) Molecular basis for pathogenicity

During replication, NDV particles are produced with a precursor glycoprotein, F0, which has to be cleaved to
F1 and F2 for the virus particles to be infectious. This post-translation cleavage is mediated by host-cell
proteases. Trypsin is capable of cleaving F0 for all NDV strains.
It would appear that the F0 molecules of viruses virulent for chickens can be cleaved by a host protease or
proteases found in a wide range of cells and tissues, and thus spread throughout the host damaging vital
organs, but F0 molecules in viruses of low virulence are restricted in their cleavability to certain host
proteases resulting in restriction of these viruses to growth only in certain host-cell types.
Most ND viruses that are pathogenic for chickens have the sequence 112R/K-R-Q-K/R-R116 at the C-
terminus of the F2 protein and F (phenylalanine) at residue 117, the N-terminus of the F1 protein, whereas
the viruses of low virulence have sequences in the same region of 112G/E-K/R-Q-G/E-R116 and L (leucine)
at residue 117. Some of the pigeon variant viruses (PPMV-1) examined have the sequence 112G-R-Q-K-R-
F117, but give high ICPI values. Thus there appears to be the requirement of at least one pair of basic
amino acids at residues 116 and 115 plus a phenylalanine at residue 117 and a basic amino acid (R) at 113
if the virus is to show virulence for chickens.
Several studies have been done using molecular techniques to determine the F0 cleavage site sequence by
reverse-transcription polymerase chain reaction (RT-PCR), either on the isolated virus or on tissues and
faeces from infected birds, followed by analysis of the product by restriction enzyme analysis, probe
hybridisation or nucleotide sequencing with a view to establishing a routine in vitro test for virulence (for a
review see ref. 2). Determination of the F0 cleavage sequence may give a clear indication of the virulence
of the virus, and this has been incorporated into the definition of ND (see Section B.1.f).
In the diagnosis of ND it is important to understand that the demonstration of the presence of virus with
multiple basic amino acids at the F0 cleavage site confirms the presence of virulent or potentially virulent
virus, but that failure to detect virus or detection of NDV without multiple basic amino acids at the F0
cleavage site using molecular techniques does not confirm the absence of virulent virus. Primer mismatch,
or the possibility of a mixed population of virulent and avirulent viruses means that virus isolation and an in-
vivo assessment of virulence will still be required.
Analyses of viruses isolated in Ireland in 1990 and during the outbreaks of ND in Australia since 1998 have
given strong evidence that virulent viruses may arise from progenitor viruses of low virulence (5, 45).
Virulent NDV has also been generated experimentally from low virulence virus by passage in chickens (39).
f) Definition of Newcastle disease

It seems likely that the vast majority of birds are susceptible to infection with ND viruses of both high and
low virulence for chickens, although the clinical signs seen in birds infected with NDV vary widely and are
dependent on factors such as: the virus, host species, age of host, infection with other organisms,
environmental stress and immune status. In some circumstances infection with the extremely virulent
viruses may result in sudden high mortality with comparatively few clinical signs. Thus the clinical signs are
variable and influenced by other factors so that none can be regarded as pathognomonic.
Even for susceptible hosts, such as chickens, ND viruses show a considerable range of virulence.
Generally, variation consists of clusters around the two extremes in the ICPI test, but, for a variety of
reasons, some viruses may show intermediate virulence.
The enormous variation in virulence and clinical signs means it is necessary to define carefully what
constitutes ND for the purposes of trade, control measures and policies. The definition of ND currently in
use in all member states of the European Union is defined in Directive 92/66/EEC (20).
The OIE definition for reporting an outbreak of ND is:
‘Newcastle disease is defined as an infection of birds caused by a virus of avian paramyxovirus serotype 1
(APMV-1) that meets one of the following criteria for virulence:
a) The virus has an intracerebral pathogenicity index (ICPI) in day-old chicks (Gallus gallus) of 0.7
or greater.
or

b) Multiple basic amino acids have been demonstrated in the virus (either directly or by deduction)
at the C-terminus of the F2 protein and phenylalanine at residue 117, which is the N-terminus of
the F1 protein. The term ‘multiple basic amino acids’ refers to at least three arginine or lysine
residues between residues 113 and 116. Failure to demonstrate the characteristic pattern of
amino acid residues as described above would require characterisation of the isolated virus by
an ICPI test.’
In this definition, amino acid residues are numbered from the N-terminus of the amino acid sequence
deduced from the nucleotide sequence of the F0 gene, 113–116 corresponds to residues –4 to –1 from the
cleavage site.’
g) Monoclonal antibodies
Mouse monoclonal antibodies (MAbs) directed against strains of NDV have been used in HI tests to allow
rapid identification of NDV without the possible cross-reactions with other APMV serotypes that may occur
with polyclonal sera. MAbs have been produced that give reactions in HI tests that are specific for particular
strains or variant NDV isolates (6, 10).
Panels of MAbs have been used to establish antigenic profiles of NDV isolates based on whether or not
they react with the viruses. This has proven to be a valuable method for grouping and differentiating isolates
of NDV, and has been particularly valuable to the understanding of the epidemiology of outbreaks (10).
h) Phylogenetic studies
Development of improved techniques for nucleotide sequencing, the availability of sequence data of more
ND viruses in computer databases and the demonstration that even relatively short sequence lengths could
give meaningful results in phylogenetic analyses have led to a considerable increase is such studies in
recent years. Considerable genetic diversity has been detected, but viruses sharing temporal, geographical,
antigenic or epidemiological parameters tend to fall into specific lineages or clades and this has proven
valuable in assessing both the global epidemiology and local spread of ND (4, 8, 19, 28, 32, 33, 38, 41, 43,
44).
Although in the past phylogenetic studies have been impracticable as a routine tool, the greater availability
and increased speed of production of results obtained using sophisticated, commercially available kits for
RT-PCR and automatic sequencers now means such studies are within the capabilities of many more
diagnostic laboratories and can give meaningful results that are contemporaneous rather that retrospective
(2). Aldous et al. (4) proposed that genotyping of NDV isolates should become part of diagnostic virus
characterisation for reference laboratories by producing a 375-nucleotide sequence of the F gene, which
includes the F0 cleavage site, routinely for all viruses and comparing the sequences obtained with other
recent isolates and 18 viruses representative of the recognised lineages and sub-lineages. Such analysis
should allow rapid epidemiological assessment of the origins and spread of the viruses responsible for ND
outbreaks.
i) Molecular techniques in diagnosis

In addition to the use of RT-PCR and other similar techniques for the determination of the virulence of ND
viruses (see Section B.1.e) or for phylogenetic studies (see Section B.1.h), there has been increasing use
of such molecular techniques to detect NDV in clinical specimens, the advantage being the extremely rapid
demonstration of the presence of virus.
Care should be taken in the selection of clinical samples as some studies have demonstrated lack of
sensitivity in detecting virus in some organs and particularly in faeces (23, 25, 30). Tracheal or
oropharyngeal swabs are often used as the specimens of choice because they are easy to process and
usually contain little extraneous organic material that can interfere with RNA recovery and amplification by
PCR. However, tissue and organ samples and even faeces have been used with some success. The
system used for RNA extraction will also affect the success of RT-PCR on clinical specimens and even with
commercial kits care should be taken in selecting the most appropriate or validated for the samples to be
analysed.
Usually RT-PCR systems have been used to amplify a specific portion of the genome that will give added
value; for example by amplifying part of the F gene that contains the F0 cleavage site so that the product
can be used for assessing virulence (3, 14, 23, 25, 29, 37, 38). Perhaps the most serious problem with the
use of RT-PCR in diagnosis is the necessity for post-amplification processing because of the high potential
for contamination of the laboratory and cross contamination of samples. Extreme precautions and strict
regimens for handling samples are necessary to prevent this (see Chapter 1.1.5 Validation and quality
control of polymerase chain reaction methods used for the diagnosis of infectious diseases).

One of the strategies used to avoid post-amplification processing is to employ real-time RT-PCR (rRT-PCR)
techniques. The advantages of such assays are that rRT-PCR assays based on the fluorogenic hydrolysis
probes or fluorescent dyes eliminate the post-amplification processing step and that results can be obtained
in less than 3 hours. The most successful application of an rRT-PCR assay was in the USA during the ND
outbreaks of 2002–2003, when the assay described by Wise et al. (46) was employed and showed a
sensitivity of 95% when compared with virus isolation for more than 1400 specimens. The assay has three
sets of primers and probes that are used in separate reactions: a matrix primer/probe set that is designed to
detect most strains of NDV, a fusion primer/probe set that can identify virulent strains of NDV (including
many PPMV-1 viruses) and a primer/probe set designed to detect low virulent strains of the virus. Samples
are first screened with the matrix primers/probe then positive specimens are tested with the low virulent and
fusion and primers/probe sets to confirm presence of low or highly virulent virus, respectively. The primers
and probes in this report were validated on lentogenic, mesogenic and velogenic strains circulating in the
United States of America. At the peak of the outbreak, between 1000 and 1500 samples were tested daily
by rRT-PCR. A disadvantage of rRT-PCR is that, at present, the special thermocyclers required are
extremely expensive and this would deter many laboratories from employing this system.
One further important problem is that while the vast majority of NDV isolates are genetically quite close,
some have been shown to be genetically distinct. For example, one group of viruses, which were placed in
genogroup 6 by Aldous et al. (4) and subsequently Class I by Czegledi et al. (24), are so different from all
the other NDV isolates, i.e. Class II viruses (24) that different primers would be necessary for their detection
in RT-PCR tests.
As with virulence determination, it is important that PCR techniques alone are not used to record a negative
result in investigations of suspected ND.
NDV may be employed as an antigen in a wide range of serological tests, enabling neutralisation or enzyme-
linked immunosorbent assays (ELISA) and HI to be used for assessing antibody levels in birds. At present, the HI
test is most widely used for detecting antibodies to NDV in birds, although many poultry producers are using
commercial ELISA kits to assess post-vaccination antibody levels.
a) Haemagglutination and haemagglutination inhibition tests

Chicken sera rarely give nonspecific positive reactions in the HI test and any pretreatment of the sera is
unnecessary. Sera from species other than chickens may sometimes cause agglutination of chicken red
blood cells (RBCs), so this property should first be determined and then removed by adsorption of the serum
with chicken RBCs. This is done by adding 0.025 ml of packed chicken RBCs to each 0.5 ml of antisera,
shaking gently and leaving for at least 30 minutes; the RBCs are then pelleted by centrifugation at 800 g for
2–5 minutes and the adsorbed sera are decanted.
Variations in the procedures for HA and HI tests are practised in different laboratories. The following
recommended examples apply in the use of V-bottomed microwell plastic plates in which the final volume for
both types of test is 0.075 ml. The reagents required for these tests are isotonic PBS (0.01 M), pH 7.0–7.2,
and RBC taken from a minimum of three SPF chickens and pooled in an equal volume of Alsever’s solution.
(If SPF chickens are not available, blood may be taken from unvaccinated birds monitored regularly and
shown to be free from antibodies to NDV.) Cells should be washed three times in PBS before use as a 1%
(packed cell v/v) suspension. Positive and negative control antigens and antisera should be run with each
test, as appropriate.
• Haemagglutination test
i) 0.025 ml of PBS is dispensed into each well of a plastic V-bottomed microtitre plate.
ii) 0.025 ml of the virus suspension (i.e. infective or inactivated allantoic fluid) is placed in the first well.
For accurate determination of the HA content, this should be done from a close range of an initial
series of dilutions, i.e. 1/3, 1/5, 1/7, etc.
iii) Twofold dilutions of 0.025 ml volumes of the virus suspension are made across the plate.
iv) A further 0.025 ml of PBS is dispensed to each well.
v) 0.025 ml of 1% (v/v) chicken RBCs is dispensed to each well.
vi) The solution is mixed by tapping the plate gently. The RBCs are allowed to settle for about 40 minutes
at room temperature, i.e. about 20°C, or for 60 minutes at 4°C if ambient temperatures are high, when
control RBCs should be settled to a distinct button.

vii) HA is determined by tilting the plate and observing the presence or absence of tear-shaped streaming
of the RBCs. The titration should be read to the highest dilution giving complete HA (no streaming);
this represents 1 HA unit (HAU) and can be calculated accurately from the initial range of dilutions.
• Haemagglutination inhibition test

i) 0.025 ml of PBS is dispensed into each well of a plastic V-bottomed microtitre plate.
ii) 0.025 ml of serum is placed into the first well of the plate.
iii) Twofold dilutions of 0.025 ml volumes of the serum are made across the plate.
iv) 4 HAU virus/antigen in 0.025 ml is added to each well and the plate is left for a minimum of 30 minutes
at room temperature, i.e. about 20°C, or 60 minutes at 4°C.
v) 0.025 ml of 1% (v/v) chicken RBCs is added to each well and, after gentle mixing, the RBCs are
allowed to settle for about 40 minutes at room temperature, i.e. about 20°C, or for about 60 minutes at
4°C if ambient temperatures are high, when control RBCs should be settled to a distinct button.
vi) The HI titre is the highest dilution of serum causing complete inhibition of 4 HAU of antigen. The
agglutination is assessed by tilting the plates. Only those wells in which the RBCs stream at the same
rate as the control wells (containing 0.025 ml RBCs and 0.05 ml PBS only) should be considered to
show inhibition.
vii) The validity of results should be assessed against a negative control serum, which should not give a
titre >1/4 (>22 or >log2 2 when expressed as the reciprocal), and a positive control serum for which the
titre should be within one dilution of the known titre.
The value of serology in diagnosis is clearly related to the expected immune status of the affected birds. HI
titres may be regarded as being positive if there is inhibition at a serum dilution of 1/16 (24 or log2 4 when
expressed as the reciprocal) or more against 4 HAU of antigen. Some laboratories prefer to use 8 HAU in
HI tests. While this is permissible, it affects the interpretation of results so that a positive titre is 1/8 (23 or
log2 3) or more. Back titration of antigen should be included in all tests to verify the number of HAU used.
HI titres may be used to assess the immune status of a flock. In vaccinated flocks that are being monitored
serologically, it may be possible to identify anamnestic responses as the result of a challenge infection with
field virus (13), but great care should be exercised as variations may occur from other causes. For example,
it has been demonstrated that APMV-3 virus infections of ND-virus-vaccinated turkeys will result in
substantially increased titres to NDV (11).

There are a variety of commercial ELISA kits available and these are based on several different strategies
for the detection of NDV antibodies, including indirect, sandwich and blocking or competitive ELISAs using
MAbs. At least one kit uses a subunit antigen. Usually such tests have been evaluated and validated by the
manufacturer, and it is therefore important that the instructions specified for their use be followed carefully.
The HI test and ELISA may measure antibodies to different antigens; depending on the system used
ELISAs may detect antibodies to more than one antigen while the HI test is probably restricted to those
directed against the HN protein. However, comparative studies have demonstrated that the ELISAs are
reproducible and have high sensitivity and specificity; they have been found to correlate well with the HI test
(1). Conventional ELISAs have the disadvantage that it is necessary to validate the test for each species of
bird for which they are used. Competitive ELISAs usually employ MAbs which, because of their specificity
for single epitopes, may not recognise all strains of APMV-1.

A detailed account of all aspects of NDV vaccines, including their production and use, has been published (13)
and should be referred to for details of the procedures outlined here. Guidelines for the production of veterinary
vaccines are given in Chapter 1.1.8 Principles of veterinary vaccine production. The guidelines given here and in
Chapter 1.1.8 are intended to be general in nature and may be supplemented by national and regional
requirements.
In this section, conventional live and inactivated vaccines will be considered, as these are still used universally.
However, it should be remembered that there has been much recent work on the application of molecular biology
techniques to the production of new vaccines, and success has been reported in obtaining protective immunity
with recombinant fowlpox virus, vaccinia virus, pigeonpox virus, turkey herpesvirus and avian cells in which the
HN gene, the F gene, or both, of NDV are expressed. Several of these recombinant viruses have been licensed
for use in certain countries.

NDV strains used in conventional commercial live virus vaccines fall into two groups: lentogenic vaccines, such
as Hitchner-B1, La Sota, V4, NDW, I2 and F, and mesogenic vaccines, such as Roakin, Mukteswar and
Komarov. Strains from both these groups have been subjected to selection and cloning to fulfil different criteria in
their production and application. The mesogenic vaccine viruses all have two pairs of basic amino acids at the F0
cleavage site and ICPI values of around 1.4. This means that infections of birds with these viruses would fall
within the intended definition of ND (Section B.1.f), but as these vaccines are used primarily in countries where
ND is endemic this may not necessarily preclude their use. Some countries have specified that only lentogenic
NDV strains can be used as vaccines (42).
The vaccine production facility should operate under the appropriate biosecurity procedures and practices. If ND,
as defined in Section B.1.f of this chapter, is used for vaccine production or for vaccine–challenge studies, that
part of the facility where this work is done should meet the requirements for Containment Group 4 pathogens as
outlined in chapter 1.1.2 of this Terrestrial Manual.
Most live virus vaccines are grown in the allantoic cavity of embryonated fowl eggs but some, notably some
mesogenic strains, have been adapted to a variety of tissue culture systems.
Live virus vaccines may be administered to birds by incorporation in the drinking water, delivered as a coarse
spray, or by intranasal or conjunctival instillation. A lentogenic vaccine for use in ovo has been licensed for use in
the United States of America. Some mesogenic strains are given by wing-web intradermal inoculation. Vaccines
have been constructed to give optimum results through application by specific routes. In general, the more
immunogenic live vaccines are more virulent, and are therefore more likely to cause adverse side-effects. For
example, vaccination with the La Sota strain will cause considerably greater problems in young susceptible birds
than the Hitchner-B1 strain, although La Sota induces a stronger immune response.
Inactivated vaccines are considerably more expensive than live vaccines, and their use entails handling and
injecting individual birds. They are prepared from allantoic fluid that has had its infectivity inactivated by the
addition of formaldehyde or beta-propiolactone. This is incorporated into an emulsion with mineral oil, and is
administered intramuscularly or subcutaneously. Individual birds thus receive a standard dose. There is no
subsequent spread of virus or adverse respiratory reactions. Both virulent and avirulent strains are used as seed
virus although, from the aspect of safety control, the use of the latter appears more suitable. As no virus
multiplication takes place after administration, a much larger amount of antigen is required for immunisation than
for live virus vaccination. A high yield of virus to produce a potent vaccine is important, and the Ulster 2C strain is
very suitable for this purpose.
The duration of immunity depends on the vaccination programme chosen. One of the most important
considerations affecting vaccination programmes is the level of maternal immunity in young chickens, which may
vary considerably from farm to farm, batch to batch, and among individual chickens. For this reason, one of
several strategies is employed. Either the birds are not vaccinated until 2–4 weeks of age when most of them will
be susceptible, or 1-day-old birds are vaccinated by conjunctival instillation or by the application of a coarse
spray. This will establish active infection in some birds that will persist until maternal immunity has waned.
Revaccination is then carried out 3–4 weeks later. It has been demonstrated that inactivated vaccines may also
be usefully employed to vaccinate 1-day-old chicks that have a degree of maternal immunity (17), and the best
results of all were obtained when 1-day-old maternally immune chicks were given a combination of live and
inactivated vaccines, compared with live or inactivated vaccines given alone (16). Vaccination of fully susceptible
1-day-old birds, even with the most mild of live vaccines, may result in respiratory disease, especially if common
pathogenic bacteria are present in significant numbers.
Vaccination after 3 weeks of age is normally practised only in breeding hens and hens laying table eggs. This
should be done at sufficiently frequent intervals to maintain an adequate immunity. Vaccination programmes
often employ slightly more pathogenic live virus vaccines to boost immunity than those used initially. These more
pathogenic live vaccines may also be used following initial vaccination with oil emulsion inactivated vaccines.
When devising a vaccination programme, consideration should be given to the type of vaccine used, the immune
and disease status of the birds to be vaccinated, and the level of protection required in relation to any possibility
of infection with field virus under local conditions (13). Two examples of vaccination programmes that may be
used in different disease circumstances are listed here. For the first example, when the disease is mild and
sporadic, it is suggested that the following order of vaccination be adopted: live Hitchner-B1 by conjunctival or
spray administration at 1 day of age; live Hitchner-B1 or La Sota at 18–21 days of age in the drinking water; live
La Sota in the drinking water at 10 weeks of age, and an inactivated oil emulsion vaccine at point of lay. For the
second example, when the disease is severe and more widespread, the same protocol as above is adopted up to
21 days of age, and this is followed by revaccination at 35–42 days of age with live La Sota in the drinking water
or as an aerosol; this revaccination is repeated at 10 weeks of age with an inactivated vaccine (or a mesogenic
live vaccine) and again repeated at point of lay (13).

1. Seed management

The first principle to consider when selecting a strain for a live NDV vaccine is whether it is to be used as a
primary or a secondary vaccine, the main consideration being its pathogenicity. The methods of application
and frequency of use are valid considerations. The use of MAbs has demonstrated considerable variation in
the antigenicity of different strains (10). This may indicate a need to tailor vaccines more carefully to relate
antigenically to any prevalent field virus.
A live vaccine based on NDV strain V4, selected for heat stability, has been introduced to combat the
specific problems associated with village chicken rearing in developing countries. The intention is that this
vaccine could be coated on food fed to scavenging chickens. To date, trials in different countries have
produced mixed results; it may well be that local factors are extremely important in affecting the success of
this strategy (40). More recently the thermostable I2 vaccine has been developed specifically for vaccinating
village chickens; it is currently recommended that this vaccine be given by eye drop (9).
Use of live vaccines may be restricted by legislation. For example, Commission Decision 93/152/EEC (21)
restricts the use of vaccines in member states of the European Union from 1 January 1995 to those for
which the master seed has been tested and shown to have an ICPI of <0.4 if no fewer than 107 mean egg
infectious doses (EID50) are administered to each bird, or <0.5 if no fewer than 108 EID50 are administered
to each bird. The OIE Standards Commission has similarly recommended that while in principle vaccines
should have an ICPI < 0.7, in order to account for interassay and interlaboratory variability a safety margin
should be allowed so that vaccine master seed virus strains should not have an ICPI exceeding 0.4 (35).
The most important consideration in selecting a seed for the preparation of inactivated vaccine is the
amount of antigen produced when grown in embryonated eggs; it is rarely cost-effective to concentrate
virus. Both virulent and lentogenic strains have been used as inactivated vaccines, but the former offer an
unnecessary risk because the manipulation of large quantities of virulent virus is involved, as well as the
dangers of inadequate inactivation and possible subsequent contamination. This risk is reflected in
Commission Decision 93/152/EEC (21), which restricts the use of viruses used for inactivated vaccine in
member states of the European Union from 1 January 1995 to those for which the master seed has been
tested and shown to have an ICPI of <0.7 if no fewer than 108 EID50 are administered to each bird. Some
lentogenic strains grow to very high titres in eggs. Exceptionally high titres can be obtained by the Ulster 2C
strain, which has been recommended as a seed for inactivated vaccine (26). However, successful
commercial inactivated vaccines are produced when the Hitchner B1, La Sota or F strains are used as
seeds.
In view of the finding that virulent NDV can emerge by mutation from virus of low virulence (5, 39, 45), the
introduction of wholly new strains of ND in live vaccines should be considered carefully and the vaccines
subjected to evaluation before use.
A master seed is established, and from this a working seed. If the strain has been cloned by limiting dilution
or plaque selection, the establishment of a master culture may only involve producing a large volume of
infective allantoic fluid (minimum 100 ml), which can be stored as lyophilised aliquots (0.5 ml).
Seed viruses of unknown pedigree should be passaged through SPF eggs and cloned before producing the
master seed. Some passage through SPF chickens may also be desirable (13). In either case, the master
seed should be checked after preparation for sterility, safety, potency and extraneous agents. Some
countries also require back passage studies for live NDV vaccine to ensure that the pathogenicity is not
increased by cycling through birds (42).
For vaccine production, a working seed, from which batches of vaccine are produced, is first established by
expansion of an aliquot of master seed to a sufficient volume to allow vaccine production for 12–18 months. It is
best to store the working seed in liquid form at –60°C or lower as lyophilised virus does not always multiply to
high titre on subsequent first passage (13).
Most ND vaccines are produced in embryonated fowl eggs, and live virus vaccines should be produced in SPF
eggs. The method of production is large-scale aseptic propagation of the virus; all procedures are performed
under sterile conditions.

It is usual to dilute the working seed in sterile PBS, pH 7.2, so that about 103–104 EID50/0.1 ml is inoculated into
the allantoic cavity of 9- or 10-day-old embryonated SPF fowl eggs. These are then incubated at 37°C. Eggs
containing embryos that die within 24 hours should be discarded. The incubation time will depend on the virus
strain being used and will be predetermined to ensure maximum yield with the minimum number of embryo
deaths.
The infected eggs should be chilled at 4°C before being harvested. The tops of the eggs are removed and the
allantoic fluids aspirated after depression of the embryo. The inclusion of any yolk material and albumin should
be avoided. All fluids should be stored immediately at 4°C and tested for bacterial contamination before large
pools are made for lyophilisation or inactivation. Live vaccines are usually lyophilised. The methodology depends
on the machinery used and the expertise of the manufacturers, but this is a very important step as inadequate
lyophilisation results in both loss of titre and a reduced shelf life.
In the manufacture of inactivated vaccines, the harvested allantoic fluid is treated with either formaldehyde (a
typical final concentration is 1/1000) or beta-propiolactone (a typical final concentration is 1/2000–1/4000). The
time required must be sufficient to ensure freedom from live virus. Most inactivated vaccines are not
concentrated; the inactivated allantoic fluid is usually emulsified with mineral or vegetable oil. The exact
formulations are generally commercial secrets.
Generally oil-based inactivated vaccines are prepared as primary emulsions of water-in-oil. The oil phase usually
consists of nine volumes of highly refined mineral oil, such as Marcol 52, Drakeol 6VR and BayolF, plus one
volume of emulsifying agent, such as Arlacel A, Montanide 80 and Montanide 888 (36). The aqueous phase is
the inactivated virus to which a non-ionic emulsifier such as Tween 80 has been added. The oil phase to
aqueous phase ratio is usually 1:1 to 1:4. Manufacturers strive to reach a balance between adjuvant effect,
viscosity and stability. Too high viscosity and the vaccine is difficult to inject; too low viscosity and the vaccine is
unstable.
Each batch of live virus vaccine should be tested for viability and potency. For those produced in eggs, the most
important process control is testing for bacterial and fungal contamination. This is necessary because of the
occasional occurrence of putrefying eggs, which may remain undetected at the time of harvest.
For inactivated vaccines, the efficacy of the process of inactivation should be tested in embryonated eggs, taking
25 aliquots (0.2 ml) from each batch and passaging each three times through SPF embryos (13).
4. Batch control
Most countries have published specifications for the control of production and testing of NDV vaccines (e.g. ref.
34), which include the definition of the obligatory tests on vaccines during and after manufacture.
It is necessary to test the infectivity of live virus vaccines to enable adequate levels of virus to be administered.
The virus is usually titrated in embryonated fowl eggs to give the EID50. This involves making tenfold dilutions of
virus; 0.1 ml of each dilution is inoculated into between five and seven 9–10-day-old embryonated fowl eggs.
After 5–7 days’ incubation at 37°C, the eggs are chilled and tested for the presence of haemagglutinin activity,
which is an indication of the presence of live virus. The EID50 end-point is calculated using a standard formula
such as Spearman–Kärber (12).
a) Sterility
b) Safety
The use of chickens for the testing of vaccines involves the inoculation of ten or more birds of stated age
that originate from an SPF flock. Ten doses of live vaccine are administered supraconjunctivally to each
bird and the birds are then observed for 21 days. No chicken should show serious clinical signs and none
should die from causes attributable to the vaccine (22). An alternative is to use the prechallenge part of the
potency test below as a safety test and if unfavourable reactions that are attributable to the product occur,
the test is declared inconclusive and the safety test is repeated. If not repeated satisfactorily, the batch is
declared unsatisfactory (42).
For inactivated vaccines, a double dose is administered by the recommended route to ten 3-week-old birds,
and these are observed for 2 weeks for the absence of clinical signs of disease or local lesions.

c) Potency
Various methods for the testing of NDV vaccines for potency have been proposed. The importance of using
a suitable challenge strain for assessment has been stressed (13). Suitable challenge strains are Herts 33
or GB Texas. For live vaccines, the method recommended involves the vaccination of 10 or more SPF or
other fully susceptible birds, some countries specify 20 birds (22), at the minimum recommended age by
the suggested route using the minimum recommended dose. After 14–21 days, each vaccinated bird and
ten control birds are challenged intramuscularly with 105 LD50 (50% lethal dose) of ND challenge virus The
vaccine passes the test if at the end of 10 days, 90% of the vaccinated chickens survive with no signs of
disease, but all controls die within 6 days.
For inactivated vaccines, 21–28-day-old SPF or susceptible chickens are used. Three groups of 20 birds
are injected intramuscularly with volumes of vaccine equivalent to 1/25, 1/50 and 1/100 of a dose. A group
of ten chickens is kept as controls. All the birds are challenged by intramuscular injection of 106 LD50 of ND
challenge virus, 17–21 days later. Chickens are observed for 21 days. The PD50 (50% protective dose) is
calculated by standard statistical methods. The test is only valid if challenged control birds all die within
6 days. The vaccine complies with the test if the PD50 is not less than 50 per dose and if the lower
confidence limit is not less than 35 PD50 per dose. Some control authorities accept a test at 1/50 only, for
animal welfare reasons.
It is not necessary to repeat the potency test on each batch if it has been shown that a representative batch
of the final product from the master seed has passed the test.
The level of immunity reached with any single dose or regimen of ND vaccination will vary enormously with
both vaccine and host species. The level of immunity required in a given host (i.e. to protect against death,
disease, meat or egg production losses) is extremely complex and difficult to evaluate. Generally some
assessment of the longevity of serum antibodies should be made and vaccine regimens adopted to
maintain these above an acceptable level (13).
e) Stability
When stored under the recommended conditions the final vaccine product should maintain its potency for at
least 1 year. Accelerated stability tests such as reduction of infectivity following incubation at 37°C for
7 days (31) may be used as a guide to the storage capabilities of a batch of live vaccine. Oil emulsion
vaccines should also be subjected to accelerated ageing by storing at 37°C, for a minimum of 1 month,
without separation of the aqueous and oil phases. The US requires real-time stability to be checked on the
first few batches of NDV vaccine. Usually three samples are checked for killed vaccine and 10 for live
vaccine. Live virus vaccines must be used immediately after reconstitution. Inactivated vaccines must not
be frozen.
f) Preservatives
In most countries, preservatives must not be included in the freeze-dried live product, but antimicrobial
preservatives may be incorporated in the diluent used to reconstitute the vaccine. An alternative used in the
US is to allow the use of certain preservatives, but they must be indicated on the labelling.
Live NDV vaccines may represent a hazard to humans. ND viruses, both virulent and of low virulence for
chickens have been reported to have infected humans, usually causing acute conjunctivitis following direct
introduction to the eye. Infections are usually transient and the cornea is not involved.
Mineral oil emulsion vaccines represent a serious hazard to the vaccinator. Accidental injection of humans
should be treated promptly by incision and washing of the site, as for a ‘grease-gun’ injury.
a) Safety
See Section C.4.b above.
b) Potency
See Section C.4.c above.

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Phylogenetic analysis of Newcastle disease virus genotypes isolated in Japan. J. Clin. Microbiol., 40, 3826–
3830.
34. MINISTRY OF AGRICULTURE, FISHERIES AND FOOD (1993). Specifications for the Production and Control of Avian
Live Virus Vaccines. Her Majesty’s Stationery Office, London, UK.
35. OFFICE INTERNATIONAL DES EPIZOOTIES (2000). Report of the meeting of the OIE Standards Commission,
November 2000. OIE, Paris, France, p. 4.
36. PALYA V. & RWEYEMAMU M.M. (1992). Live versus inactivated Newcastle disease vaccines. Proceedings of
the FAO Symposium Newcastle Disease Vaccines for Rural Africa, Debre Zeit, Ethiopia, April 1991, 107–
119.

37. PARK N.-Y., CHOI H.-I., CHO H.-S., KANG S.-K., CHO K.-O. & BROWN C. (2002). Development of diagnostic
techniques for Newcastle disease in chickens by in situ RT-PCR and in situ hybridization. Korean J. Vet.
Res., 42, 351–362.
38. SEAL B., KING D.J. & BENNETT J.D. (1995). Characterisation of Newcastle disease virus isolates by reverse
transcription PCR coupled to direct nucleotide sequencing and development of sequence database for
pathotype prediction and molecular epidemiological analysis. J. Clin. Microbiol., 33, 2624–2630.
39. SHENGQING Y., KISHIDA N., ITO H., KIDA H., OTSUKI K., KAWAOKA Y. & ITO T. (2002). Generation of velogenic
Newcastle disease viruses from a nonpathogenic waterfowl isolate by passaging in chickens. Virology, 301,
206–211.
40. SPRADBROW P.B., ED. (1992). Newcastle Disease in Village Chickens. Proceedings No. 39, Australian
Centre for International Agricultural Research, Canberra, Australia, 189 pp.
41. TAKAKUWA H., ITO T., TAKADA A., OKAZAKI K. & KIDA H. (1998). Potentially virulent Newcastle disease viruses
are maintained in migratory waterfowl populations. Jpn J. Vet. Res., 45, 207–215.
42. UNITED STATES DEPARTMENT OF AGRICULTURE (USDA) (2000). Code of Federal Regulations, Title 9, Parts 1–
199. US Government Printing Office, Washington DC, USA.
42. W EHMANN E., CZEGLEDI A., W ERNER O., KALETA E.F. & LOMNICZI B. (2003). Occurrence of genotypes IV, V, VI
and VIIa in Newcastle disease outbreaks in Germany between 1939 and 1995. Avian Pathol., 32, 157–163.
44. W EINGARTL H.M., RIVA J. & KUMTHEKAR P. (2003). Molecular characterisation of avian paramyxovirus 1
isolates collected from cormorants in Canada from 1995 to 2000. J. Clin. Microbiol., 41, 1280–1284.
45. W ESTBURY H. (2001). Commentary. Newcastle disease virus: an evolving pathogen. Avian Pathol., 30, 5–
11.
46. W ISE M.G., SUAREZ D.L., SEAL B.S., PEDERSEN J.C., SENNE D.A., KING D.J., KAPCZYNSKI D. & SPACKMAN E.
(2004). Development of a real-time reverse-transcription PCR for detection of Newcastle disease virus RNA
in clinical samples. J. Clin. Microbiol., 42, 329–338.
*
* *
NB: There are OIE Reference Laboratories for Newcastle disease (see Table in Part 3 of this Terrestrial Manual

CHAPTER 2.3.15.
TURKEY RHINOTRACHEITIS
(avian metapneumovirus)
SUMMARY
Avian metapneumovirus (aMPV) causes an acute highly contagious upper respiratory tract
infection primarily of turkeys and chickens. The disease produced by aMPV was originally referred
to as avian pneumovirus infection and avian rhinotracheitis; it also has been referred to as turkey
rhinotracheitis in turkeys and swollen head syndrome in chickens. Avian metapneumoviruses are
single-stranded non-segmented negative-sense RNA viruses belonging to the family
Paramyxoviridae, genus Metapneumovirus. The disease can cause significant economic losses in
turkey and chicken flocks, particularly when exacerbated by secondary pathogens. The only avian
species known to support the replication of aMPVs, other than turkeys and chickens, are
pheasants, Muscovy ducks and guinea fowl. The disease has global distribution in poultry-
producing regions with only Oceania and Canada reported to be free of aMPV infection. Four
antigentically distinct subtypes, A, B, C and D, of aMPV have been identified by neutralisation with
monoclonal antibodies and sequence analysis of the attachment protein, G.
In susceptible turkeys, infection of the respiratory tract can occur at any age although young poults
appear to be more severely affected. The severity of disease and mortality rate is largely
influenced by secondary bacterial infection. Infection with aMPV can result in serious egg
production problems in turkey breeding flocks. Clinical signs include sneezing, tracheal rales, nasal
and ocular discharge and swollen infraorbital sinuses and conjunctivitis. The onset of clinical signs
and spread of infection through a flock can be rapid occurring as quickly as 2–4 hours. In chickens,
the disease is characterised by apathy and swelling of the face and infraorbital sinuses, leading to
mild respiratory signs, egg production problems and swollen head syndrome (SHS). Cerebral
disorientation, torticollis and opisthotonus frequently follow. In broilers, aMPV is not a primary
pathogen, but is involved with other agents in SHS or other respiratory disease complexes.
Identification of the agent: Virus isolation in cell cultures, embryonated eggs, and tracheal organ
cultures as well as molecular methods for identification of the nucleic acid have all been used
successfully to detect aMPV, but the degree of success depends on the strain of virus, type and
timeliness of sample collection, as well as storage and handling of specimens. Electron
microscopy, virus neutralisation and molecular techniques are widely used to identify the virus.
Virus detection and identification can be difficult unless samples are taken early in the course of
the disease. Both attenuated and virulent strains of aMPV replicate to the highest titre in the upper
respiratory tract tissues of young turkeys, but only for approximately 10 days.
Monoclonal antibodies to the G glycoprotein have been used in virus neutralisation tests to
differentiate subtypes A and B, while neutralisation tests using polyclonal antiserum have shown
that subtypes A and B belong to a single serotype. Subtype C is neutralised poorly by subtype A or
B monospecific antiserum, and not by monoclonal antibodies that differentiate subtype A and B.
These data suggest that subtype C represents a second serotype of aMPV. Monospecific
antiserum and monoclonal antibodies can be used for agent identification by virus neutralisation
and immunofluorescence staining of infected cell cultures; however antigenic characteristics need
to be considered. The immunodiffusion test has also been used to confirm aMPV isolates.
Molecular procedures based on the F, G, M and N genes of aMPV have been used for the
detection and or genomic subtyping of aMPV. Proteins coded by the F and G genes are major
immunogens and, as such, present nucleotide variability. Nucleotide sequence analysis of the G
gene has been used to confirm previous virus neutralisation studies differentiating subtypes A and

Chapter 2.3.15. — Turkey rhinotracheitis (avian metapneumovirus)
B. Conventional reverse-transcriptase polymerase chain reaction (RT-PCR) procedures can also

be used for aMPV A and B genomic subtyping. Moreover, nucleotide sequence analysis of the
gene encoding the matrix protein (M), further supports the subtype A and B division. Sequence
analysis of the M gene of subtype C aMPV shows the United States of America isolate to be
distinct from subtype A and B viruses. RT-PCR assays directed to the F and M genes are subtype
specific and useful for detection of aMPV when subtype identity of the virus is known. However, a
single set of RT-PCR primers directed to the N gene have been shown to detect subtypes A, B and
could possibly be used as universal primers for the detection of aMPV.
Serologic tests: Due to difficulties in isolating and identifying aMPV, confirmation of infection is
often achieved by serological methods, particularly in unvaccinated flocks. The most commonly
employed method is the enzyme-linked immunosorbent assay (ELISA). Other methods that have
been used are virus neutralisation (VN), immunofluorescence and immunodiffusion tests. The VN
test can be performed in tracheal organ cultures, chicken embryo fibroblast (CEF), chicken embryo
liver (CEL) or Vero cell cultures. However, the VN and ELISA show similar sensitivity and the
ELISA is the most commonly used assay. Numerous commercial ELISA kits as well as in-house
assays have been developed. Differences in sensitivity and specificity between tests have been
reported to occur between commercial kits. A homologous strain of aMPV should be used as
antigen due to variations in antigenicity. In many countries where the disease is endemic,
vaccination is practised complicating interpretation of the results. Ideally, serum samples from
animals in the acute phase of disease and from convalescent animals should be obtained for
testing. In chickens the serological response to aMPV infection is weak when compared with the
response in turkeys.
Requirements for vaccines and diagnostic biologicals: This section is under study, it is
anticipated that information on vaccines will be added to the online version of the Terrestrial
Manual from June 2008, subject to approval by the OIE International Committee.
A. INTRODUCTION
Avian metapneumovirus (aMPV) previously referred to as avian pneumovirus (APV) and avian rhinotracheitis
(ART) virus causes an acute, highly contagious upper respiratory tract infection of turkeys and chickens. In
turkeys, the virus causes a disease known as turkey rhinotracheitis (TRT). The aetiological agent is an
unsegmented single-stranded negative-sense RNA virus of approximately 15 kilo bases with helical symmetry
(17). The virus is well characterised as a pneumovirus, but differs from mammalian pneumoviruses at the
molecular level and has recently been classified as the type strain of a new genus, Metapneumovirus, in the
family Paramyxoviridae (35). Recent reports have indicated that similar viruses have been detected in humans
associated with respiratory tract infection in children (30, 48). Avian metapneumovirus has no non-structural NS1
and NS2 proteins and the gene order (3’-N-P-M-F-M2-SH-G-L-5’) is different from that of mammalian
pneumoviruses (3’-NS1-NS2-N-P-M-SH-G-F-M2-L-5’) (46). Avian metapneumovirus has been classified into four
subtypes; A, B, C and D based on nucleotide sequence analysis and neutralisation with monoclonal antibodies
(8, 14). Other subtypes may exist but have not yet been detected and identified.
Infection with aMPV can occur from a very young age in turkeys and is characterised by snicking, rales,
sneezing, nasal discharge, foaming conjunctivitis, swelling of the infraorbital sinuses and submandibular edema
(37). Secondary adventitious agents can dramatically exacerbate the clinical signs. In an uncomplicated
infection, recovery is rapid and the birds appear normal in approximately 14 days. When husbandry is poor or
secondary bacterial infection occurs, airsacculities, pericarditis, pneumonia, and perihepatitis may also cause
increasing morbidity and mortality rates (12, 28). Secondary agents that have been shown to exacerbate and
prolong clinical disease are Bordetella avium, Pasteurella-like organisms, Mycoplasma gallisepticum,
Ornithobacterium rhinotracheale and Escherichia coli (1, 12, 21, 41). Morbidity can be as high as 100%, with
mortality ranging from 0.5% in adult turkeys to 80% in young poults (5, 17, 51). Clinical signs of infection in
chickens are less characteristic than those in turkeys. Severe respiratory distress may occur in broiler chickens
particularly when exacerbated by secondary pathogens such as infectious bronchitis virus, mycoplasmas, and
Escherichia coli (33, 34). Unlike subtype A and B, the United States of America (USA) strain (subtype C) has not
been shown to naturally induce disease in chickens although experimentally infected chickens were shown to be
susceptible to a subtype C turkey isolate of aMPV (43). Different strains of aMPV have been shown to have a
specific tropism for chickens or turkeys (15). Evidence shows other species of birds can be infected with aMPV,
however clinical signs have rarely been reported (18). Viruses similar to subtype C aMPV have been reported to
occur in ducks in France associated with respiratory signs and egg production problems (47). Retrospective
molecular analysis of viruses isolated in the 1980s from turkeys in France indicates the presence of a fourth
subtype of aMPV designated subtype D (3). The results of experimental studies suggest that direct contact is

necessary for bird-to-bird spread of the virus (1, 12). In commercial conditions transmission is also likely to be
airborne as the disease is restricted to the respiratory tract. Following experimental infection of 2-week-old
turkeys the virus was detected in the respiratory tract for only a few days (2) in birds inoculated with aMPV alone.
However, in birds inoculated with aMPV and B. avium virus was detected for up to 7 days post-inoculation (dpi)
(7). There is no evidence that aMPV can result in a latent infection and no carrier state is known to exist.
Although neonatal turkeys are occasionally infected (42) there are no reports of vertical transmission of aMPV.
In growing turkeys virus replication is limited to the upper respiratory tract and is short in duration. Replication of
both attenuated and virulent strains of aMPV persist for approximately 10 dpi (12, 51). Limited replication occurs
in the trachea and lung, but virus has not been shown to replicate in other tissues following natural infection (9,
10). Sequential histopathological and immunocytochemical studies have shown viral replication in the turbinates
causing a serous rhinitis with increased glandular activity, epithelial exfoliation, focal loss of cilia, hyperaemia and
mild mononuclear infiltration in the submucosa at 2 dpi. A catarrhal rhinitis with mucopurulent exudate, damage
to the epithelial layer and a copious mononuclear inflammatory infiltration in the submucosa was seen 3–4 dpi.
Transient lesions were seen in the trachea, with little or no lesions present in the conjunctiva and other tissues
(24). Respiratory infection is less severe in laying turkeys; however, there may be a drop in egg production of up
to 70% (45) and the quality of eggs during the recovery period, up to 3 weeks, may be poor.
In chickens there is strong evidence to suggest aMPV is one of the aetiological agents of swollen head syndrome
(SHS). The syndrome is characterised by respiratory disease, apathy, swelling of infra-orbital sinuses and
unilateral or bilateral facial swelling, extending over the head. These signs are frequently followed by cerebral
disorientation, torticollis and opisthotonos. Although mortality does not usually exceed 1–2%, morbidity may
reach 10%, and egg production is frequently affected (19, 29, 33, 34, 46).
Serological evidence suggests aMPV is widespread throughout the world and of considerable economic
importance, particularly in turkeys. Oceania and Canada are the only regions that have not reported aMPV (9,
10, 17). There is serological and molecular evidence that aMPV occurs in a variety of other avian species,
including pheasants, guinea fowl, ostriches, passerines and various waterfowl (4, 17, 44), but there is no
evidence of serious disease.
To maximise the chances of successfully isolating the virus, a multiple approach to diagnosis is recommended.
This is particularly relevant when dealing with different subtypes or genotypes that may require varied in-vitro
methods to isolate the virus. This was illustrated in the USA with the failure to culture subtype C aMPV, which is
not associated with ciliostasis, in tracheal organ cultures (13, 41). The agent was cultured following multiple
embryo and cell culture passages. This was in contrast to the European experience and elsewhere in which
tracheal organ cultures were shown to be the most reliable method for the primary isolation of subtype A and B
aMPV (11).
• Collection and selection of diagnostic specimens

It is very important to take samples for attempted virus isolation in the early stages of infection as the virus
may be present only in the sinuses and turbinates for a short period. Ideally, the upper respiratory tract of
live birds in the acute phase of the disease should be sampled using sterile swabs (17, 45). The most
successful samples have been nasal exudates, choanal cleft swabs and scrapings of sinus and turbinate
tissue. The virus has also been isolated from trachea and lungs, and occasionally viscera of affected turkey
poults (5, 13). Isolation of virus is rarely successful from birds showing severe chronic signs as the extreme
clinical signs are usually due to secondary adventitious agents. This certainly applies to SHS of chickens in
which the characteristic signs appear to be due to secondary Escherichia coli infection. Furthermore, for
reasons that are unclear, virus isolation from chickens appears to be more difficult than from turkeys.
It is essential that samples requiring dispatch to a diagnostic laboratory be sent immediately on ice. When
delays of more than 3 days are expected, the samples should be frozen prior to dispatch. Swabs for
attempted virus isolation should be sent on ice fully immersed in viral transport medium, but those for
polymerase chain reaction (PCR) analysis can be sent dry.
For virus isolation, a 20% (v/v) suspension of the nasal exudate or homogenised tissue is made in
phosphate-buffered saline (PBS) or brain–heart infusion (BHI) broth containing antibiotics at pH 7.0–7.4.
This is then clarified by centrifugation at 1000 g for 10 minutes and the supernatant is passed through a
450 nm membrane filter.

• Culture and Identification of avian metapneumovirus (aMPV)

The best method for primary virus isolation from infected birds is in tracheal organ cultures or embryonating
turkey or chicken eggs and subsequent cultivation in cell cultures (5, 13). The original isolation of aMPV in
South Africa in the late 1970s and the more recent Colorado aMPV were isolated in embryonating eggs,
however subtype A and B aMPV isolations have routinely been conducted in tracheal organ cultures (11).
Subtype C aMPV, and perhaps other non-identified APV, do not cause ciliostasis in organ cultures and for
this reason embryonated eggs and subsequent cultivation in cell culture is the preferred method of primary
virus isolation (17, 41).
Tracheal organ cultures are prepared from turkey embryos or very young turkeys obtained from flocks free
of specific antibodies to aMPV. Tracheas from chicken embryo or 1-to-2-day-old chicks may also be used.
Transverse sections of trachea are rinsed in PBS (pH 7.2), placed one section per tube in Eagles medium
with antibiotics, and held at 37°C. For inoculation with infective material, the tubes are drained, and 0.1 ml
of bacteria-free inoculum is added. After incubation for 1 hour at 37°C growth medium is added and the
cultures are incubated at 37°C on a roller apparatus rotating at 30 revolutions per hour. Cultures are
examined daily after agitation on a laboratory mixer to remove debris from the lumen. Ciliostasis may occur
within 7 days of inoculation on primary passage but usually is produced rapidly and consistently only after
several blind passages (17).
Six-to-8-day-old embryonating chicken or turkey eggs from flocks known to be free of aMPV antibodies are
inoculated by the yolk-sac route with 0.2–0.3 ml of bacteria-free material from infected birds and incubated
at 37°C. Yolk sac material should be processed for a second blind embryo passage if there is no evidence
of infection (embryo stunting or mortality) with the first passage. Within 7–10 days, there is usually evidence
of stunting of the embryos with few deaths. Serial passage is often required before the agent causes
consistent embryo mortality. Isolation in embryonated eggs is slow, expensive, labour intensive, and
requires multiple subsequent cell culture passages for identification (17).
Various cell cultures have been used for the primary isolation of aMPV, including chicken embryo cells,
Vero cells and more recently the QT-35 cells, with varying degrees of success. Primary isolation of subtype
C has been conducted with multiple (5–6 serial passages) in Vero cell cultures (4). However, once the virus
has been adapted to growth in embryonating eggs or tracheal organ cultures, in which it grows only to low
titres, the virus will readily replicate to moderate titres following multiple passages in a variety of primary
chicken or turkey embryo cells, Vero cells, and QT-35 cells (5, 9, 10, 20, 38). The virus produces a
characteristic cytopathic effect (CPE) with syncytial formation in QT-35 and chicken embryo fibroblast cells
within 7 days. Identification of virus-infected cell culture is conducted by immunofluorescence staining of
infected cells or by plaque reduction neutralisation assay (PRNA) in QT-35 cells (39, 40).
By negative-contrast electron microscopy, the virus has a paramyxovirus-like morphology. Pleomorphic

fringed particles, roughly spherical and 80–200 nm in diameter are commonly seen. Occasionally much
larger filamentous forms are present, which may be up to 1000 nm in length. The surface projections are
13–14 nm in length and the helical nucleocapsid that can sometimes be seen emerging from disrupted
particles is 14 nm in diameter with an estimated pitch of 7 nm per turn (7).
• Molecular Identification
Reverse-transcriptase PCR (RT-PCR) is a significantly more sensitive and rapid method for the detection of
aMPV than standard virus isolation methods because of the fastidious nature of aMPV (11, 17). RT-PCR
procedures targeted to the F, M, N and G genes are used for the detection of aMPV. However, because of
molecular heterogeneity between aMPV strains, most RT-PCR procedures are subtype specific or do not
detect all subtypes (2, 11, 35, 36). Subtype specific assays are successfully used for the detection and
diagnosis of endemic strains (3, 11, 26, 31, 36). However, limitations of subtype-specific assays need to be
recognised when conducting diagnostic testing for respiratory disease. Primers directed to conserved
regions of the N gene have been shown to have broader specificity, detecting representative isolates from
A, B, C, and D subtypes (3). RT-PCR assays directed to the G gene have also been successfully used for
genotype or subtype identification (22, 23, 25). A variety of RT-PCR techniques have been developed and
evaluated and these have been extensively reviewed elsewhere (11, 32).
Nasal exudates, choanal cleft swabs, and turbinate specimens collected 2–7 days post-exposure are the
preferred specimen (15, 17, 36, 45). It is imperative to collect specimens when clinical signs are first
exhibited as recent studies have shown that the maximum amount of virus is present in the trachea and
nasal turbinates at 3 days post-inoculation and viral RNA persists for 9 days in the trachea and up to
14 days in the nasal turbinates (49). It has been shown that aMPV can be detected from specimens
collected 7–10 days post-exposure, however the viral concentration is considerably less thus reducing
success of detection (1, 36). Swabs from a single flock can be pooled in groups of five to increase recovery
rate.

Template RNA for RT-PCR can be extracted from homogenised tissue, dry swabs or wet swab pools with
silica column or magnetic bead commercial RNA extraction reagents according to manufacturer’s protocol.
Tracheal swab supernatant and sinus fluid (140 µl/600 µl lysis buffer) specimens can also be processed
with the RNeasy® (Qiagen, Valencia, CA) procedure.
Target Primer ID Sequence 5’–3’ Position Product size
N Gene Nc 5’–TTC-TTT-GAA-TTG-TTT-GAG-AAG-A–3’ 632–653 RT primer
Nx 5’–CAT-GGC-CCA-ACA-TTA-TGT-T–3’ 830–812 115
Nd 5’–AGC-AGG-ATG-GAG-AGC-CTC-TTT-G–3’ 716–737 115
i) Synthesis of the cDNA can be carried out in 20 µl volume with the Nc RT primer and SuperScript II®
Rnase H-RT (Invitrogen, Carlsbad, CA) enzyme. Heat 1 µl RT primer (2 pmol), 1 µl dNTP mix (10 mM
each), with extracted RNA and sterile distilled water (QS to 20 µl) to 65°C for 5 minutes.
ii) Chill quickly and pulse centrifuge.
iii) Add 4 µl 5× First-Strand buffer, 2 µl 0.1 M DTT, and 1 µl RNaseOUT® (Invitrogen, Carlsbad, CA).
iv) Heat contents to 42°C for 2 minutes and add 1 µl (200 units) of SuperScript II®, mix gently.
v) RT is conducted at 42°C for 50 minutes followed by 70°C for 15 minutes for inactivation of RT enzyme.
vi) PCR amplification can be conducted with AmliTaq Gold® polymerase (Applied Biosystems, Foster
City, CA) according to manufacturer’s instructions. Amplification conditions are as follows: 94°C for
15 minutes and 30 cycles of 94°C for 20 seconds, 51.0°C for 45 seconds, 72°C for 45 seconds with a
final extension of 72°C for 10 minutes.
Several RT-PCR assays directed to the F, G and M genes have been successfully used for subtype
identification and detection or diagnosis of endemic aMPV (20, 21, 24). Nucleotide sequence and
phylogenetic analysis of the G gene has been used to genotype subtype A, B, and C aMPV and is the
recommended procedure for subtype identification of an unidentified virus. Recommended RT-PCR
procedures for sequence analysis of the G gene have been described (22, 23). Procedures for the
identification of subtype A and B RNA in diagnostic specimens have also been described (31), as have
procedures for the detection of subtype A and C viruses (36). Isolation of aMPV from chickens is difficult
and has succeeded only in a limited number of cases; for this reason, molecular tests have been used
primarily for the detection of aMPV in chickens (11, 25). It is important to remember that PCR detects viral
RNA, not live virus, so the significance of a positive PCR result in terms of detecting an active infection
remains to be established, particularly if live vaccination is employed routinely.
2. Serologic tests
Due to difficulties in isolating and identifying aMPV, serology is the most common method of diagnosis of
aMPV infections, particularly in unvaccinated flocks. The most commonly employed method is the ELISA;
however, virus neutralisation, microimmunofluorescence and immunodiffusion tests have been used (39,
40). A number of commercial and in-house ELISA kits are available that are suitable for testing both turkey
and chicken serum; however, differences in sensitivity and specificity between commercial kits have been
reported (27, 28). Competitive or blocking ELISA kits incorporating an aMPV-specific monoclonal antibody
have been developed. These kits claim to have a broad spectrum of sensitivity and specificity for all
subtypes of aMPV and can be used for testing sera from a variety of avian species. In-house ELISA
antigens, as described below, have been prepared in a variety of substrates including various cell cultures
and tracheal organ cultures (6, 11). Generally, aMPV antibodies are less well detected when a heterologous
strain of aMPV is used as antigen, even though the strains appear closely related by virus neutralisation
test (11). The situation is further complicated by discrepancies in the ability of different ELISAs to detect
vaccinal antibody when different aMPV strains are used as coating antigens (16, 40). In-house assays
using a homologous antigen have been used extensively for the surveillance of endemic aMPV strains.
Ideally, both acute and convalescent serum samples should be obtained for testing. In chickens, the
serological response to aMPV infection is weak when compared to the response in turkeys (12).
• Enzyme-linked immunosorbent assay

Virus is propagated in chicken embryo fibroblast (CEF) or Vero cell cultures until 70–100% of the monolayer
is simultaneously infected (3–4 days). The cell culture fluid is decanted and the monolayer washed with
PBS (pH 7.2). The monolayer is lysed with 0.5 ml (per 75 cm2 flask) of a 0.5% non-ionic detergent solution
(IGEPAL CA-630 or Nonidet P-40) on a rocking platform for 1 hour at 4°C. Following physical disruption of
lysed cells, the whole virus antigen lysate is clarified at 3000 g for 15 minutes. Uninfected cell cultures are

treated in the same manner for a negative control antigen. Serial dilutions of antigen are tested against
serial dilutions of anti-species IgG horseradish peroxidise conjugate in a checker-board fashion to
determine the optimal antigen/conjugate dilution. A working dilution of the aMPV antigen and normal
antigen (100 µl) are coated onto flat-bottom microtitre plates with a carbonate/bicarbonate coating buffer
(6). Each serum is tested against aMPV and normal antigen for determination of the S/P ratio. Coated
plates are incubated at 4°C overnight and washed a total of five times with a Tween 20 wash solution (6)
prior to use or three times prior to long-term storage at –70°C. Residual wash solution remains on the plate
when the plates are frozen. Following storage and equilibration to room temperature, the plates are washed
twice and blotted dry prior to use.
i) Dilute test sera 1/40 in dilution/blocking buffer (6).

ii) Apply 50 µl test sera and working dilutions of positive and negative sera to aMPV antigen and normal
antigen-coated wells.
iii) Incubate at room temperature for 1 hour.
iv) Wash plates five times with Tween 20 wash solution
v) Apply 50 µl of the working dilution of anti-species IgG horseradish peroxidise conjugate to each well
and incubated for 1 hour at room temperature.
vi) Wash plates five times with Tween 20 wash solution
vii) Apply 100 µl of the prepared ortho-phenylenediame (OPD) substrate solution to each well and
incubated for 10 minutes in the dark. Combine the following reagents for preparation of OPD
substrate: 243 ml 0.1 M citric acid, 257 ml 0.2 M disodium hydrogen phosphate, adjust pH to 5.0 and
QS to 1 liter with distilled water.
viii) Stop the reaction with 25 µl/well of 2.5 M sulphuric acid.
ix) Read the OD at 490/450 nm.
The results are expressed as the OD difference between the virus antigen-coated and negative control
antigen-coated wells. Determine the mean OD490 reading for each duplicate set of wells with the positive
and negative antigen for each serum. A sample with an OD490 difference between the antigen-coated and
negative control antigen-coated wells of more than 0.2 is considered positive. Sporadic non-specific positive
reactions are inherent with the ELISA test, especially with chicken serums, and immunofluorescence may
be used for confirmation testing.

This section is under study, it is anticipated that information on vaccines will be added to the online version of the
Terrestrial Manual from June 2009, subject to approval by the OIE International Committee.
REFERENCES
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and N-based RT-PCR protocols with conventional virological procedures for the detection and typing of
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protein genes of two non-A/non-B avian pneumoviruses (APV) reveal a novel APV subgroup. J. Gen. Virol.,
81, 2723–2733.
4. BENNETT R.S., NEZWORSKI J., VELAYUDHAN B.T., NAGRAJA K.V., ZEMAN D.H., DYER N. GRAHAM T., LAUER D.C.,
NJENGA M.K. & HALVORSON D.A. (2004). Evidence of avian pneumovirus spread beyond Minnesota among
wild and domestic birds in central North America. Avian Dis., 48, 902–908.
5. BUYS S.B. & DU PREEZ J.H. (1989). The isolation and attenuation of a virus causing rhinotracheitis in turkeys
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*
* *
NB: There is an OIE Reference Laboratory for Turkey rhinotracheitis (see Table in Part 3 of this Terrestrial

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What is the purpose of the OIE Terrestrial Manual?

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WORLD ORGANISATION FOR ANIMAL HEALTH

MANUAL OF DIAGNOSTIC TESTS AND VACCINES

OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals

Manual of Recommended Diagnostic Techniques and Requirements for Biological Products:

OIE Terrestrial Manual 2008 iii

Doctor Bernard Vallat Professor Steven Edwards

iv OIE Terrestrial Manual 2008

OIE Terrestrial Manual 2008 v

PART 1 GENERAL INFORMATION

SECTION 1.1. INTRODUCTORY CHAPTERS

PART 2 OIE LISTED DISEASES AND OTHER DISEASES OF IMPORTANCE TO

SECTION 2.1. MULTIPLE SPECIES

OIE Terrestrial Manual 2008 vii

Chapter 2.1.18. Tularemia.................................................................................................................. 361

SECTION 2.2. APIDAE

SECTION 2.2. AVES

viii OIE Terrestrial Manual 2008

• Arrangement of the Terrestrial Manual

There is an alphabetical index of the diseases at the end of Volume 2.

• Explanation of the tests described and of the table on pages xi–xiv

OIE Terrestrial Manual 2008 ix

• List of OIE Reference Laboratories

x OIE Terrestrial Manual 2008

Chapter No. Disease name Prescribed tests Alternative tests

2.1.5. Foot and mouth disease ELISA *, VN CF

2.1.6. Heartwater – ELISA, IFA

2.1.11. Paratuberculosis (Johne’s disease) – DTH, ELISA

2.1.16. Trichinellosis Agent id. ELISA

* Please refer to Terrestrial Manual chapters to verify which method is prescribed.

OIE Terrestrial Manual 2008 xi

Chapter No. Disease name Prescribed tests Alternative tests

2.2.3. European foulbrood of honey bees – –

xii OIE Terrestrial Manual 2008

Chapter No. Disease name Prescribed tests Alternative tests

2.4.15. Malignant catarrhal fever – IFA, PCR, VN

2.5.10. Equine viral arteritis Agent id. (semen –

2.5.12. Horse mange – Agent id.

OIE Terrestrial Manual 2008 xiii

Chapter No. Disease name Prescribed tests Alternative tests

2.8.4. Nipah virus encephalitis – –

2.9.9. Salmonellosis – Agent id.

Agent id. Agent identification HI Haemagglutination inhibition

xiv OIE Terrestrial Manual 2008

ABTS 2,2’-azino-di-(3-ethyl-benzthiazoline)-6- FBS Fetal bovine serum

OIE Terrestrial Manual 2008 xv

MSV Master seed virus RBC Red blood cell

xvi OIE Terrestrial Manual 2008

OIE Terrestrial Manual 2008 xvii

• Dilutions used in virus neutralisation tests

• Final product (lot)

xviii OIE Terrestrial Manual 2008

• Inter-laboratory comparison (ring test)

• Master cell (line, seed, stock)

• Master seed (agent, strain)

• Predictive value (negative)