FEDERAL UNIVERSITY OF TECHNOLOGY, OWERRI

P.M.B 1526, OWERRI

IMO STATE

INCIDENCE OF ESCHERICHIA COLI AND

SALMONELLA IN DIFFERENT BRNDS OF POULTRY
FEEDS

BY

ONWUCHURUBA REMIJUS OBINNA

REG. NO: 20161945905

DEPARTMENT OF MICROBIOLOGY
SCHOOL OF BIOLOGICAL SCIENCES
SUPERVISED BY

MR O. I OGUOMA

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR

THE AWARD OF BACHELOR OF TECHNOLOGY (B.TECH) IN
MICROBIOLOGY

OCTOBER 2022
 CERTIFICATION
I certify that this project ‘incidence of Escherichia coli and salmonella in different brands of
poultry feeds’ is an original work by Onwuchuruba, Remijus O. (20161945905) of the school of
biological sciences, Federal University Of Technology Owerri in accordance with the
requirements of the school of biological sciences.

…………………….. ………………………
Onwuchuruba, Remijus O. Date

i
 APPROVAL PAGE
It is hereby certified that this thesis titled incidence of Escherichia coli and salmonella in
different brands of poultry feeds was written by Onwuchuruba, Remijus O. (20161945905)and
has been read and approved as meeting the requirements of the school of Biological sciences ,
Federal University of Technology Owerri for the award of Bachelor of
Technology(B.Tech)Degree in Microbiology.

………………………. ……………………….
Mr. O.I. Oguoma Date
Supervisor

………………………. ……………………….
Prof. Ifechukwu Enyinna Adieze Date

Head of Department

………………………. ……………………….
Prof. Chinwe S. Alisi Date
Dean, school of Biological Sciences

………………………. ……………………….
Prof. N.I. nwanyanwu Date
External Examiner

ii
 DEDICATION
This study is dedicated to the Onwuchuruba families.

iii
 Acknowledgements

Firstly I thank the Almighty creator who is the source of my living and inspirations, to him alone be the glory
for he gives understanding wisdom and ideas which led in the writing of this work and its successful
completion in the midst of obvious challenges.
I would love to express my profound gratitude to Prof. Ifechukwu Enyinna Adieze
(Head of Department), Department of microbiology in the faculty of biological sciences, Federal University
of Technology Owerri and all the staffs in the department.
My gratitude goes also to Mr. O.I. Oguoma my project Supervisor who in his busy schedule brought out time
to go through my work, I sincerely thank Prof. Braide for his assistance and support throughout the
experimental procedures, words will not be enough to express my gratitude to my loving parents Mr. and
Mrs. R. Onwuchuruba and my aunt Dr. Mrs. Augustina Anaeme and my family for standing beside me all
these time. To my fellow course mates and friends Duru Sandra, nze oziomajesus, Divinelove oluebube,
Nnewdimma, Dr. coins, Ebuka, Ifeanyi, Jane, Faustina, Marvis chimezie, Walter, success and so many others
who contributed to the work, May God Almighty Bless You All.

iv
 TABLE OF CONTENTS

COVER PAGE

TITLE PAGE

CERTIFICATION PAGE i

APPROVAL PAGE ii

DEDICATION iii

ACKNOLEDGEMENTS iv

TABLE OF CONTENTS v

LIST OF TABLES viii

ABSTRACT ix

CHAPTER ONE: INTRODUCTION 1

1.1 Background to the study 1

1.2 Statement of the problem 4

1.3 Scope of the study 5

1.4 Purpose of the study 5

1.5 Significance of the study 5

1.6 Research Questions 5

1.7 Hypotheses 5
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CHAPTER TWO: REVIEW OF LITERATURE

2.1 Major Concepts of the study 8

2.2 Theoretical Framework 31

2.3 Review of Empirical Studies 34

2.4 Summary of literature Reviewed 35

CHAPTER THREE: METHODOLOGY 36

3.1 Collection of samples 36

3.2 Preparation of media and diluents 38

3.3 Sample collection and preparation 39

3.4 Determination of microbial population 39

3.5 Characterization and identification of microbial isolates 40

3.6 Microscopic characterization 41

3.6.1 Gram staining test 41

3.6.2 Biochemical characterization of bacterial isolates 41

3.6.3 Catalase test 41

3.6.4 Coagulase test 42

3.6.5 Oxidase test 42

3.6.7 Sugar fermentation/oxidation 43

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3.6.8 Hydrogen sulphide production test 43

3.6.8 Urease test 43

3.6.9 Indole test 43

3.6.10 Citrate utilization test 43

CHAPTER FOUR: RESULTS 45

CHAPTER FIVE: DISCUSSION OF FINDINGS AND SUMMARY OF THE

STUDY 49

5.1 Discussion Of The Findings 49

5.2 Conclusion 49

REFERENCES 50

Apppendices 53

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 LIST OF TABLES

Table 1: Total counts and colonial characteristics of Bacteria isolated on Salmonella Shigella
Agar (SSA) Page 43.

Table 2: Colonial and Microscopic Characteristics of Bacteria isolated on Nutrient Agar and
MSA Page 45.

Table 3: Biochemical Characteristics of Bacteria Isolated from Meat sample on NA and MSA
Page 45.

Table 4: Distribution of Salmonella and Shigella sp species in samples. Page 45.

Table 5: Percentage Distribution of Salmonella sp in samples. Page 46.

viii
 ABSTRACT

Poultry birds which include the chickens, ducks, turkey and goose reared domestically either for
meat, egg production or feathers are of tremendous importance to man and these birds demand
the best quality of feed in other to yield the expected results.

As poultry feeds are being produced they can potentially be contaminated with food borne
bacteria either during processing at the mill or during storage and harvesting in this case corn
piles weather covered or uncovered attract birds including migratory species known to carry
pathogens like avian influenza virus, when they feed on these grains in the open field some of
them deficate shedding pathogens in their feaces on these grains which eventually leads to the
contamination of these grains unknown to the farmer.

Consumption of some of these commonly monitored bacteria like Salmonella, listeria

monocytogens and pathogenic Escherichia coli which have found their way into our poultry
feeds through artificial of natural means by animals can present a great risk to the health of this
animals and man alike.

Further more Since the movement of feed ingredients including micro ingredients is not always
completely transparent and the process might introduce some microorganisms as brought forth
this research to determine the presence of E coli and salmonella in different brands of poultry
feeds sold in relief market Owerri Imo state.

ix
 CHAPTER ONE

INTRODUCTION

1.1 Background Of The Study

Poultry feed is food for farm poultry birds which include chickens, ducks, geese and other
domestic birds and in this project the researcher focused on four different brands of poultry feeds
obtainable in the poultry market to serve different categories of poultry birds classified into
starters, growers and finishers. These feeds are as follows; Hybrid, layers, vital, and top.
Before the twentieth century, poultry were mostly kept on general farms and foraged for much of
their feed, eating insects, grain spilled by cattle and horses, and plants around the farm. This was
often supplemented by grain, household scraps, calcium supplements such as oyster shell, and
garden waste.
As farming became more specialized many farms kept flocks too large to be fed in this way and
nutritionally complete poultry feed was developed. Modern feeds for poultry consists largely of
grain, protein supplements such as soybean, oil meal, mineral supplements, and vitamin
supplements. The quantity of feed and the nutritional requirements of the feed depend on the
weight and age of the poultry bird, their rate of growth, their rate of egg production, the weather
and the amount of nutrition the poultry obtain from foraging. This results in a wide variety of
feed formulations. These substitution of less expensive local ingredients introduces additional
variations(G. F. Heuser, 1955).
The feed must remain clean and dry (James R. Gillespie; Frank B. Flanders, 2010) because
contaminated feed can infect poultry, Damp feed and encourage fungal
growth. Mycotoxin poisoning as an example is one of the most common and certainly most
under reported causes of toxicoses in poultry (Mark Pattison, 2008).
Diseases can be avoided with proper maintenance of the feed and feeder. A feeder here is the
device that supplies the feed to the poultry (Robert Blair,2008) or privately raised chickens,
or chickens as pets, feed can be delivered through jar, trough or tube feeders. The use of poultry
feed can also be supplemented with food found through foraging (Malcolm F. Fuller, 2004).
Furthermore the researcher was able to find out that As reports have indicated, feeding makes up
the major cost in raising poultry animals as birds, In general poultry birds require feeding more
than any other animal particularly due to their faster growth rate and high rate of productivity.
Feeding efficiency is reflected on the bird’s performance and their products. According to
National Research Council (1994), poultry requires at least 38% components in their feed. The
ration of each feed components, although differing for each different stage of birds, must include
carbohydrates, fats, proteins, minerals and vitamins. Carbohydrates, which are usually supplied
by grains including corn, wheat, barley, etc. serves as a major energy source in poultry feed.
Fats, usually from tallow, lard or vegetable oil are essentially required to provide important fatty
acid in poultry feed for membrane integrity and hormone synthesis.

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Proteins are important to supply the essential amino acids for the development of body tissues
like muscles, nerves, cartilage, etc. Meals from soybean, canola, and corn gluten are the major
sources of plant protein in poultry diets. Supplementation of minerals are often required because
grains, which are the main component of commercial feed contain very little amounts of these.
Calcium, phosphorus, chlorine, magnesium, potassium and sodium are required in larger
amounts by poultry. Vitamins, such as vitamin A, B, C, D, E, and K, on the other hand, are the
components that are required in lower amounts by poultry animals.
Fanatico ( 2003) reported that the easiest and most popular way to feed birds is to use pelleted
feeds. Aside from the convenience to the farmer, pelleted feeds enable the bird to eat more at a
time. In addition, some researchers also found improvement of feed conversion, decreased feed
wastage, improved palatability and destruction of pathogens when birds were fed with pellet feed
as compared to birds fed with mash feed.
Commercial manufacturing of pelleted feed usually involves a series of major processes
including grinding, mixing and pelleting. The produced pellets are then tested as to pellet
durability index (PDI) to determine quality. To enhance good health and growth, antibiotics are
often added to the pelleted feed.
Researchers have concluded that smaller particle-sized feed will improve digestion due to the
increased surface area for acid and enzyme digestion in the gastrointestinal tract. However, some
researchers have recently brought to our attention the necessity of coarse particles for poultry
feed to complement the natural design and function of the gastrointestinal tract (GIT). Hetland et
al (2002) and Svihus et al. (2004) discussed that the GIT retention time decreased due to lack of
gizzard function that eventually gave a negative impact on live performance. Zanotto & Bellaver
(1996) compared the performance of 21-day-old broilers fed with different feed particle size;
0.716 mm and 1.196 mm. They found that the subject fed with larger particle size feed showed
better performance. Parsons et al. (2006), evaluating different corn particle sizes in the broiler
feed found that the largest particle size (2.242 mm) gave better feed intake than the other particle
sizes tested (0.781, 0.950, 1.042 and 1.109 mm). Nir et al. (1994), however, argued that the
development of broilers was influenced by changing particle sizes. However, variation in particle
size between 0.5–1 mm usually did not have any effect on the broilers. Very fine particles
(<0.5 mm) may impair the broilers performance due to the presence of dust that cause respiratory
problems, increased water intake, feed presence in the drinkers and increased litter moisture.
(Chewning et al. (2012), in their recent study, concluded that although fine particle sizes (0.27
mm) enhanced broilers live performance, the pelleted feed did not.
All of this data shows that both fine and coarse particle sizes do have different functions in
poultry feed. Appropriate proportions of these two ingredients must be used with respect to the
live performance of the broilers. Xu et al. (2013) compared the performance of non-pelleted feed
to pellets with fine particles and found that the addition of coarse particles improved feed
conversion and body weight. Similar results were also obtained by other researchers
like Auttawong et al. (2013) and Lin et al. (2013). As research go on to improve poultry feed
Animal feeds can potentially become contaminated with food borne bacteria either during
harvesting, processing at the feed mill or during storage. Animal feeds are also potential
reservoirs for cross contamination from environmental sources while being fed to animals.
Microbial contamination from bacteria and viruses to molds and mycotoxins access poultry feed
and feed ingredients through a variety of routes. Some routes are naturally occurring, while
2
others are caused by exposure during the manufacturing process. Microbes as Contaminants
being Invisible to the naked eye, microbes are uniquely able to contaminate living hosts as well
as inanimate objects and have proven effective at evading detection. As part of a wider drive for
biosecurity, producers and suppliers screen feed and feed ingredients for certain bacteria, such
as Enterobacteriaceae and mycotoxins. Consumption of commonly monitored
bacteria, Salmonella, Listeria monocytogenes and pathogenic Escherichia coli, by animals can
present a health risk to animals and to humans alike.
“There's always a possibility of contamination when purchasing feed or feed ingredients,” said
Enrique Montiel, Anitox DVM and Director of Nutrition and Live Production. “However, some
ingredients are more welcoming to contamination than others. Corn piles, whether covered or
uncovered, attract wild birds, including migratory species known to carry pathogens including
the Avian Influenza (AI) virus. While they’re there to feed, birds defecate, shedding pathogens in
their feces. That’s a particular concern because AI virus, for example, has been shown to survive
in both feces and feed materials for long periods of time. Then that feed ingredient will make its
way to the feed mill and ultimately into a farmer’s feed supply.”

Despite recognition of the challenge, it is not uncommon to see piles of raw feed materials such
as corn, soybeans or rapeseed accessible to wild birds with no protective covering. That’s
especially true at harvest time and in countries importing or exporting high volumes of grains
which lack proper storage capacity. All feed and feed ingredients are vulnerable to contamination
and spoilage. Some ingredients are more prone to certain kinds of contamination, whether that be
corn growing mold, or high protein materials sustaining bacteria such as Salmonella.

“You have a higher risk of Salmonella in certain ingredients and not just Salmonella,” he notes.
“For example, the major United States AI outbreak six years ago resulted in researchers
exploring routes through which Avian Influenza could be transmitted from wilds birds to
commercial poultry. It was theorized that since commercial birds are effectively secured inside
buildings that perhaps feed could more easily be contaminated and serve as a fomite. Further
research has shown virus survival in feed, and proposed that once introduced, the infectability of
a virus could escalate with every bird and feed interaction, spreading throughout the flock. "

Contaminants can also include naturally occurring components of certain food and feed
ingredients like glucosinolates and toxins produced by microorganisms.

Molds can grow on grains, seeds and complete animal feed and growth is influenced by
environmental factors, like temperature, humidity and rainfall. This can occur during the
growing season, harvest and/or storage.

“It is commonly assumed that grain exposed to the elements for a few days is not a big deal, but
contamination can happen very quickly and can result in animal health problems and losses of
productivity,” said Dr. Montiel.

Mold growth leads to the production of secondary metabolites called mycotoxins. Once the
mycotoxins exist on the surface or inside the feed ingredient, they remain throughout the
different phases of feed processing and are very difficult to eliminate. When ingested by animals,
3
they can negatively impact metabolic processes in the animal and significantly reduce
productivity.

The most concerning mycotoxins include:

 Aflatoxins
 Deoxynivalenol
 Fumonisin
 Ochratoxin A
 Zearalenone

“If the general growing conditions are conducive to mold growth, then it is important to monitor
crops and determine the risk of mycotoxins,” Dr. Montiel explains. “Ingredients, such as corn
and DDGs, are susceptible to Aspergillus contamination. These molds produce types of
aflatoxins that not only negatively impact bird production but can also impact consumer health.
Ultimately, the allowance specification for aflatoxins is low, which makes mold management a
crucial intervention strategy.”

To further evaluate the project topic samples of different brands of feed where collected and
tested to determine the presence of escherinchia coli and shamonella species in these poultry
feeds.

1.2 Statement of problem

Since the movement of feed ingredients including micro ingredients is not always completely
transparent and the process might introduce some micro-organisms.

Escherichia coli as a case study is a Gram-negative, facultative anaerobic, rod-shaped, coliform

bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-
blooded organisms Most E. coli strains are harmless, but some serotypes can cause serious food
poisoning in their hosts, and are occasionally responsible for food contamination incidents that
prompt product recalls. E. coli is expelled into the environment within fecal matter. The
bacterium grows massively in fresh fecal matter under aerobic conditions for three days, but its
numbers decline slowly afterwards.

E. coli and other facultative anaerobes constitute about 0.1% of gut microbiota, and fecal–oral
transmission is the major route through which pathogenic strains of the bacterium cause disease.

According to the Centers for Disease Control and Prevention, National Center for Emerging and
Zoonotic Infectious Diseases (NCEZID), Division of Foodborne, Waterborne, and
Environmental Diseases (DFWED) in 2004 Escherichia coli (E. coli) bacteria normally live in
the intestines of people and animals. Most E. coli are harmless and actually are an important part
of a healthy human intestinal tract. However, some E. coli are pathogenic, meaning they can
cause illness, either diarrhea or illness outside of the intestinal tract. The types of E. coli that can
4
cause diarrhea can be transmitted through contaminated water or food, or through contact with
animals or persons. E. coli consists of a diverse group of bacteria. Pathogenic E. coli strains are
categorized into pathotypes. These Six pathotypes are associated with diarrhea and collectively
are referred to as diarrheagenic E. coli.

Shiga toxin-producing E. coli (STEC)—STEC may also be referred to as Verocytotoxin-

producing E. coli (VTEC) or enterohemorrhagic E. coli (EHEC). This pathotype is the one most
commonly heard about in the news in association with foodborne outbreaks.

it is not possible to know the exact climate and storage conditions that feed ingredient were
exposed to ,therefore Feed sanitation should be considered to prevent disease and support food
chain biosecurity. Sanitizing treatments and introduction of antibiotics can be applied as part of a
feed formulation as well as to individual ingredients.

1.3 Purpose of the study

The purpose of these study is to ascertain the presence of salmonella shigella and escherinchia
coli in different brands of poultry feeds. The specific objective is as follows:

1. The presence of escherinchia coli and salmonella in different poultry feeds sold in the
market.
2. The presence of escherinchia coli and salmonella in different poultry feeds distributed in
the poultry farms.
3. The presence of escherinchia coli and salmonella in different poultry feeds distributed in
their feeding trough

1.4 hypothesis

There is presence of salmonella and other organisms but no Escherichia coli was found in this
research

1.5 significance of the study

This research is mapped out to encourage agriculturist and poultry rearers on safer production of
uncontaminated poultry feeds and how to reduce the imtroduction of this organisms into our
poultry farms.
1.6 scope of the study
This research will be carried out by collecting samples from a retail store in a general market at
random in owerri imo state where the samples will be through a serial dilution method be
inoculated into a growth media to check for most occurring colonies

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1.7 operational definition of terms

Pelletted feed: Feed pelleting can be defined as conversion of finely ground mesh feed into dense,
free flowing pellets or capsules, in a process that involves steam injection and mechanical pressure.
Beaked jaws: The beak is an external anatomical structure found mostly in birds, it is used for
eating, preening, manipulating objects, killing prey, fishing, probing food, courting and feeding
young ones.
Oviparous: Producing young ones by means of eggs which are hatched after they have been laid
by the parents, as in birds.
Bird Flu: Strains of the influenza virus that primarily infect birds, but can also infect humans.
Parasitic Disease: A parasitic disease also known as parasitosis is an infectious disease
caused by parasites. Parasites are organisms which derive sustenance from its host.
Jungle Fowl: Jungle fowl are the only five living species of bird from the genus Gallus in the
bird order Galliformes and occur in parts of South and Southeast.
Monophyletic: Not comparable of or pertaining to, or affecting a single phylum of organisms.
Descending from a single ancestral species.
Broiler: A broiler is any chicken of the family Gallus gallus domestica that is bred and raised
specifically for meat production.
Domestic birds: a domestic bird is any bird that has been bred within the country it resides in.
Mycotoxins: mycotoxins are naturally occurring toxins produced by certain moulds(fungi) and
can be found in food. The moulds grow on a variety of different crops and foodstuffs including
cereals, nuts, spices, dried fruits, apples and coffee beans, often under warn and humid
conditions.
Cereals: A cereal is any grass cultivated for the edible component of its grain, composed of the
endosperm, germ and bran. Cereal grain crops are grown in greater qualities and provide more
food energy worldwide than any other type of crop.
Anti microbial therapy: An anti microbial theraphy kills or inhibits the growth of
microorganisms such as bacteria, fungi or protozoans. Therapies that kill microorganisms are
called microbiocidal therapies and therapies that only kill only inhibit the growth of
microbiology are called microbiostatic therapies.
Stock: A culture of a microorganism maintained solely to keep it viable for subculture into fresh
medium.
Eosin methylene blue agar(EMB): EMB is a selective and differential medium used to isolate
fecal coliforms. Eosin Y and methylene blue are pH indicator dyes which combines to form a
dark purple precipitate at low pH; they also serve to inhibit the growth of most Gram positive
organisms.

6
Salmonella Shigella agar(SSA): salmonella shigella agar is moderately selective and
differential medium for the isolation, cultivation and differention of salmonella species and some
strains of shigella species. It is a modification of the desoxycholate citrate agar.

Lactose fermenters: lactose fermenting species will grow pink colonies. Lactose fermentation
will produce acidic by products that lower the pH indicator to pink. Example of Lac positive
species are Escherichia coli, Enterobacteria and Klebsiella.

Colony count: plates with over 200 colonies are usually counted by dividing the plates into
equal sectors from ½ up to 1/8. After counting one sector, the count is multiplied with the total
number of sectors to estimate the whole plate CFU. The number of colonies on a plate is the
colony count.

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 CHAPTER TWO

REVIEW OF RELATED LITERATURE

This chapter deals with the review of literature considered important to this study. The literature
is review is discussed under the following sub-headings: Conceptual Framework, Theoretical
Frame work, Empirical studies using relevant sub-headings, Appraisal of the Reviewed
literature.

Major Concepts Of The Study

Birds
Birds are a group of warm blooded vertebrates constituting the class Aves, characterized by
feathers, toothless beaked jaws, the laying of hard shelled eggs, a high metabolic rate, a four
chambered heart and a strong yet lightweight skeleton. Birds live worldwide and range in size
from the 5.5cm(2 in) bee hummingbird to the 2.8m (9 ft 2 in) ostrich. There are about ten
thousand species, more than half of which are perching birds.

Birds have wings whose development varies according to species; the only known groups
without wings are extinct birds like the meo and the elephant birds, their wings which are
modified forelimbs gave the ability to fly, although further evolution has led to the loss of flight
in some birds including ratites, penguins and diverse endemic island species.

The digestive and respiratory systems of birds are also uniquely adapted for flight. Some bird
species of aquatic environments, particularly seabirds and some water birds have further evolved
for swimming.

Birds produce offsprings by laying of eggs which are fertilized through sexual reproduction.
Many species of birds are economically important as food for human consumption and raw
material in manufacturing, with domesticated birds being important sources of eggs, meat, and
feathers.(Wikipedia.org/wiki/bird).

Poultry farming
According to professor Wesley Patterson Garrigus he stated in one of his publications that
raising of birds domestically or commercially, primarily for meat, eggs and feathers is a
highly perfected science that ensures a maximum intake of energy for growth and fat
production.

The essential minerals necessary for their growth are calcium, phosphorus, sodium, chlorine,
potassium, sulfur, magnesium and zinc coupled with some vitamins which include vitamins A,C,

8
D, E and K.

9
In commercial setting according to the professor antibiotics are widely used to either stimulate
appetite, control harmful bacteria and prevent diseases.

To prevent diseases he stated a well hygienic and controlled environment that avoid crowding,
chilling, over heating or frightening should be avoided .

The feeding, watering and cleaning operations are highly mechanized as the birds are usually
housed in wire cages with two to three animals per cage.

According to research cage laying birds have been found to increase production, lower
mortality, reduce cannibalism, lower feeding requirements, reduce diseases and parasites,
breeding of these birds is an outstanding example of the application of basic genetic principles
of inbreeding and cross breeding as well as of intensive mass selection to effect Faster and
cheaper gains in meat and maximum egg laying strains.

Among the world’s agricultural industries chicken breeding in the U.S. is one of the most
advanced intensive nutritional and research application, highly improved breeding stock,
intelligent management and scientific disease control has gone into the effort to give a modern
broiler(meat chicken) of uniformly high quality produce at ever-lower cost.

Poultry are quite susceptible to a number of diseases. Some of the more common are fowl
typhoid, pullorum, fowl cholera, chronic respiratory disease, infectious sinusitis, infectious
coryza, avian infectious hepatitis, infectious synovitis, bluecomb, Newcastle disease, fowl pox,
avian leukosis complex, coccidiosis, blackhead, infectious laryngotracheitis, infectious
bronchitis, and erysipelas. Strict sanitary precautions, the intelligent use of antibiotics and
vaccines, and the widespread use of cages for layers and confinement rearing for broilers have
made it possible to effect satisfactory disease control.

Furthermore Outbreaks of bird flu, or avian influenza, which was first detected in humans in
1997, have led to the culling of millions of poultry animals since the late 20th century.
Waterfowl such as wild ducks are thought to be primary hosts for all bird flu subtypes. Though
normally resistant to the viruses, the birds carry them in their intestines and distribute them
through feces into the environment, where they infect susceptible domestic birds. Sick birds pass
the viruses to healthy birds through saliva, nasal secretions, and feces. Within a single region,
bird flu is transmitted readily from farm to farm by airborne feces-contaminated dust and soil, by
contaminated clothing, feed, and equipment, or by wild animals carrying the virus on their
bodies. The disease is spread from region to region by migratory birds and through international
trade in live poultry. Humans who are in close contact with sick birds like the poultry farmers
and slaughterhouse workers are at the greatest risk of becoming infected.

1
Parasitic diseases of poultry, including hexamitiasis of turkeys, are caused by roundworms,
tapeworms, lice, and mites. Again, modern methods of sanitation, prevention, and treatment
provide excellent control.

TYPES OF POULTRY

Chickens
Chickens were originated as junglefowl in Asia and were domesticated over 3000 years ago, and
are known now as chicken; Gallus gallus domesticus (Moreng and Avens, 1985; Crawford,
1990a; Sullivan, 1991; Siegel et al., 1992; Fumihito et al., 1994; Romanov and Weigend, 2001;
Hillel et al., 2003; Vaisanen et al. 2005, Columbia Encyclopedia, 2006). There was a debate on
whether the domestic fowl is monophyletic or polyphyletic origin (Crawford, 1990a). It was
indicated by many research studies that the red junglefowl is the direct ancestor of the domestic
chicken (Gallus gallus domesticus) used in commercial production for meat and eggs (Moreng
and Avens, 1985; Crawford, 1990a; Sullivan, 1991; Siegel et al., 1992; Fumihito et al., 1994;
Romanov and Weigend, 2001; Hillel et al., 2003; Vaisanen et al., 2005; Columbia Encyclopedia,
2006). Fumuhito et al. (1994) reported that G. g. gallus of the red junglefowl is the origin of all
domestic breeds. Collias and Collias (1996) reported that the red junglefowl is the principal and
perhaps the sole ancestor of the domestic fowl. Moreover, the use of molecular genetics and
micro-satellite techniques provided evidence that the origin of domestic fowl is monophyletic.
Hillel et al. (2003) evaluated the gene pool of 52 chicken populations from a wide range of
origins using the micro-satellite markers technique. They found that the red junglefowl is the
main progenitor of the domestic chickens. The current poultry is a domesticated fowl used for
both meat and egg production. This includes birds such as chicken, turkey, duck, goose, ostrich,
quail, pheasant, guinea fowl, and peafowl. Chickens are the most popular poultry worldwide
irrespective of culture and religion (Roenigk, 1999; Aho, 2001; Aho, 2004). This is because
poultry products have very high nutritive values. Chicken meat and eggs are the major protein
source for consumers in most of the countries around the world. Chicken meat consumption has
been increasing during the last few years due to the rise in health awareness of consumers all
over the world.

Broilers dominate the world poultry market, consisting of about 70% of the market. Turkeys
account for about 8% and other poultry provides 22% of the global market (Roenigk, 1999;
2004; USDA, 2006). This indicates that broiler meat continues to be desired over other poultry
meats.

All commercial chickens are the descendents of the red junglefowl species (Crawford, 1990a;
Sullivan, 1991; Siegel et al., 1992; Fumihito et al., 1994; Romanov and Weigend, 2001; Hillel et
al., 2003; Vaisanen et al., 2005).

1
Chickens are classified as order Galliformes, family Phasianidae, genus Gallus (junglefowl)
which is distinguished from all other phasianidae species in having the comb and the wattles
associated with it (Crawford, 1990a; USDA/ ITIS, 2006 a-j). It is important to select such criteria
to maintain differences from other species. Four species of junglefowl are recognized. These are:
Red junglefowl, grey junglefowl, green junglefowl and Ceylon junglefowl. There are no sub-
species for grey, green or Ceylon junglefowl. However, geographic variation is very marked in
red junglefowl and this has been recognized by designating several sub-species for red
junglefowl (Moreng and Avens, 1985; Crawford, 1990a; Collias and Collias, 1996; Moiseyeva et
al., 2003; USDA/IT IS, 2006a-j; Wikipedia, 2006; Wikispecies, 2006). The geographic variation
implies the adaptation of the different sub-species to certain environmental conditions. Species
nomenclature has undergone some changes. Red junglefowl was called G. bankiva and G.
ferrugineus. The Ceylon junglefowl was named G. stanleyi, and the green junglefowl was named
G. furcatus (Crawford, 1990a). It should be noted that classification of animals and plants was
based on morphological, geographical and behavioural characteristics. The taxonomy and
classification were done before the development of sequencing and molecular techniques (Bush
and Strobeck, 2003; Wikipedia, 2006). Bush and Strobeck (2003) investigated phylogenetic
relationships of the Phasianidae. They used nucleotide sequences from the mitochondrial
cytochrome b gene of 27 pheasants and 6 non-pheasant species. They found that red junglefowl
is located outside the pheasant lineage between the grey peacock (Polyplectron bicalcaratum)
and the koklass (Pucrasia macrolopha macrolopha). The authors concluded that it is unclear
whether the grey peacock, red junglefowl or koklass are pheasants, and since their respective
positions were unstable it cannot be ruled out that they are pheasants. More studies and research
in this subject is suggested using all the different species of junglefowl. It is clear that the science
of molecular genetics will be fully utilized to provide more accurate classification to the different
sub-species.

Rhode Island Red rooster

Small farms and backyard flocks utilize a much wider variety of breeds and hybrids. Common
American breeds include the Plymouth Rock, the Wyandotte, the Rhode Island Red, and the
New Hampshire, all of which are dual-purpose breeds that are good for both eggs and meat. The
Asiatic Brahma, thought to have originated in the United States from birds imported from
China, is popular for both its meat and its large brown eggs.

Turkeys
After World War II, turkey production became highly specialized, with larger flocks
predominating. Turkeys are raised in great numbers in Canada where their ancestors still live
wild, as well as in some parts of Europe, the United States, Mexico, and Brazil. A hybrid white
turkey dominates commercial production, while the Broad Breasted Bronze, the Broad Breasted
White, the White Holland, and the Beltsville Small White are common breeds for smaller farms.

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In breeding flocks, one tom is required per 8 or 10 hens, though the modern hybrid turkey is too
large for natural breeding and must be artificially inseminated.

Modern turkey breeding and farming practices have significantly reduced both the amount of
feed and the time required to produce a pound of turkey meat. In 12–14 weeks a hen turkey eats
about 16 kg (35 pounds) of feed and reaches 6–9 kg (14–20 pounds). Toms require some 36 kg
(80 pounds) of feed to reach a market weight of 16–19 kg (35–42 pounds) in 16–19 weeks.
Smaller turkey broilers are marketed from 12 to 15 weeks of age. Turkeys can be raised on open
land with automatic waterers, self-feeders, range shelters, heavy fencing, and rotated pastures;
however, they are often “grown out” commercially in rearing houses under environmentally
controlled conditions.

DUCKS AND GEESE

PEKIN DUCK

Duck raising is practiced on a limited scale in nearly all countries, usually as a small-farm
enterprise, though some commercial plants do exist. Ducks are easily transported, can be raised
in close confinement, and convert some waste products and scattered grain (e.g., by gleaning
rice fields) to nutritious and very desirable eggs and meat. Khaki Campbell and Indian Runner
ducks are prolific layers, each averaging 300 eggs per year. The Pekin duck, one of the most
popular breeds in the United States, is used for both egg and meat production. Although the
white- fleshed Aylesbury was once the favoured meat duck in England, disease and market
competition from the yellow-fleshed Pekin duck have led to its decline.

DOMESTIC GOOSE

Goose raising is often a minor farm enterprise, though some European countries have large-scale
goose-production facilities. The two outstanding meat breeds are the Toulouse, predominantly
gray in colour, and the Embden (or Emden), which is white. The birds are raised for meat and
eggs as well as for their down feathers. Geese do not appear to have attracted the attention of
geneticists on the same scale as the meat chicken and the turkey, and no change in the goose
industry comparable to that in the others has occurred or seems to be in prospect. In some
commercial plants, geese are fattened by a special process of force-feeding, resulting in a
considerable enlargement of their livers, which are sold as the delicacy foie gras.

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GUINEA FOWL AND SQUABS
GUINEA FOWL

Guinea fowl are raised as a sideline on a few farms in many countries and are eaten as gourmet
items. In Italy there is a fairly extensive industry. The birds are often raised in yards with open-
fronted shelters, and a number of varieties and species are utilized throughout the world. Guinea
fowl are marketed in England at 16–18 weeks of age and in the United States at about 10–12
weeks. The market weight is usually about 1–1.5 kg (2.5–3.5 pounds), but food conversion is
poor.

PIGEONS
Pigeons are raised not only as messengers and for sport but also for the meat of their squabs
(nestlings). Squab production, carried on locally, is rare in most countries with established
poultry industries, though the meat is often marketed as a gourmet item.

GALLUS GALLUS (Red Jungle Fowl) Red junglefowl is the common name of the Gallus
gallus species (Figure 2). The red junglefowl still can be found in India, China, Java, Malaysia,
Indonesia and the Philippine. The red junglefowl has the largest natural range among the four
species of the junglefowls. There are marked geographic variations recognized by its division
into several subspecies. Most literature recognized only five subspecies of red junglefowl based
on geographic variation, which are as follows: 1) Cochin-Chinese or Indochina red junglefowl;
Gallus gallus gallus (Figure 3), 2) Javan red junglefowl; Gallus gallus bankiva, (Figure 4) 3)
Tonkinese red junglefowl; Gallus gallus jabouillei (Figure 5), 4) Indian red junglefowl; Gallus
gallus murghi (Figure 6) and 5) Burmese red junglefowl; Gallus gallus spadiceus (Figure 7)
(Crawford, 1990a; Collias and Collias, 1996; Moiseyeva et al., 2003; USDA/ ITIS, 2006a-g).
These five subspecies vary in size of facial wattles and combs and length and colour of the neck
hackles in males (Cowell, 2006a). In recent literature, four more sub-species were considered
(Wikispecies, 2006). These are Gallus gallus domesticus which is the one that the domestic fowl
was developed from (Moreng and Avens, 1985; Sullivan, 1991; Siegel et al. 1992; Fumihito et
al., 1994; Romanov and Weigend, 2001; Hillel et al., 2003; Vaisanen et al., 2005; Columbia
Encyclopedia, 2006). The other three sub-species are considered as feral G. gallus since they
were successfully introduced in many places such as the Philippines, Micronesia, Melanesia and
Polynesia (Crawford, 1990a). These were considered in some literature as subspecies and were
added to the list of the red junglefowl subspecies as follows: Gallus gallus gallina, Gallus gallus
micronesiae, and Gallus gallus philippenisis (Wikispecies, 2006). Red junglefowl habitat varies
and they used most types of forests in Southeast Asia, field edges, groves and scrubland.
Breeding season is March through June. Clutch size is 5-6 eggs with an incubation period of 18-

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21 days (Cowell, 2006a). Physical description of red junglefowl is shown in Table 1. It is
important to note that red junglefowl in the pure wild form is still uncommon in captivity. There
are only few subspecies that are pure without any interbreeding from domestication or within the
races (Cowell, 2006a; Gautier, 2006).

GALLUS VARIUS (green junglefowl) Green junglefowl is the common name of this
species (Figure 8), also it is known as the fork tail. It inhabits the island of Java. It is also found
in Bali, Sumbawa, Komodo, Lombok and the surrounding islands. Green junglefowl also
inhabits coastal regions and semi-arid cliff habitat and they forage in mangrove swamps, along
beaches, in rice paddies and deep inside subterranean sea caves (Crawford, 1990a; Zanon, 2003).
It is capable of sustained flight over open water. It has no sub-species (Blackwood, 2006a;
Crawford, 1990a; USDA/ ITIS, 2006h; Zanon, 2003;). Its primary foods include species of
semi terrestrial crustaceans called copepods or sand fleas, small crabs and marine insects
(Blackwood, 2006a; USDA/ITIS, 2006h). Breeding season is mid April to June. Clutch size is 5
to 10 eggs with incubation period of 21 days (Cowell, 2006b). Physical description of green
junglefowl is shown in fig 9.

GALLUS SONNERATII (grey junglefowl) Grey junglefowl is the common name of this
species (Figure 10). It is native to southern and western India. It lives in mountain areas
sometimes as high as 1500 – 2000 meters and it can easily fly. They feed on seasonal pulp seed
fruits like pumpkin and hot peppers. It has no sub-species (Blackwood, 2006b; Crawford, 1990a;
USDA/ ITIS, 2006i; Zanon, 2003). Breeding season is March to July. Clutch size is 4 to 6 eggs
with incubation period of 21 days (Cowell, 2006c). Physical description of grey junglefowl is
shown delow.

GALLUS LAFAYETII (Ceylon Junglefowl) The common name of this species is Ceylon
junglefowl (Figure 11). It is endemic to Sri Lanka in Ceylon Island. It has no sub-species
(Crawford, 1990a; Zanon, 2003; Cowell, 2006d; USDA/ ITIS, 2006j). It is considered as Sri
Lanka national bird and is widely spread throughout the Island. It is known in Sinhala as Wali
Kukula and Katu Koli in Tamil (Jayawardene, 2006). These birds fly only when it is necessary
and roost at night. They feed on small fruits, berries, termites and other insects which they find
on the forest floor. It is seen regularly in forest areas (Crawford, 1990a; Jayawardene, 2006).
Breeding season varies on climates generally begins in April and lasts through June. Clutch size
3–6 eggs with an incubation period of 20-21 days (Cowell, 2006d). Physical description of
Ceylon junglefowl is shown below.

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Poultry diseases
Handling of live birds brings perhaps the greatest risk of exposure to bacteria and viruses for
farmers, their families and poultry workers.

In areas where highly pathogenic avian influenza (HPAI) is present. A study in Guangzhou,
China in 2007 to 2008 found that 15 percent of poultry workers in live poultry markets, where
the birds are also slaughtered, had antibodies against HPAI. This compares with only 1 percent
in the general population (Wang, Fu and Zheng, 2009).

Unless all the necessary precautions are taken along the poultry production, marketing and
processing chains, poultry meat and eggs can be contaminated by infectious agents that are
harmful to humans. Poultry products can also be contaminated with the antimicrobial and anti-
parasitic drugs or pesticides used on farms. The ingestion of antimicrobials can cause
antimicrobial-resistant bacteria to develop in humans. Campylobacter and Salmonella infections
are among the most important food safety hazards. These bacteria account for more than 90
percent of all reported cases of bacteria-related food poisonings worldwide. Most of these cases
are related to the consumption of poultry and poultry products, but all domestic livestock are
potential reservoirs of infection. Reported cases of Campylobacter and Salmonella infections are
believed to represent only a fraction of the true number of cases. Consuming raw or undercooked
poultry or poultry products has been implicated as a potential risk factor for human cases of
influenza H5N1 infection (HPAI). Poultry meat should be well cooked, with the core
temperature reaching 70°C for at least one second during cooking. Data on food-borne diseases
in low-income countries are scarce. There is no precise and consistent global information about
the full extent of the occurrence of food poisoning and the costs related to unsafe food.
Symptoms are often mild and cases are not reported, but their importance is thought to be
substantial.

Other microbial contaminants of the poultry birds and farm caused by bacteria include:
Salmonelloses, Escherinchia Coli Infections, Fowl Cholera, Paratyphoid Infections,
Cholangiohepatitis In Broiler Chickens, Riemerella Anatipestifer Infections, Necrotic Enteritis,
Mycoplasma, Gangrenous Dermatitis, Chicken Tuberculosis, Infectious coryza

Salmonellosis Causative agent: Salmonella Salmonella is an enteric microorganism that can

contaminate practically all creatures including people. Salmonellosis in poultry is brought about
by Gram-negative microorganisms from the class Salmonella [Barbour E et al 1985] Signs
include Intestine hemorrhages, Off Feed .

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E.coli Infection Causative agent: E.coli Disease or infection in which accumulation body cells
and tissue which is due to E.coli bacteria such disease is called E.coli Infection . Signs Liver
damage, Laziness, Off feed, Infection on liver (Blanco, J. E et al 1997)

Fowl Cholera Causative agent: Pasteurella multocida Fowl cholera is a infectious disease of
Avian Group which is due to a bacteria named as Pasteurella multocida. (Carr, D., D. Shaw et al
1996, Chaka, H. et al 2012, Chamanza, R et al 1999) Less mortality caused by this disease
.Having some signs Sign Laziness, Swelling of wattle, Pneumonitis,Twisting neck Paratyphoid
Infections Causitive agent: Salmonella Parathyrioid is major disease of poultry which is due to
unguided staff in feed industry as it is due to Salmonella. Feed is contaminated by salmonella..
[Blackall PJ 1999] Major source of Salmonella are feces by which salmonella is transferred to
egg shell and then in the germinal disk. ( Cloud, S. S., et al 1985, Avian Dis 2017, Corney BG, et
al 2008) Sign Stress, Reduce in growth, Laziness, diarrhea, water deficiency in the body

Cholangiohepatitis (CAH) in broiler chickens Causative agent: Unknown Cholangiohepatitis

(CAH) in broiler chickens is a condtion in which liver swells and become hard etiological agent
is unknown yet but Clostridium perfringens was found in liver some other bacteria like
Еscherichia coli, Pasteurella haemolytica, Streptococcus equisimilis, Campylobacter sp Also
found in liver and bile duct of infected birds [Addis Ababa; 2014, Dashe Y et al 2003, . Dawit
Alemu et al 2003, . Eriksen, J., et al 2010] Signs Enlargement of liver, Yellow liver, Hardness of
liver, Loss of weight, Off feed

Riemerella Anatipestifer Infections Causative agent: Riemerella anatipestifer It is an infectious

disease which effects ducks goose and other avian species.It is due to a gram negative bacteria
Riemerella anatipestifer I [FAO 2004, Filip Van Immerseel et al 2004, Fossum, O. et al 1988]
Signs Cough , Sneezing, Discharge from nose, Watery green feces,Nervous signs

Necrotic Enteritis Causative agent: Clostridium perfringens Necrotic enteritis is a poultry disease
which is due to extra growth of Clostridium perfringens type A, and to a less extent type C,
(Havelaar, A.H. et al 2015, Heller, E. D. and M. Perek 1968, Heller, E. D. and N. Drabkin. 1977)
in the avian small intestine. C produce toxins. The intestinal wall would be damage by
perfringens. Normally it effects the chickens at the age 2- 6 weeks. Signs Severe type of Stress,
Loose motions, Deficiency of water, Off feed, Disarranged feathers

Mycoplasma Causative Agent:- Mycoplasma gallisepticum Mycoplasma gallisepticum (MG) is

common respiratory disease of chickens [Jolles, A.E. et al 2006, Jordan, F. T. W. 1996,
Kaldhusdal, M. et al 2001] Signs Water in eyes, Lesions on trachea, Runny nose, Cough, Off
feed,Weight Loss

Gangrenous dermatitis Causative Agent: Clostridium septicum, C, perfringens type A, and

Staphylococcus It is disease in hens and other avian groups caused by Clostridium septicum, C,

1
perfringens type A, and Staphylococcus in which high ratio of mortality and Narcosis of skin and
thigh occur Signs ,Dark color of skin, wing and breast, Loss of weight ,Off feed

Chicken Tuberculosis Causative agent: Mycobacterium avium subsp avium It is disease of birds
which usually effects the small birds like pigeons and pheasant and also effect bird in free range
Signs Weight gain, Loss in production, stress Infectious Coryza Causative agent : Avibacterium
paragallinarum

Infectious Coryza is a respiratory disease. Infectious Coryza occur due to a bacteria named as
Avibacterium paragallinarum [Berhe, N., et 2012].Clinical signs of Infectious coryza are Runny
nose, laziness and face swelling. Effected bird could be recover by using antibiotics at initial
stage. We can prevent birds from infectious coryza by adopting appropriate measures of
biosecurity, good managemental practices and doing vaccination of birds. Infectious Coryza seen
in many country and it effect especially multistage farm.[ Kaper JB et al 2004, Lovland, A. and
M. Kaldhusdal. 2001] Signs Swelling of Face, Watery discharge from eyes and nose of bird,
Swelling of wattles,Wheezin, Poor respiration , 10-40% decrease in egg production, Anorexia.
[Lutful KS. 2010].

Poultry Feeds And Types

Poultry feed is food for farm poultry, including chickens, ducks, geese, and other domestic birds.
Before the twentieth century, poultry was mostly kept on general farms, and foraged for much of
their feed, eating insects, grain spilled by cattle and horses, and plants around the farm. This was
often supplemented by grain, household scraps, calcium supplements such as oyster shells, and
garden waste. As farming became more specialized, many farms kept flocks too large to be fed
in this way, and nutritionally complete poultry feed were developed. Modern feeds for poultry
consists largely of grain, protein supplements such as soybean oil meal, mineral supplements,
and vitamin supplements. The quantity of feed, and the nutritional requirements of the feed,
depending on the weight and age of the poultry, their rate of growth, their rate of egg production,
the weather (cold or wet weather causes higher energy expenditure), and the amount of nutrition
the poultry obtain from foraging. This results in a wide variety of feed formulations. The
substitution of less expensive local ingredients introduces additional variations. Healthy poultry
requires a sufficient amount of protein and carbohydrates, along with the necessary vitamins,
dietary minerals, and an adequate supply of water. Lactose-fermentation of feed can aid in
supplying vitamins and minerals to poultry. Egg-laying hens require 4 grams per day of calcium
of which 2 grams are used in the egg. Oyster shells are often used as a source of dietary calcium.
Certain diets also require the use of grit, tiny rocks such as pieces of granite, in the feed. Grit aids
in digestion by grinding food as it passes through the gizzard. Grit is not needed if the
commercial feed is used. Calcium iodate is used as a supplement to iodine. The feed must remain
clean and dry; contaminated feed can infect poultry. Damp feed encourages fungal growth.
Mycotoxin poisoning, as an example, is "one of the most common and certainly most under-

1
reported causes of toxicoses in poultry". Diseases can be avoided with proper maintenance of the
feed and feeder. A feeder is a device that supplies the feed to the poultry. For privately raised
chickens or chickens as pets, the feed can be delivered through jar, trough, or tube feeders. The
use of poultry feed can also be supplemented with food found through foraging. In industrial
agriculture, machinery is used to automate the feeding process, reducing the cost and increasing
the scale of farming. For commercial poultry farming, feed serves as the largest cost of the
operation.

Requirements Of A Poultry Feed

In order to provide Poultry with necessary nutrients to meet their requirements for maintenance,
growth, to reduce the risks of Poultry health and to minimize excretions and emissions into the
environment, the processed Poultry feed required.

Energy Sources Of Poultry Feed:

These are described under the following categories: Grains and seeds, Milling by-products,
Molasses, Roots and tubers. Grains and seeds Grains are seeds from cereal plants, members of
the grass family called Gramineae. Cereal grains are essentially carbohydrates, the main
component of the dry matter being starch, which is concentrated on the endosperm. All cereal
crops are annuals (Kharif). By-products of harvested grains as chaff, Stover, and straw are
utilized as low quality forages for ruminant Poultry. Moreover, many of the grains are milled or
processed in some manner thereby creating additional by-products that can be fed to livestock
with varying degrees of nutritive values. In India except for poultry, swine, and lactating dairy
animals, grains are not usually fed for livestock production, because of high cost due to high
demand by human beings.

Nutrient Composition Of Grains: The name cereal is given to those members of the
Gramineae which are cultivated for their seeds. The dry matter content of grain depends on the
method of harvesting and storage conditions but is generally within the range of 80-90%. Protein
constitutes 85-90% of the nitrogenous compounds. The protein occurs in all tissues of cereal
grains, but higher concentrations are found in the embryo and aleuronic layer than in the starchy
endosperm pericarp and testa. The protein content of grain though variable normally ranges from
8-12%. The lipid content of cereal grains also varies with species, normally ranges from 1-6%.
Maize and oat contain 4-6% oil, while sorghum 3-4% and wheat, barley, and rice contain 1-3%
oil. The embryo or germ contains more oil than the endosperm. Cereal oils are unsaturated, the
main acids being linoleic and oleic and because of this, they tend to become rancid quickly.

1
Cereal starch consists of about 25% amylase and 75% amylopectin, although waxy starches
contain greater proportions of amylopectin.

The Major Grains Used:

Maize (Zea mays): Maize contains about 70% starch, 85-90% TDN, 4% oil and about 8-12%
protein. Sorghum (Sorghum bicolor): Sorghum contains about 65% starch, 80-85% TDN, 2-3%
oil and about 8-12% protein.

Wheat (Triticum aestivum): wheat is a good source of energy containing 75-80% TDN,

crude protein content ranges from 8-14, Lysine, threonine and methionine are the major limiting
amino acids in wheat grain.  Barley (Hordeum vulgare): The crude protein varies from 11-16%
and TDN from 78-80%. The lipid content of barley grain is low; usually less than 2.5% of dry
matter.

Oat (Avena sativa): Oats of high hull content are richer in crude fibre and have a lower
metabolisable energy value than low hulled oats. The soft physical nature of the hull and high oil
content contribute to the high palatability of oats. The crude protein content ranges from 8-12%
and TDN 70-73%.

Rice (Oryza sativa): Unprocessed rough rice contains about 8-10% crude protein, 9% rude fibre,
1.9% ether extract and 6.5% ash. The TDN content varies from 78- 82%.

Rye (Secale cereale): Protein content of rye varies from 10-14% and TDN 75- 80%. It is
regarded as the least palatable of the cereal grains. 1.5.2. Milling by-products

Wheat bran: The crude protein ranges from 13-16% and TDN from 65-70%. The bran has
amino acid balance superior to that of wheat.

Wheat middling: It has 96% of the energy value of barley and 91% of the energy value of corn.
Midds are palatable feedstuffs and can be included in the grain mixture at high levels.

Rice bran de-oiled: The crude protein ranges from 13-16% and TDN from 55-65%. It is a good
source of proteins, vitamins and minerals.

Rice polish /raw rice bran: The oil content of rice polish varies from 13-19%. The crude protein
ranges from 13-16% and TDN from 70-90% depending on the oil content.

Chunies: Generally they are high in CP and low in TDN value than that of the parent pulse
grain. The CP value of different chunies varies from 15-20% and TDN value ranges from 55-
65%.

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 Molasses Cane molasses: Cane molasses is a by-product during manufacture of sugar from
sugarcane. From each ton of sugarcane approximately 25-50 kg of molasses are 10 produced.
Cane molasses must contain at least 43% sugars and have a density of not less than 79.5o brix.

Beet molasses: Beet molasses is a by-product during manufacture of sugar from sugar beet. Beet
molasses contain about 48-53% sugars and have a density of not less than 79.5o brix.

Citrus molasses: Citrus molasses is produced from the juice of citrus wastes. Citrus molasses
contain about 41-43% sugars and have a density not less than 71.0o brix. Moisture content is
higher ranging from 27-30%. The crude protein ranges from 10-14% and TDN from 65-75%.

Roots and tuber: Turnips (Brassica rapa), Cassava root (Manihot esculenta), Potato (Solanum
tuberosum), Sweet potato (Ipomoea batatas), Carrot (Daucus carota)

Salmonella
Salmonella enterica is the most important food borne pathogen, and it is often associated with the
contamination of poultry products. Annually, Salmonella causes around 93 million cases of
gastroenteritis and 155,000 deaths worldwide. Antimicrobial therapy is the first choice of
treatment for this bacterial infection; however, antimicrobial resistance has become a problem
due to the misuse of antibiotics both in human medicine and animal production. It has been
predicted that by 2050, antibiotic-resistant pathogens will cause around 10 million deaths
worldwide, and the WHO has suggested the need to usher in the post-antibiotic era. The purpose
of this review is to discuss and update the status of Salmonella antibiotic resistance, in particular,
its prevalence, serotypes, and antibiotic resistance patterns in response to critical antimicrobials
used in human medicine and the poultry industry. Based on our review, the median prevalence
values of Salmonella in broiler chickens, raw chicken meat, and in eggs and egg-laying hens
were 40.5% ( interquartile range [IQR] 11.5-58.2%), 30% (IQR 20-43.5%), and 40% (IQR 14.2-
51.5%), respectively. The most common serotype was Salmonella Enteritidis, followed by
Salmonella Typhimurium. The highest antibiotic resistance levels within the poultry production
chain were found for nalidixic acid and ampicillin. These findings highlight the need for
government entities, poultry researchers, and producers to find ways to reduce the impact of
antibiotic use in poultry, focusing especially on active surveillance and finding alternatives to
antibiotics.

Salmonella enterica subsp. enterica remains the leading cause of infectious gastroenteritis. Cases
are often related to the consumption of food of animal origin, mainly poultry products, such as
eggs and raw chicken [Braden C.R. (2006), Donado-Godoy et al (2012) and Heng Y et al
(2013)]. Globally, non-typhoidal Salmonella causes around 93 million cases of gastro enteritis
and 155 000 deaths each year [CDC. (2013), Cosby et al (2015), Majowicz et al (2010)]. The
disease manifestation depends on the serotype involved, virulence factors, infective dose, and
host immunity. Immunocompromised patients, children, and elderly people tend to be more
2
susceptible and suffer more serious clinical symptoms, including sepsis [Coburn et al (2007),
Fierer et al (2001), Kothary, M. and Babu, U. (2001), Luquez, J.L. (2017), WHO. (2017) ]. In
other cases, the infection can cause a chronic state of asymptomatic carriage in the host [ Coburn
et al (2007)]. The serotypes Salmonella Enteritidis and Salmonella Typhimurium are the most
frequent causes of salmonellosis in humans [Gantois et al (2009), Liljebjelke et al (2017), Rajan
et al (2017)]. However, emerging serotypes such as Salmonella Heidelberg, Salmonella Javiana,
Salmonella Infantis, and Salmonella Thompson have been reported to infect humans in the
United States and are becoming more prevalent in certain segments of the poultry production
chain [Donado-Godoy et al (2012) and Foley et al (2011)].

PATHOGENESIS AND TISSUE INVASION

Salmonella spp. possesses an arsenal of genetic determinants responsible for colonization,

adhesion, invasion and proliferation in host cells, including fimbriae, flagella, toxins, surface
lipopolysaccharides (LPS), etc. Virulence genes are organized in clusters and spread throughout
the chromosome, such as Salmonella Pathogenicity Islands 1 and 2 (SPI-1, SPI-2), or located on
virulence plasmids, such as spv genes (associated with invasive strains). Salmonella
pathogenicity genomic islands carry genes that are required for successful infection in poultry
(Wisner et al., 2012). Noninvasive strains cause gastroenteritis, while invasive strains may cause
systemic bacteremia in humans and animals. The outcome of infection depends on virulence
factors, the pathogenesis of Salmonella spp. and their interaction with the host organism (Foley
et al., 2013). Unlike noninvasive strains, invasive Salmonella strains penetrate through the
epithelial lining to the lower parts of the lamina propria. Also, invasive strains are commonly
isolated from parenhymatous organs (spleen, liver, ovaries) and a small number of bacteria
become internalized by macrophages (Berndt et al., 2007). The survival in the acidic
environment of the stomach is enabled by the activation of more than 50 acid tolerance response
proteins (Bearson et al., 2006). The first phase of the infection has to provide a chance for the
bacteria to invade intestinal epithelial cells. This process is accomplished by proteins encoded by
Salmonella Pathogenicity Island (SPI-1) type III secretion system (T3SS). These organelles
produce a special structure in the bacterial envelope called “the needle complex” which delivers
toxins and other effector proteins and injects them into the host cells (Kubori et al., 2000).
Bacterial effector proteins modulate the host actin cytoskeleton and initiate the signal
transduction pathways required for the internalization of the bacteria. In addition, invasive strains
recruit their own systems responsible for survival in macrophages. Salmonella spp. become
internalized in a specific membrane bound compartment called “Salmonella containing vacuole”
(SCV). The maturation of the SCVs and their migration to the basal membrane disable the
destruction of the bacteria by phagolysosomes. Such intracellular trafficking and intracellular
pathogenesis is also accomplished by the activation of the second T3SS encoded by the SPI-2.
Hence, the type III secretory system encoded by SPI-1 and SPI-2 enables the attachment,
invasion and survival of the pathogen within the host cell, as well as the avoidance of

2
antimicrobial compounds (Hensel, 2000; Foley et al., 2013). Most of Salmonella serovars
contain SPI-1 to -5, while other pathogenicity islands are not so common. The colonization of
the gastrointestinal tract and of the internal organs of poultry is enabled by the type VI secretion
system encoded by the SPI-19 locus present in serovar Gallinarum (Blondel et al., 2010). In mice
infected with serovar Typhimurium, the SPI-6 was necessary for the intracellular replication of
the pathogen in macrophages and its systemic dissemination. The experimental work indicates
that T6SS encoded by both SPI-6 and SPI-19 gene clusters are genetically involved in bacterial
pathogenesis and that T6SS-SPI-6 play a role in gastrointestinal colonization and systemic
spread of serovar Typhimurium in chickens (Pezoa et al., 2013). Besides Salmonella
pathogenicity islands-1 and 2, Salmonella strains involved in extraintestinal non typhoid
disease with bacteremia carry additional virulence genes in a spv locus, contained on virulence
plasmids (Guiney and Fierer, 2011). Genes spv were found in serovars Typhimurium, Enteritidis,
Choleraesuis, Abortusovis, Dublin, Gallinarum/ Pullorum and in subspecies arizonae. The
plasmid genes in the spv locus include spvABCD operon which is positively regulated by the
upstream spvR gene. Only spvR, spvB and spvC are responsible for spv related virulence
phenotype. In spite of having different biochemical pathways of action, SpvB and SpvC proteins
are eventually involved in late apoptosis of macrophages, enabling the intracellular proliferation
of Salmonella spp. Subsequent uptake of apoptotic macrophages by surrounding macrophages,
facilitates cell to cell spread of Salmonella spp. (Guiney and Lesnick, 2005; Derakhshandeh et
al., 2013). Consequently, it potentiates the systemic spread of the pathogen instead of causing a
self limited gastroenteritis. Salmonellae have different invading capacities in the poultry intestine
and parenchymatous organs. They trigger systemic and local immune response which is in good
correlation with their virulence. Experimental work was conducted by Berndt et al., (2007) to
measure the immune response in cecum after the infection of White Leghorn day old chickens
with serovars Enteritidis, Typhimurium, Hadar and Infantis. At 2, 4 and 7 days post infection (pi)
serovars Hadar and Infantis showed diminished invading capabilities for liver, compared to
serovars Enteritidis and Typhimurium. S. Enteritidis was the best invader of the lower zones of
the lamina propria, while S. Infantis was found in epithelial lining and subepithelial region. The
increase of granulocytes, TCR1 gd and CD8α+ in chicken cecum was most prominent for
serovar Enteritidis, followed by serovars Typhimurium and Hadar, while Infantis provoked less
significant immune cell influx. In the same study the reorganization of the extracellular matrix
proteins, notably the increase of total fibronectin and tanascin-C, has been more pronounced
after the infection of day old chickens with serovar Enteritidis comparing to the infection with
the non invasive Salmonella Infantis. Furthermore, enhanced Salmonella spp. entry and the
ability to disseminate in the gut epithelium support the concept that the most virulent strains
utilize distinctive genetic mechanisms to invade the intestine and disseminate through the body,
showing an important ability to provoke better immune responses in infected birds, as well
(Berndt et al., 2009).

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ESCHERICHIA COLI

Escherichia coli (E.coli) is a bacterium that causes disease in different animal species, mainly
localized in the intestine of mammals, but also they can cause septicemic or special type disease
like ‘Oedema disease’ in pigs and ‘watery mouth in calves’ (Quinn et al., 2011; Tseng et al.,
2014; Bashahun and Amina, 2017; Luise et al., 2019). Poultry is also affected by E.coli which
causes serious clinical disease named colibacillosis. Those E.coli strains are called avian
pathogenic Escherichia coli (APEC) and more often they can cause disease outside of the
intestinal tract in poultry (extraintestinal APEC) (Dho-Moulin and Fairbrother, 1999; Ewers et
al., 2009). Escherichia coli is described as the most frequent bacterial agent that causes general
health and produc tion problems in poultry (Barnes et al., 2008; Kabir, 2010; Zhuang et al.,
2014; Landman and van Eck, 2015). Avian pathogenic E.coli strains seem to have re stricted
role in causing clinical disease in humans. However, some human uropathogenic E.coli strains
are found to share similar genetic factors with APEC indicating a relationship between them
(Skyberg et al., 2006; Ewers et al., 2007; Zhu Ge et al., 2014; Stromberg et al., 2017; Jorgensen
et al., 2019). Fur thermore, APEC antibioresistance can be a source of resistance for human
pathogens (Singer and Hofacre, 2006; Johnson et al., 2007; Mellata, 2013; Singer, 2015).

Escherichia coli is a natural inhabitant of the poultry intestinal tract. It returns in the bird
environent through droppings and can insert to other birds by the oral-feacal route (Dho-Moulin
and Fairbrother, 1999). It seems that bird’s intestine and environment are the most important
reservoirs for Escherichia coli strains capable of causing infection (Ewers et al., 2009). Studies
has reported that E.coli strains isolated from the intestine of healthy birds and from their
environment are phylogenetically similar to APEC strains that have been isolated from
coliseptisemic birds. Therefore, they have the zoonotic potential to cause colibacillosis in poultry
(Ewers et al., 2009; Kabir, 2010). Vectors like darkling beetle (Goodwin and Waltman, 1996),
flies, insects, mites (Wales et al., 2010), rats and wild birds can play a role in the spreading of
Escherichia coli (Barnes et al., 2008). Escherichia coli can be transmitted either horizontal ly,
directly or indirectly, or vertically from breeders carrying the organism in their reproductive tract
to their progeny (Giovanardi et al., 2005). The respiratory route seems to be also very
impor tant for the appearance of clinical disease, while the oviduct can also be another route of
infection for Es cherichia coli concerning layers and breeders (Antao et al., 2008; Ozaki and
Murase, 2009; Landman et al., 2013). Finally, penetration of E.coli through the skin can produce
an inflammation in the form of cellulitis (Norton et al., 2000) identified in specific genes which
are responsible for coding the production of specific components of the bacterial cell, the
virulence factors. These factors can either increase the ability of E.coli to attach to the host cells,
increase the bacterium ability to multiply and attack the bird cell or can protect E.coli against the
immune response of the host (Dho-Moulin and Fair brother, 1999). The most important
virulence factors described in E.coli bacteria cells are adhesins like the type 1-F adhesin (La

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Ragione and Woodward, 2002) and the P-adhesin , iron related factors, like aerobactin system
consisting of iuc, iut factors (Dziva and Stevens, 2008), protectins like the K1 and the iss factor
(Mellata, 2013), invasins like ibeA (Cortes et al., 2008; Flechard et al., 2012), toxins/bacteriocins
like stx1,2, hlyD and hlyF and miscellaneous factors (Barnes et al., 2008)

Clinical disease

Ε.coli can infect and damage different bird systems causing various syndromes and localised or
systemic disease. In day old chicks, a very common type of E.coli infection is the yolk sack
infection (omphalitis) which is characterized by inflammation of the yolk sack and high
mortality (Dinev, 2007). In birds of different ages, E.coli can cause severe infection of the upper
or lower respiratory system through the nasal route or the trachea. In cases of localised infection
of the upper respiratory system, facial oedema and sinuses swelling can be presented
and described as swollen head syndrome. However, in case of lower respiratory
infection, colibacillosis is characterized by the presence of fibrinous whitish yellow exudates in
the air sacks and fibrinous pneumonia (Barnes et al., 2008). Another type of localized
colibacillosis is cellulitis that is characterized by in flammation of the skin and presence
of subcutaneous fibrinous exudates especially in broilers. Cellulitis is a very common
reason for carcass condemnations in the slaughterhouse (Norton, 1997). In layers and breeders,
the most common type of colibacillosis is the infection of reproductive system; which is known
as salpingitis, oophoritis and perito nitis (Jordan et al., 2005; Barnes et al., 2008; Brugere-Picoux
et al., 2015). Regarding the macroscop ic findings in necropsy, fibrin in different quantities can
be present inside the peritoneum, the oviduct or around the ovaries (Dinev, 2007). Egg
peritoni tis syndrome caused by APEC is of great economic significance in layer production
because of its high incidence, the induced flock mortality and drop in egg production (Landman
et al., 2013). Furthermore, there is an added cost of flock antibiotic treatment. In males, E.coli
infection can cause reduced fertility due to testicles inflammation (Barnes et al., 2008) The
septicemic or systemic form of colibacillosis is mainly found in broilers but can also be present
in layers. It is characterized by polyserositis and presence of fibrin in various organs
(pericarditis, peri hepatitis, air-sacculitis, peritonitis) (Randall, 1985; Dinev, 2007). If birds
survive the acute phase of coli septisemia, colibacillosis can revert into a chronic localized type
of disease. In such cases, coli infection can be present in unusual sites like brain, eyes,
joints and bones (Barnes et al., 2008).

HOW ESCHERICHI COLI AND SALMONELLA COME IN CONTACT WITH

POULTRY BIRDS,FEEDS AND MAN

Escherichia coli (E.coli) is a common bacterium that can be naturally found in the intestinal tract of birds
and as a result in their environment. However, it can cause clinical disease called colibacillosis which is
regarded as one of the most common and important diseases in poultry. Strains of E.coli that have the
ability to cause clinical disease are described as Avian Pathogenic Escherichia Coli (APEC).

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Colibacillosis can affect birds of all ages and different types of poultry production including broiler and
commercial layers and breeders. The ability of E.coli to cause colibacillosis is not always the same; that is
why its role as primary or secondary pathogen triggered by various predisposing factors is contradictory
and differs from case to case. Antibiotics have been used as the main tool against colibacillosis for many
decades. However, the emergence of increased antibiotic resistance has posed the need of alternative
treatment to colibacillosis as well as emphasizing on preventive measures to avoid disease.

The incidence of Salmonella on the other hand has contributed to food poisoning which has increased
world wide remarkably in the last few years. Although the proportion of food poisoning outbreaks and
cases in which the source of infection can be positively identified is small, poultry and poultry products
are repeatedly implicated in human outbreaks. Salmonella organisms from poultry sources currently enter
the human food chain mainly as a result of carcass contamination from infected faecal material or eggs.
Several Salmonella serovars have been isolated from poultry. The exact number, however, is difficult to
estimate. Some serovars may be predominant for a number of years in a region or country. Then, they
disappear and are replaced by another serovar. Previously, the dominant type worldwide of salmonella
food poisoning was S. Typhimurium but since 1982 S. Enteritidis has been challenging for dominance.

2.2 THEORITICAL FRAMEWORK

This study relies on the following theories for its notional underpinning;

THE USEFULNESS OF PREBIOTICS AND PROBIOTICS IN MODERN POULTRY

NUTRITION
Besides quality attributes, special attention in recent years has been paid by the consumers to
safety of animal products. Considering some evidence that the use of antibiotic growth promoters
(AGP) may cause pathogen resistance (Phillips et al., 2004), the application of antibiotics as
animal growth enhancers had already been prohibited in the European Union since 2006.
Today’s animal farming, especially the poultry industry have been greatly intensified with
respect to both large number of animals and modern feeding systems. Concerns about the losses
in animal performance and thus sustainability of production and its profitability coupled with this
ban have led to an increase in research on the alternative supplements to AGP and strategies for
food-producing animals. Today, the most accepted definition states that probiotics are mono or
mixed cultures of live microorganisms which, when administered in adequate amounts, confer a
health benefit on the host (FAO/WHO, 2002). Unlike probiotics, prebiotics are not
microorganisms – they are a sort of nourishment source for existing flora, allowing the natural
colony of gut to grow naturally and replicate. Prebiotics were defined as non-digestible food
(feed) ingredients that beneficially affect the host by selectively stimulating the growth and/or
activities of one or a limited number of bacteria in the gut, thereby improving host health
(Gibson and Roberfroid, 1995). However, more recent definitions stated that a prebiotic is a
selectively fermented ingredient that allows specific changes, in both the composition and
activity in the gastrointestinal microbiota which confers benefits to the host (FAO/WHO, 2002).
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The common point of these definitions is that prebiotics are characterized by a selective effect on
the microbiota, and thus can improve the host health. Prebiotics include mainly oligosaccharides,
sugar molecules of three to six chains, and soluble fibre. These carbohydrates are found naturally
in fruit and vegetables (Charalampopolus and Rastall, 2009). Nutritional supplements combining
probiotics and prebiotics are referred to as synbiotics, which are a combination of “a probiotic
and a prebiotic that beneficially affects the host by improving the survival and establishment of
live microbial dietary supplements in the gastrointestinal tract” (Trachoo et al., 2008). The main
importance of this form of synergism is that a probiotic alone, i.e. without a source of
nourishment which can be represented by a prebiotic, cannot survive well in the digestive system
(Bhupinder and Saloni, 2010). Synbiotics are gaining popularity and scientific credibility as
functional food (feed) supplements at nutritional and therapeutic levels. It is believed that they
can ensure a high level of viable probiotic cells once ingested (Trachoo et al., 2008). Some
studies have shown the importance and benefits of this kind of synergy between probiotics and
prebiotics and the effectiveness in helping young animals to achieve better growth performance
(Patterson and Burkholder, 2003). Probiotics, prebiotics and synbiotics are gaining importance
because they seem to exert their nutritional benefits in various animal species and their concept
is more and more comprehensible. Poultry studies have demonstrated clear benefits in
performance and health status of birds. They affect the intestinal microbiota and immune system
to decrease colonization by pathogens in some conditions. As with growth promoting antibiotics,
environment and stress may have an impact on the efficiency of prebiotics and probiotics. These
feed supplements can present immense potential as alternatives for antibiotics to totally eliminate
antibiotic use, because they do not cause microbial resistance. The beneficial consequences of
these supplements may generally translate into improved health and performance parameters.
More studies are now focusing on their symbiotic effects that apparently can be more efficient
than a separate use. This represents an important perspective of applied biotechnological
research that must be better understood to reveal more mechanisms through which they could
positively impact poultry, but these will depend largely on the regulatory developments in the
area that can bring new products for poultry and livestock, which are currently limited because of
time and generated expenses of safety testing.

THEORY ON THE PATHOGENICITY OF ESCHERICHIA COLI O157 IN

COMMERCIAL BROILER CHICKENS

According to the researcher he stated that In poultry, E. coli infections include

egg peritonitis, omphalitis, coligranuloma, swollen head syndrome, cellulitis, and
colisepticaemia. Colisepticaemia is a severe systemic form of infection (Dho-Moulin and
Fairbrother, 1999, Barnes et al., 2003).Omphalitis is a major factor responsible for early chick
mortality during the first few days after hatching (Fasenko and O’Dea, 2008). It is accounting for
2
large economic losses in poultry by causing mortality rates up to 25% during the first week of
life (Cortes et al, 2010). The infection is aggravated by poor hygiene in breeding farms and
faulty management at the hatchery (Kim and Kim, 2010). Other factors that favor rapid bacterial
growth include the fact that the yolk contains a lot of fat and water, favored nutrients for bacteria
(Shaw and Halvorson, 1993)

In poultry, E. coli infections include egg peritonitis, omphalitis, coligranuloma, swollen head
syndrome, cellulitis, and colisepticaemia. Colisepticaemia is a severe systemic form of infection
(Dho-Moulin and Fairbrother, 1999, Barnes et al., 2003).Omphalitis is a major factor responsible
for early chick mortality during the first few days after hatching (Fasenko and O’Dea, 2008). It is
accounting for large economic losses in poultry by causing mortality rates up to 25% during the
first week of life (Cortes et al, 2010). The infection is aggravated by poor hygiene in breeding
farms and faulty management at the hatchery (Kim and Kim, 2010). Other factors that favor
rapid bacterial growth include the fact that the yolk contains a lot of fat and water, favored
nutrients for bacteria (Shaw and Halvorson, 1993) in conclusion the used serogroup of E.
coli O157 was pathogenic to chicks when experimentally infected So particular attention must be
directed toward E. coli O157 not only as a pathogen infect bird but also as a possible threat to
public health. Probiotics was of value in preventing the infection and improvement of
performance parameters of chicks. Therefore we recommended the routine use of probiotics in
drinking water. Also ciprofloxacin can be used in treatment of E. coli.

Salmonella spread in broiler production

The researcher stated in this research that The presence of Salmonella spp. in broiler production
is a food safety concern as the bacterium can be transmitted to humans via contaminated meat
and derived products. Salmonella detection in litter at the pre-slaughter period has been linked to
increased odds of contaminated broiler carcasses and meat derived products. To determine risk
factors related to farm and broiler house characteristics and management practices, this study
uses a unique longitudinal data set from a Brazilian integrated broiler enterprise, which contains
official results of Salmonella spp. isolation from drag swabs collected at the end of the grow-out
period. A Bayesian hierarchical spatio-temporal model found significant spatial and time
influence on the odds of isolating Salmonella spp. from litter as well as significant effects from
the size of a broiler house, total housing area per farm, type of broiler house, and number of litter
recycles. Results indicate that recycling litter beyond 6 rearing cycles significantly increased the
odds of isolating Salmonella before slaughter, and the bacterium was more likely to persist in
conventional broiler houses, compared to broiler houses with controlled environment. Evidence
of a potential principal-agent problem was also found in setting strategies to control the
bacterium from litter, which suggests strong incentives to adopt the strategies aiming to reduce
prevalence of the bacterium in the integrated enterprise. Our findings could be used to develop

2
alternative measures to reduce the risk of persistence of the bacterium in the broiler production
chain.

2.3 REVIEW OF EMPERICAL STUDIES

This section deals with the summary of previous researchers that are related to the present
study. As this section deals with related studies, the researcher made efforts to point out one or
two differences(gaps) between the studies and the present study at the end of the empirical
review. Later in the summary of this chapter the broad gap was explored.

EMPIRICAL STUDIES ON MULTI-DRUG RESISTANT (MDR) ORGANISMS IN

SOME POULTRY FEEDS

This study reports on the incidence of multi-drug resistant (MDR) organisms in poultry feeds
sold in Calabar metropolis, Nigeria. According to the researcher Twenty samples of poultry
feeds were purchased from different locations and analyzed microbiologically using standard
methods. Significant enough to note is the microbial loads of these poultry feeds which were
quite high 1.232 x 109 cfu/g (Top feeds) and 1.03 x 108 cfu/g (Vitals feeds). Eight bacteria
isolates were obtained and identified as Bacillus sp [3(15.0%)], Escherichia coli [2(10.0%)],
Nocardia sp [2(10.0%)], Salmonella sp [3(15.0%)], Proteus mirabilis [2(10.0%)], Pseudomonas
aeruginosa [4(20.0%)], Staphylococcus aureus [2(10.0%)], and Streptococcus pyogenes
[2(10.0%)]. The antibiotics susceptibility profile showed that S. aureus and S. pyogenes were
more susceptible (75%) to the test antibiotics, followed by E. coli (72.7%), Nocardia sp (58.3%)
and Proteus mirabilis (54.5%). All gram positive isolates were resistant to ampiclox (100%) and
sensitive to streptomycin and ciprocin (100%) while all gram negative isolates were resistant to
tetracycline and ampicillin (100%). However, all isolates satisfied the most common multidrug
resistance patterns (>3 antibiotics resistant). Generally, significantly higher number of multidrug-
resistant Pseudomonas [10(90.1%)], Bacillus sp. [9(83.3%)], Salmonella sp [8(72.7%)], P.
mirabilis [5(45.5%)], and Nocardia sp [5(41.7%)] were noted in this study. Low resistance rates
was observed for E. coli [3(27.3%)], S. aureus and S. pyogenes [3(25%)] was found in the
poultry feeds. The MIC values of tetracycline of the isolates ranged from 0.13 – 8.00mcg/ml.
Among all the test organisms only S. aureus (25%) was susceptible to tetracycline at an MIC
value of 0.13mcg/ml. This showed that 75% of the bacterial species exhibited an MIC value of
0.25-8.00mcg/ml. To reduce the effect of these MDR organisms in poultry feeds; antibiotics
incorporated into feeds should be in synergistic combinations as this will prevent the possibility
of resistance development. The findings of this study confirm the presence of multi-drug
resistant organisms in animal feeds sold in Nigeria. It significantly points to the great need to
evaluate and monitor the incidence rate of multi-drugs (antibiotics) resistant organisms in poultry
feeds.

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 Two trials were conducted to evaluate the presence of salmonella, campylobacter, and generic
Escherichia coli on broilers raised on Poultry Litter Treatment (PLT )-enhanced litter in
comparison with those raised on untreated litter. Two Company A farms included three houses
on each farm as the treated group and three houses per farm as controls. Two complete growouts
were evaluated on each farm. The Company B study included 10 farms with two paired houses
per farm, one house as the treated group and one house as the control. One growout was
evaluated per farm. The pathogen sampling consisted of litter sampling and whole bird rinses on
the farm and in the processing plant. Litter pH, ammonia concentration, total litter bacteria,
temperatures, and humidity were also recorded. The study with Company A resulted in lower
mean levels of pH, ammonia concentration, total litter bacteria, litter E. coli, and bird rinse
counts for salmonella and E. coli in houses treated with PLT. The results for Company B
closely resembled those for Company A, but also included campylobacter data, which showed no
difference between treated and control groups. The data indicate that PLT may be a beneficial
component for on-farm pathogen reduction.

3.4 SUMMARY OF LITERATURE REVIEW

In this chapter the researcher made review under major concepts of the study, theoretical
framework, review of empirical studies and summary of literature review. Subheadings under the
major concepts of the study include birds , poultry farming, types of poultry , poultry diseases,
poultry feeds and types, salmonella, Escherichia coli and how salmonella and E coli come in
contact with poultry birds. It was based on the review that the researcher deducted that Poultry
feed is food for farm poultry including chickens, ducks, geese and other domestic birds. It is
essential that we feed the poultry birds with the feeds that contain all essential nutrients so as to
enable these birds yield maximum production either in meat, egg production or feather
production. But this feeds can be exposed to some certain microorganisms which possess a threat
to our poultry birds and to humans as the consume them to ensure that this feeds are not
contaminated, feeds must remain dry because contaminated feed can infect poultry , dampen
feed and encourage fungal growth. in summary all this diseases caused by either fungal, bacterial
or viral organisms can be avoided with proper maintanance of the feed and the feeder coupled
with the birds and their housing system.

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 CHAPTER THREE

METHODOLOGY
3.0 Materials and Methods

Thirty six (36) samples of poultry feed from randomly selected stores in relief market Owerri
Imo State was collected by the researcher.1 gram of each of the samples where weighed and
mixed with 9 ml of distilled water and a 10 fold serial dilution process was carried out after
which 1ml of the stock was inoculated and spread using a sterile glass rod on the media prepared
SSA for salmonella and EMBA for E coli, the media was incubated for 24hrs after which results
where taken. The Colonial morphology of E coli on EMBA will be a 2-3 diameter colonies with
a greenish metallic sheen in reflected light, dark or even black center in transmitted light while in
salmonella the will not ferment lactose but produce hydrogen sulfide(H2S) gas. The resulting
bacterial colonies will appear colorless with black centers, shigella do not ferment lactose or
produce hydrogen sulfide gas, so the resulting colonies will be colourless.

3.1 Collection of samples

The feed samples collected where different brands depending on the particular poultry bird
involved (starters, growers, finishers and layers) the feed brands involved are vital, top and
hybrid.

3.2 Preparation of media and diluents

Salmonella Shigella Agar (SSA) was prepared according to manufacturer’s specification. SSA
is highly selective for cultivation of Salmonella species.

3.3 Sample collection and preparation

One gram (1 g) of each of the sample was weighed into different 9 ml of sterile distilled water to
obtain 101 dilution. Ten fold dilutions was done by transferring 1 ml into 9 ml until the desired
dilution was obtained. One-tenth millilitre (0.1 ml) of appropriate dilution was inoculated into
the medium, spread evenly with sterile glass spreader to ensure uniformity. This was then
Incubated at 370C for 24 hours in an incubator.

3.4 Determination of microbial population

3
Colony counts obtained on the media were expressed as colony forming units per gram (CFU/g)
to obtain total population

3
3.5 Characterization and identification of microbial isolates

Microbial isolates were characterized based on cultural (colonial), microscopic and biochemical
methods with reference to standard manuals. The identities of the isolates were cross-matched
with reference to standard manuals for the identification of bacteria.

3.6 Characterization of Microbial

Isolates Microscopic Characterization

3.6.1 Gram Staining Test

The Gram staining technique was used for the bacterial isolates as described by Cheesbrough, M
(2000). A smear of the isolate was made on grease free glass slide with a drop of water and
allowed to dry. The smear was fixed by mild heating, flooded with crystal violet and allowed to
stand for 30 seconds. The crystal violet was rinsed off with water; Lugol’s iodine was added and
allowed to stand for 30 seconds. This was washed off with water and acid alcohol, till
discoloration. It was counter stained with Safranin for 10 seconds and rinsed with water. The wet
slide was allowed to air dry. A drop of oil immersion was added on the slide and viewed using
X100 objective lens of the microscope.

Biochemical Characterization of Bacterial Isolates

Microorganisms that were not identified by the colonial and microscopic characteristics were
further subjected to few biochemical tests described by Cheesbrough (2000) and Beishir (1987).

3.6.2 Catalase Test

The enzyme catalase is present in most cytochrome containing aerobic and facultative anaerobic
bacteria. Catalase has one of the highest turnover numbers of all enzymes such that one molecule
of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen in a
3
second. Catalase activity can be detected by adding the substrate H₂O₂ to an appropriately
incubated (18-24 hours) tryptic soy agar slant culture. Organisms which produce the enzyme
breakdown the hydrogen and the resulting O₂ production produces bubbles in the reagent drop
indicating a positive test. Organisms lacking the cytochrome system also lack the catalase
enzyme and are unable to breakdown peroxide into O₂ and water and are catalase negative.

3.6.3 Coagulase Test

Coagulase is enzymes that clot blood plasma by a mechanism that is similar to normal clotting.
The coagulase test identifies whether an organism produces this exoenzyme. This enzyme clots
the plasma component of blood. The only significant disease causing bacteria of humans that
produce coagulase are Staphylococcus aureus. Thus this enzyme is a good indicator of S. aureus.
In the test, the sample is added to rabbit plasma and held at 37 ⁰C for a specified period of time.
Formation of clot within four hours is indicated as positive result and indicative of a virulent
Staphylococcus aureus strain. The absence of coagulation after 24 hours of incubation is a
negative result indicative of an avirulent strain.

3.6.4 Oxidase Test

Oxidase test is an important differential procedure that should be performed on all gram negative
bacteria for their rapid identification. The test depends on the ability of certain bacteria to
produce indophenol blue from the oxidation of dimethyl-p-phenylenediamine and ∞-naphthol.
This method uses N, N-dimethyl-p-phenylenediamine oxalate in which all Staphylococci are
oxidase negative. In the presence of the enzyme cytochrome oxidase (gram negative bacteria) the
N, N-dimethhyl-p-phenylenediamine oxalate and ∞-naphthol react to indophenol blue.
Pseudomonas aeruginosa is an oxidase positive organism.

3.6.5 Sugar Fermentation/Oxidation

This test is used to differentiate between bacteria groups that oxidize carbohydrate such as
members of Enterobacteriaceae. One milliliter (1ml) of 10% glucose, maltose, lactose, fructose,
mannitol, and sucrose were separately under aseptic conditions transferred into duplicate tubes

3
containing 9ml of sterile Hugh and Leifson’s medium to obtain a final concentration of 1% of
each of sugar. The tubes were stab-inoculated in duplicates while two uninoculated tubes serve
as control. Vaseline was used to cover one set of the duplicate tubes, one control to discourage
oxidative utilization of sugar. All tubes were incubated at 37oc for 48h. After the incubation, they
were observed for acid production in the culture. Yellow colouration indicates acid production in
the open tubes only suggesting oxidative utilization of the sugar while acid production in the
sealed tubes suggests a fermentative reaction.

3.6.6 Hydrogen Sulphide Production (H2S) Test

The test isolates were aseptically inoculated into a tube containing triple sugar iron agar started
by stabbing the agar to the bottom and streaking the surface of the slant. The inoculated tube was
incubated at 37oC for 72 h and was examined daily. Black precipitation and yellow colouration
was checked for. Black precipitate indicates H 2S production and yellow colouration for sucrose,
lactose and glucose fermentation.

3.6.7 Urease Test

Urease Agar slant in McCartney bottle was inoculated with the bacteria isolate at 30 oC for 4 h
and then overnight. A pink colour in the medium indicated a positive result.

3.6.8 IMViC Test

This test consists of four different test; they are Indole production, Methyl-Red test, Voges

Proskaeur test and Citrate utilization test. This test is specifically designed to determine the

physiological properties of microorganism. They are especially useful in the differentiation of

Gram-negative intestinal bacilli, particularly Escherichia coli and the Enterobacter-Klebsiella

group.

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3.6.9 Indole Test

This test demonstrates the ability of certain bacteria to decompose the amino acid-Tryptophan to
Indole. The bacteria isolates were inoculated into the medium and incubated at 37°C for 48 h. At
the end of incubation period, 3 drops of kovac’s reagents (see appendix) was added and then
shaken. A red colour ring at the interface of the medium denotes a positive result.

Methyl red and Voges-Proskaeur test must be considered together since they are physiologically
related. Opposite test is usually obtained from the MR and VP terst, that is, MR+, VP-, or MR-,
VP+.

Methyl red test was preformed to demonstrate the capacity of different organisms to produce
acid from the fermentation of sugar (dextrose). Methyl-red positive organisms produce a red
colouration when five drops of methyl-red indicator is added into 48 h old MR-VP broth culture.

The Voges-Proskaeur test demonstrates the ability of organisms to produce acetoin from glucose
metabolism. Some organisms metabolise glucose to produce pyruvic acid which is further
broken down to yield Butane-diol and acetyl-methyl carbinol as an intermediate product.

Into one milliliter of the culture add one milliliter of six percent alcoholic solution of alpha-
naphtol and one milliliter of 16% KOH and stand for 15-20 minutes. Development of red to pink
colour is a positive test.

Citrate Utilization Test

This is one of the several techniques used to assist in the identification of Enterobacteria.
Principle of the test is based on the ability of an organism to use citrate as its only source of
carbon. The test was carried out using Simmon’s citrate agar.

The slopes of the media were prepared in bijou bottles as recommended by the manufacturers. A
sterile straight wire was used to the slope with a saline suspension of the test organisms before
stabbing the butt. The bottles are incubated at 35oC for 48 h. Bright blue colours in the medium

3
means positive test while no change in colour of medium indicates negative citrate test
(Cheesbrough, 2000).

3
 CHAPTER FOUR

RESULTS

Table 1

Total counts and colonial characteristics of Bacteria isolated on Salmonella Shigella

Agar (SSA)

Sample codes Total Colony Colonial Characteristics Most Probable Identity

counts Types
(Cfu/g)
A - - - -
B 1.0 x 103 Bx Moist and shiny black fish Salmonella sp
eye colonies
C 3.0 x 103 Moist and shiny black fish Salmonella sp
eye colonies
D 5.3 x 104 Dx Moist and shiny black fish Salmonella sp
eye colonies

Light pink/colourless moist

Dy Shigella sp
and shiny colonies
E 1.2 x 104 Moist and shiny black fish Salmonella sp
eye colonies
G - - - -
H 2.1 x 104 Light pink/colourless moist Shigella sp
and shiny colonies
I - - - -
J 1.3 x 104 Light pink/colourless moist Shigella sp
and shiny colonie
K 2.0 x 103 Moist and shiny black fish Salmonella sp
eye colonies

3
Sample codes Total Colony Colonial Characteristics Most Probable Identity
counts Types
(Cfu/g)
L 2.7 x 104 Light pink/colourless moist Shigella sp
and shiny colonies.
M 1.2 x 104 Moist and shiny black fish Salmonella sp
eye colonies.
N TNTC Light pink/colourless moist Shigella sp
and shiny colonies.
O 1.0 x 103 Light pink/colourless moist Shigella sp
and shiny colonies.
P 1.0 x 103 Light pink/colourless moist Shigella sp
and shiny colonies.
Q 7.9 x 104 Moist and shiny black fish Salmonella sp
eye colonies.
R 1.0 x 103 Light pink/colourless moist Shigella sp
and shiny colonies.
S 1.5 x 104 Light pink/colourless moist Shigella sp
and shiny colonies.
T 1.0 x 103 Light pink/colourless moist Shigella sp
and shiny colonies.
U 3.0 x 103 Light pink/colourless moist Shigella sp
and shiny colonies.
V 2.0 x 103 Light pink/colourless moist Shigella sp
and shiny colonies.
W TNTC Light pink/colourless moist Shigella sp
and shiny colonies.
X 7.0 x 104 Moist and shiny black fish Salmonella sp
eye colonies.
Y 901 x 104 Moist and shiny black fish Salmonella sp
eye colonies.

3
Sample codes Total Colony Colonial Characteristics Most Probable Identity
counts Types
(Cfu/g)
Z 6.6 x 104 Moist and shiny black fish Salmonella sp
eye colonies
F 4.3 x 104 Light pink/colourless moist Shigella sp
and shiny colonies

4
 Table 2
Colonial and Microscopic Characteristics of Bacteria isolated on Nutrient Agar and MSA

Colonial characteristics Spore Motility Gram Morphology Most Probable

Formation Identity
Moist and shiny black fish - + Gram negative rods in Salmonella sp
eye colonies singles and short chains
Small circular moist and shiny - - Gram negative rods in Shigella sp
light pink to colourless short chains
colonies

4
 Table 3
Biochemical Characteristics of Bacteria Isolated from Meat sample on NA and MSA

Oxi Cat Coag In MR VP S L G M NO3 H2S URE Identity of isolates

- + - - - + + + + - + + - Salmonella sp
- + - - + - - - - + + - - Shigella sp
Oxi, Oxidase; Cat, Catalase; Coag, Coagulase; In, Indole Test; MR, Methyl Red Test; VP,
Voges Proskaeur Test; S, Sucrose; L, Lactose; G, Glucose; M, Maltose; NO3, Nitrate reduction;
URE, Urease production; +, Positive Test; -, Negative Test

4
 Table 4
Distribution of Salmonella and Shigella species in samples

Sample Distribution Salmonella sp in samples Distribution Shigella sp in samples

code
A - -
B Salmonella sp
C Salmonella sp
D Salmonella sp Shigella sp
E Salmonella sp
F - Shigella sp
G - -
H - Shigella sp
I - -
J - Shigella sp
K Salmonella sp -
L - Shigella sp
M - Shigella sp
N - Shigella sp
O - Shigella sp
P - Shigella sp
Q - Shigella sp
R - Shigella sp
S - Shigella sp
T - Shigella sp
U - Shigella sp
V - Shigella sp
W - Shigella sp

X Salmonella sp -

Y Salmonella sp -

Z Salmonella sp -

4
 Table 5

Percentage Distribution of Salmonella sp in samples

Bacterial isolates Percentage Distribution

Salmonella sp 10 (38.5%)
Shigella sp 16 (61.5%)

4
 CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 Discussion of the Findings

After the incubation and biochemical test taken it was noticed that Salmonella and Shigella
where present in the different samples provided but no Escherichia coli was observed. Obiageli
Roseline Ezeigbo in 2004 reported a similar case in her research work where different brands of
feed and feacal samples where collected from different commercial distribution centres and
poultry farms in Abia, According to the researcher her results revealed that no salmonella species
were obtained from the commercial distribution however, all the samples the researcher collected
from different poultry farms were positive for salmonella species, giving prevalence rate of
100%.

Futher researches has been carried out to investigate the presence of salmonella spp, In the
Brazilian journal of poultry science published in 2003 laid emphasis on how salmonella species
occur in laying hens, the researcher Barrow in 2000 stated how salmonella are introduced into
the poultry farm including through day old infected chicks, domestic animals, humans, water
and feed.

Lastly in a research by Ezemba Constance et al in 2021 on the incidence of Salmonella and

Escherichia coli in poultry feeds, he stated that raw poultry and meat products consumption
remains the principal source of Salmonella and E. coli in many countries, He further stated in his
result gotten from eight different feed samples labeled from A-H in Anambra state, the sample
with the highest coliform count was sample H and the lowest was seen in sample A, sample D
had the highest viable bacteria count and was too numerous to count. In summary the bacteria
percentage of Salmonella was 62% and E. coli was 100% from different feed samples he further
stated in his research that with the presence of pathogens in feeds that there is need for good
manufacturing practices, handling and retailing methods to enhance the microbiological quality
of feeds.

4
The morphological characteristics was Moist and shiny black fish eye colonies and Small
circular moist and shiny light pink to colourless colonies which indicate the presence of shigella
and salmonella.

Other researches done by other researchers like ezemba constance et al 2021 shows both the
presence of E. coli and Salmonella.

5.2 Conclusion
Our poultry birds are always exposed to contamination from the environment, without necessary
sanitary measures been observed, even during the process of the poultry feed production. Further
research work should be carried out in other to curb the rate at which the poultry feed products
are produced. This will help in minimizing the effect of these organisms on human consumption
of this poultry products(birds).

4
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 APPENDIX

Fig 1. Birds FIG 2. Poultry birds

(SOURCE:https://images.app.goo.gl/ob (SOURCE
oTRwgmmQMcJkdA8) https://images.app.goo.gl/DNYhSTSnJbFv
gLwi9)

Fig 3 Rhode Island Red rooster

(Sourcehttps://upload.wikimedia.org/ Fig 4: turkey
wikipedia/commons/thumb/8/83/Rho (source: en.wikipedia.org)
de_Island_Red_cock.)

5
 Fig.5. Duck and gesse Fig. 6. Domestic goose
(source: https://petkeen.com/wp- (source:
content/uploads/2021/12/.jpg)
https://images.app.goo.gl/W9

Fig.8. (source:
Fig.7. Pigeon https://images.app.goo.gl/cH8vhgqA
(source: https://images.app.goo.gl/cH8vhgqA4qFPViC56 4qFPViC56))
Fig.7. Pigeon

5
 Fig.9. Gallus Varius Fig.10. Gallus Sonneratii (Source:
(source: ebird.org) https://images.app.goo.gl/j2rGnKqgGtC
UnuHD6)

Fig.11. Gallus Lafayetii

(Source:https://www.google.com/url?sa=i&url=https%3 Fig.12. Poultry Diseases
A%2F%2Fwww.travelblog.org%2FPhotos%2F9024318 (sources: Thepoultrysite.Com)
&psig=AOvVaw3fHGYh7SxRevhGBynFZQEL&ust=1
665931180948000&source=images&cd=vfe&ved=0CA
0QjRxqFwoTCPCstaW74voCFQAAAAAdAAAAABA
D)

5
 Fig. 13. Poultry feed source: (Source:
https://poultryreporternews.files.wordpress.com/2019/12/poultry-feed-formulation-
ingredients 1502246523_182-64-162-199.jpg)

Fig.14. pathogenic invasion of diseases Fig. 15. The administration of prebiotics

(source: en.wikipedia.org) and probiotics in poultry
(source: en.wikipedia.org)

5
Fig.16. Growthers,Top Feed and Layers, Top Top Feed
Samples(shop 1)

Fig.17. Starters,Top Feed and Finishers,Top Feed

Samples(shop 1)

5
Fig.18. At a retail store in Relief Market

Fig.19. sample collection

6
Fig. 20. Serial Dilution Stock Samples and media preparation

Fig. 21. Preparation of media

6
 .

Fig.22. Colony Counting