The Armed Forces Institute of Pathology

Department of Veterinary Pathology

WEDNESDAY SLIDE CONFERENCE
2004-2005

SLIDE 1
CONFERENCE 1 / CASE I – N03-516 (AFIP 2933954)

Signalment: 12 year-old, female, Alpine Swiss, caprine, goat.

History: This 12 year-old goat presented with coughing, dyspnea and white mucous
membranes. Radiographs and ultrasound revealed a 1.5-inch diameter mass on the left
lung lobe. A CBC revealed evidence of anemia due to blood loss. A transtracheal wash
indicated reactive respiratory epithelial hyperplasia and hemorrhage. The animal died.

Gross Pathology: The lungs were moist and poorly collapsed. Several white nodules
up to 2 cm in diameter were present on the surface of all lobes. On cut section, the lung
was gelatinous and bulged. Small numbers of Haemonchus contortus were present in
the abomasum. Caseous material exuded from an abscess over the medial aspect of
the right hock joint.

Laboratory Results: No parasite ova were present on fecal examination. Alpha-

hemolytic streptococci were isolated from the hock joint abscess. On AGID the serum
was positive for CAE virus antibody.

Contributor’s Morphologic Diagnosis: Pneumonia, lymphocytic, bronchointerstitial,

diffuse, severe, with alveolar proteinosis, lung.

Contributor’s Comment: The histologic appearance of the lung is consistent with

pneumonia caused by caprine arthritis-encephalitis (CEA) virus disease, with findings of
type II pneumocyte proliferation, lymphoid infiltration and proteinaceous fluid within
alveoli (alveolar proteinosis). Electronmicroscopy has shown that the proteinaceous
fluid found in CAE is lung surfactant.

CEA syndrome is a viral disease of domestic goats that manifests as chronic

proliferative synovitis and periarthritis and progressive pneumonia in adult goats and
afebrile leukoencephalomyelitis in goat kids.1 The causative agent, a Lentivirus, is
transmitted from adult goats to kid goats via colostrum or by lateral transmission. The
CAE virus has a worldwide distribution. All breeds and ages of goats are susceptible to
infection and once established the infection persists throughout the animal’s life. A
diagnosis is established on clinical signs, demonstration of serum antibodies on ELISA
and pathological changes such as diffuse pulmonary consolidation or hypertrophic,
proliferative synovitis with intra-articular rice bodies. There is a microscopic component
of interstitial mastitis leading to induration and agalactia.

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Viral RNA can be identified in macrophages where viral transcription occurs. Infected
macrophages may be detected with immunohistochemistry techniques in tissues such a
2,3
lung, udder, and lymph nodes.

Interestingly, the mass identified radiographically in the left lung lobe turned out to be a
secondary granulomatous pleuritis with intralesional Cryptococcus neoformans yeast
organisms.4

AFIP Diagnosis: Lung: Pneumonia, interstitial, proliferative, lymphocytic, diffuse,

moderate, with alveolar proteinosis and secondary acute bronchopneumonia, Alpine
Swiss, caprine.

Conference Comment: Conference attendees discussed slide differences with some

sections containing distinct lymphoid nodules, while others contained variable numbers
of interstitial and perivascular lymphoid infiltrates. However, all slides were
characterized by prominent type II pneumocyte hyperplasia and abundant alveolar
proteinosis, which are characteristic of CAE virus.5 Most slides also had features
consistent with bronchopneumonia; bronchioles and alveoli filled with an exudate
composed of degenerate neutrophils, necrotic debris, and proteinaceous fluid. This is
not surprising, as secondary bacterial infections are common in animals with CAE.5
However, no organisms were seen with H&E, B&B, B&H, or PAS.

CAE virus is a lentivirus (subfamily Lentivirinae, family Retroviridae). Lentiviruses are

non-oncogenic retroviruses with clinical disease characterized by long incubation
periods, persistent infection, and a progressive course. Other lentiviruses include:
maedi-visna virus in sheep (ovine progressive pneumonia); equine infectious anemia
virus in horses and, human, simian, bovine and feline immunodeficiency viruses.

Other causes of pneumonia in sheep and goats include:

Viral:
Maedi-visna virus (ovine progressive pneumonia)
Caprine Morbillivirus (Peste des petits ruminants)
Bacterial:
Mannheimia (Pasteurella) haemolytica (ovine pneumonic pasteurellosis)
Mycoplasma ovipneumoniae (chronic enzootic pneumonia)
Mycoplasma mycoides spp. mycoides large colony (contagious caprine pneumonia)
Mycobacterium bovis (tuberculosis)
Mycobacterium avium (tuberculosis)
Parasitic:
Dictyocaulus filaria
Muellerius capillaris
Protostrongylus rufescens

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Contributor: University of Florida College of Veterinary Medicine, Dept of
Pathobiology, Box 110880, Gainesville, FL 32610

References:
1. Ellis, TM, Robinson, WF, Wilcox, GE.: The pathology and aetiology of lung lesions in
goats infected with caprine arthritis-encephalitis virus. Aust Vet J 65:69-73,1988
2. Zink, MC, Yager,JA, Myers,JD: Pathogenesis of caprine arthritis encephalitis virus.
Cellular localization of viral transcripts in tissues of infected goats. Am J Pathol 136:843-
854.1990
3. Storset AK, Evensen, O, Rimstad E. Immunohistochemical identification of caprine
arthritis-encephalitis virus in paraffin-embedded specimens form naturally infected
goats. Vet Pathol 34:180-188,1997
4. Gutierrez M, Garcia Marin JF. Cryptococcus neoformans and Mycobacterium bovis
causing granulomatous pneumonia in a goat. Vet Pathol 36:445-448, 1999
5. Lopez A: Respiratory system, thoracic cavity, and pleura. In: Thompson’s Special
Veterinary Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp.174-178.
Mosby, St. Louis, MO, 2001

SLIDE 2
CONFERENCE 1 / CASE II – 03N0138 (AFIP 2936140)

Signalment: 2-year-old male castrated Great Dane.

History: The dog presented with a several day history of weight loss, vomiting,
diarrhea, and urinary incontinence. The dog had been treated for six months with
immunosuppressive doses of prednisone (1 mg/kg PO q12h for 2 weeks) for chronic
lymphocytic-plasmacytic enteritis (inflammatory bowel disease).
The dog continued to have weight loss and diarrhea but also developed profound
lethargy and hematuria. Despite treatment, the clinical signs worsened and two weeks
later, the dog presented to the hospital recumbent, comatose, and with intermittent
seizures and rotary nystagmus. Due to the poor prognosis, the owners elected to
euthanize the dog.

Gross Pathology: Necropsy revealed numerous small, randomly distributed

granulomas restricted to the kidneys and brain. In both kidneys, the lesions spread
from the cortico-medullary junction to the cortex. The kidney lesions consisted of 0.3 to
1.0 cm in diameter soft raised tan well-demarcated raised nodules that were distributed
randomly throughout the parenchyma. In the brain, lesions were found in the right
occipital lobe and right caudal cerebellar peduncle. The brain lesions were well-
demarcated unencapsulated gelatinous tan foci.

Laboratory Results: Blood cultures were negative and the kidney culture revealed one
colony of hemolytic E. coli.

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Contributor’s Morphologic Diagnosis: Kidney: Multifocal severe granulomatous and
necrotizing nephritis with intralesional amoeba organisms.

Contributor’s Comment: The histologic features observed throughout this kidney

consisted of marked interstitial edema and tubular necrosis associated with marked
perivascular inflammatory infiltrates mainly comprised of macrophages, lymphocytes,
Langerhans type giant cells, plasma cells and small numbers of neutrophils. Admixed
with these cells were large numbers of amebic organisms in two different life stages,
trophozoites and cysts. The trophozoites ranged in size from 15-45 _m in diameter, and
were round to oval with occasional short plump cytoplasmic processes (pseudopodia).
Their cytoplasm was pale, eosinophilic, and partially vacuolated and contained a 4-6 _m
in diameter round, pale-staining vesicular nucleus. In contrast, the cysts were smaller,
more uniformly round, and surrounded by an undulating outer wall and an internal thick
round basophilic inner wall. The cytoplasm was scarce and contained multiple,
hyperchromatic basophilic granules. The nucleus was centrally located with 1-2
prominent eosinophilic karyosomes. The cystic forms were distributed throughout the
renal interstitium, but were most prominent within collecting tubules. The trophozoites
were more abundant near blood vessels.

In the brain, lesions associated with this case had histologic features of fibrinoid
necrosis of the blood vessels and a florid perivascular infiltration of neutrophils and
macrophages admixed with lakes of fibrin. There were also extensive areas of malacia
associated with astrogliosis, surrounding the affected vessels. Large numbers of
trophozoites were concentrated primarily in the perivascular regions of the gray matter,
the meninges and the choroid plexus. The cystic forms were located further away from
the blood vessels and were associated with less inflammation and necrosis.

The amoeba in this case were diagnosed as Balamuthia mandrillaris based on positive
immunohistochemical results (performed at San Bernardino) and PCR, which used
specific primers designed for this case at Dr. Sykes’ laboratory (UC Davis, VMTH).

Balamuthia mandrillaris is a free-living ameba of the order Leptomyxida capable of

causing fatal granulomatous amebic meningoencephalitis (GAE) in humans and
animals1. It was first isolated from a mandrill1,6 (Papio sphinx) at San Diego Zoo Wild
Animal Park, then subsequently in gorillas2 (Gorilla gorilla gorilla), an orangutan3 (Pongo
pygmaeus) and Old World primates, including a colobus monkey4 (Colobus guereza
kikuyuensis) and a gibbon (Hylobates concolor leucogenys). In recent years, B.
mandrillaris has also been increasingly identified as a cause of GAE in humans.1,5,6 So
far, the only reports in a non-primate species have involved a horse7 and a sheep.8 B.
mandrillaris has been recently isolated from environmental samples suggesting it
occupies similar habitats as other opportunistic ameba such as Naegleria fowleri and
Acanthamoeba spp..5,6 The route of invasion is still unknown, however penetration of
skin or respiratory tract and subsequently hematogenous spread has been postulated.6
Infections with B. mandrillaris are reported to be more common in immunosuppressed
hosts such as patients with AIDS, although immunocompetent individuals also have
been affected.6 Reports of canine disease caused by free -living amebas are rare.

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There is a single report of kidney lesions associated with dissemination of
Acanthamoeba castellani.9

There are two forms of B. mandrillaris, a trophozoite form and a cyst form. The
trophozoite amoebic form ranges in size from 15-60 _m in diameter and has a round
nucleus and a dense nucleolus. More than one nucleolus can be observed, which aids
as a distinguishing feature between B. mandrillaris and Acanthamoeba.6 The
trophozoite has pale, eosinophilic, and partially vacuolated cytoplasm that can
occasionally be observed branching into short plump cytoplasmic processes
(pseudopodia). In contrast, the cyst form, which is the dormant form, is smaller, more
spherical, uninucleated, measuring 15-60 _m, and has a prominent bilayer wall with
granules positioned beneath the inner cell wall.6

The precise pathogenesis of B. mandrillaris is unknown. However, a recent review

discusses the life cycle and pathogenesis of Entamoeba histolytica. Initial infection is
through ingestion of contaminated food or water with E. histolytica cysts. Cysts are
ingested and excysted in bowel lumen. The invasive trophozoite form invades intestinal
epithelium and spreads to other sites.10 In the case of B. mandrillaris, infection may
occur through ulceration in skin or lower respiratory tract infection. Spread to other
sites, especially the brain, is thought to be hematogenous.

AFIP Diagnosis: Kidney: Nephritis, interstitial, necrotizing, pyogranulomatous,

multifocal to coalescing, moderate, with amebic trophozoites and cysts, Great Dane,
canine.

Conference Comment: This case was reviewed in consultation with Dr. Chris
Gardiner, AFIP consultant in veterinary parasitology. The contributor provides a
thorough description of the gross and histologic lesions associated with B. mandrillaris.
Other important amebic organisms to consider include: Acanthamoeba spp.;
Entamoeba histolytica; Naegleria fowleri; and, Hartmannella spp..

Acanthamoeba spp. is a free-living organism found in fresh water, soil, or sewage.

Transmission is via inhalation with the lung being the primary target organ. However,
the central nervous system and other organs may be affected through hematogenous
spread. This organism is also known to cause granulomatous amebic encephalitis
(GAE) in humans and other species. Trophozoites are 10-30 _m in diameter, contain
an eccentric nucleus, a single nucleolus, and eosinophilic cytoplasm with glycogen
vacuoles. Cysts are generally rare.11

Entamoeba histolytica is often a non-pathogenic inhabitant of the large intestine, only

occasionally causes amebic dysentery in humans and nonhuman primates, and rarely
affects other species. Transmission is via ingestion resulting in characteristic flask-
shaped intestinal ulcers. Hematogenous and lymphatic dissemination to the brain, liver,

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or other organs may occur. Trophozoites are 6-50 _m in diameter, often surrounded by
a clear halo, and contain an eccentric nucleus with a distinct karyosome.12

Naegleria fowleri is a free-living ameba that causes acute and fulminating primary
amebic meningoencephalitis (PAM) primarily in young healthy humans, with rare reports
in animals. Naegleria is commonly found in fresh water, soil, and sewage.
Transmission is via inhalation with invasion of the olfactory neuroepithelium.
Trophozoites are 6-12 _m in diameter with a centrally located nucleus and a large single
nucleolus. Unlike Acanthamoeba and Balamuthia, cysts are generally not found in
neural tissue.13

Hartmannella sp. is a non-pathogenic, free-living amoeba. Older isolates of

Hartmannella suspected of being opportunistic pathogens (like Acanthamoeba and
Naegleria) have now been reclassified as Acanthamoeba. True Hartmannella sp. are
not known to cause CNS infections in humans or other species.5

Contributor: University of California, Davis, Dept. of Veterinary Pathology, Vet. Med.

Teaching Hospital,1 Garrod Drive, Davis, CA 95616

References:
1. Visvesvara GS, Schuster FL, Martinez AJ: Balamuthia mandrillaris, N.G.,N.Sp.,
agent of amebic meningoencephalitis in humans and other animals. J Eukaryot
Microbiol 40:504-514, 1993
2. Anderson MP, Oosterhuis JE, Kennedy S, Benirschke K: Pneumonia and
meningoencephalitis due to ameba in a lowland gorilla. J Zoo Anim Med 17:87-91, 1986
3. Canfield PJ, Vogelnest L, Cunningham MI, Visvisvara GS: Amoebic
meningoencephalitis caused by Balamuthia mandrillaris in an orang utan. Aust Vet J
75:97-100, 1997
4. Rideout BA, Gardiner CH, Stalis IH, Zuba JR, Hadfield T, Visvisvara GS: Fatal
infections with Balamuthia mandrillaris (a free-living amoeba) in gorillas and other Old
world primates. Vet Path 34:15-22, 1997
5. Martinez AJ, Visvisvara GS: Free-living, amphizoic and opportunistic amebas. Brain
Pathol 7:583-598, 1997
6. Martinez AJ, Visvisvara GS: Balamuthia mandrillaris infection. J Med Microbiol
50:205-207, 2001
7. Kinde H, Visvisvara GS, Barr BC, Nordhausen RW, Chiu PH: Amebic
meningoencephalitis caused by Balamuthia mandrillaris (leptomyxid ameba) in a horse.
J Vet Diagn invest 10:378-381, 1998
8. Fuentealba IC, Wikse SE, Read WK, Edwards JF, Visvesvara GS: Amebic
meningoencephalitis in a sheep. J Am Vet Med Assoc 200:363-365, 1992
9. Pearce JR, Chandler FW, Visvesvara GS: Amebic meningoencephalitis caused by
Acanthamoeba castellani in a dog. JAVMA 187:951-52, 1985
10. Hague R, Huston CD, Hughes M, Houpt E, Petri WA: Current Concepts –
Amebiasis. The New England Journal of Medicine 348 (16), pp., 15651573. 2003
11. Greene, CD: Acanthamebiasis. In: Clinical Microbiology and Infectious Diseases of
the Dog and Cat. 2nd ed., pp.491-493. WB Saunders, Philadelphia, PA, 1998

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12. Barker IK, Van Dreumel AA, Palmer N: The Alimentary System. In: Pathology of
Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 2, pp. 315-316.
Academic Press, San Diego, CA, 1993
13. Lozano-Alarcon F, Bradley GA, Houser BS, Visvesvara GS: Primary amebic
meningoencephalitis due to Naegleria fowleri in a South American tapir. Vet Pathol
34:239-243, 1997

SLIDE 3
CONFERENCE 1 / CASE III – L02-3969-1A or -1B or L04-3969-1C (AFIP 2936455)

Signalment: Adult, unknown sex, Boa constrictor.

History: History of lethargy, regurgitation, head tremors and death.

Gross Pathology: Uric acid deposits in multiple visceral organs (visceral gout).

Contributor’s Morphologic Diagnoses:

Liver: There are multifocal, large, circular areas of acute hepatocellular necrosis
surrounded by minimal inflammation. Within the center of most foci are shards of clear
spaces surrounded by cellular debris, histiocytes and acidophilic fibrillar material
(dissolved uric acid tophi). Approximately 80% of hepatocytes contain single to
multiple, prominent, eosinophilic, circular 5-10 micron diameter, intracytoplasmic
inclusion bodies. Randomly scattered individual to small clusters of hepatocytes are
shrunken, hypereosinophilic and have fragmented nuclei (necrosis).

Kidney: There is severe architectural distortion by scattered large aggregates of

necrotic cellular debris, mineralized basophilic material and dilated tubules containing
shards of clear material and mineralized debris. Dilated tubules are lined by
fragmented, shrunken, hypereosinophilic epithelial cells, many containing large
eosinophilic prominent intracytoplasmic inclusion bodies (as previously described).
Tubule lumens are filled with mineralized basophilic granular debris and shards of clear
material surrounded by histiocytes and lymphocytes. The interstitium is widened by
bands of edema fluid mixed with scattered lymphocytes and histiocytes.

Stomach: Lining epithelial cells contain large numbers of 5-15 micron diameter, deeply
eosinophilic intracytoplasmic inclusion bodies.

Lung: Epithelial cells lining bronchi and alveoli contain single to multiple, brightly
eosinophilic 5-7 micron diameter, intracytoplasmic inclusion bodies.

1. Hepatitis, granulomatous with uric acid deposition and multifocal hepatocellular

necrosis.
2. Urate nephrosis, severe, chronic with tubular dilatation and mineralization.
3. Inclusion bodies, multifocal/multiorgan, epithelial cells and hepatocytes, diffuse.

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Contributor’s Comment: Histologic lesions indicate the cause of death is visceral gout
secondary to Boid inclusion body disease (IBD). Gout is a common sequela to chronic
infection or debilitating conditions in reptiles with dehydration often being a primary
compounding issue. Underlying viral infections (IBD) also lead to secondary bacterial
infections due to immunosuppression and debilitation.

Boid inclusion body disease (IBD) is a fatal disorder of boid snakes caused by a
retrovirus that was described in 1994.1 Clinical signs of IBD include chronic
regurgitation and central nervous system disorders manifested in head tremors,
disorientation and paresis and/or paralysis. Histologic examination reveals numerous
eosinophilic intracytoplasmic inclusion bodies in epithelial cells of all major organs, in
hepatocytes and within neurons of the CNS. Inclusion bodies contain an antigenically
distinct 68 kDa protein.2 In all snakes with CNS disease, nonsuppurative
meningoencephalitis with neuronal degeneration and perivascular cuffing was present
and viral particles resembling type C retrovirus were detected in the brain, pancreas,
and kidney as well as in cultured kidney cells. The disease was shown to be
transmissible by cell free primary kidney culture supernatants from infected Boa
constrictor snakes to young Burmese pythons (Python molurus bivittatus) or by liver
homogenates from Boa constrictor.2 These data imply that a C type retrovirus may
indeed be the causative agent of IBD.

AFIP Diagnoses: 1. Liver: Mineral deposition (gouty tophi), multifocal, with minimal
granulomatous inflammation, boa constrictor (boa constrictor), reptile.
2. Liver, hepatocytes: Inclusion bodies, eosinophilic, intracytoplasmic, multifocal.
3. Liver, hepatocytes: Degeneration, multifocal, moderate, with random single cell
necrosis.

Conference Comment: The contributor provides a thorough overview of boid inclusion

body disease (IBD), a fatal disorder of boid snakes, thought to be caused by a
retrovirus. It is well established that snakes with IBD have increased susceptibility to
secondary infections and a variety of neurological signs. It is not surprising this snake
likely developed gout secondary to IBD.

Gout is a common clinical finding in reptiles and may also affect birds, humans, non-
human primates, and the Dalmatian dog. There are two forms of gout in birds and
reptiles: articular and visceral. Articular gout is less common and presents as white
deposits on tendon sheaths. Visceral gout is more common and presents as chalky
white patches on visceral surfaces. Gout results from hyperuricemia (elevated plasma
uric acid concentration), which may be caused by impaired renal function and clearance
of urates, nephrotoxic drugs, dietary excesses (protein and calcium) and deficiencies
(vitamin A), and dehydration.3 Nucleic acids ingested in foods undergo enzymatic
hydrolysis to yield free purine (adenine, guanine) and pyrimidine bases. In humans, the

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formation of uric acid from purine degradation has been extensively studied and
requires xanthine oxidase:

Adenine>hypoxanthine>xanthine>uric acid>allantoin>allantoic acid>urea

Guanine>xanthine>uric acid>allantoin>allantoic acid>urea

In order to conserve water, birds and reptiles excrete uric acid rather than urea.4,5 The
majority of uric acid excretion is via renal tubular secretion and is largely independent of
urine flow rate. When hyperuricemia occurs, uric acid crystals may be deposited in
joints, viscera, or extra-visceral sites.6 The crystals that are deposited in tissues (urate
tophi) are often dissolved during processing. Histologically, all that remains are large
aggregations of clear acicular clefts, frequently surrounded by granulomatous
inflammation.

In humans, gout may be the result of a deficiency in hypoxanthine guanine

phosphoribosyl transferase (HGPRT), leading to increased production of uric acid due
to synthesis of purine nucleotides from non-purine precursors. A complete lack of
HGPRT occurs in the uncommon X-linked Lesch-Nyhan syndrome seen in males that is
characterized by hyperuricemia, severe neurologic deficits, and occasionally gouty
arthritis.7

Contributor: Utah Veterinary Diagnostic Laboratory, 950 E 1400 N, Logan, UT, 84322
(www.usu.edu)

References:
1. Huder, J B, Boni J, Hatt, JM, et al. Identification and characterization of two closely
related unclassifiable endogenous retroviruses in pythons. J Virol 76(15): 7607-7615,
2002
2. Wozniak E, McBride J, Denardo D, et al. Isolation and characterization of an
antigenically distinct 68 kd protein from non viral intracytoplasmic inclusions in Boa
constrictors chronically infected with the inclusion body disease virus (ibdv:
Retroviridae). Vet Pathol 37: 449-459, 2000
3. Jones TC, Hunt RD, King NW: Veterinary Pathology, 6th ed., pp.60-61. Williams and
Wilkins, Baltimore, MD, 1997
4. Mader DR. Gout. In: Reptile Medicine and Surgery, pp.374-379. WB Saunders Co.,
Philadelphia, PA, 1996
5. Mader DR: Reptilian metabolic disorders. In: Laboratory Medicine: Avian and Exotic
Pets, ed. Fudge AM, pp.213-215. WB Saunders Co., Philadelphia, PA, 2000
6. Lumeij JT: Nephrology. In: Avian Medicine: Principles and Application, ed., Ritchie
BW, Harrison GJ, Harrison LR, pp.539-542. Wingers Publishing, Lake Worth, FL, 1994
7. Rosenberg AE: Bones, joints, and soft tissue tumors. In: Robbins and Cotran
Pathologic Basis of Disease, eds. Kumar V, Abbas AK, Fausto N, 7th ed., pp.1311-1314.
Elsevier Saunders, Philadelphia, PA, 2005

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SLIDE 4
CONFERENCE 1 / CASE IV – 0404975 (AFIP 2937765)

Signalment: Approximately 16 month-old female B6C3F1 mouse (Mus musculus)

History: The mouse was a sentinel animal and did not receive any treatment. It was
found dead at approximately 16 months of age.

Gross Pathology: Macroscopic examination revealed about 5 cc of a red fluid in the

abdominal cavity. The spleen was enlarged (32x6x3 mm) and the mediastinal lymph
node was enlarged (3x) and dark. A red mass was found on the right ovary (47x31x19
mm).

Contributor’s Morphologic Diagnosis: Yolk Sac (Carcinoma) Tumor

Contributor’s Comment: The tumor is composed of nests, clusters or ribbons of

discrete cells embedded within an abundant eosinophilic matrix. The cells vary in size
from small to large and have distinct borders and hyperchromatic nuclei.1,2,3

AFIP Diagnosis: Ovary: Yolk Sac Carcinoma, B6C3F1 mouse, rodent.

Conference Comment: Conference attendees had difficulty with tissue identification.

Some sections contain rare follicles at various stages of development. Tumors arising
from ovarian tissue are divided into three broad categories based on embryological
origin: tumors of surface epithelium, tumors of gonadal stroma, and tumors of germ
cells. Tumors may also arise from more than one of the three embryological lineages
(mixed tumors) or from nongonadal support tissues, including smooth muscle, vascular,
or fibroblastic tissue.4

Epithelial tumors may arise from ovarian surface epithelium, subsurface epithelial
structures (SES) in canines, and the rete ovarii. These tumors commonly appear as
unilateral or bilateral multinodular, cystic outgrowths extending from the ovarian surface.
Examples of epithelial tumors include papillary or cystic adenoma, or less frequently
papillary or cystic adenocarcinoma. These tumors are common only in the dog as SES
are unique to this species.5

Gonadal stromal tumors, also known as sex cord-stromal tumors, arise from granulosa,
theca, or interstitial cells. Granulosa cell tumors (granulosa-theca cell tumor) are the
most common ovarian tumor in the cow and mare. Granulosa cell tumors in the mare
frequently produce inhibin, which is thought to cause atrophy of the contralateral ovary.
Theca cells may be present in these tumors and either cell population may be
luteinized. Thecomas (theca cell tumor) are rare tumors composed of lipid-containing
cells of stromal origin, resembling theca interna cells. Interstitial cell tumors (luteoma,

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lipid cell tumor, steroid cell tumor) are composed of large rounded cells with round
central nuclei, resembling Leydig, luteal, or interstitial gland cells. However, the origin
of these cells has not been clearly identified.6

Germ cell tumors may fail to differentiate (dysgerminoma), differentiate into somatic
tissue (teratoma), or differentiate into extraembryonic structures (yolk sac carcinoma,
choriocarcinoma). The dysgerminoma is an uncommon tumor, which is comparable to
the more common seminoma of the testicle. This malignant tumor is composed of cells
that resemble primordial germ cells. Teratomas are rare tumors thought to arise from
totipotential germ cells that have differentiated into two or more embryonic layers
(endoderm, mesoderm, ectoderm). Most of these tumors are composed of a variety of
tissues and are generally benign.6 Yolk sac carcinomas can be distinguished
histologically by nests and cords of neoplastic polygonal to cuboidal cells embedded in
abundant amounts of a characteristic eosinophilic PAS-positive matrix.2

Contributor: Toxicology Battelle Columbus, Pathology Dept, Room 7-0-37A, Battelle

Columbus Labs, 505 King Ave, Columbus, OH, 43201-2693 (www.battelle.org)

References:
1. Davis BJ, Dixon D, Herbert RA: Ovary, Oviduct, Uterus, Cervix, and Vagina. In:
Pathology of the Mouse, eds. Maronpot RR, Boorman GA and Gaul BW, pp. 428-430.
Cache River Press, Vienna, Illinois, 1999
2. Davis B, Harleman JH, Heinrichs M, Maekawa A, McConnell RF, Reznik G, Tucker :
Female Genital System. In: International Classification of Rodent Tumors: The Mouse,
edr. Mohr U, pp. 236-237. Springer-Verlag, New York, 2001
3. Maekawa A, Maita K, Harleman, JH: Changes in the Ovary. In: Pathobiology of the
Aging Mouse, eds. Mohr U, Dungworth DL, Capen CC, Carlton WW, Sundberg JP and
Ward JM, vol 1, pp. 465-466. ILSI Press, Washington DC, 1996
4. Kennedy PC, Miller RB: The female genital system. In: Pathology of Domestic
Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., pp.364-370. Academic Press,
San Diego, CA, 1993
5. MacLachlan NJ, Kennedy PC: Tumors of the genital systems. In: Tumors in
Domestic Animals, ed. Meuten DJ, 4th ed., pp.547-556. Iowa State Press, Ames, IO,
2002
6. Kennedy PC, Cullen JM, Edwards JF, Goldschmidt MH, Larsen S, Munson L,
Nielsen S: Tumors of the ovary. In: World Health Organization, International Histological
Classification of Tumor of Domestic Animals, Histological Classification of Tumors of the
Genital System of Domestic Animals, ed. Schulman FY, vol IV, pp.24-29.

SLIDE 5
CONFERENCE 2 / CASE I – N04-188 (AFIP 2937770)

Signalment: 27 year-old, female, mongoose lemur (Eulemur mongoz), non-human

primate.

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History: The lemur presented with a history of rapidly progressive neurological
symptoms, marked polyuria and polyphagia. A neurological examination revealed a
wide-based stance, circling, ataxia and falling to the right, as well as a weak grip in the
right hand and right foot. Cranial nerve reflexes and anal tone were within normal limits.
Additional findings included patchy truncal alopecia and a cataract in the left eye. The
very thin and debilitated lemur was euthanized due to poor prognosis and advanced
age.

Gross Pathology: The submitting veterinarian reported multifocal to coalescing

atheromatous plaques on the intima of the proximal abdominal aorta. On gross
examination of the brain after formalin fixation, there were multifocal random (approx.
10), pinpoint to 1 mm, red-brown foci in the gray and white matter of the cerebrum,
cerebellum and brain stem.

Laboratory Results: Serial serum chemistry profiles revealed marked persistent

hyperglycemia and on the day of euthanasia, the blood glucose was >700 mg/dL and
the urine glucose was >500 mg/dL.

Contributor’s Morphologic Diagnosis: Brain: 1) Severe, regionally extensive to

diffuse, granulomatous meningoencephalitis with intralesional myriad fungal yeast forms
(Cryptococcus sp.).
2) Mild to moderate, multifocal atherosclerosis with vascular thrombosis and acute and
chronic infarcts.

Contributor’s Comment: The brain contains a combination of lesions that are

associated with persistent diabetes mellitus, namely exacerbation of atherosclerosis
with vascular thrombosis and opportunistic fungal infection.1 The fungal
meningoencephalitis dominates the tissue changes. The yeast forms within these
lesions are highlighted with Gomori’s methenamine silver (GMS) stain and have
morphology consistent with Cryptococcus. (Although cultures were not performed,
infection with C. neoformans is suspected.) The precise mechanisms associated with
susceptibility to infection and immune compromise in diabetic patients is unclear, but
appear to involve both humoral and cellular mechanisms.2 In human patients with
diabetes mellitus, the common opportunistic fungal infections include sino-orbital
aspergillosis, rhinocerebral mucormycosis, and cryptococcal meningitis.3

Vascular thromboses and/or infarcts are present in the majority of brain sections; some
lesions are prominent and acute, while others are subtle and may be evidenced by focal
accumulations of hemosiderin. The cause of vascular disease in diabetes mellitus is
due in part to accelerated atherosclerosis, hypertension, and thickening of small vessels
(microangiopathy).1 Vascular thromboses and infarcts in the brain are recognized as a
complication of diabetes mellitus; although, there is evidence that cerebral infarction
may occur in cases of chronic meningitis.4,5

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AFIP Diagnoses: 1. Brain, cerebrum: Meningitis, granulomatous, multifocal,
moderate, with numerous yeast, etiology consistent with Cryptococcus neoformans,
mongoose lemur (Eulemur mongoz), primate.
2. Brain, cerebrum: Infarcts, multifocal, acute and chronic.

Conference Comment: Cryptococcus neoformans is a saprophytic fungus that causes

disease in a wide variety of animals, but most frequently in cats, dogs, horses and
humans. Infected animals are often immunosuppressed. Lesions can occur in any
organ, but are most common in the central nervous and respiratory systems, followed
by the integumentary system and eyes. Cryptococcus neoformans is a cause of
mastitis in cattle. Cryptococcosis is the most frequent systemic mycoses in cats, and
often occurs in the nasal cavity.

Gross lesions of Cryptococcus neoformans, are usually gelatinous due to the

organism’s mucopolysaccharide capsule. The capsule hinders phagocytosis and is a
major diagnostic feature of the organism. However, acapsular forms do exist.
Histologically, the yeasts are round, 5-20 µm in diameter, reproduce by narrow-based
budding and are usually surrounded by a 2-8 µm mucopolysaccharide capsule that
stains with mucicarmine and Alcian blue.6,7 The immune response varies from sparse
to granulomatous depending on the presence of a capsule and the host’s immune
status.
Chronic diabetes mellitus (DM) results in immunosuppression and damage to multiple
organ systems, especially the kidneys, eyes, nerves, and blood vessels. In humans,
diabetic macrovascular disease is a common complication of DM and is characterized
by accelerated atherosclerosis that often leads to infarction. Atherosclerotic vessels
were not evident in the sections of brain examined during the conference, which is
attributed to variation among slides noted by the contributor.

Contributor: North Carolina State University, College of Veterinary Medicine, Raleigh,

NC 27606

References:
1. Crawford JM, Cotran RS: The pancreas. In: Robbins Pathologic Basis of Disease,
eds. Cotran RS, Kumar V, Collins T, 6th ed., pp. 915-926. WB Saunders, Philadelphia,
PA 1999
2. Currie BP, Casey JI: Host defense and infections in diabetes mellitus. In: Ellenberg
and Rifkin’s Diabetes Mellitus, eds. Porte D, Sherwin RS, Baron A, 6th ed., pp. 601-604.
McGraw-Hill, 2003
3. Kauffman CA, Hedderwick S: Opportunistic fungal infections: filamentous fungi and
cryptococcosis. Geriatrics 52(10):40-2, 47-9, 1997
4. Karpinsky NC, Powell H. Case of the month: July 1997—diabetic male with transient
ischemic attacks. Brain Pathol 8(1):235-6, 1998
5. Lan SH, Chang WN, Lu CH, Lui CC, Chang HW. Cerebral infarction in chronic
meningitis: a comparison of tuberculous meningitis and cryptococcal meningitis. Q J
Med 94: 247-253, 2001

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6. McGavin MD, Carlton WW, Zachary JF: Nervous system. In: Thomson’s Special
Veterinary Pathology, 3rd ed., pp. 444. Mosby, Inc., Philadelphia, PA, 2001
7. Lichtensteiger CA, Hilf LE: Atypical cryptococcal lymphadenitis in a dog. Vet Pathol
31(4):493-496, 1994
8. Maitra A, Abbas AK: The endocrine system. In: Robbins and Cotran Pathologic
Basis of Disease, eds. Kumar V, Abbas AK, Fausto N, 7th ed., pp.1189-1205. Elsevier
Saunders, Philadelphia, PA, 2005
9. Frosch MP, Anthony DC, De Girolami U: The central nervous system. In: Robbins
and Cotran Pathologic Basis of Disease, eds. Kumar V, Abbas AK, Fausto N, 7th ed.,
pp.1361-1365. Elsevier Saunders, Philadelphia, PA, 2005

SLIDE 6
CONFERENCE 2 / CASE II – ND-1 (AFIP 2935562)

Signalment: 1-year-old female domestic shorthair cat (Felis domesticus).

History: This animal developed a sinus infection following surgery (procedure not
specified). Following antibiotic therapy the cat improved for a week then regressed to
prior condition. The owners requested euthanasia.

Gross Pathology: There was marked, bilateral, pulmonary stiffness with mottling of
the tissue. Fibrin strands were present on the pleural surface. Mucopurulent exudate
was present at the opening of both nares.

Laboratory Results: Aerobic culture of lung resulted in no growth. Fluorescent

antibody (FA) examination of frozen sections of lung was positive for feline herpesvirus-
1.

Contributor’s Morphologic Diagnosis: Pneumonia, necrotizing and fibrinous, diffuse,

acute, severe with intranuclear eosinophilic inclusion bodies.

Contributor’s Comment: Feline herpesvirus-1 (FHV-1) infections in cats

typically result in keratoconjunctivitis, upper respiratory tract disease and abortion.
Along with Chlamydophila psittaci and feline calicivirus, FHV-1 is part of an upper
respiratory disease complex referred to as “feline rhinopneumonitis”. Young animals
are more severely infected. Transmission is accomplished through direct contact with
infectious secretions or excretions. Vaccination does not prevent illness, but will limit
clinical signs. The disease tends to be more common where crowding, poor
management and carrier animals exist.

Acutely infected animals shed large amounts of virus for several weeks. The virus is
typically transmitted horizontally, but can be passed from mother to fetus as well. Most
infections are restricted to the upper respiratory tract; however, less common
involvement of the trachea and lung can occur. Systemic infections can lead to

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reproductive tract disease and reproductive failure. Kittens infected in utero may be
aborted or delivered normally and develop “fading kitten” syndrome. More typical
presentations of the disease result in sneezing, oculonasal discharge, rhinitis,
conjunctivitis, fever, anorexia, ulceration of the tongue and hard palate and herpetic
keratitis. Healthy cats recover in one to two weeks.

Multifocal necrosis typical of herpesvirus infections may be seen microscopically, often

leading to secondary bacterial infections. Acidophilic intranuclear inclusion bodies can
be detected up to a week after infection. This case is an example of the less common
necrotizing pneumonia seen in fulminant viral infections.

AFIP Diagnosis: Lung: Bronchopneumonia, necrotizing, acute, diffuse, severe, with

syncytia and epithelial eosinophilic intranuclear inclusion bodies, domestic shorthair,
feline.

Conference Comment: The contributor gives a thorough overview of feline

herpesvirus-1. Although upper respiratory tract infections are common in cats,
pneumonia is uncommon except when there is immunosuppression.4 Some
differentials that conference attendees considered for pneumonia in a cat included feline
calicivirus, Toxoplasma gondii, aspiration pneumonia, and toxins.

Feline caliciviral disease in cats has clinical and pathological similarities to feline
herpesviral infection. Clinical signs include oculonasal discharge, rhinitis, conjunctivitis,
and ulcerative stomatitis. Feline calicivirus has an affinity for epithelium, and additional
lesions include interstitial pneumonia and necrotizing bronchiolitis. The primary viral
infection is transient, but secondary bacterial infections are common.4

Toxoplasmosis is a worldwide disease that is often triggered by immunosuppression

and affects humans, dogs, cats, and many wild mammals. Pulmonary lesions include
severe necrotizing interstitial pneumonia, with prominent proliferation of type II
pneumocytes and infiltrates of histiocytes and neutrophils.4

Aspiration pneumonia, resulting from vomiting, regurgitation, dysphagia, or anesthetic

complication, is a common condition in cats. Lesions may be unilateral or bilateral and
most often affect the right cranial lobe. Histologically, the severity of the lesion often
depends on the chemical and microbial composition of the aspirated material.4

Toxins, such as paraquat, a broad-spectrum herbicide, can cause severe and often fatal
interstitial pneumonia in dogs, cats, humans and other species. Gross lesions include
interstitial emphysema, bullous emphysema and pneumomediastinum. Histologically,
there is extensive necrosis of alveolar epithelial and endothelial cells, interstitial and
alveolar edema, intraalveolar hemorrhage and proliferation of type II pneumocytes.4

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Contributor: North Dakota State University, Veterinary Diagnostic Laboratory, Fargo,
ND 58105 (www.vdl.ndsu.edu)

References:
1. Wolf AM: Other feline viral diseases. In: Textbook of Veterinary Internal Medicine,
Diseases of the Dog and Cat, eds. Ettinger SJ, Feldman EC, 5th ed., vol. 1, pp. 446-448.
W. B. Saunders Company, Philadelphia, PA, 2000
2. Dungworth DL: The respiratory system. In: Pathology of Domestic Animals, eds.
Jubb KV, Kennedy PC, Palmer N, 4th ed., vol. 1, pp. 558-559. Academic Press, San
Diego, CA, 1993
3. Stiles J: Feline herpesvirus. In: The veterinary clinics of North American, Small
animal practice, Infectious disease and the eye, ed. Stiles J, pp.1001-10014. W.B.
Saunders Company, Philadelphia, PA, 2000
4. Lopez A: Respiratory system, thoracic cavity, and pleura. In: Thompson’ s Special
Veterinary Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 139-140,
185-188. Mobsy, St. Louis, MO, 2001

SLIDE 7
CONFERENCE 2 / CASE III – 04-0825 (AFIP 2936447)

Signalment: 14-month-old female Angus, Bos taurus, bovine

History: This cow was from a herd of 32 cattle maintained on a partially forested
pasture in west-central Georgia. The cattle had been purchased in October 2003 and
had shown no clinical signs of disease until April 2004, when two adult cows were found
dead without precipitating clinical signs. This animal displayed depression, ataxia,
anorexia, and ptyalism. Clinical examination revealed central blindness, tremors, and
reduced rumen motility. Therapy included thiamine, vitamin B12, oral electrolytes, and
antibiotics. The cow was euthanized for diagnostic purposes. Differential diagnoses
included polioencephalomalacia (PEM), thromboembolic meningoencephalomyelitis
(TEME), plant toxicosis, infectious bovine rhinotracheitis (IBR), rabies, and lead
toxicosis.

Gross Pathology: The body of a 14-month-old female Angus bovine in good body
condition weighing 635 kg was presented for necropsy. Excessive saliva was present in
the oral cavity. A thick cloudy discharge exuded from the nares. The reticulum and
rumen contained approximately 10-15 liters of white to light-green fibrous contents,
which incorporated many angular irregularly shaped fragments of metal measuring 5-15
mm. Upon further examination, metal fragments were found not to be attracted to a
magnet. Scattered pieces of black plastic were scattered throughout the rumen
contents.

Laboratory Results:
Clinical Pathology:

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WBC: 14,990 cells/_l
Neutrophils: 5,097 cells/_l (some toxic neutrophils present)
Lymphocytes: 8,095 cells/_l
Basophils: 300 cells/_l
Fibrinogen: 700 mg/dl
Aspartate aminotransferase (AST): 317 U/L
Creatine kinase (CK): 3,631 U/L
Calcium: 8.2 mg/dl
Phosphorus: 11.3 mg/dl
Magnesium: 1.5 mg/dl
Glucose: 80 mg/dl
Iron: 175 mg/dl

Fluorescent antibody testing of brain for rabies virus: Negative

Lead analysis of kidney: 85ppm (wet weight)
Arsenic analysis of kidney: <1ppm (wet weight)
Lead analysis of metallic rumen contents: 17%

Contributor’s Morphologic Diagnosis: Neuronal necrosis, acute, locally extensive

(laminar), severe, with capillary endothelial hypertrophy and astrocytosis, cerebral
cortex.

Contributor’s Comment: Lead levels in the kidney of this case (85 ppm) were within
the reported toxic range for cattle (5-700 ppm).1 A diagnosis of acute lead toxicosis was
confirmed by the presence of lead-containing metal fragments within the rumen and a
laminar pattern of neuronal necrosis and capillary endothelial swelling within the
cerebral cortex. Further examination of the pasture revealed the recently destroyed
remnants of an automotive battery. Acid-fast intranuclear inclusion bodies were
demonstrated within the renal tubular epithelial cells.

Cattle are usually exposed to lead through the ingestion of automotive batteries,
petroleum products, roofing felt, or lead-based agricultural products. Clinical signs are
indicative of central nervous system dysfunction, and include depression, tremors,
ataxia, blindness, seizures, and dementia.2 Gross lesions are uncommon after acute
lead toxicosis. Histologically, lesions include laminar cortical necrosis of the cerebrum
with swelling of capillary endothelium, cerebral edema, congestion, and astrogliosis.
These lesions are thought to reflect ischemic-anoxic injury, and are therefore not
specific for lead toxicity. Ischemia may be induced by capillary endothelial swelling, the
pathogenesis of which is not understood. Lead does not accumulate appreciably in
CNS tissue. Renal tubular epithelial necrosis is evident in some cases; tubular
epithelial cells often contain acid-fast intranuclear inclusion bodies.3

Lead is present in the environment in three forms: metallic lead, lead salt, and organic
lead.2 Many man-made products incorporate metallic lead or lead salts; these include
automotive batteries, lead weights, lead-based paints, lead shot, various plumbing
waste products, computer equipment, and lead arsenate pesticides.1,2 The industrial

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process of lead smelting has resulted in livestock exposure through the airborne
contamination of pasture. Organic lead (tetraethyl- and tetramethyl-lead) is found
primarily in leaded petroleum products.

The toxicity of lead is attributable to multiple mechanisms, including binding and

inactivation of enzymatic sulfhydryl groups, competition with calcium ions, and alteration
of vitamin D metabolism. Sulfhydryl binding is most evident in enzymes involved in
heme synthesis, such as gamma-aminolevulinic acid dehydratase (ALAD) and
ferrochelatase; hence, toxicity often results in red blood cell abnormalities. Inhibition of
heme synthesis may contribute indirectly to neurological abnormalities through the
increased production of serotonin, resulting in aberrant neurotransmission in the brain.
Lead competes with calcium ions in bone resulting in the formation of lead precipitates
(“lead line”) in the long bones of young animals. Lead also competes with calcium at
the neuromuscular junction, resulting in tremors and paresis.2

The absorption of lead is dependent on its form and the route of exposure. In general,
organic lead is better absorbed than lead salts and metallic lead. Ingested lead is most
readily absorbed from the acidic environment of the stomach, yet the majority of
ingested lead is passed in the feces. Once absorbed, more than 90% of absorbed lead
is bound to erythrocytes, making whole blood the tissue of choice for the clinical
diagnosis of lead toxicosis. Blood lead levels in excess of 0.6 ppm (60 _g/dl) are
diagnostic for lead toxicosis. Other tests include the detection of reduced blood ALAD
activity (<50 nmol porphobilinogen/ml erythrocytes/hr in adult cattle) or excessive
urinary ALA levels (>500 _g/dl).2 Upon post-mortem examination, kidney, liver, and
bone harbor the highest concentrations of lead. As in this case, liver and kidney levels
in excess of 10 ppm (wet weight) are considered diagnostic for lead toxicosis.1,2

AFIP Diagnosis: Brain, cerebrum: Neuronal necrosis, laminar, with gliosis and
hypertrophic endothelial cells, Angus, bovine.

Conference Comment: The majority of the sections exhibit laminar neuronal necrosis
in the middle to deep layers of the cerebral grey matter. Additionally, scattered
throughout all layers, there is neuronal necrosis evidenced by shrunken, angulated,
hypereosinophilic and often karyorrhectic or pyknotic neurons.

The contributor gives a thorough and complete overview of lead toxicity in cattle. Lead
toxicosis, or plumbism, has been reported in many different mammals, birds, and
reptiles.2 Unlike cattle, equine lead poisoning is usually chronic and affected individuals
characteristically present with laryngeal and pharyngeal paralysis, and ingestion of large
amounts can produce generalized paralysis.3 Lead toxicity is not uncommon in humans.
Humans are exposed to lead by two main routes, occupational and non-occupational.
Occupational exposures include inhalation during spray painting, foundry work, mining
and extracting lead, and battery burning. Non-occupational exposures, which may be
more difficult to track in relation to individual exposure, include ingestion of

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contaminated water due to lead plumbing, lead solder in food and soft drink cans, paint
dust and flakes in homes with interior lead paint, soil contaminated with exterior lead
paint, newsprint, automotive exhaust, and illegally produced alcoholic beverages
4
(moonshine).

Similar to cattle, intestinal absorption in humans is enhanced by calcium, iron, or zinc

deficiency. Children have a greater absorption capacity than adults and thus are more
vulnerable to lead toxicity. Absorbed lead clears rapidly from the blood, but is often
deposited in bones where it has a half-life of 30 years. Therefore, high blood lead levels
indicate a recent exposure but are not indicative of total body burden.4

As with cattle, lead toxicosis is a multisystemic disease in humans, affecting the central
and peripheral nervous systems, hematopoiesis, gastrointestinal system, kidneys, and
bones. Symptoms include headache, dizziness, memory deficits, decreased nerve
conduction velocity, a microcytic hypochromic anemia with characteristic basophilic
stippling of erythrocytes, colic, anorexia, and damage to the proximal renal tubules with
classic intranuclear lead inclusions. Chronically, lead may cause diffuse interstitial
fibrosis, gout and renal failure, in addition to infertility and hypertension.4

Contributor: Auburn University, College of Vet Med, Auburn AL 36849-5519

(www.vetmed.auburn.edu)

References:
1. Puls R: Mineral Levels in Animal Health, 2nd ed., pp. 147-149. Sherpa International,
Clearbrook, B.C., Canada, 1994
2. Gwaltney-Bryant S: Classes of toxicants, metals and minerals, lead. In: Clinical
Veterinary Toxicology, ed. Plumlee KH, pp. 204-210. Mosby, Inc., St. Louis, MO, 2004
3. Jubb KVF, Huxtable CR: The nervous system. In: Pathology of Domestic Animals,
eds. Jubb KVF, Kennedy PC, and Palmer N, 4th ed., vol. 1, pp. 348-351. Academic
Press, Inc., San Diego, CA, 1993
4. Kane AB, Kumar V: Environmental and nutritional pathology. In: Robbins and Cotran
Pathologic Basis of Disease, eds. Kumar V, Abbas AK, Fausto N, 7th ed., pp.432-433.
Elsevier Saunders, Philadelphia, PA, 2005

SLIDE 8
CONFERENCE 2 / CASE IV – 04F36 (AFIP 2935878)

Signalment: Adult 4-6 kg Chinook salmon (Oncorhynchus tshawytscha), mixed

gender.

History: During June, 2004, mortalities at a salt water net pen facility along the coast of
British Columbia increased from 0.5 to 10% over a week span. These fish had
previously had episodes of low-grade bacterial kidney disease and sporadic losses due
to vibriosis (Listonella anguillarum).

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Fish initially presented with inappetence and lethargy, which progressed over 1-2 days
to listlessness, opercular flaring, swimming high in the water column and increased
mortality.

Gross Pathology: The most significant lesions involved the gills. Involving multiple gill
arches, randomly throughout the primary, and to a much lesser extent secondary
lamellae, there were 2-3 mm diameter, firm, glistening white to grey spherical cysts with
scattered foci of acute hemorrhage. Within a small proportion of fish, there were miliary
granulomas within the anterior and posterior kidneys as well as scattered foci of acute
hemorrhage within the mesentery.

Laboratory Results: Aerobic culture yielded variably mixed isolates of Vibrio spp,
Pseudomonas spp, and Escherichia spp. Immunofluorescence of pooled liver, kidney
and spleen was positive for Renibacterium salmoninarum and polymerase chain
reaction of pooled gill tissue was positive for Loma salmonae.

Contributor’s Morphologic Diagnosis: Gills: Branchitis, moderate, multifocal to

coalescing, fibrinohemorrhagic and necrotizing, acute, with telangiectasia, respiratory
epithelial hyperplasia and intralesional protozoal xenomas morphologically consistent
with Loma salmonae.

Contributor’s Comment: In British Columbia, because of the emergence of infectious

hematopoietic virus (IHNV) and Kudoa spp in farmed Atlantic salmon (Salmo salar),
producers are opting to further domesticate and raise Pacific salmon species
(Oncorhynchus spp). With expansion and intensification of Pacific salmon aquaculture,
there has been an emerging concern about a branchial microsporidian parasitic
infection, Loma salmonae. This parasite has a narrow host range and is a recognized
pathogen of Chinook (O. tshawytscha) and to a much lesser extent, Coho salmon (O.
kisutch). Atlantic salmon, brook trout (Salvelinus fontinalis) and Arctic char (Salvelinus
alpinus) appear refractory to experimental infection.

Parasites are acquired through ingestion of infective stages. The sporoplasm either
invades between or through enterocytes, localizing within the lamina propria by 24
hours post infection. In experimental models of rainbow trout (O. mykiss) maintained at
15C, the merogonic stages are consistently detected within the heart as early as 2 days
post infection and remain for 2 weeks.1 After 2 weeks, and up to 5 weeks post infection,
parasitic xenomas (which have undergone sporogony) are detected within the gills and
spleen.

Electron microscopy has revealed uni- and binucleate meronts in sub-intimal host cells
of the capillary channels of secondary lamellae and lamellar arteries. Involvement of
phagocytic pillar cells has also been reported. Localization to these cells may be due to
embolism, receptor mediated internalization, or evasion (by directly transferring from
monocytes or lymphocytes). Respiratory distress is attributed to dissolution of the

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xenoma with inflammation directed against the chitin-rich wall of spores, and necrosis.
Horizontal transmission is believed to result from rupture of the xenomas.

Temperature has a profound influence on the kinetics of infection; at 5C or 20C xenoma

formation is interrupted, but branchial infections can still occur. At 5C, parasites localize
to the heart in 7 days with no detectable xenomas within the gills to 4 weeks post
exposure. At 20C, parasites are detected within the spleen, heart, and gills 3 days post
exposure with no apparent xenoma formation. Maintaining fish in ambient water
temperatures less than 5 C and greater than 20C appears to interrupt the life cycle of
this parasite and stock that recover are resistant to re-infection.2 Ectoparasitic
copepods, such as sea lice (Lepeophtheirus salmonis) appear to enhance susceptibility
of stock to Loma salmonae infection.

At present there is no recognized treatment for Loma salmonae infection. Efforts are
currently underway for vaccine development and possible management schemes to
limit development of clinical disease.

AFIP Diagnosis: Gill: Branchitis, necrotizing and proliferative, multifocal, moderate,

with mucus cell metaplasia, and numerous protozoal cysts, Chinook salmon
(Oncorhynchus tshawytscha), piscine.

Conference Comment: Microsporidians are generally taxonomically specific

intracellular parasites with a direct life cycle. After spore ingestion by the host, the
sporoplasm is discharged and migrates to the target organ, begins the proliferative
phase (merogony), producing large numbers of cells (meronts) by binary and multiple
fission. Meronts then give rise to sporonts that undergo sporogony, producing mature
spores. These spores are then released from lesions on the body surfaces or after
death of the host.3

Nodular microsporidial lesions may grossly resemble those of other pathogens including
myxozoans, ich, lymphocystis, and dermal metacercariae, bacterial granulomas, or
neoplasia. However, these can easily be differentiated histologically. Microsporidial
spores are typically 7 µm or less, egg-shaped to elliptical, and contain a posterior
vacuole. Myxozoans, except during autogamy (sexual reproduction), all have
multinucleated forms that have enveloping (primary) cells that contain enveloped
(secondary) cells.3 Their spores contain two polar capsules which stain intensely blue
with Geimsa.6 Ich, or white spot disease, is caused by Ichthyophthirius multifilis. The
trophonts are large and characterized by a uniform layer of external cilia and a unique
horseshoe-shaped macronucleus.4 Lymphocystis is caused by a piscine iridovirus that
preferentially infects dermal fibroblasts and inhibits mitosis, producing tremendous
cellular hypertrophy.5 Dermal metacercariae of digenean trematodes, result in white to
yellow or black raised nodules that contain the parasite.3

Contributor: Animal Health Center, Abbotsford, British Columbia, Canada V3G 2M3

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References:
1. Sanchez J, Speare D, Markham R, Wright G, Kibenge F: Localization of the initial
developmental stages of Loma salmonae in rainbow trout (Oncorhynchus mykiss). Vet
Path 38:540-546, 2001
2. Kent M, Dawe S, Speare D: Resistance to reinfection in chinook salmon
Oncorhynchus tshawytscha to Loma salmonae (microsporidian). Dis Aqua Org 37:205-
208, 1999
3. Noga EJ: Problems 59 through 66. In: Fish Disease: Diagnosis and Treatment, ed.,
Noga EJ, pp.173-191. Mosby, St. Louis, MO, 1996
4. Powell DB: Common diseases and treatment. In: The Laboratory Fish, ed. Ostrander
GK, pp. 80-84. Academic Press, San Diego, CA, 2000
5. Kurkjian KM, Latimer KS, Rakich PM: Lymphocystis in Marine and Freshwater
Fishes. Website (http://www.vet.ugs.edu/vpp/clerk/Kurkjian)
6. Gardiner C, Fayer R, Dubey J: An Atlas of Protozoan Parasites in Animal Tissues,
pp. 14-15. United States Department of Agriculture, Washington, DC, 1988

SLIDE 9
CONFERENCE 3 / CASE I – CRL2 (AFIP 2936451)

Signalment: Female, 4-month-old, RAG1 mouse (Mus musculus).

History: Formalin-fixed tissues from a 4-month-old female RAG1 mouse were

submitted for evaluation because of increased mortality in the colony.

Gross Pathology: None given

Contributor’s Morphologic Diagnoses: Lung: 1. Acidophilic macrophage

pneumonia, multifocal, moderate.
2. Intraalveolar Pneumocystis carinii, multifocal, mild.
3. Bronchopneumonia, subacute, suppurative, multifocal, marked.

Contributor’s Comment: In the sections of lung, many alveoli are filled with
macrophages, some multinucleated, with prominent intracytoplasmic eosinophilic
acicular crystals. Although in this case the crystals are easily visualized on the H&E-
stained section, their appearance can be enhanced with Grocott’s methenamine silver
stain or Gram stain. There is some alveolar septal thickening, type II pneumocyte
hyperplasia, and a mild to moderate neutrophilic, histiocytic, and eosinophilic infiltrate in
the areas with the acidophilic macrophage pneumonia. Scattered among the foci of
acidophilic macrophage pneumonia, as well as in unaffected regions of the lung, is
intraalveolar granular eosinophilic extracellular material typical of Pneumocystis carinii,
with minimal associated inflammation. Bronchi are distended with an exudate of
neutrophils and fewer macrophages with multifocal necrosis of epithelium, and
extension into the adjacent pulmonary interstitium. On some of the sections, there is a

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well-developed abscess with mineralization where an airway has been destroyed. A
tissue Gram stain demonstrates intralesional gram-negative bacilli.

RAG1 [V(D)J recombination activating protein 1]-deficient mice do not produce mature
B or T lymphocytes, and are therefore deficient in both humoral and cell-mediated
immunity.1 Loss of the RAG1 gene prevents rearrangement of both immunoglobulin
and T cell receptor genes early in lymphocyte differentiation. RAG1-deficient mice have
a greatly increased susceptibility to murine pathogens of all types.

Pneumocystis carinii is an opportunistic fungal pathogen of many species, which can

cause fatal pneumonia in immunocompromised individuals. There are two tissue forms
of Pneumocystis carinii – trophozoites, and cysts containing sporozoites, which can be
demonstrated in tissues sections by immunohistochemistry or special stains, such as
GMS.2 This case is typical of the minimal inflammation associated with Pneumocystis
carinii pneumonia in an immunodeficient mouse.

Acidophilic macrophage pneumonia is characterized by alveolar accumulations of

macrophages containing eosinophilic crystals. The crystals may also be found free in
alveoli and in the cytoplasm of airway epithelial cells. The macrophage infiltrates are
accompanied by variable numbers of eosinophils, neutrophils and lymphocytes.
Acidophilic macrophage pneumonia is often found in association with other pulmonary
lesions, such as Pneumocystis carinii pneumonia or lung tumors. The development of
this lesion is strain and age dependent, with lesions commonly found in nude mice,
C57BL/6 or Sv/129, or genetically engineered mice, particularly those generated on a
C57BL/6 or Sv/129 background. Ultrastructurally the crystals are indistinguishable from
Charcot-Leyden crystals. However, biochemical analysis indicates that they are
composed of Ym-1 protein, a member of the chitinase family. This protein is also
known as T lymphocyte-derived Eosinophilic Chemotactic Factor (ECF-L).3,4

Bacterial culture could not be performed, as the tissues were fixed in formalin.
Pasteurella pneumotropica has been associated with bronchopneumonia in association
with Pneumocystis carinii in immunocompromised mice,2 but other gram negative bacilli
are possible etiologies as well.

AFIP Diagnoses: 1. Lung: Pneumonia, acidophilic macrophage, diffuse, mild to

marked, RAG1 mouse, rodent.
2. Lung: Bronchopneumonia, suppurative, lobar, severe.
3. Lung: Intra-alveolar fungal organisms, multifocal, etiology consistent with
Pneumocystis murina.

Conference Comment: Studies have recently show that the Pneumocystis found in
laboratory mice is phylogenetically distinct from P. carinii; it was named Pneumocystis
murina sp. nov., and was formerly known as Pneumocystis carinii f. sp. muris.5

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The first transgenic (introduction of ectopic DNA) mouse was created in 1980 by
random insertion, a process that remains common today. However, as technology has
evolved, transgenes are now often targeted to specific sites on the genome for either
gain of function (knock in) or loss of function (knockout or null mice). Gene alteration,
both random and targeted, can lead to unexpected phenotypes, severe
immunodeficiency, and embryonic or fetal death.2

Primary and opportunistic pathogens often significantly affect transgenic mice, not only
because they cause overt disease, but they may also alter the biological responses of
the mice to experimental pathogens. The pathologist must be aware of strain-related
patterns of pathology in relation to spontaneous and infectious disease, developmental
and comparative pathology, and the predicted and unexpected outcomes of gene
alteration.2

Contributor: Charles River Laboratories, 251 Ballardvale Street, Wilmington, MA

01887 (www.criver.com)

References:
1. JAX®Mice Database - B6.129S7-Rag1tm1Mom-J
http://jaxmice.jax.org/jaxmice-cgi/jaxmicedb.cgi?objtype=pricedetail&stock=002216
2. Percy DH, Barthold SW: Mouse. In: Pathology of Laboratory Rodents and Rabbits,
2nd ed, pp. 10-14, 71-73. Iowa State University Press, Ames, IO 2001
3. Guo L, Johnson RS, Schuh JCL: Biochemical characterization of endogenously
formed eosinophilic crystals in the lungs of mice. J Biol Chem 275:8032-8037, 2000
4. Ward JM, Yoon M, Anver MR, Haines DC, Kudo G, Gonzalez FJ, Kimura S:
Hyalinosis and Ym1/Ym2 gene expression in the stomach and respiratory tract of
129S4/SvJae and wild-type and CYP1A2-null B6, 129 mice. Am J Pathol 158:323-332,
2001
5. Keely SP, Fischer JM, Cushion MT, Stringer JR: Phylogenetic identification of
Pneumocystis murina sp. nov., a new species in laboratory mice. Microbiology 150(Pt
5):1153-65, 2004

SLIDE 10
CONFERENCE 3 / CASE II – RT04-1470 (AFIP 2937327)

Signalment: 14 week-old, male, Norwegian Rat, (Rattus norvegicus), Strain: Sprague-

Dawley

History: This 14 week-old rat underwent surgery using an anesthetic combination of

pentobarbital, magnesium sulfate and chloral hydrate of an unknown concentration.
The anesthetic, "Equithesin", was injected intraperitoneally. The procedure implanted a
microdialysis guide cannula in the ventral tegmental area, a region of the
mesencephalon. Over the next five days, the rat's weight dropped from 273 grams to
247 grams. Four days after the operation, the animal looked depressed with "ruffled

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fur". Palpation revealed dilated gut loops, especially in the left ventral abdominal
quadrant. Five days following the operation, an eight-hour period passed with no fecal
pellets. The animal was euthanized. The attending veterinarian performed the
necropsy and submitted tissue in formalin.

Gross Pathology: Gross pathology findings reported by the clinician were as follows:
The animal had very little fat stores and the stomach and duodenum had very little
content. The ileum, cecum and colon were dilated and filled with ingesta. Contents of
the colon were dry and firm. The dilated portion of digestive tract ended abruptly at an
area of adhesion between body wall and colon. The terminal colon and rectum were
only moderately dilated. The anterior surface of the liver had round, blunted edges and
multiple adhesions to the stomach and diaphragm. The lungs were moderately
congested.

Contributor’s Morphologic Diagnosis: Colon: Peritonitis, fibrosing, diffuse, with

moderate leiomyositis.

Etiologic Diagnosis: Chloral hydrate induced peritonitis.

Contributor’s Comment: Chloral hydrate administered intraperitoneally to rodents can

injure abdominal organs. Documented effects include gastric mucosal injuries,
adynamic ileus, and peritonitis,1,2 fibrosis of the serous membranes, and steatitis.3 A
study in 1999 documented a marked decrease in intestinal peristalsis and acetylcholine-
induced contractions in rats following intraperitoneal injections of chloral hydrate.4

In this case, histopathological changes include fibroplasia of the colonic serosa and
muscularis, giving it a pleated form. Individual myofibers of the muscularis externa are
necrotic. There is inflammation of the serosa of the colon with neutrophils,
lymphocytes, macrophages and plasma cells with small numbers of inflammatory cells
extending into the muscularis externa. Hemorrhage is present in muscular layers.
Some sections of colon have ulcers. Gastric ulcers often accompany intraperitoneal
(IP) injections of chloral hydrate.2 However, no gastric sections were submitted in this
case.

Differential diagnoses included peritonitis secondary to perforating ulcer of the colon, or

a needle perforation of the bowel with leakage of contents. A perforating ulcer or other
cause of leakage of intestinal contents would be expected to cause more suppurative
inflammation with peritoneal effusion and large numbers of intraperitoneal bacteria.
Although not used in this case, the anesthetics tribromoethanol (Avertin) and
pentobarbital have been reported to produce peritonitis and ileus in rats.5, 6

In 1961, The Walter Reed Army Institute of Research reported losing some rats and
guinea pigs to ileus and intestinal blockage. They attributed it to a side effect of
intraperitoneal injections of chloral hydrate.1

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A 1977 study reporting chloral hydrate toxicity is commonly cited in literature. The study
involved the implantation of brain electrodes into rats. Several rats became ill and died.
Symptoms were lethargy, anorexia, abdominal distension, and constipation. After an
infectious etiology was ruled out, chloral hydrate was administered in the second leg of
the experiment. Of 27 rats, 20 developed typical signs of gastric distress seen in the
initial study. Fourteen of the 20 rats died.7

Chloral hydrate usage is controversial but it remains widely used in surgical procedures.
The drug is often chosen for intra-cranial surgeries because inhalation anesthetics
present access problems. Barbiturates often depress respiratory and cardiovascular
systems and require long recovery times.8 Regardless of the drug, intraperitoneal
injections in rats are often utilized because of their small muscular mass and relatively
inaccessible vasculature.6

The pathogenesis of colonic ileus is unclear but it may involve a decrease in

parasympathetic activity coupled with an increase in sympathetic inhibition of the colon.
The most common cause of paralytic ileus is abdominal surgery. Abdominal trauma,
serum electrolyte imbalance, septicemia, and intrathoracic conditions such as
pneumonia, myocardial infarction and lower rib fractures have contributed to paralytic
ileus. Narcotics, calcium channel blockers, and anticholinergic medications can also
cause this condition. Treatment usually involves discontinuing use of narcotic and
anticholinergic drugs and correction of electrolyte imbalance.9

Chloral hydrate is thought to be a chemical irritant. In a study that tested chloral

hydrates' anesthetic effect, 83% of rats that were injected intraperitoneally had mild to
moderate inflammation of the abdominal area involving the splenic capsule and
peritoneal surface of the body wall.6 Intestinal inflammation was not reported in this
study.

Some studies attribute disease to the concentration of the drug. However, ileus has
been reported to occur sporadically even at lower concentrations.4 Preceding its use
with a low dose of barbiturates, opioids, alpha-2 agonists, or phenothiazine tranquilizers
has been reported to reduce negative side effects of chloral hydrate.2 A study in 1995
concluded that "Equithesin" without chloral hydrate is an effective anesthetic that can be
maintained for several hours by supplemental doses.8

AFIP Diagnosis: Colon, serosa and tunica muscularis: Serosal fibrosis, diffuse, mild,
with leiomyocyte degeneration and necrosis, and neutrophilic inflammation, Sprague-
Dawley rat, rodent.

Conference Comment: As many pathology residents realize, it is often more difficult to

recognize the absence of cells or a structure than the addition of cells or a change in
morphology. This is especially true in this case. Many conference participants did not
recognize the focally extensive loss of smooth muscle that normally comprises the

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external longitudinal layer of the tunica muscularis and replacement by fibrous
connective tissue. This histologic feature is highlighted with a Masson’s trichrome stain.
Of interest to many of the conference participants, is the relative paucity of
inflammation. This is somewhat surprising given the extent of myocyte degeneration
and necrosis present in most tissue sections and the short clinical history of only five
days post injection. Whether these changes are due to species, strain, individual,
anesthetic, or inflammatory mediator differences is uncertain. Nonetheless, these
findings are consistent with those in published reports.4,5,6,7

Contributor: National Institute of Health, Diagnostic and Research Services Branch,

Division of Veterinary Resources, 28 Service Road West, Building 28A--Room 115,
Bethesda, MD

References:
1. Valenstein E: A note on anesthetizing rats and guinea pigs. J Exp Anal Behavior
4:6,1961
2. Silverman J, Muir WW: Special Topic Overview: A review of laboratory animal
anesthesia with chloral hydrate and chloralose. J Lab Anim Sci 43(3):210-216, 1993
3. Field KJ, White WJ, Lang CM: Anesthetic effects of chloral hydrate, pentobarbital
and urethane in adult male rats. Lab Anim 27(3):258-269, 1993
4. Vachon P, Faubert S, Blais D, Comtois A, Beinvenu JG: A pathophysiological study
of abdominal organs following intraperitoneal injections of chloral hydrate in rats:
comparison between two anesthesia protocols. Lab Anim 34:84-90, 2000
5. Reid WC, Carmichael KP, Srinivas S, Bryant JL: Pathologic changes associated with
use of tribromoethanol (Avertin) in the Sprague Dawley rat. Lab Anim Sci 49(6):665-7,
1999
6. Spikes SE, Hoogstraten-Miller SL, Miller GF: Comparison of five anesthetic agents
administered intraperitoneally in the laboratory rat. Con Topics in Lab Anim
Sci:35(2):50-53, 1996
7. Fleischman RW, McCracken D, Forbes W: Adynamic ileus in the rat induced by
chloral hydrate. Lab Anim Sci 27:238-243, 1977
8. Deacon RMJ, Rawlins, JNP: Equithesin without chloral hydrate as an anesthetic for
rats. Psychopharmacology 124:288-290, 1996
9. Snape WJ: Pathogenesis, diagnosis, and treatment of acute colonic ileus. Medscape
General Medicine eJournal 1(3), 1999

SLIDE 11
CONFERENCE 3 / CASE III – CF13 (AFIP 2935883)

Signalment: 4.33 year-old male rhesus macaque (Macaca mulatta).

History: This animal was one of a group used to evaluate viral dynamics in acute
simian immunodeficiency virus (SIV) and recombinant simian-human immunodeficiency
virus (SHIV) infection and the role of CD8+ T cells. It was inoculated with SHIV162p3

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six months prior to necropsy. Multiple biopsies of peripheral lymph nodes were
performed at determined intervals prior to euthanasia. Due to the development of
severe emaciation, weight loss, and dehydration this animal was euthanized.

Gross Pathology: Approximately 10 ml of pale yellowish fluid was present in the

peritoneal cavity. Fibrous adhesions of the colon to the abdominal wall were observed
in moderate severity. Yellow fluid feces were observed in the colon. The walls of the
gallbladder and bile ducts were extremely thickened, and folds of mucosa protruded into
the lumina, which contained cloudy bile. The pancreas was slightly firmer than normal.
Mesenteric, colonic and iliac lymph nodes were enlarged four times normal size, and
the palatine tonsils were prominent.

Contributor’s Morphologic Diagnoses: 1. Gallbladder and liver: Marked diffuse

chronic lymphoplasmacytic and suppurative cholecystitis with adenomatous hyperplasia
and intralesional protozoal parasites (Cryptosporidium sp.).
2. Moderate widespread chronic lymphoplasmacytic and neutrophilic cholangiohepatitis
with bile duct hyperplasia.
3. Marked focal intimal myocyte hyperplasia with minimal lymphocytic and neutrophilic
phlebitis.

Contributor’s Comment: Major bile ducts are markedly expanded by a proliferation of

epithelium that occludes the lumen, by cellular inflammatory infiltrates of the lamina
propria, and by variable degrees of periductal fibrosis. The epithelium is thrown into
deep folds and crypts are lengthened, giving the tissue a multilocular appearance. The
tall columnar ductular epithelium has more variability in its height, more prominent
nucleoli, and markedly increased mucus production when compared to normal macaque
bile duct epithelium. Segmentally, the apical epithelial surface has numerous 2-6 _m
diameter lightly basophilic spherical bodies interpreted as Cryptosporidium sp. The
lumina of the ducts and crypts contain increased mucus and scattered foci of
neutrophils and necrotic cellular debris. The lamina propria is markedly expanded by
infiltrates of plasma cells, neutrophils, lymphocytes, and eosinophils. Scattered
lymphoid nodules are present. The tunica muscularis is incorporated into hyperplastic
folds and is discontinuous. There is variable fibrosis surrounding the muscularis.
Within the hepatic parenchyma there are portal infiltrates of lymphocytes, histiocytes,
eosinophils, and plasma cells that rarely cross the limiting plate and there is multifocal
marked hyperplasia of bile ducts. Cryptosporidia were not detected in small bile ducts.
Rare random foci of lymphocytic inflammation are present in sinusoids. A prominent
hepatic vein has a well-demarcated plaque-like thickening of the intima due to myocyte
hyperplasia and deposition of matrix with a high content of ground substance. A few
scattered lymphocytes and neutrophils are present within the lesion. In tissues not
represented on the distributed slide, there was minimal cryptosporidiosis of the
pancreatic duct, mild chronic myelitis, and lymphoid depletion. There was also mild
chronic colitis, characterized by lymphoplasmacytic infiltration of the lamina propria,
crypt dilatation, and the accumulation of necrotic debris in crypt lumina, but no
cryptosporidia were detected. Colitis is a frequent finding in SIV-infected macaques.

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The proliferative cholangitis and cholecystitis observed in this SHIV-infected macaque
resembles the cholangiopathy previously reported in SIV-infected monkeys1,2 and
described in human AIDS patients.3 AIDS-cholangiopathy, characterized by sclerosing
cholangitis and acalculous cholecystitis is presumptively due to Cryptosporidium
infection. Unlike primary cryptosporidial infections in immune competent human hosts,
which are usually limited to the small intestine, cryptosporidial infections in AIDS result
in persistent infections and extension beyond the intestine to include the stomach, and
biliary and pancreatic ducts, lung, and inner ear.3 Among human immunodeficiency
virus (HIV)-positive persons with diarrhea, Cryptosporidium infection was present in 14-
24%.4 Cryptosporidium infection of nonhuman primates immunosuppressed by either
SIV or simian type D retrovirus occurs in the small intestine, biliary and pancreatic
tracts, conjunctiva, and lung.5,6,7 Intestinal infection is associated with a profuse
persistent watery diarrhea leading to dehydration and mortality.8,9,10

Primary intestinal cryptosporidial infections occur in neonatal macaques11, as in many

other mammalian species, and are associated with diarrhea that resolves within 16
days. Neonatal cryptosporidiosis is an important cause of diarrhea in cattle and
humans worldwide and is usually due to sporocyst-containing fecal contamination of
water supplies. Reportedly, 20% of US adolescents have been infected while over 90%
of children living in an urban shantytown in Northeast Brazil were seropositive.4 The
resistance of sporocysts to chlorination, their small size (4-6_m), and their low infective
dose (as low as 10 sporocysts) challenge the ability of water treatment plants to remove
this agent from drinking water supplies.

Cryptosporidium spp. cause primary infections in immunocompetent hosts across many,

if not all, vertebrates.12 The separation of Cryptosporidium into species remains a
controversial task and is based on morphology, host specificity, virulence, and genomic
characterization. Currently, at least two mammalian (C. parvum, C. muris), two avian
(C. meleagridis, C. baileyi), one reptilian (C. serpentis), and one fish (C. nasorum)
species are established, although mammalian infections with avian cryptosporidia are
documented.4,13 The gastric cryptosporidia, C. muris, C. serpentis, and C. baileyi have
larger cysts and are distinct from other cryptosporidia. C. parvum is divided into two
genotypes where type 1 is restricted in host range to humans and nonhuman primates
and type 2 is more commonly associated with bovine infections.13

Resolution of cryptosporidial infection depends upon interferon gamma (IFN-gamma),

interleukin 12 (IL-12) and CD4+ T cells in mouse models.14 However, differences in
immune responses to Cryptosporidium infection between species limit further
generalizations regarding the mechanisms of immune response. Secretory
immunoglobulins can provide protection at the sporozoite attachment and entry
stages.14 The lack of effective chemotherapeutics has led to investigation of maternal
vaccines for protection through maternal antibody transfer in cattle and humans and
immunoglobulin therapeutics for immunocompromised patients.

The recent sequencing of the genome for Cryptosporidium parvum type 2 revealed a
paucity of metabolic and mitochondrial enzymes, indicating that the organism depends

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heavily on the host cell for nutrients. However, the identification of metabolic enzymes
that resemble enzymes of plant and bacterial origin has provided new targets for drug
development.15

Cryptosporidial infection causes epithelial hyperplasia in a variety of species, but the

mechanism remains unknown. Cryptosporidial infection also results in villus blunting,
which may be due in part to induction of apoptosis in epithelial cells.16 Recently,
oligonucleotide microarrays identified host cell genes which are differentially regulated
during infection of epithelial cells in culture. Several genes involved in control of cell
proliferation and apoptosis were significantly modulated17, which may lead to a better
understanding of this characteristic pathological effect of Cryptosporidium sp. infection.

The intimal myocyte hyperplasia and minimal phlebitis are interpreted as a SIV-related
change, similar to the arteriopathy that has been described.18

AFIP Diagnoses: 1. Liver: Choledochitis, proliferative, chronic-active, diffuse, severe,

with mucus cell metaplasia, apical protozoa, and multifocal mild cholangiohepatitis with
bile duct hyperplasia, etiology consistent with Cryptosporidium sp., rhesus macaque,
primate.
2. Large muscular vein, intima: Fibromyxomatous proliferation, focally extensive,
moderate.

Conference Comment: The contributor provides a thorough overview of

cryptosporidiosis in several species. Transmission is direct with ingestion or inhalation
as the main routes of infection. Upon ingestion of oocysts, sporozoites excyst, invade
mucosal epithelial cells, and undergo both asexual (merogony) and sexual
(gametogony) replication resulting in meronts or macro- and microgametocytes
respectively. Oocytes sporulate internally to contain four sporozoites that then either
contribute to autoinfection or exit the body.19

The ultrastructure of Cryptosporidium sp. in tissue is unique. In the intestine, the

organism attaches to enterocytes via a specialized feeder organelle, displaces microvilli,
and is surrounded by a host cell derived membrane (parasitophorous vacuole). This
results in the organism being intracellular but extracytoplasmic and is characteristic for
Cryptosporidium sp.20

Contributor: Tulane National Primate Research Center, Division of Comparative

Pathology, 18703 Three Rivers Road, Covington, LA
(http://www.tpc.tulane.edu)

References:
1. Baskerville A, Ramsay AD, Millward-Sadler GH, Cook RW, Cranage MP, Greenaway
PJ: Chronic pancreatitis and biliary fibrosis associated with cryptosporidiosis in simian
AIDS. J Comp Pathol 105:415-421, 1991

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2. Kaup F-J, Kuhn E-M, Makoschey B, Hunsmann G: Cryptosporidiosis of liver and
pancreas in rhesus monkeys with experimental SIV infection. J Med Primatol 23:304-
308, 1994
3. Chen X-M, LaRusso NF: Cryptosporidiosis and the pathogenesis of AIDS-
cholangiopathy. Semin Liv Dis 22(3):277-289, 2002
4. Dillingham RA, Lima AA, Guerrant RL: Cryptosporidiosis: epidemiology and impact.
Microbes Infect 4:1059-1066, 2002
5. Blanchard JL, Baskin GB, Murphey-Corb M, Martin LN: Disseminated
cryptosporidiosis in simian immunodeficiency virus/delta-infected rhesus monkeys. Vet
Pathol 24:454-456, 1987
6. Yanai T, Chalifoux L, Mansfield KG, Lackner AA, Simon MA: Pulmonary
cryptosporidiosis in simian immunodeficiency virus-infected rhesus macaques. Vet
Pathol 37(5):472-475, 2000
7. Baskin GB: Cryptosporidiosis of the conjunctiva in SIV-infected macaques. J
Parasitol 82:630-632, 1996
8. Kaup FJ, Matz-Rensing K, Kuhn EM, Hunerbein P, Stahl-Hennig C, Hunsmann G:
Gastrointestinal pathology in rhesus monkeys with experimental SIV infection.
Pathobiology 66:159-164, 1998
9. Klumpp SA, Novembre FJ, Anderson DC, Simon MA, Ringler DJ, McClure HM:
Clinical and pathologic finding in infant rhesus. J Med Primatol 22:169-176, 1993.
10. Osborn KG, Prahalada S, Lowenstine LJ, Gardner MB, Maul DH, Henrickson RV:
The pathology of an epizootic of acquired immunodeficiency in rhesus macaques. Am J
Pathol 114:94-103, 1984
11. Lackner AA, Wilson DW: Cryptosporidiosis, intestines, pancreatic duct, bile duct,
gall bladder, Macaca mulatta. In: Nonhuman Primates II, eds. Jones TC, Mohr U, and
Hunt RD, vol. 1, pp. 41-46. Springer-Verlag, Berlin, Germany, 1994
12. Fayer R, Ungar LP: Cryptosporidium spp. and cryptosporidiosis. Microbiol Rev
50:458-483, 1986
13. Widmer G, Lin L, Kapur V, Feng X, Abrahamsen MS: Genomics and genetics of
Cryptosporidium parvum: the key to understanding cryptosporidiosis. Microbes Infect
4:1081-1090, 2002
14. Riggs MW: Recent advances in cryptosporidiosis: the immune response. Microbes
Infect 4:1067-1080, 2002
15. Abrahamsen MS, Templeton TJ, Enomoto S, Abrahante JE, Zhu G, Lancto CA,
Deng M, Liu C, Widmer G, Tzipori S, Buck GA, Xu P, Bankier AT, Dear PH, Konfortov
BA, Spriggs HF, Iyer L, Anantharaman V, Aravind L, Kapur V: Complete genome
sequence of the apicomplexan, Cryptosporidium parvum. Science 304:441-445, 2004
16. Tzipori S, Ward H: Cryptosporidiosis: biology, pathogenesis and disease. Microbe
Infect 4:1047-1058, 2002
17. Deng M, Lancto CA, Abrahamsen MS: Cryptosporidium parvum regulation of
human epithelial cell gene expression. Int J Parasitol 34:73-82, 2004
18. Chalifoux LV, Simon MA, Pauley DR, MacKey JJ, Wyand MS, Ringler DJ:
Arteriopathy in macaques infected with simian immunodeficiency virus. Lab Invest
67:338-349, 1992
19. Gardiner C, Fayer R, Dubey J: An Atlas of Protozoan Parasites in Animal Tissues,
pp. 37-38. American Registry of Pathology, Washington, DC, 1998

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20. Cheville NF: Pathogenic protozoa. In: Ultrastructural Pathology: An Introduction to
Interpretation, pp. 742-742. Iowa State Press, Ames, IA, 1997

SLIDE 12
CONFERENCE 3 / CASE IV – PA-4039-1 or PA-4039-4 (AFIP 2888624)

Signalment: 8 week-old New Zealand White (NZW) rabbit (Oryctolagus cuniculus).

History: Because of recent unexplained deaths, 6-week-old sentinel animals were

placed in this rabbit room. They were exposed to dirty bedding from other cages and
euthanized after two weeks. At the time of their sacrifice the animals were clinically
healthy and normal in appearance.

Gross Pathology: The terminal ileum was enlarged and prominent in appearance.
The mucosal surface was markedly thickened and had a corrugated appearance.

Contributor’s Morphologic Diagnoses: 1. Enteritis, proliferative, diffuse, moderate-

marked, with severe crypt epithelial hyperplasia and focal crypt abscesses.
2. Villous blunting, focal, mild (some sections).
3. Central lacteal dilatation, patchy, mild.
4. Intraepithelial protozoa consistent with Eimeria, villous epithelium, multifocal,
minimal-mild (some sections).
Contributor’s Comment: Multiple sections of bowel were cut to provide slides for the
conference. Lesions vary slightly from block to block. Histological changes were limited
to the ileum and consisted of a significant thickening of the mucosa associated with
hyperplasia of both villous and crypt epithelium. The crypt epithelium was severely
crowded with a marked increase in mitotic rate and crypts occasionally demonstrated
branching. Focal areas of crypt luminal dilatation with attenuated epithelium and central
necrotic debris were observed (crypt abscesses). A moderate mixed inflammatory
infiltrate was observed in the deep mucosa that in areas included a significant histiocytic
component. Areas of villous shortening & blunting (some sections) and dilatation of the
central lacteal structure were occasionally observed.

In some of the H&E stained sections, numerous small punctate structures consistent
with coccoid and small bacilliform bacteria were observed adjacent to villous and crypt
epithelium, extending in some sections into the submucosal regions.

A Warthin-Starry silver stain revealed the presence of myriads of short, curved rod-
shaped argyrophilic bacterial organisms primarily within the apical cytoplasm of
hyperplastic crypt epithelium. These organisms were strongly positive on
immunohistochemical staining with a porcine Lawsonia intracellularis -specific
monoclonal antibody. Additionally, the presence of this organism was further confirmed
by amplification of 319-base-pair-(bp) and 182-bp products specific for porcine L.
intracellularis chromosomal DNA and 16S rRNA genes, respectively.

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The gross and microscopic findings, in conjunction with the immunohistochemical and
molecular verification are consistent with a diagnosis of Lawsonia intracellularis induced
subclinical proliferative enteritis.

Lawsonia intracellularis is a recently identified intracellular pathogen, phylogenetically

unrelated to other pathogens.1 It is an obligate intracellular organism and has been
associated with hyperplastic intestinal processes in a growing number of mammalian
and avian species. Although the disease can manifest sub-clinically in many animals,
moderate to severe clinical disease is often seen in a wide variety of hosts, most
notably in pigs and hamsters.

Infection in rabbits with Lawsonia has been well documented,2,3,4,5 including dual
involvement with enteropathogenic E. coli. Some of these reported outbreaks have
been associated with high mortality and the presence of lesions including severe
necrosis, ulceration and suppurative inflammation in association with the proliferative
changes. In some reports, histiocytic enteritis is described as a result of Lawsonia
infection.2,6 In other sentinel animals from this cohort sacrificed at later time points, a
distinctly granulomatous pattern of inflammation was noted - suggesting that the
histiocytic response may be associated with a later stage/resolution of infection.

The Eimeria organisms noted in this case were thought to be incidental. Concurrent
infection of this animal with other enteric organisms and agents was not excluded.

AFIP Diagnoses: 1. Small intestine: Enteritis, proliferative, histiocytic and

heterophilic, diffuse, moderate, New Zealand White rabbit, lagomorph.
2. Small intestine: Intraepithelial protozoa, multifocal, few, etiology consistent with
Eimeria sp.

Conference Comment: As the contributor mentioned, Lawsonia intracellularis has

been identified as the causative agent of a proliferative enteropathy in a number of
species, including rabbits, hamsters, guinea pigs, rats, ferrets, non-human primates,
swine, sheep, horses, white-tailed deer, dogs, Arctic and blue fox, emus, and
ostriches.1,7 Regardless of the species affected, two hallmarks are constant, namely
proliferation and intracellular bacteria.1

The gross lesions in pigs, hamsters, foals, white-tailed deer, and guinea pigs are
similar. They predominately occur in the distal ileum, beginning as small raised opaque
islands that progress to a confluent, irregularly nodular, surface which may have areas
of hemorrhage and/or necrosis. Histologically, the thickened epithelium results from
expansion and elongation of the crypts, with actively dividing epithelial cells, and an
absence of goblet cells.1

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Lesions in other species differ in location and histologic changes. In ferrets, rabbits,
and blue foxes lesions are most commonly found in the cecum and proximal colon. In
ferrets, histologic changes may include organisms that penetrate the muscular tunics
and appear in the serosa or draining lymph nodes. In rabbits, infiltrating histiocytes may
expand the lamina propria, and the organism often has a single polar flagellum and is
located in a membrane-bound vacuole, a feature not found in pigs. 1

Contributor: University of Pittsburgh, Div. of Lab Animal Resources, S 1040

BioMedical Science Tower, 3500 Terrace Street, Pittsburgh, PA (http://www.pitt.edu)

References:
1. Lawson, GHK and Gebhart CJ: Proliferative enteropathy J. Comp. Pathol 122: 77-
100, 2000
2. Duhamel GE, Klein EC, Elder RO and Gebhart CJ: Subclinical proliferative
enteropathy in sentinel rabbits associated with Lawsonia intracellularis. Vet Path
35:300-303,1998
3. Hotchkiss CE, Shames B, Perkins SE and Fox JG: Proliferative enteropathy of
rabbits: the intracellular Campylobacter-like organism is closely related to Lawsonia
intracellularis. Lab Anim Sci 46(6):623-627, 1996
4. Schauer DB, McCathey SN, Daft BM, Jha SS, Tatterson LE, Taylor NS and Fox JG:
Proliferative enterocolitis associated with dual infection with enteropathogenic
Escherichia coli and Lawsonia intracellularis in rabbits. J of Clin Micro 36(6):1700-1703,
1998
5. Schoeb TR and Fox JG: enterocecocolitis associated with intraepithelial
Campylobacter-like bacteria in rabbits (Oryctolagus cuniculus). Vet Path 27:73-80, 1990
6. Umemura T, Tsuchitani M, Totsuka M, Narama I and Yamashiro S: Histiocytic
enteritis of rabbits. Vet Path 19:326-329, 1982
7. Watarai M, Yamato Y, Horiuchi N, Kim S, Omata Y, Shirahata T, Furuoka H:
Enzyme-linked immunosorbent assay to detect Lawsonia intracellularis in rabbits with
proliferative enteropathy. J Vet Med Sci 66(6):735-737, 2004

SLIDE 13
CONFERENCE 4 / CASE I – 04-19232-7 (AFIP 2936303)

Signalment: Four and a half year-old, castrated, male Vizsla, canine.

History: This dog had a history of chronic back pain (localized to the thoracolumbar
junction) and generalized skin lesions (consistent with pyogranulomatous dermatitis
/panniculitis on biopsy) for 9 months and was treated with cyclosporine, antibiotics and
corticosteroid treatment with no clinical improvement. One week prior to death, the dog
presented clinically with a head tilt and mild ataxia, but otherwise normal behavior and
mentation. Medications were continued (prednisone, tetracycline, niacinamide,
safflower oil for back pain and skin condition). About six days later, the dog became

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acutely anorexic and depressed in the morning, progressing to lateral recumbency by
evening, and died.

Gross Pathology: The auricles, pericardium, epicardium and endocardium have

numerous, multifocal to coalescing, pinpoint to approximately 0.5 cm in diameter, white
to tan, firm, slightly raised foci (Fig.1). The foci are diffusely tan on cut section and
extend approximately 2-3 mm into the myocardium.

Laboratory Results: Aspergillus sp. was cultured in small numbers mixed with heavy
Cladosporium sp. growth (likely environmental contaminant).

Contributor’s Morphologic Diagnosis: Severe, multifocal, pyogranulomatous

myocarditis with intralesional septate fungal hyphae (Aspergillus spp.)

Contributor’s Comment: Histologic evidence of fungal hyphal structures were found

in the heart (Figs. 2, 3), brain, lungs, liver, adrenal glands, lymph nodes, kidney,
adipose tissue, subcutaneous tissues and vasculature. The gross and histologic lesions
in this dog are consistent with a severe, disseminated fungal infection. The histologic
appearance of the fungal hyphae with hematoxylin & eosin and special fungal stains
(GMS, PAS) is compatible with Aspergillus sp. (Fig. 4). The central nervous system
involvement and multiple organ failure resulted in clinical neurologic disease and
ultimately led to this animal’s demise. It was possible that the fungal infection occurred
secondarily to chronic steroid-induced immune suppression and antibiotic therapy, and
may not be associated with the inciting cause for the initial presenting complaint 9
months prior. The cause of the back pain was undetermined. There were no histologic
indications of spinal cord lesions or fungal infiltration in sections from the thoracolumbar
junction.

Aspergillus sp. is a saprophytic, ubiquitous mold that ordinarily occurs as an opportunist

rather than a pathogen.1 Two forms of aspergillosis have been described in dogs –
localized infection of the nasal cavity and paranasal sinuses (primarily caused by
Aspergillus fumigatus or A. flavus) and disseminated mycotic infection.2 In dogs,
disseminated aspergillosis is relatively rare. Most cases of aspergillosis have been
reported in the German Shepherd Dog suggesting a possible inherent inability to mount
an effective immune response against the organism.2,3 In people, disseminated
aspergillosis has been seen exclusively in immune suppressed or immune-deficient
individuals; however, the same association has not been documented in infected dogs.2
The use of corticosteroids and other immunosuppressive drugs may increase the
susceptibility and incidence of systemic aspergillosis in treated dogs.4

AFIP Diagnosis: Heart: Epicarditis and myocarditis, necrotizing and granulomatous,

multifocal to coalescing, marked, with vasculitis, and pigmented and non-pigmented
fungal hyphae, Vizsla, canine.

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Conference Comment: Mycotic diseases may be broken down into two groups: those
caused by opportunistic fungi and those caused by primary pathogens associated with
systemic “deep” mycoses. 5 Examples of both primary and opportunistic fungi are listed
below:

Opportunistic fungi infections Primary pathogenic fungi infections

1. Aspergillosis (Aspergillus sp.) 1. Blastomycosis (B. dermatitidis)
2. Zygomycosis 2. Histoplasmosis (H. capsulatum)
Mucorales: Rhizopus sp. 3. Cryptococcosis (C. neoformans)
Absidia sp. 4. Coccidioidomycosis (C. immitis)
Mucor sp.
Entomophthorales: Basidiobolus sp.
Conidiobolus sp.
3. Pythiosis (Pythium insidiosum)
4. Phaeohyphomycosis (Cladosporium sp.)

In the slides examined by conference attendees there are high numbers of non-
pigmented fungal hyphae (Aspergillus sp.) and low numbers of pigmented fungal
hyphae (Cladosporium sp.) with some sections containing a prominent vasculitis. As
mentioned by the contributor, Aspergillus spp. are ubiquitous fungi. They are an
important cause of disease in birds but are opportunistic pathogens in
immunosuppressed domestic animals and humans. Typical gross findings include
multifocal to coalescing pale nodules. Histologically, aspergillosis is characterized by
fungal granulomas or pyogranulomas composed of a central area of necrosis containing
hyphae that are 3-5 _m wide, with regularly septate parallel walls, and dichotomous
acute angle branching, surrounded by variable numbers of neutrophils, lymphocytes,
epithelioid macrophages, and fibroblasts. Many cases also demonstrate vasculitis.6

The pigmented fungal hyphae are consistent with Cladosporium sp. with 2-6 _m wide,
long, closely septate hyphae with non-parallel walls, non-dichotomous branching, and
occasional thick-walled vesicular swellings.6 Phaeohyphomycoses are uncommon
opportunistic infections caused by a number of dematiaceous (pigmented) fungi.
Cladosporium spp. primarily affect dogs and cats with a predilection for the central
nervous system, only occasionally causing systemic disease. Grossly, the granulomas
may appear pigmented. Histologically, the fungal elements may be very pale to very
dark depending on the amount of pigment. The pigment is melanin and generally stains
with Fontana-Masson. As with other opportunistic mycoses, conformation of the
etiology is based on concomitant demonstration of hyphae in tissue and culture of a
morphologically compatible organism from properly obtained tissue specimens.7

Contributor: University of Illinois, College of Veterinary Medicine, Department of

Veterinary Pathobiology, 2001 S. Lincoln Avenue, Urbana IL
(http://www.cvm.uiuc.edu/vp)

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References:
1. Jones TC, Hunt RD, King NW: Diseases caused by fungi. In: Veterinary Pathology,
th
6 ed., pp. 506-507. Williams and Wilkins, 1997
2. Watt PR, Robins GM, Galloway AM, O’Boyle DA: Disseminated opportunistic fungal
disease in dogs: 10 cases (1982-1990). JAVMA 207:67-70, 1995
3. Robinson WF, Connole MD, King TJ, Pitt JI, Moss SM: Systemic mycosis due to
Aspergillus deflectus in a dog. Aust Vet J 78:600-602, 2000
4. Pastor J, Pumarola M, Cuenca R, Lavin S: Systemic aspergillosis in a dog. Vet Rec
132:412-413, 1993
5. Lopez A: Respiratory system, thoracic cavity, and pleura. In: Thompson’s Special
Veterinary Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp.184-185.
Mosby, St. Louis, MO, 2001
6. Chandler FW, Kaplan W, Ajello L: Color Atlas and Text of the Histopathology of
Mycotic Diseases, pp.34-37, 92-95,144-157, 253-255. Year Book Medical Publishers,
Inc., Chicago, IL, 1980
7. Foil CS: Miscellaneous fungal infections. In: Infectious Diseases of the Dog and
Cat, ed. Greene CE, 2nd ed., pp. 426-428. W.B. Saunders Company, Philadelphia, PA,
1998

SLIDE 14
CONFERENCE 4 / CASE II – CP04-1187-02 (AFIP 2938293)

Signalment: Six month-old, male, p53 -/- Background strain: C56BL/6;129SJ mouse.

History: The mouse was hunched, lethargic and had a rough hair coat.

Gross Pathology: The thymus was white, soft and markedly enlarged. There was a
small, slightly dark, raised nodule in the heart.

Contributor’s Morphologic Diagnoses: Thymic lymphoma; Cardiac

hemangiosarcoma

Contributor’s Comment: Multiple neoplasms are present. The thymus is effaced by a

monomorphic population of densely packed sheets of lymphocytes. The cells have
round to oval nuclei with finely dispersed chromatin and one prominent nucleolus.
Individual cells have a small rim of cytoplasm and discernable cell borders.
Anisokaryosis and anisocytosis are moderate. The mitotic rate is high, 8-12 per 400x
field of view. Apoptotic cells are common, giving the mass a starry sky appearance.
Focally in the intraventricular septum there is a multinodular poorly circumscribed
proliferation of plump polygonal cells that line blood-filled spaces. Neoplastic
lymphocytes can also be seen along the epicardium in some sections.

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Mutation of the p53 tumor suppressor gene occurs in a high percentage of human
tumors including, colon, breast, lung and brain. In most cases the p53 mutation is
acquired in somatic cells. However, some individuals inherit a mutant p53 allele and
are predisposed to develop a malignancy when a “second hit” or loss of the normal
allele occurs. This is referred to as Li-Fraumeni syndrome.

The p53 protein (P53) is localized to the nucleus and functions to prevent the
proliferation of cells with DNA damage due to irradiation, UV light, or mutagenic
chemicals by way of repair or apoptosis. When damage occurs, P53 levels rapidly
increase and arrest the cell cycle by transcription of p21, an inhibitor of cyclin
dependent kinases that in turn prevent the phosphorylation of Rb, preventing entry into
the S phase of the cell cycle. Transcription of GADD45, (Growth Arrest and DNA
Damage) by p53, assists in DNA repair. If DNA damage is repaired, P53 activates
mdm2 whose product binds to and down-regulates p53 releasing the cell cycle arrest. If
DNA repair is unsuccessful, p53 initiates apoptosis through bax and IGF-BP3. IGF-BP3
binds insulin-like growth factor receptor and bax antagonizes bcl-2.3

Mice deficient in p53 are prone to the development of tumors. The tumor spectrum in
p53-mutant mice includes thymic lymphoma (T-cell type), rhabdomyosarcoma,
fibrosarcoma, hemangiosarcoma, teratoma, anaplastic sarcoma, osteosarcoma, lung
adenocarcinoma, hair matrix tumor, leiomyosarcoma, and rarely brain tumors. Mice
with a homozygous null mutation of p53 have a higher incidence of lymphomas usually
in the thymus. In mice with a heterozygous mutation, sarcomas predominate. Loss or
decline of the wild type P53 activity has been demonstrated in tumors of p53
heterozygotes. Mice homozygous for p53 mutation have an accelerated rate of
malignancy with the majority of the animals dying by six months of age. Occurrence of
two distinct tumor types, like this case, is not uncommon. Lymphoma with a sarcoma or
a teratoma is the most common observation.1,2

AFIP Diagnoses: 1. Thymus: Lymphoma, p53 -/- C56BL/6;129SJ mouse, murine.

2. Heart, myocardium: Hemangiosarcoma.

Conference Comment: Not all sections contained the myocardial hemangiosarcoma

and only some sections contained neoplastic lymphocytes within the epicardium.
The contributor provides a thorough overview of p53 and its critical role in cell cycle
modulation. Carcinogenesis is a multistep process at the phenotypic and genetic level.
The fundamental changes in cell physiology that determine malignancy include: self-
sufficiency in growth signals (proliferation without external stimuli, often due to
oncogene activation); insensitivity to growth-inhibitory signals (especially transforming
growth factor beta (TGFß) and direct inhibitors of cyclin-dependent kinases); evasion of
apoptosis (often through inactivation of p53); defects in DNA repair; limitless replicative
potential (associated with maintenance of telomere length); sustained angiogenesis
(induced primarily by vascular endothelial growth factor (VEGF)); escape from immunity
and rejection; and the ability to invade and metastasize.4

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Simply put, a malignant neoplasm is the result of acquired (environmental) DNA
damage that is not repaired or an inherited mutation in genes affecting DNA repair, cell
growth or apoptosis. Regardless of the initiating events, the end result is a mutation in
the genome of somatic cells leading to activation of growth-promoting oncogenes,
inactivation of tumor suppressor genes, and/or alterations in genes that regulate
apoptosis. The mutated cells then undergo unregulated cell proliferation and
sometimes decreased apoptosis. Within this clonal expansion, there may be cells that
undergo additional mutations, eventually resulting in tumor progression, invasion and
metastasis.4

Contributor: St. Jude Children's Research Hospital, Dept. of Pathology, Room 5017C,
332 N. Lauderdale, Memphis TN

References:
1. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA, Butel JS,
Bradley A: Mice deficient for p53 are developmentally normal but susceptible to
spontaneous tumours. Nature 356(6366):215-221,1992
2. Jacks T, Remington L, Williams BO, Schmitt EM, Halachmi S, Bronson RT,
Weinberg RA: Tumor spectrum analysis in p53-mutant mice. Curr Biol 4(1):1-7, 1994
3. Cotran RS, Kumar V, Collins T: Neoplasia. In: Pathologic Basis of Disease, 6th ed.,
pp. 290-292. WB Saunders Company, Philadelphia, PA, 1999
4. Kumar V, Abbas AK, Fausto N: Neoplasia. In: Robbins and Cotran Pathologic Basis
of Disease, 7th ed., pp. 288-290. Elsevier Saunders, Philadelphia, PA, 2005

SLIDE 15
CONFERENCE 4 / CASE III – 423102 (AFIP 2938284)

Signalment: Eleven-month-old, spayed female, Cocker Spaniel, Canis familiaris, dog.

History: After an elective ovariohysterectomy in January 2004, the patient developed

waxing and waning fevers with persistent neutrophilic leukocytosis. The dog was re-
examined by the referring veterinarian in June 2004. A mass was identified in the
patient’s lower left abdominal quadrant, and a fine needle aspirate of the mass revealed
suppurative inflammation. Exploratory laparotomy revealed an omentum-encased
surgical sponge.

Gross Pathology: The mass was submitted as a surgical biopsy specimen. It was 6.5
cm x 5.5 cm x 3.5 cm, lobulated, firm, tan to pale brown and was covered by flecks of
clotted blood. In the center of the mass there was entrapped surgical sponge material.

Laboratory Results: Abnormal hematology and serum biochemistry test results were
as follows:

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1. Leukocytosis (25.5 x 103/µl; Reference range - 6.0 to 14.3) with neutrophilia (20.4 x
103/µl; Reference range - 3.3 to 10.1) and monocytosis (1.4 x 103/µl; Reference range -
0.1 to 0.9)
2. Normocytic normochromic anemia
• Low erythrocyte count - 5.50 x106/µl (Reference range - 5.8 to 8.9)
• Low hemoglobin – 12.7 g/dl (Reference range - 14.3 to 21.1)
• Low hematocrit - 37.8% (Reference range - 41.7 58.1)
• Normal Mean Corpuscular Volume - 68.7 fl (Reference range - 63.2 to
76.8)
• Normal Mean Corpuscular Hemoglobin Concentration - 33.6 g/dl
(Reference range - 32.4 to 38.4)
3. Hypoalbuminemia (3.0 g/dl; Reference range - 3.1 to 4.2)
4. High serum alkaline phosphatase (202 Units/liter; Reference range - 7 to 128)
5. Low serum alanine aminotransferase (14 Units/liter; Reference range – 21 to 97)

There was scant growth of beta hemolytic Streptococcus serotype group G on aerobic
culture.

Contributor’s Morphologic Diagnosis: Adipose tissue, omentum: Marked chronic

regionally extensive pyogranulomatous omentitis with intralesional surgical sponge
material (gossypiboma).

Contributor’s Comment: The technical term for a surgical sponge accidentally left
inside a patient's body is “gossypiboma.” The word is derived from the Latin word
Gossypium for cotton and the Swahili word boma for "place of concealment".3 Retained
surgical sponges seem to be fairly common in human surgical practice and a higher
incidence of retained laparotomy sponges has been reported in association with
gynecological procedures.1,3 Intraperitoneal “forgotten” foreign bodies tend to create
adhesions and become encapsulated, with or without an accompanying bacterial
infection.1 Clinical presentations for patients with a retained surgical sponge may be
acute or chronic. Acute presentations generally follow a typical septic course with
abscess or granuloma formation.3 Delayed presentations may occur months or even
years after the original surgical procedure and be heralded by adhesion formation and
encapsulation.3 Occasionally there may be intestinal obstruction and rarely fistulation,
perforation or even extrusion.3 The animal of this report was clinically normal within 36
hours of its exploratory laparotomy, and the dog’s post surgical recovery continues to be
uneventful.

AFIP Diagnosis: Omentum (per contributor): Omentitis, pyogranulomatous,

sclerosing, marked, focally extensive, centered on abundant fibrillar anisotropic foreign
material (surgical sponge), Cocker Spaniel, canine.

Conference Comment: Conference attendees discussed the clinical pathology

findings and how these relate to the molecular basis of acute and chronic inflammation.

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The normocytic normochromic anemia is likely due to the chronic inflammatory
condition and was probably non-regenerative (anemia of inflammatory disease, AID).
AID occurs in chronic infectious, inflammatory, or neoplastic disorders and is mediated
by a variety of cytokines, including tumor necrosis factor (TNF), IL-1, and interferon
gamma. The resultant anemia is due to decreased bone marrow responsiveness to
erythropoietin, decreased release of erythropoietin, and impaired availability of iron to
the erythron. When the primary cause of AID is removed, the anemia will resolve.4

Other systemic affects of inflammation include fever, release of acute phase proteins,
and leukocytosis. Fever is produced in response to pyrogens that act by inducing
prostaglandin synthesis in the hypothalamus. Exogenous pyrogens (bacterial products)
stimulate leukocytes to release IL-1 and TNF (endogenous pyrogens) that result in
increases in cyclooxygenases, which then convert arachidonic acid (AA) to
prostaglandins. Prostaglandins, especially PGE2, stimulate the production of
neurotransmitters that reset the temperature set-point at a higher level. Acute phase
proteins are plasma proteins that are primarily synthesized in the liver and often function
as opsonins and fix complement. Three of the best-known examples include C-reactive
protein (CRP), fibrinogen, and serum amyloid A protein (SAA). Cytokines such as IL-6
(for CRP and fibrinogen) and IL-1 and TNF (for SAA) induce hepatocytes to upregulate
production of these acute phase proteins. A common feature of inflammatory reactions,
especially those induced by bacterial infection, is a leukocytosis. Initially the increase in
WBCs is due to accelerated release of cells from the bone marrow postmitotic pool,
induced by IL-1 and TNF. Prolonged infection induces production of colony stimulating
factors (CSFs) and results in increased bone marrow output.5,6 The neutrophilia and
monocytosis in this case is due to the inflammatory reaction and release of cytokines as
a result of the foreign material in the abdomen.

Contributor: Angell Animal Medical Center, Pathology Service, 350 South Huntington
Avenue, Boston, MA

References:
1. Lauwers PR, Van Hee RH: Intraperitoneal gossypibomas; the need to count sponges.
World J Surg 24(5):521-527, 2000
2. Papazoglou LG, Patsikas MN: What is your diagnosis? Retained surgical sponge. J
Small Anim Pract 42(7):325, 363, 2001
3. Zbar AP, Agrawal A, Saeed IT, Utidjian MR: Gossypiboma revisited: a case report
and review of the literature. J R Coll Surg Edinb 43(6):417-418, 1998
4. Brokus CW, Andreasen CB: Erythrocytes. In: Duncan and Prasse’s Veterinary
Laboratory Medicine Clinical Pathology, eds. Latimer KS, Mahaffey EA, Prasse KW, 4th
ed., pp.38-39. Iowa State Press, IA, 2003
5. Latimer KS, Prasse KW: Leukocytes. In: Duncan and Prasse’s Veterinary Laboratory
Medicine Clinical Pathology, eds. Latimer KS, Mahaffey EA, Prasse KW, 4th ed., pp.51-
52. Iowa State Press, IA, 2003
6. Kumar V, Abbas AK, Fausto N: Neoplasia. In: Robbins and Cotran Pathologic Basis
of Disease, 7th ed., pp. 84-85. Elsevier Saunders, Philadelphia, PA, 2005

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SLIDE 16
CONFERENCE 4 / CASE IV – NIAH-1 (AFIP 2937643)

Signalment: 3-week-old, male, white leghorn, specific-pathogen-free chicken.

History: This chicken was inoculated by eye drop with 0.1ml of inoculum containing
107 plaque-forming units (PFU) of viscerotropic velogenic Newcastle disease virus. The
chicken died three days after inoculation, and was necropsied.

Gross Pathology: There was bilateral reddening and swelling of the lower conjunctiva.
The lesions on the inoculated side were more severe than on the opposite side. There
were multifocal white foci on the spleen, and occasionally hemorrhages in the mucosa
of the proventriculus, duodenum, and cecal tonsil in the chicken.

Laboratory Results: Newcastle disease virus was isolated from the conjunctiva using
a cell culture originating from chicken kidney cells.

Contributor’s Morphologic Diagnosis: Conjunctiva: Conjunctivitis, acute, severe,

with fibrinoid vascular necrosis, fibrinous thrombi, hemorrhage, and edema, white-
leghorn, chicken.

Contributor’s Comment: Newcastle disease (ND), a serious world-wide poultry

disease, is caused by ND virus (NDV), a member of the genus Rubulavirus, family
Paramyxoviridae. ND is divided into five pathotypes.1 Viscerotropic velogenic ND
(VVND) (Doyle’s form), is a very acute and lethal form of ND with hemorrhagic lesions
of the digestive tract. Neurotropic velogenic ND (NVND) (Beach’s form) has
neurological and respiratory lesions. Mesogenic ND (Beaudette’s form) is an acute
respiratory and sometimes lethal nervous infection of young chicks. Mortality is rare in
older birds. Lentogenic ND (Hitchner’s form) is a mild or inapparent respiratory infection
of chickens. Asymptomatic-enteric form (Ulster type) manifests chiefly as gut infections
with lentogenic viruses, causing no obvious disease.

Lymphoid, vascular, respiratory, neural, and reproductive lesions are seen in the
chickens as pathological features of ND. 1 It is also well known that NDV causes
conjunctivitis1-4,6 and induces conjunctivitis in humans.5 In these human cases, an
epidemic of Newcastle disease occurred in turkeys in 1965 and 1966 in the United
States, and several workers in close contact with the turkeys at the processing plant
developed a follicular conjunctivitis with a rise of antibodies against NDV. 5 Therefore,
ND is one of a few chicken zoonotic diseases. Clinically, conjunctivitis is a significant
sign of ND.

Virulent avian influenza virus infection and VVNDV infection, acute fatal diseases with
systemic hemorrhages in the chickens, are very important diseases of the poultry

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industry. It is necessary to diagnose and differentiate them as rapidly and correctly as
possible. There are few reports on conjunctivitis induced by virulent avian influenza
except one report of avian-influenza-virus-induced conjunctivitis,7 while there are many
1-4,6
reports on macroscopical NDV-associated –conjunctivitis. Detection of conjunctivitis
with vascular necrosis can be important in the diagnosis of VVND infection in the
chickens, although more pathological studies of chickens infected with NDV are
necessary.

AFIP Diagnosis: Eye, conjunctiva: Conjunctivitis, acute, focal, moderate, with

necrotizing vasculitis, white leghorn chicken, avian.

Conference Comment: The contributor provides a brief overview of the various

pathotypes of Newcastle Disease (ND). Viscerotropic velogenic ND (VVND) is the most
severe form of ND and is likely the most serious disease of poultry throughout the world.
VVND affects numerous species of exotic pet and exposition birds, waterfowl, and
domestic poultry. Transmission between birds is via aerosolization and transmission
between premises is often via fomites. In affected birds, morbidity rates approach 100
percent and mortality often reaches as high as 90 percent. Neurologic and
gastrointestinal signs are most common. However, non-vaccinated birds may be found
dead without prior signs of illness. Gross lesions include subcutaneous and periocular
edema, hemorrhagic and catarrhal tracheitis, airsacculitis, necrohemorrhagic enteritis
(often with Peyer’s patch and cecal tonsil ulceration), petechial hemorrhagic
proventriculitis, and yolk peritonitis.1,8 Histologic findings include necrotizing vasculitis,
thrombosis, diffuse lymphoid depletion, and nonsuppurative encephalomyelitis.1

ND belongs to the Paramyxoviridae virus family, and like other members of this group,
has two important surface proteins, hemagglutinin/neuraminidase (HN) and fusion (F)
protein. HN is important in initial attachment of the virus to the host cell receptor. F
protein has a critical role in virus and cell fusion and penetration of the host cell
membrane. Other viruses in the family Paramyxoviridae include the following: 9

Subfamily Paramyxovirinae Subfamily Pneumovirinae

Genus Paramyxovirus Genus Pneumovirus
Bovine parainfluenza virus-3 Bovine respiratory syncytial virus
Sendai virus (mouse parainfluenza-1) Pneumonia virus of mice
Human parainfluenza virus-1 and –3 Turkey rhinotracheitis virus
Genus Rubulavirus Human respiratory syncytial virus
Avian paramyxovirus-1 (NCDV)
Simian virus-5
Mumps virus
Human parainfluenza virus-2
Genus Morbillivirus
Canine distemper virus
Rinderpest virus

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 Peste-des-petits-ruminants virus
Dolphin Morbillivirus
Phocine distemper virus
Measles virus

Contributor: National Institute of Animal Health, Laboratory of Chronic Diseases

Pathology, 3-1-5, Kannondai, Tsukuba, Ibaraki 305-0856, Japan
(http://www.niah.affrc.go.jp/index-j.html)

References:
1. Alexander DJ: Newcastle disease and other avian paramyxoviridae infections.
In: Diseases of Poultry, eds. Saif YM, Barnes HJ, Glisson JR, Fadly AM, McDougald
LR, Swayne DE, 11th ed., pp.64-87. Iowa State University Press, Ames, IA, 2003
2. Brown C, King DJ, Seal BS: Pathogenesis of Newcastle disease in chickens
experimentally infected with viruses of different virulence. Vet Pathol 36:125-132, 1999
3. Cheville NF, Stone H, Riley J, Ritchie AE: Pathogenesis of virulent Newcastle
disease in chickens. J Am Vet Med Assoc 161:169-179, 1972
4. Katoh H: Pathological Studies on Newcastle disease: Laryngotracheal and
conjunctival lesions caused by so-called Asian type Newcastle disease virus. Jap J Vet
Sci 39:15-26, 1977
5. Hales RH, Ostler HB: Newcastle disease conjunctivitis with subepithelial
infiltrates. Brit J Ophthal 57:694-697, 1973
6. Spalatin J, Hanson RP, Jones TD: Edema of the eyelid and face of chickens
exposed to the viscerotropic type of Newcastle disease virus. Avian Dis 17:623-628,
1973
7. Uys CJ, Becker WB: Experimental infection of chickens with influenza
A/Tern/South Africa/1961 and chicken/Scotland/1959 viruses. II. Pathology. J Comp
Pathol 77: 167-173, 1967
8. Website: http://www.vet.uga.edu/vpp/gray_book/pdf/VND.htm
9. Lamb RA, Kolakofsky D: Paramyxoviridae: The viruses and their replication. In:
Fundamental Virology, eds., Fields VN, Knipe DM, Howley PM, 3rd ed., pp. 577-588.
Lippincott-Raven, Philadelphia, PA, 1996

SLIDE 17
CONFERENCE 5 / CASE I – 04-113 (AFIP 2942979)

Signalment: 9-year-old, neutered male, Rottweiler/Shepherd dog mix.

History: The dog had a one-month history of “not doing right”. On presentation,
lethargy and vomiting had developed. The clinician noted anemia (PCV – 27%) and
increased liver enzymes. On physical exam a palpable abdominal mass was noted and
by imaging was determined to be an enlarged spleen. Peripheral lymph nodes were not
enlarged. On exploratory laparotomy, the spleen was confirmed to be extremely large
with irregular borders and a mottled surface. A splenectomy was performed and the

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spleen submitted for histopathology exam. The left lateral and medial lobes of the liver
appeared mildly enlarged with rounded edges. No biopsy was taken of the liver. No
other gross abnormalities were found within abdomen.

Gross Pathology: The spleen was markedly, diffusely enlarged (approximately 2-3
times normal size) and firm.

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Laboratory Results: CBC day of surgery:

CBC Patient Value Reference Range Units Percentage

WBC 66 5.5 – 16.9 X 103/µl
Neutrophils 7.06 2.0 – 12.0 X 103/µl 10.5
Monocytes 19.28 0.1 – 1.4 X 103/µl 28.8
Lymphocytes 39.48 0.7 – 4.9 X 103/µl 58.9
Basophils 0.36 0.0 – 0.1 X 103/µl 0.5
Eosinophils 0.81 0.1 – 1.49 X 103/µl 1.2
RBC 2.72 5.5 – 8.5 X 106/µl
Hb 7.2 12.0 – 18.0 g/dl
Hct 21.6 37.0 – 55.0 %
Reticulocytes 147.3 X 103/µl 5.4
MCV 79.5 60.0 – 77.0 Fl
MCHC 34.1 31.0 – 37.0 g/dl
MCH 27.07 19.5 – 24.5 Pg
RDW 16.3 14.7 – 17.9 %
PLT 154 X 103/µl
MPV 20.38 Fl
PCT 0.3 %
PDW 20.9 %

Contributor’s Morphologic Diagnosis: Spleen: T-cell LGL (large granular

lymphocyte) lymphoma/leukemia, Rottweiler/Shepherd mix, canine.

Contributor’s Comment: The splenic red pulp is diffusely expanded by numerous

medium to large neoplastic round cells with scant amounts of eosinophilic cytoplasm
and round to indented nuclei. Scattered germinal centers in the white pulp are still
evident but are mildly atrophic. Minimal extramedullary hematopoiesis is present.
Immunohistochemical staining demonstrates neoplastic cells to be strongly positive for
CD3 and negative for CD79a and lysozyme.

Venous blood smears reveal a leukemic blood profile with a moderate regenerative
anemia. Neoplastic lymphocytes are characterized by medium to occasionally large
cells with moderate amounts of basophilic cytoplasm, round to irregularly indented
nuclei with moderately clumped chromatin and variably visible nucleoli. Many cells
have low numbers of variably sized, azurophilic granules that are often perinuclear.
Occasional larger blast cells are noted. A rare mitotic figure is present. A manual WBC
differential count on the blood smear results in 97% of cells being neoplastic
lymphocytes, with 2% segmented neutrophils and 1% eosinophils. When compared to
the automated differential count, it is apparent many of the neoplastic lymphocytes were
erroneously classified as monocytes by the hematology analyzer.

Large granular lymphocyte (LGL) leukemias/lymphomas are either of cytotoxic T-cell

(CD8+CD3+) or natural killer (NK) (CD3-) cell origin.1 LGLs have characteristic
azurophilic granules in their cytoplasm often in a perinuclear location; ultrastructurally,
these granules appear as membrane-bound structures with an electron-dense core. In
normal dogs up to 10% of peripheral lymphocytes in the blood can be LGLs.2 Unlike

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acute lymphocytic leukemias, which originate in the bone marrow, canine LGL
+
lymphoma/leukemias are thought to originate from αdβ2 lymphocytes in the red pulp of
the spleen.3

In addition to surface antigens, cytotoxic granule proteins such as TIA-1, granzyme, and
perforin are used immunologically and in molecular studies to identify LGL leukemic
cells in people.4 Perforin-like immunoreactivity has been demonstrated in feline LGL
lymphomas5 as well as rats, mice, and guinea pigs.2

LGL lymphoma/leukemia in some dogs can be an aggressive disease involving spleen,

liver, and bone marrow. Leukemic blood profiles with lymphocyte counts of up to 138 x
106/ml have been reported, with the majority (80-97%) being LGLs. In other dogs, the
disease progress is slower, behaving like chronic lymphocytic leukemias.2 LGL
lymphoma in cats is usually associated with the gastrointestinal tract and tends to be an
aggressive disease that spreads rapidly to the spleen, liver, and lymph nodes.5, 6
Leukemia is variably seen in cats. LGL lymphoproliferative disorders have also been
reported in horses7, ferrets, birds,2 and is common in Fischer 344 rats. Non-neoplastic
proliferations of LGL in dogs have been reported with chronic ehrlichiosis.3

In humans, LGL leukemia is thought to arise from apoptosis dysregulation due to

abnormalities in the Fas/Fas ligand pathway.8 LGL leukemia cells are resistant to Fas-
mediated apoptosis and express high levels of both Fas and Fas ligand.8 Multiple
genes involved in cytotoxic functions are upregulated in LGL leukemia including
granzymes, cathepsin, calpain, perforin, and capase-8, similar to that seen in activated
cytotoxic T-cells.8 LGL lymphoma/leukemia is often associated with autoimmune
disorders, particularly rheumatoid arthritis, and other lymphoproliferative disorders.
Clonal proliferations of LGLs in humans have been associated with Epstein-Barr virus,
human immunodeficiency virus, and human T cell lymphotropic virus.2 Type C
oncovirus has been reported associated with a cell line derived from peripheral
lymphocytes from a dog with LGL leukemia.9

AFIP Diagnoses: 1. Spleen, sinusoids: Large granular lymphocyte leukemia, mixed

breed, canine.
2. Spleen, white pulp: Lymphoid atrophy, diffuse, moderate.

Conference Comment: Some sections contain discrete nodules of white pulp that are
unaffected by the neoplastic cells suggesting this neoplasm originates from the red pulp
rather than the white pulp. It can be difficult (if not impossible) to differentiate lymphoma
from leukemia when there is advanced disease with infiltration of neoplastic cells into
widespread tissues. In these instances, special methods such as immunophenotyping
of the cells are necessary to help differentiate the conditions.

Lymphoma is a neoplasm of lymphocytes arising as a solid tissue mass in organs other

than bone marrow.10 Lymphoma can be classified into subtypes according to anatomic

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distribution, histiologic pattern, cellular morphology, cytochemistry, and expression of
cluster designation (CD) markers. Subtypes of lymphoma according to distribution
include: multicentric, alimentary, mediastinal, cutaneous, and miscellaneous.
Multicentric lymphoma is the most common type of lymphoid neoplasia in dogs, cattle,
and horses, while alimentary lymphoma is the most common form in cats. The
histologic pattern in most cases is diffuse and characterized by sheets of neoplastic
lymphocytes that efface and replace normal tissue architecture.11

Leukemia is defined as hematopoietic neoplasia with neoplastic cells in the blood and/or
bone marrow. Lymphocytic leukemia arises from the bone marrow and may be either
acute or chronic. Acute lymphocytic leukemia (ALL) is most common in younger
animals, arises from undifferentiated lymphocytes in the bone marrow that are often of
B-cell origin, and usually presents as large blastic cells. Chronic lymphocytic leukemia
(CLL), is more common in older animals, arises from relatively differentiated
lymphocytes that “home” to secondary lymphoid organs such as the spleen. These
cells resemble small lymphocytes and are often of T-cell origin, specifically the CD8+
subset.11

There are several accepted classification schemes for hematopoietic tumors, including
the REAL (Revised European and American Lymphoma) system and WHO (World
Health Organization) system. The REAL system categorizes tumors based on their
histogenetic derivation and biological behavior, while the WHO system also includes
acute and chronic myeloproliferative diseases as well as myelodysplastic syndromes.
Although the classification schemes may appear complex, the differentiation of specific
hematopoietic tumors is important for veterinary oncologists to provide optimal tumor
management.1

The contributor provides a thorough overview of LGL lymphoma/leukemia in domestic

animals and humans. Although the azurophilic granules are difficult to appreciate on
the H&E slides, granules can be seen in lymphocytes particularly around the
fibromuscular trabeculae. When LGL lymphoma/leukemia is suspected, it is often
easier to diagnose using cytologic touch imprints of affected organs or peripheral blood
smears.

Contributor: Comparative Pathology, AFRL/HEDV, 2509 Kennedy Circle, Brooks AFB,

TX

References:
1. Valli VE, Jacobs RM, Parodi, AL, Vernau W, Moore PF: WHO Histological
Classification of Hematopoietic Tumors of Domestic Animals, ed. Shulman FY, 2nd
series, vol VIII, pp. 11-15, 28, 39-42, Armed Forces Institute of Pathology, Washington
DC, 2002
2. Wellman M: Lymphoproliferative disorders of large granular lymphocytes. In:
Schalm's Veterinary Hematology, ed. Feldman BF, et al, 5th ed., pp. 642-647, Lippincott
Williams & Wilkins, Philadelphia, PA, 2000

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3. McDonough SP, Moore PF: Clinical, hematologic, and immunophenotypic
characterization of canine large granular lymphocytosis. Vet Pathol 37(6):637-46 2000
4. Greer JP, Kinney MC, Loughran TP Jr.: T cell and NK cell lymphoproliferative
disorders. Hematology (Am Soc Hematol Educ Program). 2001; 259-81. Review.
5. Kariya K, Konno A, Ishida T: Perforin-like immunoreactivity in four cases of
lymphoma of large granular lymphocytes in the cat. Vet Pathol 34(2):156-9,1997
6. Darbes J, Majzoub M, Breuer W, Hermanns W: Large granular lymphocyte
leukemia/lymphoma in six cats. Vet Pathol 35(5):370-9,1998
7. Herraez P, Berridge B, Marsh P, Weeks B, Ramiro-Ibanez F: Small intestine large
granular lymphoma in a horse. Vet Pathol 38(2):223-6, 2001
8. Rose MG, Berliner N: T-cell large granular lymphocyte leukemia and related
disorders. Oncologist 9(3):247-58, 2004
9. Ghernati I, Corbin A, Chabanne L, Auger C, Magnol JP, Fournel C, Monier JC, Darlix
JL, Rigal D: Canine large granular lymphocyte leukemia and its derived cell line produce
infectious retroviral particles. Vet Pathol 37(4):310-7 2000
10. Aster JC: Diseases of white blood cells, lymph nodes, spleen, and thymus. In:
Robbins and Cotran Pathologic Basis of Disease, eds. Kumar V, Abbas AK, Fausto N
7th ed., pp. 666-670, 685-686. Elsevier Saunders, Philadelphia, PA, 2005
11. Bienzle D: Hematopoietic neoplasia. In: Duncan and Prasse’s Veterinary
Laboratory Medicine: Clinical Pathology, eds. Latimer KS, Mahaffey EA, Prasse KW, 4th
ed., pp. 80-87. Iowa State Press, Ames, IA, 2003

SLIDE 18
CONFERENCE 5 / CASE II – N04-95 (AFIP 2937501)

Signalment: 3.5-year-old, female, French Alpine goat, Capra hircus, caprine.

History: This doe was presented with a 2-month period of dyspnea and progressive
emaciation after parturition. The animal was euthanized and necropsied.

Gross Pathology: The doe was in poor body condition and exhibited minimal fat
stores. There were multifocal fibrous adhesions between the parietal and visceral
layers of the pleura (Fig. 1). Both lungs exhibited multifocal, green yellow nodules
(abscesses) ranging from 0.5-2 cm in diameter. These nodules were primarily located
in the cranial lobes. The abdominal cavity contained approximately 1 litre of clear, light
yellow watery fluid (ascites). The liver showed multifocal confluent nodules ranging
from 0.5-5 cm in diameter. Abundant pale green viscous material (pus) oozed freely
from the nodules on cut surface (Fig.2). The rumen had a focal, approximately 1.5 cm
in diameter, area of hemorrhage located in the serosal surface near the cardia. At this
site, the mucosa exhibited blunting and erosion of the ruminal papillae. The mesenteric
lymph nodes were enlarged twice their normal size. Both kidneys displayed numerous,
miliary white foci scattered throughout the cortex. The rest of the internal viscera were
unremarkable.

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Laboratory Results: An hour previous to euthanasia, a complete blood count revealed
a leukocytosis, neutrophilia, and mild monocytosis associated with chronic active
inflammation. A biochemical profile revealed azotemia, hyperproteinemia,
hyperglobulinemia, hypoalbuminemia, hypocalcemia, hyperkalemia, hyponatremia, and
hypochloremia and elevated GGT and CK.

CBC/Chem Patient Value Reference Range Units

WBC 30.2 4.0 – 13.0 X 109/L
Neutrophils 22.7 1.2 – 2.7 X 109/L
Monocytes 0.6 0.0 – 0.55 X 109/L
BUN 19.9 3.5 – 6.66 mmol/L
Creatinine 643 97 – 159 mmol/L
Protein 87 59 – 76 g/L
Globulin 78 20 – 60 g/L
Albumin 9 27 – 39 g/L
Calcium 1.08 2.22 – 2.91 mmol/L
Potassium 7.82 3.5 – 6.7 mmol/L
Sodium 127 142 – 155 mmol/L
Chloride 94 99 – 110 mmol/L
GGT 60 < 56 U/L
CK 3351 < 8.9 U/L

Bacteriology: A heavy growth of Arcanobacterium pyogenes was isolated in pure

culture from the lung and liver lesions.

Parasitology (fecal flotation): Eimeria spp.

Contributor’s Morphologic Diagnosis: 1. Liver: Multifocal confluent abscesses.

2. Liver: Hepatitis, necrosuppurative, plasmacytic and histiocytic, multifocal, severe,
chronic, with myriads of intralesional bacteria, and abundant, diffuse, intercellular,
intrahistiocytic, radiating to homogeneous, eosinophilic material consistent with
Splendore-Hoeppli material.
3. Kidney: Glomerulonephritis, proliferative, neutrophilic, fibrinous, global, diffuse,
severe, with glomerular hemorrhage, tubular dilation, proteinosis and degeneration, and
scant, multifocal, extracellular, radiating to homogeneous, eosinophilic material
consistent with Splendore-Hoeppli material.
4. Kidney: Nephritis, tubulointerstitial, neutrophilic and lymphoplasmacytic, multifocal,
severe, subacute.
5. Lung: Multifocal abscesses and fibrous pleural adhesions (slides not submitted).
6. Rumen: Rumenitis, necrosuppurative and hemorrhagic, focal, acute, moderate
(slides not submitted).

Contributor’s Comment: The section of liver shows large, multifocal areas of

liquefactive necrosis containing myriad bacteria intermixed with numerous degenerate
neutrophils and cellular debris. These areas are surrounded by large numbers of
plasma cells, with lesser numbers of histiocytes, neutrophils and rare lymphocytes.
Multifocal aggregates of neutrophils, plasma cells and lymphocytes are scattered

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throughout the parenchyma. The presence of abundant, homogeneous, eosinophilic,
hyaline material often with radiating projections (spiculated), diffusely distributed among
the parenchymal cells and sometimes within Kupffer cells is remarkable. This material
ranges approximately from 20 to 30 microns in diameter, is usually surrounded by
moderate numbers of neutrophils and is considered as consistent with Splendore-
Hoeppli substance. Same material was ruled out as amyloid through Congo red
staining (not submitted).

The section of kidney exhibits increased cellularity of the glomerular tufts caused by
proliferation of epithelial and mesangial cells, with hypertrophy of the epithelium and
focal adhesions between the glomerular tuft and Bowman’s capsule (synechiae). Most
of the glomeruli have small clusters of neutrophils and small deposits of fibrin involving
both the glomerular tufts and the urinary space. In some urinary spaces the
inflammatory cells are mixed with abundant red blood cells. Most of the tubules exhibit
marked dilation and contain abundant, intraluminal, pale eosinophilic, proteinaceous
material. The tubular epithelium has multifocal vacuolar degeneration with occasional
intracytoplasmic hyaline droplets. Multifocal small aggregates of neutrophils, plasma
cells and lymphocytes are scattered throughout the interstitium. There are also
numerous intraluminal clusters of neutrophils mainly located in the medullary region.
Occasional deposits of Splendore-Hoeppli material are present in some glomerular
tufts.

The order Actinomycetales includes several families of pathogenic organisms. These

organisms, referred to as higher bacteria, often generate lesions that resemble those
produced by fungi. The genera Actinomyces, Nocardia, Rhodococcus,
Corynebacterium, Dermatophilus, Streptomyces, and Mycobacterium are included in
this order.1 Arcanobacterium pyogenes, formerly known as Corynebacterium pyogenes
and Actinomyces pyogenes, is widespread throughout the word as a common cause of
pyogenic processes in cattle, sheep, swine, goats, and wild ungulates.1,2 It is a
facultatively anaerobic, small, pleomorphic, gram-positive bacterium, which is
considered a normal inhabitant of the mucous membranes of several domestic species.
Arcanobacterium pyogenes can induce purulent inflammation almost anywhere in the
body. Usually this takes the form of localized abscesses, but the lesions may be more
diffuse in internal viscera, joints, or tendon sheaths.2 In goats, purulent pneumonias
and abscesses in the upper respiratory tract,1 as well as mandibular osteomyelitis3 have
been described as lesions associated with A. pyogenes infection.

A distinctive morphologic feature of certain fungal and bacterial diseases is the

presence of a homogeneous, brightly eosinophilic substance, known as Splendore-
Hoeppli material, surrounding individual organisms or colonies of organisms. It exists
as a surrounding collar of radially arranged clubs, or as a variably-sized rim often with a
serrated edge. The exact nature of this material is unknown, but it appears to be a
product of the host, most likely antigen-antibody complexes. Splendore-Hoeppli material
is a relatively consistent feature of coccidioidomycosis and sporotrichosis, as well as
certain diseases caused by higher bacteria, such as actinomycosis.4 Other bacterial

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infections associated to this phenomenon are those produced by Actinobacillus
lignieressi5 and Staphylococcus aureus (Botryomycosis).

To our knowledge, the Splendore–Hoeppli phenomenon is not a common

histomorphologic feature in infections caused by Arcanobacterium pyogenes. However,
it was a remarkable finding in the liver section of this goat. It is also interesting that the
material described in this case was not surrounding bacterial colonies, as it has been
described in the literature.

AFIP Diagnoses: 1. Liver: Abscesses, multifocal and coalescing with myriad bacilli
and, moderate, random, portal, neutrophilic and plasmacytic hepatitis, with abundant
eosinophilic spiculated material, French Alpine goat, caprine.
2. Kidney: Glomerulonephritis, necrotizing, hemorrhagic and neutrophilic, global,
diffuse, severe, with moderate multifocal, neutrophilic and plasmacytic, tubulointerstitial
nephritis, numerous fibrin thrombi, and multifocal eosinophilic spiculated material.

Conference Comment: Within the liver sections, two prominent features are present,
the focal areas of necrosis with large colonies of bacteria and the abundant eosinophilic
spiculated material.

There are several gram-positive and gram-negative bacteria that form large colonies in
tissue. They are known by the pneumonic YAAACCSS5,6,7,8:
Yersinia sp. Gram-negative
Actinomyces sp. Gram-positive
Actinobacillus sp. Gram-negative
Arcanobacterium sp. Gram-positive
Clostridium sp. Gram-positive
Corynebacterium sp. Gram-positive
Staphylococcus sp. Gram-positive
Streptococcus sp. Gram-positive

There is abundant eosinophilic, often spiculated material surrounding areas of necrosis

and within sinusoids in unaffected areas. Conference attendees discussed whether this
could be fibrin thrombi, Splendore-Hoeppli material or amyloid. Fibrin thrombi often
create a long cast of the vascular lumina and tend to contain more enmeshed
erythrocytes. PTAH (Phosphotungstic Acid Hematoxylin) stains fibrin dark blue, but did
not stain the material. The spiculated morphology is most consistent with Splendore-
Hoeppli material. Although the exact nature of Splendore-Hoeppli material is not
known, it is thought to be antigen-antibody complexes. These complexes are often
found closely associated with the bacteria. In this case, a significant amount of the
material was not associated with bacteria and often not associated with inflammation.
Tissue Gram stains did not reveal bacteria within the eosinophilic material. Amyloid is a
pathologic proteinaceous substance that histologically appears as an amorphous,
eosinophilic, hyaline, extracellular material. Within the liver, amyloid first appears in the

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space of Disse, and with progressive accumulation, produces pressure atrophy of
adjacent cells. It is congophilic, and exhibits green birefringence when polarized. After
staining with Congo Red locally, the eosinophilic material is multifocally congophilic with
green birefringence. There are several forms of amyloid, but the most common are AL
(amyloid light chain) and AA (amyloid associated). AL protein is composed of partial or
complete immunoglobulin light chains and is most commonly associated with B
cell/plasma cell dyscrasias. AA protein is produced in the liver from SAA (serum
amyloid-associated), which is an acute phase protein, and is most commonly seen in
association with chronic inflammatory disorders.9 Without further testing, it is not
possible to definitively determine the composition of the eosinophilic material in this
case.

Contributor: Departamento de Patología, Facultad de Medicina Veterinaria y

Zootecnia, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad
Universitaria, Delegación Coyoacán, MEXICO, D.F. 04510.
http://www.veterin.unam.mx

References:
1. Jones TC, Hunt RD, King NW, eds.: Diseases caused by bacteria. In: Veterinary
Pathology, 6th ed., pp. 479-482. Lippincot Williams & Wilkins, Baltimore, MD, 1997
2. Wobeser G: Miscellaneous bacterial infections (Actinomyces and
Arcanobacterium infections). In: Infectious diseases of wild animals, eds. Williams ES,
Barker IK, 3rd ed., pp. 487-488. Iowa State University Press, Ames, IA, 2001
3. Seifi HA, Saifzadeh S, Fairshid AA, Rad M, Farrokhi F: Mandibular
pyogranulomatous osteomyelitis in a Sannen goat. J Vet Med A 50:219-221, 2003
4. Jones TC, Hunt RD, King NW, eds.: Diseases caused by fungi In: Veterinary
Pathology, 6th ed., pp. 505-506. Lippincot Williams & Wilkins, Baltimore, MD, 1997
5. Gelberg HB: Integumentary system. In: Thomson’s Special Veterinary Pathology,
3rd ed., pp. 12. Mosby, St. Louis, MO, 2001
6. Gelberg HB: Alimentary system. In: Thomson’s Special Veterinary Pathology, 3rd
ed., pp. 565-566. Mosby, St. Louis, MO, 2001
7. Baker IK, Van Dreumel AA, Palmer N: The alimentary tract. In: Pathology of
Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., pp.19, 227-228.
Academic Press, San Diego, CA, 1993
8. Kelly WR: The liver and biliary system. In: Pathology of Domestic Animals, eds. Jubb
KVF, Kennedy PC, Palmer N, 4th ed., pp.371-374. Academic Press, San Diego, CA,
1993
9. Abbas AK: Diseases of immunity. In: Robbins and Cotran Pathologic Basis of
Disease, eds. Kumar V, Abbas AK, Fausto N, 7th ed., pp. 258-264. Elsevier Saunders,
Philadelphia, PA, 2005

SLIDE 19
CONFERENCE 5 / CASE III – 04-437 (AFIP 2933948)

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Signalment: 3-year-old, intact female, Duncan Hartley (strain HsdPoc:DH) guinea pig
(Cavia porcellus).

History: This colony guinea pig was a short-haired albino with a recent history of
muscle mass loss and a palpable intra-abdominal mass. There had been no recent
experimental manipulations or breeding; this group of guinea pigs was kept mainly for
environmental enrichment. The guinea pig was found dead and presented for necropsy
on the same day.
Gross Pathology: At necropsy, there was marked muscle thinning on the cervical,
thoracic and abdominal regions. The lungs were diffusely firmer than normal and the
sternal lymph nodes were wet and red. The liver was diffusely red/brown, friable and
granular and the spleen contained a 1 cm diameter soft, red nodule. Both kidneys had
a granular appearance. Both ovaries were markedly enlarged by fluid-filled, thin walled
cysts, probably accounting for the abdominal mass palpated prior to death.

Contributor’s Morphologic Diagnoses: 1. Liver, lung: Cavian leukemia.

2. Liver: Mild centrilobular hepatocellular fatty change.

Contributor’s Comment: Microscopically, the hepatic sinusoids were expanded by a

fairly monomorphous population of round, lymphoid cells with scant to small amounts of
pale eosinophilic cytoplasm and centrally placed euchromatic to hypochromatic, faintly
stippled nuclei. Nuclei ranged from oval to round or indented; nucleoli were
inconspicuous. Mitotic figures averaged 6 per 10 hpf (40X) and scattered tingible body
macrophages created a “starry sky” effect, particularly in portal areas, where the
lymphoid cells were more numerous and solidly packed. The capsular surface was a
little irregular due to the subcapsular accumulation of lymphoid cells. Larger veins
throughout the liver contained similar round cells. Mild, centrilobular hepatocellular fatty
change was noted. In the lung, there was diffuse interstitial thickening due to the
presence of lymphoid cells similar to those described above. These also occupied
larger, congested veins. Incidental foci of heterotopic bone were also present.

Other infiltrated organs included lymph nodes (although peripheral lymphadenopathy

was not appreciated at gross necropsy), the spleen, kidneys, intestinal tract, adrenal
and thyroid glands. Most blood vessels contained similar cells. Bone marrow cellularity
was 95-100% but the cells were still mixed, although there was a predominance of
larger, non-segmented cells. Due to decalcification, it was difficult to be certain if these
were the same population as was present throughout the rest of the body. However,
reportedly there is lesser involvement of the bone marrow.1

Cavian leukemia is a spontaneous form of hematopoietic neoplasia that can occur in

inbred (strains 2/N and 13/N) and non-inbred lines. Disease tends to arise in young
adults under three years of age. The incidence varies in different reports, ranging from
under 0.01% in a 16-year period2 to 0.2% over a three-year period.3 Early experimental
work4 demonstrated transmission of a “leukemogenic agent” via cell-free inoculation or
cell transplantation to hybrid guinea pigs. Electron microscopy revealed Type C virus
particles in neoplastic lymphoid cells, suggesting a role for retrovirus infection.

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Experimentally, this is also a 100% transmissible (transplantable) form of neoplasia in
inbred strains and some hybrids (e.g. strain 2/Hartley), usually by intraperitoneal or
subcutaneous inoculation of fresh or thawed frozen whole blood or tissue. This has
allowed maintenance of leukemic lines and further study of the pathogenesis, although
it is not entirely clear if more recent spontaneous cases reflect exactly the same disease
process.2 The degree of transmissibility decreases quickly with age in non-inbred
strains but can be facilitated by prior corticosteroid treatment.1 While this disease is
typically thought of as retrovirus-associated, guinea pig herpes-like virus may also
contribute to the development of the leukemia. Herpes-like virus particles have been
isolated from lymphoid cells derived from leukemic guinea pigs but only in vitro5 and the
cells were also derived from guinea pigs infected with retrovirus. Cavian leukemia has
been postulated as a model for acute lymphoblastic leukemia in humans.1

AFIP Diagnosis: Lung; liver: Leukemia, lymphoblastic, Duncan Hartley guinea pig,
cavian.

Conference Comment: Cavian leukemia is a rare condition that occurs spontaneously

in various inbred and non-inbred strains of guinea pigs. Affected animals are usually
young adults and may present with either a generalized lymphadenomegaly and/or a
leukocytosis (up to 180,000 mm3) with a significant number of circulating lymphoblastic
cells. At necropsy, in addition to enlarged peripheral lymph nodes, there is marked
splenomegaly and hepatomegaly. Histologically, as was noted in this case, there is
diffuse infiltration of lymphoblastic cells in the liver, interstitium of the lung, as well as,
spleen, bone marrow, thymus, alimentary tract lymphoid tissue, heart, eyes, and
adrenal glands. As the contributor mentions, an endogenous retrovirus, is associated
with, but not proven to cause Cavian leukemia. Although a retrovirus appears to play
an important role in this disease, C-type virus particles have been noted in lymph node
germinal centers from normal guinea pigs.6

Many viruses have been associated with tumor induction in both animals and humans.
Most of these are DNA viruses and include the following7:

Family/Genus Virus Associated Tumor

DNA Viruses
Poxviridae
Leporipoxvirus Rabbit fibroma virus Myxoma
Squirrel fibroma virus Fibroma
Yatapoxvirus Yaba monkey tumor virus Histiocytoma in monkeys

Family/Genus Virus Associated Tumor

Herpesviridae
Alphaherpesvirinae Marek’s disease virus Lymphoma in fowl

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 Gammaherpesvirinae Ateline herpesvirus-2 Lymphoma
Saimirine herpesvirus-2 Lymphoma
Epstein-Barr virus Lymphoma in monkeys
Baboon herpesvirus Lymphoma in baboons
Cottontail rabbit herpesvirus Lymphoma in rabbits
Ungrouped Lucké frog herpesvirus Renal adenocarcinoma
Adenoviridae
Mastadenovirus Many adenoviruses Solid tumors in rodents

Papovaviridae
Papillomavirus Cottontail rabbit papillomavirus Papillomas in rabbits
Bovine papillomavirus-4 Papilloma, carcinoma of
intestine/urinary bladder
Bovine papillomavirus-7 Papilloma, carcinoma of
the eye
Human papillomavirus-5, 8 SCC
Human papillomavirus-16, 18 Genital carcinoma
Polyomavirus Murine polyomavirus Solid tumors in rodents

Reverse Transcribing Viruses

Hepadnaviridae (DNA virus)
Orthohepadnavirus Woodchuck hepatitis virus Hepatocellular carcinoma
Human hepatitis virus Hepatocellular carcinoma
Avihepadnavirus Duck hepatitis virus Hepatocellular carcinoma

Retroviridae (RNA virus)

Alpharetrovirus Avian leukosis virus Lymphoma/leukemia
Rous sarcoma virus Sarcoma in fowl
Avian erythroblastosis virus Erythroblastosis in fowl
Avian myeloblastosis virus Myeloblastosis in fowl
Betaretrovirus Mouse mammary carcinoma Mammary carcinoma
virus
Mason-Pfizer monkey virus Sarcoma and
Immunodeficiency
Gammaretrovirus Feline leukemia virus Leukemia
Feline sarcoma virus Sarcoma
Murine leukemia virus Lymphoma/leukemia
Murine sarcoma virus Sarcoma
Avian reticuloendotheliosis Reticuloendotheliosis
virus
Family/Genus Virus Associated Tumor

Deltaretrovirus Bovine leukemia virus Leukemia

Jaagsiekte virus Adenocarcinoma of the
lung in sheep
HTLV-1 and –2 Human adult T cell

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 leukemia and hairy cell
leukemia
Simian HTLV virus Leukemia in monkeys

RNA Viruses
Flaviviridae
Hepacivirus Hepatitis C virus Hepatocellular carcinoma
in humans

Contributor: University of Tennessee, Dept. of Pathobiology, College of Veterinary

Medicine, P.O. Box 1071, Rm. A201, Knoxville, TN
http://www.vet.utk.edu/departments/path/

References:
1. Kaplow LS and Nadel E: Animal Model: Transplantable guinea pig L2C Leukemia.
Am J Pathol 95:273-276, 1979
2. Ediger RD and Rabstein MM: Spontaneous Leukemia in a Hartley Strain Guinea Pig.
JAVMA 153:954-956, 1968
3. Hong CC, Liu P and Poon KC: Naturally Occurring Lymphoblastic Leukemia in
Guinea Pigs. Lab Animal Sc 30:222-226, 1980
4. Jungeblut CW and Opler SR: On the Pathogenesis of Cavian Leukemia. Am J
Pathol 51:1153-1160, 1967
5. Nayak DP: Isolation and Characterization of a Herpesvirus from Leukemic Guinea
Pigs. J Virol 8:579-588, 1971
6. Percy DH, Barthold SW: Guinea pig. In: Pathology of Laboratory Rodents and
Rabbits, 2nd ed., pp. 244-245. Iowa State Press, Ames, IA, 2001
7. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ: Mechanisms of viral
oncogenesis. In: Veteriary Virology, 3rd ed., pp.177-179. Academic Press, San Diego,
CA, 1999

SLIDE 20
CONFERENCE 5 / CASE IV – R 18 (AFIP 2937814)

Signalment: 13-month-old, male, Cavalier King Charles Spaniel.

History: A porto-azygos shunt was diagnosed by the veterinarian and 2 surgical

interventions were undertaken within an 11 week interval.
Before the second intervention, the basal portal pressure was 9 mm Hg. After the
ligature, it was 24 mm Hg and euthanasia was elected.

Jan 29th 2004, convulsive seizure:

Abdominal echography: very small liver, porto-azygos shunt, cholelithiasis,
increased kidney size.

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March 18th 2004, first surgery

June 2nd 2004, echography

Early signs of portal hypertension and persistent shunt.
Anorexia, polyuria polydipsia, convulsive seizure.

June 10th 2004, second surgery

Ascites
Euthanasia

Gross Pathology: Porto-azygos shunt, small-sized liver

Laboratory Results: Jan 29th 2004, convulsive seizure:

Alkaline phosphatases: 495 UI
ALAT: 540 UI
Pre-prandial bile acids: 110 mmol/L
Post-prandial bile acids: 261 mmol/L

Contributor’s Morphologic Diagnosis: Liver: Arteriolar hyperplasia, portal, diffuse,

moderate, with terminal hepatic vein hypoplasia, lobular atrophy, periportal and bridging
fibrosis, consistent with congenital portosystemic vascular shunt.

Contributor’s Comment: The liver architecture is modified, as the hepatic lobules are
difficult to identify due to loss of hepatocytic plates and terminal hepatic veins. The
lobules seem small as evidenced by a subjective decrease in distance between portal
triads, and there is slight periportal and bridging fibrosis. Serpentine arrangements of
arteriolar smooth muscle cells that are disposed in a perilobular pattern represent portal
arteriolar hyperplasia. Strikingly, portal veins are still discernible. Hepatocytes are
small, dissociated, and a proportion of them show some medium-sized vacuoles. Ito
cells are abundant and show a large and unique vacuole. Brown pigment is found in
scattered Kupffer cells and bile canaliculi (cholestasis).

Portosystemic shunts are congenital or acquired communications between the portal

and systemic vasculature.4 Congenital vascular shunts are described more often in
dogs, less frequently in cats, and sporadically in other domestic animals.1 Rarely, young
dogs may have arteriovenous (arterioportal) fistulae and develop portal hypertension,
ascites, and acquired shunts.3

Clinically, the animals may be small for their age and show neurological signs due to
hepatic encephalosis resulting from inadequate clearance of enterically derived toxins in
portal blood.2 In addition to neurological signs, animals with shunts may suffer from
renal, cystic, or urethral calculi due to increased urinary excretion of ammonia and uric
acid. The formation of ammonium biurate crystals in urine is frequent.

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Biologically, increased post-prandial bile acids are typical of this condition. Dogs with
portosystemic shunts often have hypoalbuminemia in the absence of proteinuria, low
blood urea nitrogen, hypoglycemia, and hypocholesterolemia due to decreased hepatic
function. Erythrocytic microcytosis is a common finding in animals with portosystemic
shunts although iron metabolism is normal.1

AFIP Diagnosis: Liver: Portal arteriolar hyperplasia and venule hypoplasia, diffuse,
moderate, with lymphangiectasia, hepatocellular atrophy and fatty change, periportal
and bridging fibrosis, and bile stasis, Cavalier King Charles Spaniel, canine.

Conference Comment: The blood flow to the liver is unique with the hepatic artery
providing oxygenated blood, the portal vein providing blood flow from the intestinal tract
and spleen, and the hepatic vein returning blood from the liver to the systemic
circulation. In health, portal blood contains constituents absorbed from the intestinal
tract, including bile acids, amino acids, glucose, ammonia, medium-length fatty acids,
and intestinal antigens that are largely removed by the liver before they reach systemic
circulation. In acquired or congenital shunts, the portal blood largely bypasses the liver,
and directly enters the systemic circulation. Therefore, systemic blood concentrations
of the substances normally removed by hepatic processing are increased (i.e. bile
acids, ammonia). Hyperammonemia may lead to CNS signs (hepatic encephalopathy).5

Portosystemic shunts often result in hepatic atrophy, often with concomitant loss of
functional mass, due to decreased concentrations of intestinal and pancreatic
hepatotrophic factors that normally reach the liver through the portal circulation.
Approximately 70% or more of the functional hepatocytes must be lost before
alterations of hepatic function are detectable by serum chemistry. When the functional
mass is significantly reduced, it may result in hypoproteinemia, hypoalbuminemia,
hypoglycemia, hypocholesterolemia, decreased BUN, and hyperbilirubinemia.5

The clinical pathology findings in this case are classic for animals with portosystemic
shunts. The fasting and postprandial bile acids are elevated due to the portal vein
largely bypassing the liver and delivering the blood to the systemic circulation. The
increased ALAT (ALT, alanine aminotransferase) indicates hepatocellular injury with
enzyme leakage from the cytosol of the hepatocytes into the blood. ALP (alkaline
phosphatase) is an inducible hepatic enzyme with several isoenzymes, including liver,
corticosteroid, bone, intestinal, and placental. These isoenzymes can be differentiated
by their electrophoretic mobility. The liver ALP isoenzyme is a sensitive indicator of
intrahepatic or extrahepatic cholestasis, whereas the bone ALP isoenzyme is frequently
observed in young, rapidly growing animals. In this case, both isoenzymes may be
contributing to the elevated ALP enzymatic activity.5

Contributor: Pfizer PGRD Amboise, Pathology Department, BP 109, 37401 Amboise,

Cedex, France

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References:
1. Kelly, WR: The liver and biliary system. In: Pathology of Domestic Animals, Jubb
KVF, Kennedy PC, Palmer N, eds., vol. 2, pp. 323-324, Academic Press, San Diego,
CA, 1993.
2. Summers BA, Cummings JF, de Lahunta A: Degenerative diseases of the central
nervous system: Metabolic and circulatory disorders. In: Veterinary Neuropathology, pp.
208-211, Mosby Yearbook, St. Louis, MO, 1995.
3. Van den Ingh TS, Rothuizen J, Meyer HP : Circulatory disorders of the liver in dogs
and cats. Vet Q. 1995 Jun;17(2):70-6.
4. Cotran RS, Kumar V, Collins T: The liver and biliary tract. In: Robbins Pathologic
Basis of Disease, 6th ed., pp. 855-856 and 881-884, WB Saunders, Philadelphia, PA,
1999.
5. Bain PJ: Liver. In: Duncan and Prasse’s Veterinary Laboratory Medicine: Clinical
Pathology, eds. Latimer KS, Mahaffey EA, Prasse KW, 4th ed., pp. 193-213. Iowa State
Press, Ames, IA, 2003

SLIDE 21
CONFERENCE 6 / CASE I – 04-3248 (AFIP 2937763)

Signalment: Two-year-old, male, Golden Retriever.

History: The dog presented with weakness, fever, weight loss, and muscle atrophy.
He was treated with multiple antibiotics for an undiagnosed condition. The patient was
euthanized due to severe emaciation and failure to respond to treatment.

Gross Pathology: A necropsy performed by the referring clinician revealed

generalized muscle atrophy and slight bilateral enlargement of the kidneys.

Contributor’s Morphologic Diagnoses:

1. Multifocal pyogranulomatous myocarditis with intralesional “zoites” and random
meronts and merozoites.
2. Diffuse lymphoplasmacytic enteritis with neutrophils, intralesional sporozoites,
blunting and fusion of villi, crypt abscesses, and mild lymphangiectasia.

Contributor’s Comment: American canine hepatozoonosis (ACH) is distinctly different

from “Old World” canine hepatozoonosis and the two entities have recently been
compared in excellent reviews.1,2 ACH is an emerging tick-borne disease caused by
Hepatozoon americanum and transmitted by Amblyomma maculatum (Gulf Coast tick).
The life cycle, route of infection, and pathogenesis are unique and require that the dog
ingest an infected tick rather than being bitten by the tick. Sporozoites released from
tick oocysts penetrate the intestine, enter the circulation and are carried to muscle and
other tissues where they undergo asexual reproduction. A Gulf Coast tick feeding on a
dog with circulating gamonts becomes infected and they undergo sexual reproduction.
The necropsy and histological findings in a series of naturally-occurring and

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experimental cases have been reported.3-5 Antemortem diagnosis is best made from
muscle biopsies from the biceps femoris or semitendinosus muscles because detection
of gamonts in neutrophils on peripheral blood smears is rare. Pelvic radiographs reveal
periosteal proliferation at muscle attachments to bone. An ELISA test has been
developed. Marked leukocytosis is a consistent finding.

The tissues in this case were selected because they contain rarely seen sporozoites in
the tips of villi (Image 2), a developing meront (Image 1) and merozoites
“zoites”/gamonts (Image 3) in pyogranulomas in heart muscle in addition to the
commonly seen cysts. The persistence of schizonts in the intestinal tract in the terminal
stages of the infection raises the question of whether this represents re-infection from a
tick, delayed/retarded migration of the schizonts, or possible reproduction in the
intestine. Re-infection by asexual stages has been shown to persist for >9 months.5
Severe cachexia has been attributed primarily to muscle pain and weakness. Intestinal,
as well as splenic (Image 5 – granuloma with zoites also present), amyloidosis has
been reported and was also present in this case. Intestinal amyloidosis can lead to
malabsorption and protein loss. In the intestinal section of this case, the lacteals were
severely dilated, even though the amyloid deposits were minimal and stained poorly.
Mesangioproliferative glomerulonephritis has been reported in cases of ACH.
Proteinuria is common in chronic cases and nephrotic syndrome can develop. In this
case, glomerular lesions were dramatic with focal accumulations of large foam cells.
Many glomeruli were partially effaced and adhesions were noted (Image 4). A major
veterinary pathology text states that glomerular lipidosis has no functional significance.

This case originated in rural Georgia and ACH is reported primarily from the
Southeastern United States. A seasonal occurrence corresponding with the prevalence
of ticks (Apr-Oct) has been seen. Most cases occur in young adults and this may be
related to their increased physical activity. Heavily infected dogs have a guarded
prognosis and the mortality rate is high. Treatment with a combination of trimethoprim
sulfa, pyrimethamine, and clindamycin has relieved clinical signs and prolonged
treatment with decoquinate has reduced relapses and prolonged survival time.

AFIP Diagnoses: 1. Heart: Myocarditis and epicarditis, pyogranulomatous, multifocal,

mild, with numerous protozoal cysts and merozoites, etiology consistent with
Hepatozoon americanum, Golden Retriever, canine.
2. Small intestine: Enteritis, subacute, diffuse, mild, with villar blunting and fusion, and
few sporozoites.

Conference Comment: The contributor provides a thorough overview of

Hepatozoon americanum. Besides the differences in regional distribution,
between H. americanum (United States, and possibly Central and South America)
and H. canis (India, Africa, Asia, South America, the Middle East, and southern
Europe), there are a few key distinctions between these apicomplexan protozoa. First,
H. canis appears to be adapted to dogs and therefore often results in subclinical

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disease or mild clinical signs. Moderate to severe clinical signs are only seen in
immunosuppressed dogs. In contrast, H. americanum is poorly adapted to dogs and
results in a more severe illness even in immunocompetent dogs. Secondly, the vectors
differ, with the brown dog tick, Rhipicephalus sanguineus serving as the vector for H.
canis and the Gulf Coast tick, Amblyomma maculatum serving as the vector for H.
americanum. Lastly, the clinical presentation, pathology, and prognosis differ. H. canis
causes anemia, and rarely an extreme leukocytosis. Radiographic bone lesions and
significant histologic lesions within the muscle are absent. Meronts are found primarily
in the spleen, bone marrow, and lymph nodes; they are rarely found in muscle and
exhibit a wheel-spoke arrangement of merorozoites. The prognosis for dogs infected
with H. canis is good. In contrast, H. americanum causes anemia, an extreme
leukocytosis, and periosteal proliferation visible with radiography. Histologically, there
are typical “onion-skin” cysts within muscle, meronts, and pyogranulomatous myositis.
The prognosis for dogs infected with H. americanum is guarded.1,7

The sporozoite stage of H. americanum has not been well described in the literature.
The contributor noted rare sporozoites within the villar tips. Although it seems logical
that the organisms identified in the small intestine are sporozoites of H. americanum,
they are very likely Sarcocystis oocysts containing multiple sporozoites. This case was
reviewed in consultation with Dr. Chris Gardiner, AFIP consultant in veterinary
parasitology, and Dr. J.P. Dubey. Dr. Dubey further described these organisms as
having an oocyst wall, with two oval to elongate sporocysts, each containing two
sporozoites. These characteristics are consistent with Sarcocystis sp. in the carnivore
definitive host. In contrast, dogs are the intermediate host of H. americanum. Dogs
become infected with H. americanum through ingestion of an adult tick containing
oocysts. These oocysts are broken down in the intestinal lumen and the individual
sporozoites then penetrate the intestinal wall.6,7

Contributor: University of Georgia-Tifton, Tifton Veterinary Diagnostic &

Investigational Lab, P.O. Box 1389, 43 Brighton Road, Tifton, GA

References:
1. Vincent-Johnson NA: American canine hepatozoonosis. Vet Clin Small Anim 33:905-
920, 2003
2. Baneth G, Mathew JS, Shkap V, Macintire DK, et al: Canine Hepatozoonosis: two
distinct syndromes caused by separate Hepatozoon spp. Vet Parasitol 19:27-31, 2003
3. Macintire DK, Vincent-Johnson NA, Dillon AR, Blagburn B, Lindsay D, Whitley EM, et
al: Hepatozoonosis in dogs: 22 cases (1989-1994). J Am Vet Med Assoc 210(7): 916-
922, 1997
4. Panciera RJ, Ewing SA, Mathew JS, Lehenbauer TW, et al: Canine hepatozoonosis:
Comparison of lesions and parasites in skeletal muscle of dogs experimentally or
naturally infected with Hepatozoon americanum. Vet Parasitol 82:261-272, 1999
5. Panciera RJ, Ewing SA, Mathew JS, Cummings CA, et al: Observation on tissue
stages of Hepatozoon americanum in 19 naturally infected dogs. Vet. Parasitol. 78:265-
276, 1998
6. Dubey JP, Gardiner C: Personal communication

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7. Ewing SA, Panciera RJ: American Canine Hepatozoonosis. Clin Microbiol Rev
16:688-697, 2003

SLIDE 22
CONFERENCE 6 / CASE II – MK04-1836 (AFIP 2937351)

Signalment: 47-month-old male rhesus macaque, (Macaca mulatta).

History: This macaque was on a clinical trial to test the effectiveness of monoclonal
antibody therapy to prolong renal graft survival after renal transplantation. Pre-
transplant treatment consisted of daily injections of an anti-CD154 antibody, IDEC131,
for three days preceding surgery. Post-transplant therapy was limited to weekly
treatments of IDEC131 for eight weeks, after which, the animal did not receive
additional immunosuppressive therapy. Renal biopsies were performed periodically to
assess the character of renal graft inflammation. Monitoring also consisted of regular
evaluation for increases in serum creatinine and blood urea nitrogen, and for the
presence of anti-donor antibodies. There was no intervention after the graft started to
fail. The animal lived 533 days post-transplant, the longest post-transplant period of
any animal on this particular clinical trial.

Gross Pathology: The animal was in thin body condition with mild subcutaneous
edema and pericardial effusion. The lungs were moderately pale. There were
multifocal mild hemorrhages on the capsular surface of the transplanted kidney and the
kidney was pale. No lesions were noted in the heart, lungs, liver, spleen, or
gastrointestinal tract.

Laboratory Results:
Day 29 post-transplant: renal biopsy, moderate tubulointerstitial nephritis.
Immunohistochemistry of the interstitial infiltrate revealed CD3+ cells.
Day 469: anti-donor antibodies detected in the serum
Day 533: BUN: 217 mg/dl Creatinine: 3.7 mg/dl

Contributor’s Morphologic Diagnosis: Kidney: Mesangioproliferative

glomerulonephritis, generalized, diffuse, moderate to marked, with periglomerular
fibrosis, synechia formation and glomerular obsolescence; tubular degeneration,
atrophy and loss and tubular proteinosis; interstitial nephritis; vascular smooth muscle
hyperplasia and fibrosis with multifocal vasculitis and fibrinoid degeneration and
medullary edema.

Contributor’s Comment: The microscopic appearance of rejection in renal allografts

generally is divided into three categories: hyperacute, acute and chronic. Hyperacute
rejection occurs shortly after transplantation of non-HLA cross-matched organs or
transplantation into patients who have been sensitized by previous allografts or

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pregnancy. Histologically, the reaction is characterized by collections of neutrophils in
arterioles, glomeruli, and peritubular capillaries as well as thrombi. This reaction is
antibody mediated and the endothelium of the donor graft, along which antigen-antibody
complexes are deposited, is the target of the immune response. Acute rejection may
occur months to years later after transplantation and immunosuppressive therapy and it
is mediated by cellular and humoral processes. Histologic changes suggestive of
cellular rejection are: interstitial accumulation of mononuclear cells, edema, and
interstitial hemorrhage.1 The primary lesion associated with humoral rejection is
vasculitis. Chronic rejection is characterized by disruption of vascular elastic lamina,
interstitial fibrosis, and tubule loss.2

The Banff Working Classification of Renal Allograft Pathology has been developed by
an international consortium of pathologists, clinicians, and investigators to serve as a
definitive resource by which inflammatory changes in renal allografts are scored. The
scoring system quantifies tubular, vascular, glomerular, and interstitial changes seen in
acute rejection and chronic allograft nephropathy. The histological changes are then
correlated with the severity of renal function deterioration seen clinically. This
classification is revised periodically and is used by pathologists to standardize the
diagnosis of renal allograft rejection, by clinicians to guide therapy for patients, and by
investigators to evaluate clinical trial results. Acute or active rejection is characterized
by tubulitis, tubulointerstitial inflammation, arteritis, and vasculitis. Mild, acute changes
are thought to be primarily T-cell mediated but more severe acute changes are likely to
involve an antibody mediated component.2 Changes suggestive of an antibody
mediated component include vasculitis with fibrinoid change, glomerular and small
vessel thrombosis, infarction, glomerulitis and margination of polymorphonuclear
leukocytes across peritubular capillaries and the presence of C4d, a component of the
complement cascade, in peritubular capillaries and circulating anti-donor antibody.3
Chronic changes due to immune reaction against renal allografts include interstitial
fibrosis and tubular atrophy and loss. Deposition of basement membrane-like material
to form “double contours” within capillary loops of glomeruli is a specific change
associated with chronic transplant glomerulopathy. Additional chronic changes seen in
allografts may be due to renal ischemia, hypertension, drug effects, infection, increased
ureteral pressure, and nonimmune inflammation.2 The changes seen in this case had
many lesions similar to those seen in human acute and chronic allograft rejection,
although interstitial fibrosis was not a significant finding.

One of the major obstacles facing successful organ transplantation is overcoming the
recipient's immune response against alloantigens. Most post-transplant therapies focus
on regulating the recipient's immune system by lifelong suppression, usually with a
combination of a calcineurin inhibitor (cyclosporin or tacrolimus), steroids, and an
antiproliferative agent, (mycophenolate, mofetil or azathioprine).4 Calcineurin inhibitors
specifically suppress T-cell activity. Calcineurin is a key signaling enzyme in T-
lymphocyte activation.5 Calcineurin inhibitors have improved short-term outcomes and
reduced rates of acute rejection in renal transplant recipients; however, these drugs are
nephrotoxic, and their use over a prolonged period of time contributes to chronic
allograft nephropathy.6 Antiproliferative agents non-selectively inhibit cell proliferation

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and can cause bone marrow suppression and hepatoxicity. Steroids inhibit T-
lymphocytes but also cause systemic immunosuppression.7 Additional undesirable side
effects of these immunosuppressive drugs include hypertension, hyperlipidemia,
4
osteoporosis, and chronic allograft nephropathy. In addition to chronic kidney damage
by therapeutic agents and the undesirable effects of these agents, an allograft recipient
receiving immunosuppressive therapy is predisposed to infection by adventitial
microorganisms and to developing neoplastic disease.4

In order to decrease the use of immunosuppressive drugs with their attendant

detrimental side effects, monoclonal antibodies are being developed to target cell
surface molecules on the cells associated with rejection. By targeting a specific
pathway, monoclonal antibodies may provide an adjunct to traditional therapy by
rendering the recipient tolerant to donor tissue while incurring minimal systemic
immunosuppression and, ultimately, leading to prolonged graft survival.4

The animal in this case was treated with ‘costimulation blockade’ monoclonal antibody
therapy, which inhibits one of the two main signals necessary for T-cell activation. The
first signal in T-cell activation is the presentation of antigen to T-cells in association with
MHC-I or MHC-II molecules. Costimulatory molecules are the second immunological
signal required for the activation of T-lymphocytes.4,8,9 These molecules may activate or
suppress T-cell response to an antigen. Monoclonal antibodies against costimulatory
molecules are used at the time of transplantation when the recipient encounters foreign
antigens from the graft for the first time. In theory, T-cells that are presented with novel
antigens, such as those found on an allograft, without costimulation are rendered
anergic or undergo apoptosis, removing the cells that potentially could mount an
immune response against the graft.4

Monoclonal antibodies to CD-154 have been used to induce long term (years) renal
allograft survival in nonhuman primates. CD-154 is found on the surface of T-cells and
its expression is increased in activated T-cells. Interaction of CD-154 with CD40 found
on antigen presenting cells (APCs) increases APC expression of B7 and MHC
molecules and increases production of cytokines. The relationship between inhibition of
CD-154:CD-40 binding and altered T-cell function has not been characterized
completely. Use of anti-CD-154 agents in humans has not been as successful as
predicted by studies in nonhuman primates. Some of these agents have had the
undesirable side affect of predisposing the recipient to thrombi formation.4

There are several centers in the United States that now offer kidney transplantation for
the treatment of end-stage renal disease in companion animals. While
immunosuppressive regimes are similar between feline and human graft recipients,
histological patterns of renal rejection differ between the two species. In a recent issue
of Veterinary Pathology, the authors attempted to classify histologic changes in feline
renal transplants using the Banff 1997 Guidelines. However, the scoring system did not
accurately reflect the severity of lesions based on serum creatinine and BUN levels.
Criteria for acute rejection in humans, tubulitis, lymphocytic glomerulitis, and vasculitis

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were seen rarely in cats. Additionally, subcapsular and interlobular phlebitis were seen
in this series of cats but have been reported rarely in man.10

The classification and treatment of allograft rejection continues to be an ongoing

challenge. New therapies, such as monoclonal antibodies targeted against effectors of
the immune response, may alleviate the need for multiple transplants by inducing life-
long tolerance in organ recipients. However, at the present time, there is still no
effective and definitive treatment or group of treatments to prevent the main cause of
late graft loss, chronic rejection. An even more urgent problem than chronic allograft
rejection is the tremendous demand for donated organs. In the United States, 52,000
people wait for a kidney each year.6 The waiting period for a kidney is in excess of 800
days, and people often die waiting for a donor.8 It may come to pass that, in refining our
knowledge of the mechanisms of allograft rejection, we may be able to understand the
causes of, and to develop effective treatments for, renal disease before transplantation
becomes the only treatment option.

AFIP Diagnosis: Kidney: Glomerulonephritis, membranoproliferative, global, diffuse,

chronic, moderate, with lymphoplasmacytic interstitial nephritis, and arteritis with intimal
fibromuscular proliferation, rhesus macaque (Macaca mulatta), primate.

Conference Comment: As the contributor discussed, one of the most important goals
of immunologic research is successful transplantation of tissues in humans without
rejection. Although the surgical techniques for transplantation of many tissues,
including kidneys, skin, heart, lungs, liver, spleen, bone marrow, and endocrine organs
are well refined, the ability to confer permanent acceptance of foreign grafts is still out of
reach. The basis of graft rejection involves differences in HLA proteins that are
expressed on cells. HLA genes are highly polymorphic and any two individuals, other
than identical twins, will express some HLA proteins that are different. Therefore all
individuals will recognize some difference in HLA molecules as foreign and mount an
immune response to them. Conference attendees discussed, in detail, the two types of
hypersensitivity, cell-mediated (Type IV hypersensitivity) and antibody-mediated (Type II
hypersensitivity) that are fundamental to transplant rejection. 11

Type II hypersensitivity is mediated by antibodies directed toward antigens present on

cell surfaces. In the case of transplant rejection, these antigens are the HLA proteins of
the donor organ. This process is called humoral rejection and can take two forms:
hyperacute and acute. Hyperacute rejection occurs when the recipient has preformed
antidonor antibodies in circulation. These can develop in multiparous women who
receive grafts from their husband, children, or unrelated individuals who share HLA
alleles with the husband; people who have received prior blood transfusions; and,
people who have already rejected a kidney transplant. The rejection is immediate, with
circulating antibodies reacting to graft endothelium, inducing complement fixation, and
resulting in thrombosis of graft vasculature. The acute humoral rejection occurs in
recipients who are not previously sensitized. The antibodies formed cause injury via

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complement-dependent cytotoxicity, inflammation, and antibody-dependent cell-
mediated cytotoxicity, targeting the graft vasculature. 11

Type IV hypersensitivity is T-cell mediated, is called cellular rejection, and is induced by

two mechanisms: CD8+ cytotoxic T-lymphocyte (CTL) mediated destruction of graft
cells, and CD4+ helper T-cell mediated delayed hypersensitivity. The direct pathway,
mediated by the CD8+ CTLs occurs when the T-cells of the recipient recognize donor
HLA (MHC) molecules on the surface of antigen presenting cells (APCs) in the graft.
However, both CD8+ and CD4+ T-cells are activated and differentiate into mature CTLs
and Th1 cells, respectively. Mature CTLs mediate cell destruction by either perforin-
granzyme-dependent killing or Fas-Fas ligand-dependent killing. Th1 cells secrete
cytokines that increase vascular permeability, are chemotactic for lymphocytes and
macrophages, and activate macrophages, resulting in graft injury.11

Contributor: National Institutes of Health, Diagnostic & Research Services Branch,

Division of Veterinary Resources, 28 Service Road West, Bldg 28A--Room 115,
Bethesda, MD

References:
1. Cotran RS, Kumar V, Collins T: Robbins Pathologic Basis of Disease, 6th ed., pp.
206-211. W.B. Saunders Company, Philadelphia, Pennsylvania, 1999
2. Racusen L: The Banff 97 working classification of renal allograft pathology. Kidney
Int 55:713-723, 1999
3. Racusen L, et. al: Antibody-mediated rejection criteria-an addition to the Banff '97
classification of renal allograft rejection. Am J of Transplantation 3:708-714, 2003
4. Dhanireddy K, Xu H, Mannon R, Hale D, Kirk A: The clinical application of
monoclonal antibody therapies in renal transplantation. Expert Opin Emerg Drugs 9
(1):23-37, 2004
5. Klee C, Ren H, Wang X: Regulation of the calmodulin-stimulated protein
phosphatase, calcineurin. J Biol Chem 273 (22):13367-13370, 1998
6. Flechner SM: Minimizing calcineurin inhibitor drugs in renal transplantation.
Transplantation Proceedings 35 (3) Supplement 1:S118-S121, 2003
7. Rang HP, Dale MM, Ritter JM: Pharmacology. 4th ed. Churchill Livingstone, Oxford,
England, United Kingdom, 1999
8. Goldsby RA, Kindt TJ, Osborne BA, Kuby J: Immunology, 5th ed., pp. 479-491.
W.H. Freeman and Company, New York, New York, 2003
9. Cruse J, Lewis R: Atlas of Immunology, 2nd ed., p. 151. CRC Press, Boca Raton,
Florida, 2004
10. De Cock A, Kyles AE, Griffey SM, Bernsteen L, Gregory CR: Histopathologic
findings and classification of feline renal transplants. Vet Pathol 41: 244-256, 2004
11. Abbas AK: Diseases of immunity. In: Robbins and Cotran Pathologic Basis of
Disease, eds. Kumar V, Abbas AK, Fausto N, 7th ed., pp. 210-221. Elsevier Saunders,
Philadelphia, PA, 2005

General References:

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1. Nankivell B, Borrows R, Fung CLS, O'Connell P, Allen RDM, Chapman JR: The
natural history of chronic allograft nephropathy. N Engl J Med
349 (24): 2326-2333, 2003
2. Colvin R: Chronic allograft nephropathy. N Engl J Med 349 (24):2288-2290, 2003
3. Halloran P: Call for revolution: a new approach to describing allograft deterioration.
Am J of Transplantation 2:195-200, 2002
4. Racusen L: Immunopathology of organ transplantation. Springer Semin
Immunopathol 25:141-165, 2003
Kirk A: Crossing the bridge: large animal models in translational transplantation re
5. Lemoine A, Goldman M: Non-classical pathways of cell-mediated allograft rejection:
new challenges for tolerance induction? Am J of Transplantation 3: 101-106, 2003
6. Kasiske BL, Danpanich E: Malignancies in renal transplant recipients.
Transplantation Proceedings 32:1499-1500, 2000
7. Kelsoe G: Therapeutic CD 154 antibody for lupus: promise for the future? J Clin
Invest 112 (10):1480-1482, 2003

SLIDE 23
CONFERENCE 6 / CASE III – 04-14567 (AFIP 2938299)

Signalment: 4-week-old, female, Golden Retriever-cross (Canis familiaris).

History: This individual was one of two puppies from a litter of seven that died
suddenly. The puppy had been nursing and then became cyanotic, dyspneic and died.

Gross Pathology: The carcass was mildly autolyzed and was in good nutritional
condition. The thoracic cavity contained a few milliliters of watery yellow fluid and the
lungs were diffusely edematous and had a rubbery consistency. There was marked
pallor of the left ventricle. Gastrointestinal contents were sparse and the urinary bladder
was contracted.

Laboratory Results: Abundant canine parvovirus antigen was detected in the

myocardium by immunohistochemistry.

Contributor’s Morphologic Diagnosis: Heart: Myocarditis, lymphohistiocytic with

cardiomyocyte degeneration, necrosis and loss and intranuclear inclusion bodies.

Contributor’s Comment: Multifocally, there are foci of cardiomyocyte degeneration

and necrosis characterized by hypereosinophilia, loss of cross striations and
fragmentation. The interstitium is mildly expanded by lymphocytes, macrophages and
rare plasma cells. Within the cardiomyocyte nuclei, there are moderate numbers of 5-8
um, amphophilic to basophilic intranuclear inclusion bodies that often cause margination
of the chromatin. Based on the lesion, this was suspected to be a case of parvoviral
myocarditis and immunohistochemistry confirmed the presence of parvovirus antigen
within cardiomyocytes.

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The Parvoviridae are non-enveloped viral particles about 18-26 nm in diameter with a
single-stranded DNA genome.1 In the late 1970’s, a new parvovirus emerged
worldwide as the cause of severe enteritis and myocarditis in dogs. The agent was
named canine parvovirus type 2 (CPV-2) to distinguish it from the less pathogenic
canine parvovirus type 1 (Minute Virus of Canines) which is associated with mild
diarrhea, fetal losses, myocarditis and fading puppy syndrome.2 Historically, it was
believed that CPV-2 evolved from the closely related feline panleukopenia virus;
however, recent work suggests that a wild carnivore may have harbored the immediate
ancestor of CPV-2.3 Since its emergence in the 1970’s, CPV-2 has been replaced by
two antigenic variants CPV-2a and CPV-2b. Interestingly, CPV-2a and CPV-2b
regained the ability to infect both domestic and large cats. It is believed that
approximately 5% of parvovirus infections in cats are caused by either CPV-2a or CPV-
2b.4

Parvoviruses may infect cells at any phase of the cell cycle but replication is dependent
on cellular mechanisms functional only in the S phase prior to mitosis. For this reason,
the effects of parvovirus infection are greatest in tissues with a high mitotic rate,
including hematopoietic tissue, intestinal crypt epithelium and neonatal cardiomyocytes
(<2 weeks of age).1 In the original outbreaks of CPV-2 in naïve populations,
myocarditis was a common finding. CPV-2 myocarditis is most often seen in puppies
under 4 weeks of age and usually all pups in the litter are affected. Pups are often
found dead or die after a brief period of dyspnea, crying and retching. Signs of cardiac
dysfunction may be preceded by the enteric form of the disease or may occur suddenly
without apparent previous illness.2 The myocardial form of the disease has virtually
disappeared as a result of population immunity. Virtually all bitches are immune and
pass immunity to their
pups via colostrum resulting in protection during the critical period when infection of the
myocardium is possible.5 Myocarditis is still occasionally found in pups born to isolated,
unvaccinated bitches or in cases where adequate colostrum is not received.2

AFIP Diagnosis: Heart: Myocarditis, lymphohistiocytic, multifocal, minimal, with

myocyte degeneration and necrosis, and basophilic intranuclear inclusion bodies,
Golden Retriever-cross, canine.

Conference Comment: The contributor provides a thorough overview of canine

parvovirus type 2 (CPV-2) and compares it to the less pathogenic canine parvovirus
type one (CPV-1). Members of the genus Parvovirus infect many other species of
laboratory and domestic animals, including cats, mink, calves, and swine.1

Canine parvovirus enteritis was first recognized in dogs because the gross and
microscopic lesions were identical to feline parvoviral enteritis, caused by feline
panleukopenia virus (FPV). FPV affects all members of the Felidae, as well as mink,
raccoons, and some other members of the Procyonidae.1 It is the cause of
panleukopenia, also known as cat distemper, feline enteritis, and mink enteritis,6 and is

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very similar to CPV-2. Panleukopenia virus is ubiquitous in environments frequented by
cats; infection is common, but generally subclinical. Transmission is primarily through
oronasal exposure and results in uptake of the virus by epithelium over the tonsils and
Peyer’s patches. The virus infects lymphoblasts and disseminates to other lymphoid
organs (spleen, bone marrow, thymus, lymph nodes). Viral infection of these organs
results in lymphocytolysis and viremia. Infection of the gastrointestinal epithelium is a
secondary event and leads to destruction of the cells in the crypts of Lieberkuhn. If
severe enough, this will result in focal or widespread villus atrophy, mucosal erosion or
ulceration. Proliferating cells in the bone marrow are also affected during viremia,
resulting in cytolysis and bone marrow depletion of both erythroid and myeloid
elements. Infection of the fetus during late prenatal life by FPV results in cerebellar
hypoplasia.

A summary of parvoviruses that affect domestic and laboratory animals are listed
below:7,8

Virus Disease
Feline parvovirus Panleukopenia, cerebellar hypoplasia, enteritis
Canine parvovirus-1 Mild diarrhea
Canine parvovirus-2 Enteritis, myocarditis, leukopenia
Porcine parvovirus Stillbirth, mummification, abortion, embryonic
death, infertility (SMAEDI)
Mink enteritis virus Panleukopenia, enteritis
Aleutian disease virus of mink Chronic immune complex disease, encephalopathy
Minute virus of mice Lymphotrophic, erythrocyte-associated anemia
Mouse parvovirus Lymphotrophic, immunomodulation
Kilham’s rat virus Multifocal hemorrhage (brain, liver, testes)
Toolan’s H-1 virus of rats Nonpathogenic
Rat parvovirus Nonpathogenic
Hamster parvovirus Domed calvaria, malformation of incisor teeth
Lapine parvovirus Mild to moderate enteritis in rabbits
Goose parvovirus Hepatitis, myocarditis
Duck parvovirus Hepatitis, myocarditis

Contributor: Department of Veterinary Pathology/ Prairie Diagnostic Services

Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus
Drive, Saskatoon, SK Website: www.usask.ca/wcvm/vetpath

References:
1. Barker IK, Van Dreumel AA, Palmer N: The Alimentary System. In: Pathology of
Domestic Animals, eds. Jubb KV, Kennedy PC, Palmer N, 4th ed., vol. 2, pp.193-196.
Academic Press, San Diego, CA, 1993
2. Hoskins JD: Canine Viral Enteritis. In: Infectious Disease of the Dog and Cat, ed.
Greene CE, 2nd ed., pp. 40-45. WB Saunders, Philadelphia, PA, 1998
3. Truyen U: Emergence and recent evolution of canine parvovirus. Veterinary
Microbiology 69: 47-50, 1999

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4. Steinel A, Parrish CR, Marshall EB, Truyen U: Parvovirus infection in wild carnivores.
J Wildl Dis 37(3): 594-607, 2001
5. Pollock RVH, Coyne MJ: Canine Parvovirus. Veterinary Clinics of North America:
Small Animal Practice 23(3): 555-567, 1993
6. Gelberg HB: Alimentary system. In: Thompson’s Special Veterinary Pathology, eds.
McGavin MD, Carlton WW, Zachary JF, 3rd ed., p. 62. Mosby, St. Louis, MO, 2001
7. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ: Veterinary Virology, 3rd ed., pp.
344. Academic Press, San Diego, CA 1999
8. Percy DH, Barthold SW: Pathology of Laboratory Rodents and Rabbits, 2nd ed., pp.
22-23, 109, 170, 251. Iowa State Press, Ames, IA, 2001

SLIDE 24
CONFERENCE 6 / CASE IV – D-04-0229 (AFIP 2936454)

Signalment: Three-month-old, female, albino rat (Rattus norvegicus).

History: This was one of a group of 12 rats and mice imported from Los Angeles to
Hong Kong four days previously. When examined subsequently they were showing
upper respiratory tract disease signs and ruffled coats. Four younger rats appeared
weak. They were all medicated with Baytril, but one rat died the next day (this case)
and a second died 2 days later. The animals had been held in a warehouse for an
extended period before departure and the flight was delayed in arriving at Hong Kong.

Gross Pathology: The submitted carcass was a 3-month-old white female rat in
normal body condition, but dehydrated. The right cranial lung lobe was completely
consolidated, dark red, and had a large amount of pale mucoid exudate in the
bronchioles. The rest of the lungs were congested and edematous. The stomach
contained no ingesta. The small intestine was distended with gas and the colon
contained several fecal pellets.

Laboratory Results: A pure growth of Streptococcus pneumoniae was isolated from

the rat’s lung and liver.
Contributor’s Morphologic Diagnosis: 1. Brain: Leptomeningitis, suppurative,
diffuse, subacute, severe, and severe focal paraventricular encephalitis with numerous
intralesional diplococcoid bacteria present.
2. Lung: Bronchopneumonia, suppurative, multifocal, severe, subacute with some
alveolar septal destruction.

Contributor’s Comment: This is a case of severe subacute suppurative

meningoencephalitis and bronchopneumonia caused by Streptococcus pneumoniae. In
addition to the lesions in lung and brain, the left ventricle of the heart had a focal area of
severe myocarditis involving the endocardium and deeper myocardium with degenerate
myocardiocytes and an infiltration of fibroblasts, macrophages and fewer lymphocytes.
There was also a mild tracheitis with an infiltration of moderate numbers of degenerate

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neutrophils and macrophages in the lamina propria with exudation into the lumen. The
tracheal mucosal epithelium showed squamous metaplasia and loss of mucous glands.

Streptococcus pneumoniae is a gram-positive, capsulated, non-motile, facultatively

aerobic coccus that occurs singly, in pairs, or in short chains in its natural habitat, the
upper respiratory tract of humans or other mammals.1 In infected tissues and pus this
bacterium commonly occurs as pairs of cocci which gave cause for its previous generic
name of Diplococcus. It is an important pathogen in humans causing primarily lobar
pneumonia (giving rise to the name pneumococcus), but also causing septicemia and
meningitis.2 In the past, this infection was recognized as a common problem in rodent
colonies with clinically normal carrier animals maintaining infection in the nasoturbinates
and tympanic bullae, but human carriers have also been implicated as a source of
infection to laboratory rodents.3 There are also reports of S. pneumoniae causing
bovine mastitis, calf septicemia and septicemia and septic arthritis in cats.4

S. pneumoniae is alpha-hemolytic in culture on blood agar but can show some variation
in colony type. Compared to other alpha-hemolytic streptococci, S. pneumoniae can be
identified by rapid lysis in bile salts and optochin (ethylhydrocupreine hydrochloride)
sensitivity.2 There are over 84 serotypes of this bacterium, but most infections are
caused by less than 10 serotypes.4

In lesions, pus, or blood smears S. pneumoniae has a distinctive polysaccharide

capsule which enables it to resist phagocytosis by host cells.2,3 S. pneumoniae is not
known to produce soluble toxins but several serotypes produce tissue damage by
activation of the alternate complement pathway.3

Other lesions that have occurred in outbreaks of this infection in rodent colonies have
included fibrinopurulent polyserositis, suppurative rhinitis, otitis media and embolic
suppurative lesions in organs such as liver, spleen and kidney.3

AFIP Diagnosis: 1. Lung: Bronchopneumonia, necrotizing, suppurative, subacute,

moderate, rat, rodent.
2. Midbrain: Meningoencephalitis, suppurative, diffuse, moderate, with myriad bacterial
diplococci.

Conference Comment: Streptococcus pneumoniae was previously a common

problem in laboratory rats. Today, outbreaks of clinical disease are rare in barrier-
maintained facilities. Clinical signs may include serosanguinous nasal discharge,
rhinitis, sinusitis, conjunctivitis, and vestibular signs consistent with middle ear
involvement. Asymptomatic animals may develop clinical disease when there is a
concurrent infection or a change in environment. Gross lesions are varied depending
on the organ system(s) involved and include: serous to mucopurulent exudates in the
nasal passages and/or tympanic bullae, fibrinopurulent pleuritis, peritonitis, pericarditis,
periorchitis, meninginitis, or polyserositis. Histologically, in the acute form of the

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disease, fibrinopurulent pleuritis and pericarditis are typical findings. Pulmonary lesions
vary from localized suppurative bronchopneumonia to acute fibrinopurulent
bronchopneumonia, with obliteration of normal architecture in the affected lobes.
Fibrinopurulent lesions may be identified in any organ affected by S. pneumoniae.
Embolic suppurative lesions have been identified in the liver, spleen, and kidney.
Differential diagnoses for S. pneumoniae in a rat include corynebacteriosis,
salmonellosis, pseudomoniasis, and pasteurellosis.3

Streptococcus pneumoniae is known to cause similar lesions in other animals, including

guinea pigs, hamsters, and non-human primates.3,5 Mice are resistant to S.
pneumoniae6 but can develop suppurative lesions when infected with beta-hemolytic
streptococcal organisms. However, SCID mice may develop systemic disease with
alpha-hemolytic streptococci.3,6 Rabbits have developed acute diplococcal infections on
rare occasions and streptococcal septemia has been reported in young rabbits.3

Contributor: Tai Lung Veterinary Laboratory, Agriculture, Fisheries and Conservation

Department, Government of Hong Kong SAR, Lin Tong Mei, Sheung Shui, New
Territories, Hong Kong SAR, China

References:
1. Austrian R: Streptococcus pneumoniae (Pneumococcus). In: Manual of Clinical
Microbiology, eds. Lennette EH, Spaulding EH, Truant JP, 2nd ed., pp.109-115.
American Society of Microbiology, Washington, DC, 1974
2. Quinn PJ, Carter ME, Markey BK, Carter GR: Clinical Veterinary Microbiology, pp.
127-136. Harcourt Publishers Limited, London, 1999
3. Percy DH, Barthold SW: Pathology of Laboratory Rodents and Rabbits, 2nd ed., pp.
65-66,134-136,182, 223-225, 280. Iowa State Press, Ames, IA, 2001
4. Carter GR, Chengappa MM, Roberts AW: Essentials of Veterinary Microbiology, 5th
ed., p. 111. Williams and Wilkin, Philadelphia, PA 1995
5. Baskin GB: Pathology of Nonhuman Primates. Pathology of Laboratory Animals
Conference Notes, pp.170-171, 2003
6. Barthold SW: The Laboratory Mouse. Pathology of Laboratory Animals Conference
Notes, pp. 34, 2003

SLIDE 25
CONFERENCE 7 / CASE I –D-04-0312 (AFIP 2936453)

Signalment: Two month old, female, large white-cross, pig.

History: A pig farm has had chronic problems for two years involving respiratory signs
in young pigs. Symptoms exhibited by this weaner were typical of affected animals, and
included labored breathing, mucoid nasal discharge and poor growth.

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Gross Pathology: There was a diffuse lesion throughout the lungs, with all lobes
discolored and consolidated. The cut surface was effusive and showed multiple white
spots that appeared to be material in airways, but there was no pus. The thoracic and
mesenteric lymph nodes were enlarged.

Laboratory Results: Microbiology results showed no significant bacterial growth on

routine aerobic culture from the lungs, liver, or large intestine.

PCR test results were negative for porcine reproductive and respiratory syndrome virus
(PRRSV) from lung tissue and a clotted blood sample. Virus culture on MARC cells
showed cytopathic effect, and the culture medium was PCR-positive for PRRSV (US
strain). The lung tissue was also PCR-positive for porcine circovirus 1 (PCV1), but the
lymph nodes were negative. Lungs and lymph nodes were negative for PCV2.

Contributor’s Morphologic Diagnosis: 1. Lungs: Pneumonia, interstitial and

proliferative, severe, non-suppurative, subacute to chronic, coalescing lesions, with
marked proliferation of type II epithelial cells.
2. Bronchioles: Bronchiolitis, severe, necro-suppurative, subacute, coalescing lesions,
with caseating material present in airways.

Contributor’s Comment: Porcine reproductive and respiratory syndrome (PRRS) has

become a world-wide problem for pig producers since slightly differing syndromes
(North America in 1987 and Europe in the 1990s) were united into one syndrome
caused by genetic relatives of the Lelystad virus, now referred to as PRRSV.1 The
American and European isolates have been found to have marked genotypic and
phenotypic differences, and variants of each have arisen even within their respective
continents. The widely varying syndromes involve late-term abortion, stillborn and weak
pigs, lowered farrowing rates, high death rates of neonates and weaned pigs, and
delayed returns to estrus.2

Infection and disease associated with PRRSV are common in Hong Kong pig farms,
and many cases are complicated by co-infection with porcine circovirus 2 (PCV2). This
case was negative for PCV2, so the interstitial pneumonia was demonstrative of
uncomplicated PRRSV infection. The pulmonary and bronchiolar lesions were given
distinct morphological diagnoses because of the likelihood that secondary bacteria were
involved in the bronchial lesion. The bacterial and mycoplasma culture results from this
laboratory were negative, but the bronchial lesion is more consistent with secondary
bacterial involvement than with pure PRRS, and many pigs in Hong Kong receive
prophylactic antibiotics in the feed. The experimental disease in the lungs associated
with uncomplicated PRRSV is limited to an interstitial pneumonia, is non-suppurative
and dominated by mononuclear reaction.2 Additional airway disease with neutrophils is
usually attributed to bacteria or Mycoplasma spp..

The lymph nodes of this pig also showed severe diffuse changes, including a general
loss of follicular architecture and replacement by histiocytes, lymphocytes and plasma
cells. The overall appearance of the lymph nodes was of diffuse granulomatous

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lymphadenitis. The periphery of the lymph nodes showed areas of necrosis. The brain
and meninges showed very mild perivascular cuffing, but this was not considered
significant enough to have contributed to the pig’s symptoms. The heart was normal but
there were a few foci of monocytic infiltration in the liver.

Unfortunately it was not possible to obtain a more detailed history from the farm as to
whether there were additional reproductive problems or other manifestations of PRRS
disease. Records are not kept at many traditional farms and there are often multiple
problems interacting at once.

AFIP Diagnosis: Lung: Pneumonia, bronchointerstitial, subacute, diffuse, moderate,

with multifocal type II pneumocyte hyperplasia, cross-bred pig, porcine.

Conference Comment: Porcine pneumonias are a major problem faced by the

contemporary swine industry. The incidence, prevalence, and mortality rates of
pneumonias in pigs are linked to many interdependent factors, including: the host (age,
genetic makeup, immune status), infectious agents (viruses, bacteria, mycoplasmas),
environmental conditions (humidity, temperature, ammonia concentrations), and
management practices (crowding, mixing of animals, air quality, nutrition, stress).3

Porcine reproductive and respiratory syndrome (PRRS) is endemic in many swine

producing countries and is a major cause of late-term abortions, stillbirths and
respiratory disease in young pigs. PRRS is caused by PRRS virus (PRRSV), an
enveloped single stranded RNA virus in the family Arteriviridae, genus Arterivirus.
Other arteriviruses include equine arterivirus (EAV) and simian hemorrhagic fever virus
(SHFV).4

PRRSV is transmitted by direct contact between infected and naïve pigs, although the
exact route of transmission (aerosol, body fluids, fecal) has not been proven
experimentally. The virus replicates in alveolar macrophages and glial cells. However,
the virus antigen or RNA has been identified in macrophages of multiple tissues,
monocytes, endothelial cells, smooth muscle cells, and fibroblasts. PRRSV infection
has been limited to domestic swine, with a single report of PRRSV infection in Mallard
ducks.4

The clinical presentation of PRRSV infection depends on the age, pregnancy status,
and trimester of gestation of the infected pig. Clinical presentation on a farm varies
from sporadic abortions to abortion storms. Individual animals may present with late-
term abortion, premature farrowing with stillborn fetuses, partially autolyzed fetuses, or
mummified fetuses. Clinical signs in infected sows or gilts vary from none to anorexia,
fever, lethargy, pneumonia, agalactia, cyanosis of the ears and vulva, edema, delayed
return to estrus, and less commonly, death.4

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Gross lesions associated with PRRSV infection vary widely from none to diffuse tan
consolidation of the lungs, and are commonly complicated by lesions resulting from
concurrent bacterial infection. Lymph nodes may be markedly enlarged and vary from
solid to polycystic. Fetuses from PRRSV abortions are late term and the body condition
ranges from fresh to autolyzed.4

Light microscopic lesions most commonly involve the lung and lymphoid tissue.
However, other lesions include vasculitis, myocarditis, and encephalitis. Lung lesions
are characterized by septal thickening by macrophages, type II pneumocyte
hyperplasia, necrotic debris, macrophages and syncytial cells within alveoli,
peribronchial lymphoid hyperplasia, and lymphoplasmacytic perivascular cuffing.
Lymphoid tissue exhibits lymphoid hyperplasia and necrosis.4 Other infectious causes
of pneumonia in swine are listed below3
Viral
Swine influenza virus
Porcine circovirus (Postweaning multisystemic wasting syndrome, PMWS)
Porcine respiratory coronavirus (PRCV)

Bacterial
Mycoplasma hyopneumoniae (Porcine enzootic pneumonia)
Actinobacillus pleuropneumoniae (Porcine pleuropneumonia)
Haemophilus parasuis (Glasser’s disease)
Pasteurella multocida
Streptococcus suis type II
Mycobacterium spp. (M. avium, M. bovis, M. tuberculosis)
Salmonella spp. (S. choleraesuis, S. typhisuis)

Parasitic
Metastrongylus spp. (M. apri, M. salmi, M. pudendotectus)
Ascaris suum

Contributor: Tai Lung Veterinary Laboratory, Agriculture, Fisheries and Conservation

Department, Lin Tong Mei, Sheung Shui, NT, Hong Kong SAR of China
http://www.afcd.gov.hk

References:
1. Wensvoort G, de Kluyver EP, Pol JMA, Wagenaar F, Moormann RJM, Hulst MM,
Bloemraad R, den Besten A, Zetstra T, Terpstra C: Lelystad virus, the cause of porcine
epidemic abortion and respiratory syndrome: a review of mystery swine disease
research at Lelystad. Vet Microbiol 33:185-193, 1992
2. Benfield DA, Collins JE, Dee SA, Halbur PG, Joo HS, Lager KM, Mengeling WL,
Murtaugh MP, Rossow KD, Stevenson GW, Zimmerman JJ: Porcine reproductive and
respiratory syndrome. In: Diseases of Swine, eds., Straw BE, D’Allaire S, Mengeling
WL, Taylor DJ, 8th ed, pp. 201-232. Iowa State University Press, Ames, IA, 1999

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3. Lopez A: Respiratory system, thoracic cavity, and pleura. In: Thompson’s Special
Veterinary Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 178-183.
Mosby, St. Louis, PA, 2001
4. Rossow KD: Porcine reproductive and respiratory syndrome. Vet Pathol 35:1-20,
1998

SLIDE 26
CONFERENCE 7 / CASE II – 15303 6a or 15303 6e (AFIP 2946723)

Signalment: Adult, female, intact, rabbit (Oryctolagus cuniculus).

History: This rabbit received intrahepatic inoculation of VX2 carcinoma cells in the
development of a tumor model to assess the response to treatment by various gene
therapy agents. One week post inoculation, the rabbit developed anorexia and was
noted to be losing weight and condition. After 2 days of supportive therapy with no
clinical improvement, the animal was euthanized.

Gross Pathology: The rabbit was thin with scant body fat. Abdominal organs,
including the liver and kidney, contained multiple tumor masses composed of pale,
yellow-white, soft tissue. There was palpable thickening of the pyloric region of the
stomach, with pale transmural tumor-like masses noted on cut section.

Contributor’s Morphologic Diagnosis: Stomach: Carcinoma, invasive, transmural,

intravascular, compatible with VX2 carcinoma, rabbit, Oryctolagus cuniculus.

Contributor’s Comment: The wall of the stomach is thickened by multiple

unencapsulated, poorly circumscribed and highly infiltrative, multilobular masses that
expand the submucosa and tunica muscularis, and extend into the serosa and mucosa.
Lobules of various sizes and shapes separate muscle bundles, connective tissue and
structural elements, and are supported on a fine to coarse fibrovascular stroma.
Coalescing areas of coagulative and liquefactive necrosis occupy 10-40% of tumor
area, depending on the section, and sometimes are associated with intravascular
thrombi and hemorrhage (infarction). Within lobules, neoplastic cells form cords and
packets supported on a fine fibrovascular stroma. The cells are polygonal with variably
distinct borders, and moderate amounts of pale eosinophilic and vacuolated cytoplasm,
a round vesiculate nucleus with dispersed chromatin and 1 to 3 small nucleoli. There
are 3 to 8 mitoses per high power field (HPF) with numerous bizarre mitotic figures.
There are scattered large karyomegalic and multinucleated atypical cells. Vascular
invasion seems to be primarily within thin-walled lymphatic vessels, but also sometimes
in blood vessels. There is mild to marked submucosal edema, depending on the
section. There is mild to moderate lymphoplasmacytic infiltration of the mucosa and
submucosa.

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VX2 carcinoma was established from a carcinoma induced in a rabbit by the Shope
cottontail rabbit papillomavirus (CRPV) in 1940.1 Papillomaviruses induce benign and
malignant tumors in humans and animals. VX2 tumor cells contain multiple copies of
CRPV genome integrated into their cellular DNA as tandem repeats. VX2 tumor is
considered to be an anaplastic carcinoma, composed of poorly differentiated
keratinocytes that do not keratinize (cornify). VX2 cells grow rapidly in adult allogenic
recipients, frequently metastasizing to the lungs. They are known for having extremely
aggressive behavior in vivo, and are used to model various types of aggressive
epithelial cancers including liver tumors and lung tumors.1 Auricular VX2 carcinoma of
the New Zealand White rabbit is an animal model for human squamous cell carcinomas
of the head and neck region (HNSCC), since both tumors tend to metastasize
lymphatically, leading to early lymph node and subsequent distant metastasis.3 It has
also been used as a model of tumor-induced hypercalcemia.4

AFIP Diagnosis: Stomach: Carcinoma, with intravascular tumor emboli, rabbit,

lagomorph.

Conference Comment: Without the above history, an obvious differential is metastatic

uterine adenocarcinoma, as it is the most common spontaneous neoplasm of rabbits.
The incidence is relatively low, approximately 4%, in younger does (2-3 years old) and
much higher, approximately 80%, in older does (5-6 years old). Grossly the tumors are
nodular, often multicentric, and usually involve both horns. On cut surface, they are
firm, often with a cauliflower-like surface and central ulcerations. Carcinomatosis and/or
metastasis to the lung and liver may occur. Histologically, cells invade the underlying
muscular tunics and form acinar and tubular structures.6

Conference attendees also discussed the multistep model of carcinogenesis, which

involves the sequential stages of initiation and promotion. Initiation causes permanent
DNA damage (mutations), is rapid and irreversible. However, initiation alone is not
sufficient for tumor formation. On the other hand, promoters can induce tumors in
initiated cells, but are nontumorigenic by themselves. In contrast to the effects of
initiators, the cellular changes resulting from promoters do not affect DNA directly and
are reversible. Promoters enhance the proliferation of initiated cells, which may lead to
additional mutations.7

Initiation involves nonlethal genetic damage that may be acquired by the action of
environmental agents, such as chemicals, radiation, or viruses, or it may be inherited in
the germ line. There are four classes of normal regulatory genes that are the principal
targets of genetic damage: the growth-promoting protooncogenes, the growth-inhibiting
tumor suppressor genes, genes that regulate programmed cell death (apoptosis), and
genes involved in DNA repair.7

Carcinogenesis is a multistep process at both the phenotypic and genetic levels.

Characteristics such as excessive growth, local invasiveness, and the ability to form

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distant metastases are phenotypic attributes that are acquired sequentially, a process
known as tumor progression. At the molecular level, progression is a result of
cumulative genetic damage that may be favored by defects in DNA repair.7

There are eight fundamental changes in cell physiology that together determine
malignancy. They include the following: self-sufficiency in growth signals, insensitivity
to growth-inhibitory signals, evasion of apoptosis, defects in DNA repair, limitless
replicative potential, sustained angiogenesis, ability to invade and metastasize, and the
ability to evade the immune system. Mutations in genes that regulate these cellular
traits are seen in every cancer.7

Contributor: Baylor College of Medicine, Center for Comparative Medicine, 600D, One
Baylor Plaza, MS BCM 145, Houston, TX
www.bcm.tmc.edu

References:
1. Georges E, Breitburd F, Jibard N, Orth G. Two Shope papillomavirus-associated
VX2 carcinoma cell lines with different levels of keratinocyte differentiation and
transplantability. J Virol. 1985 Jul;55(1):246-50.
2. Chen JH, Lin YC, Huang YS, Chen TJ, Lin WY, Han KW. Induction of VX2 carcinoma
in rabbit liver: comparison of two inoculation methods. Lab Anim. 2004 Jan;38(1):79-84.
3. Miao Y, Ni Y, Bosmans H, Yu J, Vaninbroukx J, Dymarkowski S, Zhang H, Marchal
G. Radiofrequency ablation for eradication of pulmonary tumor in rabbits. J Surg Res.
2001 Aug;99(2):265-71
4. van Es RJ, Franssen O, Dullens HF, Bernsen MR, Bosman F, Hennink WE, Slootweg
PJ. The VX2 carcinoma in the rabbit auricle as an experimental model for intra-arterial
embolization of head and neck squamous cell carcinoma with dextran microspheres.
Lab Anim. 1999 Apr;33(2):175-84.
5. Doppelt SH, Slovik DM, Neer RM, Nolan J, Zusman RM, Potts JT Jr. Gut
mediated hypercalcemia in rabbits bearing VX2 carcinoma: new mechanism for
tumor-induced hypercalcemia. Proc Natl Acad Sci U S A. 1982 Jan;79(2):640-4.
6. Percy DH, Barthold SW: Rabbit. In: Pathology of Laboratory Rodents and Rabbits,
2nd ed., pp. 303-304. Iowa State Press, Ames IA, 2001
7. Kumar V, Abbas AK, Fausto N: Neoplasia. In: Robbins and Cotran Pathologic Basis
of Disease, 7th ed., pp. 288-289, 319. Elsevier Saunders, Philadelphia, PA, 2005

SLIDE 27
CONFERENCE 7 / CASE III – 04-11575 (AFIP 2937341)

Signalment: 1 month old, female, Spotted Saddle Horse (Equus caballus).

History: Apparently healthy filly became acutely ill and died overnight.

Gross Pathology: The liver was slightly swollen and icteric. The kidney was 20%
swollen. The spleen was 3X normal size with petechial hemorrhages.

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Laboratory Results: Replicate sections of liver stained with Steiner silver stain reveal
clusters of long bacterial rods within adjacent hepatocytes. Low numbers of Listeria
monocytogenes as well as Salmonella typhimurium were isolated from the liver of this
foal. Listeria monocytogenes was also isolated from the kidney in slightly higher
numbers. Replicate sections of liver stained with Brown and Brenn (tissue gram stain)
reveal rare extracellular short bacterial rods, most of which are gram positive.

Contributor’s Morphologic Diagnosis: Liver: Severe, acute, multifocal random,

necrotizing hepatitis with intracellular bacteria.

Etiology: Tyzzer’s disease (Clostridium piliformis) with concurrent Listeria

monocytogenes and Salmonella typhimurium infection.

Contributor’s Comment: Tyzzer’s disease is a fatal necrotizing hepatitis caused by

Clostridium piliforme (previously named Bacillus pilliformis). This disease is most
commonly encountered in young foals1,2 and laboratory rodents3, but occasional cases
in cats and dogs are observed.2 The disease is characterized by multifocal to miliary
hepatic necrosis with the adjacent hepatocytes containing intracellular 10 to 40 micron
long, spore forming, gram-negative clostridial organisms4 arranged into “match stick”
bundles. The organism is difficult to culture and diagnosis is dependent upon
demonstration of the organism within the lesions.3,4,5 It is principally a disease of foals
1-5 weeks of age1 and is characterized by sudden onset of fever, shock, terminal coma
and death within a few hours to two days. Clinical symptoms include tachycardia,
tachypnea, jaundice and severe diarrhea. Affected foals are usually leukopenic and
have highly elevated liver enzymes.

Although clinical disease is rare, the prevalence of antibody in horses suggests that
Clostridium piliforme infection is common. Why only certain foals are susceptible to
fatal infections is not known. Adult inapparent carriers can infect newborn foals, which
are normally coprophagous. Spores survive moderate heating, freezing and thawing.
Contaminated litter remains infective for months.6

Salmonellosis and listeriosis are uncommon forms of septicemia in the horse.7

AFIP Diagnosis: Hepatitis, necrotizing, acute, random, severe, with intrahepatocytic

bacilli, etiology consistent with Clostridium piliforme, Spotted Saddle Horse, equine.

Conference Comment: Tyzzer’s disease was first reported in Japanese waltzing mice
by Ernest Tyzzer in 1917. The organism is now recognized to produce disease in a
wide variety of other species including rats, gerbils, hamsters, guinea pigs, rabbits, and
horses, and rarely in cats, birds and humans with AIDS. The etiologic agent,
Clostridium piliforme, is a gram-negative, spore-forming, filamentous, obligate

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intracellular bacterium that is difficult to isolate using standard bacteriologic
techniques.8,9

The organism is shed in the feces and spores may persist in the environment for up to
one year. Transmission is primarily through ingestion, although intrauterine infection
has been produced experimentally in mice. Outbreaks of Tyzzer’s disease are
characterized by low morbidity and high mortality in affected animals. Animal strain,
age, and immune status are important factors in susceptibility to the disease. Typically,
the organism invades the intestinal mucosal epithelium and disseminates to other
organs, particularly the liver and heart.9

Gross lesions include multifocal coagulative to suppurative hepatic necrosis, segmental

necrotizing enteritis, primarily in the terminal ileum and cecum (except for the rabbit, in
which the cecum and colon are the target organs),10 and multifocal necrotizing
myocarditis. Foci of necrosis may also be found in the mesenteric lymph nodes.
Histologically, there are intracytoplasmic bundles of bacilli within enterocytes and
hepatocytes adjacent to necrotic foci. The organisms are easily identified in Warthin-
Starry 4.0 stained tissue sections.9

Contributor: C.E. Kord Animal Disease Diagnostic Laboratory, P.O. Box 40627,
Melrose Station, Nashville, TN
http://www.state.tn.us/agriculture/regulate/labs/kordlab.html

References:
1. Knottenbelt DC, Pascoe RR: Diseases and Disorders of the Horse, pp.87. Mosby,
London, England, 1999
2. Kelly WR: The liver and biliary system. In: Pathology of Domestic Animals, eds. Jubb
KV, Kennedy PC, Palmer N, 4th ed., vol. 2, pp. 373-374. Academic Press, San Diego,
CA, 1993
3. Percy DH, Barthold SW: Rat. In: Pathology of Laboratory Rodents and Rabbits, 2nd
ed., pp. 121-123. Iowa State University Press, Ames, IA, 2001
4. Greene CE: Infectious Diseases of the Dog and Cat, 2nd ed., pp. 242-243. W.B.
Saunders Company, Philadelphia, PA, 1990
5. St. Denis KA, Waddel-Parks N, Belanger, M: Tyzzer’s disease in an 11-day-old foal.
Can Vet J 41:491-492, 2000
6. Tyzzer’s Disease. In: The Merck Veterinary Manual, eds. Aiello SE, Mays A, 8th
ed.,pp. 123-125. Merck & Company, Inc., Whitehouse Station, NJ,1998
7. Hirsch DC, Zee YC: Veterinary Microbiology, pp. 77 and 226. Blackwell Science, Inc.,
Malden, MA, 1999
8. Fosgate GT, Hird DW, Read DH, Walker RL: Risk factors for Clostridium piliforme
infection in foals. JAVMA 220(6):785-790, 2002
9. Percy DH, Barthold SW: Mouse. In: Pathology of Laboratory Rodents and Rabbits,
2nd ed., pp. 49-50. Iowa State Press, Ames IA, 2001
10. Percy DH, Barthold SW: Rabbit. In: Pathology of Laboratory Rodents and Rabbits,
2nd ed., pp. 268-270. Iowa State Press, Ames IA, 2001

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SLIDE 28
CONFERENCE 7 / CASE IV – N03-912 (AFIP 2946701)

Signalment: 11 year old adult female cynomolgus monkey (Macaca fascicularis).

History: This female cynomolgus presented to the clinician for marked lethargy, and
with a previous history of seizures of unknown etiology. Examination and laboratory
work-up revealed an inflammatory leukogram and low protein. The animal’s condition
did not respond over time to treatment and she was subsequently euthanized.

Gross Pathology: Gross postmortem examination revealed clear fluid in the thorax
(hydrothorax).

Contributor’s Morphologic Diagnosis: Pancreas and adjacent fibrovascular

connective tissue: Arteritis, necrotizing, subacute to chronic, multifocal.

Contributor’s Comment: The full range of the severity and duration of the polyarteritis
in this animal was not evident in all sections. Mildly affected arteries were characterized
by mixed populations of inflammatory cells located primarily in the tunica adventitia.
More severely affected vessels displayed transmural inflammation, intimal proliferation
and fibrinoid necrosis within the tunica media. The disease in this animal involved
multiple tissues including pancreas, heart, kidney and mesentery.

Polyarteritis nodosa (PAN) -like diseases have been described in several species
including humans, dogs, cats, pigs and rodents, but only rarely in nonhuman primates.
The contributor is aware of four cases of idiopathic PAN reported in cynomolgus
macaques. The disease in humans is multisystemic and characterized by necrotizing
lesions in small and medium-sized arteries. Other shared features of the disease as it
is observed in both cynomolgus monkeys and people include segmental distribution in
affected vessels, predilection for areas of arterial branching, and coexistence of acute
and chronic lesions. Although the pathogenesis of PAN is not well understood, it is
generally thought to involve an immune-mediated disease process. Immune complex
deposition with complement activation may play a role in initiating the disease, while
cell-mediated immune interactions are likely to contribute to the progression of the
lesion.

In rats, polyarteritis nodosa (polyangiitis, panarteritis) is a well-described entity and the

incidence varies among different strains. Grossly, the disease typically presents as
nodular lesions affecting the pancreatic, mesenteric and/or spermatic arteries.
Depending on the chronicity of the lesion, microscopic features include fibrinoid
necrosis of muscular arteries, mixed inflammatory cell infiltrate of vessels and
surrounding tissue, and fibrosis. Spontaneous arterial diseases are also described in
dogs including idiopathic necrotizing polyarteritis in Beagle dogs (Beagle pain
syndrome) and idiopathic extramural coronary arteritis. Drug administration can be

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associated with both non-necrotizing (often hypersensitivity induced) and necrotizing
inflammatory vascular lesions. Some examples of drug induced vascular injury include
the mesenteric vasculitis in rats treated with phosphodiesterase III inhibitors and the
coronary and systemic arteritis associated with endothelin A receptor antagonists in
dogs and monkeys.

AFIP Diagnosis: Pancreas: Arteritis, histiocytic and lymphocytic, necrotizing,

multifocal, with intimal fibromuscular proliferation, cynomolgus macaque (Macaca
fascicularis), primate.

Conference Comment: As the contributor mentions, there is slide variability, with the
full range of severity and duration of the polyarteritis not evident in all slides. Within all
slides the pancreatic arteries are affected to varying degrees. Within some sections,
islet capillaries are also affected and occasionally contain thrombi. The arteries in the
peripancreatic adipose tissue of some slides are also affected.

In rats, the disease most frequently occurs in the Sprague-Dawley and spontaneous
hypertensive rat (SHR) strains, and in rats with late-stage chronic nephropathy.
Classically, at necropsy, vessels are enlarged and thickened in a segmental pattern,
with marked tortuosity. The lesions occur most frequently in the pancreaticoduodenal
artery and medium-sized arteries of the mesentery, pancreas, and testis. Histologically,
there typically is fibrinoid degeneration and thickening of the tunica media, and
infiltration by monocytes and fewer neutrophils. There is marked variation in the luminal
size of affected vessels, which are often thrombosed, and occasionally recanalized.5
These features and the intimal proliferation are best appreciated with appropriate
special stains and immunohistochemistry (IHC). The modified Movat’s pentachrome
method is very helpful as it stains elastic laminae black, collagen and reticular fibers
yellow, ground substance and mucin blue, fibrin intense red, and muscle fibers red.6
With the Movat’s pentachrome method, the quantity of intimal proliferation is readily
apparent as are disruptions of the elastic laminae. Other stains and IHC, such as
Masson’s trichrome and smooth muscle actin aid in differentiating increased amounts of
intimal connective tissue from smooth muscle hyperplasia. Immunohistochemistry for
CD68 confirms that most of the infiltrating leukocytes are macrophages.

Contributor: Southwest Foundation for Biomedical Research, PO Box 760549,

San Antonio, TX 78245

References:
1. Porter BF, Frost P, Hubbard GB: Polyarteritis in a cynomolgus macaque (Macaca
fascicularis). Vet Pathol 40:570-573, 2003
2. Albassam MA, Lillie LE, Smith GS: Asymptomatic polyarteritis in a cynomolgus
monkey. Lab Animal Science 43:628-629,19933. Registry for Toxicologic Pathology:
Monkey: Spontaneous necrotizing vasculitis/polyarteritis (R145), RTPA Toxicologic
Histopathology Web Slide Conference, Feb 2004, Case #1

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4. Schoen FJ, Cotran RS: Blood vessels. In: Robbins Pathologic Basis of Disease, eds.
Cotran RS, Kumar V, and Collins T, 6th ed., pp. 493-542. WB Saunders, Philadelphia,
PA, 1999
nd
5. Percy DH, Barthold SW: Rat. In: Pathology of Laboratory Rodents and Rabbits, 2
ed., pp.153. Iowa State Press, Ames IA, 2001
6. Prophet EB, Mills B, Arrington JB, Sobin LH: Laboratory Methods in Histotechnology,
pp 128-130. American Registry of Pathology, Washington DC, 1994

SLIDE 29
CONFERENCE 8 / CASE I – XN2922 (AFIP 2940469)

Signalment: Adult male, ferret (Mustela putorius furo).

History: An adult male ferret of unknown age was one of several housed in a council
animal park open to the public. It developed progressively worsening dyspnea, with
lethargy and mild abdominal distension, in early June 2004 and was euthanized.

Gross Pathology: The adult male ferret weighed 1120 g and had a length from the
nose to the tail tip of 60 cm. The pleural cavity contained 50 ml of light brown, opaque
fluid. The heart was enlarged and globular, with a cross-section of 25 mm by 20 mm at
mid-ventricular level. At this level, the thickness of the left ventricular free wall was 4 to
5 mm, the thickness of the right ventricular free wall was 2 mm and the interventricular
septum was 3 to 4 mm thick. The heart had patchy areas of pallor on the epicardium
and there were irregular, locally extensive, white to pale yellow areas of myocardium in
the inner third of the left ventricle. The right ventricle was affected to a lesser degree.
The subendocardial myocardium and the bases of the papillary muscles were most
severely affected. The lungs had patchy, red to purple atelectasis affecting 60% of the
total lung volume. The abdomen was mildly distended and contained 5 ml of slightly
yellow, clear, transparent fluid. The liver was enlarged, with rounded borders, an
irregular, granular capsule and exudation of fibrin on the surface. The lobular pattern of
the liver was accentuated, with red-purple mottling. The kidneys and spleen were
congested.

Contributor’s Morphologic Diagnosis: Heart: Myocardium, degeneration, necrosis,

fibrosis, nonsuppurative inflammation, subendocardial and subepicardial, severe,
extensive, consistent with dilatative cardiomyopathy, ferret (Mustela putorius furo).

Contributor’s Comment: Histologically, the subendocardial myocardium of this ferret

exhibits degeneration and necrosis of cardiomyocytes, with extensive replacement by
fibrous connective tissue. The bases of papillary muscles and the inner third of the
myocardium of the left ventricle, including the left ventricular free wall and the
interventricular septum, are most severely affected. Subepicardial degeneration and
fibrosis tend to have a perivascular distribution. Mild cardiomyocyte degeneration and
fibrosis are also evident in the subendocardial myocardium of the right ventricle,

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especially in the interventricular septum. There are mild, diffuse and locally extensive
infiltrates of macrophages, lymphocytes and plasma cells, as well as occasional
neutrophils, in areas of degeneration and fibrosis. Other tissues had evidence of
congestive heart failure. Congestion, atelectasis and edema were evident in the lungs.
There was congestion, periacinar fibrosis, mild hemosiderosis and atrophy of
subcapsular hepatocyte cords in the liver, reflecting chronic hepatic venous congestion.
The kidney was congested and hemosiderosis was evident in renal tubular epithelial
cells. The adrenal and thyroid glands were unremarkable. The gross and histological
findings are consistent with dilatative (dilated or congestive) cardiomyopathy of ferrets.1-
3

Cardiomyopathy is the most common cause of heart failure in the ferret.2-5 The usual
form of this condition in ferrets is dilatative (dilated or congestive) cardiomyopathy,
although hypertrophic and restrictive cardiomyopathies have also been described in this
species.2,5 Ferrets are most frequently affected from 5 to 7 years of age, although some
severe cases can develop clinical signs as early as 1 year of age.4,5

Clinical signs of dilatative cardiomyopathy in ferrets include weight loss, weakness,

lethargy, dyspnea, tachypnea, tachycardia, coughing and abdominal distension.4,5 Heart
and lung sounds are muffled on auscultation of the thorax and moist rales may be
evident. A holosystolic murmur is usually most intense on the left side of the thorax at
the level of the seventh to eighth intercostal space. Ascites and pleural effusions may
be present. Hepatomegaly and splenomegaly may be evident on palpation of the
abdomen. Diagnosis of cardiomyopathy in ferrets is usually based on clinical signs,
radiography and echocardiography.2-4 Ancillary diagnostic tests may include
electrocardiography2,6 and cytological examination of fluids obtained by thoracocentesis
or abdominocentesis. Hematology and biochemical testing including endocrinology are
useful for evaluation of concurrent disease.

The cause of cardiomyopathy in the ferret is unknown. A genetic basis has been
suspected in some lines of ferrets in North America.5 Cardiomyopathy has been
associated with hyperadrenocorticism in ferrets.7,8 In one study, 10% of ferrets with
hyperadrenocorticism had concurrent cardiomyopathy.8 A ferret with meningitis due to
Cryptococcus neoformans had concurrent congestive cardiomyopathy.9

Treatment of cardiomyopathy in ferrets usually includes diuretics such as furosemide,

vasodilators such as enalapril, positive inotropes such as digoxin and a low sodium
diet.2-4,10 The long term prognosis for ferrets with cardiomyopathy is poor. There is
insufficient evidence to determine if supplementation with taurine or carnitine is
beneficial in preventing or treating the disease.2,11

AFIP Diagnosis: Heart, myocardium: Degeneration, necrosis, and loss, with

replacement fibrosis, multifocal, marked, ferret (Mustela putorius furo), mustelid.

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Conference Comment: Cardiac disease is common in older ferrets and is usually due
to dilated cardiomyopathy, hypertrophic cardiomyopathy or valvular disease. The
contributor provides a thorough overview of dilated cardiomyopathy. Hypertrophic
cardiomyopathy has not been extensively studied in ferrets. Unlike cats, there is no
known association with hyperthyroidism or hypertension. Grossly the interventricular
septum and left-ventricular free walls are abnormally thickened with decreased internal
dimensions, and often an enlarged left atrium. Histologically, fibrous connective tissue
is present throughout the myocardium. Valvular heart disease is reported with
increasing frequency, with gross lesions consisting of abnormally thickened valves and
dilated atria. Histologically, there is myxomatous degeneration of the valve as with dogs
with endocardiosis. Other, less common causes of heart disease in ferrets include
myocarditis, which may be due to Toxoplasma-like organisms, parvovirus (Aleutian
mink disease), septicemia, and Dirofilaria immitis (heartworm disease).2

Cardiomyopathies have been reported in a number of other species including the cat,
dog, pig, cow, hamster, turkey, mouse, and man and are classified as primary or
secondary. Primary cardiomyopathies, those without a known etiology, are further
subdivided into dilated, hypertrophic, and restrictive. Secondary cardiomyopathies are
associated with known etiologies, such as viral myocarditis.1

Dilated cardiomyopathy is an important cause of congestive heart failure in dogs and

cats. Often cats will have low tissue concentrations of taurine and supplementation with
taurine has reversed the clinical signs of cardiac failure. Affected dogs are often males
of large breeds, such as Doberman Pinschers, Irish Wolfhounds, and Newfoundlands.
Grossly the heart is rounded due to biventricular dilatation, often with a diffusely white,
thickened endocardium. Histological changes are non-specific, may be mild or absent,
and include interstitial fibrosis and myocyte degeneration. Hypertrophic cardiomyopathy
occurs frequently in middle-aged male cats. Animals often present with congestive
heart failure and approximately 10-20% will have posterior paresis from a concurrent
thromboembolism of the caudal abdominal aorta (“saddle thrombus”). Grossly the heart
is enlarged with prominent hypertrophy of the left ventricle and interventricular septum,
the left ventricular cavity is small, and the left atrium is dilated. Histologically there is
prominent disarray of cardiac myocytes, with interweaving rather than parallel fibers,
interstitial fibrosis, and myocyte degeneration. Restrictive cardiomyopathy occurs
infrequently and includes such conditions as endocardial fibrosis in certain strains of
rats and congenital endocardial fibroelastosis in Burmese cats.12

Causes of secondary cardiomyopathy include infectious, hereditable, nutritional, toxic,

physical injuries, endocrine disorders, neoplasia, and systemic hypertension in cats.
Some infectious agents include:12

Viral:
Canine parvovirus (canine parvovirus type 2)
Encephalomyocarditis (cardiovirus)
Foot-and-mouth disease (picornavirus)
Pseudorabies (porcine herpesvirus)

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 Canine distemper (canine morbillivirus)
Cytomegalovirus (porcine betaherpesvirus)
Newcastle disease (avian paramyxovirus)
Eastern and western equine encephalomyelitis (alphavirus)
West Nile Virus (flavivirus)
Bacterial:
Clostridium chauvoei (Blackleg), C. piliforme (Tyzzer’s disease)
Listeria monocytogenes (Listeriosis)
Fusobacterium necrophorum (Necrobacillosis)
Mycobacterium spp. (Tuberculosis)
Corynebacterium pseudotuberculosis (Caseous lymphadenitis)
Actinobacillus equuli
Staphylococcus sp.
Streptococcus pneumonia
Parasitic:
Toxoplasma gondii
Sarcocystis sp.
Encephalitozoon cuniculi
Trypanosoma cruzi
Cysticercus cellulosae
Trichinella sp.

Contributor: University of Glasgow Veterinary School, Institute of Comparative

Medicine, Division of Pathological Sciences, Glasgow G61 1QH, Scotland, United
Kingdom
http://www.gla.ac.uk/faculties/vet/.

References:
1. Lipman NS, Murphy JC, Fox JG: Clinical, functional and pathologic changes
associated with a case of dilatative cardiomyopathy in a ferret. Lab Anim Sci 37(2):210-
212, 1987
2. Stamoulis ME: Cardiac disease in ferrets. Sem Avian Exotic Pet Med 4(1):43-48,
1995
3. Fox JG: Other systemic diseases. In: Biology and Diseases of the Ferret, ed. Fox JG,
2nd ed., pp. 316-318. Williams & Wilkins, Baltimore, MD, 1998
4. Atkinson RM: Case reports on cardiomyopathy in the domestic ferret, Mustela
putorius furo. J Sm Exotic Anim Med 2(2):75-78, 1992
5. Williams BH: Pathology of the domestic ferret (Mustela putorius furo L.).
http://www.afip.org/ferrets/ferret.path.html (Accessed July 1, 2004)
6. Smith SH, Bishop SP: The electrocardiogram of normal ferrets and ferrets with right
ventricular hypertrophy. Lab Anim Sci 35(3):268-271, 1985
7. Fox JG, Goad ME, Garibaldi BA, Wiest LM Jr: Hyperadrenocorticism in a ferret. J Am
Vet Med Assoc 191(3):343-344, 1987
8. Weiss CA, Scott MV: Clinical aspects and surgical treatment of hyperadrenocorticism
in the domestic ferret: 94 cases (1994-1996). J Am Anim Hosp Assoc 33(6):487-493,
1997

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9. Greenlee PG, Stephens E: Meningeal cryptococcosis and congestive
cardiomyopathy in a ferret. J Am Vet Med Assoc 184(7):840-841, 1984
10. Ensley PK, van Winkle T: Treatment of congestive heart failure in a ferret (Mustela
putorius furo). J Zoo Anim Med 13(1):23-25, 1982
11. Moneva-Jordan A: What is your diagnosis? J Sm Anim Pract 39:263,303, 1998
12. Van Vleet JF, Ferrans VJ: Cardiovascular system. In: Thompson’s Special
Veterinary Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 217-220.
Mosby, St. Louis, PA, 2001

SLIDE 30
CONFERENCE 8 / CASE II – 39080 (AFIP 2936322)

Signalment: 1.5 year old, male-neutered, Domestic Shorthair cat.

History: Cat exhibited lethargy and anorexia, and presented with a temperature of 105
degrees Fahrenheit and respiratory harshness. Feline Leukemia Virus and Feline
Immunodeficiency Virus tests in clinic were negative. All in-house lab work was normal.
Cat declined despite antibiotic treatment and IV fluids. Within the last 12 hours
preceding death, it became very agitated and aggressive. Clinical diagnosis was
unknown.

Gross Pathology: The cat presented in fair flesh and poor postmortem preservation.
There was moderate dilatation of the colon, with a dark red serosa. The descending
colon’s content was dark red.

Laboratory Results: No clinical pathology data was available. Bacteriologic

examination: Lung, intestine: Nonpathogenic bacteria were isolated. Liver: No growth
was observed.

Contributor’s Morphologic Diagnosis: Protozoal schizonts, intravascular,

multisystemic, feline.

Contributor’s Comment: Multifocal intravascular protozoal schizonts were in the

brain, heart, lung, intestine, spleen and kidney. Abundant acidophilic material admixed
with RBC was in the colon. Tapeworms were present in the duodenum.

Cytauxzoonosis is a rare disease. In the U.S. it is most common in free-roaming felines

in the south and is usually, but not always, fatal. Diagnosis is made by finding the
piroplasms of Cytauxzoon felis in blood smears (although parasitized erythrocytes are
seen in low numbers, or are absent in 50% of cases) or by identification of schizonts in
tissues. Cytauxzoonosis should be considered in animals with anemia, icterus and
fever. Cytauxzoonosis is probably spread by arthropod vectors (ticks). Clinical signs
include anorexia, conjunctival/scleral injection, constant or increased vocalization,
dehydration, dullness, dyspnea, fever, generalized weakness, hepatosplenomegaly,

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hypothermia, icterus, increased respiratory rate, lymphadenopathy, mucoid nasal
discharge, pale mucous membranes, palpably enlarged kidneys, petechiae or
ecchymoses, prolapsed third eyelid, red or brown urine, reluctance to move and
tachycardia.

AFIP Diagnosis: Brain, cerebrum; lung: Intramonocytic protozoal schizonts, many,

etiology consistent with Cytauxzoon felis, domestic shorthair, feline.

Conference Comment: Cytauxzoon felis is an apicomplexan intracellular organism in

the family Theileriidae that usually causes fatal disease in domestic and exotic cats in
the south central and southeastern United States. The natural reservoir is the North
American bobcat (Lynx rufus), which typically has only a subclinical infection. Domestic
cats are considered dead-end hosts because the disease is rapidly fatal. In
experimental infections, transmission has been through ingestion or through inoculation
of infected blood or tissue or via ticks which have previously fed on infected bobcats.8

Cytauxzoon felis has both a leukocytic phase and an erythrocytic phase. The leukocytic
(tissue) phase begins when C. felis infects mononuclear cells or macrophages and
undergoes asexual reproduction, producing schizonts. As the schizonts accumulate
and mature, leukocytes enlarge up to 75 µm, often resulting in blood flow obstruction in
the liver, lung, lymph nodes, spleen, and bone marrow, leading to severe circulatory
impairment. These schizonts then bud, forming merozoites, which lead to further host
cell engorgement and cell rupture. Once the merozoites rupture from the host cells,
they infect erythrocytes, leading to the erythrocytic phase, which often results in
hemolytic anemia. During the erythrocytic phase, the piroplasms are approximately 1-2
µm in diameter, and are ring shaped (signet-ring pattern), and can be identified on
peripheral blood smears usually 1-3 days prior to death. Normally only 1-2% of
erythrocytes will be affected; however, in moribund cats up to 25% of erythrocytes may
be affected.8

Gross necropsy findings may include pallor, icterus, petechial and ecchymotic
hemorrhages over the surface of the lungs and heart, excessive clear yellow fluid in the
pericardial sac, enlarged dark spleen, prominent distended intra-abdominal veins, and
swollen, edematous, hyperemic, and sometimes petechiated lymph nodes. The
characteristic histologic lesion of cytauxzoonosis, as is present in this case, is the
occurrence of numerous intravascular large monocytes with intracytoplasmic schizonts
containing multiple small, basophilic, granular bodies (cytomeres) that represent
merozoites in various stages of development. The infected monocytes may be found in
association with the endothelial lining of venous channels and sinusoids in most major
organs, but are usually more abundant in the lung, spleen, lymph nodes, and bone
marrow.5

Contributor: Livestock Disease Diagnostic Center, University of Kentucky, 1429

Newtown Pike, P.O. Box 14125 Lexington, KY

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http://fp1.ca.uky.edu/lddc/

References:
1. Allsopp MT, Cavalier Smith, T, De Waal DT, Allsopp BA: Phylogeny and evolution of
the piroplasms. Parasit 108(Pt 2): 147-52, 1994
2. Butt MT: Diagnosing Erythrocyte Parasitic Diseases in Cats: Comp Cont Ed Prac Vet
12:628-638, 1990
3. Cowell RL, Fox JC, Panciera RJ, Tyler RD: Detection of anticytauxzoon antibodies in
cats infected with a Cytauxzoon organism from bobcats. Vet Parasit 28(12): 43-52,
1988
4. Glenn BL, Kocan AA, Blouin EF: Cytauxzoonosis in bobcats. J AmVet Med Assoc
183(11): 1155-8, 1983
5. Glenn BL, Stair EL: Cytauxzoonosis in domestic cats: report of two cases in
Oklahoma, with a review and discussion of the disease. J AmVet Med Assoc 184(7):
822-5, 1984
6. Hoover JP, Walker DB, Hedges JD: Cytauxzoonosis in cats: eight cases (1985 1992).
J AmVet Med Assoc 205(3): 455-60, 1994
7. Kier AB, Wagner JE, Kinden DA: The pathology of experimental cytauxzoonosis. J
Comp Path 97(4): 415-32, 1987
8. Meier HT, Moore LE: Feline cytauxzoonosis: A case report and literature review. J
Am Anim Hosp Assoc 36: 493-496, 2000

SLIDE 31
CONFERENCE 8 / CASE III – CB04-13 (AFIP 2947494)

Signalment: An approximately 37 year-old, sexually intact female rhesus macaque,

(Macaca mulatta), non-human primate.

History: This macaque was singly housed indoors and tested for 19 years in a
neurobiology study involving interactions between catecholamine receptors in the
prefrontal cortex and cognitive function. Seven months prior to presentation, a
diagnosis of severe spinal osteoarthritis was made. After a 5 month period of biweekly
intramuscular injections of a polysulfated glycosaminoglycan (Adequan®) treatment was
changed to one month trials of oral nonsteroidal anti-inflammatories, carprofen and
deracoxib. Seroconversion to Simian retrovirus was detected 10 weeks prior to
presentation. Clinical signs at presentation included a sudden onset of profound
lethargy, weakness, hypoglycemia and hypothermia. Treatment with intravenous
dextrose, as well as fluid and thermal support resulted in a dramatic rapid improvement.
Due to a poor prognosis for extended use, the investigator elected euthanasia and
perfusion 4 days later.

Gross Pathology: A 2 cm round, slightly red mass is present within the body of the
pancreas adjacent to the duodenum. Within the dorsal aspect of the uterine body a 4
mm round, white, firm raised myometrial mass extended to the perimetrium (serosa). A

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0.5 cm paraovarian cyst was present. The liver was rounded with three large foci of
hepatic nodules. Scattered, 0.1-0.2 mm, translucent pleural foci were present
throughout all lung lobes.

Laboratory Results: Serum biochemistry: results from a blood sample taken at

presentation revealed marked hypoglycemia (9mg/dl; reference range 84-131mg/dl) and
relative hyperinsulinemia (51.1 _IU/ml).
Electronmicroscopy: neoplastic cells contain numerous polymorphic granules
containing a dense, rectangular, crystalline core separated from the limiting membrane
by a distinct, wide halo. Granules are consistent with those found in beta cells.
Immunohistochemistry: neoplastic cells stained negatively for glucagon and
somatostatin; were diffusely moderately positive for chromogranin A and rarely weakly
positive for insulin.

Contributor’s Morphologic Diagnosis: Pancreas: Islet cell (beta) tumor; insulinoma.

Contributor’s Comment: Within the pancreas there is a 2 cm round, discrete,

expansile mass compressing the adjacent normal pancreatic tissue. Arranged in
variably sized (20 - 500 _m) disorganized nests and clusters, neoplastic cells are
suspended in a highly vascular, fatty, loose connective tissue stroma. These cells are
10-20 _m in diameter, round to oval with indistinct cell boundaries. They contain
predominantly one and occasionally two, 5 _m diameter, basally aligned, basophilic
nuclei with one or more small, indistinct nucleoli. The cytoplasm is amphophilic,
abundant and vacuolated. Less than one mitotic figure is seen per 40 power field.

The most frequently described spontaneous neoplasms in nonhuman primates involve

those of the digestive system.1 Although reports of pancreatic tumors are limited, the
most commonly described neoplasms involve islet cell adenomas; few of which present
with gross lesions.2 Clinical signs indicative of pancreatic disease have not been seen
in any instance of nonhuman primate pancreatic tumors. Neoplasms of the pancreatic
islets have been observed in several species including cattle3, ferrets4, cats5, and mice6.
In dogs, beta cell tumors have been observed in many different breeds and are more
frequently carcinomas exhibiting clinicopathological evidence of hyperinsulinism rather
than adenomas.3 In humans, insulinomas, are exceedingly rare (1-4 reported cases per
million yearly), but constitute 70-80% of the clinically symptomatic endocrine pancreatic
tumors. Greater than 80% of these are solitary masses and, unlike other endocrine
neoplasms of the pancreas where malignancy predominates, insulinomas are benign in
more than 85% of cases.

The usual histological criteria for malignancy (nuclear pleomorphism, mitotic activity,
infiltration of surrounding tissues) are considered unreliable markers in endocrine
pancreatic tumors. The accepted parameter for classification of malignancy is local or
capsular extension to adjacent organs or a demonstration of vascular invasion and
metastases.

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Insulinoma cells contain less insulin than do normal beta cells, yet neoplastic tissue has
a higher concentration of insulin and proinsulin than the surrounding normal pancreatic
cells. The presence of higher serum proinsulin levels in human patients with insulinoma
has been attributed to a decreased storage capacity leading to inappropriate insulin
release. Such release at times of low to normal blood glucose levels leads to
hypoglycemia and associated neuroglycopenic symptoms (e.g. mental dullness,
weakness, abnormal behavior and seizures).

Immunohistochemistry for Chromogranin A, a well established marker for tissues of

neuroendocrine origin, is useful in classifying pancreatic neoplasms. Islet cell tumors
can stain positive for several immunohistochemical stains and are named according to
the hormone responsible for producing clinical signs (insulin for insulinoma).

This rhesus macaque exhibited clinical signs of acute hypoglycemia and a response to
appropriate therapy. Concomitant hypoglycemia and elevated blood levels of insulin
confirmed hyperinsulinism. In addition, a distinct solitary nodule was present with
histological, immunohistochemical, and ultrastructural characteristics consistent with an
insulinoma.

AFIP Diagnosis: Pancreas: Islet cell tumor, Rhesus macaque, primate.

Conference Comment: The contributor provides a thorough overview of insulinomas

in various animal species and humans. Pancreatic islets are composed of six distinct
cell types, each with specific secretory products that are best distinguished by
immunohistochemical techniques. The alpha cells, which produce glucagon, compose
approximately 15% of cells in the islets and are usually located at the periphery, but
may not be found in all islets. The beta cells, which produce insulin, compose
approximately 70% of cells in the islets, and are distributed throughout all islets. The
delta cells are present in all islets and have two subtypes; one produces somatostatin,
the other produces vasoactive intestinal peptide. Both the gamma cells, which produce
pancreatic polypeptide, and the enterochromaffin cells, which produce serotonin, are
sparsely and variably distributed.

Most islet tumors comprise a variety of peptide-producing cells and not all islet tumors
are associated with clinical manifestations of hormone excess. There is poor correlation
between the immunohistochemical profile of islet tumors and clinical disease,7 with the
exception of the adenomas/carcinomas of the insulin secreting beta cells, which are
frequently endocrinologically active and are associated with functional disturbances
related to hypoglycemia.8 Another noteworthy exception is the gastrinoma. Although
islet cell tumors are rarely responsible for the production of polypeptides with gastrin
activity, they have been described in dogs and are associated with the Zollinger-Ellison
syndrome. Gastrin is normally produced in the gastric and duodenal mucosa, where it
stimulates glandular secretion. However, pancreatic gastrinomas producing gastrin

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result in hypergastrinemia, which leads to gastric hyperacidity, mucosal hyperplasia of
the antral region, and gastric and duodenal ulceration (Zollinger-Ellison syndrome). 7

Contributor: Yale University, Section of Comparative Medicine, P.O. Box 208016,

New Haven, CT
http://info.med.yale.edu/compmed/compmed/index.htm

References:
1. Beniashvili DS: An Overview of the World Literature on Spontaneous Tumors in
Nonhuman Primates. J Med Primatol 18:423-437, 1989
2. McClure HM, Chandler FW: A Survey of Pancreatic Lesions in Nonhuman Primates.
Vet Pathol 19(Suppl 7):193-209, 1982
3. Capen, CC: Endocrine glands. In: Tumors in Domestic Animals, ed. Moulton JE, 3rd
ed., pp.616-622. University of California Press, Berkeley, CA, 1990
4. Goad MEP, Fox JG: Neoplasia in ferrets. In: Biology and Diseases of the Ferret, ed.
Fox JG, pp. 281-284. Lea & Febiger, Philadelphia, PA, 1988
5. Carpenter JL, Andrews LK, Holzworth J: Tumors and tumor-like lesions. In: Diseases
of the Cat, vol. 1, pp. 550-552. W. B. Saunders Company, Philadelphia, PA, 1987
6. Frith CH, Sheldon WD: Hyperplasia and neoplasia. In: Endocrine System, eds. Jones
TC, Mohr U, Hunt RD pp. 297-303. Springer-Verlag, New York, NY, 1983
7. Jubb KVF: The pancreas. In: Pathology of Domestic Animals, eds. Jubb KVF,
Kennedy PC, Palmer N, 4th ed., vol. 2, pp. 407-408, 423-424. Academic Press, San
Diego, CA, 1993
8. Capen CC: Tumors of the endocrine glands. In: Tumors in Domestic Animals, ed.
Meuten DJ, 4th ed., pp. 684. Iowa State Press, Ames IA, 2002

SLIDE 32
CONFERENCE 8 / CASE IV – S 3019/03 (AFIP 2936461)

Signalment: 3 week-old, male, Merino sheep (ovis aries), ovine.

History: This newborn lamb was nourished with colostrum only several hours after
birth. The lamb suffered from omphalitis and shortening of the flexor tendons of the
forelimbs. The animal was referred to the Clinic of Pigs and Small Ruminants, School
of Veterinary Medicine, at one day after birth. It developed severe respiratory disease
and was euthanatized at 2.5 weeks of age because of poor prognosis.

Gross Pathology: The lungs failed to collapse and exhibited a generalized reddish
color and a firm consistency. The cranial lobes and ventral portions of the caudal lobes
showed lobular atelectatic areas. A focal fibrinous exudate was present on the
pulmonary pleura. On the cut surface, moderate amounts of a grey viscous fluid were
present extruding from the large airways. The bronchial and mediastinal lymph nodes
were moderately enlarged. The forelimbs could not be extended completely. One
omphalic artery showed subacute, purulent inflammation.

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Laboratory Results: Bacteriologic culture of the lung yielded a low degree of non-
hemolytic streptococci, Bacteroides fragilis, Pseudomonas aeruginosa, enterococci and
Escherichia coli.

Contributor’s Morphologic Diagnosis: Lung: 1. Bronchitis and bronchiolitis,

proliferative and necrotizing, diffuse, subacute, marked, with large basophilic
intranuclear inclusion bodies and cytomegalic cells, Merino sheep, ovine, etiology
consistent with ovine adenovirus infection.
2. Bronchopneumonia, suppurative, multifocal, subacute, moderate.
3. Pneumonia, interstitial, lymphohistiocytic, multifocal, subacute, moderate.

Contributor’s Comment: A large number of bronchiolar and alveolar epithelial cells in

this lung section contained large basophilic intranuclear inclusion bodies measuring up
to 50 µm in diameter often resulting in cytomegaly with marked enlargement of the cell.
In some areas, bronchioles and alveolar spaces are filled with moderate amounts of
neutrophils, cellular debris, and numerous sloughed epithelial cells, with few containing
inclusion bodies. Hyperplasia of type II-pneumocytes and bi- or multinucleated syncytial
cells were found in the alveolar spaces. A severe infiltration of lymphocytes and
macrophages was observed in the interstitium. In addition, interstitial hemorrhage and
epithelial necrosis appeared in areas, where inflammatory infiltration and sloughing of
epithelium was most severe.

Electron microscopical examination revealed intranuclear virus particles arranged in a

paracrystalline array consistent with adenovirus infection.

Adenovirions are naked icosahedral particles measuring 60-90 nm in diameter. The

genome is a single linear molecule of double-stranded DNA of a molecular weight
between 20 and 25 x 106.1 A typical paracrystalline array of virus particles in the
nucleus can be found by transmission electron microscopy.2

Ovine adenoviruses have been classified into 7 serotypes, six of them are included in
the genus Mastadenovirus and one in the new genus Atadenovirus, both are members
of the family Adenoviridae.3

After natural infection of adenoviruses via the oronasal route, primary virus replication
occurs in the respiratory and intestinal epithelium. Lesions in other organs, like nasal
mucosa, lymph nodes, spleen, kidney and liver are due to a viremia appearing as early
as 4 days post infection (p.i.). The virus is shed in the feces, urine and nasal discharge,
starting on day 2-3 p.i.. Direct contact between animals is the most important factor for
virus spread. Permanent shedding of the virus by recovered animals may occur and
contributes to endemics in large farms.4

The serotypes differ in virulence and tissue tropisms. In naturally occurring infections,
mild respiratory and enteric disease or a subclinical course can be observed.4,5 Infection
of other organs rarely leads to clinic or gross pathologic changes. Histopathologically,

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hyperplasia of lymphoid follicles in lymph nodes, lung and intestine, tubular
degeneration in the kidney, and activation of the reticuloendothelial system in liver and
spleen has been described. 4

The respiratory effects are more severe in experimental intranasal or intratracheal

infections. Lesions are characterized by cytomegaly, large intranuclear inclusions and
necrosis of epithelial cells in the upper and lower respiratory tract. This is accompanied
by early infiltration of neutrophils followed by accumulation of mononuclear cells.6
Secondary infection with Mannheimia haemolytica may complicate signs and lesions.

In natural infections, severe lesions are most likely due to immunodeficiency, and are
described in young animals which are deprived of colostral antibodies or which are
exposed to environmental stress or other concurrent diseases.5

AFIP Diagnosis: Lung: Pneumonia, bronchointerstitial, proliferative, subacute, diffuse,

moderate, with multifocal airway epithelial cell and pneumocyte cytomegaly, syncytia,
and large basophilic intranuclear inclusion bodies, Merino sheep, ovine.

Conference Comment: Conference attendees discussed differences among slides,

with some slides containing plant material in the bronchioles and alveoli. All slides have
moderate numbers of both syncytia and multinucleated giant cells, which are not typical
of adenovirus pneumonia. The multinucleated giant cells, primarily of the Langhans’
type, may be due to a secondary bacterial infection, which is not uncommon with
adenoviral pneumonia. However, no organisms were identified with tissue Gram stains.

Adenoviruses have been isolated from most animal species and humans. They have
differing virulences and tissue tropisms, but frequently cause respiratory and enteric
disease. Severe naturally occurring disease is usually seen only in immunodeficient
animals. The most prominent lesion of the pneumotropic strains is a necrotizing and
proliferative bronchiolitis.5

The family Adenoviridae now contains four recognized genera. The genus
Mastadenovirus contains most of the mammalian adenoviruses. The genus
Aviadenovirus contains the group I avian adenoviruses. The genus Siadenovirus
contains the group II avian adenoviruses and frog adenovirus. The genus Atadenovirus
contains the group III avian adenovirus and several mammalian viruses.7 Some
recognized adenoviruses include the following:3,7

Virus Disease______________________________
Mastadenovirus
Canine adenovirus-1 Infectious canine hepatitis
Canine adenovirus-2 Infectious canine tracheobronchitis
Equine adenoviruses-1,2 Mild respiratory disease (except CID foals)
Bovine adenoviruses-1,2,3,9,10 Enzootic pneumonia (one of many agents)

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Ovine adenoviruses-1-6 Mild respiratory and enteric disease
Goat adenoviruses-1,2 Mild respiratory and enteric disease
Porcine adenoviruses-1-5 Enteritis and encephalitis
Guinea pig adenovirus-1 Adenoviral pneumonitis
Mouse adenovirus-1,2 Enteritis and encephalitis
Simian adenoviruses-1-25 Mild respiratory and enteric disease
Human adenoviruses-1-51

Aviadenovirus
Fowl adenoviruses-1-11
Fowl adenovirus-1 Inclusion body hepatitis (chickens)
Fowl adenovirus-4 Hydropericardium syndrome (chickens)
Goose adenoviruses-1-3

Siadenovirus
Frog adenovirus-1
Turkey adenovirus-3 Hemorrhagic enteritis (turkeys)
Pheasant adenovirus-1 Marble spleen disease (pheasants)

Atadenovirus
Ovine adenovirus-7 Mild respiratory and enteric disease
Bovine adenoviruses-4-8 Enzootic pneumonia (one of many agents)
Black tail deer adenovirus-1 Pulmonary edema, hemorrhage, vasculitis
Duck adenovirus-1 Egg drop syndrome (chickens)
Contributor: Hannover School of Veterinary Medicine, Department of Pathology,
Bünteweg 17, D-30559 Hannover, Germany

References:
1. Dinter Z, Morein B: Adenoviridae In: Virus Infections of Vertebrates, Vol. 3, Virus
Infections of Ruminants, ed. Hrozinek MC, pp. 159-160, Elsevier Science Publishers
B.V., Amsterdam, 1990
2. Ghadially FN: Intranuclear viral inclusions and virus-like particles In: Ultrastructural
Pathology of the Cell and Matrix, Vol. 1, 4th ed., pp. 136-145, Butterworth-Heinemann
Publishing, Boston, 1997
3. ICTV db version 3 (2002). The Universal Virus Database of the International
Committee on Taxonomy of Viruses. http://www.ictvdb.rothamsted.ac.uk
4. Belák S: Ovine Adenoviruses In: Virus Infections of Vertebrates, Vol. 3, Virus
Infections of Ruminants, ed. Hrozinek MC, pp. 171-185, Elsevier Science Publishers
B.V., Amsterdam, 1990
5. Dungworth DL: The Respiratory System In: Pathology of Domestic Animals, eds.
Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 2, pp. 626-628, Academic Press, San
Diego, 1993
6. Cutlip RC, Lehmkuhl HD: Pulmonary lesions in lambs experimentally infected with
ovine adenovirus-5 strain RTS-42. Vet Pathol 23: 589-593, 1986
7. McFerran JB, Adair BM: Adenovirus infections. In: Diseases of Poultry, Saif YM, 11th
ed., pp. 214-221, 227-229, 2003

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SLIDE 33
CONFERENCE 9 / CASE I – 03B5515 (AFIP 2941535)

Signalment: An adult American alligator (Alligator mississippiensis).

History: Between September and December of 2003, American alligators from several
alligator farms in Louisiana died suddenly or showed various neurological signs
including circling, ataxia, head and muscle tremor, and head tilt.

Gross Pathology: With the exception of gas-filled, empty gastrointestinal tracts and a
diffuse thin pseudo-membrane on the colonic mucosa, no significant gross findings
were noted.

Laboratory Results: All brain specimens taken from 13 alligators that showed the
clinical signs were positive for West Nile virus (WNV) by RT-PCR. Viral isolation was
successful from 10 out of the 13 brain specimens. Real-time PCR revealed a high viral
load in a fecal sample. Immunohistochemistry for WNV showed strong
immunoreactivity on the sections of brain, liver, pancreas, and small and large
intestines.

Contributor’s Morphologic Diagnosis: Colon: Colitis, heterophilic and

granulomatous, multifocal, acute, moderate.

Contributor’s Comment: The colonic mucosa is multifocally attenuated and fused with
irregular paucity of colonic glands. The lamina propria is expanded with infiltration of
moderate numbers of lymphocytes, plasma cells, and macrophages as well as
heterophils. The inflammation often extends to the submucosa. The individual
glandular epithelial cells are occasionally necrotic with pyknosis. Heterophilic
exocytosis is evident. Some colonic glands are dilated with mucus and exfoliated
degenerate epithelial cells. The submucosal lymphoid follicles are hyperplastic with
heterophilic infiltration. There is a luminal core composed of a large amount of mucus
admixed with exfoliated degenerate epithelial cells, heterophils, various bacteria, and a
small amount of fibrin. Some tissue sections contain severe heterophilic infiltration
surrounding bacterial colonies in the submucosal lymphoid follicle.

Other major microscopic findings were heterophilic meningoencephalitis, necrotizing

and heterophilic hepatitis, heterophilic and histiocytic splenitis, and necrotizing,
heterophilic pancreatitis, and generalized heterophilic lymphoid folliculitis.

Since it first emerged in 1999, West Nile virus (WNV) infection has been established as
a seasonal epidemic in North America. WNV generally circulates between mosquitoes
and birds. The infected birds commonly have a high level viremia and serve as
reservoir hosts. WNV infection has been reported in various species but primarily in

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warm-blooded animals. Recently, epizootic WNV infections in American alligators have
been described from the states of Florida (2002) and Georgia (2003). Although the role
of alligators in the transmission cycle of WNV infection is still largely unknown, there are
concerns that these reptiles may be important regionally because they can develop high
viremia and shed the virus in their feces. Immunohistochemistry for WNV confirmed
that the viral antigens were located within the macrophages, intravascular monocytes,
and intestinal epithelial cells in the colon. It is believed to be that alligators are initially
infected by mosquito bites on the oral mucosa and then epizootics occur rapidly by
orofecal transmission. In this case, the virus was detected from a fecal sample. In the
report of the cases from Florida, the authors speculated that WNV in the horsemeat fed
to the alligators might be the direct source of the infection among the alligators.
However, it is considered unlikely because horses are the dead-end hosts of WNV
infection with low numbers of viruses in the body after a short period of viremia. There
have been 3 human cases of WNV infection, among workers on an alligator farm in
Louisiana.

AFIP Diagnosis: Colon: Colitis, histiocytic and heterophilic, subacute, diffuse,

moderate, American alligator (Alligator mississippiensis), reptile.

Conference Comment: West Nile virus (WNV) was first isolated from a woman in
1937 in the West Nile district of Uganda. Since then it has been reported in western
Asia, the Middle East, Europe, southern Russia, and in 1999 in the United States. WNV
is a member of the genus Flavivirus, family Flaviviridae, and is known to cause
encephalitis in a wide variety of species, including humans, birds, horses, other
mammals and reptiles.3 Natural infections have been reported in bats, a chipmunk, a
skunk, a domestic rabbit, reindeer5 and several species of squirrels.4 Mice and rhesus
monkeys have been infected experimentally. Interestingly, dogs, rabbits, guinea pigs,
hedgehogs, and sheep do not develop encephalitis after experimental inoculation with
WNV.5

As mentioned by the contributor, mosquito vectors transmit the virus among reservoir
bird populations, and susceptible mammalian species are infected incidentally. A wide
variety of native and exotic birds are susceptible, although infection typically does not
cause clinical signs in most birds. However, it does result in unusually high mortality in
crows. Gross lesions in birds include meningeal hemorrhage, multifocal pale
myocardial foci, splenomegaly, mucosal hemorrhage in the small intestine, and white
foci in the kidneys. In birds, the most severe histological lesion is hemorrhage in the
cerebellar folia, with degeneration and necrosis of the cerebellar molecular layer and
Purkinje cells.4 Other lesions include lymphoplasmacytic meningoencephalitis,
necrotizing myocarditis, and lymphoplasmacytic enterocolitis. Subacute inflammation
may be seen in the spleen, kidneys, liver, adrenal glands, or pancreas.6 In horses,
WNV exhibits a pronounced, if not exclusive, CNS tropism. As with WNV infection in
humans and squirrels, the brainstem is the most severely affected area in horses.4
Horses typically have polioencephalomyelitis, with prevalent involvement of the lower

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brain stem and ventral horns of the thoracolumbar spinal cord.7 WNV infections in
humans are usually mild, with affected individuals exhibiting non-specific flu-like
symptoms. Less than 15% of infected humans develop more severe forms of the
disease, such as meningoencephalitis, hepatitis, pancreatitis, or myocarditis. Fatalities
are more common in humans over the age of 50 and often a result of severe central
nervous system disease.8

Contributor: Louisiana State University, School of Veterinary Medicine, Department of

Pathobiological Sciences, Baton Rouge, LA.
References:
1. Miller DL, Mauel MJ, Baldwin C, et al: West Nile virus in farmed alligators. Emerging
Infectious Diseases 9(7): 794-889, 2003
2. Steinman A, Banet-Noach C, Tal S, et al: West Nile virus infection in crocodiles.
Emerging Infectious Diseases 9(7): 887-889, 2003
3. Storts RW, Montgomery DL: The nervous system. In: Thompson’s Special Veterinary
Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 433-434. Mosby, St.
Louis, PA, 2001
4. Heinz-Taheny KM, Andrews JJ, Kinsel MJ et al.: West Nile virus infection in free-
ranging squirrels in Illinois. J Vet Diag Invest 16:186-190, 2004
5. Palmer MV, Stoffregen WC, Rogers DG et al.: West Nile virus infection in reindeer
(Rangifer tarandus). J Vet Diag Invest 16:219-222, 2004

6. Steele KE, Linn MJ, Schoepp RJ, Domar N, Geisbert TW, et al: Pathology of fatal
West Nile virus infections in native and exotic birds during the 1999 outbreak in New
York City, New York. Vet Pathol 37:208-224, 2000
7. Catile C, Del Piero F, Di Guardo G, Arispici A: Pathologic and immunohistochemical
findings in naturally occurring West Nile virus infection in horses. Vet Pathol 38:414-
421, 2001
8. Ritchie BW: West Nile virus—a recent immigrant to the United States. Compendium
on Continuing Education for Practicing Veterinarians 22:576-587, 2000

SLIDE 34
CONFERENCE 9 / CASE II – SA787-04 (AFIP 2937490)

Signalment: Twenty-day-old Ross broiler chicken (Gallus domesticus).

History: A large number (exact number unknown) of chickens died with symptoms of
paralysis and incoordination. Parent flock young (less than 30 months old).

Gross Pathology: Negative, except for dehydration and nephrosis.

Laboratory Results: Serum from the affected chicks as well as from the parent flock
tested negative for the presence of antibodies against Avian Encephalomyelitis virus
(AEV) using the Enzyme Linked Immunosorbent Assay (ELISA) test.

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Contributor’s Morphologic Diagnoses: Multiple tissues presented for examination,
including cerebrum, cerebellum, medulla oblongata (some sections), proventriculus,
ventriculus (some sections) and pancreas.
1. Brain: Encephalitis, lymphocytic, subacute, multifocal to coalescing, severe with
neuronal degeneration and necrosis, gliosis and perivascular lymphocytic infiltration.
Neuronal degeneration and necrosis are characterized by chromatolysis and
eosinophilic discoloration of some nuclei, which was particularly severe in the Purkinje
cells of the cerebellum.
2. Proventriculus and ventriculus (some sections): Lymphocytic infiltration, multifocal,
mucosal, submucosal and intermuscular, mild to moderate.
3. Pancreas: Lymphocytic infiltration, interstitial, multifocal, mild to moderate.

Contributor’s Comment: The history of a high morbidity and a high mortality in young
20-day-old chickens, with neurological signs of paralysis and incoordination as well as
severe neural lesions characterized by lymphocytic encephalitis with marked neuronal
degeneration and necrosis, are consistent with the lesions described in the central
nervous system of birds due to avian encephalomyelitis virus (AEV), a Picornavirus.
The lymphocytic infiltrations in the proventriculus, ventriculus and pancreas provide
further support for the diagnosis.1

The high mortality and severity of the lesions was probably the result of a high
susceptibility in the affected chicks and their parents as supported by the negative
ELISA test results.1

The most sensitive method, and the method of choice, for the detection of AEV appears
to be the inoculation of serologically negative chickens at 2 weeks of age with infected
brain tissue culture cells. These chickens should be observed for typical symptoms and
positive serological reactions with the ELISA or immunodiffusion (ID) tests. In addition,
brain, proventriculus and pancreas can be examined by indirect immunofluorescence
and for the presence of typical histopathological changes.1

Picornavirus particles in crystalline array of 22-25 nm in diameter have been identified

within the cytoplasm of Purkinje cells from infected chickens.2,3 The AEV, a single-
stranded RNA virus, has the capacity to induce nuclear DNA fragmentation with
apoptosis in embryonal nerve cells through its structural VP3 –and non-structural 2C
proteins.4,5

Avian encephalomyelitis (AE) should be differentiated from a number of diseases with

similar clinical signs in young chicks such as Newcastle disease, equine
encephalomyelitis virus infection, as well as from certain nutritional imbalances such as
rickets, encephalomalacia and riboflavin deficiency. Newcastle disease in 1-3 week-old
chicks can be distinguished from AE by peripheral chromatolysis in neurons; in contrast,
AE results in central chromatolysis within neurons and multifocal infiltrations of
lymphocytes within the proventriculus and the pancreas.1 Rickets, encephalomalacia

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and riboflavin deficiency cause very dissimilar lesions which cannot be confused with
AE.1

Although AE occurs almost worldwide, the virus has a limited host range, and the
incidence is very low due to vaccination and relatively high levels of immunity in
commercial chicken and turkey production systems. Fatal natural infections have been
reported in pheasants and quail1 and also recently in pigeons in Turkey with typical
symptoms and lesions.6 Experimental infections of guinea fowl and ducklings have also
been reported.1

Avian encephalomyelitis virus is most closely related to the human hepatitis A virus.7 All
isolates of AEV are serologically similar, but two distinct pathotypes of the virus exist.
The natural field strains are enterotropic, and infection occurs via the oral route with
fecal excretion of the virus. These strains have a low pathogenicity and only cause
neurological signs in vertical or horizontal infection of susceptible chicks at a young age.
Embryo-adapted strains represent the second group of pathotypes which are highly
neurotropic by intracerebral inoculation and parenteral infection. These require high
doses for oral infection and do not spread horizontally.1

AFIP Diagnoses: 1. Brain: Encephalitis, lymphoplasmacytic, multifocal, mild, with

gliosis, and neuronal degeneration and necrosis, domestic chicken, avian.
2. Pancreas: Lymphoid infiltrates, multifocal, moderate.

Conference Comment: Avian encephalomyelitis virus (AEV) is a non-enveloped

icosahedral single-stranded RNA virus of the Picornaviridae family and is pathogenic to
young chickens, pheasants, quails, and turkeys, resulting in reduced hatching, ataxia,
and tremors in 1-7 day old chicks.5 Transmission may be vertical, fecal-oral, or via
fomites. The only gross lesions associated with avian encephalomyelitis (AE) are areas
of pallor in the tunica muscularis of the ventriculus. Histological lesions occur in the
central nervous system (not the peripheral nervous system) and some viscera. Lesions
in the CNS include diffuse nonsuppurative encephalomyelitis, Purkinje cell degeneration
and gliosis in the molecular layer, and central chromatolysis, especially of large neurons
in the midbrain. Dense aggregates of lymphocytes in the muscular wall of the
proventriculus are pathognomic.1 Similar lesions occur in the ventriculus muscle,
myocardium, and pancreas. Although lymphoid nodules are normally found in the
pancreas, in animals with AE, these nodules will be increased in number two to three
fold.1 Some common Picornaviruses include the following:8

Virus Primary species affected Disease__________

Aphthovirus
FMD viruses Ruminants, swine Foot-and-mouth disease

Enterovirus

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Swine vesicular disease virus Swine Swine vesicular disease
Porcine enterovirus-1 Swine Polioencephalomyelitis
Avian enteroviruses Chickens Avian encephalomyelitis
Ducks , turkeys Hepatitis
Coxsackieviruses Humans Aseptic meningitis,
myocarditis, poliomyelitis

Cardiovirus
Encephalomyocarditis virus Swine, elephants Encephalomyocarditis
Theiler’s murine Mice Murine
encephalomyelitis virus polioencephalomyelitis

Rhinovirus
Bovine rhinovirus Cattle Mild rhinitis
Human rhinovirus Humans Common cold

Hepatovirus
Simian hepatitis A virus Monkeys Hepatitis
Human hepatitis A virus Humans Hepatitis

Contributor: Pathology Section, Department of Paraclinical Sciences, Faculty of

Veterinary Sciences, University of Pretoria, Priv. Bag X4, Onderstepoort 0110, Republic
of South Africa.
http://www.up.ac.za/academic/veterinary

References:
1. Calnek BW: Avian encephalomyelitis. In: Diseases of Poultry, eds. Saif YM, Barnes
HJ, Glisson JR, Fadly AM, McDougald LR, Swaine DE, 11th ed., pp. 271-282. Iowa
State Press, Ames, IA, 2003
2. Cheville NF: The influence of thymic and bursal lymphoid systems in the
pathogenesis of avian encephalomyelitis. Am J of Pathol 58:105-125, 1970
3. Hishida N, Yoshiharu O, Kotani T, Horiuchi T: Morphological changes of neurons in
experimental avian encephalomyelitis. Jpn J of Vet Sci 48(1):169-172, 1986
4. Liu J, Wei T, Kwang J: Avian encephalomyelitis virus induces apoptosis via major
structural protein VP3. Virology, 300:39-49, 2002
5. Liu J, Wei T, Kwang J: Avian encephalomyelitis virus nonstructural protein 2C
induces apoptosis by activating cytochrome c/caspase-9 pathway. Virology 318:169-
182, 2004
6. Toplu N, Alcigir G: Avian encephalomyelitis in naturally infected pigeons in Turkey.
Avian Pathol, 33(3):381-6, 2004
7. Marvil P, Knowles NJ, Mockett PB, David T, Brown K, Cavanagh D: Avian
encephalomyelitis virus is a picornavirus and is most closely related to hepatitis A virus.
J of Gen Virol 80:653-662, 1999
8. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ: Picornaviridae. In: Veterinary
Virology, 3rd ed., pp. 519. Academic Press, San Diego, CA, 1999

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SLIDE 35
CONFERENCE 9 / CASE III – PA 4121 (AFIP 2943312)

Signalment: 8 year old, male castrate Pug dog.

History: This dog presented with a progressive history of weight loss and poor doing.
Diarrhea developed later in the clinical course. Despite aggressive clinical supportive
measures, the animal continued to deteriorate and was euthanized.

Gross Pathology: The kidneys were bilaterally enlarged, pale, firm and somewhat
gritty on cut surface. No additional gross findings were specified.

Laboratory Results: Proteinuria was reported without additional

specification/quantification.

Contributor’s Morphologic Diagnoses: 1. Glomerular amyloidosis, global and

diffuse, marked, with additional mild, patchy, interstitial (peri-tubular) amyloid deposits.
2. Tubular proteinosis, patchy to diffuse, moderate.
3. Interstitial nephritis, lymphoplasmacytic, patchy, mild.
4. Interstitial fibrosis, patchy, mild-moderate.
5. Tubular mineralization, multifocal, mild-moderate.
6. Vascular thrombosis, multifocal (some sections).

Contributor’s Comment: The pale, amorphous, eosinophilic material effacing the

architecture of most glomeruli was confirmed as amyloid via strong positivity under
fluorescence with Thioflavin T staining.

Amyloidosis is well described in the canine. Although familial renal amyloidosis is

reported in several breeds (Shar Pei, Beagle), concurrent neoplasia or chronic
inflammatory disease is detected in over half of affected animals. Dogs often present
with progressive protein-losing nephropathy and frequently demonstrate the nephrotic
syndrome, including ascites, peripheral edema and hypercholesterolemia. A
thromboembolic phenomenon is seen in up to 40% of affected dogs, and this lesion is
seen in some of the submitted sections. Amyloid was not noted in a limited number of
other organs submitted with this case, nor was an underlying chronic inflammatory or
neoplastic disorder recognized.

The primary clinical differentials in dogs that present with renal associated protein loss
include primary congenital glomerulopathy, described in the Samoyed, Bull Terrier and
English Cocker Spaniel, immune-mediated glomerulonephritis, also known to have
breed predilection, including Bernese Mountain Dogs and Soft-coated Wheaten Terriers
and occasional functional renal tubular transport disorders.

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The prognosis for dogs with renal amyloidosis is quite guarded, and most dogs die or
are euthanized shortly after diagnosis.

AFIP Diagnoses: 1. Kidney: Amyloidosis, glomerular, diffuse, global, severe and

interstitial, multifocal, mild, with tubular proteinosis, Pug, canine.
2. Kidney: Nephritis, interstitial, lymphoplasmacytic, chronic, multifocal, moderate.
3. Kidney, large pelvic vein: Thrombus, organizing, with mineralization.

Conference Comment: Amyloid, an insoluble fibrillar protein with a beta-pleated sheet

conformation, is deposited between cells in various tissues. With progressive
accumulation it encroaches on and produces pressure atrophy of adjacent cells. On
standard tissue sections stained with H&E, amyloid appears as an amorphous,
eosinophilic, hyaline, extracellular substance. It is congophilic, with apple green
birefringence when polarized.5

There are three main types of amyloid protein: amyloid light chain (AL); amyloid-
associated (AA); and beta-amyloid (Aß). AL protein is derived from plasma cells and
contains immunoglobulin light chains, which may be complete immunoglobulin light
chains, the NH2-terminal fragments, or both. It is commonly seen in association with
immunocyte dyscrasias, particularly multiple myeloma. AA protein is derived from
serum amyloid-associated (SAA) protein that is synthesized in the liver, and is
associated with chronic inflammatory conditions. Aß amyloid is found in the cerebral
plaques of human patients with Alzheimer disease. Amyloidosis, a heterogeneous
group of disease processes that result in the deposition of amyloid, may be classified as
localized or systemic (generalized). Examples of localized amyloidosis include cerebral
plaques in Alzheimer disease, islet associated amyloid polypeptide (IAPP) with type II
diabetes, and some prion diseases. Systemic or generalized amyloidosis can be further
classified as primary or secondary. Primary amyloidosis is associated with immunocyte
dyscrasias, while secondary amyloidosis occurs as a complication of an underlying
chronic inflammatory disease. 5

Regardless of the inciting cause or type of amyloid, amyloidosis results from the
abnormal folding of proteins, which are deposited as fibrils in extracellular tissue and
disrupt normal tissue function. In this case, glomerular filtration was almost certainly
severely compromised and resulted in proteinuria. Glomerular amyloid deposition
initially causes selective loss of albumin, but as the disease progresses, large protein
molecules (globulins) also may be lost. If protein loss is severe, then hypoproteinemia
and edema develop.6 As a result of the protein-losing nephropathy, animals can
become deficient in antithrombin III, resulting in an increased tendency for thrombosis,7
which may have been the cause of the thrombus formation in this case.

Contributor: University of Pittsburgh, Division of Laboratory Animal Resources, S-

1040 BioMedical Science Tower, 3500 Terrace Street, Pittsburgh, PA
http://www.pitt.edu/

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References:
1. DiBartola SP, Tarr MJ, Parker AT, Powers JD: Clinicopathologic findings in dogs with
renal amyloidosis: 59 cases (1976-1986). JAVMA 195(3):358-364, 1989
2. DiBartola SP, Tarr MJ, Webb DM, Giger U: Familial renal amyloidosis in Chinese
Shar Pei dogs. JAVMA 197(4):483-48, 1990
3. Bowles MH, Mosier DA: Renal amyloidosis in a family of Beagles. JAVMA
201(4):569-574, 1992
4. Lees GE: Congenital renal diseases. In: The Veterinary Clinics of North America
Small Animal Practice Volume 26, No. 6, ed., Polizin DJ, pp. 1379-1389.
5. Abbas AK: Diseases of immunity. In: Robbins and Cotran Pathologic Basis of
Disease, 7th ed., pp. 258-264. Elsevier Saunders, Philadelphia, PA, 2005
6. Latimer KS, Mahaffey EA, Prasse KW: Case studies. In: Duncan and Prasse’s
Veterinary Laboratory Medicine, Clinical Pathology, 4th ed., pp.386-388. Iowa State
Press, Ames, IA, 2003
7. Slauson DO: Disturbances of blood flow and circulation. In: Mechanisms of Disease,
A Textbook of Comparative General Pathology, eds. Slauson DO, Cooper BJ, 3rd ed.,
pp. 98. Mosby, St. Louis, MO, 2002

SLIDE 36
CONFERENCE 9 / CASE IV – KL-8 M3 (AFIP 2936433)

Signalment: 2-year-old, male North American opossum (Didelphis virginiana).

History: Wild opossum rescued by a rehabilitation program. The opossum presented

with dyspnea and pleural and abdominal effusions. He died post-thoracocentesis.

Gross Pathology: Heart, lungs, adrenal gland and portions of kidney and liver were
submitted in 10% neutral buffered formalin. Gross lesions were restricted to the lungs
and liver. All lung lobes were collapsed, firm and rubbery. On cut section, there were
multifocal to coalescing, pale tan to reddish-brown, 3 mm diameter, perivascular and
peribronchial nodules. On cut sections of the liver, an enhanced reticular pattern was
apparent, somewhat resembling a nutmeg liver.

Contributor’s Morphologic Diagnosis: Lung, granulomatous pneumonia and

catarrhal bronchitis, chronic, multifocal, marked with intralesional Didelphostrongylus
hayesi, pulmonary smooth muscle hyperplasia and widespread atelectasis.

Contributor’s Comment: The majority of the pulmonary parenchyma is atelectatic.

Within alveoli, bronchioles and bronchi are numerous transverse and longitudinal
sections of nematodes characterized by a cuticle, coelom, gastrointestinal tract with
intraepithelial black pigment, and reproductive tract with ova, sperm and developing
larvae. Associated with scattered free larvae in alveolar spaces are variable numbers of
multinucleated giant cells, lymphocytes, plasma cells and fewer eosinophils. Numerous

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bronchial lumina contain large amounts of mucus admixed with free larvae. There are
increased goblet cells distended with mucus lining most bronchial lumina. These
bronchi are also associated with increased peribronchial mucous glands and prominent
lymphoid nodules. Marked smooth muscle hyperplasia involving terminal bronchioles,
alveolar ducts and less frequently, arterioles is apparent. Interstitial spaces around
blood vessels are distended by proteinaceous fluid (edema). There are multiple foci of
heterotopic bone in the pulmonary parenchyma, and mesothelial hyperplasia is present
on all pleural surfaces. Some sections contain subpleural aggregates of lipid-laden
macrophages with acicular (cholesterol) clefts. Additional histologic findings included
chronic, multifocal, marked centrilobular hepatocellular degeneration and coagulation
necrosis with fibrosis.

Didelphostrongylus hayesi, named in honor of Professor Frank A. Hayes who made

great contributions to the study of North American wildlife diseases, is a common
metastrongyloid lungworm in opossums.1 In one retrospective study, pulmonary
parasites were documented in 18/27 (67%) opossum necropsies with 13/18 (72%)
being D. hayesi.2 In another, D. hayesi larvae were found in 13/20 (65%) resident and
10/13 (77%) newly arrived opossums in an opossum care program.3 Finally,

D. hayesi was documented in 11/20 (55%) opossum necropsies, and contributed

to the cause of death in 8/11 (73%) of these cases.4 The parasite has an indirect
lifecycle similar to other metastrongyloid nematodes, with the intermediate hosts being
the terrestrial snails Triodopsis albolabris and Mesodon perigraptus.1

Histopathologic findings reported with D. hayesi infection are as seen in this case.2-6
Pulmonary inflammation is associated with free larvae rather than intact adult
nematodes. A feature of D. hayesi infected lungs is bronchiolar and alveolar ductular
smooth muscle hyperplasia, which is reminiscent of the marked pulmonary arteriolar
smooth muscle hyperplasia seen in Aelurostrongylus abstrusus infected cats. The
hepatic lesions in this opossum and reported in others were consistent with right-sided
heart failure secondary to verminous pneumonia-induced pulmonary hypertension.4
Foci of heterotopic bone have also been reported in the lungs of unparasitized
opossums and are considered to be incidental findings.5

Although a complete blood cell count and differential were not performed in this case,
opossums with D. hayesi display a regenerative erythrogram characterized by
nucleated red blood cells and polychromasia.4,6 Similar findings are reported in cats
with A. abstrusus. However, it is unknown whether this is due to hypoxia or a direct
stimulus from the lungworms.6 In spite of the heavy parasitic burden in these
opossums, a peripheral eosinophilia has not been reported.4

Evidence of endogenous lipid pneumonia was present in some slides. This lesion was
present in 19/27 (70%) opossums from Louisiana, with 13/19 (68%) of these animals
having pulmonary parasites.2 In addition to D. hayesi, the pulmonary parasites included
Capillaria sp. and Besnoitia darlingi. It is hypothesized that these parasites induce

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pulmonary irritation which results in type II pneumocyte hyperplasia and surfactant
overproduction with subsequent accumulation in alveolar macrophages.

AFIP Diagnosis: Lung: Bronchopneumonia, histiocytic, multifocal, mild, with many

adult and larval metastrongyles, bronchiolar mucus cell hyperplasia with abundant
catarrhal exudate, atelectasis, and bronchiolar and arteriolar smooth muscle
hypertrophy, North American opossum (Didelphis virginiana), marsupial.

Conference Comment: The contributor provides a thorough overview of

Didelphostrongylus hayesi infection in opossum. Conference attendees discussed lung
parasites in other species as listed below:7

Species/Parasite Location
Lesion/Comment________
Canine
Filaroides osleri Tracheal bifurcation Luminal nodules
Filaroides hirthi Bronchioles, alveoli Subpleural nodules
Paragonimus kellicotti Bronchioles Subpleural nodules
Angiostrongylus vasorum Vasculature Chronic arteritis
Dirofilaria immitis Vasculature Chronic arteritis
Crenosoma vulpis Sm. bronchi/bronchioles Catarrhal bronchitis

Feline
Aelurostrongylus abstrusus Bronchioles, alveoli Subpleural nodules
Paragonimus kellicotti Bronchioles Subpleural nodules
Dirofilaria immitis Vasculature Chronic arteritis
Syngamus laryngeus Larynx “gapeworm”

Equine
Dictyocaulus arnfieldi Bronchi Eosinophilic bronchitis
Parascaris equorum Interstitium Migrating larval stages

Bovine
Syngamus laryngeus Larynx Asia and S. America
Dictyocaulus viviparous Intrapulmonary bronchi Bronchitis, BALT
hyperplasia, edema
Ascaris suum Bronchioles, alveoli Interstitial pneumonia

Ovine/caprine
Dictyocaulus filaria Small bronchi Catarrhal bronchitis;
BALT hyperplasia

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Muellerius capillaries Subpleural alveoli Pulmonary nodules
Protostrongylus rufescens Bronchioles Pulmonary nodules

Porcine
Metastrongylus apri Bronchioles Catarrhal bronchitis
Ascaris suum Subpleural alveoli Subpleural hemorrhage

Contributor: Weill Medical College of Cornell University / Memorial Sloan-Kettering

Cancer Center / Rockefeller University, 1300 York Avenue, Box 40, RARC, Room C-
701, New York, NY
http://www.mskcc.org; http://www.med.cornell.edu/; http://www.rockefeller.edu

References:
1. Prestwood AK: Didelphostrongylus hayesi gen. et sp. n. (Metastrongyloidea:
Filaroididae) from the opossum, Didelphis marsupialis. J Parasitol 62(2):272-275, 1976
2. Brown CC: Endogenous lipid pneumonia in opossums from Louisiana. J Wildl Dis
24(2):214-219, 1988
3. Baker DG, Cook LF, Johnson EM, Lamberski N: Prevalence, acquisition, and
treatment of Didelphostrongylus hayesi (Nematoda: Metastrongyloidea) infection in
opossums (Didelphis virginiana). J Zoo Wildl Med 26(3):403-408, 1995
4. Lamberski N, Reader JR, Cook LF, Johnson EM, Baker DG, Lowenstine LJ: A
retrospective study of 11 cases of lungworm (Didelphostrongylus hayesi) infection in
opossums (Didelphis virginiana). J Zoo Wildl Med 33(2):151-156, 2002
5. Prestwood AK, Nettles VF, Farrell RF: Pathologic manifestations of
experimentally and naturally acquired lungworm infections in opossums. Am J Vet Res
38(4):529-532, 1977
6. Duncan RB Jr, Reinemeyer CR, Funk RS: Fatal lungworm infection in an
opossum. J Wildl Dis 25(2):266-269, 1989
7. Lopez A: Respiratory system, thoracic cavity, and pleura. In: Thompson’s Special
Veterinary Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 145,
168, 173-174, 177, 182, 186, 187. Mosby, St. Louis, PA, 2001

SLIDE 37
CONFERENCE 10 / CASE I – 03-1893 (AFIP 2948659)

Signalment: 6-7 month gestation, female, Holstein, bovine fetus.

History: Normal pregnancy. Cow was euthanatized because of luxated proximal left
femur.

Gross Pathology: None in the fetus.

Contributor’s Morphologic Diagnosis: Normal plexiform cortical bone, radius and

ulna.

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Contributor’s Comment: Normal appositional growth (widening) of cortical bone can
be circumferential lamellar, simple primary osteonal, plexiform, and saltatory.1 Laminar
bone is composed of lamellae deposited circumferentially in layers parallel to the
convex surface of the cortex. Primary osteonal bone consists of anastomosing vascular
Haversian canals surrounded by concentric lamellae forming Haversian systems.
Plexiform bone is formed by multiple relatively widely spaced lamina of periosteal
woven. The spaces between these woven laminae subsequently fill in (compact) with
lamellar bone. In the horse, the compaction can form osteons (saltatory formation) with
the orientation of the osteon and its vessels being perpendicular to the long axis of the
bone.1 In calves, the compaction (and blood vessels) are oriented parallel with the
convex surface of the bone2 without formation of osteons. Many species exhibit multiple
patterns of primary periosteal cortical bone, dependent upon regional rate of growth,
pattern of vascularization, and functional requirements.3,4

AFIP Diagnosis: Bone, radius and ulna (per contributor): Normal fetal bone, Holstein,
bovine.

Conference Comment: There are structural variations in the microscopic organization

of bone tissue in different animal species depending on the growth and remodeling
processes for that species. Bones undergo change in size and shape as well as
remodeling of the internal architecture during normal growth and due to changes in
functional stresses throughout life. These processes result in bone deposition,
resorption, and remodeling, which ultimately affect the amount of bone, the amount of
mineralization, and type of bone tissue present within a given bone.1

Fetal bone development occurs in two ways, both of which involve the replacement of
primitive collagenous supporting tissue by bone. The long bones, vertebrae, pelvis, and
bones of the base of the skull are formed through a continuously growing cartilage
model that is progressively replaced by bone (endochondral ossification). The bones of
the vault of the skull, the maxilla and most of the mandible are formed by the deposition
of bone within primitive mesenchymal tissue (intramembranous ossification).
Regardless of the method of ossification, initially the bone that is formed is immature, or
woven bone. The developing bone is then extensively remodeled by resorption and
appositional growth to form mature, or lamellar bone.5

Contributor: The Ohio State University, College of Veterinary Medicine,

Department of Veterinary Biosciences, 1925 Coffey Rd., Columbus, Ohio
http://www.vet.ohio-state.edu/docs/biosci/index.html

References:
1. Stover, S.M., Pool, R.R., Martin R.B., Morgan J.P. “Histological Features of the
Dorsal Cortex of the Third Metacarpal Bone Mid-Diaphysis during Postnatal Growth in
Thoroughbred Horses” J. Anat. 181(Pt 3):455-69, 1992

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2. Mori R, Kodaka T., Sano T., Yamagishi N., Asari M., Naito Y. “Comparative
Histology of the Laminar Bone between Young Calves and Foals” Cells Tissues Organs
175(1):43-50, 2003
3. Enlow, DH: A Study of the Post-Natal Growth and Remodeling of Bone. Am J Anat
110:79-101, 1962
4. Enlow, D.H.: Functions of Haversian System. Am J Anat 110:269-305, 1962
5. Young B, Heath JW: Skeletal tissues. In: Wheater’s Functional Histology, A Text and
Color Atlas, 4th ed., pp. 183. Churchill Livingstone, Philadelphia, PA, 2000

SLIDE 38
CONFERENCE 10 / CASE II – X7764-6 or X7764-7 (AFIP 2946673)

Signalment: A juvenile female Black-crowned night heron (Nycticorax nycticorax).

History: Found dead. This was an individual among a wild flock that nests around the
bird house at the National Zoo.

Gross Pathology: Poor nutritional condition; hepatomegaly with miliary hepatitis;

splenomegaly; parasitism, roundworm, ventriculus and small intestine; arthritis,
tibiotarsal-metatarsal joint, chronic.

Laboratory Results:
West Nile virus PCR – negative
Liver culture – Salmonella typhimurium
Liver cytology – Marked, subacute inflammation with intracellular, gram-negative
coccobacilli
Heart blood culture – coagulase-negative Staphylococcus sp.

Contributor’s Morphologic Diagnosis: Bone, tibiotarsus: Osteomyelitis,

granulomatous, with trabecular resorption, periosteal new bone formation, marrow
cavity bone and cartilage sequestrum, and intralesional coccobacilli, Black-crowned
night heron (Nycticorax nycticorax), Ciconiiformes.

Contributor’s Comment: Salmonellosis is a worldwide disease affecting a wide range

of species from humans to birds to reptiles to insects. This bacterium used to be
employed in the control of rodents, but the practice was banned in the US when the
public health risk was realized. Clinical presentations most commonly linked to
Salmonella include enteritis, colitis, abortion, and septicemia. Additionally, carrier states
can be established with Salmonella sp. in a non-clinical or subclinical host. In our
experience, infection in these hosts cannot be eliminated through antibiotic
administration, and may only serve to create resistant strains of the bacterium.

In birds, salmonellosis is usually fatal in the animal’s first 2-3 weeks of life. Older birds
may survive the infection to become carriers of the pathogen. The two most common

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Salmonella diseases in domestic fowl are Pullorum Disease and Fowl Typhoid, caused
by S. pullorum and S. gallinarum respectively. In either disease, young birds may have
splenomegaly, hepatomegaly, and caseous material within the yolk sac. Joints,
especially the hock, may become enlarged and filled with viscous, yellow fluid. A
common gross lesion in older, female birds is misshapen, firm, and discolored ovarian
follicles. Pericarditis and hydropericardium may also be seen. Microscopically,
inflammation is initially heterophilic and lymphocytic, and progresses to necrotizing and
granulomatous lesions in target organs.
Wild waterfowl are less susceptible to Salmonella-induced disease. Pullorum Disease
and Fowl Typhoid are only rarely reported in waterfowl. The most common Salmonella
strain isolated from wild waterfowl is S. typhimurium, the strain isolated from this heron.
In this case, infection was of utmost concern, as potential shedders of the organism live
in and around the wetlands exhibit and defecate in close proximity to collection animals
and the public.

AFIP Diagnoses: 1. Bone, tibiotarsus (per contributor): Osteomyelitis, granulomatous

and heterophilic, multifocal, marked, with medullary bone and cartilage sequestrum,
trabecular resorption, periosteal new bone formation, and colonies of coccobacilli,
black-crowned night heron (Nycticorax nycticorax), avian.
2. Tendons, leg: Tenosynovitis, chronic-active, proliferative, multifocal, minimal to
moderate.

Conference Comment: There are two species of Salmonella: S. enterica, which is

very common and comprised of over 2000 serotypes; and S. bongori, comprised of 10
serotypes, all of which are rare. The Kauffmann-White classification system for
serotypes is based on differences among somatic (O), capsular (Vi), and flagellar (H)
antigens. Each serotype is named by where it was first isolated or by the clinical
syndrome it produces in a particular host. In conventional terminology, the serotypes
are treated as species. Based on host specificity, serotypes can be divided into two
groups: those that are highly adapted to a specific host species, and those that affect a
wide range of species. Most serotypes fall into the latter category, although there can
be marked differences in virulence of a serotype in various hosts.3

Some common and important animal diseases caused by Salmonella sp. include the
following:4,5

Species Salmonella sp.

Disease/Lesions__________________________
Porcine S. choleraesuis Septemia (piglets) (cyanosis of the skin,
turkey-egg kidney); necroulcerative

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 enterocolitis (button ulcers); hepatic
paratyphoid nodules
S. typhimurium Enterocolitis; rectal stricture
S. typhisuis Ulcerative enterocolitis; caseous tonsillitis
and lymphadenitis

Equine S. typhimurium Septemia (foals); enterocolitis (older horses)

S. abortus-equi Abortions (6-9 months of gestation); orchitis

Bovine S. dublin Fibrinous cholecystitis

S. typhimurium Septemia (calves); fibrinonecrotic enteritis
and necrosis of the Peyer’s patches

Ovine S. abortus-ovis Abortion

S. typhimurium Septemia (lambs); fibrinonecrotic enteritis
S. dublin Fibrinous cholecystitis
S. enteritidis Enterocolitis

Avian S. typhimurium Septemia

S. pullorum Pullorum disease: necrotizing typhilitis and
necrotic foci in the liver, lung, myocardium,
gizzard (chicks); oophoritis (adults)
S. gallinarum Fowl-typhoid: catarrhal enteritis and necrotic
foci in the liver, myocardium, intestine,
pancreas

Carnivores S. dublin Rare; associated with septemia in puppies

S. typhimurium Rare; gastroenteritis in immunosuppressed
kittens

Lab animals S. typhimurium Uncommon; mice, gerbils, hamsters, rabbits,

guinea pigs; non-human primates
S. enteritidis Uncommon; rats, hamsters, rabbits, guinea
pigs; non-human primates

Contributor: Smithsonian National Zoological Park, Department of Pathology, 3001

Connecticut Ave, NW, Washington, DC
http://nationalzoo.si.edu/default.cfm

References:
1. Gast RK: Salmonella infections. In: Diseases of Poultry, ed. Saif YM, 11th ed., pp.
567-599. Iowa State Press, Ames, IA, 2003
2. Wobeser GA: Diseases of Wild Waterfowl, 2nd ed., pp. 75-78. Plenum Press, New
York, NY, 1997

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3. Morner T: Salmonellosis. In: Infectious Diseases of Wild Mammals, eds. Williams
ES, Barker IK, 3rd ed., pp. 505-507. Iowa State Press, Ames, IA, 2001
4. Barker IK, Van Dreumel AA, Palmer N: The alimentary system. In: Pathology of
th
Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4 ed., vol. 2, pp. 213-227.
Academic Press, San Diego, CA, 1993
5. Percy DH, Barthold SW: Pathology of Laboratory Rodents and Rabbits, 2nd ed., pp.
53-54, 123-124, 180-181, 199-201, 218-219. Iowa State Press, Ames IA, 2001

SLIDE 39
CONFERENCE 10 / CASE III – N2004-0345 (AFIP 2942032)

Signalment: 10 day-old, male, boat-billed heron (Cochlearius cochlearius).

History: This animal was the first offspring produced from a group of boat-billed
herons, and presented after falling from the nest. On physical exam, all long bones
were very flexible. Radiographs revealed multiple fractures of the long bones with very
poor bone density. Due to a poor prognosis, humane euthanasia was elected.

Gross Pathology: Gross findings confirmed moderate to severe flexibility of all bones,
with multiple folding fractures. Additionally, there was a 0.5 cm diameter focus of
subdural hemorrhage within the right frontal region of the cerebrum (consistent with
trauma associated with the recent fall).

Laboratory Results:
Serum calcium: 6.0 MG/DL
Serum phosphorus: 10.5 MG/DL
Serum 25-hydroxy Vitamin D: 36 nmol/L*
*Laboratory comment: Reference values for this test are not defined for this species in
our laboratory. However, this concentration is similar to values seen in chickens,
turkeys, and conures.
Serum Vitamin A: Retinol = 610 ng/ml**
**Laboratory comment: We do not have serum vitamin A reference ranges for boat-
billed herons. For comparison, in adult mammals, adequate serum vitamin A (retinol)
concentrations range between 175 to 500 ng/ml.

Contributor’s Morphologic Diagnoses: Long bone: Elongation and thickening of the

zone of hypertrophic cartilage, with excess osteoid (unmineralized matrix), fibroplasia,
and folding fracture (rickets).
Parathyroid (not submitted): Hyperplasia, diffuse, moderate.

Contributor’s Comment: The gross and histologic findings within the long bones of
this bird are consistent with rickets. Rickets is caused by the failure of mineralization of
newly deposited osteoid and may be caused by deficiencies of vitamin D, calcium, or
phosphorus, as well as excess calcium or phosphorus. Grossly, affected birds have

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swollen joints, soft bones, and flared metaphyses. Curved deformities and folding
fractures may occur.1

Much research has been done on the different histologic lesions associated with rickets
depending on the specific nutrient imbalances present. With calcium deficiency, there is
disorganization and thickening of cartilage within the zone of proliferation, with poor
physeal vascularization. Only a small zone of hypertrophic cartilage is present in this
type of rickets. Calcification of the hypertrophic cartilage is reduced, and there is little
vascular invasion of this cartilage. At the base of the cartilage, few calcified bone
trabeculae are present, with a loss of the normal longitudinal arrangement.1,2 The
defect of matrix calcification appears to be due to lack of continuation of mineral
deposition, rather than failure of its initiation. In one study, electron micrographic
studies revealed that failure of progression of matrix deposition is likely due to the
presence of hypertrophic cells (responsible for the initiation of matrix deposition).3 The
bone marrow is often replaced by fibrous tissue, and osteoclasts are abundant. The
defect in calcium-deficiency rickets is thought to be impaired hypertrophy of
chondrocytes, rather than increased cell replication. In calcium-deficiency rickets, the
parathyroid glands are often hypertrophied.

Conversely, with phosphorous deficiency, the zone of proliferation is unchanged, but the
hypertrophic zone is elongated and thickened, and there is defective mineralization of
the hypertrophic cartilage cells resulting in long columns of cartilage, surrounded by
wide unmineralized osteoid seams extending into the primary spongiosa. There is
normal invasion by metaphyseal blood vessels. Osteoclasts are reduced, osteoblasts
are increased, and amounts of osteoid are limited.1,2 This condition is thought to be due
to decreased resorption of hypertrophic cartilage by chondroclasts, rather than an
increased proliferation of chondrocytes.4 Birds with phosphorus-deficiency rickets often
have atrophy of the parathyroids.2

Vitamin D-deficiency rickets results in lengthening and disorganization of the

proliferating zone and variable lengthening and dysplasia of the mineralizing zone. The
primary spongiosa is short, thick cartilage columns.1 As in calcium-deficiency rickets,
there is often parathyroid gland hyperplasia.2

It is interesting to note that the morphologic appearance of rickets in this case is most
consistent with decreased phosphorous. However, the presence of parathyroid
hyperplasia and relatively low serum calcium with high serum phosphorous is
suggestive of disease due to hypocalcemia. It should be stressed that the previously-
described histologic patterns are general guidelines only, and may vary according to
deficiency. Other causes of rickets, such as magnesium toxicity, excess vitamin A or fat
in the diet, mycotoxins, and enteritis have also been reported, and may cause
modifications in the typical morphology.2

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AFIP Diagnoses: 1. Long bone: Failure of endochondral ossification and retained
cartilaginous cores, with increased osteoid seams (rickets), boat-billed heron
(Cochlearius cochlearius), avian.
2. Long bone: Fracture with callus formation.

Conference Comment: As mentioned by the contributor, rickets in avian species may

be the result of a deficiency in vitamin D, calcium, or phosphorus as well as an excess
of calcium or phosphorus. Although there may be histological differences in rachitic
bones depending on the cause, the common denominator is the failure of mineralization
of newly deposited osteoid (failure of endochondral ossification), which results in bone
deformities and fractures.1

In domestic animals, rickets is most commonly caused by a deficiency in vitamin D or

phosphorus. However, it may also result from chronic renal disease or chronic
fluorosis. Histologically, the lesions in domestic animals are similar to those seen in
birds. There is abnormal endochondral ossification characterized by failure of
mineralization of the growth plate cartilage and osteoid. However, there is also
disorganization of chondrocytes within the zone of hypertrophy, which may or may not
be present in birds, depending on the etiology. It is unclear if the disorganization of
chondrocytes in vitamin D-deficiency rickets is due to a primary affect of vitamin D
metabolites or a mechanical consequence of the failure of endochondral ossification.5

Contributor: Wildlife Conservation Society, Department of Pathology, 2300 Southern

Blvd., Bronx, NY
http://wcs.org/

References:
1. Schmidt RE, Reavill DR, Phalen DN: Pathology of Pet and Aviary Birds, pp.155-156.
Iowa State Press, Ames, IA, 2003
2. Riddel C: Skeletal system. In: Avian Histopathology, ed. Riddel C, 2nd ed., pp. 252-
253. American Association of Avian Veterinarians, Kennet Square, PA, 1996
3. Takechi M, Itakura C: Ultrastructural studies of the epiphyseal plate of chicks fed a
vitamin D-deficient and low-calcium diet. J Comp Pathol 113(2):101-11, 1995
4. Grone A, Swayne DE, Nagode LA: Hypophosphatemic rickets in rheas (Thea
Americana). Vet Pathol 32(3):324-27, 1995
5. Weisbrode SE, Doige CE: Bones and joints. In: Thompson’s Special Veterinary
Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 512-513. Mosby, St.
Louis, PA, 2001

SLIDE 40
CONFERENCE 10 / CASE IV – AFIP 04 Case 1 (AFIP 2936425)

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Signalment: Approximately 8-10 weeks of age, intact males, DBA/1LacJ mice (Mus
musculus) from Jackson Laboratories.

History: Mouse with collagen-induced arthritis as a model for rheumatoid arthritis was
used to investigate the therapeutic effect of a drug under development.

Gross Pathology: Swelling and redness of the fore and hindpaw joints with visible
phalangeal distortion.

Contributor’s Morphologic Diagnosis: Front and rear paw joints (with associated soft
tissues): Polyarthritis, suppurative, severe, chronic with pannus formation, articular
cartilage degeneration, periosteal bone remodeling and surrounding tissue involvement
(bursitis, tendinitis and myositis), DBA/1LacJ mice (Mus musculus), rodent.

Contributor’s Comment: The tissue section on the slide can be either the front or rear
paw of the mouse. Microscopically, all joints (not present in all submitted slides) of the
paws are severely disrupted with replacement of the synovium and capsule by severe
fibrosis and marked infiltrations of neutrophils and a small number of macrophages and
lymphocytes. Across the synovial cavity bridging the synovial capsule, a pannus was
usually noted and consisted of a fibrous structure usually poorly vascularized,
containing basophilic debris, and infiltrates of neutrophils and a few macrophages. The
pannus formation is present with a partial to complete erosion and/or degeneration of
the articular cartilage extending in the subchondral bone and occasionally in the
underlying bone marrow containing mixed inflammatory cells and slight fibrosis. Along
the metaphyseal and diaphyseal bone, a marked periosteal reaction is noted and
consists of a marked thickening of the periosteum by a mild infiltration of mostly
neutrophils, fibrous connective tissue, bone remodeling (cartilage metaplasia, osteoid
matrix and/or woven bone formation with intense osteoclastic activities), and/or
sometimes marked osteoblastic hyperplasia (i.e., cells with a large vesicular nucleus
and abundant pale cytoplasm). Furthermore, in the surrounding tissues, a marked
chronic multifocal suppurative bursitis, tendinitis and myositis are noted.

This submission is a severe case (i.e., grade 4) of arthritis induced by collagen

[collagen-induced arthritis (CIA)] in mice as a model for rheumatoid arthritis (RA). The
severity of the arthritis was scored on a scale of 0 to 4 by histological assessment as
noted in Wooley and Wooley et al.1,2 CIA is induced in MHC-susceptible mice
immunized with bovine type II collagen emulsified in complete Freund's adjuvant.
Typically the mice are boosted 3 weeks after immunization with bovine type II collagen
in incomplete Freund’s adjuvant. The resultant response is dependent on T cells, B
cells, and cytokines. About 4 weeks after immunization, clinical signs of disease
develop in the paws. Active inflammation of the paws remains for about 2-3 weeks and
usually results in joint destruction and deformity.

In response to joint injury, hypertrophy and hyperplasia of synoviocytes of the synovial

membrane, and villous and/or pannus formation are observed. Pannus is an
intraarticular fibrovascular structure with inflammatory cell infiltrates that arises from the

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synovial membrane and spreads over neighboring cartilage subsequent to chronic
infectious nonsuppurative synovitis and/or immune-mediated diseases such as RA.
With time, as observed in this case, opposing cartilaginous surfaces are united by
fibrous tissue resulting in fibrous ankylosis of the joint. The pannus can act as a
physical barrier between the synovial fluid and the cartilage preventing chondrocyte
nutrition. The pannus macrophages with the proteolytic enzyme-producing neutrophils
and the collagenase-producing fibroblasts enhance the cartilage degeneration that
commonly extends into adjacent subchondral bone.3

CIA in the mouse and adjuvant-induced arthritis (AIA) in the rat are two models used to
mimic the clinical manifestations of RA due to similarities in the development of synovial
and cartilage lesions. RA is a chronic, erosive polyarthritic disease observed in both
humans and dogs, but rarely observed in the latter. The cause of RA is not yet fully
understood, but may involve both immune mediated processes i.e., humoral and cell-
mediated immunity. IgG or IgM classes known as rheumatoid factors are produced in
response to an unknown stimulus. The factors that might be involved are alterations in
the stearic configuration of IgG, persistent bacterial cell wall components that cross-
react with normal proteoglycans, anticollagen antibodies, and/or defective suppressor T
cell activity. Immune complexes formed are ingested by neutrophils that release
lysosomal enzymes (e.g., IL-1 promotes secretion of prostaglandins, nitric oxide, and
neutral proteases inhibiting proteoglycan synthesis), which are responsible for the
inflammatory reaction and destroy intraarticular structures.3,4 The loss of proteoglycans
from cartilage results in alterations in the hydraulic permeability of the cartilage, thus
interfering with joint lubrication and leading to further mechanically-induced injury to the
cartilage. TNF-_ has similar effects to IL-1: increasing concentrations of agents that will
decrease matrix synthesis and increase matrix destruction.3,4 Furthermore, matrix
metalloproteinases (gelatinases, collagenases) activated by products of degenerating or
reactive chondrocytes and inflammatory cells result in digestion of the matrix.3

AFIP Diagnosis: Paw, bones and joints: Polyarthritis and osteomyelitis, chronic-
active, diffuse, severe, with articular cartilage erosion, subchondral pannus, cortical
resorption, periosteal fibroplasia, reactive bone formation, and extensive soft tissue
inflammation, DBA/1LacJ mouse, murine.

Conference Comment: The contributor provides a thorough overview of collagen-

induced arthritis (CIA) and its use as an animal model for human rheumatoid arthritis
(RA). There is significant variation in slides due to the necessary use of multiple
animals and multiple paws. Therefore, as mentioned by the contributor, not all aspects
of the described lesions are present on all slides.

Arthritis in the dog is often classified as erosive or nonerosive. Rheumatoid arthritis in

the dog is a chronic, erosive polyarthritis that resembles RA in humans. The cause is
unknown, but the process is immune-mediated, and as described by the contributor,
involves both humoral and cell-mediated immunity, as well as inflammatory mediators

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and fibroblasts. In dogs, RA is characterized by progressive lameness due to
involvement of the peripheral joints of the limbs. Grossly, the lesions consist of marked
villous hypertrophy of the synovial membrane, erosion of the articular cartilage, pannus
formation, periarticular osteophytes, and occasionally fibrous ankylosis of affected
joints.3

Nonerosive arthritis occurs in dogs with systemic lupus erythematosus (SLE), or chronic
disease processes such as pyometra or otitis externa. In dogs with SLE, in addition to
arthritis, these animals often present with anemia, thrombocytopenia, polymyositis, or
glomerulonephritis. Immune complexes (type III hypersensitivity) can localize in the
synovium and lead to synovitis. In contrast to erosive arthritis, in joints with nonerosive
arthritis there is usually minimal villous hypertrophy, no pannus formation, no articular
cartilage destruction, and the exudate in the synovial fluid is neutrophilic.3

Contributor: Wyeth Research, Department of Pathology, Chazy, New York

References:
1. Wooley PH: Collagen-induced arthritis in the mouse. Methods Enzymol 162:361-373,
1988
2. Wooley PH, Luthra HS, Stuart JM, David CS: Type II collagen-induced arthritis in
mice. I. Major histocompatibility complex (I region) linkage and antibody correlates. J
Exp Med 154:688-700, 1981
3. Weisbrode SE, Doige CE: Bone and joints. In: Thomson’s Special Veterinary
Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 526-530. Mosby,
Inc., St. Louis, Missouri, 2001
4. Bolon B, Campagnuolo G, Zhu L, Duryea D, Zack D, Feige U: Interleukin-1beta and
tumor necrosis factor-alpha produce distinct, time-dependent patterns of acute arthritis
in the rat knee. Vet Pathol 41:235-243, 2004

SLIDE 41
CONFERENCE 11 / CASE I – 02-1123 (AFIP 2933943)

Signalment: 1-year-old, male castrated, Singapura, cat (Felis domesticus).

History: Presented with lethargy, persistent fever, enlarged mesenteric lymph nodes,
enlarged kidneys, abdominal and thoracic effusions. No history of diarrhea except
when fed pumpkin/goat milk diet recommended by breeder.

Gross Pathology: Very thin, icteric cat with 20 ml of gelatinous serosanguinous

thoracic fluid and 80 ml of similar abdominal fluid; enlarged lymph nodes (sternal,
cranial mediastinal, mesenteric, colonic); multifocal to coalescing yellow-white nodules
in liver, spleen, lung, lymph nodes, and kidneys; multiple tan-green 3 to 6 mm irregularly
shaped raised ulcerated foci on mucosa of cecum.

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Laboratory Results: Anemia, thrombocytopenia, hyperbilirubinemia, elevated alkaline
phosphatase, negative feline leukemia virus (FeLV) and feline immunodeficiency virus
(FIV) titers; Feline coronavirus titer 1:1600

Contributor’s Morphologic Diagnoses: 1. Severe multifocal ulcerative typhlitis with

innumerable intralesional trichomonads (Tritrichomonas foetus).
2. Cecum: Severe multifocal necrotizing and lymphoplasmacytic vasculitis (FIP).

Contributor’s Comment: This is a section of cecum with areas of mucosal necrosis

and ulceration. The ulcers are filled with innumerable, irregularly pyriform to elliptical, 5
x 10 _m, pale-staining basophilic protozoal organisms (trichomonads). These
organisms extend into the underlying submucosa and muscularis in some sections.
There is granulation tissue and neutrophilic inflammation associated with the ulcers, but
not always with the organisms. Many of the adjacent mucosal crypts contain
trichomonads as well. Several blood vessels in the submucosa, muscularis, and serosa
are surrounded or obscured by neutrophilic and lymphoplasmacytic inflammation.
There are also areas of fibrinoid necrosis characterized by karyorrhectic debris (nuclear
dust) and fibrin replacing and expanding the walls of blood vessels. There are mixed
populations of bacteria (predominantly filamentous) on the ulcerated surface and
extending into the areas of mucosal necrosis.

Tritrichomonas foetus is a recently recognized primary enteric pathogen of cats.1

Natural infections have been associated with large bowel diarrhea and ulceration of the
colonic mucosa.2 Although the organism was previously identified as Pentatrichomonas
hominis3, rRNA gene sequence analysis, restriction enzyme digest mapping, and light,
transmission, and scanning electron microscopy have identified the agent as
Tritrichomonas foetus, the same organism that causes reproductive disease in cattle.1
Experimental infection of domestic shorthair cats resulted in diarrhea which resolved
after 7 weeks followed by persistent infection.4 In previously reported natural and
experimental cases, invasion through the mucosa has not occurred. In this case, the
combination of feline infectious peritonitis and trichomonosis may have synergistically
resulted in more severe enteric lesions.

AFIP Diagnosis: Cecum (per contributor): Typhlitis, necrotizing, histiocytic,

neutrophilic and lymphoplasmacytic, transmural, multifocal, severe, with ulceration,
granulation tissue and myriad protozoa, Singapura, feline.

Conference Comment: There is considerable variation among slides. In some

sections, there is a perivascular inflammatory infiltrate suggestive of feline infectious
peritonitis while in others the inflammation is more diffuse. Precise identification of
protozoa in histologic sections is often impossible without utilization of special
techniques. Methods of identification of intestinal flagellates include direct cytologic
examination of feces suspended in saline solution, protozoal cultures,
immunohistochemistry, electron microscopy and PCR. Characteristic cytologic features

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of Tritrichomonas foetus include three anterior flagella, one posterior free flagellum, an
undulating membrane, a single nucleus, a stout axostyle, and a stout costa.1 In this
case, the identity of the organism was confirmed by PCR (personal communication, Dr.
Jody L. Gookin, North Carolina State University).

Genital trichomoniasis is a contagious venereal disease of cattle. Bulls can be carriers

with early infections resulting in balanoposthitis with a purulent discharge. As the
infection becomes chronic, there is no discharge, organisms are present in low numbers
and often concentrated in the glans penis. Cows are infected during coitus, and a
vaginitis with mucoid floccular discharge develops within a few days. Following the
vaginitis, the organism localizes to the uterus and cervix, causing endometritis and
cervicitis, and results in repeat breedings, abortion, or pyometra. There are no specific
lesions in the aborted fetus. However, large numbers of organisms can be isolated from
the fetal fluids and stomach. The placental lesions are not characteristic, and include a
white to yellow flocculent exudate, placental thickening, and hemorrhagic cotyledons.5

T. foetus has been recently demonstrated to be a feline pathogen that causes large
bowel diarrhea in young cats. The factors that result in the rare instances of invasive
infections such as this one have yet to be determined.

Contributor: University of Tennessee, College of Veterinary Medicine, Department of

Pathobiology, 2407 River Drive, Knoxville, Tennessee
http://www.vet.utk.edu/departments/path/

References:
1. Levy MG, Gookin JL, Poore M, Birkenheuer AJ, Dykstra MJ, Litaker RW:
Tritrichomonas foetus and not Pentatrichomonas hominis is the etiologic agent of feline
trichomonal diarrhea. J Parasitol 89(1):99-104, 2003
2. Gookin JL, Breitschwerdt EB, Levy MG, Gager RB, Benrud JG: Diarrhea associated
with trichomonosis in cats. J Am Vet Med Assoc 215:1450-1454, 1999
3. Romatowski J: Pentatrichomonas hominis infection in four kittens. J Am Vet Med
Assoc 216:1270-1272
4. Gookin JL, Levy MG, Law JM, Papich MG, Poore MF, Breitschwerdt EB:
Experimental infection of cats with Tritrichomonas foetus. Am J Vet Res 62:1690-1697,
2001
5. Kennedy PC, Miller RB: The female genital system. In: Pathology of Domestic
Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 3, pp. 426-427. Academic
Press, San Diego, CA, 1993

SLIDE 42
CONFERENCE 11 / CASE II – HB3973 (AFIP 2943308)

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Signalment: 5-year-old, female (spayed), Shiba-dog, canine.

History: The dog was presented for hematuria. By ultrasonography, a polypoid round
mass, approximately 2 cm in diameter, protruding into the lumen of bladder was noted
on the mucosal surface at the cranio-ventral wall of the bladder. Partial cystectomy was
performed to remove the affected bladder wall.
Gross Pathology: The firm, 2 x 2 x 1.5 cm mass protruded on the mucosal surface.
The polypoid mass had a smooth surface but was extensively ulcerated. The cut
surface was solid, white and mildly edematous. The border on the cystic wall was well
defined.

Laboratory Results: No atypical cells were detected by fine needle aspiration.

Contributor’s Morphologic Diagnosis: Urinary bladder: Cystitis, eosinophilic,

chronic, Shiba-dog, canine.

Contributor’s Comment: Sections were obtained from either of two paraffin-

embedded blocks.

The mass was located in the submucosa and was composed of a diffuse proliferation of
fibrous tissue that contained fibrocytes, fibroblasts, lymphocytes, plasma cells,
abundant blood vessels, and numerous eosinophils. Most of the mucosal epithelium on
the mass was ulcerated and numerous neutrophils infiltrated the superficial layer of the
mass. There was mild epidermal hyperplasia with Brunn’s nests, mild proliferation of
blood vessels, mild focal edema and hemorrhage with hemosiderin-laden macrophages
in the mucosa adjacent to the mass. These histological findings are consistent with
those of eosinophilic cystitis previously described in dogs.2

The lesion is considered a variant of polypoid cystitis, one in which eosinophils are the
predominant component.1 The alternative name of inflammatory fibrous polyps has
been suggested.3 A differential diagnosis includes fibroma, which lacks eosinophilic
infiltration. There appears to be some overlap between the diagnosis of fibrous polyps
and fibroma and the interpretations of such lesions are not unanimous.3

The etiology and pathogenesis of eosinophilic cystitis are still unknown.2 The lesions
occur in a variety of breeds of dogs ranging in age from 8 months to 15 years, with an
average of 8 years.3 Hematuria is the most common clinical sign in dogs. The lesions
usually have a benign clinical course and surgical excision is curative.

AFIP Diagnosis: Urinary bladder: Polypoid eosinophilic cystitis, Shiba, canine.

Conference Comment: As the contributor noted, a variety of names have been utilized
for similar lesions including fibroma, fibrous polyp, eosinophilic cystitis, polypoid
eosinophilic cystitis, cystitis with fibroplasia, and mesenchymal tumor with inflammation.

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Although pathologists may not agree on nomenclature, the histological features include:
hyperplastic, often ulcerated transitional epithelium; a nodule of fibrous connective
tissue confined to the propria/submucosa; abundant vascular supply; inflammatory cells
with a predominance of eosinophils; and occasionally foci of granulopoiesis,
eosinophilopoiesis, cystitis glandularis and Brunn’s nests. The mesenchymal cells are
surrounded by a material that stains as collagen with Masson’s trichrome.
Immunohistochemically, the mesenchymal cells are not immunoreactive for desmin and
muscle specific actin.3,4 Definitive differentiation of inflamed fibrous neoplasms from
proliferative fibrous inflammatory lesions is often problematic.

Contributor: Hokkaido University, Graduate School of Veterinary Medicine, Laboratory

of Comparative Pathology, Sapporo, Japan
http://www.hokudai.ac.jp/veteri/index-e.html

References:
1. Esplin DG: Urinary bladder fibromas in dogs: 51 cases (1981-1985). J Am Vet Med
Assoc 190:440-444, 1987
2. Fuentealba IC, Illanes OG: Eosinophilic cystitis in 3 dogs. Can Vet J 41:130-131,
2000
3. Meuten DJ: Tumors of the urinary system. In: Tumors in Domestic Animals, ed.
Meuten DJ, 4th ed., pp. 541-544. Iowa State Press, Ames, IA, 2002
4. Meuten DJ, Everitt J, Inskeep W, Jacobs FM, Peleteiro M, Thompson KG:
Histological Classification of Tumors of the Urinary System of Domestic Animals, 2nd
series, vol. XI, ed. Schulman FY, pp. 36. The Armed Forces Institute of Pathology,
Washington, DC, 2004

SLIDE 43
CONFERENCE 11 / CASE III – 2004903384 (AFIP 2937492)

Signalment: Five-year-old, female, ferret (Mustelae putorius furo).

History: An intact female domestic ferret was evaluated for bloody stool. Clinical signs
included emaciation, loss of body weight and abdominal swelling due to ascites.
Generalized alopecia and anemia were also observed. Blood biochemical examinations
revealed increased enzyme activities of aspartate aminotransferase (AST), alanine
aminotransferase (ALT), lactose dehydrogenase (LDH) and alkaline phosphatase
(ALP), and increased level of total bilirubin. Mild hypoglycemia was also detected.
Despite various supportive treatments, the ferret died three months after first
presentation to the animal hospital.

Gross Pathology: The liver was yellow to light tan and slightly enlarged, with a coarse
multi-nodular appearance (Fig.1).

Laboratory Results:

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At first presentation:
WBC 10400/_l ALT over 1000 IU/l T-Cho 252.2 mg/dl
Ht 26.3% AST 540 IU/l TG 87.6 mg/dl
Hb 7.8g/dl ALP 200 IU/l BUN 33 mg/dl
T-Bil 0.1 mg/dl Glu 118.5 mg/dl

About 1 week prior to death:

WBC 8800/_l ALT 267 IU/l T-Bil 4.3 mg/dl
Ht 29.7% AST 154 IU/l BUN 51.1 mg/dl
Hb 10.1g/dl ALP 125 IU/l Glu 136.3 mg/dl

Contributor’s Morphologic Diagnosis: Massive hepatocellular necrosis and various

degenerative changes in hepatocytes (ie; eosinophilic granules, lipid and bile pigment
accumulations, severe vacuolation). Regenerative nodules of hepatocytes and
cholangiocellular (bile ducts) proliferation (so-called toxic hepatopathy or chronic
progressive hepatitis, consistent with Copper toxicosis).

Contributor’s Comment: Copper storage hepatopathies in Bedlington terriers and

West Highland White terriers are well documented in the literature. The disorder in
Bedlington terriers is an inherited autosomal recessive defect. In West Highland White
terriers, although the disorder is also thought to be an inheritable liver disease, not all of
the affected dogs show signs of liver disease. In domestic ferrets, an identical disease
condition with similar clinical features and histopathological changes is reported,1 and
thought to have an inheritable component.

The main histopathological changes in this specimen are massive to focal necrosis of
hepatocytes, with mild fatty change and severe vacuolation. In addition to these
changes, there are eosinophilic granules in the cytoplasm of degenerative hepatocytes.
These granules stain positive for rhodanine, a copper-specific stain. Accumulation of an
intracytoplasmic eosinophilic material is also present in Kupffer cells and macrophages.

Other lesions include mild fibrosis with mononuclear cell infiltration, reactive proliferation
of cholangiolar epithelial cells, bile duct obstruction (bile thrombus or cholestasis), and
regenerative hepatocellular nodules. This ferret also had bilateral adrenocortical
adenomas and an islet cell tumor that are thought to be the cause of the generalized
alopecia and hypoglycemia.
The genetic defect in the Bedlington terriers causes expression of an abnormal hepatic
metallothionein. Defective metallothionein results in reduced biliary excretion of copper
and excessive copper becomes sequestered in hepatocellular lysosomes. The
association between an increased hepatic copper concentration and its relationship to
tissue injury is controversial in dog breeds other than the Bedlington terrier.

Occlusion of the major bile duct has been shown to cause elevation of hepatic copper
concentration in cats, but not in dogs. Chronic hepatitis, chronic cholestasis and
cirrhosis have been shown to lead to increased tissue copper concentrations in dogs,

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but these findings are not known in ferrets. The cause of copper toxicosis in this ferret
is unknown, and a genetic predisposition is also unknown.

AFIP Diagnosis: Liver: Cirrhosis, characterized by multifocal necrosis, regenerative

nodular hepatocellular hyperplasia, fibrosis, biliary hyperplasia, canalicular cholestasis,
hepatocellular lipidosis, extramedullary hematopoiesis and eosinophilic refractile
hepatocellular cytoplasmic granules, ferret, mustelid.

Conference Comment: Copper-associated liver diseases are well recognized in

animals and humans. Copper toxicosis results from disruption of normal copper
homeostasis or accumulation of copper in excess of metabolic requirements and may
be either primary or secondary. Primary copper toxicosis results from an inherited
metabolic defect. Secondary copper toxicosis results from an underlying pathologic
process that leads to abnormally high intake, increased absorption, or reduced
excretion of copper. To better understand copper-associated diseases, one must first
understand copper homeostasis in mammals.2

Dietary copper is absorbed primarily in the proximal small intestine where transport from
the lumen into the intestinal mucosa is a carrier-mediated process, with a saturable
transport component, which may be influenced by other dietary factors. Most of the
copper within the intestinal epithelium is found within the cytosol bound to
metallothioneins. From here, copper enters the portal circulation bound to albumin or
other carrier proteins, and is primarily transported to the liver, with small amounts
entering the kidney. Once in the liver, copper undergoes three processes:
hepatocellular uptake, intracellular distribution and utilization, and copper export, with
each of these steps tightly regulated by transporters, chaperones, and other proteins.
ATP7B protein is required for copper incorporation into ceruloplasmin in the liver, for
biliary excretion, and possibly for transport of copper into a vesicular compartment,
where it may be delivered to lysosomes and interacts with metallothionein. The main
route of excretion of copper is in the bile. While some copper is excreted into plasma as
a complex with ceruloplasmin, very little copper crosses the glomerular capillaries.2

Familial copper storage disorders occur in Wilson’s disease in humans, Long Evans
Cinnamon (LEC) rats, toxic milk mice, Bedlington terriers, and West Highland White
terriers. Wilson’s disease is an autosomal recessive inherited disorder of copper
metabolism and results in copper accumulation in the liver, cornea, and brain. The
gene defect has been localized on human chromosome 13 and codes for ATP7B, a
copper transporting P-type ATPase. The mutations occur throughout the entire gene,
leading to variable clinical presentations. The resulting liver disease may mimic a wide
variety of common liver conditions, including fulminant hepatic failure, chronic hepatitis,
and cirrhosis. The LEC rats and toxic milk mice are the only known valid animal models
of Wilson’s disease. The canine copper toxicosis locus in Bedlington terriers has been
mapped to canine chromosome region CFA 10q26, and recently a mutated MURR1
gene was discovered in animals with the disease.2

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Secondary copper toxicosis, characterized by copper retention secondary to an
underlying disease has been documented in primary biliary cirrhosis in humans, chronic
active hepatitis in Doberman pinschers, and Skye Terrier hepatitis. Primary biliary
cirrhosis is a chronic, progressive, often fatal liver disease that results in cirrhosis and
liver failure. Although the pathogenesis is unknown, an immune-medicated mechanism
is suspected, and copper accumulation is a secondary event. Similarly, the cause of
chronic active hepatitis in Doberman pinschers is unknown, but is also thought to be
immune-mediated, with copper accumulation within the centroacinar (portal) areas
occurring secondarily. Skye Terrier hepatitis is characterized by intracanalicular
cholestasis, with copper accumulation, hepatocellular degeneration, and ultimately
cirrhosis. In this disease, copper accumulates primarily in the periacinar (centrolobular)
areas.2

Regardless of the cause, cirrhosis and hepatic failure are often the end result. Initially,
as hepatocytes degenerate, become necrotic, and are lost, the liver will become
hyperplastic, leading to both micro- and macro-nodular regeneration. These nodules
form at varying times and therefore, as in this case, have varying histological
appearances. Some regenerative nodules are composed of hepatocytes with
intracytoplasmic lipid vacuoles while others are not. As this process continues, normal
hepatic architecture is lost and, as is seen in this case, it may be difficult to identify a
“normal” hepatic lobule.

Contributor: Setsunan University, Faculty of Pharmaceutical Sciences, Department of

Pathology, 45-1 Nagaotouge-cho, Hirakata, Osaka, Japan

References:
1. Fox JG, Zeman DH, Mortimer JD: Copper toxicosis in sibling ferrets. J Am Vet Med
Assoc 205(8):1154-1156, 1994
2. Fuentealba IC, Aburto EM: Animal models of copper-associated liver disease. Comp
Hepatol 2(1):5-17, 2003

SLIDE 44
CONFERENCE 11 / CASE IV – D04-23121 (AFIP 2938283)

Signalment: 12-year-old, spayed female, Labrador Retriever mixed breed dog (Canis
familiaris).

History: The dog presented to a local practitioner for a soft tissue swelling, of unknown
duration, that extended from the left metacarpal pad distally over the left forepaw.
Systemic antibiotics in combination with epsom salt soaks failed to resolve the swelling.
Incisional biopsies from the left metacarpal pad and adjacent interdigital skin were
submitted for histopathological examination.

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Gross Pathology: In a single biopsy, nests, islands, acinar and tubular arrangements
of tumor cells extend from the dermal-epidermal junction to all surgical borders. Tumor
cells have indistinct cell borders, are cuboidal to columnar to polygonal with a high
nuclear to cytoplasmic ratio. One to two layers of cells line the tubular structures. The
nucleus is centric, oblong to oval to round with finely stippled chromatin and a single
nucleolus. The cytoplasm is scant and pale pink. Anisokaryosis and anisocytosis are
moderate. Mitoses are present (0 to 1 per 40X objective field). Binucleate and
trinucleate cells and karyomegaly are rare. Occasional angular nucleolar forms are
noted. The arrangements of tumor cells are surrounded by an abundant fibrous
connective tissue stroma (scirrhous reaction). A moderate infiltrate of plasma cells,
small lymphocytes and eosinophils is present in the supporting stroma. Several normal
eccrine glands are surrounded by the arrangements of tumor cells.

Contributor’s Morphologic Diagnosis: Eccrine carcinoma

Contributor’s Comment: Eccrine carcinoma is a malignant tumor showing

differentiation to eccrine secretory epithelium.1 The tumor is rare but has been reported
in the footpads of the dog and cat where these glands are normally located.1,2,3 Lesions
may affect multiple toes in cats.2 Affected areas are swollen and often the overlying
epidermis is ulcerated.1,2 There may be invasion of adjacent bones of the digit. Eccrine
carcinomas are highly aggressive tumors that exhibit rapid metastasis to lymph nodes
and subcutaneous tissues of the affected limb.2,3 Visceral metastasis has not been
reported for eccrine carcinomas.1,3 Most cases are treated by excision of the tumor with
wide margins.1 If the spread of the tumor is confined to the leg then amputation of the
leg is a reasonable option.3

AFIP Diagnosis: Foot pad: Adenocarcinoma, Labrador Retriever cross, canine.

Conference Comment: Exocrine glands are composed of highly specialized epithelial

cells and discharge their secretory product via a duct onto an epithelial surface.
Exocrine glands can be classified according to two major characteristics: the
morphology of the gland, and the means of discharge of the secretory product.
Exocrine glands are either simple, with a single, unbranched duct, or compound, with a
branched duct system. These glands secrete their product in one of three ways:
merocrine (eccrine) secretion involves the process of exocytosis; apocrine secretion
involves the release of membrane-bound vesicles; and holocrine secretion involves the
discharge of the whole secretory cell.4

The eccrine glands develop independently of the hair follicle, with the duct opening
directly onto the surface of the epithelium.5 They are found only on the glabrous skin,
such as the footpad of dogs and cats, the frog region of ungulates, the carpus of pigs,
and the nasolabial region of ruminants and pigs.5,6 These glands have been designated
eccrine based on their mode of secretion. However, it is now known that these glands

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excrete their substances by a variety of secretory modes, including microapocrine
blebbing. Some authors now prefer the term atrichial glands.5

The apocrine glands develop embryologically as part of the hair follicle complex and are
found in all haired skin areas, although only associated with the primary hair follicles.
The ducts of the apocrine glands open in the superficial portion of the hair follicle.5
Apocrine gland activity is rarely visible in domestic animals, except in the horse. Other
apocrine glands include the interdigital glands of small ruminants, glands of the external
ear canal and eyelids of domestic animals, anal sac glands of dogs and cats, and the
mental organ of pigs.6 Previously, the mode of secretion was thought to involve the
pinching off of apical blebs of cytoplasm. However, more recent studies have shown
this to be largely artifact with sweat production resulting from a combination of
processes including holocrine secretion, vesicle exocytosis, active ion and water
transport, and a minor contribution from microapocrine blebbing. Due to this fact, some
authors now prefer the term epitrichial or paratrichial glands.5

Differentiation of eccrine carcinoma from apocrine carcinoma is exceedingly difficult and

requires knowledge of the site of origin of the tumor (footpad in the dog or cat). In dogs,
the normal eccrine gland is composed of a secretory coil, a ductular segment that
courses through the dermis and an intraepidermal segment. The secretory coil consists
of a single layer of cuboidal to columnar epithelial cells and a single layer of fusiform
myoepithelial cells. The ductular segment is composed of two layers of non-secretory
cuboidal epithelial cells. The duct opens on the footpad surface.7 Carcinoembryonic
antigen (CEA) is present in both the ductular and secretory portions of the gland8 and
has been reported to be useful in identification of eccrine carcinomas in animals.2,7 In
this case, neoplastic cells are immunohistochemically negative for CEA and no unique
features of eccrine differentiation were found. This neoplasm may be an eccrine
carcinoma but the possibility of metastasis from another site should be excluded.
Particularly in cats, occult pulmonary adenocarcinomas sometimes metastasize to one
or more footpads and may be misdiagnosed as eccrine carcinomas.

Contributor: University of Saskatchewan, Western College of Veterinary Medicine and

Prairie Diagnostic Services, Department of Veterinary Pathology, 52 Campus Drive,
Saskatoon, Saskatchewan, Canada
www.usask.ca/wcvm/vetpath
www.usask.ca/pds

References:
1. Meuten DJ: Tumors in Domestic Animals, 4th ed., pp. 76-78. Iowa State Press,
Ames, 2002
2. Gross TL, Ihrke PJ, Walder EJ: Veterinary Dermatopathology: A Macroscopic
and Microscopic Evaluation of Canine and Feline Skin Disease, pp. 396-397. Mosby
Yearbook, St.Louis, 1992
3. Kusters AH, Peperkamp KH, Hazelwinkel HA: Atrichial sweat gland
adenocarcinoma in the dog. Vet Dermatol 10 (1): 51-54, 1999
4. Young B, Heath JW: Wheater’s Functional Histology: A text and colour atlas, 4th

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ed., pp. 93. Churchill Livingston, Philadelphia, PA, 2002
5. Yager JA, Scott DW: The skin and appendages. In: Pathology of Domestic Animals,
eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 1, pp. 536. Academic Press, San
Diego, CA, 1993
6. Hargis AM, Ginn PE: Integumentary system. In: Thompson’s Special Veterinary
Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 539. Mosby, St.
Louis, PA, 2001
7. Scott DW, Miller WH, Griffin CE: Muller & Kirk’s Small Animal Dermatology, 6th ed.,
pp. 51-53, 1275-1276. W.B. Saunders Company, Philadelphia, PA, 2001
8. Murphy GF, Elder DE: Atlas of Tumor Pathology: Non-Melanocytic Tumors of the
Skin, 3rd Series, Fascicle 1, pp. 64. The Armed Forces Institute of Pathology,
Washington, DC, 1991

SLIDE 45
CONFERENCE 12 / CASE I – 04-514 (AFIP 2941213)

Signalment: 6 year-old Rhodesian Ridgeback, neutered male, dog (Canis familiaris).

History: This dog initially presented with a chief complaint of vomiting. Over a period
of several days its condition declined, and it developed lingual ulcers. Six days after
initial presentation the dog developed tachypnea, a cough and excessive salivation. It
fatigued easily and developed cyanosis after exercise. Therapy included fluids,
antibiotics and oxygen via a nasal line. The dog’s condition worsened over the next
day. Due to its declining condition the dog was euthanatized. A postmortem
examination was carried out at the veterinary hospital (on the day following the death of
the animal) and tissues were submitted to the Veterinary Diagnostic Laboratory at
Oregon State University for examination.

Laboratory Results: Chemistry panels showed elevated lipase, at > 6000. BUN and
creatinine were not elevated. Multiple blood gases were performed over the last 3 days
before euthanasia. Despite therapeutic oxygen insufflation pO2 fell from initial values of
170 mm Hg to 69, while pCO2 rose from initial values of 42.4 to 64 mm Hg.

Contributor’s Morphologic Diagnosis: Diffuse pulmonary fibrosis with hemorrhage

and edema and multifocal hyperplasia of type II pneumocytes.

Contributor’s Comment: No gross description of lesions was available. A full set of

tissues was submitted for histopathologic examination. Lesions were confined to the
lung and kidney, which are presented in this submission.
In the lung there is diffuse hemorrhage and pulmonary edema, with alveoli containing
eosinophilic proteinaceous fluid. Macrophages are present in many alveoli. There is
diffuse thickening of alveolar walls and many alveoli are lined discontinuously by large,
hypertrophied cells, presumably hyperplastic type II pneumocytes. There is increased
collagenous tissue on the pleural surface. Pleural mesothelial cells are hypertrophied

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and most appear to be ciliated. There is vascular congestion and some pulmonary
vessels are thrombosed (in some sections). Trichrome stains reveal moderate, diffuse
interstitial fibrosis. In the kidney there is diffuse congestion and scattered acutely
necrotic tubules within the cortex.

This is one of seven cases presented to the Veterinary Diagnostic Laboratory within a
period of about one week with similar clinical histories. All affected dogs had visited a
particular park in Portland, Oregon. All had been seen to either ingest some material or
to have vomited shortly after the visit. All developed gastrointestinal signs and
progressive dyspnea. Some had elevated BUN and creatinine levels early in the clinical
course, although that was not true for this case. Based on clinical signs and
histopathology a presumptive diagnosis of paraquat intoxication was made. This was
confirmed by the detection of paraquat in the urine of some of the cases.

Paraquat (1,1’-dimethyl-4,4’-bipyridillium dichloride; CAS # 1910-42-5) is a contact

herbicide and its use is restricted in the US. Despite its restricted use, poisoning of pet
animals remains a problem and in this series of cases is presumed to have been
malicious.

The lung is the primary tissue affected in paraquat toxicity.1 However, other tissues,
including the kidney, liver and thymus also may be affected. In this series of cases
lesions were consistently present in both lung and kidney. Experimentally paraquat
produces pulmonary edema and hemorrhage with necrosis and loss of both type I and
type II pneumocytes. Alveolar capillary endothelium is spared. These lesions may be
followed by hyperplasia of type II cells. The extent of loss of pneumocytes and, thus,
the capacity for regenerative hyperplasia of type II cells are dose dependent. In time
diffuse interstitial fibrosis develops. The pulmonary lesions of paraquat toxicity are not
unique and are similar to those induced by other agents that cause diffuse alveolar
injury.

The targeting of the lung in paraquat toxicity is explained by its selective uptake in that
tissue. Uptake is an energy dependent process that can occur against a concentration
gradient. It utilizes the same biological uptake mechanisms that transport other
polyamines, such as putrescine and spermidine. Toxicity is considered to result from
cyclic oxidation-reduction reactions of the paraquat molecule, which result in the
generation of large quantities of reactive oxygen species, including superoxide anion
(O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH·). Toxicity is dependent on
molecular oxygen and tissue damage is probably mediated, at least in part, by lipid
peroxidation. Depletion of cellular NADPH may also play a direct role in toxicity.

Lesions in this case are consistent with paraquat toxicity. The discontinuous
appearance of the hyperplastic type II pneumocytes is most likely explained by the dose
received by this dog. Severe depletion of type II cells by the toxin would be expected to
result in “patchy” hyperplasia. Renal lesions are relatively acute compared to those in
the lung and the relative contributions of direct paraquat renal toxicity and hypoxia are

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not clear. The presence of cilia, or rudimentary cilia, on pleural mesothelial cells has
been reported in hyperplastic, metaplastic or neoplastic conditions affecting these cells.

AFIP Diagnoses: 1. Lung: Pneumonia, interstitial, hemorrhagic, diffuse, moderate,

with type II pneumocyte hyperplasia, multifocal interstitial fibrosis, and emphysematous
change, Rhodesian Ridgeback, canine.
2. Kidney: Nephritis, interstitial, lymphoplasmacytic, chronic, multifocal, minimal, with
glomerular sclerosis.

Conference Comment: The contributor provides a thorough overview of paraquat

toxicity in dogs. Conference attendees also considered other differential diagnoses for
pulmonary edema and hemorrhage including warfarin toxicity, oxygen toxicity, nitrogen
toxicity, ionizing radiation, and a neoplastic induced clotting deficiency.

Both paraquat and diquat are bipyridyl herbicides that have caused numerous deaths in
humans and various animal species. Intoxication involves either carelessness or
malicious poisoning and has been reported in cattle, sheep, horses, pigs, poultry, dogs,
rabbits, and fish. Clinical signs and lesions vary with the species, dose received and
route of administration.2

The most common route of administration for most species is ingestion. Cattle often
present with neurological signs including dullness, incoordination, and staggering that
progress to prostration, convulsions, coma and death. Oral lesions have also been
reported in horses after grazing on freshly sprayed pasture. The lesions described in
poultry and swine are similar to those seen in dogs, with dyspnea and pulmonary
edema noted in both species. In sheep, intravenous administration of paraquat resulted
in a dose-dependent decrease in glomerular and tubular function. The lesions in fish
include massive desquamation of gill epithelial cells.2

In humans, accidental or intentional ingestion is the most common route of exposure;

however, inhalation and dermal exposure have also been reported. Acute toxicity
results in severe damage to all organ systems and death within 24 hours. Chronic
toxicity results in progressive pulmonary fibrosis and pulmonary edema resulting in
dyspnea and asphyxia.3

Contributor: Oregon State University, Veterinary Diagnostic Laboratory, 105 Magruder

Hall, Corvallis, Oregon
http://www.vet.orst.edu/

References:
1. Bus JS, Gibson JE: Paraquat: Model for Oxidant-initiated Toxicity. Environmental
Health Perspectives 55:37-46, 1984
2. Humphreys DJ: Veterinary Toxicology, 3rd ed., pp. 134-135. Bailliere Tindall, London,
UK, 1988

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3. Wexler P: Encyclopedia Toxicology, vol. 2, pp. 475-476. Academic Press, San
Diego, CA, 1998

SLIDE 46
CONFERENCE 12 / CASE II – 98-1713 (AFIP 2694678)

Signalment: Tissue from a bovine.

History: Four of 21 cattle died suddenly during July 1998, while one recovered on a
small farm close to Bloemfontein in the Free State Province of South Africa. When the
farm was visited two days after the mortalities occurred, Cestrum spp. plants were
found in an adjacent camp to which the cattle had gained access through a break in the
fence. The tops of the plants had died off due to the effects of frost but at the bottom of
the plants there were tufts of luscious green foliage which showed evidence of having
been browsed.

Necropsies were performed on 2 of the 4 animals by the provincial state veterinarian

and a range of organ specimens from the second case were submitted in unbuffered
formalin to the Onderstepoort Veterinary Institute for histopathological examination.
The plant involved was identified as a Cestrum sp. at the local herbarium.

Gross Pathology: Both of the animals necropsied presented with multiple

hemorrhages throughout the serosal surfaces as well as on the endo- and epicardium,
swollen and friable livers with marked hepatic lobular accentuation, hemorrhage and
edema of the wall of the gall bladders, and colonic and cecal hemorrhages.

Contributor’s Morphologic Diagnoses: 1. Necrosis and hemorrhage, coagulative,

severe, acute, liver with hepatocyte vacuolation and presence of intracellular and loose
intrasinusoidal segrosomes, midzonal, severe, acute.
2. Bile ductular proliferation, subacute, portal, moderate.

Contributor’s Comment: As the tissues were fixed in unbuffered formalin, they

became very fragile and several moderate to severe artifactual lesions are present in
many sections:
• Acid hematin pigment accumulation
• Tears along edges
• Folds
• Presence of pink fluid in lumens of larger vessels
• Tearing and unraveling of walls of larger vessels
The liver revealed extensive periacinar coagulative necrosis and hemorrhage. Midzonal
hepatocytes were extremely swollen and vacuolated, and many contained
intracytoplasmic, single or multiple bright eosinophilic droplets of varying size,
presumably cytosegrosomes. A few segrosomes were also present loose within the
sinusoids in this region.

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The history of ingestion of Cestrum spp. plants and the macroscopical- and
histopathological changes of multiple hemorrhages and an acute hemorrhagic hepatosis
correspond to the lesions described for acute to subacute cases of Cestrum
poisoning.1,2 The most likely differential diagnoses for this lesion in South Africa include
acute seneciosis and acute algal toxicity.1 No Senecio spp. plants could be found on
the property and algal toxicity was unlikely in this incident as no algal blooms were
noted in the stream from which the cattle drank, nor were mortalities reported in other
groups of cattle drinking from the stream, either up- or downstream from the affected
farm.

Species of the genus Cestrum are evergreen, ornamental shrubs which have been
imported into South Africa from South America where they occur indigenously. In Africa
suspected cases of poisoning of livestock due to C. laevigatum, C. parqui and C.
aurantiacum have been reported from South Africa, Zimbabwe, Kenya and the island of
St. Helena. In South Africa, outbreaks of poisoning by C. laevigatum have traditionally
occurred in the Kwazulu-Natal Province.1 The outbreak described above is one of 3
outbreaks attributable to Cestrum spp. toxicity, all of which occurred in the Free State
Province of South Africa, an area not traditionally associated with Cestrum toxicity in
South Africa. The Cestrum spp. are known for their ability for rapid establishment and
spread, particularly along river banks and in recent years it appears that in South Africa,
these plants have spread beyond the Kwazulu-Natal Province northwards into the Free
State and Gauteng Provinces despite all 3 species having been proclaimed as weeds in
South Africa.

AFIP Diagnosis: Liver: Hepatocellular degeneration and necrosis, centrilobular and

midzonal, diffuse, acute, severe, bovine.

Conference Comment: Hepatoxicity may be acute or chronic, but often is somewhere

in between, and the agent’s effect is usually a function of dose-rate. Nonetheless,
agents that most often cause acute hepatotoxicity in cattle include: blue-green algae,
Cestrum sp., cocklebur, poison peach, and sawfly larvae. Agents that most often cause
chronic hepatotoxicity in cattle include: aflatoxin, pyrrolizidine alkaloids, sporidesmin,
lantana, and nitrosamines.

There are several species of the genus Cestrum. In the United States, pathologists are
most familiar with Cestrum diurnum as a cause of enzootic calcinosis in cattle. The
species known to be hepatotoxic include: C. parqui, C. laevigatum, and
C. aurantiacum, all of which cause similar hepatic disease. However, in the field,
speciation within the genus is often uncertain due to hybridization.

Cestrum spp., the ink-berry plants, cause acute hepatotoxicity in South America,
southern and central Africa, and Australia. Cattle are most frequently affected;
however, sheep, goats, and fowl are susceptible. The young leaves and unripened

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berries are the most toxic parts of the plant. The toxin, an atractyloside, has been
recently identified. The histological lesions are consistently centrilobular (periacinar)
and are identical to lesions caused by poison peach (Trema aspera) and cocklebur
(Xanthium pungens).

Contributor: Onderstepoort Veterinary Institute, Pathology Section, P.O. Box 12502,

Onderstepoort, South Africa
http://www.arc.agric.za/institutes/ovi/main/divisions/path2.htm

References:
1. Kellerman TS, Coetzer JAW, Naude TW: Plant Poisonings and
Mycotoxicoses of Livestock in Southern Africa, 1st ed., pp.13-15. Oxford University
Press, Cape Town, 1998
2. Van Der Lugt JJ, Nel PW, Kitching JP: The pathology of Cestrum laevigatum
(Schleschtd.) poisoning in cattle. Onderstepoort Journal of Vet Res 58:211-221, 1991
3. Kelly WR: The liver and biliary system. In: Pathology of Domestic Animals, eds. Jubb
KVF, Kennedy PC, Palmer N, 4th ed., vol. 2, pp.384-401. Academic Press, San Diego,
CA, 1993

SLIDE 47
CONFERENCE 12 / CASE III – S04-66 (AFIP 2948647)

Signalment: One-year-old, male, mixed-breed dog, canine.

History: The dog had clinical signs of gradual weight loss, vomiting, anorexia, and was
fed with commercial dog food for several months.

Gross Pathology: The submitted samples were formalin fixed kidney, stomach and
spleen. Prominent, white, powder-like deposits were observed on the kidney sample.
The other samples had no significant gross lesions.

Laboratory Results: Moderate increases in WBC (15.2 × 103 /µl), neutrophils (95% of
WBC), markedly increased BUN (407 mg/dl) and creatine (18.3 mg/dl).

Contributor’s Morphologic Diagnosis: Kidney: Interstitial nephritis, fibrosing,

lymphocytic, diffuse, severe, subacute, with severe tubular necrosis, mineralization,
intratubular and pelvic oxalate crystals, canine.

Contributor’s Etiologic Diagnosis: Oxalosis associated nephrotoxicosis.

Contributor’s Comment: The lesions in the kidney were characterized by prominent

interstitial fibrosis, crystal accumulation, atrophy, calcification, and mild lymphocytic
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infiltration. The outline of the kidney was rough. Multifocally renal tubular lumina had
accumulation of light yellow crystals. These crystals were round with many lines
radiating from their center and had a birefringent appearance with polarized light.
Affected tubular epithelium was degenerate to necrotic with mineral deposition on some
tubular basement membranes and in the tubular epithelium. Some glomerular atrophy
was also noticed. The interstitium had prominent fibrous connective tissue proliferation.
The Alizarin red S stain was positive in the mineralized areas. Histopathologically, this
case has marked renal tubular injury, prominent connective stroma proliferation and
combined with the results of clinical pathology, renal failure is suspected clinically. The
finding of renal tubular lumina with radially arranged crystal deposition could be induced
by many causes; the most probable is calcium oxalate deposition. In a recent outbreak
(March 2004) estimated at 3000 cases of canine renal failure in Taiwan, there was no
sex, age or breed predilection. The ages of affected dogs ranged from several months
to adult. The owners of the dogs told clinicians that their pets only ate a certain brand
and type of commercial dry dog food. After eating this dog food for several days, dogs
were presented with various degrees of renal failure. Finally, most of the affected dogs
had gradual weight loss.

Oxalate is a simple organic carboxylic acid, which is excreted as calcium oxalate by

many fungi. Oxalate also occurs as byproducts in various plant tissues, in urine, and in
mantles of certain bivalves. In dogs, the most common cause of nephrotoxicosis is
exposure to ethylene glycol. Ethylene glycol is rapidly absorbed from the gut, with peak
plasma concentration achieved 2 to 3 hours after ingestion. It is predominantly
metabolized in the liver in the following sequence: ethylene glycol to glycoaldehyde to
glycolate to glyoxylate. Glyoxylate is converted to several final metabolites, including
oxalate, glycine, and formate. Oxalate then binds with calcium and forms a soluble
complex which is filtered by glomeruli. As water is reabsorbed by the tubules and the
pH of the filtrate decreases, calcium oxalate precipitates and forms crystals. This
results in nephrosis, and hypocalcemia if enough calcium is complexed. Other sources
of oxalate may come from plant origin foods. Plants which may contain toxic amounts
of oxalate are Halogeton glomeratus (halogeton), Sarcobatus vermiculatus
(greasewood), Rheum rhaponticum (the common garden rhubarb), Oxalis cernua
(soursob), Rumex spp. (sorrel and dock). In tropical and subtropical areas, certain
grasses that are cultivated widely (genera of Cenchrus, Panicum and Setaria),
accumulate large amounts of oxalate, and have been associated with renal oxalosis in
cattle and sheep and with skeletal disease in the horse, the latter due to conditioned
calcium deficiency.

The fungi Aspergillus niger and A. flavus can produce large quantities of oxalates on
feedstuffs. Large doses of ascorbic acid have caused oxalate nephrotoxicosis in
humans and in a goat; ascorbic acid is a metabolic precursor of oxalate. Primary
hyperoxaluria, a rare inherited metabolic condition, occurs in humans, cats and perhaps
dogs (Tibetan spaniels). Pyridoxine (vitamin B6) deficiency and methoxyflurane
anesthesia can also cause renal oxalosis.

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Calcium oxalate is precipitated in the renal tubules during the process of elimination. A
fatal outcome may occur from renal insufficiency and uremia after the earlier symptoms
have abated. Conversely, recovery is possible, with blood urea levels slowly subsiding
after about one month. Cystitis and urethritis may be a part of this syndrome.

AFIP Diagnosis: Kidney: Tubular degeneration, necrosis, and loss, diffuse, moderate,
with interstitial fibrosis, and multifocal tubular mineralization and oxylate crystal
deposition, mixed-breed, canine.

Conference Comment: The contributor provides a thorough overview of the most

common causes of oxalosis, as well as the pathophysiology of calcium oxalate
deposition in the kidney. As mentioned by the contributor, ethylene glycol is the most
common cause of oxalosis in dogs and cats.

Intoxication with ethylene glycol is usually seasonal and coincides with the changing of
antifreeze solutions in the spring and fall. Many antifreeze solutions are composed of
up to 95% ethylene glycol. Toxicosis is common due to the sweet taste and very low
minimal lethal dose (1.5 ml/kg for cats and 6.6 ml/kg for dogs). Ethylene glycol itself is
of low toxicity. It is rapidly absorbed from the gastrointestinal tract and most is excreted
unchanged in the urine. Only a small percentage is metabolized in the liver to the
primary toxic metabolite glycolic acid (glycolate).1,5

The clinical signs of depression, ataxia, and osmotic diuresis develop within a few hours
of ingestion of ethylene glycol. The nervous signs are due to the effects of aldehydes
and severe metabolic acidosis, which develops due to accumulation of lactic acid,
glycolate, and glyoxylate. Pulmonary edema, tachypnea, and tachycardia develop
sequentially over the next 12 hours and are likely due to the systemic effects of the
osmotic diuresis. If the animal survives for 1-3 days after ingestion, acute renal failure
develops as a result of renal tubular damage caused by glycoaldehyde, glycolic acid,
glyoxylic acid, and oxalate. The oxalate precipitates with calcium in the renal tubular
lumina resulting in intrarenal obstruction, and tubular epithelial degeneration and
necrosis. Using polarized light, the microscopic identification of large numbers of
birefringent crystals in renal tubules is virtually pathognomonic for ethylene glycol
toxicity in dogs and cats.1,5 Alizarin red S stain will stain calcium oxalate crystals red at
a pH of 7.0, but not at a pH of 4.2. In contrast, calcium phosphate and calcium
carbonate stain red at a pH of both 7.0 and 4.2. In addition, calcium oxalate can be
confirmed by its insolubility in 2M acetic acid, since both calcium phosphate and calcium
carbonate are soluble.6

Contributor: Animal Technology Institute Taiwan, Division of Animal Medicine, P.O.

Box 23, Chunan, Miaoli, Taiwan

References:

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1. Kelly WR: The urinary system. In: Pathology of Domestic Animals, ed. Jubb KVT,
Kennedy PC, Palmer N, 4thed, vol. 2, pp. 491-494. Academic Press Inc., San Diego, CA
1993
2. Jones TC, Hunt RD and King ND: Diseases due to extraneous poisons. In:
Veterinary pathology, 6thed., pp. 725-726. Williams and Wilkins, Baltimore, MD, 1997
3. Thrall MA, Dial S M and Winder DR: ldentification of calcium oxalate monohydrate
crystal by X-ray diffraction in urine of ethylene glycol-induced dogs. Vet Pathol
22(6):625-628, 1985
4. Lekcharoensuk C, Osborne CA, Lulich JP, et al: Associations between dietary
factors in canned food and formation of calcium oxalate uroliths in dogs. Am J Vet Res
63(2):163-169, 2002
5. Confer AW, Panciera RJ: The urinary system. In: Thompson’s Special Veterinary
Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 251. Mosby, St.
Louis, MO, 2001
6. Proia AD, Brinn NT: Identification of calcium oxalate crystals using alizarin red S
stain. Arch Pathol Lab Med 109(2):186-189, 1985.

SLIDE 48
CONFERENCE 12 / CASE IV – UFSM-2 (AFIP 2940460)

Signalment: 7 year-old, castrated male, crossbred, bovine (Bos taurus).

History: The owner reported that for the last 60 days this draft ox had chronic bloat,
regurgitation of food and loss of weight. The rumen had been punctured several times
during this period to alleviate the bloat. At clinical examination the mucous membranes
were pale, there was marked bloat, and subcutaneous emphysema extended from the
ruminal area to the shoulder, lumbar and cervical areas on the left side. This ox was
from an area where squamous cell carcinoma (SCC) of the upper digestive tract is
endemic in cattle. There was heavy infestation of bracken fern (Pteridium aquilinum) in
the pasture where this ox was kept. A clinical diagnosis of SCC of the upper digestive
tract was made and the ox was euthanatized due to a poor prognosis.

Gross Pathology: Depletion of body fat deposits and pale mucous membranes were
observed at necropsy. The extensive subcutaneous emphysema of the left side of the
body was interpreted as being secondary to the ruminal punctures. Small papillomas (2-
5 mm in diameter) were present in the surface of epiglottis, soft palate and pharynx.
There was 1 liter of serous, clear yellow, partially coagulated fluid in the peritoneal
cavity. The rumen was markedly distended by gas. The distal portion of the esophagus
was distended (approximately 3 times its normal diameter), firm and contained a dark
green plug of packed ingesta (grass) in its lumen. The ruminal contents were markedly
dry and a mass, formed by multiple nodules (some edematous), protruded from the
ruminal mucosa. The ruminal wall, just at and immediately distal (8 cm) to the
esophageal entrance, was thickened (approximately 5 cm) and its cut surface was firm,
gray-white, and speckled with yellow (keratinization) and dark brown (necrosis) foci.

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This mass caused stenosis of the ruminal entrance. There was hypertrophy of the
muscular layer of the walls of rumen and reticulum. Clear translucent, gelatinous
edema was noted in the abomasal folds and multiple nodules due to
Oesophagostomum sp. were present in the wall of the small intestine. There was
atrophy of the left liver lobe and a dry, friable (sand-like) gallstone was located within
the gallbladder.

Contributor’s Morphologic Diagnoses: 1. Rumen, squamous cell carcinoma, well

differentiated, with dystrophic mineralization, bovine, 7-year-old, castrated male,
crossbred, bovine.
2. Rumen, squamous epithelial papillomas, bovine, 7-year-old, castrated male,
crossbred, bovine (slides not included).
3. Pharynx and esophagus, squamous epithelial papillomas, bovine, 7-year-old,
castrated male, crossbred, bovine (slides not included).

Contributor’s Comment: Pteridium aqulinum (bracken fern) is a plant of worldwide

distribution that causes intoxication in livestock in several regions of the world. Bracken
fern is one of the more important toxic plants for cattle in Brazil, causing severe losses
in the southern and southeastern regions. The plant also grows in other Brazilian
regions including the states of Bahia, Amazonas, Acre, Mato Grosso and Pernambuco.8

There are three clinical manifestations of the poisoning by bracken fern and they were
recently reviewed.8 When cattle graze large amounts of the plant (between 10 and 30
g/kg/bw/day or more) for relatively short periods of time (weeks to few months, generally
until the weight of ingested plant equals the weight of the animal) bone marrow aplasia
develops, which results in an acute, usually fatal, clinical disease characterized by
fever, hemorrhagic diathesis, thrombocytopenia and neutropenia. When cattle ingest
less than10 g/kg/bw/day for longer periods (one year or more), a chronic disease
characterized by intermittent hematuria is observed. This form is known as enzootic
hematuria and is related to the development of tumors in the urinary bladder.
Hemangioma is frequently found in these cases but several other types of benign and
malignant neoplasms may occur.7 Enzootic hematuria eventually leads to chronic
anemia and death. As in this case, the development of SCC in the upper digestive tract
of cattle is the third clinical manifestation related to the ingestion of bracken fern.8 The
occurrence of SCC in the digestive system of cattle is rare6 and is virtually not observed
in cattle grazing pastures where bracken fern is absent.8 The clinical course associated
with the SCC of the upper digestive tract in cattle is rather chronic (months to years)
and the deleterious effects of the tumor are mainly mechanical and related to the
interference with feeding and rumination. This leads to extreme malnutrition. Affected
cattle are usually 5 years-old or older. It is presumed that the development of the SCC
occurs when cattle ingest small amounts of bracken fern for extended periods (years) of
time. The neoplasm occurs in one or more of the following anatomical sites in the
bovine digestive tract:5,8 base of the tongue, esophagus, cardia and rumen. Clinical
signs include coughing, regurgitation of food, bloat, diarrhea and progressive loss of
weight which eventually culminate in death. The clinical signs vary depending on the

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location of the tumor, for example, coughing is related to tumors located in the base of
the tongue and as in this case, bloat is related to tumors located in the esophagus or
cardia. Metastasis occurs in some cases, mainly to the regional lymph nodes and
lungs. However, SCC of the rumen may metastasize to the liver through the portal
circulation.

A recent survey carried out by our lab that included necropsies of 14 cattle with SCC of
the upper digestive tract (yet unpublished data) revealed multiple neoplasms in 57% of
the cases and a single mass in 43%. The main affected anatomical sites were base of
tongue (5/14), pharynx (3/14), epiglottis (6/14), proximal esophagus (3/14), distal
esophagus (2/14), middle esophagus (3/14), entrance of the rumen (“cardia”) (3/14) and
rumen (2/14). Metastasis were observed in 50% of the cases and were identified in
retropharyngeal (4/14), mediastinal (2/14), gastric/ruminal (3/14), hepatic (1/14),
paravertebral (1/14) and mesenteric (1/14) lymph nodes; in the lungs (1/14) and in the
liver (1/14). Usually a few or several papillomas were observed in the proximities of the
malignant masses (SCC). Histological evidence of transition between benign
(papilloma) and malignant (SCC) growths were occasionally found. The consistent
finding of papillomas in the sites where SCC develop led to the suspicion that bovine
Papillomavirus (BPV) has a role in the pathogenesis of bracken fern associated SCC in
the upper digestive tract of cattle and this was later confirmed.3,4

There are six subtypes of BPV (BPV-1-6) that induce lesions with specific
characteristics and distributions. SCC of the upper digestive tract in cattle is associated
with prolonged ingestion of bracken fern and concomitant infection with BPV-4; whereas
BPV-2 is associated with bladder tumors and enzootic hematuria in cattle feeding on
bracken fern invaded pastures.3,4 The BPV induced papillomas are benign growths that
occasionally undergo malignant transformation due to genetic or environmental factors.
In cattle, infection of the upper digestive tract with BPV-4 leads to the formation of
papillomas which eventually regress (within approximately one year) due to a host-
derived cell mediated immune response.2 However, in cattle grazing bracken fern,
which contains immunosuppressants, the papillomas persist for longer periods and may
be transformed to carcinomas.4 In vitro studies indicate that bracken fern is the co-
carcinogen of BPV-4 in the pathogenesis of the SSC of the digestive tract in cattle. In
addition, the flavonoid quercetin, a well known mutagenic compound of bracken fern,
synergizes with BPV-4 in malignant transformation of papilloma cells.3 The combination
of increased viral BPV-4 transcriptional activity, the failure of cell arrest at G1 and the
malfunction of the tumor suppressor protein p53 are thought to be the events
contributing to transformation of the cell.1,3 In fact, a strong epidemiological correlation
between bracken fern consumption, high incidence of persistent papillomas, and SCC
of the upper digestive tract has been noted. The progression from papilloma to SCC
was experimentally reproduced in cattle fed bracken fern.4

In sheep, bracken fern causes progressive retinal degeneration referred to as bright

blindness and in horses and pigs it has been described as causing a nervous disease
due to thiamin deficiency.5 However despite the high incidence of bracken fern
poisoning in cattle, none of these conditions in sheep, pigs or horses were documented

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in Brazil. The feeding of horses with high amounts of bracken fern failed to induce the
neurological disease (Gava, personal communication).

Tumors of the digestive tract are reported in humans consuming the crosiers and
rhizomes of bracken fern. In southeastern Brazil, a case-control study showed a 3.4
and 3.45-fold increased relative risk respectively for developing esophageal and gastric
cancer in people who ingested bracken fern. Ptaquiloside, another carcinogenic
substance isolated from bracken is present in the milk of cows fed this plant and it has
been demonstrated that milk from these cows causes tumors in mice.5

AFIP Diagnosis: Rumen (per contributor): Squamous cell carcinoma, crossbred,

bovine.

Conference Comment: The contributor provides a thorough overview of bracken fern

toxicosis and the three clinical syndromes recognized in cattle, which are dependent on
the dose and duration of ingestion. As mentioned by the contributor, bovine
Papillomavirus (BPV) has a role in the pathogenesis of bracken fern associated SCC in
the upper digestive tract of cattle.

Many viruses, both DNA and RNA, can cause neoplasia in humans and animals. The
AFIP comments on conference 5, case III (October 2004) includes a list of common
virally induced neoplastic diseases. At the cellular level, one of two consequences may
follow infection with potentially oncogenic DNA viruses. If the infection is productive, the
cells produce infective virus particles and the cells are lysed in the process. If the
infection is non-productive, the cells survive and may be transformed, with introduction
of specific gene sequences or gene products. Oncogenic DNA viruses contain specific
viral oncogene products that are responsible for neoplastic transformation. The viral
oncogene products are often virus specific with particular host cell targets. From the list
below, it is apparent that many oncogenic DNA viruses share common mechanisms of
cellular transformation mediated by their specific oncogene protein products. A
prominent shared mechanism is the interaction with and functional inactivation of the
tumor-suppressor gene products, Rb and p53, both of which play critical roles in
processes responsible for cellular homeostasis, such as the cell cycle and apoptosis.9
Virus Viral Oncogene Product Cellular Target_____
Adenovirus E1A (289aa) Rb
E1A (243aa) Rb
E1B (495aa) p53
E1B (175aa) unknown

Polyomavirus Large T-antigen Rb

Middle T-antigen src
Small T-antigen unknown

SV40 Large T-antigen Rb, p53

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 Small T-antigen unknown

Papillomaviruses
BPV-1 E5 PDGF receptor
HPV-16 E6 p53
E7 Rb

Contributor: Universidade Federal de Santa Maria, Departamento de Patologia, Santa

Maria, RS, Brazil

References:
1. Beniston RG, Morgan IM, O’Brien V, Campo MS: Quercetin E7 and p53 in
papillomavirus oncogenic cell transformation. Carcinogenesis 22:1069-1076, 2001
2. Borzacchiello G, Ambrosio V, Roperto S, Poggiali F, Tsirimonakis E, Venuti A,
Campo MS, Ropero F: Bovine papillomavirus type 4 in esophageal papillomas of cattle
from the south of Italy. J Comp Path 128: 203-206, 2003
3. Campo MS, Beniston RG, Connolly JA, Grindlay GJ: Synergism between
papillomavirus and bracken fern in carcinogenesis of the upper gastrointestinal tract in
cattle and humans: quercetin and cell transformation, p. 116-122. In: Taylor JA, Smith
RT (eds) Bracken Fern: Toxicity, Biology and Control. Proceedings of the Bracken Fern
Group Conference, IV International Bracken Conference, Manchester, August, 2000
4. Campo MS: Bovine Papillomavirus and cancer. Vet J 154: 175-188, 1999
5. Gava A, Neves DS, Gava D, Saliba TM, Schild AL, Riet-Correa F: Bracken fern
(Pteridium aquilinum) poisoning in cattle in southern Brazil. Vet Human Toxicol 44: 362-
365, 2002
6. Head KW, Else RW, Dubielzig RR: Tumors of the alimentary tract. In: Tumors in
domestic animals, ed. Meuten DJ, 4th ed., pp. 443-449. Iowa State Press Ames, IO,
2002
7. Peixoto PV, França TC, Barros CSL, Tokarnia CH: Histopathological aspects of
bovine enzootic hematuria in Brazil. Pesq Vet Bras 23:65-81, 2003.
8. Tokarnia CH, Dobereiner J, Peixoto PV: Plantas Tóxicas do Brasil [Poisonous Plants
from Brazil], pp. 178-187. Helianthus, Rio de Janeiro, Brazil, 2000 (In Portuguese)
9. Cockerell GL, Cooper BJ: Disorders of cell growth and cancer biology. In:
Mechanisms of Disease, A Textbook of Comparative General Pathology, eds. Slauson
DO, Cooper BJ, 3rd ed., pp. 367-369. Mosby, St. Louis, MO, 2002

SLIDE 49
CONFERENCE 13 / CASE I – 1178/03 (AFIP 2948699)

Signalment: Four month-old, male, New Zealand White rabbit.

History: This rabbit was one of 20 rabbits in an experimental study inducing chronic
cardiomyopathy. Doxorubicin (3 mg/kg) was given intravenously in the lateral ear vein

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of three month-old male rabbits once a week for a six week period. The presented
animal died after the fourth dose and was autopsied.

Gross Pathology: Mild alopecia was visible at the neck. There was 15 ml of watery
fluid in the thorax. The myocardium was grayish-brown and the left ventricle of the
heart was mildly dilated. The lungs showed moderate diffuse acute emphysema and
edema. The testes were small and soft, and bone marrow was light-red but of normal
consistency. The spleen and lymph nodes were light-brown, small and inactive. The
gastrointestinal tract showed moderate diarrhea. The liver, kidneys and brain were
without any specific morphological lesions.

Contributor’s Morphologic Diagnoses: 1. Heart: Myocardial degeneration,

eosinophilic and vacuolar, diffuse, with mild fibrosis, and multifocal, mild, mononuclear
inflammation.
2. Bone marrow: Hypocellularity, diffuse, severe.
3. Testis: Degeneration and atrophy of the seminiferous tubular epithelium, diffuse,
moderate.
4. Skin: Epidermal and adnexal atrophy, diffuse, moderate. (not submitted)
5. Intestine: Atrophy of the intestinal mucosa, moderate, with diffuse mild
lymphoplasmacytic infiltration. (not submitted)
6. Lymph nodes, spleen: Hypocellularity, moderate. (not submitted)

Contributor’s Comment: Anthracycline antibiotics, such as doxorubicin (adriamycin),

epirubicin and daunorubicin, are effective anti-neoplastic agents which are widely used
in cancer chemotherapy. However, administration of these agents is associated with a
dose-related cardiomyopathy,1 atrophy of hematopoietic tissues,1 nephrotoxicosis,2 and
atrophy of skin and testes1,3 as well as intestinal alterations.4

Cellular mechanisms of anthracyclines inducing heart failure are multifactorial and

include local release of vasoactive substances,5 cytotoxic effects of local free radicals,6
inhibition of nucleic acid and protein synthesis,7 and disturbed Ca2+ metabolism in
cardiomyocytes.8

Acute doxorubicin toxicity is reflected by increased cytoplasmic eosinophilia and mild to

moderate vacuolization of cardiomyocytes. Ultrastructurally, numerous vacuoles,
swollen mitochondria with “onion ring” shaped cristae, and swollen sarcoplasmic
reticulum occur. Myofibrillar loss as well as separation of intercalated discs and
dilatation of the sarcotubular system is described.3 In later stages small numbers of T-
lymphocytes and histocytes surround the degenerating/necrotic myocytes9 and
interstitial fibrosis1 is evident. Chronic effects may occur several weeks or months after
repetitive exposure of cardiomyocytes to anthracycline. In humans, cardiovascular
signs indicative of chronic cardiotoxicity include severe congestive heart failure.10

Further pathological systemic side effects due to anthracycline administration are

described in organs with high turn over rate of cells leading to bone marrow depression,
alopecia and atrophy of the skin, and testicular atrophy1 as observed in the present

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case. Additionally, in long term studies at about 17 weeks, necrosis and calcification of
the liver, skeletal muscles and pancreas were observed.1
3,11
The lesions of the present case are typical findings induced by anthracyclines. Since
it is often used in experiments studying therapeutic models of cardiomyopathy, intensive
investigations of this cardiac disease are available in literature. However, the effects on
other organs are described sparsely3,11 but may be responsible for the death of animals
during experiments due to diarrhea, hemorrhagic diathesis and opportunistic infections.

AFIP Diagnoses: 1. Heart: Myocardial vacuolar degeneration, necrosis, and loss,

diffuse, mild to marked, with multifocal mild fibrosis, New Zealand White, rabbit,
lagomorph.
2. Bone marrow, hematopoietic cells: Hypocellularity, diffuse, marked.
3. Testis, spermatogenic epithelium: Degeneration and atrophy, diffuse, severe.

Conference Comment: The contributor provides a thorough overview of the dose-

dependent cardiotoxicity of adriamycin. Both nutritional and toxic myopathies tend to
result in degeneration and necrosis with little to no inflammation. However, if the animal
survives long enough, inflammation may be present in response to the necrosis.
Common causes are listed below:12,13

Disorder Cause Primary Species Affected_

Ionophore toxicity Monensin Horses, pigs
Lasalocid Many

Plant toxicity Cardiac glycosides Many

(Nerium oleander)
Lantana camara Small ruminants
Gossypol Young ruminants, pigs
Cassia occidentalis Many
Hairy Vetch (Vicia villosa) Many
Calcinogenic plants Many
(Cestrum diurnum)
(Trisetum flavescens)
(Solanum malacoxylon)

Nutritional Vitamin E/Selenium deficiency Many

Other Blister beetle (Epicauta sp.) Horses

Many conference attendees considered nutritional causes of cardiomyopathy. Vitamin

E and selenium deficiency is well recognized and has been described in many species,
including rabbits. However, with rigid quality control standards for commercial feed,
nutritional myopathy is relatively rare in laboratory colonies. Occasionally researchers

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prepare specialized diets for specific research protocols, and deficiencies can result
from errors in these formulations. Affected rabbits may present with stiffness and
muscle weakness, infertility, or increased neonatal mortality. At necropsy, the
musculature, especially of the diaphragm, paravertebral regions, and the hind limbs,
may be pale with mineralized streaks. Microscopically, there is typically hyaline
degeneration of affected myofibers and clumping and mineralization of the sarcoplasm.
Interstitial fibrosis frequently occurs in lesions of longer duration.14

Contributor: Universität Leipzig, Institut für Veterinär-Pathologie, An den Tierkliniken

33, Leipzig, Germany

References:
1. Van Fleet JF, VJ Ferrans: Clinical and pathologic features of chronic adriamycin
toxicosis in rabbits. Am J Vet Res 41:1462-1469, 1980
2. Fajardo LF, JR Eltringham, JR Stewart, MR Klauber: Adriamycin nephrotoxicity. Lab
Invest 43:242-253, 1980
3. Suzuki T, S Minamide, T Iwasaki, H Yamamoto, H Kanda: Cardiotoxicity of a new
anthracycline derivative (SM-5887) following intravenous administration to rabbits:
Comparative study with doxorubicin. Invest New Drugs 15:219-225, 1997
4. Van Fleet JF, LA Greenwood, VJ Ferrans: Pathologic features of adriamycin
toxicosis in young pigs: nonskeletal lesions. Am J Vet Res 40:1537-1552, 1979
5. Arnolda L, B McGrath, M Cocks, E Sumithran, C Johnston: Adriamycin
cardiomyopathy in the rabbit: an animal model of low output cardiac failure with
activation of vasoconstrictor mechanism. Cardiovascular Research 19:378-382, 1985
6. Gille L, H Nohl: Analyses of the molecular mechanism of adriamycin-induced
cardiotoxicity. Free Radical Biol Med 23:775-782, 1997
7. Arai M, K. Tomaru, T Takizawa, K Sekiguchi, T Yokoyama, T Asuzuki, R Nagai:
Sarcoplasmic reticulum genes are selectively down-regulated in cardiomyopathy
produced by doxorubicin in rabbits. J Mol Cell Cardiol 30:243-254, 1998
8. Huang X, W Zhu, M Kang: Study the effect of doxorubicin on expressions of genes
encoding myocardial sarcoplasmic reticulum Ca2+ transport proteins and the effect of
taurine on myocardial protection in rabbits. J of Zhejiang University Science 4(1):114-
120, 2003
9. Gaudin PB, RH Hruban, WE Beschorner, EK Kasper, JL Olson, KL Baughman, GM
Hutchins: Myocarditis associated with doxorubicin cardiotoxicity. Anat Pathol 100:158-
163, 1993
10. Herbay A, B Dörken, G Mall, M Körbling: Cardiac damage in autologous bone
marrow transplant patients: an autopsy study. Klin Wochenschr 66:1175-1181, 1988
11. Maral RJ, M Jouanne: Toxicology of daunorubicin in animals and man. Cancer
Treat Reports, 65 (Suppl 4):9-18, 1981
12. McGavin MD, Valentine BA: Muscle. In: Thompson’s Special Veterinary Pathology,
eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 214, 479. Mosby, St. Louis,
MO, 2001
13. Robinson WF, Maxie MG: The cardiovascular system. In: Pathology of Domestic
Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 3, pp. 27-29. Academic
Press, San Diego, CA, 1993

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14. Percy DH, Barthold SW: Rabbit. In: Pathology of Laboratory Rodents and Rabbits,
2nd ed., pp. 300. Iowa State Press, Ames IA, 2001

SLIDE 50
CONFERENCE 13 / CASE II – 420230 (AFIP 2948690)

Signalment: Mixed breed dog, approximately 6 weeks of age.

History: Died acutely without any clinical signs.

Gross Pathology: The pup was in good to moderate body condition. The most
prominent findings were ecchymoses in the mucosa of the digestive tract. The liver was
congested and slightly enlarged. The gallbladder wall was edematous. The spleen and
lymph nodes were edematous and congested.

Contributor’s Morphologic Diagnosis: Liver: Hepatitis, necrotizing, centrilobular,

severe, with occasional intranuclear inclusion bodies consistent with Canine Adenovirus
type 1 (CAV –1), mixed breed dog.

Contributor’s Comment: Infectious canine hepatitis (ICH) caused by canine

adenovirus -1 (CAV-1) was recognized as a specific viral disease of dogs in 1947.1 The
virus is a medium-sized DNA virus without a lipoprotein envelope. There is antigenic
relationship between CAV-1 and CAV-2, and they provide cross-protective immunity.2
Infection can cause severe disease in dogs, other canids, and also in bears (Family
Ursidae).3 The virus is ubiquitous, and is excreted in the urine of affected dogs for long
periods of time. As with other adenoviruses, CAV-1 is resistant to environmental
inactivation with chemicals such as chloroform, ether, acid and formalin. The virus
survives for days at room temperature and remains viable for months at temperatures
below 4o C. CAV-1 is inactivated after 5 minutes at 50 to 60o C. Chemical disinfection
is successful using iodine, phenol and sodium hydroxide.3

Vaccination has greatly reduced the incidence of the disease and it is now rare in many
countries. Infection with CAV-1 probably occurs in nature via the oral route.4 The
incubation period is from 4 to 7 days. Virus multiplication occurs first in the tonsils
leading to tonsillitis and local lymphadenitis, and the infection reaches the blood via the
thoracic duct. Viremia lasts between 4 to 8 days after infection and results in rapid
dissemination of the virus to other tissues and body secretions, including saliva, urine
and feces.3

The clinical signs caused by CAV-1 infections are due to cellular damage as a result of
direct effects of viral replication. The virus of ICH has a special tropism for endothelium,
mesothelium and hepatic parenchyma, and it is injury to these tissues that is
responsible for the pathologic features of edema, hemorrhage and hepatic necrosis.

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AFIP Diagnosis: Liver: Hepatocellular necrosis and loss, centrilobular and midzonal,
diffuse, with marked congestion and hemorrhage, and basophilic hepatocellular
intranuclear inclusion bodies, mixed-breed, dog.

Conference Comment: As mentioned by the contributor, due to vaccination infectious

canine hepatitis is now rare in many countries in which it was once endemic.

Clinically, affected dogs may have inapparent infection, mild illness, or severe disease
with vomiting, melena, high fever, abdominal pain, blanched mucus membranes with
petechia, and occasionally icterus.3 Virus-induced endothelial damage may lead to
disseminated intravascular coagulation and hemorrhagic diathesis.5 In the peracute
form of the disease, the animal may be found dead without previously observed clinical
signs.3 Some recovering dogs will develop an immune complex uveitis (type III
hypersensitivity) resulting in unilateral or bilateral corneal edema (blue eye).5

The virus has a special tropism for endothelium, mesothelium, and hepatic parenchyma,
resulting in gross and microscopic lesions due to cellular injury. Grossly, the classic
lesion is marked edema of the gallbladder wall. If the edema is mild, it may only be
evident in the attachments of the gallbladder. The gallbladder may also be darkened by
intramural hemorrhages.3 Other lesions include edema and hemorrhage of the
superficial lymph nodes, linear (paintbrush) hemorrhages on the serosa of the stomach,
3
widespread petechia and ecchymoses, fluid in serous cavities, fibrin strands on the
surface of an enlarged, turgid and friable liver, or small foci of hepatocellular necrosis.5
Gross lesions in other organs are inconsistent. Histologically, there may be
hemorrhages in many tissues due to the endothelial tropism and the resultant
destruction. At low magnification, the histologic changes in the liver appear similar to
those caused by acute hepatotoxins producing a prominent centrilobular (periacinar)
pattern. The virus is known to produce large, amphophilic to basophilic, solid,
intranuclear inclusion bodies that often have a “smudgy” appearance and fill the
nucleus. They may be found in hepatocytes, endothelial cells, Kupffer cells, renal
tubular epithelium, bronchial epithelium, and primitive reticulum cells.3 Ultrastructurally,
adenoviruses are nonenveloped, 70-90 nm, icosahedral particles that form
characteristic paracrystalline arrays within the nuclei of affected cells.6

Contributor: Kimron Veterinary Institute, Pathology Department, P.O. Box 12, Beit-
Dagan, Israel

References:
1. Greene CE: Infectious canine hepatitis and canine acidophil cell
hepatitis. In: Infectious Diseases of the Dog and Cat, ed. Greene CE, pp. 22-27. W.B.
Saunders Co., Philadelphia, PA, 1999
2. Swango LJ: Canine viral diseases. In: Textbook of Veterinary Internal
Medicine, ed. Ettinger SJ, pp. 303-305. W.B. Saunders Co., Philadelphia, PA, 1989

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3. Kelly WR: The liver and biliary system. In: Pathology of Domestic Animals, eds. Jubb
KVF, Kennedy PC, Palmer N, 4th ed., vol. 2, pp. 364-366. Academic Press, San Diego,
CA, 1993
4. Hoskins JD: Canine viral diseases. In: Textbook of Veterinary Internal Medicine, eds.
Ettinger SJ, Feldman EC, pp. 418-419. W.B. Saunders Co. Philadelphia, PA, 2000
5. Cullen JM, MacLachlan NJ: Liver, biliary system, and exocrine pancreas. In:
Thompson’s Special Veterinary Pathology, eds. McGavin MD, Carlton WW, Zachary JF,
3rd ed., pp. 102-103. Mosby, St. Louis, PA, 2001
6. Cheville NF: Cytopathology of viral diseases. In: Ultrastructural Pathology, An
Introduction to Interpretation. pp. 514-515. Iowa State Press, Ames, IA, 1994

SLIDE 51
CONFERENCE 13 / CASE III – 200404 (AFIP 2942014)

Signalment: Three-year-old, female, Holstein, bovine.

History: This animal was kept in a herd of cattle experimentally infected with bovine
leukemia virus (BLV). Clinical examination did not reveal any abnormalities.

Laboratory Results: BLV serology: positive; nested PCR positive for BLV-proviral
DNA

Hematology
Hct 24.0 %
Hb 10.0 g/dl
RBC 6.1 x 106/µl
MCV 39.6 fl
MCH 16.5 pg
MCHC 41.7 %
RBC morphology: normal (echinocytes)
Platelets: 347.0 x 103/µl
MPV: 7.6 fl

WBC: 16.9 x103/µl H (normal morphology)

Segm. Neu: 1.1 x103/µl (6.0 %)
Band Neu: 0.1 x103/µl (1 %)
Lymp: 14.5 x103/µl (86 %) H
3
Mono: 0.9 x10 /µl (5.5 %)
Eos: 0.3 x103/µl (1.5 %)
Baso 0.0 x103/µl (0 %)
Contributor’s Morphologic Diagnosis: Pappenheim’s stained peripheral blood
smear: Parasitemia, numerous flagellated protozoa, etiology consistent with
Trypanosoma sp.

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Contributor’s Comment: The blood smear contains on average in each 400x field two
spindle-shaped curved protozoan organisms that are between erythrocytes and
sometimes in proximity to aggregated platelets. The 30-35 µm long protozoa are
characterized by tapered ends, an undulating membrane with a flagellum up to 30 µm
long, a central nucleus and a large marginal kinetoplast. These morphological features,
and the fact that they were isolated from a cow within Germany, are most likely
consistent with Trypanosoma (Megatrypanum) theileri. These protozoan parasites
occur with worldwide distribution and can be isolated from 50 to 70% of blood cultures
from clinically healthy cattle. However, singular cases of higher parasitemia, serious
clinical disease or even death are recorded in cattle severely stressed from concurrent
disease or in newborn calves.1 Parasitemia is common in cattle herds with concurrent
BLV-infection.2 T. theileri is transmitted mechanically by many biting fly species
(Tabanus, Haematopota) and probably by ticks (Rhipicephalus, Boophilus, Ixodes). In
general, the parasitemia is very low, and the trypanosomes are found incidentally in
smears of blood or blood cell cultures. In many cases, detection is only possible after
repeated blood culture (blood-agar-plates, Eagle’s medium, BHJ-agar with 10% rabbit
blood and incubation at 28 °C or 37 °C). Therapeutic approaches are usually not
necessary.
The observed lymphocytosis in this case is related to the experimental infection of this
animal with BLV and was shown by flow cytometry to be caused by an expansion of
CD5+ sIgM+ B-lymphocytes. This is considered as characteristic for the persistent
lymphocytotic (PL) stage of the disease.

AFIP Diagnosis: Peripheral blood smear: Trypomastigotes, numerous, and a relative

lymphocytosis, Holstein, bovine.

Conference Comment: Trypanosoma theileri is classified within the phylum

Sarcomastigophora, class Zoomastigophorea, order Kinetoplastida, family
Trypanosomatidae, genus Trypanosoma, subgenus Megatrypanum. It is one of the
largest mammalian trypanosomes and is commonly an incidental finding in clinically
healthy cattle, but may cause serious disease in immunocompromised animals.3

Blood sucking arthropods, especially tabanid flies such as the common horsefly, serve
as vectors. Following ingestion during a blood meal, trypomastigotes undergo cyclic
development in the insect’s gut. The infective stages are then excreted during
subsequent feedings and enter the host through the bite wound or abrasions in the
skin.4 Other Trypanosoma spp. are listed below:3,5

Organism Transmission Species Disease / Lesions______________

T. cruzi Reduviid bugs Dogs “Chagas’ disease”; myocarditis
T. brucei Tsetse fly Ruminants “Nagana disease”; anemia
T. congolense Tsetse fly Ruminants “Nagana disease”; anemia
T. vivax Tsetse fly Ruminants Anemia
T. evansi Biting fly Many “Surra”; edema, emaciation

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T. equinum Biting fly Horses “mal de Caderas”
T. equiperdum Coitus Horses “Dourine”; genital plaques
T. gambiense Tsetse fly Humans “African Sleeping Sickness”
T. rhodesiense Tsetse fly Humans “African Sleeping Sickness”
T. cervi Horsefly Deer Nonpathogenic
T. melophagium Sheep ked Sheep Nonpathogenic

As mentioned by the contributor, T. theileri, may be pathogenic in animals that are

stressed or otherwise immunocompromised. This animal was experimentally infected
with bovine leukemia virus (BLV), the cause of enzootic bovine lymphoma. BLV is a
retrovirus and has a high incidence in dairy cattle. Transmission is primarily horizontal
via blood-sucking arthropods or by fomites. Clinical expression peaks at 6-8 years post-
infection with animals often presenting with enlargement of one or more superficial
lymph nodes. Clinical signs reflect the location of the lesions: unilateral or bilateral
exophthalmus with involvement of retrobulbar lymphoid tissue; diarrhea with
gastrointestinal involvement; congestive heart failure if the heart, most commonly the
right atrium, is affected; or posterior paresis or paralysis with nervous system
involvement. In addition, animals may have lesions in the liver and spleen with
involvement of the bone marrow and leukemia during the terminal stages of the
disease. A persistent lymphocytosis develops in approximately 30% of animals and
may occur without, or prior to, clinical expression of the disease.6

Contributor: Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal

Health, Boddenblick 5A, Greifswald – Insel Riems, Germany
www.bfav.de

References:
1. www.afip.org/vetpath/WSC/wsc01/01wsc08.pdf
2. Mammerickx M, Dekegel D. Presence of Trypanosoma theileri in herds with a
high incidence of enzootic bovine leukosis. Ann Soc Belg Med Trop 56:47-53,
1976
3. Gardiner CH, Fayer R, Dubey JP: An Atlas of Protozoal Parasites in Animal
Tissue, 2nd ed., pp. 3-4. The Armed Forces Institute of Pathology, Washington, DC,
1998
4. Schlafer DH: Trypanosoma theileri: A literature review and report of incidence in
New York cattle. Cornell Vet 69:411-425, 1979
5. Bowman DD, Lynn RC: Georgis’ Parasitology for Veterinarians, 6th ed., pp. 83-
85. W.B. Saunders Company, Philadelphia, PA, 1995
6. Searcy GP: The hemopoietic system. In: Thompson’s Special Veterinary Pathology,
eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 372-373. Mosby, St. Louis,
MO, 2001

SLIDE 52
CONFERENCE 13 / CASE IV – N04-46 (AFIP 2937499)

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Signalment: 8-year-old, female, mongrel dog, canine (Canis familiaris).

History: This animal was presented with a one-week history of hematuria and mild
constipation. A mass was palpated in the caudal abdomen during physical examination.
A pneumocystogram revealed a tumor within the urinary bladder. The growth was
located on the ventral surface near the trigone area. It was removed surgically and
submitted for examination.

Gross Pathology: A papillary mass measuring 5.5 x 5 x 2.5 cm was submitted for
histological assessment. The papillary projections were gray white and intermixed with
blood clots. The bladder wall underneath the tumor was thickened and sclerotic.

Laboratory Results: A complete blood count revealed lymphocytosis,

granulocytopenia, and polycythemia.

Lymphocytes= 57% (10.0-40.0)

Granulocytes= 37.7% (50.0-80.0)
RBC= 9.24 m/mm3 (5.5-8.5)
MCV= 75.1 fl (58.0-73.0)
Hct= 69.3% (35.0-55.0)
Hb= 19.6 g/dl (10.0-18.0)

Contributor’s Morphologic Diagnosis: Urinary bladder: Transitional cell carcinoma,

papillary, infiltrating.

Contributor’s Comment: Sections of the submitted tissue reveal multiple, tall papillary
growths covered by multiple layers of closely-packed, columnar to polygonal cells
supported by thin stalks of fibrovascular connective tissue. Tumor cells have moderate
amounts of pale eosinophilic, often vacuolated cytoplasm, with well-defined cell borders.
Tumor cell nuclei are round to oval, euchromatic, with finely granular chromatin, usually
single prominent nucleoli, and show moderate anisokaryosis. Mitotic figures are
common (1-3 per random 40x field). Small clusters of neoplastic cells infiltrate into the
stalks of the tumor, the lamina propria, submucosa, and muscle layers of the bladder
wall. Multifocally, variably sized aggregates of lymphocytes mixed with lesser numbers
of eosinophils are scattered throughout the stalks and lamina propria. Gross and
histomorphologic features of this case are characteristic of the papillary and infiltrating
variant of transitional cell carcinoma.

Neoplasia of the urinary bladder is common in dogs, relatively frequent in cats, and rare
in all other species. Cattle rarely develop urinary bladder tumors spontaneously but
have a high prevalence (as high as 25%) in endemic areas where bracken fern
(Pteridium spp.) grows.1 The etiology of bladder cancer in dogs is unknown. However,
topical insecticides containing inert petroleum products commonly used for fleas and
ticks, in addition to obesity, appear to increase the risk of bladder cancer in this species.
Tryptophan, its metabolites, and the cytotoxic drug cyclophosphamide have also been

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incriminated in the development of bladder cancer in dogs.2 In humans, occupational
exposure from employment as aniline dyes manufacturers, painters, farmers, rubber
workers, electrical workers, pesticide applicators, hairdressers, truck drivers, petroleum
and other chemical industry workers have a well-established higher risk of bladder
cancer.2 Cigarette smoking, chronic cystitis, schistosomiasis of the bladder, and certain
drugs (cyclophosphamide) are also believed to induce a higher risk of bladder tumors.3

Transitional cell carcinoma (TCC) is the most commonly diagnosed tumor in the urinary
bladder of domestic animals. Approximately, 75-90% of primary epithelial urinary
bladder neoplasms in dogs are TCC.1 This is a neoplasm of older dogs (average 9-11
years) and apparently, females are more susceptible (2:1 ratio of female to male).4,5
Breeds that may have a greater risk include Airedales, beagles, and Scottish terriers.
Nearly 90% of affected dogs present with clinical problems referable to the urinary
system such as hematuria, pollakiuria, or dysuria.4 Paraneoplastic diseases associated
with bladder tumors include hypercalcemia, cachexia, hyperestrogenism, hypertrophic
osteopathy, and, as in this case, polycythemia.1

The most common location of TCC in dogs is in the trigone area of the urinary bladder.
Most tumors are solitary and only rarely are multiple on gross examination. These
tumors are divided based on their patterns of growth as papillary (project into the
lumen), or nonpapillary (sessile or flat) and infiltrating (90% in dogs) or noninfiltrating
(10% in dogs).1

Transitional cell carcinomas are one of the most malignant neoplasms in domestic
animals. Metastases are present in the majority (50-90% of cases) of dogs at
necropsy;4,5 lungs and lymph nodes are the two most common sites, but bones6 are
frequently involved. In dogs, reported rates to regional lymph nodes are 48% and for
distant sites 51%.4,5 Some features have been associated with survival such as sex
and treatment selection. Some investigators found that spayed females survive
significantly longer than castrated males (358 days versus 132 days) and dogs that
received doxorubicin or mitoxantrone in addition to a platinum based chemotherapeutic
(either cisplatin or carboplatin) lived significantly longer than those that received only a
platinum compound (358 days versus 132 days).7

This bitch was clinically normal one month after surgical removal of the mass.
Unfortunately, the owner rejected the option of chemotherapy and there was not further
clinical follow up.

AFIP Diagnosis: Urinary bladder: Transitional cell carcinoma, papillary and infiltrating,
mixed-breed, canine.

Conference Comment: The contributor provides a thorough overview of transitional

cell carcinoma, including etiology, location, and histomorphologic categorization.

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Conference attendees also discussed common causes of a positive urine occult blood
test and how to differentiate hematuria from hemoglobinuria and myoglobinuria.

Hematuria is an increased erythrocyte concentration in the urine sediment and may be

due to pathologic urinary system hemorrhage, iatrogenic hemorrhage, or genital tract
hemorrhage. Causes of pathologic hemorrhage include the following: vascular damage
due to trauma, inflammation, or renal infarcts; poor repair of small vessels due to
thrombocytopenia, thrombocytopathia, or von Willebrand disease; or acquired or
congenital coagulopathies. Iatrogenic hemorrhage may result from trauma during
bladder palpation, cystocentesis, or catheterization. Genital tract hemorrhage is often
associated with estrus in voided samples. Hemoglobinuria is most often due to
intravascular hemolysis, while myoglobinuria is a result of muscle disease.8

With hematuria, the urine appears red and cloudy and will usually clear with
centrifugation. Erythrocytes will be present in the urine sediment. There should not be
clinical or laboratory evidence of hemolytic anemia or muscle disease. With
hemoglobinuria, the urine is red to brown and does not clear with centrifugation and
excessive numbers of erythrocytes will not be present in the sediment. There is a
concomitant hemoglobinemia as free hemoglobin will discolor plasma before it saturates
serum haptoglobin or causes hemoglobinuria. Clinically, there may be evidence of
intravascular hemolytic anemia. With myoglobinuria, the urine is also red to brown,
does not clear with centrifugation, and excessive numbers of erythrocytes will not be
present in the sediment. Unlike hemoglobinemia, the plasma will be clear and of normal
color. Clinical or laboratory evidence of muscle disease should be present rather than
evidence of anemia. To differentiate urine hemoglobin from myoglobin in the laboratory,
the addition of saturated ammonium sulfate solution will remove the color by
precipitating the hemoglobin. Conversely, ammonium sulfate solution will not
precipitate myoglobin and the urine will remain discolored. A better technique to
differentiate hemoglobin from myoglobin is spectrophotometric analysis.9

Contributor: Departamento de Patología, Facultad de Medicina Veterinaria y

Zootecnia, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad
Universitaria, Delegación Coyoacán, Mexico
http://www.veterin.unam.mx

References:
1. Meuten D.J, ed.: Tumors of the urinary system. In: Tumors in Domestic Animals, 4th
ed., pp. 524-537. Iowa State Press, Ames, Iowa, 2002
2. Morrison WB, ed.: Cancers of the urinary tract. In: Cancer in dogs and cats, pp. 569-
579. Williams & Wilkins, Baltimore, Maryland, 1998
3. Kumar V, Cotran RS, Robbins SL, eds.: The kidney and its collecting system. In:
Basic Pathology, 6th ed., pp. 468-469. WB Saunders Company, Philadelphia,
Pennsylvania, 1997
4. Norris AM, Laing EJ, Valli VEO, Withrow SJ, Macy DW, Ogilvie GK, Tomlinson J,
McCaw D, Pidgeon G, Jacobs RM: Canine bladder and urethral tumors: A retrospective
study of 115 cases (1980-1985). J Vet Intern Med 6(3):145-153, 1992

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5. Valli VE, Norris A, Jacobs RM, Laing E, Withrow S, Macy D, Tomlinson J, McCaw D,
Ogilvie GK, Pidgeon G, Henderson RA: Pathology of canine bladder and urethral
cancer and correlation with tumor progression and survival. J Comp Path 113:113-130,
1995
6. McCaw DL, Hogan PM, Shaw DP: Canine urinary bladder transitional cell carcinoma
with skull metastasis and unusual pulmonary metastases. Can Vet J 29:386-388, 1988
7. Rocha TA, Mauldin GN, Patnaik AK, Bergman PJ: Prognostic factors in dogs with
urinary bladder carcinoma. J Vet Intern Med 14:486-490, 2000
8. Stockham SL, Scott MA: Fundamentals of Veterinary Clinical Pathology, pp. 322-
333. Iowa State Press, Ames, IA, 2002
9. Gregory CR: Urinary system. In: Duncan and Prasse’s Veterinary Laboratory
Medicine, Clinical Pathology, 4th ed., pp. 240-241. Iowa State Press, Ames, IA, 2003

SLIDE 53
CONFERENCE 14 / CASE I – 25078 (AFIP 2936460)

Signalment: One-year-old, male, Basset Hound, dog.

History: The dog was admitted at the Universidade Federal de Minas Gerais (UFMG)
Veterinary Hospital with a history of lameness of the left hind limb for the past 4 months,
and progressive weight loss. Radiological changes included a periosteal proliferative
reaction in the metaphysis of the left femur suggestive of either an inflammatory or a
neoplastic lesion (in spite of the age of the dog). Samples were obtained by fine needle
aspiration for cytological exam, and a serum sample was processed for serological
diagnosis of leishmaniasis.

Gross Pathology: Soon after euthanasia, fragments of the proximal metaphysis of the
left femur were submitted for histopathology and a necropsy was not performed.

Laboratory Results: The serological tests for leishmaniasis yielded the following
results: Indirect immunofluorescence: reactive 1/160; Complement fixation: reactive
1/160; ELISA: positive

Non-neoplastic mesenchymal cells and macrophages containing intra-cytoplasmic

amastigotes were observed by microscopic examination of fine needle aspirates from
the bone lesion. Immunohistochemical staining was strongly positive for Leishmania
sp..

Contributor’s Morphologic Diagnoses: 1. Bone, periosteum: Periostitis, histio-

plasmacytic with periosteal proliferation and immature bone formation, osteoblastic
hyperplasia and hypertrophy, intense osteoclasia, and innumerous intra-cytoplasmic
amastigotes in macrophages, chronic, focal, moderate.
2. Bone marrow: Fibrosis, chronic, multifocal; and accumulation of macrophages
containing intra-cytoplasmic amastigotes, diffuse, moderate.

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Etiologic diagnosis: Protozoal periostitis

Etiology: Leishmania chagasi

Contributor’s Comment: Visceral leishmaniasis (VL) is a disease caused by protozoa

from the genus Leishmania, particularly L. donovani, L. infantum, and L. chagasi.5 VL is
a significant public health concern in several Brazilian States, including the State of
Minas Gerais. The dog is considered the most important reservoir for human VL,
particularly in urban areas.1,2,3

The clinical manifestation of canine VL is usually chronic, and associated with cachexia,
cutaneous lesions, hepatomegaly, splenomegaly, and lymphadenopathy.5 Osteo-
articular involvement in cases of VL has been described in dogs,4,6,8 and is usually due
to either the inflammatory response to the parasite or accumulation of immune
complexes in the joints.6

Although VL is widespread in Brazil, periosteal proliferation with the intensity and

localization described here is extremely uncommon. As in several other areas in Brazil,
VL is considered an emerging disease in the State of Minas Gerais. In spite of the large
number of dogs with VL presented to the UFMG Veterinary Hospital, this was the first
case in which the primary complaint was a skeletal condition due to periostitis
associated with Leishmania sp.

Joint lesions occur in approximately 37.5% of VL cases. These changes are often
associated with reluctance to walk, arthralgia, and periosteal proliferation in the
periphery of the joint.7 Interestingly, in this case the dog had no clinical signs of VL
such as lymphadenopathy, which is one of the most frequently observed clinical signs.5

The diagnostic approach employed in this case allowed us to establish the etiology of
the process to the level of classification as Leishmania sp.. However, considering the
geographic distribution of the donovani complex Leishmania species, it can be assumed
that the agent involved in this case was Leishmania chagasi, which is the agent of VL in
the New World.

Considering the significance of the dog as a major reservoir for human VL, particularly
in urban areas, it is important for clinicians to keep unusual clinical manifestations of VL
including skeletal changes in their list of differentials.

AFIP Diagnosis: Bone, proximal metaphysis of left femur (per contributor):

Osteomyelitis and periostitis, plasmacytic and histiocytic, multifocal, moderate, with
reactive bone formation, periosteal fibroplasia, and myriad intrahistiocytic amastigotes,
etiology consistent with Leishmania sp., Basset Hound, canine.

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Conference Comment: Leishmaniasis is a zoonotic disease caused by many
pathogenic species of the genus Leishmania. Histologically, the amastigotes are 2-4
_m, round to oval, with clear cytoplasm and a kinetoplast perpendicular to the nucleus.
9
The kinetoplast is a specialized mitochondrion. Organisms are usually located in the
cytoplasm of macrophages, but have also been reported in neutrophils, eosinophils,
endothelial cells and fibroblasts.6 Clinical pathology findings in cases of leishmaniasis
include hypergammaglobulinemia, hypoalbuminemia, nonregenerative anemia,
thrombocytopenia, uremia and proteinuria.7

Differential diagnoses include Histoplasma capsulatum, Sporothrix schenckii,

Trypanosoma cruzi, and Toxoplasma gondii. H. capsulatum and S. schenckii can be
differentiated by special stains for fungal organisms, such as GMS or PAS. Unlike
Leishmania sp., T. cruzi is located primarily within muscle and its kinetoplast is parallel
to the nucleus.

As the contributor stated, periosteal proliferation to this extent is an unusual finding with
leishmaniasis. More common causes of periosteal proliferation in dogs include
hypertrophic osteopathy, Hepatozoon americanum infection, craniomandibular
osteopathy, osteosarcoma and osteomyelitis.11

Although endemic throughout much of the world, there are only rare reports of
leishmaniasis in dogs in the southern and midwestern United States. In 1999, L.
infantum was diagnosed in an outbreak of foxhounds in the northeastern US. Beagles
and Bassett hounds housed in the same kennel and with a similar travel history as the
foxhounds were seronegative. The cause of the increased susceptibility of foxhounds
to leishmaniasis is unknown.10 The clinical disease depends largely on whether the
animal mounts a predominantly Th1 or Th2 response to the parasite. The development
of a Th1 immune response is important in the control of leishmanial infections. Th1
cells secrete interferon-gamma, which activates macrophages to kill the parasites.
Whereas a predominantly Th2 response results in the release of IL-4, IL-10 and IL-13
which inhibit the activation of macrophages thereby preventing the killing of leishmanial
organisms, and stimulate immunoglobulin production which may result in immune
complex deposition.7,9

Contributor: Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária,

Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
http://www.vet.ufmg.br

References:
1. Ashford DA, David JR, Freire M, David R, Sherlock I, Eulálio MC, Sampaio DP,
Badaro R: Studies on control of visceral leishmaniasis: impact of dog control on canine
and human visceral leishmaniasis in Jacobina, Bahia, Brazil. Am J Trop Med Hyg
59(1):53-57, 1998
2. Ashford RW: Leishmaniasis reservoirs and their significance in control. Clin
Dermatol 14:523-532, 1996
3. Boelaert M, Criel B, Leeuwenburg J, Van Damme W, Le Ray D, Van der Stuyft

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P: Visceral leishmaniasis control: a public health perspective. Trans R Soc Trop Med
Hyg 94(5):465-471, 2000.
4. Buracco P, Abate O, Guglielmino R, Morello E: Osteomyelitis and arthrosynovitis
associated with Leishmania donovani infection in a dog. J Small Anim Pract 38:29-30,
1997
5. Marsella R, Gopegui RR: Leishmaniasis: a re-emerging zoonosis. Int J Dermatol
37:801-814, 1998
6. McConkey SE, López A, Shaw D, Calder J: Leishmanial polyarthritis in a dog.
Can Vet J 43:607-609, 2002
7. Slappendel RJ: Canine leishmaniasis. A review based on 95 cases in The
Netherlands. Vet Q 10:1-16, 1988
8. Spreng D: Leishmanial polyarthritis in two dogs. J Small Anim Pract 34:559-
563, 1993
9. McAdam AJ, Sharpe AH: Infectious diseases. In: Pathological Basis of Disease, eds.
Kumar V, Abbas AK, Fausto N, 7th ed., pp. 403-405. Elsevier Saunders, Philadelphia,
PA, 2005
10. Gaskin AA, Schantz P, Jackson J, Birkenheuer A, Tomlinson L, Gramiccia M, Levy
M, Steurer F, Kollmar E, Hegarty BC, Ahn A, Breitschwerdt EB: Visceral leishmaniasis
in a New York foxhound kennel. J Vet Intern Med 16:34-44, 2002
11. Panciera RJ, Mathew JS, Ewing SA, Cummings CA, Drost WT, Kocan AA: Skeletal
lesions of canine hepatozoonosis caused by Hepatozoon americanum. Veterinary
Pathology 37:225-230, 2000

SLIDE 54
CONFERENCE 14 / CASE II – 03-03451 (AFIP 2948752)

Signalment: 4 month-old Arabian foal.

History: This foal had a fever for 2 days and a 4 day history of dyspnea, coughing, and
mild subcutaneous edema of the head and neck. Euthanasia was elected due to a poor
prognosis. No clinical signs appeared in other foals or the adult horses of the same
farm.

Gross Pathology: The cranioventral pulmonary areas (approximately 60-70% of total

lung surface area) were bilaterally consolidated, failed to collapse, and had a corrugated
surface. On cut section, there were random multiple and variably-sized (0.2 to 2 cm),
multifocal to coalescing abscesses that contained casseated white necrotic material.

Laboratory Results: High numbers of Rhodococcus equi were grown on culture and
seen on smears.

Contributor’s Morphologic Diagnosis: Lung: Subacute Bronchopneumonia,

multifocal to coalescing, suppurative and histiocytic with intralesional intrahistiocytic
rods consistent with Rhodococcus equi.

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Contributor’s Comment: Rhodococcus equi (R. equi) is a Gram positive facultative
intracellular pathogen that causes significant respiratory disease with an occasional
1
enteric form in foals less than 6 months of age. Histology is characterized by chronic
suppurative/pyogranulomatous bronchopneumonia and ulcerative enteritis. Although
rare, infections are also recorded in other mammals including goats. Transmission is
primarily by inhalation and rarely by ingestion and may be facilitated by poor dusty
conditions. R. equi is characterized by the presence of virulent and avirulent strains.
Despite the fact that most environmental isolates are avirulent, the isolates from
diseased foals are always virulent.2 The virulent isolates are characterized by the
presence of an 85 or 90 kb virulence-associated plasmid (Vap).3 Virulent strains can
survive within the macrophages likely due to the products of Vap genes.4

AFIP Diagnosis: Lung: Bronchopneumonia, pyogranulomatous, multifocal, severe,

with myriad intrahistiocytic coccobacilli, Arabian foal, equine.

Conference Comment: R. equi causes pyogranulomatous pneumonia with

abscessation, lymphadenitis, ulcerative enterocolitis, and less commonly, osteomyelitis
in foals. R. equi has been reported to cause lymphadenitis in swine, sheep, cattle,
llamas and cats. Disseminated infections are reported in goats, primarily causing
hepatic and pulmonary abscesses. Unlike foals, avirulent strains of R. equi may cause
disease in goats.5 Although all foals are susceptible to R. equi, those with compromised
immune systems from failure of passive transfer or combined immunodeficiency (CID),
are particularly vulnerable to diseases such as those caused by R. equi, Pneumocystis
carinii and equine adenovirus.

Contributor: Department of Veterinary Pathology-Alexandria University, Egyptian

Society of Comparative and Clinical Pathology, Alexandria, Egypt

References:
1. Lopez A: Respiratory system, thoracic cavity, and pleura. In: Thompson’s Special
Veterinary Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 167.
Mosby, St. Louis, PA, 2001
2. Haites RE, Muscatello G, Begg AP, Browning GF: Prevalence of the virulence-
associated gene of Rhodococcus equi in isolates from infected foals. J Clin Microbiol
35:1642–1644, 1997
3. Takai S, Hines SA, Sekizaki T, Nicholson VN, Alperin DC, Osaki M, Takamatus D,
Nakamura M, Suzuki K, Ogino N, Kakuda T, Dan H, Prescott JF: DNA sequence and
comparison of virulence plasmids from Rhodococcus equi ATCC 33701 and 103. Infect
Immun 68:6840–6847, 2000
4. Benoit S, Benachour A, Taouji S, Auffray Y, Hartke A: Induction of vap genes
encoded by the virulence plasmid of Rhodococcus equi during acid tolerance response.
Res Microbiol 152(5):439-49, 2001

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5. Davis WP, Steficek BA, Watson GL, Yamini B, Madarame H, Takai S, Rander JA:
Disseminated Rhodococcus equi infection in two goats. Veterinary Pathology 36:336-
339, 1999

SLIDE 55
CONFERENCE 14 / CASE III – 0303046 (AFIP 2937764)

Signalment: Approximately 6 month-old female laboratory Beagle (Canis familiaris) (9

kg).

History: Five day duration of sudden onset muscle rigidity especially noticeable in the
neck and shoulder. Muscle mass appeared to be increased.

Gross Pathology: Most proximal muscles in each limb were pale and firmer than
normal, especially in the thoracic limbs and neck. Fresh samples of affected muscles
20x10x5mm would stand out straight horizontally when held by one end (compared to
samples from normal dogs, which hang down vertically).

Laboratory Results: Elevated CPK (762 U/l compared to lab normal of 242 U/l),
normal liver enzymes, total protein, WBC normal (with no eosinophilia), RBC
parameters normal.

Contributor’s Morphologic Diagnosis: Myopathy, characterized by degeneration,

necrosis, and regeneration, with minimal to mild histiocytic inflammation.

Contributor’s Comment: The proximal muscles of the thoracic limbs were most
severely affected, but pelvic limb muscles, diaphragm, temporalis and pharyngeal
muscles were affected to a lesser extent. Section provided is from the rhomboideus or
serratus ventralis muscle, both of which were markedly affected. Microscopically, most
muscles examined, including several muscle groups that appeared grossly normal (e.g.
diaphragm and esophagus), were characterized by differing degrees of myopathy
(degeneration and regeneration). The myopathy was characterized by nuclei located
centrally instead of peripherally (minimal in mildly affected muscles), wide variation in
cross-sectional diameter, and in more extensively affected muscles by multifocal
cytoplasmic hypereosinophilia (usually in central sarcoplasm), granularity, and loss of
cross-striations. Granularity of sarcoplasm was due to abnormal organization of
myofibrils (myofibril disarray). Occasional fibers were necrotic. Inflammation
(predominantly histiocytic, with occasional neutrophils) was present in the most severely
affected muscles, but was not a prominent feature (i.e. was considered a response to
necrosis). Rarely, ring fibers were noted. Regeneration was present and characterized
by rowing of nuclei, which were plump, euchromatic, and had prominent nucleoli;
associated cytoplasm was usually slightly basophilic.

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Based upon the intense rigidity of the muscles of this dog clinically and immediately
post mortem, and on the microscopic findings, the myopathy in this dog is consistent
with a diagnosis of “myotonic dystrophy”. Many myotonic dystrophies are inherited (e.g.
in Chow Chows, Miniature Schnauzers, Staffordshire terriers, myotonic goats, and
humans), although myotonia can also occur following administration of cholesterol
lowering agents, corticosteroids, and rarely in cases of hypothyroidism.

Myotonic Dystrophies:
Myotonic dystrophy is the most common form of muscular dystrophy in humans, with an
estimated incidence of 1 in 8,000. The two types of heritable myotonic dystrophies in
people are designated as type 1 (DM1, Steinert’s disease) and type 2 (DM2, proximal
myotonic myopathy or PROMM). Both are dominantly inherited, multiorgan diseases.

Muscle pathology in both DM1 and DM2 includes central nuclei (sometimes in chains),
angular/atrophic fibers, hypertrophic fibers (hence wide variation in fiber diameter),
necrotic fibers, fibrosis, and deposition of adipose tissue.1 DM2 is known as proximal
myotonic myopathy because muscle symptoms (pain, stiffness, myotonia, and
weakness) characteristically involve proximal limb muscles.

Both DM1 and DM2 also cause a variety of extramuscular effects in a proportion of
patients. These include cardiac conduction abnormalities, cataracts, diabetes, testicular
failure, and hypogammaglobulinemia. DM1 also results in mental retardation and
skeletal abnormalities in the congenital form.1

DM1 is due to DNA (CTG)n repeats that cause a “gain-of-function” at the RNA level,
wherein (CUG)n RNA transcripts accumulate, resulting in aberrant splicing of chloride
channel pre-mRNA, loss of CIC-1 (chloride channel) protein from the membrane
surface, and therefore reduced membrane conductance to chloride.3 DM2 results from
(CCTG)n repeats having similar effects to DM1.1 Reduced membrane conductance
results in membrane hyperexcitability, with subsequent degeneration, necrosis, and
attempts at regeneration.

Naturally-occurring animal models of the human disease include the myotonic goat and
various dog breeds in which myotonia is inherited. An autosomal dominant mutation in
the goat CIC-1 gene results in reduced channel conductance and hyperexcitability.
Similarly, in miniature Schnauzer dogs, a missense mutation in CIC-1 has been
identified causing recessive myotonia congenita by a similar mechanism.4 These
models have assisted in understanding the electrophysiology and function of the
chloride channels, whereas transgenic mouse models of the human disease were used
to elucidate the RNA splicing regulation abnormalities and “gain-of-function” mechanism
of the (CTG)n and (CCTG)n repeats observed in humans.2,5

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AFIP Diagnosis: Skeletal muscle: Myocyte degeneration and necrosis, multifocal,
moderate, with regeneration, variation in fiber size, satellite cell proliferation, and
endomysial fibrosis, Beagle, canine.

Conference Comment: Muscular dystrophies (MD) are a heterogeneous group of

inherited disorders that cause progressive muscle weakness and wasting. In humans,
MDs are divided into several groups, the most common of which are X-linked MD,
autosomal MD, and myotonic dystrophy. The MDs all have similar histological lesions
of muscle degeneration, necrosis and regeneration. Clinical correlation, genetic testing
and electromyography are often used for definitive diagnosis of a specific MD.6

Myotonia refers to a sustained involuntary contraction of a group of muscles. Humans

affected with myotonic dystrophy describe “stiffness” and an inability to release their grip
after a handshake. The contributor has provided an excellent overview of the
pathogenesis of myotonic dystrophy. Alterations in the CIC-1 (chloride channel) protein
results in reduced chloride conductance, membrane hyperexcitability, and ultimately
muscle degeneration. There are several animal models of myotonic dystrophy.
Mutations in the CIC-1 (chloride channel) protein have been documented in the
myotonic (or “fainting”) goat and the miniature Schnauzer.4 Myotonic dystrophy has
also been reported in the Chow Chow and the Staffordshire Terrier. Gross lesions are
variable, primarily depending on the stage of the disease, and range from muscular
atrophy to hypertrophy. In this case, the clinical and gross findings of muscle rigidity
correlate with the diagnosis of myotonic dystrophy.

The best known example of X-linked MD in humans is Duchenne MD. The cellular
defect occurs in the gene encoding for the dystrophin protein. Dystrophin connects the
intracellular contractile apparatus and the extracellular connective tissue matrix. Animal
models include the xmd dog, mdx mouse and cats. In most species, X-linked MD
causes muscular atrophy. However, affected cats develop muscular hypertrophy and
the condition is known as “Hypertrophic feline muscular dystrophy”. The muscles of the
neck, tongue, diaphragm and pectoral girdle are most commonly affected.7

Contributor: Toxicology Battelle, Pathology Dept, Room 7-0-37A, Batelle Columbus

Labs, 505 King Ave, Columbus, Ohio
www.battelle.org

References:
1. Day JW, Ricker K, Jacobsen JF, Rasmussen LJ, Dick KA, Kress W, Schneider C,
Koch MC, Beilmen GJ, Harrison AR, Dalton JC, & Ranum LPW: Myotonic dystrophy
type 2. Neurology 60:657-664, 2003
2. Kanadia RN, Johnstone KA, Mankodi A, Lungu C, Thornton CA, Esson D, Timmers
AM, Hauswirth WW, & Swanson MS: A muscleblind knockout model for myotonic
dystrophy. Science 302:1978-1980, 2003
3. Mankodi A, Takahashi MP, Jiang H, Beck CL, Bowers WJ, Moxley RT, Cannon SC,
& Thornton CA: Expanded CUG repeats trigger aberrant splicing of CIC-1 chloride

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channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy.
Molecular Cell 10:35-44, 2002
4. Rhodes TH, Vite CH, Giger U, Patterson DF, Fahlke C, & George AL Jr.: A missense
mutation in canine CIC-1 causes recessive myotonia congenita in the dog. FEBS
Letters 456:54-58, 1999
5. Wansink DG & Wieringa B: Transgenic mouse models for myotonic dystrophy type 1
(DM1). Cytogen Genome Res 100:230-242, 2003
6. Anthony DC, Frosch MP, De Girolami U: Peripheral nerve and skeletal muscle. In:
Pathological Basis of Disease, eds. Kumar V, Abbas AK, Fausto N, 7th ed., pp. 1336-
1339. Elsevier Saunders, Philadelphia, PA, 2005
7. McGavin MD, Valentine BA: Muscle. In: Thompson’s Special Veterinary Pathology,
eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 489-497. Mosby, St. Louis,
MO, 2001

SLIDE 56
CONFERENCE 14 / CASE IV – 201602-1 (AFIP 2956376)

Signalment: 4 week-old female Simmental cow (Bos bovis).

History: This calf had watery and mucoid diarrhea over a long period of time. The calf
was presented with dehydration and acidosis. It had lesions between the digits and on
the muzzle.

Gross Pathology: At necropsy, especially in the esophagus, rumen and small intestine
there were multiple linear erosions. In the small intestine, Peyer's patches were
moderately depleted. Multiple erosions in the interdigital clefts and on the lower lip were
seen.

Laboratory Results: Serology for Bovine Viral Diarrhea virus: antigen positive /
antibody negative.

Contributor’s Morphologic Diagnosis: Rumen: Rumenitis, erosive, multifocal,

subacute, mild to moderate with single cell necrosis and hydropic degeneration of
epithelial cells, bovine.

Contributor’s Comment: Multifocally, mostly the basal layer of the epithelium and
occasionally the upper layers show hydropic degeneration (swollen cells characterized
by intracellular edema and clear pale eosinophilic vacuoles within the cytoplasm) and
there are scattered apoptotic cells (characterized by hypereosiniphilia and condensed or
karyorrhectic nucleus). There is some accumulation of lymphocytes surrounding these
degenerated epithelial cells (satellitosis). In a focal extensive area, on the epithelium,
there is a mild hyperkeratotic, orthokeratotic sometimes parakeratotic, layer with mild
serocellular crusts and there are scattered small pustules (not on all slides). The
epithelium is mildly eroded and mildly and irregularly hyperplastic.

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Etiology: Bovine pestivirus

The Bovine Viral Diarrhea virus belongs to the family Flaviviridae, genus Pestivirus. It is
a small, enveloped, positive strand RNA virus. The Bovine Viral Diarrhea virus (BVDV)
is found in cattle; but can also infect sheep, goats and pigs and has been isolated in
many wild and captive African species including the rhinoceros, giraffe and eland.
There are two biotypes--cytopathic (CP) and noncytopathic (NCP), each with two
serovars.

For the pathogenesis, the virus is shed in fluids (saliva, blood, oculonasal discharge,
urine, feces, semen, uterine secretions, amniotic fluid, fetal tissue and blood). The
primary replication takes places in the tonsils and oropharyngeal lymphoid tissues. The
virus enters circulating monocytes. It is transported to lymphoid tissues and the
subepithelial connective tissues of the dermis and the GI tract and spreads locally to the
overlying epithelial cells. The outcome depends on viral strain and virulence, immune
status of the host, whether or not the animal is pregnant, and the stage of pregnancy.
Infection with some NCP-BVDV may produce a thrombocytopenia and hemorrhage.
The virus can also inhibit macrophage chemotaxis and neutrophil function (animals may
have concurrent pneumonia or mastitis).

Acute Bovine Virus Diarrhea:

Most BVDV infections in immunocompetent, nonpregnant cattle are subclinical and of
the NCP type. Clinical disease is usually seen in seronegative, immunocompetent
cattle from 6-months to 2-years old.

Transplacental infections can occur with CP strains:

Between 50-100 days: fetal death, abortion, mummification
Between 100-150 days: congenital defects (microencephaly, cerebellar
hypoplasia, hydranencephaly, hydrocephalus, microphthalmia, thymic aplasia,
hypotrichosis, alopecia, brachygnathism, growth retardation, pulmonary hypoplasia)

Transplacental infections can also occur with NCP strains:

Before 100-125 days: immunotolerance and persistent infection (bovine fetuses
become immunocompetent at 150-200 days)
After 150-200 days: calves may be born with neutralizing antibodies and may be
unthrifty, slow growers or show no clinical signs

Persistent Infection:
Several factors have influenced the persistence of BVDV in cattle. Non-lytic infections
produced by non-cytopathic BVDV strains, and the ability to evade the host immune
response, are the primary mechanisms of persistence. In addition, some man-made
factors have provided opportunities for BVDV to persist in cattle populations. Others
mechanisms unique to BVDV probably result from its adaptation to cattle as a primary
host.

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The ability to induce fetal persistent infections is a unique aspect of BVDV
pathogenesis. An additional requirement of this mechanism is a non-lytic infection with
BVDV, which does not adversely affect fetal development and maturation. This unique
phenomenon is the primary mechanism whereby BVDV is maintained in cattle
populations providing for direct and indirect transmission. Although persistently infected
(PI) animals may represent approximately one percent of the cattle population, they
shed virus and initiate further virus replication and genetic variation. Therefore, control
and prevention programs must focus on prevention of persistent infections and
identification and removal of PI animals. Breaking the cycle of exposure of pregnant
animals in the first 125 to 150 days of gestation is the key to preventing persistent
infections.

As an RNA virus, BVDV generates mutations that precipitate antigenic changes.

Changes in the E2 glycoprotein are the primary sites of variation in neutralizing
epitopes. Recently, the phylogenetic classification of BVDV isolates as type Ia, Ib, and
II has emphasized the significance of genetic variation. Infection of immunocompetent
animals with BVDV stimulates cross-reactive antibody and provides protection from
disease due to infection with diverse strains. Due to the ease with which BVDV crosses
the placenta, the fetus may remain susceptible to infection although the pregnant dam is
protected by cross-reactive antibody. Variations in BVDV have led to vaccine failures
against fetal infection due to differences between vaccine virus and field virus.
However, continued genetic and antigenic variation is responsible for the circulation of
BVDV in susceptible cattle and the development of persistent fetal infections in
susceptible pregnant animals. The move toward multivalent vaccines is in response to
the recognition of the importance of genetic variation of BVDV.

Recently, Voges et al. reported a chronic BVDV infection in the testicles of a bull that
was previously acutely infected with the virus. The bull was not viremic and possessed
high levels of anti-BVDV antibody while shedding approximately 103CCID50 of virus/ml
of semen. Currently, studies are being conducted to determine the prevalence and
potential of chronic persistent infections that may follow acute BVDV infections. The
establishment of chronic infections would provide an additional mechanism for BVDV to
persist in cattle populations.

Recognition of the pathogenic mechanism of immunotolerant fetal persistent infections

was an important step in the evolution of BVDV control and prevention. The
identification and removal of PI animals is an important component of current prevention
and control methods. Due to the prevalence of PI animals and their shedding of BVDV
they represent a high risk and are justifiable targets of control methods. However, it is
clear that BVDV has many mechanisms at its disposal to ensure that it can persist and
be maintained in cattle populations. When this is considered, the slow progress in
preventing and controlling BVDV infections is understandable. In addition, this aspect
will be an important consideration as increased emphasis is placed on the eradication of
BVDV from cattle.

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Mucosal disease (MD) develops when immunotolerant cattle (infected with a NCP strain
in utero) are infected with a CP strain, or it may occur with introduction of an exogenous
CP virus or a mutation of the endogenous NCP virus that becomes CP. Cattle with MD
can infect other animals in the herd. The greater the genetic homogeneity between the
CP and NCP strains, the shorter the clinical course. Less similar viruses produce a
disease with a more protracted clinical course. Case fatality rates approach 100%.

Typical gross findings are:

With BVD: Erosions or shallow ulcerations of the oral cavity
With early MD: Erosions in oral and nasal mucosa, esophagus (linear), rumenal
papillae, abomasum, omasum, cecum, and colon; ulcers at the interdigital cleft, vulva
and testis; blunting of the oral papillae; Peyer's patches swollen, necrohemorrhagic +/-
diphtheritic membrane
With chronic MD: Alopecia and hyperkeratosis (especially on the neck), chronic
erosive lesions in the mouth and skin at mucocutaneous junctions, and around hooves
and horns

Typical histological findings are:

Severe, acute inflammation in intestinal mucosa, especially overlying Peyer's
patches, destruction of underlying crypts, stromal collapse, lymphocytolysis
Hyaline degeneration or fibrinoid necrosis of blood vessels; vasculitis in multiple
organs accompanied by a mild-to-moderate mononuclear cell infiltrate in the vessel
walls and perivascular tissues
Mesenteric lymph nodes and spleen: lymphocytolysis and lymphoid depletion
Erosions in the skin similar to those in the mucosa

Differential diagnoses are:

Pestiviral thrombocytopenia: similar clinical signs, diarrhea less pronounced, with
profound thrombocytopenia
Rinderpest (Paramyxoviridae-Morbillivirus): intranuclear/intracytoplasmic inclusion
bodies, syncytia
Malignant catarrhal fever (Herpesviridae - gammaherpesvirus): similar gross
findings plus conjunctivitis and corneal edema; lymphoblastic and lymphocytic
necrotizing vasculitis
Infectious bovine rhinotracheitis (Herpesviridae - alphaherpesvirus): Similar gross
findings; epithelial necrosis, intranuclear inclusions.
Diseases with oral lesions only: foot and mouth disease (Picornaviridae -
Aphthovirus); vesicular stomatitis (Rhabdoviridae-Vesiculovirus); bluetongue
(Reoviridae-Orbivirus); bovine papular stomatitis (Poxviridae-Parapoxvirus); and
necrotic stomatitis or oral necrobacillosis (Fusobacterium necrophorum)
Diseases with diarrhea only: salmonellosis (Salmonella dublin and S. typhimurium),
winter dysentery (“coronavirus”), paratuberculosis (Mycobacterium avium
paratuberculosis), and intestinal parasitism

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AFIP Diagnosis: Rumen: Rumenitis, erosive, subacute, multifocal, moderate, with
epithelial degeneration and necrosis, Simmental, bovine.

Conference Comment: The contributor has provided an excellent overview of Bovine

Viral Diarrhea virus (BVBV). BVDV is a common cause of erosive and ulcerative
lesions on epithelial and mucosal surfaces. Histopathology and additional laboratory
testing can be used to differentiate BVDV from the list of differential diagnoses that the
contributor provided. Although there is some variation among slides, the majority of
them have the erosions described by the contributor. Additional pestiviral diseases
include Border Disease in sheep and Classical Swine Fever.

Contributor: Institute of Animal Pathology, University of Bern, Laenggassstrasse 122,

Postfach, CH-3001 Bern, Switzerland
www.unibe.ch

References:
1. Ames TR: Bovine Virus Diarrhea; Mucosa Disease. In: Large Animal Internal
Medicine, ed. Smith BP, pp. 806-14, 1996
2. Baginski SG, Pevear DC, Seipel M, Sun SCC, Benetatos CA, Chunduru SK, Rice
CM, Collett MS: Mechanism of action of a pestivirus antiviral compound. PNAS
97(14):7981-6, 2000
3. Barker IK, Van Dreumel AA, Palmer N: The Alimentary System. In: Pathology of
Domestic Animals, eds., Jubb KVF, Kennedy PC, Palmer N, 4th ed. Vol. 2, pp. 149-159,
1993
4. Fischer-Tenhagen C, Hamblin C, Quandt S, Frolich, K: Serosurvey for selected
infectious disease agents in free-ranging black and white rhinoceros in Africa. J of Wildl
Dis 36(2):316-23, 2000
5. Liebler-Tenorio EM, Lanwehr A, Greiser-Wilke I, Loehr BI, Pohlenz J:
Comparative investigation of tissue alterations and distribution of BVD-viral antigen in
cattle with early onset versus late onset mucosal disease. Vet Microbiol 77(1-2):163-74,
2000
6. Murphy FA, Paul E, Gibbs J, Horzinek MC, Studdert MJ: Veterinary Virology, 3rd ed.
pp. 563-567. Academic Press, San Diego, CA, 1999
7. Voges H, Horner GW, Rowe S, Wellenberg GJ: Persistent bovine pestivirus
infection localized in the testes of an immuno-competent, non-viremic bull. Vet Microbiol
61:165–175, 1998
8. Brock KV: The persistence of bovine viral diarrhea virus. Biologicals: 31(2):133-
135, 2003

SLIDE 57
CONFERENCE 15 / CASE I – A040870057 (AFIP 2940514)

Signalment: Five year-old, female, mixed breed farm dog (Canis domesticus) weighing
approximately 23 kg.

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History: Over a period of several weeks, the dog developed progressive rear leg ataxia
that worsened on exercise and then extended to the front legs. The dog also had a
head tilt to the left and would stumble and fall to the left. During the course of the
illness, the dog was alert, but unable to rise at times (astasia). The owner elected
euthanasia, as the dog did not respond to treatment.

Gross Pathology: No overt gross findings were present on gross examination except
for slight congestion of meningeal vessels.

Laboratory Results: Fluorescent antibody testing for rabies was negative.

Immunohistochemistry using polyclonal antibody against both Toxoplasma gondii and
Neospora caninum was positive.

Contributor’s Morphologic Diagnosis: Brain, cerebrum at the level of hippocampus;

meningoencephalitis, necrogranulomatous and eosinophilic, multifocally extensive,
chronic, moderate to severe with intralesional protozoan cysts and tachyzoites.

Contributor’s Comment: Several, smooth and thin-walled, nonseptate cysts

(containing 1-2 _m bradyzoites) varying in size from 15-40 _m in diameter and individual
tachyzoites are scattered in the vicinity of necrotic foci and inflamed leptomeningeal
vessels. Meninges and Virchow-Robin spaces are moderately expanded by infiltration
of moderate to large numbers of eosinophils, macrophages, lymphocytes and plasma
cells. Glial cells including microglia and astrocytes surround the necrotic foci and
vacuolated neuropil.

Naturally occurring or experimental disease caused by Neospora caninum or a

Neospora-like coccidian has been recognized in a variety of animals including the dog,
cat, cattle, sheep, and horse as well as laboratory rodents. The dog has been recently
identified as the definitive host for the organism but other hosts may exist. The organism
has some features similar to Toxoplasma gondii, including division of tachyzoites by
endodyogeny and has both a proliferative (tachyzoite) and tissue cyst (bradyzoite)
phase.1 Although there are morphologic differences between the organisms (N.
caninum has a thicker cyst wall), differentiation based on light microscopy is problematic
and definitive diagnosis necessitates immunohistochemistry or electron microscopy. N.
caninum does not develop within a parasitophorous vacuole as does T. gondii.
Tachyzoite multiplication in both infections results in focal necrosis, followed by
inflammation. Postnatal neosporosis is less common than toxoplasmosis. Felids (both
domestic and wild) are the only definitive hosts for T. gondii. Toxoplasma can be
transmitted to intermediate hosts via oocysts in feline feces, via cysts in host tissue
(meat), and via tachyzoites transplacentally (vertical transmission).2,3

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AFIP Diagnosis: Brainstem and cerebrum, at the level of the hippocampus:
Meningoencephalitis, lymphoplasmacytic and eosinophilic, multifocal, moderate, with
protozoal cysts and tachyzoites, mixed breed, canine.

Conference Comment: Dr. J.P. Dubey, USDA, Animal Parasitic Diseases Laboratory,
performed immunohistochemistry using monoclonal antibody against both Toxoplasma
gondii and Neospora caninum. The organisms in this case are positive for N. caninum
and negative for T. gondii.

Neospora caninum is a recently recognized apicomplexan and until 1998, was

misdiagnosed as Toxoplasma gondii.2 N. caninum is a major pathogen for cattle and
dogs, and occasionally causes clinical infections in goats, sheep, horses, and deer.
Domestic dogs can be both the intermediate and the definitive host; canids are the only
known definitive host.4

N. caninum has three infectious stages: tachyzoites, tissue cysts, and oocysts. The
tachyzoites and tissue cysts are intracellular and found in the intermediate hosts.
Tachyzoites are approximately 6 x 2 _m, while cysts are round to oval, up to 107 _m
wide, and found primarily in the central nervous system. The tissue cyst wall is up to 4
_m thick and the enclosed bradyzoites are 8 x 2 _m.
N. caninum can be transmitted transplacentally in several hosts and transplacental is
the main mode of transmission in cattle. Carnivores can acquire infection by ingestion
of infected tissues. Domestic dogs will shed unsporulated oocysts in the feces, which
sporulate and become infective outside of the host. Sporulated oocysts can be found in
the soil, water, or food and are subsequently ingested by the intermediate host (cattle,
sheep, goats, horses, and dogs). Upon ingestion, sporozoites excyst, multiply, spread
to many tissues as tachyzoites, and eventually encyst as bradyzoites.4

N. caninum is a major pathogen of cattle, causing abortion and neonatal mortality. T.

gondii is a major pathogen in sheep and humans, and not of cattle. In dogs, the most
severe cases of neosporosis occur in young, congenitally infected pups. The disease
may be localized or generalized and virtually all organs may be involved, including the
skin. Neurologic signs depend on the site parasitized, but often the hind limbs are more
severely affected and often in rigid extension. Subclinically infected bitches can
transmit the parasite to their fetuses, and successive litters from the same bitch may be
born infected.4

Another differential diagnosis considered by conference attendees was Sarcocystis

canis, a related protozoan known to cause systemic illness in dogs. S. canis has been
documented in fatal visceral and neural disease in dogs. Although there are subtle
histomorphological differences between N. caninum, T. gondii, and S. canis, electron
microscopy or immunohistochemistry should be employed to positively identify the
organism.5

Contributor: Texas Veterinary Medical Diagnostic Laboratory, Texas A & M University,

6610 Amarillo Blvd. West, Amarillo, Texas

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References:
1. Storts RW, Montgomery DL: The nervous system. In: Thomson’s Special Veterinary
rd
Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3 ed., pp. 439- 440. Mosby,
St. Louis, MO, 2001
2. Gardiner GH, Fayer R, Dubey JP: An Atlas of Protozoan Parasites in Animal
Tissues, 2nd ed., pp. 53-60. Armed Forces Institute of Pathology, American Registry of
Pathology, Washington, DC, 1998
3. Summers BA, Cummings JF, De Lahunta: Veterinary neuropathology, pp. 163-169.
Mosby, St. Louis, MO, 1995
4. Dubey JP: Review of Neospora caninum and neosporosis in animals. The Korean J
of Parasitology 41(1):1-16, 2003
5. Trasti SL, Dubey JP, Webb DM, Blanchard TW, Britt J, Fritz D, Lewis RM: Fatal
visceral and neural sarcocystosis in dogs. J Comp Path 121:179-184, 1999

SLIDE 58
CONFERENCE 15 / CASE II – CASE 1 (AFIP 2942328)

Signalment: 2.5 year old, white and brown, male Boer goat (Capra hircus)

History: A 2.5 year old buck presented with a 4-month history of multifocal
proliferative, ulcerative and exudative epidermal lesions that were most severe on the
right hind limb. Initial physical exam revealed a slightly thin, mildly dehydrated goat that
was bright, alert and responsive. Orf, with a secondary bacterial infection, was
considered the most likely diagnosis. Attempted treatments included systemic
antibiotics, pain medications as well as topical antibiotics and antivirals applied to the
skin lesions. No improvement was noted over several weeks and the owner elected
humane euthanasia.

Gross Pathology: The main lesions were confined to the skin on the limbs and trunk.
The skin on these areas displayed numerous and extensive proliferative epidermal
lesions that consisted of firm, irregular, raised, gray, verrucous plaques or nodules with
extensive superficial crusting, frequent ulceration and occasional purulent exudation.
The lesions were limited to the epidermis and dermis and did not involve the
subcutaneous tissues.

Other findings at postmortem examination include:

Focal spondylosis at the level of tenth thoracic vertebral body.
Mild diffuse pulmonary edema.
Multifocal moderate peripheral lymphadenopathy.

Laboratory Results: CBC and chemistry panel showed moderate anemia with
severely decreased albumin levels. Aerobic bacterial culture resulted in isolation of
Proteus mirabilis and Pseudomonas aeruginosa. Anaerobic bacterial culture resulted in

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isolation of Fusobacterium necrophorum and Bacteroides spp.. Tissues were submitted
to Dr. A. Dela Concha at Texas A&M and viral isolation and PCR identified parapoxvirus
(orf).

Contributor’s Morphologic Diagnosis: Skin: Locally extensive epidermal

hyperplasia and intraepidermal vesiculo-pustular proliferative dermatitis with severe
superficial perivascular to diffuse lymphoplasmacytic, histiocytic and suppurative
dermatitis and dermal edema.

Contributor’s Comment: The section consists of large, exophytic, papillary projections

covered by thick serocellular crusts within which there are numerous degenerate
neutrophils, red blood cells and colonies of coccoid bacteria. The epidermis is
irregularly thickened with marked parakeratosis, occasional pustules and ballooning
degeneration of numerous keratinocytes. Individual keratinocyte necrosis and
occasional intracytoplasmic, eosinophilic inclusion bodies are present. Elongation of
rete pegs is evident. The connective tissue core within papillary projections is loose,
edematous, hemorrhagic and contains numerous small capillaries. It is heavily
infiltrated by lymphocytes, plasma cells, macrophages and neutrophils.

Contagious ecthyma is also known as contagious pustular dermatitis, infectious labial

dermatitis, scabby mouth, soremouth, lippengrind and orf (Old English for "rough").2 It
is a contagious viral skin disease of sheep and goats caused by a parapoxvirus related
to those causing pseudocowpox and bovine papular stomatitis. It can be transmitted to
humans.4 The disease is usually more severe in goats than in sheep, where it affects
primarily the lips of young animals.1,2

Typical contagious ecthyma lesions heal spontaneously over 3-4 weeks, and infection
results in partial immunity to reinfection. Atypical contagious ecthyma infections have
been described and the lesions are extremely severe and generalized and do not
spontaneously regress.1,2,3 These atypical cases have been described in Boer or Boer-
crossed goats. The virus isolated from these cases was orf virus-San Angelo 2000
(OV-SA00). This is the same type of virus isolated from this case (Dr. Dela Concha,
personal communication).

It has not been elucidated if Boer goats have a particular susceptibility to the virus or if
they are immunosuppressed in some way.2 However, the lesions described in the initial
report include lymph node depletion.5 In this case, lymph nodes were moderately
enlarged with variable sized white to pale tan areas on cut section. Microscopically, the
pale areas corresponded to large accumulations of amyloid, partially effacing normal
lymphoid follicles (lymphoid depletion).

AFIP Diagnosis: Haired skin: Dermatitis, proliferative, lymphoplasmacytic and

neutrophilic, chronic, diffuse, severe, with hyperkeratosis, intracorneal pustules,

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epidermal intracellular edema, and epidermal intracytoplasmic eosinophilic inclusion
bodies, Boer goat, caprine.

Conference Comment: Ovine parapoxvirus, the cause of contagious ecthyma,

belongs to the family Poxviridae, and genus Parapoxvirus. Contagious ecthyma is an
important disease of sheep and goats causing high morbidity and low mortality.
Transmission occurs through direct contact or indirectly through fomites. It is zoonotic;
however, lesions in humans are usually circumscribed, solitary, and confined to the
hands. Parapoxviruses infect a wide range of species, generally causing only localized
lesions. Infections of cattle, goats, sheep, and camels can be of economic importance.
Parapoxviruses also infect several terrestrial and marine wildlife species (chamois, red
deer, seals).6
In animals, gross lesions of typical ovine parapoxvirus consist of papules, pustules, and
thick crusts that occur primarily on the muzzle and mouth. However, lesions may
appear in the oral cavity, and on the eyelids, feet, or teats. Orf may prevent lambs from
suckling and severely affected animals may loose weight and be predisposed to
secondary infections.6 Histologically, the pathognomic changes include marked
proliferation of keratinocytes, extreme cell swelling resulting in ballooning degeneration,
nuclear shrinkage, and eosinophilic cytoplasmic inclusions. The virus is highly
keratinolytic, and inclusions appear to be floating in the fluid remains of the cytoplasm.
Virions average 320 x 125 nm, but vary in size and shape. Small cytoplasmic inclusions
must be differentiated from deeply basophilic keratohyaline granules and moderately
eosinophilic, larger, intracellular keratin bodies.7

Other parapoxviruses of ruminants include bovine parapoxvirus (bovine papular

stomatitis) and pseudocowpox virus. Bovine papular stomatitis (BPS) is a disease of
calves characterized by proliferative lesions in the oral cavity and esophagus, with little
to no systemic disease. BPS is transmissible to humans and results in lesions
resembling those of orf. Pseudocowpox virus causes pox lesions on the teats of cattle
and is the agent of milkers’ nodules in humans.7

Contributor: Department of Biomedical Sciences, Section of Anatomic Pathology,

College of Veterinary Medicine, Cornell University, Ithaca, New York
www.vet.cornell.edu

References:
1. de la Concha-Bermejillo A, Guo J, Zhang Z, Waldron D: Severe persistent orf in
young goats. J Vet Diag Invest 15(5):423-31, 2003
2. Guo J, Zhang Z, Edwards JF, Ermel RW, Taylor C Jr, de la Concha-Bermejillo A:
Characterization of a North American orf virus isolated from a goat with persistent,
proliferative dermatitis. Virus Res 93(2):169-79, 2003
3. Moriello KA, Cooley J: Difficult dermatologic diagnosis. Contagious viral pustular
dermatitis (orf), goatpox, dermatophilosis, dermatophytosis, bacterial pyoderma, and
mange. J Am Vet Med Assoc 218(1):19-20, 2001
4. Buttner M, Rziha HJ: Parapoxviruses: from the lesion to the viral genome. J Vet Med
B Infect Dis Vet Public Health 49(1):7-16, 2002

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5. Smith GW, Scherba G, Constable PD, Hsiao V, Behr MJ, Morin DE: Atypical
parapoxvirus infection in sheep. J Vet Intern Med 16(3):287-92, 2002
6. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ: Poxviridae. In: Veterinary
rd
Virology, 3 ed., pp. 289-291. Academic Press, San Diego, CA, 1999
7. Cheville NF: Cytopathology of viral diseases. In: Ultrastructural Pathology An
Introduction to Interpretation. pp. 497. Iowa State Press, Ames, IA, 1994

SLIDE 59
CONFERENCE 15 / CASE III – PM03-90 (AFIP 2937350)

Signalment: 4 year old, neutered female, English springer spaniel (Canis familiaris).

History: Variety of bizarre neurological and behavioral signs. These included

stumbling and a periodic tremor. She was also reported to tire quickly. MRI showed
changes suggestive of fucosidosis including swelling of the trigeminal nerve. Following
laboratory investigations she was euthanized and submitted for full necropsy.

Gross Pathology: The cadaver weighed 19 kg. No gross abnormality of the brain was
present but the trigeminal ganglia were twice the expected size. The vagosympathetic
trunk and vagus nerves were also enlarged along with the cervical dorsal root ganglia.
No other gross abnormalities were noted.

Laboratory Results: PCR screening on genomic DNA confirmed the animal was
homozygous for the mutant gene producing alpha-L-fucosidase deficiency (Animal
Health Trust, Newmarket, Suffolk, England).

Contributor’s Morphologic Diagnosis: Trigeminal ganglion/Dorsal root ganglion.

Neuronal degeneration with cytoplasmic vacuolation. Ganglionopathy, canine
fucosidosis.

Contributor’s Comment: Canine fucosidosis is a lysosomal storage disease which

affects English springer spaniels. It occurs as a result of a frameshift mutation involving
a 14 base pair deletion of exon 1 of the canine fucosidase gene.1,2 The disease is
inherited recessively. There is deficiency of the enzyme, which is present in plasma,
leukocytes and other tissues including brain, which results in defective degradation of
water-soluble glycoproteins containing fucose. Homozygotes have less than 5% of
normal enzyme activity. Heterozygotes have intermediate activity.3

Clinical signs include behavioral changes as well as motor abnormalities such as wide-
based stance and hypermetria. Male dogs may be infertile. Visual impairment has also
been reported. The onset is usually around 6 months of age and is progressive.
Affected individuals rarely survive beyond 4 years.

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Microscopically there is neuronal swelling with cytoplasmic vacuolation. The vacuoles
are large, single and displace the Nissl substance. Some are empty. Others contain
fine floccular material. Similar changes were present throughout the CNS. In the
springer spaniel, most symptoms are related to CNS pathology. It was first described in
this breed in 1982.
Fucosidosis also occurs in humans. Mental retardation is a common sign. Twelve
different mutations have been described in people and there is more widespread
involvement of organs. The condition in dogs most resembles the intermediate form of
the human disease and has been used as an experimental model for the CNS
pathology. In addition to the neuronal changes in dogs, vacuolated macrophages are
found in the meninges, perivascularly in the CNS, and in thickened peripheral nerves.4

Bone marrow transplantation has been shown to limit the severity and progression of
the disease. Enzyme activity levels rise in a range of tissues including the CNS. If
symptoms are already manifested, transplant is less effective.5 Age at marrow
transplantation has been shown to be important for survival, disease progression and
the level of enzyme activity attained.6 Gene therapy and recombinant enzymes have
also been proposed as treatment modalities for humans.

AFIP Diagnosis: Ganglion: Vacuolar change, neuronal, multifocal, marked, with

multifocal mild lymphoplasmacytic ganglionitis, English Springer Spaniel, canine.

Conference Comment: The contributor provides a thorough overview of fucosidosis,

which is a lysosomal storage disease of complex carbohydrates, specifically a defect in
the gene encoding the alpha-L-fucosidase enzyme, resulting in accumulation of fucose-
containing sphingolipids and glycoprotein fragments.

Lysosomes are key components of the “intracellular digestive tract” and contain many
hydrolytic enzymes that function as the acid milieu of the lysosomes. These lysosomal
enzymes (acid hydrolases) are synthesized in the endoplasmic reticulum and then
uniquely processed in the Golgi apparatus. Within the Golgi complex, these enzymes
undergo post-translation modification, which involves the addition of terminal mannose-
6-phosphate groups to some of the oligosaccharide side chains. This is an “address
label” that is recognized by specific receptors found on the inner surface of the Golgi
membrane. Lysosomal enzymes bind to these receptors, are segregated from other
secretory proteins, and are delivered to lysosomes in transport vesicles.7

Lysosomal acid hydrolases catalyze the breakdown of a variety of complex

macromolecules, from both metabolic turnover of intracellular organelles (autophagy)
and from phagocytosis (heterophagy). With an inherited deficiency of a functional
lysosomal enzyme, catabolism of its substrate remains incomplete, leading to
accumulation of the partially degraded insoluble metabolite within the lysosomes. As
this accumulation progresses, organelles increase in number and become enlarged,
eventually interfering with normal cell functions.7

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Lysosomal storage diseases may result from the lack of any protein essential for the
normal function of lysosomes. Defects may include reduced synthesis of lysosomal
enzymes, synthesis of a catalytically inactive protein that cross-reacts with the normal
enzyme, defects in post-translational processing of the enzyme protein, lack of an
enzyme activator, lack of a substrate activator protein, or lack of a transport protein.7

In general, the distribution of the organs affected is determined by two factors: the
tissue where most of the material to be degraded is found; and, the cells or location
where most of the degradation normally occurs. Lysosomal storage diseases can be
divided into categories bases on the biochemical nature of the accumulated metabolite:7

Disease Enzyme Deficiency Accumulating Metabolites

Glycogenosis
Type 2—Pompe disease alpha-1,4-glucosidase Glycogen

Sphingolipidoses
GM1 gangliosidosis GM1 ganglioside ß-galactosidase GM1 ganglioside,
Galactose-containing
oligosaccharides
Disease Enzyme Deficiency Accumulating Metabolites
GM2 gangliosidosis
Tay-Sachs disease Hexosaminidase-alpha subunit GM2 ganglioside
Sandhoff disease Hexosaminidase-beta subunit GM2 ganglioside, globoside
Variant AB Ganglioside activator protein BM2 ganglioside

Sulfatidoses
Metachromatic leukodystrophy Arylsulfatase A Sulfatide
Krabbe disease Galactosylceramidase Galactocerebroside
(Globoid cell leukodystrophy)
Gaucher disease Glucocerebrosidase Glucocerebroside
Niemann-Pick disease Sphingomyelinase Sphingomyelin

Mucopolysaccharidoses (MPS)
MPH I H (Hurler) alpha-L-Iduronidase Dermatan sulfate,
heparan sulfate
MPH II (Hunter) L-Iduronosulfate sulfatase

Mucolipidoses (ML)
I-cell disease (ML II) Deficiency of phosphorylating Mucopolysaccharide,
enzymes essential for the glycolipid
formation of mannose-6-phosphate
recognition marker

Other Disease of Complex Carbohydrates

Fucosidosis alpha-fucosidase Fucose-containing
sphingolipids and
glycoprotein fragments
Mannosidosis alpha-mannosidase Mannose-containing
oligosaccharides
beta-mannosidase Mannose-containing
oligosaccharides

Other Lysosomal Storage Diseases

Wolman disease Acid lipase Cholesterol esters,
triglycerides

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Neuronal Ceroid-Lipofuscinosis Unknown Unknown

Contributor: University of Liverpool, Department of Veterinary Pathology, Crown

Street, Liverpool, England
www.liv.ac.uk

References:
1. Skelly BJ, Sargan DR, Herrtage ME, Winchester BG: The molecular defect
underlying canine fucosidosis. J Med Genet 33 (4):284-288, 1996
2. Occhiodoro T, Anson DS: Isolation of the canine alpha-L-fucosidase cDNA and
definition of the fucosidosis mutation in English Springer Spaniels. Mammalian Genome
7:271-274, 1996
3. Keller CB, Lamarre J: Inherited lysosomal storage disease in an English Springer
Spaniel. J Am Vet Med Assoc 200:194-5, 1992
4. Jubb KVF, Huxtable CR: The Nervous System. In: Pathology of Domestic Animals,
eds Jubb KVF, Kennedy PC, Palmer N, 4th ed., pp. 314-315. Academic Press, San
Diego, CA 1993
5. Taylor RM, Farrow BR, Stewart GJ: Amelioration of clinical disease following bone
marrow transplantation in fucosidase-deficient dogs. Am J Med Genet 42(4): 628-632,
1992
6. Ferrara ML, Taylor RM, Stewart GJ: Age at marrow transplantation is critical for
successful treatment of canine fucosidosis. Transplantation Proceedings 24:2282-2283,
1992
7. Kumar V, Abbas AK, Fausto N: Genetic disorders. In: Robbins and Cotran
Pathologic Basis of Disease, 7th ed., pp. 158-161. Elsevier Saunders, Philadelphia, PA,
2005

SLIDE 60
CONFERENCE 15 / CASE IV – P04-5855 (AFIP 2956372)

Signalment: 5 year-old, castrated male, European domestic shorthair cat (Felis

domesticus).

History: A 5 year-old, European domestic shorthair, castrated, male cat was presented
at a veterinary clinic in The Netherlands with progressive abnormal behavior, anorexia
and loss of weight, ataxia, ocular mydriasis and nystagmus. The cat deteriorated over
the next 2 months, was euthanized, and admitted to the Pathobiology Department of the
Veterinary Faculty in Utrecht, The Netherlands.

Gross Pathology: At necropsy, gross pathologic findings were limited to the central
nervous system (CNS). Between both cerebral hemispheres there was a large mass (3
x 1.5 cm diameter). This process was well demarcated and had a yellowish aspect with
some empty spaces in it.

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Laboratory Results: Earlier PCV, blood chemistry and FIV/FeLV tests showed no
specific remarks.

Contributor’s Histopathologic Description: Microscopically, between both

hemispheres, there is a well demarcated, expansive growth that is partly encapsulated.
This mass contains many cholesterol clefts and a large amount of foamy macrophages
filled with lipophilic material. Many multinucleated giant cells with the same appearance
are seen particularly surrounding the cholesterol clefts. Between these cells there are
some fibrous strands and areas of mineralization. Multifocally, in the margins of the
process, some lymphocytes and plasma cells are present. The nervous tissue around
this mass is moderately compressed. Locally the process is continuous with the
meninges.

Contributor's morphologic diagnosis: Brain: Xanthomatous meningioma, European

domestic shorthair, (Felis domesticus).

Contributor’s Comment: Meningioma is the most common primary CNS tumor in the
cat. It rises within the meninges, has a mesodermal character and is composed of
arachnoid cap cells and occasionally pial cells.1 Usually it is in close association with
the dura, and grows expansively, compressing but seldom invading the brain. Malignant
meningiomas, which invade the surrounding brain tissue or metastasize, are rare in
cats.1,2 Meningiomas are seen more often in older cats (between 9 and 15 years old)
and there is a slight predominance towards the male gender.3 Meningiomas are quite
common incidental findings at autopsy in the aged feline.1 In cats, meningiomas have
the tendency to be multiple and often arise from the tela choroidea of the third ventricle.
They can be soft or firm and may be gritty on cut surface. They grow slowly (except for
the malignant variant) and the clinical signs associated with the tumor reflect the
neuroanatomical location of the tumor and the severity of any secondary pathology,
such as edema, hemorrhage, brain herniation or hydrocephalus.4

Meningiomas show a remarkable diversity in histopathology probably due to the fact

that both the mesoderm and neural crest contribute to the formation of the meninges.
The range of patterns recognized is: meningothelial, fibrous, transitional,
psammomatous, angiomatous, papillary, granular cell, myxoid and anaplastic
(malignant) meningioma. Most meningiomas exhibit vimentin, and less often
cytokeratin and S-100 protein immunoreactivity.5

In cats most are meningothelial or psammomatous and many have cholesterol

deposits.1 In humans xanthomatous infiltration in meningiomas has been described as
metaplastic changes and rarely xanthomatous meningiomas are documented.6,7,8 In
2004 a human case was presented with extensive xanthomatous change with focal
lymphoplasmatoid infiltration and foci of necrosis with nuclear debris and cholesterol
clefts. There were many epithelioid cells surrounding the areas of necrosis, forming
granulomas. The xanthomatous change is often the result of lipid accumulation in
meningeal cells, rather than infiltration by foam macrophages-lipid laden
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“xanthomatous” cells. They have been shown to be meningeal in nature by
immunohistochemistry and electron micrography but they also demonstrated the
presence of the macrophage marker CD68. In between the xanthomatous zones there
was presence of typical meningioma areas and there was a gradual transition from
areas of typical meningioma to xanthomatous zones.9 This suggests metaplastic
changes are occurring in the meningothelial cells and that changes are not only from
the entering of blood borne histiocytes from the bloodstream to ingest necrotic tumor
cells.6

In this cat, next to the xanthomatous changes as could be found in a cholesterol

granuloma, there are zones with evidence of neoplastic meningeal cells consistent with
a meningioma. Those areas are positive in vimentin expression and negative for S100
protein, PAS and cytokeratin. Only three cases of a xanthomatous meningioma in a cat
are documented: the case described here, in Veterinary Neuropathology and one case
earlier described as a granular cell tumor, which in our opinion, is also a xanthomatous
meningioma. It is remarkable that all three cases appear at the same site in the
meninges.1,10

AFIP Diagnosis: Brain, cerebrum and meninges: Cholesterol granuloma, focally

extensive, European domestic shorthair, feline.

Conference Comment: Although we gave careful consideration to the contributor’s

diagnosis of meningioma, we interpret the lesion in the provided sections as a
cholesterol granuloma. Many feline meningiomas contain cholesterol deposits,1 but to
our knowledge a meningioma with diffuse xanthomatous metaplasia has not been
reported in the veterinary literature. When present in feline meningiomas,
xanthomatous/cholesterinic granulomatous inflammation is often located in areas of
necrosis and hemorrhage. Although the lesion in this case is continuous with the
meninges in some sections, transition of the xanthomatous /cholesterinic
granulomatous inflammation to recognizable meningioma is not evident in the sections
examined by the conference attendees. This case was reviewed by the Department of
Neuropathology of the Armed Forces Institute of Pathology, which concurred with the
diagnosis of cholesterol granuloma. Meningiomas usually affect cats significantly older
than the one that had this lesion. Interestingly, meningiomas have been reported in
young cats with mucopolysaccharidosis I.11

In veterinary medicine, cholesterol granulomas, also known as cholesteatomas or

cholesteatosis, are most commonly seen as incidental findings in aged horses. In
horses, they frequently occur in the fourth ventricle; those that occur in the lateral
ventricle may be very large, leading to obstruction and hydrocephalus, which results in
dilation and pressure atrophy of the walls of the ventricles. Possible causes of
cholesterol granulomas include necrosis and hemorrhage or hyperlipidemia.12

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Cholesterol granulomas are also occasionally documented in other animals, including a
great plated lizard,13 and meerkats,14,15 as well as in humans.

Contributor: University of Utrecht, Faculty of Veterinary Medicine, Department of

Pathobiology, Pathology Division, Yalelaan 1, 3508 TD Utrecht, The Netherlands

References:
1. Summers BA, Cummings JF & De Lahunta A: Tumors of the central nervous system.
In: Veterinary Neuropathology, 1st ed, pp. 355-362. Mosby, St Louis, MO 1995
2. Jubb KV, Huxtable CR: The nervous system. In: Pathology of Domestic animals,
eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol 1, pp. 430-432. Academic Press,
San Diego, CA, 1993
3. Troxel MT, Vite CH, Van Winkle TJ, et al: Feline intracranial neoplasia:
Retrospective review of 160 cases (1985-2001). J Vet Intern Med 17:850-859, 2003
4. Lu D, Pocknell A, Lamb CR, Targett MP: Concurrent benign and malignant multiple
meningiomas in a cat: clinical, MRI and pathological findings. Vet Rec 152:780-782,
2003
5. Koestner A, et al: Tumors of the meninges. In: Histological classification of tumors of
the nervous system of domestic animals, 2nd ed, pp. 27-30. Armed Forces Institute of
Pathology, Washington, DC, 1999
6. Germano A, Galatioto S, La Rosa G, et al: Xanthomatous posterior pyramid
meningioma in a 2-year-old girl. Child’s Nerv Syst 13:406-411, 1997
7. Ijiri R, Tanaka Y, Hara M, Sekida K: Radiation-associated xanthomatous
meningioma in a child. Child’s Nerv Syst 16:304-308, 2000
8. Kepes JJ: Lipidized meningothelial tumor cells in “xanthomatous” meningioma
express macrophage antigen. J Neuropathol Exp Neurol 53:384-388, 1994
9. Vlodavsky E, Konstantinesku M, Pery-Eran A, Zaaroor M: Meningioma with
extensive necrotizing granulomatous changes: possible mimic of inflammatory dural
lesion. Histopathology 44:406-408, 2004
10. Mandara MT, Ricci G, Sforna M: Cerebral granular cell tumour in a cat. In: Abstract
from the 22nd meeting of the European society of veterinary pathology, 1st ed, pp.137,
University of Warmia and Mazury, Olsztyn, 2004
11. Haskins M, McGrath J. Meningiomas in young cats with mucopolysaccharidosis I. J
Neuropathol Exp Neurol. 42:664-70, 1983
12. Jubb KVF, Huxtable CR: The nervous system. In: Pathology of Domestic Animals,
eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 1, pp. 334-335. Academic Press,
San Diego, CA, 1993
13. Gyimesi ZS, Stedman NL, Crossett VR: Cholesterol granulomas in a Great Plated
Lizard Gerrhosuarus major. J Herpe Med Surg 12(3):36-39, 2002
14. Sladky KK, Dalldorf FG, Steinberg H, Wright JF, Loomis MR: Cholesterol
granulomas in three meerkats (Suricata suricatta). Vet Pathol 37:684-686, 2000
15. Blanchard TW, Lipscomb TP, Inskeep W: Department of Veterinary Pathology,
Wednesday Slide Conference 1997-1998, Conference 2, Case III, Microslide #7, pp.15-
17. The Armed Forces Institute of Pathology, Washington, DC, 1998

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SLIDE 61
CONFERENCE 16 / CASE I – 030928-41 (AFIP 2941564)

Signalment: 16-year-old, female, rhesus macaque (Macaca mulatta), nonhuman

primate.

History: This adult female rhesus monkey was originally acquired from the Delta
Primate Center, Louisiana, in 1990 by the United States Army Medical Research
Institute of Chemical Defense (USAMRICD), Aberdeen Proving Ground, Maryland and
transferred to the United States Army Medical Research Institute of Infectious Diseases
(USAMRIID) in 1992. She served as a subject on several Ebola virus protocols, and
most recently as a blood donor for a whole-blood transfer study conducted in May 2003.
The monkey was clinically normal during the study. The only anomaly noted was a
slight change in eating habits that developed in November 2002, described as being
“very picky” in her choice of foods. Although her weight was normal during the short
course of the study, she became noticeably thinner throughout the month. A routine
blood draw and clinical exam in early June demonstrated a significant weight loss of
three kilograms. By late June, the monkey was still active, but began losing hair, did not
eat regularly, and occasionally vomited. On July 7, 2003 the monkey had difficulty
standing, demonstrated abdominal discomfort, and was shaking. She was euthanatized
the following day.

Gross Pathology: The carcass was very thin, with significant loss of muscle mass and
only a small amount of subcutaneous fat over the abdominal region. The fat had a
white, chalky appearance (interpreted as steatitis). There was marked alopecia and
moderate flaking of the skin, primarily over the back and the back of the head. Mild hair
loss was present over the remainder of the body. Within the abdominal cavity there
were multiple strictures in the distal colon and distal ileum, decreasing the lumen by
60% and 85%, respectively. Both the jejunum and the ileum proximal to the stricture
were dilated up to five times normal and contained liquid fecal material (interpreted as
reflux from the cecum and colon). The liver was yellow-brown and friable (hepatic
lipidosis), and the gallbladder was distended by inspissated bile.

Gross Diagnoses: 1. Whole body: cachexia of chronic disease, moderate.

2. Subcutaneous fat: steatitis, moderate.
3. Colon and ileum: strictures, multiple, marked.
4. Liver: lipidosis, diffuse, severe.
5. Gallbladder: inspissated bile, moderate.

Contributor’s Morphologic Diagnoses: 1. Colon, serosa: Endometriosis, multifocal,

mild to moderate, with low numbers of hemosiderin-laden macrophages.
2. Uterus; ileum; and jejunum, serosal surfaces: Endometriosis, with few hemosiderin-
laden macrophages (not submitted).

Contributor’s Comment: The cause of this adult female monkey’s weight loss,
anorexia, and vomiting over the period before euthanasia was severe strictures of the

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intestinal tract, especially the ileum, which significantly decreased the passage of
ingesta. The pathogenesis of the intestinal strictures was attributed to chronic
endometriosis. The intestinal strictures resulted from constant cyclical hemorrhage and
menstruation from the ectopically located endometrial tissue within the abdominal
cavity, followed by formation of abdominal adhesions. The endometrial tissue was
present primarily on the serosa of the ileum, and to a lesser degree on the colon,
uterus, and jejunum.

Endometriosis is the condition in which normal endometrial glands and stroma occur in
abnormal locations outside the uterine cavity.1 The ectopically located endometrial
tissue physiologically responds to normal ovarian hormonal influences associated with
the menstrual cycle. The aberrant endometrial tissue undergoes monthly desquamation
and hemorrhage (menstruation), with the exception that the process occurs within the
lower abdominal cavity rather than in the uterine lumen. The entrapped hemorrhagic
menstrual fluid provokes an intense inflammatory reaction in the abdomen, often
leading to fibrosis and adhesions among the pelvic organs. Infertility, abdominal pain,
and occasionally bowel and/or urinary tract obstruction occur secondarily to the
adhesions and strictures.2

Endometriosis occurs only in humans and animal species that menstruate.2 In humans,
endometriosis affects 10% of women and often causes dysmenorrhea, pelvic pain,
infertility, and other problems; it is primarily a disorder of those in the active reproductive
stage of life, especially during the third and fourth decades.3

Similarly, endometriosis is one of the most common reproductive disorders in Old World
nonhuman primates, and has been proposed as a naturally occurring model of the
disease in humans. While the disease occurs as a spontaneous condition, it is
frequently a complication of repeated hysterotomies or caesarean sections.2 As in
humans, the most common clinical signs in affected monkeys and apes include
abdominal discomfort and infertility; in some cases, the disease is asymptomatic. Other
clinical signs include cyclical anorexia, depression, weight loss, and absence of feces
for several days; there may be palpable masses within the abdominal and pelvic
cavities.1

In nonhuman primates, endometriosis has been reported at various anatomical sites,

although lesions most commonly occur in the pelvic cavity. Macroscopically, gross
lesions often appear as soft, red-brown or white, masses of tissue adherent to the
serosa of the pelvic organs, or masses of dense connective tissue containing fluid-filled
cysts distended with brown menstrual blood (“chocolate cysts”).2 Common sites and
organs involved include the ovaries, uterine tubes, urinary bladder, and, as in this case,
the bowel. In some cases unilateral or bilateral hydroureter and hydronephrosis
develop as a result of adhesions that develop among the lower abdominal organs which
impinge upon the pelvic ureters. Infrequently, endometriosis may result in bowel
infarction.1

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Microscopically, endometriotic lesions consist of variably sized foci of normal appearing
uterine glands surrounded by typical endometrial stroma and thick bands of fibrous
connective tissue; scattered aggregates of hemosiderin-laden macrophages are often
2
present throughout the bands of connective tissue. In human pathology, the
histological diagnosis of endometriosis is satisfied if two of the three following features
are identified: endometrial glands; endometrial stroma; and hemosiderin pigment.3
While present in a few locations, the number of hemosiderin-laden macrophages
admittedly is less than overwhelming in the submitted histologic sections from this
female monkey.

Although the pathogenesis of endometriosis is not understood, three potential but not
mutually exclusive theories have been offered to explain both the origin and dispersion
of the lesions:3
1. Regurgitation theory: retrograde menstruation or reflux of endometrial tissue
through the fallopian tubes, with subsequent implantation and proliferation of
viable endometrial fragments in the abdominal cavity.
2. Metaplastic theory: endometrial tissue arises directly from coelomic
epithelium (itself the origin of the endometrium).
3. Vascular or lymphatic dissemination theory: this would explain the presence
of lesions in the lungs and lymph nodes (described in both humans and
nonhuman primates), which is not explained by the two previous hypotheses.

AFIP Diagnosis: Colon; mesentery: Endometriosis, multifocally extensive, Rhesus

macaque (Macaca mulatta), primate.

Conference Comment: The contributor provides a thorough overview of endometriosis

in human and non-human primates. Although the gross lesions of endometriosis are
often distinctive, other differentials to consider, especially in markedly fibrotic lesions,
are adenocarcinoma and retroperitoneal fibromatosis.

In nonhuman primates, intestinal adenocarcinoma occurs most commonly in the cotton-

top tamarin, which is the animal model for ulcerative colitis and associated carcinoma in
humans. Approximately 50% of colony-maintained animals develop active colitis, with
disease in 25-40% of those with active colitis progressing to colonic adenocarcinoma
after 2-5 years of captivity. Grossly the lesions are nodular to annular, firm, gray-white,
transmural, stenotic masses, often with proximal intestinal dilation or muscular
hypertrophy. Histologically, there are four subtypes of adenocarcinoma based on the
predominant cell type and growth pattern: papillary, tubular, mucinous, and signet ring.
Although the pathogenesis is not completely understood, it is thought that chronic
inflammation leads to hyperplasia and dysplasia, which may eventually progress to
adenocarcinoma.4

Retroperitoneal fibromatosis is a disorder of macaques that primarily occurs in young

animals (1-3 years of age), and is characterized by an aggressive proliferation of highly

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vascular fibrous connective tissue, usually involving the ileocecal junction. This disorder
is associated with a gammaherpesvirus - Retroperitoneal fibromatosis-associated
herpesvirus (RFHV), and with an oncovirus, Simian type D retrovirus (SRV-2). SRV-2 is
unique in its ability to induce both Simian Acquired Immunodeficiency Syndrome
(SAIDS) and retroperitoneal fibromatosis. Lesions may be localized or progressive.
Gross lesions in the localized syndrome include 1-4 cm single to multiple, firm, pale
nodules beneath the peritoneum. In the progressive syndrome, lesions may encircle
the intestines and adjacent lymph nodes leading to obstruction. Histologically, there are
proliferating fibroblasts arranged in ill-defined bundles with occasional interweaving
patterns within a disorganized matrix of collagen and reticulum fibers.5

Contributor: U.S. Army Medical Research Institute of Infectious Diseases

(USAMRIID), Pathology Division, 1425 Porter Street, Ft. Detrick, MD
http://www.usamriid.army.mil/

References:
1. Ford EW, Roberts JA, Southers JL: Urogenital system. In: Nonhuman Primates in
Biomedical Research: Diseases, eds., Bennett BT, Abee CR, Henrickson R, pp. 69-71.
Academic Press, San Diego, CA, 1998
2. Jones TC, Hunt RD, King NW: The genital system. In: Veterinary Pathology, pp.
1168-1170, 6th edition, Williams and Wilkins, Baltimore, MD, 1997
3. Crum CP: The female genital tract: Body of uterus and endometrium. In: Robbins
Pathologic Basis of Disease, eds., Cotran RS, Kumar V, Collins T, 6th edition, pp. 1057-
1058, Saunders Company, Philadelphia, PA, 1999
4. Saunders KE, Shen A, Dewhirst FE, Paster BJ, Dangler CA, Fox JG: Novel intestinal
Helicobacter species isolated from cotton-top tamarins with chronic colitis. J of Clin
Microbiol 37(1):146-151, 1999
5. Greensill J, Sheldon JA, Renwich NM, Beer BE, Norley S, Goudsmit J, Schulz TF:
Two distinct gamma-2 herpesviruses in Africa green monkeys: a second gamma-2
herpesvirus lineage among Old World primates? J of Virol 74(3):1572-1577, 2000

* Research was conducted in compliance with the Animal Welfare Act and other Federal
statutes and regulations relating to animals and experiments involving animals and
adheres to principles stated in the Guide for the Care and Use of Laboratory Animals,
National Research Council, 1996. The facility where this research was conducted is
fully accredited by the Association for Assessment and Accreditation of Laboratory
Animal Care International.

**Opinions, interpretations, conclusions, and recommendations are those of the

author(s) and are not necessarily endorsed by the U.S. Army.

SLIDE 62
CONFERENCE 16 / CASE II – CASE 2 (AFIP 2942335)

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Signalment: Female,1.5-year-old miniature donkey (Equus asinus).

History: The animal had a chronic history of crusty skin lesions near her vulva and on
her flanks. A skin biopsy was taken (tissue submitted). A month after the biopsy, the
animal was noted to be lame, with a painful, swollen knee. The animal was humanely
euthanized and submitted for a necropsy.

Gross Pathology: At the postmortem examination the animal was in poor body
condition with minimal fat stores. The skin was thickened, irregular and crusty with
variably-sized areas of alopecia and hypotrichosis. When sectioned, the skin, subcutis
and superficial fascia had hundreds of small pinpoint white granular organisms. The
scleral conjunctiva and vaginal mucosa contained dozens of similar foci. The distal
phalanges of the left and right forelimb and right hind limb were ventrally rotated 32, 20
and 40 degrees, respectively, from the hoof wall.

Contributor’s Morphologic Diagnosis: Skin Biopsy: Locally extensive, chronic

lymphoplasmacytic and granulomatous dermatitis with intradermal protozoal cysts
(Besnoitia bennetti, presumptive).

Contributor’s Comment: Throughout the dermis there are dozens of randomly

distributed, 0.5 to 1 mm diameter protozoal cysts within greatly distended and flattened
fibroblasts (vimentin positive, smooth muscle actin negative cells). The cysts have a 25
µm thick hyaline internal capsule and are engorged by hundreds of fusiform
bradyzoites, 8-9 µm long and 1-2 µm wide. There are variable numbers of
macrophages around the intact cysts and larger numbers of macrophages mixed with
lymphocytes and lesser numbers of plasma cells surround ruptured cysts. The
epidermis is multifocally ulcerated, with granulation tissue and necrotic cell debris mixed
with lymphocytes and plasma cells. The superficial dermis is moderately and diffusely
expanded by dense fibrous connective tissue, with a mild to moderate perivascular to
interstitial infiltrate of lymphocytes, macrophages, plasma cells and rare eosinophils.

Cutaneous besnoitiosis is a serious skin condition of cattle and horses characterized by

painful swellings and thickening of the skin with loss of hair. Besnoitiosis is a rare
disease in equines. It has been reported only in animals with a history of individual or
herd travel outside the United States. Besnoitia besnoiti has been reported from
southern Europe, Africa, Asia, and South America, but it has not been reported in cattle
in the USA. Besnoitia bennetti has been reported from Africa, southern France, and in
two imported burros in the USA.5 However, this is the second case in a donkey from
the northeastern USA that our service has diagnosed in the last two months.

Besnoitiosis is a contagious protozoal disease of various domestic and wild animals,

including horses, burros, cattle, rodents, goats, antelope, reindeer, caribou and mule
deer.1 It is caused by members of the genus Besnoitia, which are characterized by
having cysts containing bradyzoites within fibroblasts. The apicomplexan parasites
under the subphylum Sporozoa, class Coccidian, and within the family Sarcocystidae
include four genera: Toxoplasma, Sarcocystis, Besnoitia and Hammondia. The genus

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Besnoitia has the following species: B. bennetti (horses and donkeys), B. besnoiti
(cattle), B. caprae (goats), B. oryctofelisi (rabbits) B. darlingi (lizards), B. jellisoni
(kangaroo and opossum), B. tarandi (reindeer and caribou), and B. wallacei (mouse).1-2
The old name of the parasite was Globidium spp , which describes the characteristic
large, thick-walled cysts filled with bradyzoites.4 The life cycle involves a definitive host
and an intermediate host. The cat has been identified as the definitive host for B.
besnoiti, B. wallacei, and B. darlingi.3 In the intermediate host, Besnoitia is found in the
dermis, subcutaneous tissues and fasciae. The parasite produces characteristic thick-
walled cysts containing bradyzoites within fibroblasts. It has been speculated that a
biting insect vector spreads Besnoitia between intermediate hosts, but this is not
proven. Besnoitia tissue cysts are characterized by hypertrophy of the infected host
cell.2

AFIP Diagnosis: Haired skin: Dermatitis, chronic-active and eosinophilic, diffuse,

moderate, with numerous intradermal protozoal cysts, etiology consistent with Besnoitia
sp., miniature donkey, equine.

Conference Comment: As mentioned by the contributor, Besnoitia sp. are

apicomplexan parasites within the class Sporozoasida, order Eucoccidiorida, family
Sarcocystidae. Other genera of the family Sarcocystidae include Toxoplasma,
Sarcocystis, Neospora, Hammondia, Cystoisospora, Frenkelia, and Atoxoplasma.6

All members of the family Sarcocystidae have a motile stage with apical complex, have
a simple resistant spore, and undergo both sexual and asexual reproduction. Sexual
reproduction results in the production of oocysts with two sporocysts in the intestine of
the definitive host, while asexual reproduction results in spore formation within the
intermediate host.6

Gross lesions caused by organisms of this family vary, but infection often results in
acute necrosis from migration and multiplication of the tachyzoites and little tissue
damage in organs with cysts containing bradyzoites. However, if the cysts rupture, the
organisms often incite a granulomatous response. Histologically, these organisms
appear very similar and immunohistochemistry or electron microscopy is needed for a
definitive diagnosis.

Ultrastructurally, bradyzoites are found in the cytoplasm within a parasitophorous

vacuole, which constitutes the innermost cyst wall layer. This is lined by a thin granular
layer, which often contains one or more host cell nuclei. The outermost layer
(secondary cyst wall) surrounds the host cell. The structures which help identify the
organism as an apicomplexan parasite include a conoid, rhoptries, and micronemes.2
The presence or absence, number, electron density, and/or location of each of these, as
well as other organelles, assist in identifying the organism to the genus level.

Contributor:

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Department of Biomedical Sciences, Section of Anatomic Pathology, College of
Veterinary Medicine, Cornell University, Ithaca NY
www.vet.cornell.edu

References:
1. Dubey JP, Sreekumar C, Lindsay DS, Hill D, Rosenthal BM, Venturini L, Venturini
MC, Greiner EC Besnoitia oryctofelisi n. sp. (Protozoa: Apicomplexa) from domestic
rabbits. Parasitology. 126(Pt 6):521-39, 2003
2. Dubey JP, Lindsay DS. Development and ultrastructure of Besnoitia oryctofelisi
tachyzoites, tissue cysts, bradyzoites, schizonts and merozoites. Int J Parasitol.
30;33(8):807-19, 2003
3. van Heerden J, Els HJ, Raubenheimer EJ, Williams JH. Besnoitiosis in a horse. J S
Afr Vet Assoc. 64(2):92-5, 1993
4. Schulz K.C.A. and Thorburn J.A. Globidiosis- A cause of dermitis in a horse. J S Afr
Vet Assoc. 26(1) 39-43, 1955
5. Besnoitia bennetti in two Mexican burros. Terrell TG, Stookey JL. Vet Pathol.
10(2):177-84, 1973
6. Gardiner CH, Fayer R, Dubey JP: An atlas of protozoan parasites in animal tissues,
2nd ed., pp. 2. The Armed Forces Institute of Pathology, The American Registry of
Pathology, Washington, DC, 1998

SLIDE 63
CONFERENCE 16 / CASE III – 030926-44 (AFIP 2940303)

Signalment: Adult, female African green monkey (Chlorocebus aethiops), nonhuman

primate.

History: This 4.3 kg female African green monkey (AGM) was procured from St.
Kitts/Primate Products. The monkey was assigned to a protocol, but had not been
exposed to any agent. Six weeks after arriving at the institute, blood was noted around
the anogenital region by animal care technicians (day 1). Menses was suspected, but
the condition was reported to Veterinary Medicine Division personnel after the animal
had decreased appetite over the weekend.

Upon examination by a veterinarian, the suspected menses had persisted for 5 days
(the normal menstrual period in AGMs is about 1-3 days, usually with scant discharge).
Under ketamine sedation, the monkey had a body temperature of 100.7°F, pulse = 180,
and respiratory rate = 28. The animal had lost 0.5 kg from the previous weight taken 7
days earlier. Formed stool with fresh blood and loose, stringy clots were noted in the
catch pan beneath the cage. There was moderate periodontal disease with worn,
stained teeth. Abdominal palpation was within normal limits. No vaginal bleeding was
found; only rectal bleeding with decreased anal tone.

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The differential diagnosis included stress-induced, dietary-related, or idiopathic
inflammatory bowel disease; hemorrhoids, polyps, or neoplasia; and infection with
intestinal pathogens such as Campylobacter, Shigella, Salmonella, Yersinia, or
enteropathogenic Escherichia coli. Although this monkey was never positive for
intestinal parasites, she was previously housed in a room where one monkey was
diagnosed with whipworms (Trichuris sp.), so all monkeys in the room were treated at
that time.

For initial work-up, a complete blood count (CBC), serum chemistry, clotting times, and
fecal exam were ordered (results below). Gingival and anal swabs were submitted for
bacterial culture. A B-vitamin injection was given to stimulate eating, and high-water
content fruit treats were offered along with the regular diet. A fruit-flavored, sweetened
water solution (Prang) replaced the monkey’s normal water supply.

On day 6, the monkey showed no signs of improvement. She remained anorexic and
continued to pass blood, but no stool. Based on the unremarkable laboratory results,
flat film abdominal radiographs and a proctoscopic examination were ordered. A
possible right side colonic stricture was noted on radiographs. Endoscopy revealed an
advanced, severe hemorrhagic colitis. Difficulty was encountered in trying to pass the
endoscope 30-33 cm (12-13 inches) proximal to the anus. The monkey was
administered a broad spectrum antibiotic (enrofloxacin), subcutaneous fluids and given
another B-vitamin injection. Exploratory surgery was planned for the following day.

Exploratory abdominal surgery revealed segmental areas of hemorrhage, restricted to

the colon, that were visible through the serosa. Within the proximal colon, a 10-13 cm
(4-5 inch) section contained markedly thickened mucosa, with hemorrhage and
sloughed pseudomembrane formation that completely occluded the lumen. Additional
colonic and gingival culture swabs were obtained. The decision to euthanize the
monkey was based on the monkey’s clinical history, the fact that she had lost 10-12% of
her body weight and continued to deteriorate, and the severity and extent of the lesion
found at surgery.

Gross Pathology: The body presented for necropsy was that of an adult, female
African green monkey (C. aethiops). The carcass was in good body condition with
adequate subcutaneous and cavitary fat. There was moderate periodontal disease with
gingivitis, gingival hyperplasia, and worn canines. The stomach was empty and the
small intestine contained a small amount of gas. Colon walls were edematous with
swollen rugae and there were segmental areas of hemorrhage with blood clots in the
lumen. Other areas in the colon were ulcerated or had a sloughed,
pseudomembranous mucosal lining with dry, adherent fecal material.

Gross diagnoses: 1. Colon: Colitis, ulcerative and hemorrhagic, segmental,

moderate, with pseudomembrane.
2. Gingiva: Gingivitis, multifocal, moderate, with mild gingival hyperplasia.

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Laboratory Results: Initial work-up (Day 5)
Serum chemistry panel values were within normal limits, except for slightly elevated
blood urea nitrogen, alkaline phosphatase, and triglycerides; albumin was slightly low,
but the total protein was within normal limits. CBC values were all within normal limits
(no anemia and no inflammatory leukogram). Fecal exam revealed no significant
findings. Clotting times were within normal limits.
Bacterial culture results: Bacteria in the genus Shigella were isolated from the rectum,
colon, and gingiva (growth was not speciated).

Contributor’s Morphologic Diagnosis: Colon: Colitis, ulcerative and hemorrhagic,

subacute, multifocal, moderate, with crypt abscesses and abundant luminal
fibrinohemorrhagic and cellular debris (pseudomembrane), African green monkey
(Chlorocebus aethiops), nonhuman primate.

Contributor’s Comment: Histologically within the submitted section of colon, there are
focally extensive areas of mucosal ulceration that are covered by a layer of sloughed
mucosal epithelial cells, fibrin, hemorrhage, and necrotic debris. The lamina propria is
expanded by many lymphocytes and plasma cells that widely separate crypts. Colonic
crypts are often dilated and filled with many viable and degenerate neutrophils, mucus,
and cellular debris (crypt abscesses). Subacute inflammation extends into the
edematous submucosa where lymphatics are ectatic. Multifocally, similar inflammation
and focal areas of hemorrhage are present within the tunica serosa.

By immunohistochemistry using a polyclonal anti-Shigella antibody, there is strong

staining of necrotic mucosal epithelial cells and luminal necrohemorrhagic debris within
affected sections of colon.

Shigella are gram-negative, non-motile, aerobic and facultatively anaerobic bacilli from
the family Enterobacteriaciae.1 S. dysenteriae, flexneri, boydii, and sonnei are highly
infectious strains that can cause dysentery in humans with an ID50 of only 100-200
bacteria.2 Nonhuman primates usually acquire the zoonotic infection from humans via a
fecal-oral route and endemic infections can be maintained in monkey colonies via
asymptomatic carriers.1 Nonenteric Shigella infections in monkeys with gingivitis, air
sacculitis, and abortion have also been reported.1 The pathogenesis of diarrhea or
dysentery among the strains is similar, with a typical incubation period of 1-4 days
followed by watery and mucoid diarrhea mixed with blood. Although clinical disease
usually requires a stressor in endemically infected monkeys, it is typically self-limiting in
adults, requiring minimal supportive care.

Studies on the pathogenesis of Shigella have revealed unique methods of mucosal

invasion that result in the lesions seen with infection. Because most lesions are often
centered on gut-associated lymphoid tissue (GALT) and spread outward, it is suspected
that the bacteria make their initial entry into the body through the normally phagocytic M
cells overlying the lymphoid tissue.2 Additional studies have revealed that through a
complex process involving multiple genes found on both a large plasmid and on the
Shigella chromosome, attachment of the bacteria to mucosal epithelial cells stimulates a

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structural alteration of the normally non-phagocytic epithelial cell cytoskeleton and actin
filaments to cause uptake of the organism in a manner similar to phagocytosis. Once
within the intracellular vacuole of the invaded cell, a hemolysin produced by Shigella
causes release of the organism into the cytoplasm. The Shigella then rapidly multiply
and migrate along polymerized actin filaments to reach the plasma membrane so that
adjacent cells can be invaded.3 Early in the course of disease, low numbers of Shigella
organisms can be found by electron microscopy within mucosal epithelial cell vacuoles.
As the disease progresses, though, fibrinous exudate replaces the dead epithelial cells.4
Death of epithelial cells and sloughing of mucosa creates the ulceration,
pseudomembrane formation, hemorrhage, and inflammatory response that typifies
shigellosis.

An additional aspect of virulence involves the production of an exotoxin, shiga toxin, by

S. dysenteriae. Released during host cell lysis, shiga toxin stops host cell protein
synthesis by inactivating the 60S ribosomal subunit (similar to the method of action of
the plant toxin, ricin). Shiga toxin exerts effects similar to enterotoxins, neurotoxins, and
cytotoxins, and can induce apoptosis in epithelial cells. Shiga toxin also enhances the
lipopolysaccharide-mediated release of cytokines, such as interleukin-1 and tumor
necrosis factor-alpha, which likely contributes to the vascular damage leading to renal
failure seen in a complication of shigellosis, hemolytic uremic syndrome.2

AFIP Diagnosis: Colon: Colitis, necrotizing, subacute, diffuse, moderate, with a

fibrinohemorrhagic pseudomembrane, African green monkey (Chlorocebus aethiops),
primate.

Conference Comment: There is variation in slides with some slides exhibiting

ulceration of the colonic mucosa, while others are only eroded. In this case,
immunohistochemistry reveals that most of the Shigella organisms are located within
the pseudomembrane and along the epithelial border, with few organisms found in the
submucosa. However, in other cases, it is not uncommon to find moderate numbers of
organisms within the submucosa.

Lesions of enteric shigellosis, as in this case, are primarily in the cecum and colon. The
intestinal walls are thickened and edematous with luminal contents varying from fluid
mucus with fibrin and cellular debris to frank hemorrhage, multifocal ulcerations and
pseudomembrane (diphtheritic membrane) formation. Nonenteric Shigella infections
have been reported, including gingivitis, abortion, and air sacculitis. With gingivitis, the
gums are swollen, hyperemic, with scattered yellow-white foci of necrosis. Severely
affected monkeys may have gingival recession and root exposure.1,4

The differential diagnosis should include yersiniosis, salmonellosis, and Campylobacter-

associated enteritis, as well as Clostridium piliforme and E. coli. Definitive diagnosis
requires culture of the organism from a rectal swab or fresh stool specimen.1,4

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Contributor: U.S. Army Medical Research Institute of Infectious Diseases
(USAMRIID), Pathology Division, 1425 Porter Street, Fort Detrick, MD
http://www.usamriid.army.mil/

References:
1. Gibson SV: Bacterial and mycotic diseases: Shigella. In: Nonhuman Primates in
Biomedical Research Diseases, eds., Bennett BT, Abee CR, Henrickson R, pp. 69-71.
Academic Press, San Diego, CA, 1998
2. Salyers AA, Whitt DD: Dysentery caused by Shigella species. In: Bacterial
Pathogenesis: A Molecular Approach, pp. 169-181. ASM Press, Washington, DC, 1994
3. Keusch GT, Thea DM: Invasive and tissue-damaging enteric bacterial pathogens:
bloody diarrhea and dysentery. In: Mechanisms of Microbial Disease, eds. Schaechter
M, Medoff G, Eisenstein BI, 2nd ed., pp. 267-272. Williams & Wilkins, Baltimore, MD,
1993
4. Brady AG, Morton DG: Digestive system: small and large intestine. In: Nonhuman
Primates in Biomedical Research Diseases, eds., Bennett BT, Abee CR, Henrickson R,
pp. 392-393. Academic Press, San Diego, CA, 1998

* Research was conducted in compliance with the Animal Welfare Act and other federal
statutes and regulations relating to animals and experiments involving animals and
adheres to principles stated in the Guide for the Care and Use of Laboratory Animals,
National Research Council, 1996. The facility where this research was conducted is
fully accredited by the Association for Assessment and Accreditation of Laboratory
Animal Care International.

**Opinions, interpretations, conclusions, and recommendations are those of the author

and are not necessarily endorsed by the U.S. Army.

SLIDE 64 & 65
CONFERENCE 16 / CASE IV – 04011035 (AFIP 2948689)

Signalment: 5-year-old, intact female, Shetland sheepdog.

History: (Per clinician) This canine patient was previously diagnosed with Ehrlichiosis
and had been on a Imidocarb therapy regimen. Five months following the initial
diagnosis of Ehrlichiosis, the dog presented with clinical signs of ataxia, vestibular
signs, lethargy and pale mucus membranes. The patient exhibited anemia and
thrombocytopenia. After a month, the neurological signs worsened and the dog started
falling while walking. The PCV and platelet count decreased sharply. Horizontal, rotary
and positional nystagmus appeared. Craniopropioceptive deficits were observed in the
rear limbs and right front limb. Based upon a neurological examination, central
vestibular disease, a cerebral cortical lesion, and a lesion between T2-T3 were
suspected. Pulmonary radiographs revealed increased interstitial opacity. The dog
continued to grow weaker with declining PCV and platelet count. The dog developed

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hypoalbuminemia (1.5 g/L) and a markedly distended abdomen. The dog died in
respiratory distress.

Gross Pathology: At necropsy, the dog was in poor nutritional condition characterized
by prominent bony protuberances and absence of visceral and subcutaneous adipose
tissue. The mucous membranes and subcutis were mildly icteric. The subcutaneous
tissue in the ventral caudal cervical and cranial thoracic region was focally wet and jelly-
like (subcutaneous edema). Large volumes of serofibrinous effusions were present
within the peritoneum (2L) and pleural cavity (1L). The liver contained multiple,
randomly scattered nodules with pale yellow margins and indented, soft, friable
(necrotic) centers. Multiple, discrete, variably-sized, slightly raised, dark brown necrotic
foci were present on the capsular surfaces and in the parenchyma of spleen, renal
cortices and pancreatic lymph node. There was mild cerebellar coning and the
leptomeninges over the frontal lobes were multifocally cloudy.

Laboratory Results:
Clinical pathology findings: January 10 – January 17; anemia (PCV-18% to low of 13%),
thrombocytopenia (15,000 to low of 13,000/mm3); hypoalbuminemia (1.5g/dL).
Ehrlichia canis serum titer: 1:40000
Fungus Testing Laboratory, The University of Texas Health Sciences Center at San
Antonio identified the fungus as Ochroconis gallopava.

Contributor’s Morphologic Diagnoses:

Submitted tissue:
1. Liver and kidney: Necrosis, multifocal, subacute, severe with vasculitis and
intralesional fungus, Shetland sheepdog, canine.
2. Liver and kidney: Plasmacytic perivasculitis, multifocal, chronic, moderate.
3. Kidney: Membranous glomerulopathy, multifocal, chronic, marked with mild
proteinuria.

Tissue not submitted:

4. Brain, spinal cord, gall bladder, pancreas: Perivasculitis, plasmacytic, multifocal,
chronic, moderate to severe.
5. Lung: Pneumonia, interstitial, chronic, moderate.
6. Liver, spleen, lung: Extramedullary hematopoiesis.

Contributor’s Comment: This patient has two overlapping disease processes. The
perivascular lymphoplasmacytic infiltrate in multiple organs, interstitial pneumonia,
glomerulopathy, multiorgan microthrombosis, anemia, thrombocytopenia and
hypoalbuminemia, which are consistent with the clinical diagnosis of Ehrlichiosis. An
unexpected finding in this case includes the necrotizing lesions in multiple organs
secondary to a systemic fungal infection.

Phaeohyphomycosis is a collective term for cutaneous and systemic diseases caused

by several genera of black molds that develop in tissue in the form of dark-walled,
septate mycelium. Phaeohyphomycotic fungi belonging to the genera Ochroconis

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(formerly Dactylaria) are known for their neurotropic potential and their predilection to
cause severe necrotizing encephalitis in humans,1 cats2 and young birds. Dactylaria
gallopava infection was first reported in 1962 in turkey poults in South Carolina.3
4
Subsequent epidemics have been reported in young birds – chickens, grey-winged
trumpeters (Psophia crepitans),5 Japanese quail (Coturnix coturnix japonica).6

The exact mechanism by which this fungus causes systemic disease is unknown.
Respiratory exposure to spores has been shown to produce the disease experimentally
in poultry.

Ochroconis is a thermophilic fungus and favors soil and decaying vegetation, which can
undergo a composting phenomenon associated with the generation of heat and an
acidic environment. It has been isolated from broiler house litter where similar
environmental conditions prevail. It is also a contaminant of effluents of hot springs and
nuclear reactors, thermal soils and self-heated coal waste piles.7

Though this fungus is more amenable to therapy, if not recognized and treated in time it
can be a cause of significant mortality. Amphotericin B is considered the antimycotic
agent of choice for systemic phaeohyphomycosis, including ochroconiosis. In a case
report by Kralovic and Rhodes in 1995, a human liver transplant patient developed
Ochroconis sp. infection despite receiving prophylactic fluconazole treatment.

AFIP Diagnoses: 1. Kidney: Necrosis, focally extensive, with moderate

pyogranulomatous inflammation, vasculitis, and dematiaceous fungal hyphae, Shetland
Sheepdog, canine.
2. Liver: Necrosis, multifocal, with neutrophilic inflammation, vasculitis, and
dematiaceous fungal hyphae.
3. Kidney: Glomerulonephritis, membranous, global, diffuse, moderate, with multifocal
mild plasmacytic interstitial nephritis.

Conference Comment: As mentioned by the contributor, this animal had a high titer to
Ehrlichia canis as well as related clinical pathology abnormalities. Histologically, a
characteristic change is generalized perivascular plasma cell infiltration. Infiltrates are
evident in the section of kidney; although increased numbers of perivascular plasma
cells might be expected in the liver, the numbers on the slides examined by conference
attendees are deemed within normal limits.

Ehrlichiosis is a tick-transmitted rickettsial disease affecting several species of animals

and humans. In dogs, disease is caused by E. canis, E. chaffeensis, E. risticii, E.
ewingii, E. equi, E. phagocytophilia, and E. platys and in horses by E. equi and E.
risticii. E. risticii is the causative agent of Potomac horse fever and E. platys is the
cause of canine cyclic thrombocytopenia.8

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The arthropod vector of E. canis is the brown dog tick, Rhipicephalus sanguineous.
Following a short incubation period, E. canis induces acute disease in which organisms
infect monocytes and spread throughout the mononuclear phagocyte system. During
this stage, the morula of Ehrlichia species may be noted on cytological examination in
neutrophils, lymphocytes, and monocytes. Endothelial invasion follows, resulting in
vasculitis. There is then a subclinical phase from which the dog either recovers or
develops pancytopenic bone marrow failure. The chronic phase is characterized by
pancytopenia with depletion of erythrocytic, granulocytic, and megakaryocytic cells, with
a persistence of plasma cells within the bone marrow. Gross findings include
widespread petechiae and ecchymoses, splenomegaly, lymphadenomegaly, and either
hyperplastic (acute disease) or hypoplastic (chronic disease) bone marrow.
Histologically there is a perivascular plasma cell infiltration, nonsuppurative
meningoencephalitis, interstitial pneumonia, and glomerulonephritis in most dogs.
Ehrlichiosis in German Shepherd dogs causes a severe hemorrhagic disorder attributed
to a depressed cell-mediated immune response to E. canis in this breed.8

Phaeohyphomycoses are uncommon opportunistic infections caused by a number of

ubiquitous saprophytic and plant pathogenic molds with the characteristic of forming
pigmented (dematiaceous) hyphal elements in tissue. The pigment is melanin and the
fungus will generally stain with the Fontana Masson method. Other special
histochemical stains commonly used to visualize the fungal morphology include
Grocott’s methenamine silver (GMS), or periodic acid-Schiff (PAS). With these stains,
the fungal hyphae of Ochroconis (Dactylaria) gallopavum are characterized by thick, 2-4
µm wide, septate, non-parallel walls, with acute and right angle dichotomous branching,
and yeastlike swellings.9

Contributor: Oklahoma State University, College of Veterinary Medicine, Department

of Pathology, Rm. 250 McElroy Hall, Stillwater, OK
http://www.cvm.okstate.edu

References:
1. Kralovic SM, Rhodes JC: Phaeohyphomycosis caused by Dactylaria (human
dactylariosis): report of a case with review of the literature. J Infect 31:107-13, 1995
2. Padhye AA, Amster RL, Browning M, Ewing EP: Fatal encephalitis caused by
Ochroconis gallopavum in a domestic cat (Felis domesticus). J Med Vet Mycol 32:141-
5, 1994
3. Georg LK, Bierer BW, Cooke WB: Encephalitis in turkey poults due to a new fungus
species. Sabouraudia 3:239-44, 1964
4. Waldrip DW, Padhye AA, Ajello L, Ajello M: Isolation of Dactylaria gallopava from
broiler-house litter. Avian Dis 18:445-51, 1974
5. Karesh WB, Russell R, Gribble D: Dactylaria gallopava encephalitis in two grey-
winged trumpeters (Psophia crepitans). Avian Dis 31:685-8, 1987
6. Shane SM, Markovits J, Snider TG 3rd, Harrington KS: Encephalitis attributed to
dactylariosis in Japanese quail chicks (Coturnix coturnix japonica). Avian Dis 29:822-8,
1985

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7. Tansey MR, Brock TD: Dactylaria gallopava, a cause of avian encephalitis, in hot
spring effluents, thermal soils and self-heated coal waste piles. Nature 242:202-3, 1973
8. Searcy GP: The hemopoietic system. In: Thompson’s Special Veterinary Pathology,
rd
eds. McGavin MD, Carlton WW, Zachary JF, 3 ed., pp. 343-344. Mosby, St. Louis,
MO, 2001
9. Foil CS: Miscellaneous fungal infections. In: Infectious Diseases of the Dog and Cat,
ed. Greene CE, 2nd ed., pp. 426-427. W.B. Saunders Company, Philadelphia, PA, 1998

SLIDE 66
CONFERENCE 17 / CASE I – S03-2084 (AFIP 2936462)

Signalment: 3.5 year-old, male, Asian Elephant (Elephas maximus).

History: This juvenile elephant from the zoological garden, Zürich, refused to eat in the
morning, showed signs of colic and depression and did not urinate. In the afternoon the
tongue appeared cyanotic, the periorbital skin was swollen and the animal collapsed
and died.

Gross Pathology: The main pathological changes were extensive hemorrhages

particularly in the heart, the intestines and mesentery as well as in the subcutaneous
tissues. The latter were also edematous in the head region. The myocardium,
particularly the right atrium, was severely hemorrhagic and dark red. The liver was
markedly swollen.

Contributor’s Morphologic Diagnosis: Generalized severe petechial to extensive

hemorrhage in nearly all organs and tissues. Intranuclear endothelial inclusion bodies
(only in capillaries) in the heart, liver and tongue.

Etiology: Endotheliotropic Herpesvirus (Elephant herpesvirus-1)

Contributor’s Comment: Histologically nearly every tissue was congested and most
had areas of hemorrhage. There were early signs of hypoxic degeneration in the liver.
Intranuclear inclusion bodies in the endothelium of the capillaries in the heart, liver and
tongue were observed.

Death due to this disease typically occurs as a result of cardiac failure following
herpesviral induced capillary injury and extensive myocardial hemorrhage. It is also
typical that inclusion bodies may be found in the endothelium of capillaries of the heart,
liver and tongue, but never in larger vessels.

The death of an Asian elephant due to Elephant Endotheliotropic Herpesvirus was first
described in a juvenile Swiss circus animal in 1990.1 The animal presented here is the
third juvenile elephant which has died as a result of a herpesvirus infection in
Switzerland.

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The significance of this disease for the survival of elephant populations in captivity
became apparent after a retrospective study published a decade later revealed that
deaths due to herpesvirus, but unrecognized at necropsy, had occurred in both Asian
2,3
and African elephants as early as 1983. So far, the virus has not been isolated.
However, molecular methods have provided evidence that several newly identified
herpesviruses are involved. The current theory is that otherwise healthy African
elephants with hyperplastic lymphatic tissue in the genital tract and nodules in the skin
and in the lung, which harbor herpesvirus, may be the source for a fatal infection in
Asian elephants.3,4,5 This obviously has great significance for the risk of disease
transmission in connection with the translocation of animals.6

AFIP Diagnosis: Liver: Hepatocellular degeneration and necrosis, centrilobular,

diffuse, mild, with congestion, hemorrhage, and endothelial cell eosinophilic intranuclear
inclusion bodies, Asian elephant (Elephas maximus), proboscidea.

Conference Comment: The Asian and African elephant endotheliotropic

herpesviruses (EEHV) are two novel, distinct, yet related viruses that are an important
cause of fatalities in young Asian elephants, and less commonly cause death in African
elephant calves 2

Clinical signs include sudden onset of lethargy, anorexia, edema of the head, neck and
thoracic limbs, cyanosis of the tongue, lymphopenia, and thrombocytopenia. Gross
lesions include pericardial effusion with widespread petechial to ecchymotic
hemorrhages primarily involving the heart, liver, intestine and tongue. Oral, laryngeal
and intestinal ulcerations often occur. Histologically there are extensive
microhemorrhages, edema, and mild lymphohistiocytic infiltrates throughout the heart
and tongue. Congestion and hemorrhage cause hepatic sinusoidal expansion with mild
hepatocellular degeneration, and endothelial cells of capillaries in the myocardium,
tongue, and liver contain amphophilic to basophilic intranuclear inclusion bodies.
Ultrastructurally, the inclusion bodies are 80-92 nm and morphologically consistent with
other herpesviruses.2

Currently it is thought that the African elephant may be the reservoir of the
herpesviruses that can cause disease in the two elephant species. African elephants
carrying EEHV have typical herpetic lesions on the skin and vulva. Transmission is
thought to be through intimate contact. However, direct proof of transmission has not
been established. Nonetheless, it is currently recommended that Asian and African
elephants be housed separately.2

The differential diagnosis for widespread necrosis and hemorrhage in elephants

includes encephalomyocarditis virus, orbivirus, salmonellosis or other bacterial
septicemia, and vitamin-E deficiency.7 The Smithsonian National Zoological Park is
developing PCR and ELISA tests to diagnose elephant herpesvirus infections.

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Additional endotheliotropic viruses of veterinary importance include equine viral arteritis
virus, equine Hendra virus, equine orbivirus (African horse sickness), cervid orbivirus
(epizootic hemorrhagic disease), ovine orbivirus (bluetongue), hamster parvovirus, rat
parvovirus (Kilham rat virus), canine adenovirus type 1 (infectious canine hepatitis),
porcine adenovirus, bovine adenovirus, and adenovirus of deer.2

Contributor: University of Zürich, Institute of Veterinary Pathology,

Winterthurerstr. 268, CH-8057 Zürich, Switzerland
www.vetpathology.unizh.ch

References:
1. Ossent P, Guscetti F, Metzler A, Lang E, Rübel A, Hauser B: Acute and fatal
herpesvirus infection in a young Asian elephant (Elephas maximus). Vet Pathol 27:131-
133, 1990
2. Richman L, Montali R, Cambre R, Schmitt D, Hardy D, Hildbrandt T, Bengis R,
Hamzeh F, Shahkolahi A, Hayward G: Clinical and pathological findings of a newly
recognized disease of elephants caused by endotheliotropic herpesviruses. J Wildlife
Diseases 36:1-12, 2000
3. Richman L, Montali R, Garber R, Kennedy M, Lehnhardt J, Hildbrandt T, Schmitt D,
Hardy D, Alcendor D, Hayward G: Novel endotheliotropic herpesviruses fatal for Asian
and African elephants. Science 283:1171-1176, 1999
4. Ehlers B, Burkhardt S, Golz M, Bergmann V, Ochs A, Weiler H, Hentschke J:
Genetic and ultrastructural characterization of a European isolate of the fatal
endotheliotropic elephant herpesvirus. J Gen Virol 82:475-482, 2001
5. Fickel J, Richman L, Montali R, Schaftenaar W, Göritz F, Hildbrandt T, Pitra C:
A variant of the endotheliotropic herpesvirus in Asian elephants (Elephas maximus) in
European zoos. Vet Microbiol 82:103-109, 2001
6. Ryan S, Thompson S: Disease risk and inter-institutional transfer of specimens in
cooperative breeding programs: Herpes and the elephant species survival plans. Zoo
Biology 20: 89-101, 2001
7. Richman LK, Montali RJ: Elephant herpesvirus infections. In: Infectious Diseases of
Wild Mammals, eds. Williams ES, Barker IK, 3rd ed., pp. 170-173. Iowa State University
Press, Ames, Iowa, 2001

SLIDE 67
CONFERENCE 17 / CASE II – ND-2 (AFIP 2935568)

Signalment: 12-year-old American bison (Bison bison) cow.

History: The animal was found dead on pasture with no prior clinical signs of illness.

Gross Pathology: Pale, hemorrhagic nodules were present in multiple tissues: nearly
all skeletal muscle groups (intercostals, epaxial and hypaxial groups, quadriceps,

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gluteals, semimembranosus, semitendinosus, deltoids, etc.), heart, liver, kidney, spleen,
walls of the forestomachs, small and large intestines, lung and brain.

Laboratory Results: Specials staining of replicate sections of tumor tissue yielded the
following results: Desmin +, Factor VIII -, trichrome -, vimentin +, PTAH +, myoglobin +,
and muscle specific actin +. Electron microscopy of tumor samples showed cross
striations consistent with myocyte origin.

Contributor’s Morphologic Diagnosis: Disseminated rhabdomyosarcoma.

Contributor’s Comment: Rhabdomyosarcomas are described as embryonal, botryoid,

alveolar and pleomorphic. The embryonal variety is classified into two types, large
round cell and myotubular. It has been reported in a variety of species, including cows
and sheep. Botryoid rhabdomyosarcomas are a distinct entity most commonly found in
the canine bladder. It is considered a variant of the embryonal type. The alveolar
subtype has been reported in large and small animals, and shows a distinct histologic
pattern of tumor cells supported by a prominent fibrovascular stroma. Least common of
the subtypes in animals is the pleomorphic variant. As the name implies, there is
considerable cellular pleomorphism with minimal connective tissue involvement.
Morphologic characteristics of the tumor in this bison cow would seem most consistent
with the more rare pleomorphic variant.

Rhabdomyosarcomas of the limbs, trunk, and neck typically present as nodules within
muscle. While usually pale on cut surface, hemorrhage and necrosis associated with
continued growth can change the appearance. Necrosis occurs when tumor cells
outgrow their blood supply. Metastatic disease occurs primarily in muscle tissue and
less commonly in other organs. Microscopically, the cells are highly pleomorphic with
unusual morphology characterized by racquet shapes, multinucleation, vacuolated
cytoplasm, and strap cells. Striated myofibrils may or may not be visible by light
microscopy. Immunohistochemical testing for specific proteins such as desmin,
vimentin, myoglobin, and actin can help with diagnosis.

A search of the literature indicates that this is the first report of a rhabdomyosarcoma in
a bison.

AFIP Diagnosis: Skeletal muscle; heart; kidney: Sarcoma, favor rhabdomyosarcoma,

American bison (Bison bison), bovine.

Conference Comment: Based on the H&E histomorphology alone, conference

attendees unanimously diagnosed a sarcoma with a differential list including
rhabdosarcoma, fibrosarcoma, leiomyosarcoma, hemangiosarcoma, and
neurofibrosarcoma.

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Several histochemical and immunohistochemical stains are helpful in narrowing the
differential diagnosis. With Masson’s trichrome, moderately abundant blue-staining
collagen is demonstrated separating neoplastic cells, which stain red. In the
contributor’s laboratory, neoplastic cells are negative for Factor VIII-related antigen,
which does not support a diagnosis of hemangiosarcoma. Neoplastic cells exhibit
diffuse immunoreactivity for vimentin, which supports mesenchymal origin, and exhibit
multifocal immunoreactivity for desmin and muscle specific actin, suggesting it is a
neoplasm of muscle origin. Neoplastic cells multifocally exhibit positive
immunoreactivity for myoglobin, which is specific for striated muscle. In addition,
phosphotungstic acid hematoxylin (PTAH) is used to highlight cross-striations, staining
them blue. Depending on the laboratory results, immunohistochemistry can assist in
determining the histogenesis of a tumor, but should never be used alone when making
a diagnosis. In this case, the contributor also evaluated the tumor utilizing electron
microscopy and results are consistent with myocyte origin. Conference attendees
discussed the potential difficulty of evaluating an electron micrograph of a
rhabdomyosarcoma within a skeletal muscle sample, especially if the sample were
taken from the periphery, in areas where the neoplastic cells separate and surround
pre-existing myocytes. In this case, ideally one would perform EM on the mass in the
kidney.

Contributor: North Dakota State University, Veterinary Diagnostic Laboratory, 1523

Centennial Boulevard, Van Es Hall, Fargo, North Dakota
www.vdl.ndsu.edu

References:
1. Cooper BJ, Valentine BA: Tumors of muscle. In: Tumors in domestic animals, ed.
Donald J. Meuten, 4th ed., pp.341-359. Iowa State Press, Ames, IA, 2002
2. Hulland TJ: Muscle and Tendon. In: Pathology of domestic animals, eds. Jubb KVF,
Kennedy PC, Palmer N, 4th ed., pp. 261-262. Academic Press, San Diego, CA 1993

SLIDE 68
CONFERENCE 17 / CASE III – 8053 (AFIP 2944793)

Signalment: Tissue from an adult female Vietnamese potbellied pig (Sus scrofa).

History: This animal came from a Vietnamese potbellied pig rescue establishment.
The animal had been recently introduced into a herd of mixed sexes, most of which had
been neutered. She had a history of hematuria for the previous 2 weeks. Ultrasound
revealed pyelonephritis, along with pus in the uterus and urinary bladder. The owner
attempted to give oral antibiotics without success and the animal was euthanized.

Gross Pathology: The animal had extensive subcutaneous and abdominal body fat.
Thick, mucopurulent exudate was present in the vaginal lumen. The mucosa of the
urethra and urinary bladder were streaked with hemorrhages, and the urine was cloudy.

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The ureters were distended and thickened. Purulent exudate could be extruded into the
pelvis of the kidneys if the ureters were compressed. The renal pelvis was bilaterally
dilated by considerable tan flocculent debris. There was also edema of the adipose
tissue of the mesentery, around the pancreas, and near the adrenal glands. The
adipose tissue in this general area contained multiple chalky white areas. Two ascarids
were present in the lumen of the duodenum; one of these protruded from the pancreatic
duct and extended 12 cm into the duct itself.

Laboratory Results: A heavy growth of Eubacterium suis was isolated from the
kidney.

Contributor’s Morphologic Diagnosis: Acute fibrinosuppurative pancreatitis and fat

necrosis, with pancreatic duct obstruction by an adult ascarid.

Contributor’s Comment: Ascaris suis is the largest enteric nematode of pigs. Adults
reside in the lumen of the small intestine, and the females may be up to 40 cm in length.
Pigs are most often exposed in dirt lots where earthworms and dung beetles may ingest
larvated eggs and serve as reservoirs. Piglets may ingest eggs attached to mammary
nipples, but prenatal infection is not thought to occur. L2 larvae leave the eggs in the
intestine, penetrate the hepatic portal system and molt to L3. These migrate via the
blood from the liver to the lungs where they exit pulmonary capillaries, molt to L4, and
migrate up airways. They are then swallowed and develop into enteric adults. Most
pathology attributed to ascarids is a result of migration of large numbers of L3 larvae
through the liver (white spot disease or milk-spotted liver) or lungs, where pneumonia
results.

Ascaris suum is a close relative of Ascaris lumbricoides, the human ascarid, with which
it shares a similar shape and size. Ascarids are sporadically responsible for blockage of
the pancreatic duct in a variety of species, including human beings, especially in
developing countries where 60% of the children and 30% of adults are parasitized. In
people, the mechanism of pancreatitis is most often compression of the pancreatic duct
by blockage of the bile duct or common duct, with obstruction of the pancreatic duct
alone being less common. This pig had rather localized pancreatitis, and pigs have
separate entrances for the pancreatic and biliary ducts at the duodenum. Living
parasites slip in and out of ducts in people with relative ease, and occasionally bypass
large obstructions such as gall stones in the ducts. Children are less commonly
affected, perhaps because of the small diameter of the hepatic and pancreatic ducts,
although they can develop severe pancreatitis. There is a 3:1 prevalence of women
over men in adult patients. Chronic pancreatitis also has been described in horses as a
result of migration of Strongylus equinus, occasionally with resulting diabetes, and there
is a single report of an ascarid becoming lodged in the pancreatic duct of a horse.
Trichospiruria leptostoma commonly inhabits the pancreatic ducts of wild-caught
common marmosets. Occasional pancreatic fibrosis and exocrine insufficiency have
been reported.

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Characteristics of ascarid nematodes include coelomyarian musculature, and
uninucleate intestinal epithelium with a low microvillous border. Numerous thick shelled
eggs are present in the coelom, as characteristic of an adult female.

AFIP Diagnosis: Pancreas; peripancreatic fat; and duodenum: Pancreatitis,

neutrophilic and eosinophilic, acute, multifocal, moderate, with vasculitis, fibrinous
peritonitis, necrotizing steatitis, focal mural duodenitis, pancreatic duct ectasia,
ulceration, and intraluminal adult ascarid, Vietnamese potbellied pig (Sus scrofa),
porcine.

Conference Comment: The contributor provides a thorough overview of the lifecycle

of Ascaris suum and the lesions associated with infestations by the nematode.

As pathologists, it can be tempting to play the “vet game” and diagnose A. suum
infestation based on finding a large nematode in the pancreas of a pig. However, with a
basic understanding of common histomorphological features of the various groups of
metazoans, one can easily identify the parasite of this case as a nematode, and further
classify it as an ascarid.

There are six groups of commonly seen metazoan parasites: nematodes,

acanthocephalans, trematodes, cestodes, arthropods, and pentastomes. When
evaluating a parasite histologically, it is important to note the following features: type of
body covering and body wall, presence or absence of a body cavity, location and type of
musculature, presence and type of digestive tract, presence and type of reproductive
organs. Below is a simple table to identify the parasite to its group:8

GROUP GENERAL BODY DIGESTIVE STRIATED SPECIAL DIAGNOSTIC

SHAPE CAVITY TRACT MUSCLE FEATURES
Cestode Flattened -- -- -- 1. calcareous corpuscles
dorso-ventrally 2. scolex
3. tegument
Trematode Flattened -- + -- 1. suckers
dorso-ventrally 2. tegument
3. blind ceca
4. yolk gland
5. hermaphroditic
Acanthocephalan Spherical + -- -- 1. hypodermis
2. lemniscus
3. two muscle layers
4. proboscis
Nematode Spherical + + -- 1. cuticle
2. musculature
Arthropod Tend to be + + + 1. chitinized exoskeleton
spherical

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 spherical 2. jointed appendages
3. tracheal tubes
Pentastomes Spherical + + + 1. chitinized exoskeleton
2. digestive glands
3. sclerotized openings

Once the organism has been identified as a nematode, it must be further classified into
one of the following groups: Aphasmids or Phasmids. Aphasmids lack a tiny pair of
sensory papillae (the phasmids) on the caudal end; however, these are not readily
identifiable on histologic section. The morphological features that distinguish them from
phasmid nematodes are hypodermal bands with associated nuclei, and prominent
esophageal glands that form a stichosome. The Phasmids consist of the Rhabditoids,
Oxyurids, Ascarids, Strongyles, Spirurids, and Filarids. Both the Rhabditoids and
Oxyurids have a rhabditoid esophagus composed of a corpus, isthmus and bulb. The
Strongyles have a cuticle, which occasionally is ridged, and all have an intestine
composed of few multinucleated cells and a prominent brush border. Spirurids can be
very diverse, but all adult females in this group produce embryonated eggs. Filarial
nematodes are small and produce distinctive larvae called microfilariae.2

Ascarids are large worms that are found, as adults, in the intestines of their host. Larval
ascarids may be found in other tissues in both the definitive and intermediate hosts.
Adult ascarids have a cuticle, a pseudocoelom, coelomyarian musculature, large lateral
chords, and less prominent dorsal and ventral chords, a simple esophagus, a large
intestine lined by uninucleate cuboidal cells and a low brush border. Adult female
ascarids produce eggs which contain a uninucleate zygote covered by a thick shell.
Larval ascarids of mammals commonly have lateral alae.2

Contributor: University of Missouri, Veterinary Medical Diagnostic Laboratory, et P.O.

Box 6023, Columbia, Missouri
References:
1. Corwin RM, DiMarco NK, McDowell AE, Pratt SE: Internal parasites. In: Diseases of
Swine, eds. Leman AD, Straw B, et al., 6th, ed., pp. 648-650. Iowa State University
Press, Ames, IA,1986
2. Gardiner CH, Poynton SL: An Atlas of Metazoan Parasites in Animal Tissues, pp. 1-
3, 14, 17, 19, 22, 30, 35, 40. Armed Forces Institute of Pathology, Washington DC,
1999
3. Breider MA, Kiely RG, Edwards JF: Chronic eosinophilic pancreatitis and ulcerative
colitis in a horse. J Am Vet Med Assoc 186: 809-811, 1985
4. Hamir AN: Verminous pancreatitis in a horse. Vet Rec 121:301-302, 1987
5. Hawkins JV, Clapp NK, Carson RL, et al: Diagnosis and treatment of Trichospirura
leptostoma infection in common marmosets (Callathrix jacchus). Contemp Topics Lab
Animal Sci 36:53-55,1997
6. Khuroo MS: Ascariasis. Gastroenterol Clin North Am 25:553-577, 1995
7. Bahy MG, Baldisseroto M, Custodio CM, et al: Hepatobiliary and pancreatic
complications of ascariasis in children: a study of seven cases. J Pediatr Gastroenterol
Nutr 33:271-275, 2001

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8. Toft JD, Ekstrom ME: Identification of metazoan parasites in tissue sections. In:
Metazoan Parasites in Tissue Sections, pp. 369-378. The Armed Forces Institute of
Pathology, Washington, DC

SLIDE 69
CONFERENCE 17 / CASE IV – 8-81-04 (AFIP 2956553)

Signalment: Male, yearling Mule Deer (Odocoileus hemionus).

History: A male, yearling mule deer was observed lying down near a rural residence in
southwest Montana. The animal was unable to stand, had tachypnea, and moist airway
sounds were audible from a distance.

Gross Pathology: Numerous tan foci occurred throughout the lungs, kidney and liver.
Tuberculosis was suspected and the carcass was buried. Two visiting veterinarians
who performed a field necropsy submitted specimens.

Laboratory Results: Yersinia pestis was cultured from the lung and kidney.

Contributor’s Morphologic Diagnoses: 1. Lung: Pneumonia, necrotizing,

suppurative, multifocal to coalescing, severe, with intralesional bacterial rods.
2. Kidney: Nephritis, necrotizing, suppurative, acute, multifocal to coalescing, severe.

Contributor’s Comment: Plague is endemic in many areas of the western United

States. Yersinia pestis is maintained in the environment by a variety of rodent species
and their associated fleas. Reports of plague are usually seasonal with the greatest
incidence between March and October. Susceptibility differs both in domestic and wild
species. In domestic animals, the cat and dog are most frequently infected. Besides
rodents and rabbits, plague has been reported in mule deer, pronghorn antelope,
mountain lions and bobcats in the western United States. There are three clinical
manifestations: bubonic, septicemic, and pneumonic. Lymphadenitis of bubonic plague
occurs after inoculation of the organism through the skin or mucous membrane by flea
bites or direct penetration. Septicemia can occur subsequent to dissemination of the
infection from the infected lymph nodes or by direct introduction into the blood
vasculature. Primary pneumonic plague occurs after inhalation of infected material.
This particular case represents the septicemic form. Yersinia pestis is occasionally
isolated from domestic cats in this region in Montana.

AFIP Diagnoses: 1. Lung: Pneumonia, necrotizing, suppurative, subacute, multifocal

and coalescing, severe, with vasculitis, and large colonies of bacilli, mule deer
(Odocoileus hemionus), cervid.

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2. Kidney: Nephritis, necrotizing, suppurative, subacute, multifocal and coalescing,
severe, with vasculitis, and large colonies of bacilli.
3. Kidney: Nephritis, interstitial, lymphoplasmacytic, chronic, multifocal, with
mineralization.

Conference Comment: Of the eleven species of Yersinia, family Enterobacteriaceae,

only four are considered to be primary pathogens: Y. pestis (plague in mammals), Y.
enterocolitica (yersiniosis in mammals and birds), Y. pseudotuberculosis (yersiniosis in
mammals and birds), and Y. ruckeri (red mouth in fish).3

Y. pestis remains endemic in certain rodent populations on five continents due to an

elaborate interaction of the agent, fleas, vertebrate hosts, and the environment.
Enzootic hosts, such as voles, mice, and rock squirrels, serve as reservoirs and do not
suffer 100% mortality. These animals may experience a transient bacteremia and
thereby transmit the infection to fleas. The fleas may then transmit bacteria to other
enzootic hosts, thereby maintaining the disease, or they may transmit it to epizootic
rodent hosts. These hosts, such as prairie dogs, have a low resistance to plague
morbidity and mortality. Subsequently there is a high death rate, which favors spread of
plague, amplifies the intensity of the epizootic, and increases the risk of human infection
as infected fleas disseminate from dead hosts.3

The primary mode of transmission of Y. pestis in mammals is via flea bite. Other modes
of transmission include ingestion of, or exposure to, another mammal infected with Y.
pestis. Another rare but effective mode of transmission is inhalation of aerosolized
bacteria by a mammal in close proximity to an animal with pneumonic plague.3

As mentioned by the contributor, bubonic, pneumonic, and septemic plague are three
clinical manifestations of Y. pestis infection. In many cases they represent a continuum
as illness, if left untreated, will often progress from one form to the next. The bubonic
form results from flea bite inoculation of bacteria and subsequent acute local
inflammation of the lymph node draining the inoculation site. The Latin root “bubo”,
meaning “swelling”, is descriptive of the lymphadenomegaly that occurs. Primary
septemic plague is defined as bacteremia without the presence of palpable buboes.
Secondary septemic plague occurs when bacteria from buboes enter the bloodstream.
Primary pneumonic plague may result from inhalation of aerosolized droplets of Y.
pestis from an animal with pneumonic plague.3 This form has a very rapid incubation
period (1-6 days) and is usually fatal if not treated within the first 24 hours of illness.4
Secondary pneumonic plague may result from hematogenous spread of bacteria to the
lungs in the septemic form. Clinically, the septicemic and pneumonic forms are much
more severe and almost always result in death.3

Y. pestis is a potential bioterrorism agent, and if aerosolized, pneumonic plague could

induce many human casualties. Accordingly, the Centers for Disease Control (CDC),
lists it as a Category A agent. Category A agents are those agents that have the
greatest potential for inflicting large numbers of human casualties, can be manufactured

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and disseminated on a large scale, require significant efforts in public health
preparedness, and are most familiar to the public.4

Contributor: Montana Veterinary Diagnostic Laboratory, P.O. Box 997, Bozeman,

Montana
www.state.mt.us/liv/lab/index.asp

References:
1. Orloski KA, Lathrop SL: Plague: a veterinary perspective. J Am Vet Med Assoc
222(4):444-448, 2003
2. Thorne ET, Quan TJ, Williams ES, Walthall TJ, Daniels D: Plague in free-ranging
mule deer from Wyoming. J Wild Dis 23(1):155-159, 1987
3. Gasper PW, Watson RP: Plague and yersiniosis. In: Infectious Diseases of Wild
Mammals, eds. Williams ES, Barker IK, 3rd ed., pp. 313-323. Iowa State University
Press, Ames, IA, 2001
4. Davis RG: The ABCs of bioterrorism for veterinarians, focusing on Category A
agents. J Amer Vet Med Assoc 224(7):1084-1095, 2004

SLIDE 70
CONFERENCE 18 / CASE I – 98-443 (AFIP 2681375)

Signalment: 10 year-old female spayed American Eskimo dog.

History: 3-4 month history of inflamed, ulcerated, and hyperkeratotic foot pads +/- oral
mucocutaneous lesions. The dog was treated with glucocorticoids, which exacerbated
lesions; then with antibiotics, dietary change, and antihistamines
• 3-4 week history of inappetance; treated with Baytril, Dicural, and Reglan
• One week history of polyuria/polydypsia, vomiting, icterus, and hyperglycemia
with glucosuria and ketoacidosis; treated with insulin, Reglan, and centrine with no
improvement
• Upon referral, a tentative diagnosis of hepatocutaneous syndrome was made
and the owners elected euthanasia

Gross Pathology: The footpads, elbows, vulva, and lips were variably ulcerated and
hyperkeratotic. The animal was severely icteric. The liver was firm, yellow-brown with
multifocal to coalescing nodules which replaced the entire normal parenchyma. (See
gross photo)

Laboratory Results:
Test Result 7-2-98 9-17-98 9-23-98
ALP 1724 NA >2400 NA=not available
ALT 158 142 45
HCT 30 38.3 24.7
WBC 16,500 22,900 28,500
PMN 14,500 18,300 27,300
GLU NA >700 221 (on insulin)

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ALB NA 3.1 2.4

Contributor’s Morphologic Diagnoses: 1. Skin, footpad: Superficial dermatitis with

basal epidermal hyperplasia, epidermal pallor, and parakeratotic hyperkeratosis.
2. Liver: Severe lobular collapse with nodular regeneration, vacuolar hepatopathy, mild
portal fibrosis and bile duct proliferation, and marked canicular bile stasis.

Contributor’s Comment: Hepatocutaneous syndrome, also known as superficial

necrolytic dermatitis, necrolytic migratory erythema, or diabetic dermatosis is the
association of a specific skin lesion (hyperplastic basal epithelium, a zone of epidermal
pallor, and parakeratotic hyperkeratosis) with severe liver disease. The liver lesion is
typically a severe vacuolar hepatopathy with parenchymal collapse and nodular
regeneration which mimics cirrhosis.

In humans the disease is most often associated with glucagon secreting tumors of the
endocrine pancreas; however, some human cases have cirrhosis or chronic pancreatic
disease with normal glucagon levels. In dogs, most cases are associated with liver
disease with normal glucagon levels, but rare cases of glucagon secreting tumors have
been reported. The pathogenesis is unknown.

AFIP Diagnoses: 1. Skin, footpad: Hyperkeratosis, parakeratotic, diffuse, severe, with

marked basal epidermal hyperplasia, stratum spinosum edema and degeneration, mild
subacute dermatitis, and focal ulcer with a serocellular crust, American Eskimo, canine.
2. Liver: Hepatocellular loss with stromal collapse, diffuse, severe, with nodular
regeneration, multifocal vacuolar degeneration, mild bridging fibrosis, and biliary
hyperplasia.

Conference Comment: Superficial necrolytic dermatitis (SND) is considered a

paraneoplastic syndrome that more commonly occurs in association with hepatopathy
than with glucagon-secreting neoplasia, giving rise to the familiar name of
hepatocutaneous syndrome.5

Often the main presenting complaints of glucanoma-associated SND are progressive

skin lesions of three weeks to many months, with concurrent lethargy and
inappentance. The main dermatologic findings include erosions and ulcerations, with
alopecia, exudation and adherent crusts on the feet, pressure points such as the elbows
and hocks, flank, perineal area, muzzle, facial mucocutaneous junctions and/or oral
cavity. Hyperkeratosis and fissuring of foot pads occur in all animals. Many dogs will
also have hyperglucagonemia, hyperglycemia, and marked hypoanimoacidemia
involving many amino acids.5

The histopathological findings of SND are distinctive and can be strongly suggestive of
the disease. The epidermis has a “red, white, and blue” appearance on H&E.
Parakeratotic hyperkeratosis and crusting create the upper eosinophilic layer. Edema

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and necrosis of keratinocytes within the stratum spinosum make up the middle “white”
layer. Hyperplasia of the stratum basale gives rise to the deep basophilic layer. In
addition, there may be secondary clefting in the devitalized middle layer, leading to
ulceration and secondary inflammation. A mixed inflammatory infiltrate may be present
in the superficial dermis.5

Although the exact pathogenesis is unknown, there are several current theories. The
first is that glucagon results in sustained gluconeogenesis and is involved in the
catabolism of amino acids, and chronic elevation of the hormone, as seen in
glucagonomas, may result in hypoaminoacidemia, leading to epidermal protein
depletion and subsequent keratinocyte necrosis. This is supported by cases of
glucagonoma-associated SND that rapidly resolve following surgical resection of the
tumor. However, there are also documented cases of SND in which glucagon
concentrations are within normal limits.5 There may be other causes of
hypoaminoacidemia. In one report, dogs with nonglucagonoma-associated SND, also
had significantly lower amino acids concentrations than the control dogs or dogs with
acute or chronic hepatitis.6 Another theory involves deficiencies in zinc or fatty acids.
However, there has been little response to replacement therapy. Finally, hepatic
impairment has also been implicated as a possible mechanism. Many, although not all
canine patients with nonglucagonoma-associated SND have elevated serum glucagon.
It may be that impaired hepatic function leads to decreased metabolism of glucagon,
resulting in increased serum levels.5

Although SND is an uncommon skin disorder, it is an important diagnosis because it is

one of the relatively few skin conditions from which one can diagnose a life-threatening
disease, with confidence, from a skin biopsy.

Contributor: University of Pennsylvania, School of Veterinary Medicine, Laboratory of

Pathology, 3800 Spruce Street, Philadelphia, PA

References:
1. Gross TL, Song MD, Havel PJ, Ihrke PJ: Superficial necrolytic dermatitis (necrolytic
migratory erythema) in dogs. Vet Path 30:75-81, 1993
2. Miller WH, Scott DW, Buerger RG, Shanley KJ, Paradis M, McMurdy MA, Angarana
DW: Necrolytic migratory erythema in dogs. JAAHA 26: 573-581, 1990
3. Miller WH, Anderson WI, McCann JP: Necrolytic migratory erythema in a dog with
a glucagon-secreting endocrine tumor. Vet Dermatology 2:179-182, 1991
4. Gross TL, O’Brien TD, Davies AP, Long RE: Glucagon-producing pancreatic
endocrine tumors in two dogs with superficial necrolytic dermatitis. JAVMA 197:1619-
1622, 1990
5. Turek MM: Cutaneous paraneoplastic syndromes in dogs and cats: a review of the
literature. Vet Dermatol 14:279-296, 2003
6. Outerbridge CA, Marks SL, Rogers QR: Plasma amino acid concentration in 36 dogs
with histologically confirmed superficial necrolytic dermatitis. Vet Dermatol 13:177-186,
2002

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SLIDE 71
CONFERENCE 18 / CASE II – 03-56361 (AFIP 2937321)

Signalment: 1 year-old male Standardbred horse.

History: This colt was purchased at an auction sale 6 weeks prior to presentation.
Nasal discharge, lethargy, and limb edema had been evident for the past 4 weeks and
the colt had been treated unsuccessfully with 2 courses of antibiotics. Physical
examination revealed peripheral lymphadenopathy, severe pneumonia with suspected
microabscesses in lung, and ventral edema.

Gross Pathology: Severe generalized lymphadenopathy involved peripheral and

many internal lymph nodes, including submandibular, internal iliac, and mesenteric
nodes. The enlarged nodes were diffusely white and nodular on sectioning, with
occasional areas of necrosis. Left lung contained 2 discrete, 2 cm white foci similar in
texture and appearance to the affected lymph nodes. Scrotal skin contained numerous
3-4 mm pale, poorly pigmented, non-haired papules that were deemed an incidental
finding.

Contributor’s Morphologic Diagnoses: 1. Multicentric lymphosarcoma

2. Equine molluscum contagiosum

Contributor’s Comment: Slides demonstrate lesions of equine molluscum

contagiosum only. Although lymphosarcoma was the most significant disease process
in relation to this horse’s clinical demise, lesions of equine molluscum contagiosum
presented an additional concurrent and interesting disease process.

Equine molluscum contagiosum (MC) is an uncommon, mildly contagious, cutaneous

poxviral disease generally involving skin of the face, neck, chest, trunk, limbs, and
genitalia.1 The virus is transmitted by contact with either an infected individual or
contaminated fomites. Multiple 2-8 mm papules develop in affected skin, and although
these lesions are benign, they are refractory to therapy and persist for months to years.
The lesions in this horse were typical of equine MC and consisted of focal, discrete
lobulated areas of epidermal hyperplasia bulging into the underlying dermis.
Keratinocytes of the stratum spinosum were swollen and contained large
intracytoplasmic inclusions (‘molluscum bodies’). In some sections, there is an
impression of a central pore in the hyperplastic lobules, through which degenerate
keratinocytes are exfoliated. Electron micrographs of the cutaneous lesions
demonstrated numerous poxviral inclusions within epithelial cells. Although MC has
been diagnosed in clinically normal horses, several literature reports describe MC in
horses with other concurrent immunocompromised conditions, such as in this case.1,2
In humans, lesions of MC are generally more severe and widespread in patients with
compromised cell-mediated immune function.3

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Equine MC bears clinical and histologic resemblance to the human skin disease of the
same name, and recent in situ hybridization experiments have shown that the viruses
causing lesions in these two species have significant nucleic acid homology, leading to
3
the hypothesis that equine MC is an anthropozoonosis. Equine MC has been identified
in horses worldwide and clinical lesions are similar to those of Uasin Gishu disease,
which has been identified in horses only in Kenya.2,3 Although the Uasin Gishu virus
can be cultivated, the virus associated with equine MC has yet to be isolated. The
poxvirus of equine MC is currently classified in the subfamily Chordopoxvirinae, genus
Molluscipoxvirus.

AFIP Diagnosis: Haired skin: Hyperplasia, epidermal, focal, marked, with large,
eosinophilic intracytoplasmic inclusion bodies (molluscum bodies), Standardbred,
equine.

Conference Comment: Equine molluscum contagiosum (MC) is caused by a

molluscipoxvirus that is either identical with, or very closely related to, its human
equivalent, molluscum contagiosum virus (MCV). MCV is a poorly characterized pox
virus which causes a human skin disease characterized by benign but persistent
papular lesions with a central opening or umbilicus. Lesions are commonly found on
the face, trunk, lower limbs, and anogenital regions. Humans with immunodeficiency
have a more severe, disseminated form of the disease.
Molluscum contagiosum has been reported in horses, chimpanzees, and kangaroos. In
all of the cases, lesions clinically and histologically were very similar to those seen in
humans. MC may represent an anthropozoonosis, wherein disease is transmitted from
humans to animals.3

In horses, affected animals commonly have hundreds of lesions, especially on the

chest, shoulders, neck, limbs, and external genitalia. Lesions on haired skin consist of
papules that are initially tufted, but usually become alopecic and covered with a
powdery crust or scale. Papules in glabrous skin may be smooth, shiny,
hypopigmented, and umbilicated, or hyperkeratotic and hyperpigmented. Lesions are
usually nonpruritic and nonpainful. Histologically, lesions are well-demarcated and
characterized by epidermal hyperplasia and papillomatosis. Keratinocytes above the
stratum basale become swollen and contain ovoid, eosinophilic, floccular
intracytoplasmic inclusion bodies (molluscum bodies). These inclusions increase in size
and density as the keratinocytes move toward the skin surface. Molluscum bodies
exfoliate through a central pore that forms in the stratum corneum and enlarges into a
central crater. Usually there is no dermal inflammatory reaction. Ultrastructural
examination reveals mature virions that are brick-shaped and approximately 150 x 300
nm, with a biconcave nucleoid and two lateral bodies, typical of poxviral inclusions.1

Contributor: University of Guelph, Laboratory Services Division, Animal Health

Laboratory, Guelph, Ontario, Canada
http://ahl.uoguelph.ca

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References:
1. Scott DW, Miller WH: Viral and protozoal skin diseases. In: Equine Dermatology,
eds. Scott DW Miller WH, pp 379-382. Elsevier Science, Philadelphia, PA, 2003
2. Van Rensburg IBJ, Collett MG, Ronen N, Truuske G: Molluscum contagiosum in a
horse. J S Afr Vet Assoc 62:72-74, 1991
3. Thompson CH, Yager JA, Van Rensburg IB: Close relationship between equine and
human molluscum contagiosum virus demonstrated by in situ hybridization. Res Vet
Sci 64:157-161, 1998

SLIDE 72
CONFERENCE 18 / CASE III – 04-2262 (AFIP 2941563)

Signalment: 10 year-old, female, mixed breed dog (Canis familiaris).

History: The dog was being treated for lymphoma and immune-mediated
thrombocytopenia with a standard chemotherapy protocol (cyclophosphamide,
doxorubicin, vincristine, prednisone). Small nodular dermal lesions developed after 11
treatments with progression to severe exudative dermatitis following the last treatment.

Gross Pathology: Variably sized irregular erythematous exudative skin lesions were
randomly distributed on the trunk.

Contributor’s Morphologic Diagnosis: Severe generalized necrotizing dermatitis and

vasculitis with intralesional protozoal organisms.

Contributor’s Comment: The slides vary somewhat in the degree of necrosis and
ulceration. The lesion is characterized by extensive epidermal ulceration and necrosis
that includes the follicular epithelium and adnexal structures. Necrosis extends from the
epithelium to the superficial hypodermis with edema, hemorrhage and an inflammatory
infiltrate of neutrophils, histiocytes, small lymphocytes and plasma cells that is diffuse to
perivascular. The perivascular inflammatory infiltrates extend into the hypodermis.
Small 1-2 µm oval organisms were found free in the necrotic tissue debris, within
macrophages and, occasionally, within endothelial cells and epithelial cells of the
epidermis, follicles and adnexal glands. Immunohistochemistry was performed and the
organisms reacted strongly with a Polyclonal rabbit anti-Toxoplasma gondii antibody
and did not react with a Polyclonal rabbit anti-Neospora caninum antibody.

Necrotizing dermatitis in the dog due to a Toxoplasma gondii – like apicomplexan

organism and Neospora caninum has been previously described.1,2 In addition to these
two organisms, the differential diagnosis for protozoal dermatitis in the dog includes
Sarcocystis canis, Leishmania sp., and Caryospora sp..2,3 Caryospora sp., Sarcocystis
canis and Leishmania sp have sufficiently distinctive morphologic characteristics to

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assist in their diagnosis (i.e. caryocyts, schizont formation and the presence of a distinct
kinetoplast, respectively).

Toxoplasma gondii and Neospora caninum are sufficiently similar in routine histological
sections that immunohistochemistry and electron microscopy (EM) are usually
employed to distinguish between these two apicomplexan organisms. The Toxoplasma
gondii-like organism described by Dr. Dubey, et al1 reacts with a polyclonal rabbit
antibody to T. gondii, but its ultrastructural characteristics differ significantly. The T.
gondii-like organism formed schizont-like organisms with a residual body that was best
appreciated on EM but could be seen on close examination of the routine histological
sections. In addition, the rhoptries in this organism were several in number and electron
dense as compared to the few electron lucent rhoptries in T. gondii. Close examination
of the sections in this case did not reveal any definitive residual bodies. However,
electron microscopic examination would be necessary to determine if the protozoa in
this case are T. gondii or the organisms described by Dr. Dubey.

Predisposing factors such as chronic ehrlichiosis, cardiomyopathy and immune-

suppressive therapies for immune-mediated disease and neoplasia were reported in
many cases of protozoal dermatitis in the dog.1,2

AFIP Diagnosis: Haired skin and subcutis: Dermatitis and vasculitis, necrotizing,
acute, diffuse, severe, with multifocal erosion, and myriad intra- and extracellular
protozoal tachyzoites, mixed-breed, canine.

Conference Comment: As mentioned by the contributor, organisms are noted both

intra- and extracellularly. The intracellular organisms are present in many different cell
types including fibroblasts, follicular and epidermal keratinocytes, sebocytes, apocrine
ductular epithelium, endothelium, macrophages, adipocytes, and myocytes of the
erector pili muscles.

This case was reviewed in consultation with Dr. J.P. Dubey, United States Department
of Agriculture, Animal Parasitic Diseases Laboratory, who performed
immunohistochemistry using polyclonal antibodies to Neospora caninum and
Toxoplasma gondii. In his laboratory, the organisms exhibit weak positive
immunoreactivity for Neospora caninum and strong positive immunoreactivity for
Toxoplasma gondii. Electron microscopy is necessary to positively identify the
organism.

Contributor: Arizona Veterinary Diagnostic Laboratory, 2831 N. Freeway,

Tucson, AZ
http://www.microvet.arizona.edu/AzVDL/index.shtml

References:

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1. Dubey JP, Pimenta AL, Abboud LCS, Ravasani RR, Mense M: Dermatitis in a dog
associated with an unidentified Toxoplasma gondii-like parasite. Vet Parasit 116:51-59,
2003
2. LaPerle KMD, Del Piero F, Carr RF, Harris C, Stromberg PC: Cutaneous
neosporosis in two adult dogs on chronic immunosuppressive therapy. J Vet Diagn
Invest 13:252-255, 2001
3. Dubey JP, Slife LN, Speer CA, Lipscomb TP, Topper MJ: Fatal cutaneous and
visceral infection in a Rottweiler dog associated with a Sarcocystis-like protozoon. J Vet
Diagn Invest 3:72-75, 1991

SLIDE 73
CONFERENCE 18 / CASE IV – 433846B (AFIP 2948686)

Signalment: Three month-old lamb, belonging to a sheep flock of 380 lambs of which
40% died.

History: The animal presented with a fever, swollen eyelids, excessive lacrimation, and
mucopurulent nasal discharge.

Gross Pathology: Skin lesions were found on the areas free from wool and were
characterized by raised, circular plaques that occasionally had congested borders.

Laboratory Results: Sheep pox antigen was identified in cryostat sections by

immunohistochemistry using monoclonal antibodies.

Contributor’s Morphologic Diagnosis: Skin: Hydropic degeneration of stratum

spinosum keratinocytes, epidermal hyperplasia, and intracytoplasmic eosinophilic
inclusion bodies, predominantly in squamous epithelium of hair follicles, consistent with
sheep pox.

Contributor’s Comment: The epidermis is hyperplastic with hyperkeratosis,

acanthosis and significant ballooning degeneration. Occasionally, within keratinocytes,
there are pale eosinophilic intracytoplasmic inclusion bodies. In the dermis, there is
multifocal infiltration of neutrophils, lymphocytes, and histiocytes.

Sheep pox is a malignant pox disease of sheep characterized by fever, multiple non-
vesicular swellings on the skin and mucous membranes, rhinitis, conjunctivitis,
respiratory distress (due to the pressure on the upper respiratory tract from the swollen
retropharyngeal lymph nodes and developing lung lesions) and death. Sheep pox
occurs in all ages of sheep but the disease is most severe in lambs, with mortality
reaching 80-100%.

Skin lesions are often less obvious on post mortem examination of acutely infected
animals than in live animals with disease. Gross findings include necrotic mucous
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membranes, enlarged and edematous lymph nodes, and typical pox lesions
characterized by papules, which may be ulcerated, on the abomasal mucosa, tongue,
hard and soft palates, trachea and esophagus, and sometimes on the wall of the rumen
and the large intestinal mucosa. Pale areas of approximately 2 cm in diameter may
occasionally be seen on the surface of the kidney and liver, and have been reported on
the testicles. Throughout the lung, but particularly in the diaphragmatic lobes, there are
numerous white-gray firm lesions up to 5 cm in diameter. The pathological lesions and
clinical signs mentioned above are pathognomonic for sheep pox.

Generalized contagious ecthyma (Orf) or parapox viral infection is rare. Confusion

could occur between mild sheep pox and orf or even insect bites. Sheep pox causes
extensive economic loss through high mortality, reduced meat, milk or wool yields,
quarantine requirements, and the cost of disease prevention programs.

Capripox is endemic in Africa, north of the equator, the Middle East, including Israel,
Turkey and Iran, and in Afghanistan, India, Nepal, parts of the Peoples Republic of
China, and since 1984, Bangladesh. Recently, it has made frequent incursions into
southern Europe.

Transmission of infection is by direct contact with diseased sheep or indirect contact via
a contaminated environment. Transmission is usually via aerosolization, but virus can
also be spread mechanically by insect bites or experimentally by intradermal or
subcutaneous injection.

The incubation period is 4-7 days and is followed by a leukocyte-associated viremia.

The virus localizes in many organs including the skin, where virus concentration peaks
at 10-14 days post infection. The skin lesions develop 1-2 days later, especially in the
sparsely wooled areas and typically involve the eyelids, cheeks, nostrils, vulva, udder,
scrotum, prepuce, ventral surface of the tail, and the medial thigh.

Sheep pox lesions have a prominent vesicular stage. The vesicles are umbilicated,
multiloculated, and yield only a small amount of fluid if punctured. Occasionally, a large
vesicle forms as a result of separation of the necrotic epidermis from the underlying
dermis. The pustule stage is characterized by the formation of a thin crust. There may
be marked gelatinous dermal edema and in severely affected animals the lesions
coalesce. Healing of the skin lesions is slow, taking up to 6 weeks and a scar may
remain. Highly susceptible animals often develop hemorrhagic papules early in the
course of the disease and later ulcerative lesions in the gastrointestinal and respiratory
tracts. Approximately one third of animals develop multiple pulmonary lesions
composed of foci of pulmonary consolidation.
The kidneys have multifocal, circular, fleshy nodules throughout the cortices.

Histologically, sheep pox lesions have the typical epithelial changes seen with
poxviruses, including marked hydropic degeneration of keratinocytes in the stratum
spinosum, microvesiculation, epidermal hyperplasia and eosinophilic intracytoplasmic
inclusion bodies. The lesions affect both surface epithelium and follicular epithelium.

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Marked dermal lesions reflect the systemic route of cutaneous involvement and may be
due to immune mediated disease as well as direct viral damage. During the papular
stage, large numbers of mononuclear cell accumulate in the increasingly edematous
dermis. These cells, first described by Borrel, are called “cellules claveleuses” or
sheep-pox cells, and are characteristic of the disease. The nuclei of sheep-pox cells
are vacuolated and have marginated chromatin. The vacuolated cytoplasm contains
single, occasionally multiple, eosinophilic intracytoplasmic inclusion bodies. Sheep-pox
cells are virus-infected monocytes, macrophages and fibroblasts, but not endothelial
cells.

Approximately 10 days post-infection, and corresponding with the peak of dermal

infectivity, a severe necrotizing vasculitis develops. Virus particles have not been
identified in endothelial cells and it is thought that the vasculitis may be due to immune
complex deposition. Ischemic necrosis of the dermis and overlying epidermis follows.
The pulmonary lesions are proliferative alveolitis and bronchiolitis with focal areas of
caseous necrosis. Alveolar septal cells contain intracytoplasmic inclusion bodies.
Histologic lesions, characterized by the accumulation of sheep pox cells, may involve
the heart, kidney, liver, adrenal glands, thyroid gland, and the pancreas.

AFIP Diagnosis: Haired skin: Dermatitis, hyperplastic, subacute, multifocal, moderate,

with epidermal and follicular keratinocyte ballooning degeneration, eosinophilic
intracytoplasmic inclusion bodies, and sheep pox cells, breed not specified, ovine.

Conference Comment: As mentioned by the contributor, intracytoplasmic inclusion

bodies may be present in several cell types. Conference attendees noted
intracytoplasmic inclusion bodies in surface and follicular epithelial cells, and in dermal
fibroblasts and macrophages.

Many strains of poxviruses are species specific and are given the name of the species
that they infect (i.e turkeypox virus, canarypox virus, cowpox virus, etc.), while others
may infect a wide range of hosts. Some pox diseases of vertebrates (family Poxviridae,
subfamily Chordopoxvirinae) include the following:5,6,7,8

Genus Virus Major Hosts Geographic

Distribution
Orthopoxvirus Variola virus (smallpox) Humans Eradicated globally
Vaccina virus Numerous: humans, cattle, Worldwide
buffalo, swine, rabbits
Cowpox virus Numerous: cattle, humans, Europe, Asia
rats, cats, gerbils, lg. felids,
elephants, rhinoceros, okapi
Camelpox virus Camels Asia, Africa
Ectromelia virus (mousepox) Mice, voles Europe
Monkeypox virus Numerous: squirrels, monkey, Western and
anteaters, great apes, central Africa
humans

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 Uasin Gishu disease virus Horses Eastern Africa
Tatera poxvirus Gerbils (Tatera kempi) Western Africa
Raccoon poxvirus Raccoons North America
Vole poxvirus Voles (Microtus californicus) California
Seal poxvirus Grey seals North Sea
Capripoxvirus Sheeppox virus Sheep, goats Africa, Asia
Goatpox virus Goats, sheep Africa, Asia
Lumpy skin disease virus Cattle, Cape buffalo Africa
Genus Virus Major Hosts Geographic
Distribution
Suipoxvirus Swinepox virus Swine Worldwide
Leporipoxvirus Myxoma virus Rabbits (Oryctolagus and Americas, Europe,
Sylvilagus spp.) Australia
Rabbit (Shope) fibroma virus Rabbits (Oryctolagus and Americas, Europe,
Sylvilagus spp.) Australia
Squirrel fibroma Gray squirrels and Eastern United
woodchucks States
Hare fibroma European hares Europe
Molluscipoxvirus Molluscum contagiosum virus Humans, horses, Worldwide
chimpanzees, kangaroos
Yatapoxvirus Yabapox virus Monkey, humans West Africa
Tanapox virus Monkey, humans West Africa
Avipoxvirus Fowlpox virus Chickens, turkeys, other birds Worldwide
Parapoxvirus Orf virus Sheep, goats, humans Worldwide
Pseudocowpox virus Cattle, humans Worldwide
Bovine papular stomatitis virus Cattle, humans Worldwide
Auzdyk virus Camels Africa, Asia
Seal parapoxvirus Seals, humans Worldwide

Contributor: Kimron Veterinary Institute, Pathology Department, P.O. Box 12,

Beit-Dagan, 50250 Israel

References:
1. Jubb KVF, Kennedy PC, Palmer N: Pathology of Domestic Animals, 4th ed. Vol.1,
pp. 637-639. Academic Press, San Diego, California, 1993
rd
2. W.B. Martin and I.D. Aitken: Diseases of sheep, 3 ed. pp 266-270. Moredum
Research Institute, Edinburg, 2000
3. M.G. Garner, S.D. Sawarkar, E.K. Brett, J.R. Edwards, V.B. Kalkarni, D.B. Boyle:
The extent and impact of sheep pox and goat pox in the state of Maharashtra, India.
Tropical Animal Health and Production 32(4):205-223, 2000
4. O.I.E, Manual of standards for diagnostic Tests and Vaccines, 4th ed. pp. 168-177,
2000
5. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ: Picornaviridae. In: Veterinary
Virology, 3rd ed., pp. 279. Academic Press, San Diego, CA, 1999
6. DiGiacomo RF, Mare CJ: Viral diseases. In: The Biology of the Laboratory Rabbit,
eds. Manning PJ, Ringler DH, Newcomer CE, 2nd ed., pp.180. Academic Press, San
Diego, CA, 1994
7. Davidson WR, Nettles VF: Field Manual of Wildlife Diseases in the Southeastern
United States, 2nd ed., pp. 249-250. University of Georgia, Athens, GA, 1997
8. Terrell SP, Forrester DJ, Mederer H, Regan TW: An epizootic of fibromatosis in gray
squirrels (Sciurus carolinensis) in Florida. J of Wildlife Disease 38(2):305-312, 2002

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SLIDE 74
CONFERENCE 19 / CASE I – 04020321 (AFIP 2948688)

Signalment: Bovine, Angus, male, 5 months old.

History: Five-month-old Angus bullcalf found acutely dead. Lung tissues (only)
submitted for microbiology and histopathology.

Laboratory Results: Aerobic culture of the lung retrieved trace numbers of

contaminant bacteria: Alpha-Streptococcus sp., Bacillus sp., Lactobacillus sp.
Moderate numbers of Mycoplasma sp. were obtained from Mycoplasma culture. Lung
tissue was positive by PCR for bovine respiratory syncytial virus (BRSV).

Contributor’s Morphologic Diagnosis: Severe acute bronchointerstitial pneumonia

with syncytia.

Contributor’s Comment: This case represents acute bronchointerstitial pneumonia

secondary to BRSV infection in a young calf. The lung is diffusely hypercellular with
widening of the interlobular septa by congestion, edema and inflammatory cells. The
inflammation is intensified around bronchi and bronchioles with flooding of adjacent
alveoli by edema. There is necrosis of bronchiolar epithelium and the bronchioles are
partially occluded by large foamy macrophages, neutrophils, edema, fibrin and
scattered multinucleated (syncytial) cells. Some bronchioles are surrounded by
follicular type aggregates of lymphocytes implicating the role of Mycoplasma in the
lesion.
The histological lesions and conspicuous syncytial cells suggest a viral pneumonia with
the main differential diagnoses of BRSV or PI-3. The PCR test on DNA collected from
lung tissue was positive for BRSV.

In the original sections obtained for diagnosis, the histological lesions were unique
compared to normal field cases submitted to our laboratory because significant lesions
from a secondary bacterial infection are not yet present. The features of the viral
pneumonia were untainted. In the recuts of the tissue for WSC submission, there is
regional variability in the purity of the viral lesion (vs. obscurity by the secondary
bacterial component) and variability in the conspicuous numbers of syncytia.

Two excellent reviews on BRSV are provided.1,2 BRSV was first isolated from an
outbreak of respiratory disease in calves from Switzerland in 1970 and first reported in
the United States in 1974. BRSV is a member of the pneumovirus genus,
Pneumovirinae subfamily, Paramyxoviridae family within the virus order of
Mononegavirales. The respiratory syncytial viruses are single stranded, negative-sense
RNA viruses. The Pneumovirinae subfamily of paramyxoviruses is unique in that its
members lack neuraminidase. The viruses attach to cells via membrane glycoprotein

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G, and following infection, viral antigen is detected in bronchiolar and alveolar
epithelium as well as alveolar macrophages. Although usually a prelude to bacterial
infections as part of the bovine respiratory disease complex, BRSV can produce
outbreaks of respiratory disease and occasional deaths on its own. Severe BRSV
respiratory disease is usually restricted to calves less than 6 months old.

AFIP Diagnoses: 1. Lung: Pneumonia, bronchointerstitial, acute, multifocal,

moderate, with necrotizing bronchitis and bronchiolitis, syncytia, and intracytoplasmic
eosinophilic inclusion bodies, Angus, bovine.
2. Lung: Peribronchitis and peribronchiolitis, lymphoplasmacytic, multifocal, mild.

Conference Comment: Bovine Respiratory Syncytial Virus (BRSV) is a pneumovirus

in the family Paramyxoviridae and is a causative agent of “enzootic pneumonia” or “calf
pneumonia”. BRSV occurs in a variety of breeds, although some reports state that
certain breeds are more susceptible. During natural outbreaks, clinical disease is most
severe in 1 to 5 month old calves, is seldom seen in calves less than 2 weeks of age,
and is virtually absent in calves over 9 months of age. The high prevalence of
antibodies against BRSV suggests that infection is endemic in most areas.2

Although the mode of transmission has not been determined, it is thought to occur
through direct contact or aerosolization over short distances. Many factors influence the
severity of disease, including: the animal’s immune status, environmental conditions,
animal management, and the presence of other infectious agents. The pathogenesis of
BRSV is not clear; however, some research indicates that immune-mediated
mechanisms play a dominant role. BRSV enhances bacterial colonization and
adherence and alters the specific and non-specific defense mechanisms of the
respiratory tract.2

Gross lesions are characterized as typical interstitial pneumonia involving the cranio-
ventral region of the lungs. In affected areas, the lungs are consolidated and bronchi
and bronchioles often are filled with mucopurulent exudate; hemorrhage and
emphysema may also be present. The interlobular septa are expanded by pronounced
edema. The cranio-dorsal and dorsal regions of the lungs often appear normal, but may
also be markedly distended due to edema and severe alveolar, interstitial, and
subpleural emphysema. Bronchial and mediastinal lymph nodes are often markedly
enlarged, edematous, and occasionally emphysematous. Histologically, there is a
bronchitis and peribronchitis accompanied by a large number of syncytial cells in the
nasal and tracheal mucosa and alveolar and bronchiolar epithelium. Viral antigen is first
detected in the bronchiolar epithelium, later in type I and type II pneumocytes, and may
also be detected in alveolar macrophages. The virus is capable of cell-to-cell
transmission, resulting in the generation of characteristic syncytial giant-cells.
Degeneration, necrosis, and hyperplasia of bronchial epithelium and of lymphoid tissue
around the bronchi are consistently present. The bronchial and bronchiolar exudates

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consist primarily of epithelial cells, neutrophils, and occasionally eosinophils, and are
often accompanied by edema and hyaline membrane formation.2

Contributor: Department of Veterinary Pathobiology, College of Veterinary Medicine,

Oklahoma State University, Room 250 McElroy Hall, Stillwater, OK 74074
http://www.cvm.okstate.edu

References:
1. Baker JC, Ellis JA, Clark EG: Bovine respiratory syncytial virus. Vet Clin North Am
Food An Pract 13:425-454, 1997
2. Larsen LE: Bovine respiratory syncytial virus (BRSV): a review. Acta Vet Scand
41:1-24, 2000

SLIDE 75
CONFERENCE 19 / CASE II – 03-21575 (AFIP 2936448)

Signalment: Unknown age, castrated male, mixed breed, Bos taurus, bovine.

History: Five out of 100 castrated male feedlot calves in the same pen died suddenly
with no clinical signs prior to death.

Gross Pathology: The heart had numerous epicardial, myocardial and endocardial
hemorrhages mixed with a few inconspicuous pale streaks.

Laboratory Results: Haemophilus somnus was isolated in pure culture from the heart,
lung, and a pericardial swab. Immunohistochemical staining of an ear notch biopsy and
the heart was negative for bovine viral diarrhea (BVD) virus.

Contributor’s Morphologic Diagnosis: Heart: Multifocal and perivascular

suppurative myocarditis and epicarditis with vasculitis, thrombosis, hemorrhage,
myocardial degeneration and necrosis, and intralesional gram-negative coccobacilli.

Contributor’s Comment: The submitted heart sections contain multiple perivascular

and random infiltrates of numerous intact and degenerate neutrophils within the
myocardium and epicardium. The tunica media of many small venules contains intact
and degenerate neutrophils with necrosis of the vascular wall. Many of the affected
venules contain fibrinous and fibrinocellular thrombi. The epicardium and myocardium
contain multiple hemorrhages, which are often associated with the affected blood
vessels. A few of the affected venules and inflammatory foci in the myocardium contain
variable numbers of small gram-negative coccobacilli. There are a few foci where the
cardiomyocytes are degenerate and necrotic, especially those entrapped within the
inflammatory foci.

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Haemophilus somnus is a small, gram-negative bacillus that can cause various and
numerous clinical disease presentations in cattle.1 The disease presentations in cattle
include, but are not limited to, pneumonia, meningoencephalitis, myocarditis, myocardial
abscesses, myositis, polyarthritis, abortion, endometritis, orchitis, epididymitis, placental
vasculitis, intestinal thrombosis, laryngeal ulceration, and a single case report of a
urachal abscess.1-9 Although some of the disease presentations can be primary
localized infections, such as pneumonia, many of the disease manifestations of H.
somnus infections in cattle are due to septicemia, such as meningoencephalitis,
polyarthritis, and myocarditis.1

Often, the septicemic form results in meningoencephalitis, but polyarthritis and

myocarditis can be seen singly or in combination with other affected organs.1 Although
the lung can be involved with H. somnus septicemia, pneumonia is an uncommon
feature of the septicemic disease. When the lung is involved, it more commonly results
in a primary fibrinous pneumonia or suppurative bronchopneumonia. The pneumonia
caused by H. somnus can be microscopically indistinguishable from that caused by
Mannheimia (Pasteurella) haemolytica.1,10 Most affected cattle develop a
fibrinopurulent polyarthritis, particularly in the atlanto-occipital joint.1 Myocarditis
supposedly following an asymptomatic episode of septicemia is a major manifestation of
H. somnus infection in some parts of North America.1 In one study performed in
Canadian feedlots, H. somnus infection was found in 70 out of 92 cases of myocarditis
in calves in the feedlot.11

The most common macroscopic lesions seen with H. somnus septicemia in cattle are
multiple foci of hemorrhage and necrosis in multiple organs.1 Microscopically, the
consistent feature of H. somnus septicemia is an intense vasculitis, usually of small
venules and veins. The inflammation can extend into the surrounding parenchyma of
the affected organ. The vasculitis often results in hemorrhage and can result in
infarction of the organ. The affected venules often contain fibrin thrombi, which
commonly contain colonies of bacteria. These colonies of bacteria are believed to
proliferate at the site of thrombosis and are believed not to be bacterial emboli.1
Although vasculitis is a common feature of H. somnus septicemia in cattle, the exact
pathogenesis of the vasculitis is not known, but it is believed to be due to H.
somnus–induced apoptosis of endothelial cells.12

AFIP Diagnosis: Heart: Myocarditis and epicarditis, suppurative, perivascular and

random, moderate, with vasculitis, thrombi, myocardial degeneration and necrosis, and
colonies of coccobacilli, mixed breed, bovine.

Conference Comment: Conference attendees discussed the histopathological

changes seen in cardiomyocyte degeneration and necrosis. Cardiac muscle is
structurally similar to skeletal muscle and is subject to the same anatomic changes
associated with degeneration. Cardiac myocyte degeneration is characterized by a
swollen vacuolated sarcoplasm, while necrosis is generally characterized by shrunken,

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hypereosinophilic or fragmented sarcoplasm, loss of cross-striations, and karyorrhexis,
karyolysis, or pyknosis. However, the gross and microscopic appearance of myocardial
necrosis is dependent on the interval between the initial insult and death.

In this case, the inflammation, degeneration, and necrosis occurred around, and
frequently obscured and disrupted vessels. Other causes of embolic myocarditis
include Salmonella sp., E. coli, and rarely Erysipelothrix rhusiopathiae.13 Clinically,
Clostridium chauvoei is another common cause of acute death in cattle with no clinical
signs prior to death. However, gross and histologic lesions of C. chauvoei differ from
those of this case. Clostridium chauvoei affects skeletal muscle and cardiac muscle,
with gross lesions characterized by focally extensive myonecrosis with edema and
emphysema; histologically, the lesions are not vasocentric, but are focally extensive.

Histophilus somni is the proposed new name for Haemophilus somnus,14 and infection
causes a variety of disease syndromes, as previously mentioned by the contributor.
However, H. somni is an opportunistic pathogen that is a relatively non-invasive
commensal of the bovine respiratory and reproductive mucosal surfaces. Nonetheless,
when factors that favor disease and compromise immunity, such as the stress of
transportation, concurrent viral infection, overcrowding, pregnancy, lactation, and harsh
weather significantly affect the animal, disease ensues.15

Vasculitis is the hallmark of systemic H. somni infections; however, the pathogenesis of

vascular damage is poorly understood.15 Identifying the mechanism by which H. somni
induces endothelial cell damage is difficult because the virulence factors are not well
characterized. In one report, the H. somni virulence factor LOS (lipo-oligosaccharide)
induced endothelial cell apoptosis in a time- and dose-dependent manner in-vitro.12
Another recent report noted virulence factors such as LOS phase variation, induction of
endothelial apoptosis, intraphagocytic survival, and immunoglobulin Fc-binding proteins
were important to survival and colonization of H. somni.15

Contributor: Kansas State University, Department of Diagnostic

Medicine/Pathobiology, 1800 Denison, Manhattan, KS
http://www.vet.k-state.edu/depts/dmp/index.htm

References:
1. Jubb KV, Huxtable CR: The nervous system. In: Pathology of Domestic Animals,
eds. Jubb KV, Kennedy PC, Palmer N, 4th ed., vol. 1, pp. 397-400. Academic Press,
San Diego, CA, 1993
2. Storts RW, Montgomery DL: The nervous system. In: Thomson’s Special Veterinary
Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 440-441. Mosby, St.
Louis, MO, 2001
3. Jones TC, Hunt RD, King NW: Diseases caused by bacteria. In: Veterinary
Pathology, eds. Jones TC, Hunt RD, King NW, 6th ed., pp 439-440. William and Wilkins,
Baltimore, MD, 1997

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4. Hulland TJ: Muscle and tendon. In: Pathology of Domestic Animals, eds. Jubb KV,
Kennedy PC, Palmer N, 4th ed., vol. 1, p. 245. Academic Press, San Diego, CA, 1993
5. Ladds PW: The male genital system. In: Pathology of Domestic Animals, eds. Jubb
th
KV, Kennedy PC, Palmer N, 4 ed., vol. 3, p. 547. Academic Press, San Diego, CA,
1993
6. Kennedy PC, Miller RB: The female genital system. In: Pathology of Domestic
Animals, eds. Jubb KV, Kennedy PC, Palmer N, 4th ed., vol. 3, pp. 413-415. Academic
Press, San Diego, CA, 1993
7. Barker IK, Van Dreumel AA, Palmer N: The alimentary system. In: Pathology of
Domestic Animals, eds. Jubb KV, Kennedy PC, Palmer N, 4th ed., vol. 2, p. 102.
Academic Press, San Diego, CA, 1993
8. Dungworth DL: The respiratory system. In: Pathology of Domestic Animals, eds.
Jubb KV, Kennedy PC, Palmer N, 4th ed., vol. 2, pp. 568-569. Academic Press, San
Diego, CA, 1993
9. Starost MF: Haemophilus somnus isolated from a urachal abscess in a calf. Vet
Pathol 38(5):547-548, 2001
10. Lopez A: Respiratory system, thoracic cavity, and pleura. In: Thomson’s Special
Veterinary Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., p. 170.
Mosby, St. Louis, MO, 2001
11. Haines DM, Moline KM, Sargent RA, Campbell JR, Myers DJ, Doig PA:
Immunohistochemical study of Haemophilus somnus, Mycoplasma bovis, Mannheimia
haemolytica, and bovine viral diarrhea virus in death loss due to myocarditis in feedlot
cattle. Can Vet J 45(3):231-234, 2004
12. Sylte MJ, Corbeil LB, Inzana TJ, Czuprynski CJ: Haemophilus somnus induces
apoptosis in bovine endothelial cells in vitro. Infect Immun 69(3):1650-1660, 2001
13. Radostits OM, Gay CC, Blood DC, Hinchcliff KW: Veterinary Medicine A Textbook
of the Diseases of Cattle, Sheep, Pigs, Goats, and Horses, 9th ed., pp. 64, 741. W.B.
Saunders Company Ltd, Philadelphia, PA, 2000
14. Angen O, Ahrens P, Kuhnert P, Christensen H, Mutters R: Proposal of Histophilus
somni gen. nov., sp. nov. for the three species incertae sedis ‘Haemophilus somnus’,
‘Haemophilus agni’ and ‘Haemophilus ovis’. Int J Syst Evol Microbiol 53:1449-1456,
2003
15. Siddaramppa S, Inzana TJ: Haemophilus somnus virulence factors and resistance
to host immunity. Anim Health Res Rev 5(1):79-93, 2004

SLIDE 76
CONFERENCE 19 / CASE III – 03-9400 (AFIP 2933956)

Signalment: 3 month old female, Jersey calf, bovine (Bos taurus).

History: Three calves from a group of approximately 70 had bloody diarrhea and were
recumbent. Animals were euthanized by the practitioner and tissues were submitted to
the diagnostic laboratory.

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Gross Pathology: The only gross lesions noted on the necropsy report by the
practitioner were ulcers throughout the abomasums of the calves.

Laboratory Results: Rare oocysts of Eimeria zuernii and Eimeria ellipsoidalis were
noted on routine fecal examination. A fluorescent antibody test of colon and an
immunohistochemical stain of colon were positive for bovine coronavirus.

Contributor’s Morphologic Diagnosis: Colon: Colitis, lymphoplasmacytic and

eosinophilic, diffuse, moderate with numerous coccidial schizonts and gametocytes,
Jersey, bovine.

Contributor’s Comment: Diffusely throughout the colon, there is mild to moderate

lymphoplasmacytic eosinophilic colitis with many second generation coccidial schizonts
and gametocytes present within epithelial cells of colonic crypts. In addition, multifocally
there is marked dilation of colonic crypts with occasional epithelial attenuation and crypt
hyperplasia. Occasionally colonic crypts are collapsed. Within sections of small
intestine examined (sections not submitted with this case), there is multifocal crypt
dilation and hyperplasia with occasional crypts containing inflammatory debris.

Histologic lesions within the colon in this case are characteristic for coccidial enteritis in
calves. The second generation schizonts of the more pathogenic coccidia enter the
crypt epithelial cells of the colon and cecum approximately 14 – 18 days after infection.2
Virtually all cells lining cecal and colonic glands can be infected.2 As cells rupture and
oocysts are released, the remaining intact glandular epithelium can become markedly
attenuated and the glands may even collapse.2 Crypt hyperplasia occurs in an attempt
to regenerate mucosal epithelium in areas where it has been ulcerated and/or
denuded.2

Eimeria zuernii and Eimeria bovis are most often implicated in cases of clinical
coccidiosis in cattle up to 2 years of age3; however, other coccidia including E.
ellipsoidalis and E. auburnensis are also known to cause less severe diarrhea.2

Additionally, laboratory testing indicated infection with bovine coronavirus in this case.
It is likely the characteristic histologic lesions of this viral infection are masked by the
coccidial colitis present; however, some of the lesions present may non-specifically
support the presence of viral enteritis. For example, colonic lesions indicative of
coronavirus infection include a mixed inflammatory reaction within the lamina propria,
dilated colonic glands with attenuated epithelium and glandular hyperplasia.1
Additionally, crypt epithelium in the small intestine may also be hyperplastic as a result
of coronavirus infection.1

AFIP Diagnosis: Colon: Colitis, lymphoplasmacytic and eosinophilic, diffuse,

moderate, with crypt loss, regenerative hyperplasia and ectasia, and myriad intracellular
coccidia, etiology consistent with Eimeria spp., Jersey, bovine.

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Conference Comment: Over a thousand species of Eimeria are known that primarily
infect intestinal epithelial cells of domestic and wild mammals and birds. The life cycle
of each species is host specific and direct. Unsporulated oocysts are shed in the feces
and sporulate in the environment to become infectious. Following ingestion,
sporozoites excyst, invade intestinal epithelial cells, form trophozoites and undergo
asexual multiplication (schizogony, merogony) within a schizont or meront. Merozoites
are released and eventually form sexual stages (micro- and macrogametes), which
unite to form oocysts.4 Some common coccidia species of domestic and wild mammals
and birds include the following:3,4,5

Animal Coccidia Organ affected

st
Cattle E. bovis 1 gen schizont – Jejunum
nd
2 gen schizont – Cecum and colon
Sheep E. ahsata Small intestine
E. bakuensis Small intestine
E. ovinoidalis Ileum/Large intestine
Goats E. christenseni Small intestine
E. arloingi Small intestine
E. ninakohlyakimovae Large intestine
Equine E. leuckarti Small intestine
Swine I. suis Small intestine
Canine I. canis Ileum, colon occasionally
Feline I. felis Small intestine, colon occasionally
Mice E. falciformis Colon
Rabbit E. stiedae Bile ducts
E. intestinalis Ileum & cecum
E. flavescens Ileum & cecum
Birds
Chickens E. acervulina Duodenum
E. necatrix Mid-intestine
E. maxima Mid-intestine
E. tenella Ceca
Turkey E. adenoeides Ceca
E. meleagrimitis Mid-intestine
E. gallopavonis Colon, rectum
Geese & ducks E. truncata Kidney
E. anseris Mid-intestine

Contributor: South Dakota State University, Veterinary Science Department, P.O. Box
2175, North Campus Drive, Brookings, SD
http://vetsci.sdstate.edu

References:
1. Barker IK, Van Dreumel AA, Palmer N: The alimentary system. In: Pathology of
Domestic Animals, eds. Jubb KV, Kennedy PC, Palmer N, 4th ed., vol.2, pp. 187–189.
Academic Press, San Diego, CA, 1993

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2. Barker IK, Van Dreumel AA, Palmer N: The alimentary system. In: Pathology of
Domestic Animals, eds. Jull KV, Kennedy PC, Palmer N, 4th ed., vol. 2, pp. 297–300.
Academic Press, San Diego, CA, 1993
th
3. Georgi JR, Georgi ME: Protozoans. In: Parasitology for veterinarians, 5 ed., pp.
84–91. WB Saunders Company, Philadelphia, PA, 1990
4. Gardiner CH, Fayer R, Dubey JP: An Atlas of Protozoan Parasites in Animal
Tissues, 2nd ed., pp. 20-30. The Armed Forces Institute of Pathology, Washington, D.C.,
1998
5. McDougald LR: Protozoal infections. In: Diseases of Poultry, ed. Saif YM, 11th ed.,
pp. 976-981, 986, 989. Iowa State Press, Ames, IA, 2003

SLIDE 77
CONFERENCE 19 / CASE IV – 04-K0227 (AFIP 2937322)

Signalment: 2 month old female polled Hereford calf.

History: This calf was found dead in the field where she was housed with her dam and
90 other cows and calves. The calf had recently (< 1 week ago) been moved to pasture
from a calving pen, where management had been of good quality (adequate colostrum,
good navel care, perinatal vitamin E / selenium injection). No previous illnesses had
been identified in this calf.
Gross Pathology: Body condition was fair. The liver was pale and mottled, with
capsular petechiation. Mesenteric lymph nodes were slightly enlarged. Lungs were
red, wet, and heavy, with obvious interlobular edema in some areas. The rumen
contained black, semi-fluid, odiferous debris.

Laboratory Results: Bacteriology: Listeria monocytogenes was isolated in large

numbers from liver and spleen.

Contributor’s Morphologic Diagnoses: 1. Acute multifocal necrotizing adrenalitis

and hepatitis with intralesional bacilli.
2. Multifocal renal intravascular bacterial emboli.
3. Multifocal microvascular pulmonary thrombosis.

Contributor’s Comment: Histologic lesions were consistent with septicemic listeriosis

but were more severe than typically seen.1 In the liver, multiple foci of acute
coagulation necrosis involved over 75% of hepatic parenchyma, and large numbers of
plump gram-positive bacilli were present within hepatocytes and extracellularly at the
margin of necrotic and viable parenchyma. Lesions were similar in the adrenal gland,
with multiple discrete foci of coagulation necrosis, involving approximately 50% of
cortical parenchyma and large numbers of bacilli present among necrotic cellular debris
within most foci. In the kidney, dense clusters of similar bacilli filled the lumens of
several cortical and medullary interstitial blood vessels and glomerular capillaries.
Pulmonary alveoli were flooded with proteinaceous edema fluid occasionally mixed with

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fibrin or low numbers of macrophages, and a moderate number of alveolar capillaries
contained luminal fibrin thrombi.

Small intestinal submucosal lymphoid aggregates were sparsely populated and rare
individual mucosal crypts had widely dilated lumens filled with neutrophils and necrotic
cellular debris. Immunohistochemistry (IHC) for bovine viral diarrhea virus (BVDV)
identified abundant viral antigen in mononuclear cells in the intestinal lamina propria,
submucosa, and Peyer’s patches; in the tunica media of submucosa blood vessels; and
in scattered mucosal epithelial cells. Abundant positive staining for BVDV was also
evident in Kupffer cells and few intact hepatocytes in liver; mononuclear cells in lymph
node; and tunica media of myocardial blood vessels. IHC was negative for bovine
herpesvirus-1 antigen in liver and adrenal gland. Immunosuppression in cattle due to
BVDV infection can promote susceptibility to other infectious agents, and BVDV may
have predisposed this calf to severe septicemic listeriosis.2

AFIP Diagnosis: Liver: Hepatitis, necrotizing, acute, random, severe, with myriad
bacilli, Hereford, bovine.

Conference Comment: Listeria monocytogenes is a small, rod-shaped, gram-positive

intracellular bacterium that causes disease in most species of animals and humans.
The organism is ubiquitous in nature and can be found in soil, vegetation, dairy
products, animal feces, and sometimes the oropharynx and tissues of healthy animals.1

Listeriosis occurs as three distinct syndromes, which ordinarily do not occur together:
systemic infection (septemia) in humans, cattle, sheep, swine, dogs, cats, and rodents;
encephalitis in humans, cattle, sheep, goats, and swine; and abortion in humans, cattle,
and sheep. Less commonly, L. monocytogenes is a cause of endocarditis and purulent
lesions in other tissues.1

Systemic infection is the more common form of listeriosis in monogastric animals and in
human infants. The most characteristic lesion in this form is focal necrosis of the liver.
However, lesions may also occur in the spleen, lymph nodes, lungs, adrenal glands,
gastrointestinal tract, and brain. Microscopically, there are areas of necrosis infiltrated
by mononuclear cells and some neutrophils. The organisms may be seen in sections
stained with H&E (Hematoxylin and Eosin) or can be easily demonstrated with B&B
(Brown-Brenn) and B&H (Brown-Hopps).1

Encephalitis is the most characteristic form of the disease in ruminants. Clinical signs
include abnormal posturing of the head and neck, walking aimlessly in a circle (“circling
disease”), nystagmus, blindness, and paralysis. The organism is thought to reach the
central nervous system by ascending peripheral nerves, particularly the trigeminal
nerve, and localizing in the brain stem, particularly the medulla oblongata, and in the
spinal cord. Gross lesions are usually absent; however, leptomeningeal opacity and
foci of necrosis in the terminal brain stem have been noted.3 Microscopically, there are

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perivascular mononuclear cell infiltrates, with or without neutrophils. Diffuse cellular
infiltration and microabscessation involving both the gray and white matter may occur,
but there is usually relatively little tissue necrosis.1 However, necrosis and
accumulation of gitter cells can be prominent in some cases. Other changes include
neuronal necrosis and leptomeningitis.3

Listeric abortion in animals is important in cattle and sheep. Abortion usually occurs in
the last quarter of gestation without signs of infection in the dam. The fetus dies in utero
and may be severely autolyzed when expelled.1 Placental lesions include severe
diffuse necrotizing and suppurative inflammation of both the cotyledons and the
intercotyledonary areas. The fetal lesion is an enlarged liver with numerous 1 mm
yellow foci. Microscopically, severe inflammation involves the mesenchyme of the villi
and the upper intercotyledonary chorion. Chorionic epithelial cells, especially in areas
between the villi, are filled with gram-positive bacilli. The cells in the areas of acute
multifocal necrotizing hepatitis are also filled with organisms.4

Contributor: University of Guelph, Laboratory Services Division, Animal Health

Laboratory, Guelph, Ontario
http://ahl.uoguelph.ca

References:
1. Jones TC, Hunt D, King NW: Diseases caused by bacteria: Listeriosis. In: Veterinary
Pathology, Jones TC, Hunt RD, and King NW, pp 461-463. Williams and Wilkins,
Baltimore, MD, USA, 1997
2. Brackenbury LS, Carr BV, Charleston B: Aspects of the innate and adaptive immune
responses to acute infections with BVDV. Vet Microbiol 96:337-344, 2003
3. Storts RW, Montgomery DL: The nervous system. In: Thompson’s Special Veterinary
Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 441-442. Mosby, St.
Louis, MO, 2001
4. Acland HM: Reproductive system: Female. In: Thompson’s Special Veterinary
Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 617-618. Mosby, St.
Louis, MO, 2001

SLIDE 78
CONFERENCE 20 / CASE I – 99-10725 (AFIP 2683903)

Signalment: 2-month-old, male, Arabian horse (Equus caballus).

History: This colt had a spastic gait, ataxia and head tremors for about two weeks prior
to elective euthanasia.

Gross Pathology: No significant gross lesions were noted in the thoracic or abdominal
viscera. The cerebrum and cerebellum appeared normal in size and development,

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externally. A sagittal section of the cerebellum revealed less prominent folia in the
cranial and dorsal areas of the vermis.

Laboratory Results: A computerized tomography (CT) scan of the brain was

inconclusive.

Contributor’s Morphologic Diagnosis: Cerebellar cortical abiotrophy.

Contributor’s Comment: The history and gross and microscopic lesions in this case
are consistent with a diagnosis of cerebellar cortical abiotrophy. Affected animals are
usually neurologically normal at birth with clinical signs developing at various times in
postnatal development. Spasticity and ataxia are observed in the gait, especially in the
forelimbs. Grossly, the cerebellar folia are best developed (or least degenerative) in the
caudal and ventral areas of the cerebellum (see gross photograph). The cerebellum
was not weighed in this case but in normal horses the cerebellum should be about 10-
12% of total brain weight. Microscopically, there are degenerative and missing Purkinje
cells with a reduction in all layers where Purkinje cells are absent. There is
accentuation of radial astrocyte processes in the molecular layer. The cause of this
condition is unknown, although a hereditary component (autosomal recessive trait) is
suspected.

AFIP Diagnosis: Brain, cerebellum: Purkinje and granular cell degeneration and loss
(cerebellar cortical abiotrophy), diffuse, moderate, with mild Wallerian degeneration in
the folia white matter, and mild gliosis of cerebellar nuclei, Arabian, equine.

Conference Comment: Conference attendees discussed the relationship between the

normal development and physiology of the cerebellum and the clinical and
histopathological changes identified in this case.

The term abiotrophy literally means the lack of a life-sustaining nutritive factor. Implicit in
its use in veterinary medicine is the presumption that the premature neuronal
degeneration is not an acquired insult, but rather the consequence of an intrinsic
metabolic disorder. However, the specific metabolic derangement may vary from
syndrome to syndrome. The hallmark of cerebellar abiotrophic diseases is the
premature demise of discrete and often functionally related populations of neurons, after
the organ has developed its full cellular complement.1

In abiotrophic conditions encountered in veterinary medicine, neurological deficits

usually begin in the first few weeks to months of life, are progressive, and are inherited
in studied populations. The clinical hallmark of cerebellar cortical abiotrophies is
neurological normality at birth, followed by the development of cerebellar deficits that
progressively worsen in the post-natal period. In contrast, in-utero damage, such as
with viral agents that may damage the developing cerebellum at a very precise stage of
fetal life, results in cerebellar ataxia from the time of birth. Because the injury is not

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ongoing, the neurological deficits are often static and may even slowly improve as the
animal learns to compensate.1

Microscopically, cerebellar cortical abiotrophies are characterized by ongoing neuronal

degeneration and loss, with reactive gliosis in the background of a normally developed
cerebellum. Neither folial dysplasia nor neuronal heterotopia occurs in cerebellar
cortical abiotrophies; they are features of in-utero viral infections that can disrupt normal
cerebellar development. Purkinje cells are usually affected first in abiotrophies, and, in
general, a reduction of the granule cell neurons follows. There is often proliferation of
astroglia (Bergmann astrocytes) in the affected folia and a mild gliosis of the molecular
layer. Due to Purkinje cell degeneration, Wallerian degeneration may be found in the
white matter of the folia and low numbers of spheroids may be present in the granular
layer, cerebellar white matter, or the nuclei of the cerebellar medulla. Cerebellar nuclei
are often gliotic.1

Cerebellar abiotrophies have been reported in a number of domestic and laboratory

species, including dogs, cattle, sheep, Yorkshire pigs and Arabian horses and Gotland
ponies. The time of onset of clinical signs varies with the species and breed affected.
Common dog breeds affected include Kerry Blue Terriers, Gordon Setters and Rough-
Coated Collies. Veterinary Neuropathology provides a more thorough list of
predisposed breeds.1 The syndrome in Kerry Blue Terriers is unique since the caudate
nucleus and the substantia nigra are also affected. Cerebellar abiotrophy of Gordon
Setters is unusual since it has a delayed onset, with clinical signs not typically
appearing until six to 24 months of age. Kerry Blue Terriers and Rough-Coated Collies
may have extracerebellar involvement, with Wallerian degeneration in the brainstem
and spinal cord.

Contributor: University of Minnesota, College of Veterinary Medicine, Veterinary

Diagnostic Laboratory, 1333 Gortner Avenue, St. Paul, MN

References:
1. Summers BA, Cummings JF, de Lahunta A: Veterinary neuropathology, pp. 300-305.
Mosby, St. Louis, MO, 1995
2. Palmer JD, Blakemore WF, Cook WR, et al: Cerebellar hypoplasia and degeneration
in the young Arab horse: clinical and neuropathological features. Vet Rec 93:62-66,
1973
3. Baird JD, MacKenzie CD: Cerebellar hypoplasia and degeneration in part-Arab
horses. Aust Vet J 50:25-28, 1974
4. Sustronck B, Mylle E, De Geest J, De Smidt M: Cerebellaire abiotrofie bij drie
arabische volbloedveulens. Vlaams Dier Tijd 59:151-154, 1990

SLIDE 79
CONFERENCE 20 / CASE II – 51304 (AFIP 2947490)

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Signalment: 14-year-old, male-castrated, Golden retriever.

History: In early spring 2003, the dog began to become bilaterally paretic in the rear
legs. This progressively got worse. In November 2003, a cervical laminectomy was
performed. The dog did not get any better following surgery. After six months, the
owner opted for euthanasia.

Gross Pathology: No gross abnormalities were noted.

Contributor’s Morphologic Diagnosis: Meninges: Pachymeningitis, ossifying,

diffuse, severe with fibrosis.

Contributor’s Comment: The case resembles closely a case described in the Journal
of the American Veterinary Medical Association,1 except that it is far more extensive,
with involvement of the entire spinal cord. A comment on their report was written by Dr.
John McGrath,2 who felt that they had over-interpreted the lesion. The case and lesion
are unusual, but the effects on the animal were severe, as are the lesions. A differential
diagnosis that might be considered is Lyme disease.

AFIP Diagnoses: 1. Leptomeninges, spinal: Fibroplasia, diffuse, with vascular

hyalinization, and multifocal arachnoid cell proliferation, Golden Retriever, canine.
2. Dura, spinal: Osseous metaplasia, focal.
3. Spinal cord, ventral funiculi: Axonal degeneration and loss, mild, with dilated myelin
sheaths, axonophagia, and rare spheroids.

Conference Comment: There is significant variation in slides and not all features may
be present on all slides. In the sections reviewed by conference attendees, the major
histological changes in the arachnoid layer included fibroplasia, vascular hyalinization,
and multifocal arachnoid cell proliferation. The osseous metaplasia noted in some
slides is within the dura mater.

The literal definition of pachymeningitis is inflammation of the pachymeninges, also

known as dura mater. Metaplastic ossification of the spinal dura occurs most frequently
in the lumbar region of large and giant breed dogs, and is sometimes referred to as
ossifying pachymeningitis. These grayish islands of lamellar bone may contain
adipocytes and myeloid elements and are an incidental, age-related change. An
association with intervertebral disk prolapse has been suggested, but this metaplastic
change can be found in the cranial dura also. In addition, thickening (sclerosis) as a
result of fibrosis, with acellular collagen deposition or hyalinization, may be found in the
leptomeninges and choroid plexus stroma of old animals.3 Residents also noted small
amounts of perivascular lipofuscin accumulation, particularly in the gray matter. This
too is a common finding in older dogs.

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Of additional interest in this case is the axonopathy in the ventral funiculi, which is likely
due to focal or diffuse disease rostral to this segment and may be the cause of the
clinical signs noted in this case. Nonetheless, the histopathological lesions in the spinal
cord are likely not the result of the meningeal lesions. The causes of the
leptomeningeal changes and the spinal cord axonopathy are not evident in the slides
examined in conference.

Contributor: Johns Hopkins University School of Medicine, Department of

Comparative Medicine, 733 North Broadway, Suite 811, Baltimore, MD

References:
1. Wilson J, Greene H, Leipold H: Osseous metaplasia of the spinal dura mater in a
Great Dane. J of Am Vet Med Assoc 167(1):75-7, 1975
2. McGrath JT. Letter: Spinal ossifying pachymeningitis. J of Am Vet Med Assoc
167(12):1045-1048, 1975
3. Summers BA, Cummings JF, de Lahunta A: Veterinary neuropathology, pp. 52-53.
Mosby, St. Louis, MO, 1995

SLIDE 80
CONFERENCE 20 /CASE III – 03-1499 (AFIP2935569)

Signalment: 4-month-old, female mixed breed goat.

History: Recumbent for one week. The animal had some tremors and stiff hind limbs.

Gross Pathology: None.

Contributor’s Morphologic Diagnoses: 1. Cerebellar abiotrophy.

2. Neuronal and axonal degeneration, spinal cord, mild.

Contributor’s Comment: The cerebellar folia have depletion of the granular cell layer
and loss of Purkinje cells. Chromatolysis is seen among the remaining Purkinje cells
and in the large neurons within the brainstem. The spinal cord has mild Wallerian
degeneration of the ventral white matter tracts and chromatolysis of neurons in the
ventral horn. The cerebellar lesion is consistent with cerebellar abiotrophy, but when
taken in consideration with the spinal cord lesions, this case is an example of a
multisystem neuronal degeneration.

Multisystem neuronal degeneration is a degeneration or abiotrophy of neurons in

multiple locations throughout the nervous system. In humans these include both familial
and acquired conditions and may affect single or multiple neuronal systems, including
motor, sensory and autonomic neurons. Neuronal system degenerations have been
reported in a pig and several breeds of dogs, including the Swedish Lapland reindeer-
herd dog, Cairn Terrier, Cocker Spaniel and Miniature Poodle. These primarily affect the

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brain and spinal cord and are inherited in all but the Miniature Poodle. Cerebellar
abiotrophy and multisystem neuronal degeneration have not been reported in the goat.

AFIP Diagnoses: 1. Brain, cerebellum: Purkinje and granule cell degeneration and
loss, diffuse, moderate, with Purkinje cell ectopia, and molecular gliosis, mixed breed,
caprine.
2. Brainstem, medulla oblongata; spinal cord, ventral column: Neuronal degeneration
(chromatolysis), multifocal, mild to moderate, with gliosis.

Conference Comment: In contrast to the first case, not only is there Purkinje cell
degeneration and loss, but there is also Purkinje cell ectopia with moderate numbers of
Purkinje cells located in the molecular layer. The latter indicates this degenerative
disease began in-utero when the Purkinje cells were still migrating from the internal
germinal layer adjacent to the fourth ventricle.2

The histopathological changes present in the cerebellum, brain stem, and the spinal
cord, suggest copper deficiency as a possible etiology. Copper deficiency can cause
two clinical neurologic disease syndromes in sheep and goats: congential (swayback)
and acquired (enzootic ataxia).

In the congenital form (swayback), the condition develops in-utero and clinical signs are
present at birth with affected animals being totally recumbent or severely ataxic. Other
signs include depression, head shaking, trembling, and most affected animals die soon
after birth. Grossly, there may be small foci of gelatinous softening, or cavitation of the
cerebral hemispheres. Microscopically there is absence or destruction of the white
matter of the cerebral hemispheres with chromatolysis of large motor neurons of the red
and vestibular nuclei. Demyelination of the motor tracts of the white matter of the spinal
cord has also been reported.3

The delayed form (enzootic ataxia) develops after birth with animals appearing normal
at birth and developing signs of the disease from one week to six months of age.
Clinical signs include incoordination, ataxia, and posterior paresis. Lesions are limited
to the large neurons of the brain stem and spinal cord. However, goats with enzootic
ataxia may have well defined lesions in the cerebellum, including patchy cerebellar
hypoplasia, necrosis, and loss of Purkinje cells and depletion of the granular cell layer.3

Swayback and enzootic ataxia may result from either primary or secondary copper
deficiency. Primary copper deficiency is caused by a diet that is low in copper.
Secondary copper deficiency results from dietary composition, which determines the
proportion of dietary copper that is absorbed. It is well known that other minerals, such
as molybdenum, sulfur, and iron can interfere with proper copper utilization. However,
food type and the interaction between food type and mineral composition will also affect
copper absorption and ultilization.3

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Contributor: Virginia Tech, College of Veterinary Medicine, Department of Biomedical
Sciences & Pathobiology, Blacksburg, VA
www.vetmed.vt.edu

References:
1. Summers BA, Cummings JF, de Lahunta, A: Veterinary Neuropathology, pp. 300-
307. Mosby, St. Louis, MO, 1995
2. De Lahunta A: Cerebellum. In: Veterinary Neuroanatomy and Clinical Neurology, 2nd
ed., pp. 255-256. W.B. Saunders Company, Philadelphia, PA, 1983
3. Banton MI, Lozano-Alarcon F, Nicholson SS, Jowett PLH, Fletcher J, Olcott BM:
Enzootic ataxia in Louisiana goat kids. J Vet Diagn Invest 2:70-73, 1990

SLIDE 81
CONFERENCE 20 / CASE IV – TAMU 04-02 (AFIP 2941201)

Signalment: 10-day-old, male Brangus calf.

History: The calf was born in a pasture and found recumbent with a “hole in the back.”
The calf never walked, and was brought into the clinic as a donation. At presentation,
the animal was moribund and considered septicemic. He had a withdrawal reflex in all
limbs but could not stand. A skin defect over a deep hole was noted on the dorsum in
the thoraco-lumbar area. No work-up was conducted, and a clinical diagnosis of spina
bifida preferred. The animal was euthanized.

Gross Pathology: Decubital ulcers were over bony prominences. A 2X1.5 cm, open,
skin defect was at the T13-L1 junction. Another depression in dorsal, axial tissues was
at the L6-S1 junction, but was not associated with a skin defect. The dorsal arch of the
T13-L1 junction was absent and a hyalinized membrane connected the spinal canal to
the “hole” seen grossly. The spinal cord became attenuated and deviated dorsally into
the membrane. No spinal cord was seen from L1 to approximately L3 (spinal
dysraphism in its broadest definition; segmental spinal cord aplasia or necrosis). The
spinal column began again and continued normally from L4 caudally. A fibrous band
was in the area of the arch at L6-S1, and the dura and spinal cord appeared normal in
this area (spina bifida occulta).

The cortex of the occipital pole of the brain extended caudally over the cranial aspect of
the cerebellar cortex at the midline with a redundant or hamartomatous growth of the
marginal gyrus (local, cerebral-cortical dysplasia/redundancy). Meninges were slightly
opacified. The lateral ventricles were twice normal volume (hydrocephalus)

Macroscopic Diagnosis: Multiple spina bifida aperta and occulta; spinal

dysraphism/focal spinal cord aplasia/necrosis; meningitis, hydrocephalus, cerebral
cortical hamartomatous dysplasia.

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Contributor’s Morphologic Diagnosis: Severe, subacute/chronic suppurative
meningomyelitis, with granulation tissue and numerous bacteria; spinal meningocele;
spinal dysraphism with agenesis of the spinal cord, OR absence of spinal cord, OR
hypoplasia of the spinal cord, OR duplication of the spinal canal AND/OR
diastematomyelia

Contributor’s Comment: Malformations offer special challenges to the diagnostician.

The most important decision is determining if the malformations are hereditary. I do not
believe this is a hereditary condition. The low incidence of such lesions and the
inadequate histories in our necropsy population usually make that determination
impossible. The variation in malformations between individuals makes comparison
between cases of malformations difficult, and usually, the diagnosis is a descriptive
exercise.1,2,3,5,7,8,12,13 Often, one malformation is accompanied by or leads to more
malformations. Sometimes, names given to cases are inaccurate. Depending on the
level of the section received, the lesion you see on your slide will vary in this presented
case. A unifying theme is subacute to chronic inflammation with granulation tissue,
neutrophilic and histiocytic exudates, and bacteria associated with fibrin and necrosis.
An occasional thrombus is noted (considered a consequence not a primary lesion). The
animal could not move and history suggested the animal’s back lesion (the myelocele)
had been pecked at by birds. Thus, with the opening to the CNS, sepsis became
rampant in this case (and other similar cases in our files). The meninges surround the
cord as the cord 1) moves dorsally, 2) becomes smaller, 3) loses its central canal, 4) is
progressively bisected to give diastematomyelia, 5) becomes a small core of
nondescript neural tissue, and then, 6) disappears from the section.

A series of images is provided for all participants to follow. The entire affected area of
cord was blocked and cut.

Figure 4. Cranial to the meningocele (2X).

Figure 5. Cord at start of the “coele” (2X).
Figure 6. Cord with ventral fibrous septum dissecting the cord (2X).
Figure 7. Fibrous septum dividing the cord (diastematomyelia) (2X).
Figure 8a. Fibrous tissue and remnant neural parenchyma (2X).
Figure 8b. Remnant neural tissue of 8a, note thrombus (10X)
Figure 9. Intact cord with two central canals distal to the meningocele (2X).

The sections where spinal cord is not present are not submitted. One could argue that
the infection destroyed the cord in this area; however, the progressive diminution of
tissue and persistence of remnant cord in caudal sections of the affected area as
described above argues against this theory. Segmental loss of spinal cord with
reappearance in both cervical and lumbo-sacral areas is described in calves.5,7,10,11
Such segmental loss in areas where the development of a canal and mesenchymal
structures is complete suggests to me that the cord was present at one time to allow
induction of somite development, and only later, the cord underwent necrosis.
Subsequently, the lumbar and sacral cord reappears as normal except for a brief caudal
lumbar segment with duplication of the central canal and the continued inflammatory

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change seen at all levels of the cord and brain. The loss of the dorsal bony arch in the
lumbo-sacral junction was associated with no defect in the associated cord and is spina
bifida occulta. The withdrawal reflex noted clinically is a spinal reflex and not
demonstrative of perception of deep pain. It is tempting to say that the premature
termination of the cord with its filamentous end not going into the meningocele may
represent tethering of the cord.

Spina bifida is a form of rachischisis/cleft vertebral canal.4,6,14 Dysraphism is failure of a

fusion of a raphe. Interestingly, the term per se is not defined in or not even used by
some current human neuropathology texts. However, some authors3 have (incorrectly I
believe) used the term more broadly to include any “myelodysplasia” including: aplasia,
hydromyelia, syringomyelia, fusion failures of the neural tube, etc.. Unfortunately, I was
taught and remember that Weimaraner dogs had a condition of spinal dysraphism,
which is an incorrect diagnosis for the condition.15 It is syringomyelia, hydromyelia, and
central canal dysplasia. I think the term, dysraphism, should be used generically when
there is a neural tube fusion disorder. Most cases of spina bifida have a closed neural
tube. We will not discuss the process of closure of the neural tube (Chapter 8 in
Greenfield’s), but it occurs as a bidirectional process occurring multifocally in both the
spinal cord and brain. The caudal spinal cord develops by secondary neurulation via a
growth of neural cells caudally, NOT from a tube. The lumbosacral spina bifida occulta
of this case is probably the result of a defect in this secondary neurulation process.
Spina bifida’s pathogenesis is thought to involve: 1) abnormal proliferation of neural
tissue in this area, 2) focal ischemic injury, or 3) an idiopathic/undefined
maldevelopment of the tail bud.

Spinal cord anomalies often are associated with brain malformations, especially Chiari II
malformations; however, the cerebellar vermis in this animal is normal. The redundant
cortex seen is not part of a described syndrome. Hydrocephalus is common in cases of
spina bifida as well.

AFIP Diagnosis: Spinal cord: Myelodysplasia, severe, with duplication of spinal roots,
chronic suppurative meningitis, granulation tissue, and numerous bacteria, Brangus,
bovine.

Conference Comment: The contributor provides an excellent description, possible

pathogenesis, and discussion of the lesions present in this case. There is considerable
variation in slides and not all lesions described may be present on all slides.

Spina bifida in its customary usage refers to absence of the dorsal portions of the
vertebrae. It is an imperfect name as the various forms of the defect largely represent
differences in the degree of defective closure of the neural tube, its separation from the
ectoderm, and its induction of a skeletal investment. Often, the defect is divided into
several classes on the basis of severity. Myeloschisis, spina bifida occulta, spina bifida
cystica with meningocele, and spina bifida with myelomeningocele apply to the vertebral

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defect. Amyelia, diastematomyelia, hydromyelia, and dysraphism apply to the spinal
cord defect. The most severe forms of myelodysplasia occur in association with spina
bifida, as seen in this case.15

Contributor: Texas A&M University, College of Veterinary Medicine, Department of

Veterinary Pathobiology, College Station TX
http://vtpb-www.cvm.tamu.edu/

References:
1. Bradley R, Kirby FD: Anomalous development of the spinal cord in a calf. Vet Pathol
16:49-59, 1979
2. Cho DY, Leipold HW: Arnold-Chiari malformation and associated anomalies in
calves. Acta Neuropath 39:129-133, 1977
3. Cho DY, Leipold HW: Spina bifida and spinal dysraphism in calves. Zbl Vet Med
Assoc 24:680-695, 1977
4. Davis RL, Robertson DM: Textbook of Neuropathology, 3rd ed. Williams & Wilkins,
Baltimore, MD, 1997
5. Doige CE: Spina bifida in a calf. Can Vet Jour 16:22-25,1975
6. Graham DI and Lantos PL: Greenfield’s Neuropathology, Vol. 1, 6th ed. Arnold,
London, England 1997
7. Goss, LJ and Hull FE: Spina bifida in a calf. Cornell Vet 29:239-240,1939
8. Gruys E: Dicephalus, spina bifida, Arnold-Chiari malformation and duplication of
thoracic organs in a calf. Zbl Vet Med Assoc 20:789-800, 1973
9. Leipold HW, Cates WF, Radostits OM, Howell WE: Spinal dysraphism,
arthrogryposis and cleft palate in newborn Charolais calves. Can Vet Jour 10:268-
273,1969
10. Leipold HW, Cates WF, Radostits OM, and Howell WE: Arthrogryposis and
associated defects in newborn calves. Am J Vet Res 31:1367-1374, 1970
11. Madarame H, Azuma K, Nozuki H, and Konno S: Perocormus associated with
segmental aplasia of the cervical spinal cord in a Japanese shorthorn calf. J Comp Path
101:225-230, 1989
12. Madarame, H, Ito, N, and Takai, S: Dicephalus, Arnold-Chiari malformation and
spina bifida in a Japanese black calf. J Vet Med Assoc 40:155-160, 1993
13. McFarland, LZ: Spina bifida with myelomeningocele in a calf. J Am Vet Med Assoc
134:32-34, 1959
14. Summers BA, Cummings JF, and de Lahunta A: Veterinary Neuropathology.
Mosby-Year Book, Inc., St. Louis, MO, 1995
15. Jubb KVF, Huxtable CR: The nervous system. In: Pathology of Domestic Animals,
eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 1, pp. 273-274. Academic Press,
San Diego, CA, 1993

SLIDE 82
CONFERENCE 21 / CASE I – 166-04 or 1250-03 (AFIP 2941192)

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Signalment: 4 year old, spayed female, Dachshund, dog (Canis familiaris).

History: The dog first presented in April 2003 for cutaneous petechiae, and was
diagnosed with immune-mediated thrombocytopenia and anemia. She was treated with
vincristine, cyclosporine and prednisone, and the anemia and thrombocytopenia
resolved. She was then prescribed long term prednisone and cyclosporine. In May, the
dog presented for an acute onset of reddening of the left eye. Ophthalmic examination
revealed fibrinous anterior uveitis and increased intraocular pressure. At that time, she
was also showing stranguria. Candida spp. was isolated from her urine, and treatment
with itraconazole was initiated for fungal cystitis. Topical prednisone and glaucoma
medications failed to control the inflammation and high intraocular pressure in the left
eye, which became buphthalmic and was enucleated in June. The signs of urinary tract
infection resolved, but the dog presented later in June, for evidence of uveitis in the right
eye. The right eye partially responded to therapy with topical steroids, until January
2004 when the dog became blind. The right eye was then buphthalmic, and was
enucleated. In the meantime, cystitis recurred several times in 2003, despite treatment
with itraconazole and ketoconazole.

Gross Pathology: The right globe was 20 mm in diameter. The cornea was
moderately thickened and opaque, and the anterior chamber was filled with friable tan
material. The lens was irregular in shape, opaque, and partially surrounded by a thick
layer of tan to brown tissue. The retina was detached, and the subretinal space was
partially filled with clotted blood. The vitreous humor was cloudy. Focally at the limbus,
the sclera was markedly thickened.

Contributor’s Morphologic Diagnoses:

Right eye (166-04):
1. Pyogranulomatous endophthalmitis, severe, chronic, with lens rupture and fungal
yeasts and pseudohyphae.
2. Keratitis, ulcerative, neutrophilic, moderate, chronic, with vascularization.
3. Retinal detachment with severe full thickness degeneration and severe retinitis.
4. Limbus: Focal suppurative scleritis and conjunctivitis, severe, acute.

Left eye (1250-03):

1. Pyogranulomatous endophthalmitis, severe, chronic, with lens rupture and fungal
yeasts and pseudohyphae
2. Keratitis, ulcerative, neutrophilic, moderate, chronic, with focal abscess and
vascularization
3. Retinal detachment with moderate degeneration of inner layers

Contributor’s Comment: Both eyes had a similar endophthalmitis with lens rupture,
and numerous fungal organisms consistent with Candida spp. in the anterior segment.
The route of ocular infection was believed to be hematogenous, originating from a
primary cystitis. Candida spp. are normal inhabitants of the gastrointestinal, upper
respiratory, and genital mucosa of dogs, and alterations in local and systemic immunity
can result in opportunistic infections of mucosae and mucocutaneous junctions.1

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Urinary tract infection by Candida spp. in dogs and cats is often associated with
concurrent diseases or drug therapy (e.g. immunosuppressive drugs, broad-spectrum
antibiotics) that may interfere with host defense mechanisms and alter normal bacterial
2 2
flora. Systemic candidiasis is an uncommon sequela of candidal cystitis.

While Candida albicans is the most common cause of fungal endophthalmitis in

humans, ocular candidiasis is rare in domestic animals.3 Fungal endophthalmitis in
dogs is most commonly caused by dimorphic fungi such as Blastomyces dermatitidis.4
Endophthalmitis caused by Candida spp. has been occasionally reported in dogs and
cats, resulting from hematogenous spread,3,6 or inoculation into the eye, secondary to
candidal keratitis.5 In our case, both eyes showed lens rupture, and organisms were
most numerous within the lens capsule and degenerate lens fibers, suggesting a
tropism for lenticular tissues. However, this particular distribution of organisms has not
been reported in published cases.

Candida spp. have a distinct morphology in histologic sections: they consist of round to
oval yeasts that are 3 to 6 microns in size, reproduce by narrow-based budding, and
form chains of elongated yeasts separated by constrictions (pseudohyphae).7 While
Candida albicans is the most common isolate of this genus, at least five other species
have been isolated from dogs and cats with urinary tract infections: C. tropicalis, C.
rugosa, C. krusei, C. parapsilosis, and C. glabrata (previously called Torulopsis
glabrata).2

AFIP Diagnosis: Eye: Endophthalmitis, pyogranulomatous, severe, diffuse, with

retinal detachment, lens rupture, intracorneal abscess, and numerous yeast and
pseudohyphae, Dachshund, canine.

Conference Comment: Candida albicans is a dimorphic, saprophytic, opportunistic

fungus that is a normal inhabitant of the gastrointestinal, upper respiratory, and genital
mucosae of dogs. Infections may develop as a result of breaks in the normal mucosal
barrier, immunosuppression, or treatment with broad-spectrum antimicrobials. Antibiotic
therapy reduces the number of anaerobic bacteria, allowing proliferation of Candida
spp., and resulting in an overall change in the mucosal flora.1,6

Candidiasis is mainly a disease of keratinized epithelium in young animals, especially

pigs, calves, and foals. In pigs, Candida spp. often invade the parakeratotic material
that accumulates on the gastric squamous mucosa. Thrush, candidiasis of the oral
cavity, occasionally occurs in young pigs. Lesions may be confined to the tongue, hard
palate, or pharynx, but often involve the esophagus, and gastric squamous mucosa as
well. In calves, lesions are most often in the ventral sac of the rumen but may also
involve the omasum and reticulum, and occasionally the abomasum.
Gastroesophageal candidiasis in foals involves the squamous epithelium and is
associated with ulceration adjacent to the margo plicatus. Grossly, the lesions are
yellow-white, smooth, or wrinkled plaques that cover the mucosa. Histologically, the

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epithelium is spongiotic and contains yeast and abundant hyphae admixed with
neutrophils and bacteria beneath the cornified layer.8

Localized candidal infections in dogs are reported in chronically immunosuppressed

dogs and include infections of the skin and nailbed, urinary tract, ears, and
gastrointestinal tract.1 Additional factors that promote candidal urinary tract infections
are thought to include an increased intestinal Candida spp. population (i.e. post-
antibiotic treatment) and local alterations in the urinary tract environment (i.e. diabetes
mellitus or aciduria).2 Systemic dissemination is by embolization from primary sites of
colonization and local invasion. Clinical signs of generalized infection include pyrexia
and erythematous skin lesions, myositis, osteomyelitis, and ocular infections.1

Candida spp. are unique fungi in that they often form yeast (blastospores,
blastoconidia), pseudohyphae, and hyphae in tissue. If only blastospores are seen,
they can be confused with morphologically similar yeast forms in tissue, such as
Histoplasma capsulatum var. capsulatum, Blastomyces dermatitidis, and poorly
encapsulated Cryptococcus neoformans. 9 Histoplasma capsulatum var. capsulatum
and Blastomyces dermatitidis only very rarely form pseudohyphae in tissue.7 In some
instances Candida hyphae and pseudohyphae may resemble dematiaceous fungi or
other filamentous fungi. However, Candida spp. are not pigmented and usually there is
more than one type of fungal element present. In mucocutaneous candidiasis, masses
of branching, septate hyphae, pseudohyphae, and round to oval budding yeast forms
measuring 3-5 _m in diameter are seen on the surface and within the epithelium. In
systemic candidiasis, either all or any combination of these fungal elements may be
seen. The presence of blastospores mixed with characteristic pseudohyphae or hyphae
in tissue enables the pathologist to identify the fungus as a species of Candida.9

Contributor: The Caspary Institute for Veterinary Research, The Animal Medical
Center, Department of Pathology, 510 East 62nd Street, New York, NY www.amcny.org

References:
1. Heseltine JC, Panciera DL, Saunders GK: Systemic candidiasis in a dog. J Am Vet
Med Assoc 223(6):821-824, 2003
2. Pressler BM, Vaden SL, Lane IF, Cowgill LD, Dye JA: Candida spp. Urinary Tract
Infections in 13 Dogs and Seven Cats: Predisposing Factors, Treatment, and Outcome.
J Am Anim Hosp Assoc 39(3):263-270, 2003
3. Linek J: Mycotic endophthalmitis in a dog caused by Candida albicans. Vet
Ophthalmol 7(3):159-162, 2004
4. Peiffer RL, Wilcock BP, Dubielzig RR, Render JA, Whiteley HE: Fundamentals of
Veterinary Ophthalmic Pathology. In: Veterinary Ophthalmology, ed. Gelatt KN, 3rd ed.,
pp. 355-425. Williams & Wilkins, Baltimore, MD, 1999
5. Gerding PA, Morton LD, Dye JA: Ocular and disseminated candidiasis in an
immunosuppressed cat. J Am Vet Med Assoc 204(10):1635-1638, 1994
6. Miller WW, Albert RA: Ocular and Systemic Candidiasis in a Cat. J Am Anim Hosp
Assoc 24(5):521-524, 1988

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7. Yager JA, Wilcock BP: Color Atlas and Text of Surgical Pathology of the Dog and
Cat, Dermatopathology and Skin Tumors, pp.128. Wolfe Publishing, London, England,
1994
8. Barker IK, Van Dreumel AA, Palmer N: The alimentary system. In: Pathology of
Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 2, pp. 256-257.
Academic Press, San Diego, CA, 1993
9. Chandler FW, Kaplan W, Ajello L: Candidiasis. In: Color Atlas and Text of the
Histopathology of Mycotic Diseases, pp. 42-46. Year Book Medical Publishers, Inc.,
Chicago, IL, 1980

SLIDE 83
CONFERENCE 21 / CASE II – 8004-476 (AFIP 2940131)

Signalment: 10 month old, neutered male Golden Retriever dog (Canis familiaris).

History: A 6 month old male Golden Retriever was presented for chronic vomiting.
Clinical chemistry showed a BUN of 83 (7-27) and creatinine of 3.3 (0.5-1.8) mg/dl with
a hematocrit of 34 (37-55)%. The animal was maintained with periodic medical
treatments and diet (Science Diet® K/D) but clinical signs and azotemia (BUN > 130
mg/dl, creatinine 13.35), hyperphosphatemia (15.43, 2.5-6.8 mg/dl) and anemia (PCV
21%) slowly progressed until the dog was euthanized at 10 months of age. The
referring veterinarian noted that both kidneys were small and nodular and these were
submitted in fixative for histological examination.

Gross Pathology: Grossly, kidneys were irregularly multinodular, firm, pale and small.

Laboratory Results: Final BUN > 130 (7-27) mg/dl, creatinine 13.35 (0.5-1.8) mg/dl,
hyperphosphatemia 15.43 (2.5-6.8) mg/dl and PCV = 21%

Contributor’s Morphologic Diagnoses: 1. Renal dysplasia.

2. Chronic suppurative pyelonephritis.
3. Multifocal mild tubular necrosis and regeneration with intratubular and intraepithelial
birefringent crystals, hyaline and granular casts.

Contributor’s Comment: In both kidneys, the cortex is irregularly contoured and

composed of radially arrayed zones of fibrosis interspersed with zones of dilated
tubules. Throughout the kidney, renal corpuscles have cystically dilated Bowman’s
spaces containing eosinophilic fluid; as well, a few small glomeruli with external nuclei
and inapparent capillary loops (immature glomeruli) are scattered throughout,
predominantly in the outer cortex. Areas of fibrosis have many large ductular structures
mainly in the medulla, with degeneration of tubules, coalescence of glomeruli and
moderate lymphoplasmacytic inflammation in the cortex. Medullary ducts have
flattened basophilic (immature) and columnar (mesonephric) epithelium. Dilated tubules
contain intraluminal eosinophilic fluid, many hyaline and fewer granular casts and

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scattered birefringent pale intratubular and intraepithelial crystals (calcium oxalate?).
Tubules are separated by expanded proliferative pale to collagenous stroma. Areas of
pale, poorly differentiated, stellate (immature) stroma are present primarily in the
medulla. Cortical tubules have variable epithelium with occasional necrosis, flattening
of epithelium and hyperplasia (regeneration). The pelvic and ureteral urothelium is
underlain by moderate mixed lymphoplasmacytic and neutrophilic inflammation and
fibrosis with varying degrees of epithelial degeneration, transmigration of neutrophils
and intraepithelial micropustules (chronic pyelonephritis). Some sections show a focus
of necrosis with exudation of fibrin and degenerating neutrophils in the renal papilla
(fibrinosuppurative pyelitis, not in all sections).

Renal dysplasia is thought to arise as a consequence of perturbations in the complex

chains of events involved in the embryological development of the kidneys resulting in
arrest of full maturation with retention of immature structures. Histological features of
renal dysplasia include fibrosis, immature glomeruli, large columnar-lined (mesonephric)
ducts, immature ducts lined with flattened hyperchromatic cells and the persistence of
pale poorly differentiated (immature) mesenchyme.1,2,3 Clinical signs of renal failure are
variable and are commonly recognized at several months age.1,2 Animals with renal
dysplasia show increased susceptibility to pyelonephritis; however, cortical fibrosis is
found independent of inflammatory disease. Renal dysplasia has been reported to be
caused by canine herpesvirus, feline panleukopenia virus, bovine virus diarrhea virus
and porcine hypovitaminosis A.3 The influence of inheritance is controversial,3 but
multiple occurrences in litters4 and breeds, notably Golden retrievers2, Lhasa apsos,
Shih Tzus, Boxers, Finnish harriers, Dutch kooiker, and Cocker spaniels1,4,5,6 have been
reported demonstrating hereditary determination or predisposition. Dogs with renal
dysplasia may suffer from renal dysfunction-related conditions such as anemia and
fibrous osteodystrophy7 (renal hyperparathyroidism). Nephrogenesis is largely
undefined but transgenic manipulations in mice indicate cytokine and other apparently
dual-purpose genes function in intercellular communication during organogenesis and
that interruption or over-expression of certain genes can lead to increased incidence of
renal dysplasia.8,9 Renal dysplasia is an important cause of renal failure necessitating
renal transplantation in children. Renal dysplasia has also been described in the adult
horse.10

AFIP Diagnoses: 1. Kidney: Dysplasia, with severe interstitial fibrosis, persistent

metanephric ducts and primitive mesenchyme, fetal glomeruli, cystic glomerular spaces,
tubular ectasia, degeneration and loss, and chronic mild lymphoplasmacytic
pyelonephritis, Golden Retriever, canine.
2. Kidney, tubules: Necrosis, multifocal, with intratubular crystals.

Conference Comment: The contributor provides a thorough overview of renal

dysplasia in animals. By definition, renal dysplasia is disorganized development of
renal parenchyma due to abnormal differentiation. Lesions associated with dysplasia
include the presence of structures inappropriate to the stage of development of the host
or the development of structures that are anomalous. Associated with and often

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obscuring dysplastic lesions are a number of secondary compensatory, degenerative,
and inflammatory changes.1

In humans, about 10% of all people are born with potentially significant malformations of
the urinary tract. Renal dysplasias and hypoplasias account for 20% of chronic renal
failure in children. Congenital renal disease can be hereditary, but is most often the
result of an acquired developmental defect in utero. Dysplasia can be unilateral or
bilateral and is almost always cystic. Grossly, the kidney is usually enlarged, extremely
irregular, and multicystic. The cysts vary in size from microscopic to several
centimeters in diameter. Microscopically, there is abnormal lobar organization and
persistence of abnormal structures, including cartilage, undifferentiated mesenchyme,
and immature collecting ductules. The characteristic histologic feature is the presence
of islands of undifferentiated mesenchyme, often with cartilage, and immature collecting
ducts.11

Although microscopic features of human renal dysplasia are present in dogs, a number
of differences are apparent. The consistent segmental cortical pattern of asynchronous
differentiation of nephrons is not a characteristic feature of human dysplasia. In man,
ducts lined by tall columnar epithelium are interpreted as persistent metanephric ducts
with no analogous structure in the normally developed kidneys. The pseudostratified
columnar epithelium lining medullary ducts in canine cases may similarly represent
persistent metanephric ducts.1

Contributor: Mississippi State University, College of Veterinary Medicine, Diagnostic &

Field Services Program, Box 9825, Mississippi State, MS www.cvm.msstate.edu

References:
1. Picut CA, Lewis RM: Microscopic features of canine renal dysplasia. Vet Pathol
24(2):156-63, 1987
2. Kerlin RL, Van Winkle TJ: Renal dysplasia in golden retrievers. Vet Pathol 32(3):
327-9, 1995
3. Maxie M: The urinary system. In: Pathology of Domestic Animals, eds. Jubb KVF,
Kennedy PC, Palmer N, 4th ed., pp. 461-463. Academic Press, San Diego, CA, 1993
4. Hoppe A, Karlstam E: Renal dysplasia in boxers and Finnish harriers. J Small Anim
Pract 41(9):422-6, 2000
5. Schulze C. et al.: Renal dysplasia in three young adult Dutch kooiker dogs. Vet Q
20(4):146-8, 1998
6. de Morais HS, DiBartola SP, Chew DJ: Juvenile renal disease in golden retrievers:
12 cases (1984-1994). J Am Vet Med Assoc 209(4):792-7, 1996
7. Lobetti RG, Pearson J, Jimenez M: Renal dysplasia in a Rhodesian ridgeback dog. J
Small Anim Pract 37(11):552-5, 1996
8. Hu MC, Piscione TD, Rosenblum ND: Elevated SMAD1/beta-catenin molecular
complexes and renal medullary cystic dysplasia in ALK3 transgenic mice. Development
130(12):2753-66, 2003

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9. El-Dahr SS, Harrison-Bernard LM, DippS, Yosipiv IV, Meleg-Smith S: Bradykinin B2
null mice are prone to renal dysplasia: gene-environment interactions in kidney
development. Physiol Genomics 3(3):121-31, 2000
10. Ronen N, van AMstel SR, Nesbit JW, van Rensburg BJ: Renal dysplasia in two
adult horses: clinical and pathological aspects. Vet Rec 132(11):269-70, 1993
11. Alpers CE: The kidney. In: Robbins and Cotran Pathologic Basis of Disease, eds.
Kumar V, Abbas AK, Fausto N, 7th ed., pp. 961-962. Elsevier Saunders, Philadelphia,
PA, 2005

SLIDE 84
CONFERENCE 21 / CASE III – Kiupel (AFIP 2942010)

Signalment: A 60 pound, 2.5 year old, female spayed, mixed breed (Border collie)
dog.

History: The dog presented to the Veterinary Teaching Hospital (VTH) at Michigan
State University for a previously diagnosed retinal detachment of the right eye. The dog
was bright, alert and responsive, with a good appetite. Physical examination revealed a
small skin lesion on the lateral surface of the hock, and bilateral retinal detachment.
CBC and serum chemistry were submitted to the clinical pathology laboratory. Fine
needle aspirate of the ocular fluid of the right eye and right hock were obtained for
cytologic examination. Fungal cultures were obtained from the ocular fluid.

Gross Pathology: This 60 pound female spayed dog presented with mild dehydration,
adequate nutrition, and minimal autolysis. The eyes had been enucleated prior to
necropsy for special processing. The primary lesions were widespread foci of
suspected granulomatous inflammation in multiple parenchymal organs. There were
hundreds of pinpoint white foci scattered across the capsular surface of the right and left
kidneys. These lesions did not extend into the inner parenchyma. There were
hundreds of multifocal to coalescing, white foci scattered across the epicardial surface
of the heart. These white foci extended throughout the myocardium of the left
ventricular free wall and the interventricular septum. There were multifocal areas of
moderate mucosal hyperemia diffusely throughout the small and large intestine. The
spleen was diffusely congested and oozed blood on cut surface. There were no other
gross lesions of diagnostic significance in this animal.

Gross Morphologic Diagnoses: 1. Kidney: Multifocal, moderate, granulomatous,

interstitial nephritis (suspected).
2. Heart: Multifocal to coalescing, granulomatous cardiomyopathy (suspected).

Histopathologic Findings: Only heart was submitted to the conference. Sections of

heart examined had multifocal areas of myocardial necrosis surrounded by moderate to
severe lymphoplasmacytic and histiocytic inflammation admixed with low numbers of
neutrophils and eosinophils. The lesions caused destruction of approximately 40-50%

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of the sections of heart examined. Within the centers of the necrotic areas were
numerous Prototheca. These organisms ranged from 5-15 µm in diameter with a thin
refractile wall. Some organisms had a granular basophilic cytoplasm with a small
central basophilic nucleus. Others had undergone endosporulation and contained as
many as 20 daughter cells. Most of the inflammation appeared centered around the
spent theca cells/mother shells, which represented the remaining empty capsule wall
following release of endospores. Many of the protothecal aggregates consisting of
spent theca cells elicited only a moderate lymphohistiocytic inflammatory response.
However, several of the larger granulomas had necrotic centers that consisted of central
mineralization surrounded by cellular debris, caseous exudates, and fragmented
Prototheca shells.

Other lesions: 1. Multifocal, mild, lymphoplasmacytic, and eosinophilic

meningoencephalitis with necrosis and intralesional Prototheca.
2. Multifocal, moderate, plasmacytic, and lymphohistiocytic interstitial nephritis with
intralesional Prototheca.
3. Diffuse, moderate to severe, plasmacytic, and eosinophilic colitis with intralesional
Prototheca.
4. Multifocal, mild to moderate, lymphoplasmacytic, periportal hepatitis with
intralesional Prototheca and moderate, diffuse, vacuolar hepatopathy.
5. Multifocal, moderate to severe, plasmacytic, and lymphohistiocytic pancreatitis with
intralesional Prototheca.
6. Multifocal, moderate, plasmacytic, and lymphohistiocytic thyroiditis with intralesional
Prototheca.
7. Bilateral, severe, pyogranulomatous panophthalmitis with complete retinal
detachment, glaucoma, and intralesional Prototheca.

Laboratory Results: CBC and serum chemistry profiles had no significant

abnormalities.
Right ocular fluid
Subretinal fluid from the right eye contained frequent spherical to oval-shaped
basophilic organisms with a thin clear wall and central 'nucleus'. The organism
measured from approximately 3-9 µm in diameter and approximately 3-10 µm in length
with a clear cell wall of approximately 0.5-1 µm thick. It appeared consistent with
Prototheca sp. A mild inflammatory response consisting of predominately moderately to
severely degenerate neutrophils was observed. Culture was recommended.

Right hock skin lesion

Numerous extracellular oval to reniform-shaped organism with thin clear walls were
seen. The organisms varied greater than two-fold in size. Frequent mildly degenerate
neutrophils were present and frequently could be seen phagocytizing the organisms.

Urinalysis
No significant findings.

Cerebral spinal fluid

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Cytospin preparations of CSF were made. They were moderately to highly cellular with
a large preponderance of eosinophils and lesser numbers of lymphocytes and non-
degenerate neutrophils and macrophages. There were occasional macrophages with
phagocytized protothecal organisms and spent organisms. Microprotein was measured
to be 50 mg/dl. A 300 cell count revealed 146 eosinophils, 65 mononuclear cells, 40
small lymphocytes, and 49 neutrophils.

Contributor’s Morphologic Diagnosis: Heart: Multifocal, moderate to severe,

granulomatous and eosinophilic, necrotizing myocarditis with intralesional Prototheca
sp. and mineralization.

Contributor’s Comment: Prototheca are saprophytic achlorophyllous algae that are

closely related to the green algae of the genus Chlorella._ They reproduce by
endosporulation and may have asymmetrical cytoplasmic and nuclear cleaving leading
to anywhere from 2 to greater than 20 endospores. The mother cells rupture releasing
the daughter spores, which are tiny replicas of their mothers. They mature and repeat
the life cycle. Empty shell casings from ruptured mother cells are usually seen amongst
the intact population of organisms._

Prototheca sp. are ubiquitous organisms and may be found in sewer treatment plants
(Fetter et al., 1971), potato skin (Negroni and Blaisten, 1940), tree flux (Fetter,
Klintworth and Nielson, 1971),_ and in freshly voided human and animal feces._
Prototheca rarely causes disease, but will adversely affect its host when the immune
system is suppressed or challenged by a pre-existing or concurrent disease. Intact
protothecal organisms normally elicit a minimal inflammatory response. Once the
mother cells rupture and release the endospores, a strong lymphoplasmacytic and
histiocytic inflammatory response is initiated against the spent theca shell. It has been
speculated that a defect in the host’s cell mediated immune system is a more important
factor in protothecal infections than a defect or decrease in the humoral immune
response._ A defect in neutrophils may allow for protothecal infections. In some hosts
the neutrophils are able to phagocytose the organisms but are unable to destroy them.
In these cases, there was no evidence of humoral or cell mediated immune deficiencies.

To date there are three recognized Prototheca species: P. stagnora, P. zopfii, and P.
wickerhamii. Only P. zopfii and P. wickerhamii are known to cause disease in animals
and humans. Both species appear morphologically similar to one another and can be
differentiated based on sugar and alcohol assimilation or fluorescent antibody tests. In
humans, both cutaneous and disseminated protothecosis has been reported. P.
wickerhamii is most commonly associated with cutaneous lesions and P. stagnora
usually results in disseminated disease in humans.

In cats and dogs, infection with Prototheca sp. is rare. There have been no reports of
disseminated protothecosis in cats. Cutaneous infections in cats are caused almost
exclusively by P. wickerhamii. Dogs, however, predominantly contract the disseminated
form of protothecosis and invariably it is caused by P. zopfii._ Collies appear to be
more susceptible (7 out of 20 cases) than other breeds._ Dogs with disseminated

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protothecosis normally present with a history of bloody diarrhea that is unresponsive to
treatment. The animal continues to eat and drink well and remains bright and
responsive. As the organism disseminates, clinical signs usually develop depending on
the organ system affected. Besides the gastrointestinal tract, the eyes, heart, brain,
liver, kidney, and skin are most commonly affected. As the disease progresses, dogs
become more depressed and develop CNS signs such as ataxia, incoordination,
paresis, deafness, circling and depression. Two-thirds of the reported cases include
bilateral or unilateral ocular involvement and the animals normally present with retinal
detachment and blindness. CBC and serum chemistry are often within reference range,
but occasionally hepatic enzymes may be increased with the involvement of the liver or
other organ systems. The predominant inflammatory response invoked by protothecal
organisms within a dog is lymphoplasmacytic and histiocytic regardless of the organ
system affected.

In this case, culture performed at MSU did not further differentiate the Prototheca
organism. It was unknown if the organism is P. zopfii or P. wickerhamii. Based on the
wide dissemination of the organisms, it was speculated that P. zopfii was the pathogen.

AFIP Diagnosis: Heart: Myocarditis, granulomatous and necrotizing, multifocal,

moderate, with numerous extracellular and intrahistiocytic algae, etiology consistent
with Prototheca sp., mixed breed, canine.

Conference Comment: As mentioned by the contributor, Prototheca spp. rarely cause

disease but occasionally infections result in severe gastrointestinal, ocular, cutaneous,
or disseminated disease. The most commonly affected domestic animals are dogs,
cats, and cows. In cows, manifestation of the disease is usually in the form of mastitis
caused by P. zopfii. Cats are most commonly affected with the cutaneous form of
protothecosis (P. wickerhamii) and present with large, firm nodules on the limbs and
feet; however, the nose, pinnae, forehead, and tailbase may also be affected. The most
common clinical presentation of protothecosis in the dog is protracted hemorrhagic
enterocolitis, with the colon being most severely affected. Grossly, the colon is diffusely
reddened with multiple raised white nodules and multifocal ulcerations with hemorrhage.
Disseminated disease involving the eyes, ears, skin, skeletal muscles, kidneys, liver,
heart, spinal cord, and brain has been reported.8

Prototheca spp. reproduce via asexual endosporulation and have a characteristic

microscopic appearance. Histologically, there are intra- and extracellular organisms
that may be either small single endospores with granular cytoplasm, or large sporangia
that are round to oval, 8-20 µm in diameter, have a clear 2-4 µm thick wall, and contain
multiple (2-20) wedge-shaped endospores arranged radially (“Mercedes Benz emblem-
like”). The cells eventually rupture leaving empty theca (mother shells) in the lesions.
Organisms may be readily identified using special stains such as PAS (Periodic Acid-
Schiff) or GMS (Grocott’s Methenamine Silver). Other organisms that reproduce via
endosporulation include Chlorella sp., Rhinosporidium seeberi, and Coccidioides

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immitis. Ultrastructurally, a paucity of chloroplasts differentiates Prototheca sp. from
Chlorella sp.10

Contributor: Michigan State University, Diagnostic Center for Population and Animal
Health, Department for Pathobiology and Diagnostic Investigation, PO Box 30076,
Lansing MI
http://www.dcpah.msu.edu
http://cvm.msu.edu/vetpath/faculty.htm

References:
1. Imes GD, Lloyd JC, Brightman MP: Disseminated protothecosis in a dog.
Onderstepoort J Vet Res 44(1):1-6, 1977
2. Greene CE: Protothecosis In: Infectious Diseases of the Dog and Cat, 2nd ed., pp.
430-436. W. B. Saunders Company, Philadelphia, PA, 1998
3. Perez J, Ginel PJ, Lucena R, Hervas J, Mozos E: Canine cutaneous protothecosis:
an immunohistochemical analysis of the inflammatory cellular infiltrate. J Comp Path
117:83-89, 1997
4. Rakich PM, Latimer KS: Altered immune function in a dog with disseminated
protothecosis. J Am Vet Med Assoc 185:681-683, 1984
5. Migaki G, Font RL, Sauer RM, Kaplan W, Miller RL: Canine protothecosis: Review of
the literature and report of an additional case. J Am Vet Med Assoc 181:794-797, 1982
6. Padhye AA, Baker JG, D'Amato RF: Rapid identification of Prototheca species by
the API 20C system. J Clin Microbiol 10:579-582, 1979
7. Sudman SM, Kaplan W: Identification of the Prototheca species by
immunofluorescence. Applied Microbiol 25:981-990, 1973
8. Strunck E, Billups L, Avgeris S: Canine protothecosis. Compendium 26(2): 96-102,
2004
9. Cook JR, Tyler DE, Coulter DB, Chandler FW: Disseminated protothecosis causing
acute blindness and deafness in a dog. J Am Vet Med Assoc 184:1266-1272, 1984
10. Schultze AE, Ring RD, Morgan RV, Patton CS: Clinical, cytologic and
histopathologic manifestations of protothecosis in two dogs. Vet Opthalmol 1:239-243,
1998
11. Blogg JR, Sykes JE: Sudden blindness associated with protothecosis in a dog. Aust
Vet J 72:147-149, 1995

SLIDE 85
CONFERENCE 21 / CASE IV – 04/05 #1 (AFIP 2948745)

Signalment: A 13-year-old male castrated Irish Setter canine.

History: This dog had a history of anorexia, fever, and septic peritonitis.

Contributor’s Morphologic Diagnosis: 1. Gallbladder: Malignant neuroendocrine

neoplasia (malignant carcinoid).

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2. Gallbladder: Cholecystitis, suppurative and lymphoplasmacytic, multifocal,
moderate.

Contributor’s Comment: The submitted specimen consists of a section of gallbladder,

which contains a portion of a non-encapsulated, invasive, multilobulated mass that
expands, effaces, and replaces up to 90 percent of the wall. Individual lobules, which
are separated by a moderate fibrovascular stroma, consist of discrete and coalescing,
variably cellular sheets and packets of mildly atypical and pleomorphic cells which are
separated by a delicate fibrovascular stroma. Cells are large, round to polygonal with
distinct cell borders and contain a moderate amount of pale eosinophilic, faintly granular
and often vacuolated cytoplasm. Nuclei are round to oval, bland, and contain coarsely
granular chromatin and single, prominent nucleoli. Anisokaryosis and anisocytosis are
mild and 0-1 mitotic figures are identified per 400X field. Throughout the mass, there
are multiple, scattered foci of hemorrhage and necrosis and rare, entrapped biliary,
mucosal crypts. The gallbladder lumen contains a variable amount of sloughed,
necrotic and degenerate tumor and epithelial cells admixed with fibrin, hemorrhage, and
varying numbers of degenerate and viable neutrophils. Within the adjacent gallbladder
tissue, the lamina propria-submucosa contains modest numbers of plasma cells and
lymphocytes with scattered, hemosiderin-laden macrophages, with mild, multifocal
cystic mucosal hyperplasia. Adherent to the serosa is a moderate cellular coagulum
composed of hemorrhage, mixed leukocytes, and fibrin.

In this case, based upon the cytomorphologic and architectural characteristics, and the
demonstration of neoplastic cell argyrophilia using the Grimelius method, a diagnosis of
malignant neuroendocrine neoplasia (malignant carcinoid) was made. Further
characterization (immunohistochemical or ultrastructural analysis) of the cells was not
conducted. Additional sections, which were not included in the submitted slide series,
demonstrated multiple nests of identical cells throughout a section of liver. Based upon
their comparatively small size and multicentric nature, these were presumed to
represent metastatic foci rather than the primary lesion.

Carcinoids are rare neuroendocrine neoplasms of both humans and domestic animals,
which arise from dispersed neuroendocrine cells located in the gastrointestinal tract and
other organ systems (liver, pancreas, urogenital, and tracheobronchial). Historically, the
term carcinoid has been used as an umbrella term in the diagnosis of all
gastroenteropancreatic neuroendocrine tumors (GEP-NET’s) regardless of biologic or
clinical behavior. Recently, in an attempt to reflect their diverse histogenesis and
biologic behavior, carcinoids were reclassified with the more neutral and inclusive
terms: neuroendocrine tumor and neuroendocrine carcinoma. However, the term
carcinoid was retained for neuroendocrine gastrointestinal tumors of both benign
(carcinoid) and malignant (malignant carcinoid) forms, thus removing pancreatic
neuroendocrine neoplasms from this group.3 Carcinoids may synthesize and secrete
either a short chain polypeptide and/or biologically active amine, including serotonin (5-
HT), somatostatin, gastrin, or histamine.2,4,7 The classic carcinoid syndrome of flushing,
hypotension, diarrhea, and wheezing is due to serotonin secretion.3

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Definitive diagnosis is based upon histologic features, cytochemical (argyrophilia) and
immunohistochemical (neuron specific enolase, chromogranin A and synaptophysin)
techniques, and the ultrastructural identification of secretory granules. Hepatic and
2,7
biliary carcinoids are typically negative for cytokeratin.
In the dog, carcinoids are most common in aged animals and have been reported in the
gallbladder, liver, lung, and throughout the gastrointestinal tract.1,2,4,6,7 Hepatobiliary
carcinoids have been described in the dog, cat, and one cow.2,7 Additionally, there are
reports of intestinal carcinoids in the horse and cow and three cases of maxillary sinus
carcinoid tumors in the horse.4 In humans, carcinoids are most commonly diagnosed in
the gastrointestinal tract (73.7%) and bronchopulmonary (25.3%) system.5

Based upon their rarity, the usual biologic behavior of gastrointestinal carcinoids in dogs
is uncertain, however in the previous reports, the vast majority of hepatic carcinoids
demonstrated aggressive, metastatic behavior, with spread most common to the
peritoneal cavity and peritoneal lymph nodes.6 In humans, the overall 5-year survival
rate of all types of carcinoid tumor was 50.4%, with a localized disease (79.7%) having
a predictably better prognosis than if regional (50.6%) or distant (21.8%) metastatic
lesions are present. The prognosis of gallbladder carcinoids (41.3% 5-year survival
rate) is poor.5

AFIP Diagnosis: Gallbladder: Carcinoid, Irish Setter, canine.

Conference Comment: Neoplasms derived from neuroendocrine cells of the

gastrointestinal mucosa are known as carcinoids because, histologically, they closely
resemble some carcinomas of intestinal epithelial origin. As mentioned by the
contributor, mucosal neuroendocrine cells can secrete several different hormones;
however, individual cells synthesize and store a single hormone, with the active
secretion being either short chain polypeptides and/or biologically active amines. It has
been shown that not all neuroendocrine cells can decarboxylate an amine precursor,
and the APUD (amine precursor uptake and decarboxylation) system concept has been
modified and renamed the diffuse endocrine system.2
In animals, alimentary tract and hepatobiliary carcinoids are considered malignant.8

Theoretically, tumors of the diffuse neuroendocrine system should invoke a

recognizable clinical syndrome related to their secretory products, but this is not
consistently observed. However, carcinoids that secrete gastrin (G cell tumors) are
responsible for the Zollinger-Ellison syndrome, characterized by severe gastric
hypersecretion and peptic ulceration, with watery diarrhea. The syndrome has been
reported in dogs and cats, usually associated with a non-beta cell pancreatic islet cell
tumor rather than a gastrointestinal tumor.2

Grossly carcinoids are yellowish or tan on cut surface and range from annular stenosing
thickenings to nodular masses and the overlying epithelium may be eroded or
ulcerated.2 Tumors often have characteristic neuroendocrine features histologically,

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with large polygonal cells arranged in nests and packets, or more solidly cellular areas,
separated by a fine fibrovascular stroma, with palisading of peripheral cells along the
stroma. Cells have distinct cell borders with abundant granular cytoplasm and
irregularly round, often centrally located nuclei. However, two other patterns have been
recognized. One is characterized by groups of rosettes or acinar-like structures that
contain eosinophilic secretions separated by similar stroma. The other is composed of
anastomosing groups and rows of cells like ribbons, consisting of mostly ovoid or
spindle cells with fibrovascular stroma. Silver stains such as modified Fontana-Masson
and Churukian-Schenk are useful. However, immunohistochemical stains, such as
neuron-specific enolase (NSE) and chromogranin, which stain almost all
neuroendocrine tumors, are now more commonly used. A varying number of neoplastic
cells may stain with antibodies such as serotonin, somatostatin, gastrin, glucagon,
synaptophysin, and calcitonin. Ultrastructurally, neoplastic cells contain a variable
number of intracytoplasmic neurosecretory granules that are typically round, composed
of an electron dense core and surrounded by an electron dense membrane.8

Contributor: Colorado State University, College of Veterinary Medicine and

Biomedical Science, Department of Microbiology, Immunology, and Pathology, Fort
Collins, Colorado
www.cvmbs.colostate.edu/mip/

References:
1. Albers TM, Alroy J, McDonnell JJ, Moore AS: A poorly differentiated gastric carcinoid
in a dog. J Vet Diagn Invest 110:116-118, 1998
2. Head KW, Else RW, Dubielzig RR: Tumors of the alimentary tract. In: Tumors in
Domestic Animals, ed. Meuten DJ, 4th ed., pp. 468-469. Iowa State Press, Ames, IA,
2002
3. Kloppel G, Perren A, Heitz PU: The gastroenteropancreatic neuroendocrine cell
system and its tumors: the WHO classification. Ann N Y Acad Sci 1014:13-27, 2004
4. Morrell CN, Volk MV, Mankowski JL: A carcinoid tumor in the gallbladder of a dog.
Vet Pathol 39:756-758, 2002
5. Modlin IM, Sandor A: An analysis of 8305 cases of carcinoid tumors. Cancer
79:813-829, 1997
6. Patnaik AK, Lieberman PH, Hurvitz AI, Johnson GF: Canine hepatic carcinoids. Vet
Pathol 18:445-453, 1981
7. Cullen JM, Popp JA: Tumors of the liver and gallbladder. In: Tumors in Domestic
Animals, ed. Meuten DJ, 4th ed., pp. 502-504. Iowa State Press, Ames, IA, 2002
8. Head KW, Cullen JM, Dubielzig RR, Else RW, Misdorp W, Patnaik AK, Tateyama S,
van der Gaag I: Histological Classification of Tumors of the Alimentary System of
Domestic Animals, 2nd series, vol. 10, pp. 94-96, 127-128. The Armed Forces Institute
of Pathology, Washington, DC, 2003 http://www.afip.org/vetpath/who/whoclass.htm

SLIDE 86
CONFERENCE 22 / CASE I – 04-2871 (AFIP 2936449)

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Signalment: Three near term fetuses, mixed sex, unknown breed, ovine (Ovis aries).

History: Six out of forty yearling ewes in the flock became mildly lethargic and would
often stand alone in a corner away from the rest of the flock. Affected ewes had slight
vaginal discharge followed by abortion. The older ewes had no clinical signs or
abortions. All ewes were vaccinated, and the yearling ewes were vaccinated twice,
including vaccinations for Campylobacter species.

Gross Pathology: The cotyledons in the placenta submitted with one of the fetuses
were edematous and mottled with multiple 1-4 mm diameter, white to tan foci. The
intercotyledonary placenta occasionally contained similar foci.

Laboratory Results: A pure culture of Campylobacter jejuni was isolated from the
abomasal fluid of all three lamb fetuses.

There was no bacterial growth on aerobic or Brucella cultures. Fluorescent antibody

testing of the lung, liver, kidney, placenta, and thyroid gland was negative for bovine
viral diarrhea (BVD) virus (used to test for border disease virus). Fluorescent antibody
testing of the placenta, lung, and liver was negative for Chlamydophila abortus. Fetal
titers were negative for BVD virus at less than 1:2, negative for Toxoplasma gondii at
less than 1:8, and negative for bluetongue virus.

Contributor’s Morphologic Diagnoses: 1. Placenta: Multifocal, necrotizing and

suppurative placentitis with mineralization, vasculitis, and thrombosis
2. Liver: Multifocal, suppurative and necrotizing hepatitis
3. Liver: Extramedullary hematopoiesis

Contributor’s Comment: There is moderate postmortem decomposition in the

submitted placenta, with loss of most of the trophoblastic epithelial cells. The chorionic
villi contain multiple foci of intact and degenerate neutrophils with necrosis and
mineralization. The chorioallantois is edematous and contains a few perivascular and
multifocal infiltrates of macrophages, neutrophils and lymphocytes. The tunica media of
a few small arterioles in the chorionic villi is infiltrated with intact and degenerate
neutrophils and the vascular wall is necrotic. A few of these affected arterioles contain
fibrin thrombi. In the submitted liver, there are multifocal areas of necrosis filled with
variable numbers of neutrophils. There is extramedullary hematopoiesis in the liver. In
the placenta of another lamb (not submitted), there is mild suppurative inflammation
with many of the trophoblasts containing numerous gram-negative coccobacilli. In the
lung of all three lambs (not submitted), there is suppurative bronchopneumonia.

Campylobacter species are small, curved, highly motile, noncapsulated,

microaerophilic, gram-negative bacilli. In sheep, the most common manifestation of
Campylobacter infections are late term abortions, stillbirths, premature births, the birth
of weak lambs, and occasional ewe fatalities due to metritis.1,2 The most common ovine
Campylobacter is Campylobacter fetus subspecies fetus, but Campylobacter jejuni can

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also infect sheep.1-4 One study showed an increase in ovine abortions caused by
Campylobacter jejuni versus Campylobacter fetus subspecies fetus in the later years
(1983-1989) of the study.5 In this same study, Toxoplasma gondii, Campylobacter
species, and Chlamydophila abortus were the most common identifiable causes of
ovine abortions. Placentitis was the most prominent lesion.5

The macroscopic and microscopic lesions in aborted fetuses and their placentas are
similar in infections with Campylobacter fetus subspecies fetus and Campylobacter
jejuni. The lesions include necrotizing edematous placentitis, fetal suppurative
bronchopneumonia, and multifocal hepatic necrosis.1-3 The most common lesion seen
with Campylobacter abortions in sheep is placentitis.4 In some cases, the placentitis is
macroscopically apparent. The liver lesions can be large enough to be seen grossly
and have a “target” appearance.1,3

The transmission of C. jejuni and C. fetus subsp. fetus is believed to be orally due to
fecal contamination of water and feedstuffs.1 The Campylobacter organisms then
become transiently bacteremic with localization of the bacteria in the gut and bile.1 In
nonimmune ewes, Campylobacter can localize in the uterus during the bacteremic
phase.1 In nonimmune ewes that are pregnant, Campylobacter first localizes in the hilar
zone of the placentomes causing vascular necrosis and thrombosis.4 This results in
separation of the chorion with invasion of the chorion and chorionic capillaries, with
subsequent necrosis.4 If the fetus survives the hypoxia secondary to the necrosis of the
placenta, then the fetus can be invaded by the Campylobacter, and in some cases,
undergoes fetal death.4

AFIP Diagnoses: 1. Chorioallantois (cotyledon): Placentitis, necrotizing, suppurative,

diffuse, severe, with multifocal vasculitis, thrombi, and mineralization, breed not
specified, ovine.
2. Liver: Hepatitis, necrotizing, neutrophilic, random, mild.

Conference Comment: The contributor provides a thorough overview of

campylobacteriosis in sheep. The placentitis is characterized by an edematous
intercotyledonary chorioallantois and friable, yellow cotyledons. Grossly, about 25% of
the fetuses have multiple, yellow, “targetoid”, areas of hepatic necrosis that are
characteristic of the disease. Flexispira rappini causes similar lesions in the placenta
and fetus, but infections are sporadic.6

Infectious causes of abortion in sheep include the following (C=cotyledonary;

IC=intercotyledonary):6,7,8

Organism Placental Lesions Fetal Lesions

Gross Histological
Campylobacter C=friable, yellow Often vasculitis, inflammation Liver: large targetoid areas
fetus fetus IC=edema, exudate severe in chorionic villi with of necrosis
gram-negative bacteria

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Toxoplasma C=pinpoint white foci of Chorionic epithelial hypertrophy Focal necrotic lesions in
gondii necrosis and hyperplasia with rare the brain, liver, kidney,
IC=edema intracellular zoites lung
Neospora C=necrosis Zoites rarely seen within Multifocal encephalitis with
caninum IC=normal trophoblasts gliosis and necrosis
Chlamydophila C=necrosis Necrotizing placentitis with Inflammatory/necrotic foci
abortus IC=brown exudate neutrophilic vasculitis and in the liver, lungs, muscle,
organisms within trophoblasts etc.
Coxiella burnetii C=less affected IC necrotizing placentitis with Inconsistent; lymphocytic
IC=thick, yellow, with gram-negative rickettsial infiltrates in the lungs,
exudate organisms within chorionic kidneys, liver
epithelium
Brucella ovis C=necrosis Vasculitis; gram-negative bacilli Nonspecific
IC=brown exudate intra- and extracellularly
Listeria C=necrosuppurative Severe diffuse necrosuppurative Hepatomegaly with
monocytogenes IC=necrosuppurative placentitis with gram-positive numerous 1mm yellow
bacteria within chorionic necrotic foci
epithelial cells
Other less common causes of ovine abortion include Salmonella dublin, S. typhimurium,
S. abortusovis, Ovine orbivirus (Bluetongue virus), Ovine pestivirus (Border disease),
and bunyaviruses (Akabane virus, Cache Valley virus, Rift Valley fever virus). 6,7,8

Contributor: Kansas State University, Department of Diagnostic

Medicine/Pathobiology, 1800 Denison, Manhattan, KS
http://www.vet.k-state.edu/depts/dmp/index.htm

References:
1. Kennedy PC, Miller RB: The female genital system: In: Pathology of Domestic
Animals, eds. Jubb KV, Kennedy PC, Palmer N, 4th ed., vol. 3, pp. 402-404.
Academic Press, San Diego, CA, 1993
2. Hedstrom OR, Sonn RJ, Lassen ED, Hultgren BD, Crisman RO, Smith BB,
Snyder SP: Pathology of Campylobacter jejuni abortion in sheep. Vet Pathol
24(5):419-256, 1987
3. Diker KS, Istanbulluoglu E: Ovine abortion associated with Campylobacter jejuni.
Vet Rec 118(11): 307, 1986
4. Jones TC, Hunt RD, King NW: Diseases caused by bacteria. In: Veterinary
Pathology, eds. Jones TC, Hunt RD, King NW, 6th ed., pp 432-33. William and
Wilkins, Baltimore, MD, 1997
5. Kirkbride CA: Diagnoses of 1,784 ovine abortions and stillbirths. J Vet Diagn
Invest 5(3): 398-402, 1993
6. Acland HM: Reproductive system: Female. In: Thompson’s Special Veterinary
Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 615-621. Mosby, St.
Louis, MO, 2001
7. Radostits OM, Gay CC, Blood DC, Hinchcliff KW: Veterinary Medicine A Textbook of
the Diseases of Cattle, Sheep, Pigs, Goats, and Horses, 9th ed., pp. 885. W.B.
Saunders Company Ltd, Philadelphia, PA, 2000
8. Kennedy PC, Miller RB: The female genital system. In: Pathology of Domestic
Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 3, pp. 400-406. Academic
Press, San Diego, CA, 1993

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SLIDE 87
CONFERENCE 22 / CASE II – UCONN 2004#2 (AFIP 2942012)

Signalment: Four-year-old, female, mixed-breed dog (Canis familiaris).

History: The dog is a free-ranging outdoor dog from the U.S. Virgin Islands. A
protruding vulvar mass of two weeks duration was surgically removed.

Gross Pathology: A section of the vulvar mass, measuring 3 cm x 1.5 cm x 1.5 cm is

submitted for histopathologic examination. The tissue has a uniform pale tan color and
is homogeneous on cut section.

Contributor’s Morphologic Diagnosis: Vulva: Transmissible venereal tumor, canine.

Contributor’s Comment: Vulva. The section is of non-haired, non-cornified, stratified

squamous epithelium and associated submucosal connective tissue, within which there
is a raised, sessile, superficially eroded mass. The mass is well-demarcated,
nonencapsulated, highly cellular and expansile, extending into the submucosa. The
mass is composed of loosely packed sheets and cords of round cells, separated by fine
strands of fibrovascular connective tissue. Cells are uniform, with scant eosinophilic
cytoplasm and distinct cytoplasmic margins. Nuclei are large, round with marginated
chromatin and a single prominent nucleolus or occasionally two nucleoli. There are
moderate numbers of mitotic figures (2 – 4/HPF). At the deep and lateral margins, the
stroma is infiltrated by small numbers of lymphocytes, fewer plasma cells, and
macrophages. Toluidine blue stain shows few mast cells in subepithelial tissues; there
are no metachromatic granules detected in the cytoplasm of tumor cells.

Canine transmissible venereal tumor (CTVT) is a naturally occurring contagious round

cell neoplasm with a primarily histiocytic immunophenotype, with immunopositivity to
lysozyme and vimentin.1 Tumors are primarily found in the mucus membranes of the
external genitalia of dogs of both sexes. Commonly, single or multiple masses are
found on the caudal part of the penis, from crura to bulbis glandis and glans penis, and,
in females, in the posterior part of the vagina and at the junction of the vestibule and the
vagina. The tumor occurs also extragenitally in the nasal and/or oral cavities. This
tumor is most often seen in young, roaming, sexually active dogs. CTVT is transmitted
only by the transplantation of viable tumor cells to mucus membranes at coitus or during
other contact. CTVT have been reported in the lymph nodes and skin, and occasionally
in the tonsils, liver, pancreas, spleen, lungs, and kidneys. These neoplasms typically
regress without treatment via an IgG-mediated immune response; however, metastasis
does occasionally occur.2,3 Distinctively, karyotyping of the cells of this tumor reveals
58-59 chromosomes, with 13-17 metacentric, compared to the normal canine
component of 78 with 2 metacentric.2

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The differential diagnosis includes mast cell tumor, histiocytoma, plasmacytoma,
cutaneous lymphoma, and, grossly, other neoplasms of the vagina and vulva including
papilloma, squamous cell carcinoma, epidermoid carcinoma, fibroma, and leiomyoma.

There is patchy worldwide distribution of this tumor, with endemic areas in the
Caribbean, where this dog lived.2 Transmission to the fox, coyote and jackal is
possible.4

AFIP Diagnosis: Vulva (per contributor): Transmissible venereal tumor, mixed-breed,

canine.

Conference Comment: Canine transmissible venereal tumor (CTVT) is the only

known naturally occurring tumor that can be transplanted as an allograft across major
histocompatibility (MHC) barriers within the same species, and to other canids, such as
foxes, coyotes, and wolves.5

The histogenesis of CTVTs is not yet certain, but immunohistochemical studies suggest
the cells are of histiocytic origin. The cells are immunohistochemically positive for
vimentin, lysozyme, ACM1 (an epitope on canine mononuclear phagocyte stem cells),
and alpha-1-antitrypsin (a good marker for benign and malignant histiocytes). These
antigens are not expressed by other mesenchymal round cells, except those of
histiocytic origin. Nonetheless, CTVT cells are unique in that they contain only 59
chromosomes. The normal diploid number of chromosomes in the somatic cell of the
dog is 78.5

Like histiocytomas, the growth pattern of CTVTs includes a progressive growth phase, a
static phase, and a regression phase. The progressive growth phase occurs after
sexual transmission and is characterized by rapid proliferation of neoplastic cells. The
static phase follows and is characterized by indolent local tumor growth or progression
with metastasis. Some tumors regress spontaneously. CTVTs evoke both humoral and
cell mediated immune responses, and as seen in this case, infiltrating lymphocytes may
be present within and around the neoplasm during the regression phase.5

Contributor: University of Connecticut, Department of Pathobiology and Veterinary

Sciences, U-3089, 61 North Eagleville Road, Storrs, CT
http://www.canr.uconn.edu/patho/

References:
1. Marchal T, Chabanne L, Kaplanski C, Rigal D, Magnol JP: Immunophenotype of the
canine transmissible venereal tumor. Vet Immunol Immunopathol 57(1-2):1-11, 1997
2. Nielsen SW, Kennedy PC: Tumors of the genital systems. In: Tumors of Domestic
Animals, ed. Moulton, JE, 3rd ed., pp. 498-502. University of California Press, Berkeley,
CA, 1990

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3. Yang TJ: Metastatic transmissible venereal sarcoma in a dog. J Am Vet Med Assoc
190(5):555-6, 1987
4. Kennedy PC and Miller RB: The female genital system. In: Pathology of Domestic
th
Animals, eds. Jubb KVP, Kennedy PC, Palmer N, 4 ed., vol. 3, pp. 451-453. Academic
Press, San Diego, CA, 1993
5. Mukaratirwa S, Gruys E: Canine transmissible venereal tumour: cytogenetic origin,
immunotype, and immunobiology. A review. Vet Quarterly 25(3): 101-111, 2003

SLIDE 88
CONFERENCE 22 / CASE III – 2004B (AFIP 2937641)

Signalment: 2-year-old, CD-1, male mouse (Mus musculus).

History: This mouse was from a control group on a 2 year carcinogenicity study. There
were no adverse antemortem findings.

Gross Pathology: At necropsy, the left lobe of the seminal vesicle was irregular, with
red/brown discoloration.

Contributor’s Morphologic Diagnosis: Seminal vesicle: Granular cell tumor, benign.

Contributor’s Comment: Granular cell tumors are generally a single mass in the male
and female genital tract of the mouse, but may occur in other organs. These benign
tumors typically grow by expansion but can include a more infiltrative pattern. Individual
cells have abundant pale cytoplasm filled with numerous eosinophilic, course granules,
and small, round to oval nuclei. The cytoplasmic granules are consistent with
secondary lysosomes (residual bodies) and are PAS-positive and diastase resistant.
The histiogenesis of the granular cells is unknown, although Schwann cells or primitive
mesenchymal cells have been proposed as the cell of origin. Synonyms include
myoblastoma and benign Abrikossoff’s tumor. 1

AFIP Diagnosis: Seminal vesicle: Granular cell tumor, mouse, murine.

Conference Comment: Granular cell tumor (GCT), once called granular cell
myoblastoma, is an uncommon neoplasm of uncertain origin. They have been most
frequently reported in the dog and horse, but also occur in laboratory rodents, cats, and
birds. In the dog, GCT occurs most commonly in the tongue, but they have been
reported in the ear, lip, palate, cerebral cortex and meninges, heart, lymph node, orbit,
and the skin. In the horse, GCT appears to be exclusively a tumor of the lung, and is
frequently found in association with the bronchi, but may be disseminated throughout
the lung.2 Granular cell tumors have been described in the genital system, brain and

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meninges in mice and rats.1,3 In cats, GCTs have been reported in the tongue, palate,
vulva, and digits.2

Grossly, the neoplasm is often nodular, whitish, and firm. Microscopically, the tumor
very characteristically consists of nests of large, round to polygonal cells with prominent,
coarsely granular, eosinophilic cytoplasm. The cytoplasmic granules are PAS (periodic
acid-Schiff) positive and diastase resistant. The circumscribed nature of many of the
tumors and a lack of mitotic activity suggests a benign course. However, some reported
GCTs were invasive and/or mitotically active. Granular cell tumors are
immunohistochemically variably positive for vimentin, S-100 protein, and neuron specific
enolase (NSE), emphasizing the heterogeneous nature of these tumors.
Ultrastructurally, the cells contain packed lysosomes and phagosomes (myelin bodies).4

Contributor: Merck Research Laboratories, Department of Safety Assessment, WP45-

227, Sumneytown Pike, West Point, PA

References:
1. Rehm S, Harleman JH, Cary M, Creasy D, Ettlin RA, Eustis SL, Goley GL, LeNet JL,
Maekawa A, Mitsumori K, McMonnell RF, Reznik GZ: Male Genital System. In:
International Classification of Rodent Tumors. The Mouse, ed. Mohr U, pp. 195-196.
Springer, Verlag Berlin Heidelberg, 2001
2. Cooper BJ, Valentine BA: Tumors of muscle. In: Tumors in Domestic Animals, ed.
Meuten DJ, 4th ed., pp. 361-362. Iowa State Press, Ames IA, 2002
3. Altman NH, Goodman DG: Neoplastic diseases. In: The Laboratory Rat Volume I
Biology and Diseases, eds. Baker HJ, Lindsey JR, Weisbroth SH, pp. 367. Academic
Press, New York, NY, 1979
4. Dungworth DL, Hauser B, Hahn FF, Wilson DW, Haenichen T, Harkema JR:
Histological Classification of Tumors of the Respiratory System of Domestic Animals,
2nd series, vol. VI, ed. Schulman FY, pp. 34-35. The Armed Forces Institute of
Pathology, Washington, DC, 1999

SLIDE 89
CONFERENCE 22 / CASE IV – CASE #2 (AFIP 2940162)

Signalment: Seven-week-old, male Sprague-Dawley rat.

History: Administered 200 mg/kg/day ethylene glycol monomethyl ether (EGME) for 4
days.

Gross Pathology: None

Contributor’s Morphologic Diagnosis: Testis: Stage-specific spermatocyte necrosis,

and spermatocyte and round spermatid loss.

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Contributor’s Comment: EGME is a well-characterized experimental testicular germ
cell toxicant in rats.1 One day following a single 200 mg/kg dose, degeneration of
spermatocytes (both those undergoing meiotic division and early pachytene stage) in
Stage XIV tubules is evident. Occasionally, spermatocyte degeneration is also evident
among Stage I and XIII tubules.

In rats examined following dosing with EGME for 4 consecutive days (such as the
submitted case), similar spermatocyte degeneration predominantly within Stage XIV
tubules is evident, as is depletion or absence of round spermatids and spermatocytes
among Stage I-V tubules. There are occasional Stage IV or V tubules having absence
of pachytene spermatocytes but retention of round spermatids, consistent with loss of
Stage XIV early pachytene spermatocytes (but not spermatocytes undergoing meiotic
division) following exposure to EGME on day 1 of dosing. Most conspicuous are
tubules having Sertoli cells, spermatogonia, and elongate spermatids present, but
lacking spermatocytes and round spermatids (presumably Stage I to VI tubules). These
represent tubules having lost all spermatocytes when at Stage XIII or XIV, some time in
the previous 4 days. Some of the affected tubules contain multinucleate germ cells
(syncytia of round spermatids). However, Stage VII through XII tubules are generally
unaffected.

Staging of the seminiferous tubular epithelium is useful for identifying temporal changes
in germ cell associations in the course of spermatogenesis.1,2,3 Each stage identifies a
morphologically distinct array of spermatogonia (proliferating diploid germ cells),
spermatocytes (meiotic [tetraploid] germ cells), and round and elongate spermatids
(differentiating haploid germ cells) at a particular phase of development, supported in
layers by basilar Sertoli cells. Staging schemes are based on light microscopic
morphologic characteristics (usually related to details of spermatid development) and
vary among species and among investigators describing them. Stages are designated
by a Roman numeral and are of variable temporal duration (ranging from 7 [e.g. Stage
IX] to 58 hours [Stage VII] in the rat). The most widely accepted staging scheme for the
rat has one 12.9-day cycle divided into Stages I through XIV. Four and a half cycles (56
days) are required for spermatogenesis - development of a mature rat (step 19)
spermatid from a type A1 spermatogonium. Maturation of sperm (spermiogenesis) is
described by morphologic changes of spermatids designated by Arabic numeral as
steps 1 through 19 over the course of one and a half cycles. Familiarity with
spermatogenic staging aids recognition and description of testicular injury in acute
toxicologic studies.

In the rat, Stage I through VII tubules are characterized by a single layer of pachytene
spermatocytes and two populations of spermatids (both round and elongate). At Stage
VIII, step 19 spermatids are released into the lumen and the round (step 8) spermatids
begin to elongate. Stage IX through XIII tubules have two layers of spermatocytes (the
luminal layer being large pachytene spermatocytes, and the basilar layer smaller
preleptotene, leptotene, or zygotene spermatocytes) and a single layer of elongating
spermatids. Stage XIV tubules have the luminal spermatocytes undergoing meiotic

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division to secondary (diploid) spermatocytes and then (haploid) round spermatids, and
basilar spermatocytes progressing to the pachytene stage.

AFIP Diagnosis: Testis, seminiferous epithelium: Degeneration, necrosis and loss,

segmental, with multinucleated germ cells, Sprague-Dawley rat, rodent.

Conference Comment: The contributor provides a thorough overview of the staging of

seminiferous tubules and the importance of stage specific changes that may occur with
testicular germ cell toxicants. For the toxicologic pathologist, the ability to identify the
tubular stages of the spermatogenic cycle and a sound understanding of the
spermatogenic process are essential in order to detect and characterize toxic effects to
the male reproductive system. There are several excellent references listed below
which cover this topic.

Contributor: Abbott Laboratories, Department of Pathology, AP13A/R 469, 100 Abbott

Park Road, Abbott Park, IL

References:
1. Creasy DM, Foster PMD: Male reproductive system. In: Handbook of Toxicologic
Pathology, eds. Haschek WM, Rousseaux CG, Wallig MA, 2nd ed., vol. 2, pp. Academic
Press, San Diego, CA, 2002
2. Creasy DM: Evaluation of testicular toxicity in safety evaluation studies: The
appropriate use of spermatogenic staging. Toxicol Pathol 25: 119-131, 1997
3. Russell LD, Ettlin RA, Sinha Hikim AP, and Clegg ED: Histological and
Histopathological Evaluation of the Testis, pp 62-118. Cache River Press, Clearwater,
FL, 1990

SLIDE 90
CONFERENCE 23 / CASE I – 03-10192 (AFIP 2942336)

Signalment: 7-year-old Polypay ewe.

History: This ewe was a chronic poor doer and was positive serologically for ovine
progressive pneumonia virus.

Gross Pathology: The ewe was in poor body condition. The lungs were heavy and
did not collapse. The cranial ventral portions were consolidated and contained several
abscesses. Hilar lymph nodes and gastrohepatic lymph nodes were enlarged and
replaced by abscesses. The kidneys also contained abscesses.

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Laboratory Results: Mycoplasma cultures and immunohistochemistry for PI3 virus
were negative. Streptococcus sp. and Staphylococcus aureus were isolated from the
lung.

Contributor’s Morphologic Diagnoses: 1. Severe, diffuse, chronic lymphoid

interstitial pneumonia
2. Multifocal, severe, chronic suppurative bronchopneumonia with abscessation

Contributor’s Comment: Ovine progressive pneumonia (OPP) and maedi-visna (from

the Icelandic words for ‘shortness of breath’ and ‘wasting’)1 are virtually identical
diseases caused by closely related ovine lentiviruses. OPPV is transmitted primarily by
close contact and droplet transmission and infects cells of monocyte-macrophage
lineage, including dendritic cells.2 Recent studies suggest transmission via milk and
transplacental transmission are of minimal significance. Certain breeds are more
susceptible to infection than others.3 Recently, researchers in Iceland have identified a
gene (vif) in maedi-visna virus responsible for infectivity in vivo and in vitro.4

The gross pulmonary lesions in this case were typical of OPP complicated by secondary
bronchopneumonia.1 Heavy, grey, non-collapsing lungs often have cranioventral
consolidation. Pulmonary and systemic abscessation in this ewe suggested a
secondary infection with Corynebacterium pseudotuberculosis, although this organism
was not isolated from the lungs.

The histologic lesions were diagnostic. The most characteristic feature was extensive
lymphofollicular proliferations around airways and pulmonary vasculature, both arteries
and veins; some had germinal center formation. Also present was the characteristic
smooth muscle hyperplasia of terminal bronchioles and alveolar ducts and
lymphohistiocytic interstitial pneumonia. Severe pulmonary fibrosis in the submitted
sections was most likely secondary to chronic, suppurative bronchopneumonia, but
‘microatelectasis’ due to collapse of alveolar spaces has also been described in chronic
cases.1 Hyperplasia of type II pneumocytes and bronchiolar epithelium was not
prominent, in contrast to cases of pulmonary adenomatosis, which has been associated
with a type B/D retrovirus.3 Other lesions attributed to OPPV infection seen in this ewe
but not represented in these tissues were diffuse lymph node cortical hyperplasia and
moderate lymphoplasmacytic synovitis in multiple joints. Also present were
membranous glomerulopathy and splenic amyloidosis, which were attributed to chronic
antigenic stimulation.

AFIP Diagnoses: 1. Lung: Pneumonia, interstitial, lymphohistiocytic, chronic, diffuse,

severe, with perivascular and peribronchiolar lymphoid hyperplasia, and smooth muscle
hyperplasia, Polypay, ovine.
2. Lung: Bronchopneumonia, suppurative, multifocal, moderate, with focally extensive
abscess.

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Conference Comment: Some sections contain a large abscess composed of a central
area of necrosis with many small aggregates of gram-positive cocci around the
periphery. This area is bounded by moderate numbers of lymphocytes, plasma cells,
and fewer epithelioid macrophages. There are reactive fibroblasts and fewer
inflammatory cells at the periphery.

The contributor provides a thorough overview of ovine progressive pneumonia virus

disease, which is caused by a lentivirus in the Retroviridae family. In addition to the
closely related ovine lentivirus known as maedi-visna virus, additional lentiviruses of
veterinary significance include simian immunodeficiency virus (SIV), feline
immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and equine
infectious anemia virus.

The gross pulmonary lesions of uncomplicated OPP include expanded, heavy, rubbery
to firm lungs that fail to collapse, and have rib impressions on the pleura. The lungs are
diffusely mottled gray to grayish-tan. The bronchial and mediastinal lymph nodes are
enlarged with soft grayish-white, homogeneous thickening of the cortical regions.
Microscopically, the most characteristic feature of OPP is lymphocytic interstitial
pneumonia with perivascular and peribronchial lymphofollicular proliferations that often
have germinal centers. Other features include smooth muscle hyperplasia in the walls
of terminal bronchioles and alveolar ducts, interstitial fibrosis, and microatelectasis.
Hyperplasia of bronchiolar epithelium and type II pneumocytes is not a prominent
feature of OPP. Other lesions associated with OPP include lymphofollicular mastitis,
chronic proliferative arthritis, nonsuppurative meningoencephalitis, and vasculitis.1

Ovine Progressive Pneumonia (OPP) shares many clinical and pathological features
with caprine arthritis-encephalitis (CAE), which is caused by a closed related caprine
lentivirus. CAE virus primarily causes nonsuppurative leukoencephalomyelitis in young
goats, and chronic proliferative arthritis and synovitis in adults. Less commonly, mastitis
and lymphocytic interstitial pneumonia occur in adult goats CAE virus often induces
two prominent pneumonic features lacking in OPP. One is extensive alveolar filling with
dense, acidophilic, proteinaceous to lipoproteinaceous material, and the other is type II
pneumocyte hyperplasia.1

Similar to goats with CAE viral pneumonia, sheep with OPP seldom have a pure viral
infection, and as in this case, often develop a secondary bacterial pneumonia. It is
important in such cases to separate the two processes and understand which agent is
likely causing which lesion(s). When OPP is complicated by bronchopneumonia, the
gross appearance should include the typical cranioventral consolidation with pus-filled
airways. Additionally, there can also be coexistent lungworm lesions.1

Contributor: Washington State University, College of Veterinary Medicine, Department

of Veterinary Microbiology and Pathology, Pullman, WA www.vetmed.wsu.edu

References:

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1. Lungs. In: Pathology of Domestic Animals, eds Jubb KV, Kennedy PC, Palmer N, 4th
ed. Vol. 2, pp. 629-631. Academic Press, San Diego, CA, 1993
2. Ryan S, Tiley L, McConnell I, Blacklaws B: Infection of dendritic cells by the maedi-
visna lentivirus. J Virol 74(21):10096-10103, 2000
http://jvi.asm.org/cgi/content/full/74/21/10096
3. Lofstedt J: Progressive bacterial and viral pneumonias of sheep and goats. In: Large
Animal Internal Medicine, ed. Smith BP, 3rd ed. pp. 581-582. Mosby, St. Louis, MO,
2002
4. Kristbjornsdottir HB, Andresdottir V, Svansson V, et al.: The vif gene of maedi-visna
virus is essential for infectivity in vivo and in vitro. Virol 318:350-359, 2004

SLIDE 91
CONFERENCE 23 / CASE II – 1140-99 (AFIP 2689105)

Signalment: 2-year-old, Quarter Horse, gelding.

History: Six, 1-2-year-old horses, out of a total of 40, died after 2-3 days of blindness
and going down. One horse and another horse head were submitted. These 40 horses
ate 5 tons of corn and oat screening from January 26-February 7 (our submission was
on February 16). Other horses on the premises were hand-fed 5 lbs of these
screenings/day/horse and were not affected.

Gross Pathology: One brain had focal, mild, yellow softening in the posterior cerebral
white matter on one side. The other brain had severe yellow softening throughout the
cerebral white matter, but limited to one side. There were no gross lesions in the other
organs, except that the stomach was filled with roughage and finely cracked corn and
oats.

Laboratory Results: Screening analysis:

Fumonisin B1: 8.3 ppm (1.0 considered “positive” and above 5.0 ppm associated
with leukoencephalomalacia) (Texas Veterinary Diagnostic Laboratory)
Aflatoxin: 5 ppb (“negative”/normal)
Rabies F.A.: negative

Contributor’s Morphologic Diagnosis: Multifocally severe cerebral

leukoencephalomalacia with vasculitis, hemorrhage, and gitter cells (Fumonisin toxicity).

Contributor’s Comment: Equine leukoencephalomalacia (ELEM) is a

neurotoxic disease caused by fumonisin production in Fusarium moniliforme-infected
corn, usually screenings. The disease is characterized by sudden onset of circling,
anorexia, head pressing, paresis, ataxia, blindness and depression that may progress to
hyperexcitability. The horses usually become comatose within 1 to 10 days but can
recover with some neurologic defects.

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Fumonisin toxicity is cumulative so the exact “safe” level is unclear. In one study, the
Fumonisin B1 (FB1) level never exceeded 8 ppm in horse feed not associated with
ELEM, whereas the FB1 ranged from less than 1 to 126 ppm in feed associated with
ELEM with most of these being above 10 ppm. Another study indicated that 5 ppm was
considered toxic. The affected horses averaged about 19 lbs/day/horse. The areas of
malacia and cavitation in the cerebral white matter may be unilateral or bilateral.
Microscopically, the malacic areas have gitter cells with degenerating axons and myelin
and adjacent multifocal hemorrhage and slight perivascular cuffing of lymphocytes and
eosinophils. Lesions occur but are less common in the spinal cord, brainstem, and
cerebellum.

Fumonisins are structurally similar to sphingosine and act by blocking enzymes in

sphingolipid synthesis and thus disrupt endothelial cell membranes. This might explain
the vasculitis and rare fibrin thrombi in dilated vessels in this case. There is species
variability in toxicity to fumonisin: horses develop neurotoxicity and sometimes
hepatotoxicity, swine develop pulmonary edema, several laboratory animals develop
hepatotoxicity and fumonisin is carcinogenic in rats and possibly in humans
(esophageal cancer).

AFIP Diagnosis: Brain, cerebrum: Necrosis, white matter (leukoencephalomalacia),

multifocal to coalescing, with vasculitis, edema, and hemorrhage, Quarter Horse,
equine.

Conference Comment: There is slide variability and not all sections contain good
examples of vasculitis and fibrin thrombi. Often it can be difficult to determine if there is
a true vasculitis, or if the vascular changes are induced by the changes in the
surrounding tissue (inflammation and necrosis). However, if fibrin thrombi are present,
the vascular changes are likely not simply a result of the surrounding necrosis.

Equine leukoencephalomalacia (ELEM), also known as moldy corn poisoning, is a

mycotoxicosis caused by fumonisin B1, and is usually associated with consumption of
moldy corn or grain contaminated with Fusarium moniliforme.5

In the horse, fumonisin causes two syndromes: neurotoxic and hepatotoxic. The
neurotoxic syndrome is most common, with clinical signs including depression, head
pressing, or seizures. Gross lesions include degeneration and liquefactive necrosis of
the subcortical white matter, especially in the frontal and parietal lobes. Lesions are
often bilateral, but are not always symmetrical. The characteristic gross lesion is yellow
gelatinous malacia and liquefaction of the affected white matter, with hemorrhage.
Microscopically, areas of liquefaction are surrounded by diffuse or perivascular edema,
hemorrhage, and small leukocytic cuffs. Blood vessels may be degenerate or necrotic
with occasional thrombi. Less characteristically, there may be edema and perivascular
cuffing in the leptomeninges and neuronal necrosis in the deeper layers of the gray
matter. The hepatotoxic syndrome is characterized by a swollen, yellow-brown liver

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with multifocal pale areas. Microscopically, there is centrilobular necrosis and fibrosis
that are similar to those seen with aflatoxicosis.5

The primary toxin isolated from F. moniliforme is fumonisin B1, although other
fumonisins have been extracted. The exact mechanism of injury has not been fully
defined; however, vascular damage has been inferred as the primary injury.
Fumonisins inhibit the enzyme ceramide synthase, interfering with the synthesis of
sphingolipids. Fumonisins disrupt cellular membranes, are associated with lipid
peroxidation of cells and cellular membranes, inhibit synthesis of macromolecules and
DNA, and may enhance production of tumor necrosis factor-alpha by macrophages.5

Other animals including pigs and avian species (chickens, ducks) are susceptible, but
clinical disease and lesions generally include pulmonary, hepatic, or renal injury. In
pigs, the disease is called porcine pulmonary edema and is characterized by severe
pulmonary edema and hydrothorax.5 Recently, meningoencephalitis secondary to
Fusarium solani has been reported in a German Shepherd Dog.6

Contributor: Arkansas Livestock & Poultry Commission Lab, P.O. Box 8505, One
Natural Resources Drive, Little Rock, AR 72205

References:
1. Flaminio J, Oehme FW: A review of equine toxicoses from dietary feedstuffs. Vet
Clinical Nutrition 4(1): 14-24, 1997
2. Ross PF, Rice LG, Reagor JC, et al: Fumonisin B1 concentrations in feeds from 45
confirmed equine leukoencephalomalacia cases. J Vet Diagn Invest 3:238-241, 1991
3. Summers BA, Cummings JF, de Lahunta A: Degenerative diseases of the central
nervous system. In: Veterinary Neuropathology, pp. 270-271. Mosby-Year Book, St.
Louis, MO 1995
4. Uhlinger C: Leukoencephalomalacia. Vet. Clinics of NA 13(1): 13-20, 1997
5. Storts RW, Montgomery DL: The nervous system. In: Thompson’s Special Veterinary
Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp. 416-417. Mosby, St.
Louis, MO, 2001
6. Evans J, Levesque D, de Lahunta A, Jensen HE: Intracranial fusariosis: A novel
cause of fungal meningoencephalitis in a dog. Vet Pathol 41:510-514, 2004

SLIDE 92
CONFERENCE 23 / CASE III – A03-286 (AFIP 2948654)

Signalment: Adult (age unknown) intact male cynomolgus macaque (Macaca

fascicularis).

History: In April 2003, the animal underwent experimental kidney transplantation and
was treated with thymoglobulin and rapamycin to inhibit transplant rejection. Three

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weeks later the animal had labored breathing and appeared to be in pain. While
sedated for a physical exam, the animal died spontaneously.

Gross Pathology: At post-mortem, the animal was thin with bilateral enlargement of
the axillary and inguinal lymph nodes. There was bicavitary effusion (hydrothorax and
hydroperitoneum) and the lungs had multifocal red, wet, heavy areas with intervening
pink, crepitant areas. On the surface of the right kidney there was a focal, radiating
white streak.

Laboratory Results: Serial Creatine values: 5/2/03 Creatine 1.9 mg/dl

5/4/03 Creatine 3.2 mg/dl
5/5/03 Creatine 3.0 mg/dl

Contributor’s Morphologic Diagnoses: 1. Body as a Whole: Disseminated

cytomegalovirus (CMV) infection.
2. Spleen: Severe acute multifocal necrosuppurative splenitis with intralesional CMV
inclusions and CMV arteritis.

Contributor’s Comment: Within the section of spleen, there is widespread loss of the
white pulp denoted by severe lymphoid depletion with a conspicuous angiocentric
distribution (most prominent around the central arteries). Discrete foci of intact and
degenerate neutrophils admixed with bland cell detritus, karyomegalic cells and
occasional small “plugs” of fibrin are predominately located within the white pulp. The
karyomegalic cells are misshapen and pleomorphic with variable amounts of
amphophilic cytoplasm and large oval to cigar-shaped eosinophilic glassy intranuclear
inclusions surrounded by a distinct halo (Cowdry type-A inclusions). Karyomegalic
inclusion-bearing cells are also scattered within the red pulp. Occasionally, endothelial
cells and smooth muscle cells of large and small caliber blood vessels are karyomegalic
and contain Cowdry type-A intranuclear inclusion bodies. In some sections, a focus of
foreign body giant cells enveloping fragments of suture material is external to the
splenic capsule.

Immunohistochemistry was performed using a polyclonal CMV antibody and

cytomegalic cells show positive nuclear staining for CMV.

Cytomegalovirus (CMV) is a herpesvirus of the subfamily Betaherpesvirus and is a

naturally occurring infection in many species of nonhuman primates. Following initial
infection, which is often asymptomatic, the virus establishes latency in its natural host.1
In immunocompromised animals the virus may reactivate and disseminate as the result
of a variety of predisposing conditions, including immunosuppressive viral infections
(SIV, type D retrovirus) and immunosuppressive drug therapy (cyclophosphamide,
corticosteroids, and antithymocyte globulin).1 Organs targeted by CMV include the eye,
lung, meninges, skin, heart, intestines and testicle.2 It is known that CMV has a
predilection for mesenchymal cells, although infected cells are frequently misshapen
and difficult to identify using conventional methods. The typical, almost pathognomonic
cytopathological lesions produced by CMV infection are cytomegaly and large, Cowdry

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type-A intranuclear inclusion bodies, although both intranuclear and intracytoplasmic
inclusion bodies can be seen with CMV infection.2 In this particular animal, a
satisfactory pathoanatomical explanation for the bicavitary effusion is provided by the
widespread endothelial injury caused by CMV infection, resulting in increased vascular
permeability and effusion into the body cavities and lungs.

AFIP Diagnosis: Spleen: Splenitis, necrotizing, acute, diffuse, moderate, with marked
lymphoid depletion, and myriad cytomegalic cells with eosinophilic intranuclear
inclusions, etiology consistent with cytomegalovirus, cynomolgus macaque (Macaca
fascicularis), primate.

Conference Comment: Cytomegalovirus (CMV) is a common asymptomatic infection

of humans and many nonhuman primates. CMV resembles other herpesviruses
ultrastructurally, but differs from alphaherpesviruses in several aspects. First, CMV is
slowly cytolytic and tends to cause enlargement of the nucleus and the cytoplasm
(cytomegaly). Cytomegaly is a result of the accumulation of enveloped virions in large
cytoplasmic vacuoles during viral replication instead of the virions being released into
intercellular spaces. Secondly, CMV tends to be restricted in its host range, unlike
many of the cytolytic alphaherpesviruses. Lastly, latent infections tend to persist in
glandular tissue, lymphoreticular cells, and kidneys rather than in neurons.1

Cytomegalovirus is transmitted horizontally in a variety of body secretions, including

saliva, blood, urine, milk, and semen. Infection is usually not associated with disease.
However, disease occurs in immunocompromised individuals or following intrauterine
infection. Cytomegalovirus persists as a latent infection and may periodically be shed in
body secretions. In immunosuppressed macaques, reactivation of the virus may be
associated with disseminated lesions in the brain, lymph nodes, liver, spleen, kidney,
small intestine, nervous system, and arteries. Disseminated CMV disease may be
initiated by a variety of immunosuppressive events, including viral infection (simian
immunodeficiency virus or type D retrovirus) and drug therapy (cyclophosphamide,
cortisone, antithymocyte globulin).1

Immunosuppressive therapy is commonly given to animals and humans undergoing

renal transplantation. Rapamycin is an immunosuppressive macrolide antibiotic that
inhibits T and B cell proliferation, while thymoglobulin is a rabbit polyclonal
antithymocyte antibody that is a potent T-cell depleting agent. Thymoglobulin is
effective in depleting T-cells in both the blood and secondary lymphoid organs through
the apoptotic pathway.3

It is important to understand the primary and secondary effects that immunosuppressive

agents will have on animals when evaluating histopathological changes in specific
organs. In this case, the splenic necrosis and lymphoid depletion are primary effects of
the immunosuppressive agents given to this nonhuman primate. The disseminated
CMV disease is likely a result of the immunosuppressive agents reducing the numbers

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of B and T cells, effectively diminishing this animal’s immune response, and therefore
allowing reactivation of the latent virus.

Contributor: Harvard Medical School, New England Regional Primate Research

Center, Division of Comparative Pathology, 1 Pine Hill Drive, PO Box 9102,
Southborough, MA

References:
1. Mansfield K, King N: Viral diseases. In: Nonhuman Primates in Biomedical
Research: Diseases, eds. Bennett BT, Abee CR, Henrickson R, pp.12-13. Academic
Press, San Diego, CA, 1998
2. Baskin GB: Cytomegalovirus infection in nonhuman primates. In: Nonhuman
Primates, eds. Jones TC, Mohr U, Hunt RD, vol. 1, pp. 32-37. Springer-Verlag, New
York, NY, 1993
3. Meuller TF: Thymoglobulin: an immunologic review. Current Opinion in Organ
Transplantation 8(4):305-312, 2003

SLIDE 93
CONFERENCE 23 / CASE IV – N2004-96 (AFIP 2942031)

Signalment: 9-year-old, female, Goeldi’s marmoset (Callimico goeldii).

History: This Goeldi’s marmoset presented with an open fracture of the proximal right
humerus. The tissue distal to the fracture was largely devitalized. On physical
examination the animal was icteric and had clinical signs of shock. Elective euthanasia
was performed.

Gross Pathology: The carcass was in moderately thin body condition. Most tissues
were discolored yellow (icterus). Both kidneys had tan mottling of the capsular surfaces
as well as mottling of the medulla and cortex on cut section. The spleen was mildly
enlarged, and the liver was diffusely pale tan. The open fracture of the humerus and
the associated extensive devitalization of the distal right arm were confirmed.

Laboratory Results: Fluorescent antibody assay demonstrated Leptospira spp.

antigen in kidney and liver. Microagglutination testing (MAT) (tested for 18 Leptospira
serotypes) of serum obtained at the time of euthanasia revealed an antibody titer of
1:12800 for serotype Ballum.

Contributor’s Morphologic Diagnoses: 1. Kidney: Nephritis, tubulointerstitial,

chronic-active, diffuse, severe, with multifocal tubular regeneration, and bilirubinuria.
2. Kidney: Mineralization, tubular epithelial, multifocal, mild.

Contributor’s Comment: The clinical and gross findings together with the pathological
changes in the kidneys, presence of rare intratubular argyrophilic spirochetal bacteria

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(Steiner’s silver stain), positive Leptospira fluorescent antibody (FA) result, and high
antibody titer are consistent with leptospirosis in an “incidental host.” In this case,
infection with serotype Ballum was demonstrated.

Histologically there was, in addition to the extensive infiltration of the renal interstitium
by lymphocytes, plasma cells, histiocytes, and scattered neutrophils, multifocal tubular
epithelial degeneration, necrosis, regeneration, and occasionally intratubular
proteinosis. Several tubules contained ongoing tubulonephritis in which infiltrating
neutrophils predominated. Intratubular and intraepithelial cytoplasmic yellow-brown,
granular pigment was present in several of the tubules (bilirubin pigment; Hall’s bilirubin
stain positive). Minimal, multifocal interstitial fibroplasia and deposition of collagen were
demonstrated by trichrome staining. Few mitotic figures in cells of possible lymphocytic
origin were seen in areas of inflammation. Mineralization of epithelial cells and
associated epithelial degeneration/necrosis were seen in some tubules.

Leptospirosis is a zoonotic disease of global importance that affects a broad range of

mammalian species. It is caused by the various serotypes of Leptospira interrogans
(old classification scheme; see below). The bacteria are ubiquitous. Although not able
to replicate in the environment, they can persist in the environment under optimal, warm
and wet conditions for months.1,2 Leptospira spp. bacteria are tightly coiled spirochetes,
0.1 _m in diameter and 6-20 _m long, with hooked ends.1 They require special methods
for visualization including silver stains, fluorescent antibody methods,
immunohistochemical stains, darkfield microscopy, or electron microscopy. Leptospires
have also been isolated from arthropods, amphibians, reptiles, and birds, including
serovars pathogenic for mammals from amphibians.1

“Maintenance hosts” are distinguished from “incidental hosts.”3 In the maintenance host
there is a stable host-parasite relationship characterized by high susceptibility of the
host species, occurrence of frequent and direct infection between individuals (usually at
an early age), little or no signs of clinical disease, relatively low antibody response, and
persistence of the bacteria in the renal tubules with chronic excretion via the urine.
Animal species can be maintenance hosts of some serovars but incidental hosts for
other serovars. Throughout the world, there are distinct variations in the maintenance
hosts and the serovars they carry.3 In domestic animals cattle may be carriers for
serovar Hardjo and Pomona; pigs may carry Pomona, Tarassovi, or Bratislava; and
dogs may carry serovar Canicola.3 Examples of maintenance hosts in wild animals
include raccoons and skunks in North America harboring serovar Grippotyphosa, and
various species of wildlife worldwide including ungulates, harboring serovar Pomona.
The most important maintenance hosts are rodents. Rats are generally thought to be
carriers of serogroup Icterohaemorrhagiae serovars and mice are generally carriers for
the serogroup Ballum serovars.3 Infection with the L. interrogans serotype Ballum was
indicated in this case by the high antibody titer.

The “incidental host” is characterized by relatively low prevalence of infection in the host
population but high pathogenicity for the host, development of acute and possibly fatal
disease, a short renal phase of infection with comparably short period of leptospira

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shedding, and a marked antibody response.4 Possible routes of infection include
contact with urine, bite wounds, and ingestion of infected tissues, as well as venereal or
placental transfer.

In zoological collections, the majority of leptospirosis cases represent incidental host

infections.4 Heated buildings, that house temperature-sensitive species (i.e., tropical
primates) provide an excellent, hard-to-control, year-round environment for both the
Leptospira bacteria and the maintenance host; in this setting usually rodents. In this
case, environmental testing to determine possible sources of infection, including water
supply and the food preparation area were negative.

The mortality rate and the extent of damage to the internal organs vary with virulence of
the serovar and host susceptibility; more animals encounter infections than will develop
recognizable disease.2,4 More than one serovar may occur in a given animal.2 The
bacteria penetrate either through mucous membranes or percutaneously through
injured skin. After a variable incubation period (4-20 days)4 there is a biphasic
progression of the disease; a septicemic phase lasting for about a week is followed by
the immune phase. In the septicemic phase, there is leptospiremia with rapid
multiplication and spread via the bloodstream. During this period, the bacteria can enter
and replicate in any tissue, e.g., liver, kidney, spleen, lung, eye, central nervous tissue
and genital tract.2 Hemorrhage, intravascular hemolysis, nephritis, pneumonia, and
meningitis are thought to be due to the action of bacterial toxins and the inflammatory
response to the bacteria that lead to damage of the vascular endothelial lining. In some
Leptospira strains hemolysins were isolated 2, some of which have been shown to
mediate hemolytic and cytotoxic activities by pore formation on the mammalian cell
membranes. The leptospiral lipopolysaccharide has lower endotoxic activity compared
to other gram-negative bacteria.2 Icterus, as seen in this case, is often due to a
combination of intravascular hemolysis and liver injury. The spirochetes are cleared
from the blood and most tissues with increasing antibody titers, but can persist and
multiply for some time in renal tubules, uvea, and brain. Within the kidney, leptospires
invade the interstitium with the help of their two periplasmic flagella 2 and subsequently
penetrate the tubular epithelium, which results in an acute inflammatory response
dominated by neutrophils. In this case, tubulointerstitial nephritis is still ongoing in some
of the renal tubules, demonstrating the chronic-active nature of the inflammatory
process. The acute phase of leptospirosis is transient and replaced by the immune
phase, in which antibody production and excretion of leptospires via the urine are key
features. Lymphocytes, plasma cells, and macrophages infiltrating the interstitium are
the predominant inflammatory cells during this phase. The organisms can persist within
the renal tubules of the incidental host, protected from antibody and other host
defenses, for a few days to several weeks. Renal insufficiency and failure are
associated with the tubular damage, and possibly with decreased glomerular filtration
and hypoxia due to the overall organ swelling that may impair renal blood perfusion.2 If
infection takes place during pregnancy, fetal infection may occur, resulting in abortion
(usually last trimester), stillbirth, birth of weak neonates, or birth of healthy but infected
neonates.4 Detectable gross lesions are often absent in infected neonates; thus, failure
to further test for leptospira may result in under-reporting of the disease.5

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In the old phenotypic classification system, the Leptospira were serologically divided
into two species. Leptospira interrogans included all the pathogenic serovars, and
3
Leptospira biflexa included all the environmental, nonpathogenic serovars. The more
recent classification, based on genotypic properties (12 named and 5 unnamed
genomospecies), is somewhat problematic in the clinical situation, since it is
incompatible with the former system of serogroups (e.g., species in the new system do
not correspond to the previous two species and their serovars, and include pathogenic
and nonpathogenic within some species).3 Therefore, the phenotypic classification is
likely to remain in place until simpler molecular identification methods are developed.3

Fluorescent antibody (FA) testing is frequently used to confirm the presence of

leptospiral antigen in fresh urine or fresh or frozen tissue collected at the time of
necropsy.5 If serum is available, microagglutination testing (MAT) may help to determine
the infecting serovar. The highest titer is considered the infecting serovar, with lower
positive titers considered to be cross-reactivity.2 Leptospires can be isolated from blood
and CSF during the first phase of illness, and from urine after the first week (data from
humans);3 overall, culture is problematic due to the fragile and fastidious nature of the
organisms.2,5 The use of PCR assays is also still limited due to the inability of most
assays to identify the infecting serovar.3 Killed leptospiral vaccines are frequently used
in domestic species and occasionally in exotic species such as the black rhinoceros, in
which leptospira infections have been associated with hemolytic crises and high
mortalities.4

AFIP Diagnosis: Kidney: Nephritis, interstitial, lymphocytic, multifocal, moderate, with

neutrophilic tubulitis, Goeldi’s marmoset (Callimico goeldii), primate.

Conference Comment: The contributor provides a very thorough overview of

leptospirosis in animals, including the epidemiology, transmission, histopathological
changes, and diagnostic techniques.

Prior to being informed of the contributor’s laboratory results, some conference

attendees considered lymphoma as a primary differential diagnosis based on the
relatively monomorphic population of interstitial lymphocytes that are associated with
moderate numbers of mitotic figures, and in some slides the apparent blurring of vessel
walls by high numbers of lymphocytes.

Although the angiotropic lymphomas were considered, there are several histopathologic
changes for which one must account. First, although the infiltrating inflammatory cells
are predominantly lymphocytes, there are low numbers of plasma cells and histiocytes
within the interstitium. Therefore, there is not truly a monomorphic population.
Secondly, lymphoma classically forms sheets of cells that obliterate normal tissue
architecture and often induce a mass effect; neither is evident in this section. And,
although the angiotropic lymphomas could be considered, the lymphocytes are not

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predominantly associated with vessels, nor are they occluding vessels resulting in
ischemic infarction. Lastly, one must account for the renal tubular inflammation,
degeneration, and necrosis.

When considering all of the changes apparent with H&E alone: tubular degeneration
and necrosis, neutrophilic tubulitis, and lymphoplasmacytic and histiocytic interstitial
nephritis, the most logical differential is leptospiral nephritis.

Contributor: Wildlife Conservation Society, Department of Pathology, 2300 Southern

Blvd, Bronx, NY
www.wcs.org

References:
1. Leighton FA, Kuiken T: Leptospirosis. In: Williams ES, Barker IK, eds. Infectious
Diseases of Wild Mammals, 3rd ed., pp. 498-502. University Press, Ames, IA, 2001
2. Langston CE, Heuter KJ: Leptospirosis - A re-emerging zoonotic disease. Vet Clin
Small Anim 33:791–807, 2003
3. Levett PN: Leptospira and Leptonema. In: Manual of Clinical Microbiology, eds.
Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, 8th ed. ASM Press,
Washington, D.C., 2003
4. Bolin CA: Leptospirosis. In: Zoo and Wild Animal Medicine, eds., Fowler ME, Miller
RE, 5th ed., pp. 699-702. Saunders, St. Louis, MO, 2003
5. McNamara T, Linn M, Calle P, et al.: Leptospirosis: an under-reported disease in zoo
animals? Proceedings American Association of Zoo Veterinarians, 1997

SLIDE 94
CONFERENCE 24 / CASE I – 98-4356 (AFIP 2681370)

Signalment: Adult, male, timber rattlesnake (Crotalus horridus).

History: Five of 7 rattlesnakes within an exhibit have died and the remaining 2 are sick.
The snakes are bloated, off feed and have seizures.

Gross Pathology: Little body fat is present. The coelom contains caseous material.
There are numerous 1 mm white foci in the coelom and the lung.

Contributor’s Morphologic Diagnoses: 1. Liver: Cytoplasmic inclusions,

hepatocytes.
2. Kidney: Nephritis, interstitial, lymphocytic, multifocal, mild with cytoplasmic
inclusions in tubular epithelial cells.
3. Lung: Pneumonia, necrotizing, purulent and granulomatous, with rare intraepithelial
cytoplasmic inclusions and numerous bacteria.

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Contributor’s Comment: This is a case of paramyxovirus infection in rattlesnakes. A
secondary bacterial pneumonia is the predominant lesion in this case and a bacterial
coelomitis was also present. Ophidian paramyxovirus infection has been recently
described in snakes and produces immunosuppression like the mammalian virus.
Secondary bacterial infections are common and are the usual cause of death. The lung
and nervous system are the usual organs affected but secondary bacterial infections
can occur in a variety of organs. Intracytoplasmic viral inclusions are seen in a variety
of epithelial cells but formation of syncytial cells, as occurs in mammalian
paramyxovirus infections, is rare.

AFIP Diagnoses: 1. Lung: Pneumonia, bronchointerstitial, granulomatous and

heterophilic, diffuse, severe, with multifocal necrosis, epithelial hyperplasia, and rare
epithelial eosinophilic intracytoplasmic inclusion bodies, etiology consistent with
ophidian paramyxovirus, timber rattlesnake (Crotalus horridus), reptile.
2. Kidney: Nephritis, interstitial, lymphocytic and histiocytic, subacute, multifocal, mild,
with tubular epithelial degeneration and necrosis.
3. Esophagus, epithelium: Necrosis, multifocal, with rare eosinophilic intracytoplasmic
inclusion bodies.

Conference Comment: The respiratory system of snakes is similar to that of mammals

with a few distinct differences. The left lung is vestigial in all snakes except boas. The
right lung has a posterior avascular portion, known as the air sac. The air sac regulates
pressure inside the body cavity. The anterior portion of the lung is composed of faveoli,
similar to mammalian alveoli, separated by relatively thin septae that are lined by
capillaries and type I and type II pneumocytes. Since snakes do not have a diaphragm,
air enters and leaves the lung due to action of the body muscles and movement of the
ribs.3

Ophidian paramyxovirus (OPMV) is a single-stranded enveloped RNA virus and is an

extremely important pathogen of viperid snakes. There have also been reports of
OPMV in colubrid, boid, and elapid snakes. Clinical signs typically include loss of
muscle tone, abnormal behavior and dilated pupils. However, in many of the outbreaks
of OPMV, the snakes are found dead with minimal or no clinical signs noted.4

The most significant gross lesion of OPMV disease is hemorrhage of the lung and air
sac with caseous necrotic debris. Additional gross lesions include pancreatic
hyperplasia, and hepatic granulomas or caseous necrosis. Typical microscopic findings
include cellular debris and exudate within airways, type II pneumocyte hyperplasia,
thickening of faveolar septa, and few epithelial cells containing intracytoplasmic
inclusion bodies.4

Like mammalian paramyxoviruses, OPMV also causes immunosuppression. Animals

infected with OPMV, as in this case, commonly have secondary bacterial infections with
gram-negative organisms. The most common gram-negative organisms isolated from

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reptiles with pneumonia include Pseudomonas spp., Providencia spp., Proteus spp.,
Salmonella spp., Aeromonas hydrophila, and Escherichia coli. Thus, it is not surprising
that snakes infected with OPMV often succumb to secondary bacterial pathogens.4

Contributor: Virginia Tech, College of Veterinary Medicine, Department of Biomedical

Sciences & Pathobiology, Blacksburg, VA

References:
1. Jacobson ER, Gaskin JM, et al.: Epizootic of ophidian paramyxovirus in a zoologic
collection: pathological, microbiological, and serological findings. J Zoo and Wildlife
Med 23: 318-327, 1992
2. Jacobson ER, Adams HP, et al.: Pulmonary lesions in experimental ophidian
paramyxovirus pneumonia of Aruba island rattlesnakes, Crotalus unicolor. Vet Path 34:
450-459, 1997
3. Website: http://animal.discovery.com/guides/reptiles/snakes/anatomy_02.html
4. Website: http://www.vetmed.ufl.edu/sacs/wildlife/Pmyx.html

SLIDE 95
CONFERENCE 24 / CASE II – PP1481D (AFIP 2936457)

Signalment: 1.5 year old, female, harbor porpoise (Phocoena phocoena), cetacean.

History: A juvenile, female harbor porpoise, weighing 41 kg, was rescued in April 1999
from a pound net close to Baaring Vig and taken to the Fjord and Belt Centre,
Kerteminde for rehabilitation. The animal was regularly treated with anthelmintics and
clinically examined. A couple of weeks later, the porpoise developed pox-like lesions on
the skin. The lesions persisted for 6 to 8 months but healed without scars. On 12th
February 2000, the porpoise developed clinical signs of illness consisting of reduced
appetite, floating on the surface and being unable to support itself in the water. The
animal had two crises, with high respiratory rate and low body temperature. The
porpoise was treated intensively, including antibiotics. In spite of intensive medical
care, the animal died on February 22nd, 2000 after 10 days of illness.

Gross Pathology: The porpoise was in a good nutritional status. There was an acute
fibrinous to suppurative pleuritis and pericarditis with approximately 0.4 to 1 liter of
exudate. One myocardial abscess, approximately 5 cm in diameter, was found in the
left ventricle with fistulous tracts to both, the endocardium and epicardium. Another
myocardial abscess, 0.5 cm in diameter, was located at the base of the heart. There
was a severe diffuse, fibrinous, suppurative peritonitis with 1 litre of exudate. In the
esophagus few ulcerations were detected. On the skin of the left side of the body there
were multiple round foci approximately 1 cm in diameter consisting of a white center
surrounded by a black rim. In the bronchial tree and pulmonary blood vessels a mild to

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moderate parasitic infection with Pseudalius inflexus and Torynurus convolutus as well
as a moderate parasitic burden of Stenurus minor in both aural peribullar cavities were
observed. In the first compartment of the stomach one nematode (Anisakis simplex)
and a few fish eyes were found. In the second compartment of the stomach few
submucosal hemorrhages were noted with a diameter of about 0.5 cm. In the right
lower jaw, the third tooth was dislocated towards the buccal gingiva.

Laboratory Results: Staphylococcus aureus was cultured from a sample taken from
the blowhole while the animal was alive. Postmortem samples from cardiac abscesses,
liver, spleen, and kidneys yielded Staphylococcus aureus. In addition, a few colonies of
streptococci and Serratia liquefaciens were found. Skin biopsies revealed moderate
cytoplasmic swelling and vacuolization of keratinocytes; however, ultrastructurally pox
virus particles could not be detected. Morbillivirus antigen was not detected by
immunohistochemistry.

Contributor’s Morphologic Diagnosis: 1. Heart: Myocarditis, pyogranulomatous,

chronic, multifocal to coalescing, severe with necrosis, colonies of cocci, homogenous
eosinophilic material (consistent with Splendore-Hoeppli material), fibrosis, harbor
porpoise (Phocoena phocoena), cetacean.
2. Heart: Myocarditis, lymphocytic, chronic, multifocal, mild.
3. Heart: Epicarditis, lymphohistiocytic, chronic, diffuse, moderate.

Contributor’s Comment: The submitted tissue section includes myocardium,

epicardium and fibrous connective tissue of a harbor porpoise. The main lesion
consists of a multifocal extensive myocardial necrosis with myriad cluster-forming cocci
which are surrounded by homogenous eosinophilic substance in a club-shaped
radiating corona (consistent with Splendore-Hoeppli material). The surrounding tissue
is markedly infiltrated with inflammatory cells, predominantly neutrophils, macrophages
and epithelioid macrophages admixed with lymphocytes and plasma cells. The
periphery of the lesion is demarcated by a poorly vascularized fibrous tissue. Multifocal
areas of the myocardium showed a mild, interfascicular infiltration of lymphocytes. The
epicardium was irregularly thickened by an infiltration of lymphocytes and histiocytes.

Upon histological examination of other tissues, a multifocal acute hepatocellular,

centrolobular necrosis without inflammatory reaction, and chronic suppurative
pericholangitis, were detected. Follicular hyperplasia was found in the spleen. In the
adrenal gland a mild focal lymphoplasmacytic infiltration with multifocal hemorrhages
was observed.

Staphylococcus aureus was isolated from the cardiac abscess and other organs.
Chronic infections caused by Staphylococcus aureus are termed botryomycosis. This is
a poorly understood pyogranulomatous bacterial disease of the skin and subcutis or,
more rarely, the viscera. In humans, cutaneous, pulmonary, and hepatic botryomycosis
have been reported. In veterinary medicine botryomycosis is recognized as a
staphylococcal wound infection in horses and pigs. In addition, pulmonary
botryomycosis has been reported in horses and guinea pigs.3 In cattle, swine and

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elephants it is an unusual manifestation of mastitis.1,2 One case of disseminated
staphylococcal botryomycosis has been described in a cat with perforating gastric
ulcer.4 Intraabdominal botryomycosis has been diagnosed in a dog.5 In two harbor
porpoises (Phocoena phocoena), one case submitted here, a pyogranulomatous
myocarditis due to Staphylococcus aureus septicemia has been reported.6 In three
harp seals (Pagophilus groenlandicus) the presence of subcutaneous and systemic
botryomycosis was described.7

The pathogenesis of botryomycosis is still unclear. An imbalance between virulence of

the organism and host resistance may result in incomplete removal of bacteria by the
host which leads to formation of bacterial granulomas.8 The exact composition of the
infiltrating population of inflammatory cells depends of the stage of the infection. At
initial examination, the lesion may have a purulent exudate with small white granules
with a diameter of less than 1 mm. These granules are indistinguishable from those of
actinomycosis, nocardiosis, and mycetoma unless special stains are used. In
botryomycosis the capsules of the grains were homogenously eosinophilic (Splendore-
Hoeppli material), periodic acid-Schiff (PAS) positive, slightly acid-fast, weakly positive
with Gomori’s methenamine silver stain, and were unstained with the MacCallum-
Goodpasture modification of the Gram stain.3

Differential diagnoses include systemic mycosis, chronic bacterial abscesses, and

foreign body reaction. Fungal hyphae are best demonstrated with silver stains (for
example Grocott’s stain). Actinomyces and Nocardia are Gram-positive filamentous
organisms, and acid-fast in the case of Nocardia (for example Ziehl-Neelsen stain).
Most reported cases of botryomycosis are caused by Staphylococcus aureus, but E.
coli, Pseudomonas aeruginosa, Actinobacillus lignieresii, Bacteroides, Streptococcus
and Proteus species have also been implicated.

Staphylococcus aureus is an uncommon finding in marine mammals. Some authors

described Staphylococcus aureus as a commensal in the blowhole flora, whereas
others classify it as a potential pathogen in clinically normal dolphins.9 In the literature
Staphylococcus aureus was described in association with septicemia in a killer whale,10
a cerebral abscess in a dolphin,11 and a septic embolic nephritis in the same species.12
Furthermore it was described as a pathogen in pneumonia in dolphins,13 cutaneous
lesions, subcutaneous abscesses, and omphalitis in pinnipeds. In this case, the origin
of the staphylococci remained unclear; the potential port of entry might be the pox-like
lesions.

AFIP Diagnosis: 1. Heart: Myocarditis, pyogranulomatous, multifocal to coalescing,

severe, with fibrosis, Splendore-Hoeppli material, and numerous colonies of cocci,
harbor porpoise (Phocoena phocoena), cetacean.
2. Heart: Epicarditis, lymphoplasmacytic and histiocytic, chronic, diffuse, moderate.

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Conference Comment: The contributor provides a thorough overview of
staphylococcal infections and botryomycosis. As mentioned above, botryomycosis has
been reported in many species and in a variety of organ systems, including the skin
(humans, horses, pigs), lungs (horses, guinea pigs), mammary gland (cattle, pigs,
elephants), stomach (cat), abdomen (dog), and most recently the heart (harbor
porpoise).

Another large group of animals commonly affected by staphylococcal botryomycosis is

laboratory animals, including mice, rats, gerbils, guinea pigs, hamsters, and rabbits.
Staphylococci are common inhabitants of the skin and mucous membranes and are
often carried asymptomatically. Many factors, such as immune status, lack of
competing bacteria, nutritional deficiencies, trauma to the skin, and prevalence of
staphylococci in the environment are recognized as contributing factors.14

Clinical signs in mice include abscessation of the cervical lymph nodes, inflammation
and abscessation of the preputial and lacrimal glands, conjunctivitis, periorbital
abscesses, superficial pyoderma, and severe ulcerative dermatitis. B6 mice are prone
to ulcerative dermatitis, while nude mice tend to develop furunculosis around their
muzzles, lacrimal gland abscesses, and preputial gland infections. In rats,
staphylococcal ulcerative dermatitis characteristically is localized to the dorsal neck and
interscapular regions. In young gerbils, S. aureus causes a diffuse moist dermatitis
involving the face, nose, feet, legs, and ventral body surface. The nasal dermatitis in
gerbils is associated with porphyrin-containing lacrimal gland secretions. When these
secretions accumulate on the external nares, they act as a chemical irritant which leads
to scratching, hair loss, and dermatitis. In guinea pigs, staphylococcal infections lead to
ulcerative pododermatitis, also known as bumblefoot. Predisposing factors include
trauma due to defective caging and poor sanitation. However, guinea pigs may also
develop acute staphylococcal dermatitis (exfoliative dermatitis), which most frequently
involves strain 13 guinea pigs. This disease is characterized by alopecia and erythema
on the ventral abdomen with exfoliation of the epidermis. In hamsters, cutaneous and
cervical abscesses are colonized by a variety of organisms including S. aureus,
Actinomyces bovis, Streptococcus spp., and Pasteurella pneumotropica. Outbreaks of
staphylococcosis occur sporadically in commercial rabbitries, with disease varying from
localized abscessation to acute, and frequently fatal, septemia. In rabbits, lesions may
occur in the skin, mammary glands, genital tract, conjunctiva, footpads, and respiratory
tract. The acute septemic form typically occurs in suckling kits during the first week of
life and leads to multifocal suppurative lesions in the subcutaneous tissue, lung, kidney,
spleen, heart, and liver.14

Contributor: Department of Pathology, School of Veterinary Medicine Hannover,

Bünteweg 17, D-30559 Hannover, Germany

References:
1. Sleeman JM, Clyde VL, Finnegan MV, Ramsay EC, Shires MG: Mammary
botryomycosis and mastectomy in an African elephant (Loxodonta africana). Vet Rec
152:54-55, 2003

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2. McGavin MD: Muscle. In: Thomson’s special veterinary pathology, eds. Carlton WW,
McGavin MD, 2nd ed., pp. 410. Mosby, St. Louis, MO, 1995
3. Bostrom RE, Huckins JG, Kroe DJ, Lawson NS, Martin JE, Ferrell JF, Whitney RA:
Atypical fatal pulmonary botryomycosis in two guinea pigs due to Pseudomonas
aeruginosa. J Am Vet Med Ass 155(7):1195-1199, 1969
4. Sheikh-Omar AR, Salam Adullah A: Perforated gastric ulcer associated with
disseminated staphylococcal granuloma (botryomycosis) in a cat. Vet Rec 117:131,
1985
5. Share B, Utroska B: Intra-abdominal botryomycosis in a dog. J Am Vet Med Assoc
220(7):1025-1027, 2002
6. Siebert U, Müller G, Desportes G, Weiss R, Hansen K, Baumgärtner W:.
Pyogranulomatous myocarditis due to Staphylococcus aureus septicemia in two harbor
porpoises (Phocoena phocoena). Vet Rec 150:273-277 2002
7. Wilson TM, Long JR: The harp seal, Pagophilus groenlandicus (Erxleben, 1777) XII.
Staphylococcal granulomas (Botryomycosis) in harp seals. J Wildl Dis 6(3): 155-159,
1970
8. Donovan GA, Gross TL: Cutaneous botryomycosis (bacterial granulomas) in dairy
cows caused by Pseudomonas aeruginosa. J Am Vet Med Assoc 184(2):197-199, 1984
9. Streitfeld MM, Chapman CG: Staphylococcus aureus Infections of Captive Dolphins
(Tursiops truncatus) and Oceanarium Personnel. Am J Vet Res 37 (3): 303-305 1976
10. Power E, Murphy S: Staphylococcus aureus septicemia in a killer whale. Vet Rec
150(26):819, 2002
11. Colgrove GS, Migaki G: Cerebral abscess associated with stranding in a dolphin. J
Wildl Dis 12(2):271-274, 1976
12. Ketterer PJ, Rosenfeld LE: Septic embolic nephritis in a dolphin caused by
Staphylococcus aureus. Aust Vet J 50(3):123, 1974
13. Medway W, Schryver HF: Respiratory Problems in Captive Small Cetaceans. J Am
Vet Med Assoc 163(6):571-573, 1973
14. Percy DH, Barthold SW: Pathology of Laboratory Rodents and Rabbits, 2nd ed., pp.
64-65, 133-134, 182, 201-202, 222. Iowa State Press, Ames IA, 2001

SLIDE 96
CONFERENCE 24 / CASE III – 05-0119 (AFIP 2964502)

Signalment: 1+ year old, male, Siamese fighting fish (Betta splendens).

History: Prior to euthanasia, Calvin had a two-month history of behavioral changes,

hiding in his rock cave, and a three-week history of progressive coelomic enlargement.

Gross Pathology: Coelomic organs are displaced ventrally and caudally by a 1.2 cm
diameter mass that occupies two-thirds of the centrodorsal portion of the coelom. The
mass is composed of a cystic center filled with a viscous slightly opaque fluid and is
surrounded by a 1-3 mm thick wall.

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Contributor’s Morphologic Diagnosis: Kidney: Nephroblastoma, Siamese fighting
fish (Betta splendens).

Contributor’s Comment: Nephroblastoma (Wilms’ tumor, embryonal nephroma)

occurs commonly in young pigs and chickens, less frequently in cattle and dogs, and is
observed in various species of fish, amphibians and reptiles. It is the most common
primary renal tumor in children (Marilyn J. Wolfe, personal communication).1-3 Since
these neoplasms arise from metanephric blastema, primitive pluripotential tissue, the
triphasic histologic features resemble the developmental stages of embryonic kidneys
with predominantly a myxomatous mesenchymal component interspersed with primitive
tubules, embryonic glomeruli, occasional nests of cells resembling metanephric
blastema, and rarely contain muscle, cartilage, bone and fat.2,3

The WT1 gene protein is a transcriptional activator of genes responsible for regulating
cell growth and is essential in renal and gonadal differentiation and for the development
of blastema into epithelium. Tissues that express WT1 are the uterus, spinal cord,
spleen, abdominal wall musculature, mesothelium lining thoracic organs and the central
nervous system. Mutations in the WT1 gene, a tumor suppressor gene, are associated
with the development of Wilms’ tumor in animals and humans. In addition, mutations in
Beta-catenin, important in the wnt (wingless) signaling pathway, are demonstrated in
15% of humans with Wilms’ tumor.1,4 Thoracolumbar spinal cord tumors of young dogs
that share similar histologic features and stain for the Wilms’ tumor gene product are
considered extrarenal nephroblastomas.3

This case was reviewed in consultation with Marilyn J. Wolfe, DVM, PhD, DACVP,
Principal Investigator, Registry of Tumors in Lower Animals, Sterling, Virginia 20166-
4311.

AFIP Diagnosis: Kidney: Nephroblastoma, Siamese fighting fish (Betta splendens),

piscine.

Conference Comment: There is significant variation among slides and not all slides
have all of the characteristic features of a nephroblastoma. As mentioned by the
contributor, the diagnostic features of a nephroblastoma include the triphasic histologic
features: myxomatous mesenchyme; interspersed primitive tubulesand/or glomerular-
like buds; and, nests of cells resembling metanephric blastemain various amounts.
Rarely, these tumors contain non-epithelial tissue such as muscle, cartilage, bone and
fat.3 In this case, the mesenchyme, primitive tubules, and blastema are present on all
slides. However, there are very few glomerular-like buds and they are not present on all
slides.

Adenocarcinoma of the swim bladder was considered by most residents. However,

unlike many other fish, the swim bladder in Siamese fighting fish is located ventral to the
kidney. Therefore, if the neoplasm originated from the swim bladder, one would expect

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that the unaffected kidney would be dorsal to the swim bladder. In all slides examined
by conference participants, the unaffected portions of the kidney are ventral to the
tumor. In many slides, it was difficult to determine if the tumor was originating within the
kidney; however, in a few slides, it is clearly arising from the kidney.

In humans, nephroblastomas (Wilm’s tumor) often present as a large, solitary, well-

circumscribed mass; however, in 10% of the cases, the tumor is either bilateral or
multicentric at the time of diagnosis. On cut section, the tumor is soft, homogeneous,
tan to gray, with occasional hemorrhage, cyst formation and necrosis. Microscopically,
the classic triphasic combination of blastemal, stromal, and epithelial cell types is
observed in most tumors, although the percentage of each component is variable.
Stromal elements are usually fibrocytic or myxoid, and skeletal muscle differentiation is
not uncommon. Rarely, other elements, such as squamous or mucinous epithelium,
smooth muscle, adipose tissue, cartilage and osteoid and neurogenic tissue are
identified.1

This case was reviewed in consultation with Dr. Isabell A. Sesterhenn, Chair,
Department of Genitourinary Pathology, The Armed Forces Institute of Pathology.

Contributor: Walter Reed Army Institute of Research, Department of Pathology

503 Robert Grant Avenue, Silver Spring, MD
http: //wrair-www.army.mil/

References:
1. Kumar V, Abbas AK, Fausto N: Robbins and Cotran Pathologic Basis of Disease, 7th
ed., pp. 305, 504-506. Elsevier Inc., Philadelphia, PA, 2005
2. MD, Carlton WW, Zachary JF: Thomson’s Special Veterinary Pathology, 3rd ed., pp.
270-271. Mosby, Inc., St. Louis, MO, 2001
3. Meuten DJ: Tumors in Domestic Animals, 4th ed., pp. 518-522. Iowa State Press,
Ames, IA, 2002
4. Nakatsuru Y, Minami K, Yoshikawa A, Zhu J, Oda H, Masahito P, Okamoto N,
Nakamura Y, Ishikawa T: Eel WT1 sequence and expression in spontaneous
nephroblastomas in Japanese eel. Gene 245(2):245-251, 2000

SLIDE 97
CONFERENCE 24 / CASE IV – G 6573/8 (AFIP 2956261)

Signalment: 1-year-old, female, Goeldi´s monkey (Callimico goeldii), non-human

primate

History: For a period of several months the animal showed oral, perioral and nasal
alterations which were characterized by swelling, edema, emphysema and incrustation.
Treatment consisted of antibiotic therapy and regular removal of the overlying crusts to

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ease breathing, but clinical recovery could not be achieved. The swollen and hyperemic
lips were still remarkable when the animal was necropsied after its accidental death.
Retrospectively, similar clinical signs were present in both parents.

Gross Pathology: At necropsy the young Goeldi´s monkey was in poor nutritional
condition and showed marked abdominal edema. Main post mortem findings were
located in the upper respiratory and gastrointestinal tracts. Both nasal and oral tissues
were swollen and edematous and the nose was covered with crusts. The overgrowing
crusts and hyperemic lesions were mainly present on the mucocutaneous membranes
of the lips and the nasal region. Numerous whitish nematodes up to 1 cm in length
could be detected macroscopically within the mucous membranes of the lips, tongue,
pharynx and intestine.

Laboratory Results: Bacteriological examination of a swab that was taken from the
altered tissue - Staphylococcus sp. was cultured. Routine flotation of a fecal sample
was negative for parasites and parasite eggs.

Contributor’s Morphologic Diagnosis: 1. Skin (mucocutaneous junction): Dermatitis,

minimal to mild, chronic, diffuse, with irregular epidermal hyperplasia and parasitic
structures consistent with Spirurida, Goeldi´s monkey, non-human primate.
2. Intestine (not present in all slides): Several parasitic structures consistent with
Spirurida within the lumen of the duodenum, Goeldi´s monkey, non-human primate.

Contributor’s Comment: Histologically, the dermatitis is characterized by an irregular

epidermal hyperplasia forming prominent rete ridges and pseudocarcinomatous
proliferations in several locations. Focal erosions are present on the skin surface. In
consequence of the parasite infection there is a mild lymphocytic inflammatory reaction
associated with spongiosis in the superficial parts of the dermis. The parasitic
structures are located within the epidermal layer where they are arranged in cystic
cavities. In sections the nematodes do not cause any inflammatory response in the
adjacent tissue. No parasites are seen inside the hair follicles.

The parasites were identified as nematodes of the order Spirurida. Characteristic

histologic features are the muscular and glandular esophagus, lateral cephalic alae, the
morphology of the lateral chords and the presence of embryonated eggs.1 In the
present case, further classification was not possible on the basis of histological
examination. Because of the gross pathologic findings and the site of infection it is
presumed that the parasites belong to the genus Gongylonema.

Spirurids of the genus Gongylonema are parasites of the upper digestive and
respiratory tract in a variety of birds and mammals including non-human primates.
Coprophagous arthropods (dung beetles and cockroaches) serve as intermediate hosts
in the indirect life cycle. Embryonated eggs are deposited by the female worms, and
liberated after epithelial desquamation with the host’s feces. The first-stage larvae
hatch in the insect’s intestines, migrate into the body cavity and develop into the second
larval stage after encapsulation. The maturation of the infectious third-stage larvae is

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completed after about four weeks. Final hosts acquire the parasite infection by
ingestion of infected intermediate hosts or by drinking contaminated water. The
migration pathway in the definitive host is still unknown for the most part, especially in
primates. The larvae are released from their capsules under the influence of gastric
acid and probably migrate within the wall of the upper intestinal tract, where they
develop into adult worms.2,3

Gongylonema are considered to be non-pathogenic in most host species. Generally

there is little tissue reaction with no extensive lesions. By contrast, reports about
gongylonemiasis in non-human primates, especially in New World monkeys, indicate
that these nematodes may cause profound oral inflammation and irritation, particularly
in times of high parasite exposure, and predispose the affected animals to fatal
secondary infections.1,4

AFIP Diagnosis: Oral mucosa, multiple sites: Intraepithelial adult spirurids, with
multifocal minimal lymphocytic inflammation, etiology consistent with Gongylonema sp.,
Goeldi’s monkey (Callimico goeldii), primate.

Conference Comment: There is significant slide variation with some slides having a
section of pancreas and attached duodenum with intraluminal and submucosal
nematode parasites.

As mentioned by the contributor, the key histologic features of spirurids include a

characteristic small, usually thick-shelled, embryonated egg; cuticular ornamentations
around the buccal cavity; coelomyarian musculature; uninucleate multicellular intestine
often lined by microvilli that form a brush border; and, lateral chords that may be quite
large. In some sections, the spirurids within the oral mucosa have cuticular bosses and
lateral alae. The cuticular bosses are characteristic for Gongylonema sp. and are
located on the anterior end of the parasite. Grossly, Gongylonema sp. are long thin
worms that often form a zigzag pattern in the mucosa and submucosa and appear
“stitched” into the tissue.5

Some sections contain pancreas and duodenum with intraluminal and submucosal
spirurids. These spirurids are Pterygodermatites sp., which have characteristic lateral
alae in a sublateral position on the anterior end of the parasite. Adult parasites may be
found in the lumen of the small intestine with their anterior ends embedded in the
mucosa. The larvae, when present, are deeper in the submucosa.6

Other intraepithelial parasites include: Capillaria sp., Anatrichosoma sp., and

Trichosomoides sp.

Contributor: German Primate Center, Department of Primate Medicine and

Husbandry, Kellnerweg 4, 37077 Gottingen, Germany

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References:
1. Duncan M, Tell L, Gardiner CH, Montali R: Lingual gongylonemiasis and
pasteurellosis in Goeldi’s monkeys (Callimico Goeldii). J Zoo Wildl Med 26:102-108,
1995
2. Brack M: Gongylonematiasis in the common marmoset (Callithrix jacchus). Lab Anim
Sci 46(3):266-270, 1996
3. Cappucci DT, Augsburg JK, Klinck PC: Gongylonemiasis. In: CRC handbook series
in zoonoses, sect. C: parasitic zoonoses, ed. Steele JH, vol.2, pp. 181-192. CRC Press,
Boca Raton, Fla, 1982
4. Craig LE, Kinsella JM, Lodwick LJ, Cranfield MR, Strandberg JD: Gongylonema
macrogubernaculum in captive African squirrels (Funisciurus substriatus and Xerus
erythropus) and lion-tailed macaque (Macaca silenus). J Zoo Wildl Med 29(3):331-337,
1998
5. Gardiner CH, Poyton SL: An Atlas of Metazoan Parasites in Animal Tissues, pp. 30-
33. The Armed Forces Institute of Pathology, Washington, D.C., 1999
6. Montali R, Gardiner CH, Evans RE, Bush M: Pterygodermatites nycticebi
(Nematoda: Spirurida) in golden lion tamarins. Lab Anim Sci 33(2):194-197, 1983

SLIDE 98
CONFERENCE 25 / CASE I – 04-01930 (AFIP 2948759)

Signalment: 2-year-old Baladi (native Egyptian breed) mare.

History: Baladi horses are used as dancing and parade horses. Two days post-
dancing parade; this mare had acute progressive muscle stiffness and considerable
distress with transient pyrexia. Shortly before euthanasia she had very stiff thigh
muscles and occasional lateral recumbence.

Gross Pathology: The paraspinal lumbosacral muscles and the thigh muscles of both
legs, especially the gluteal muscles, were moist, mildly swollen, and had massive
patchy to regionally extensive pale streaks.

Contributor’s Morphologic Diagnosis: Skeletal muscles: Acute degenerative and

necrotizing myopathy with intermyofibrillar edema, moderate, multifocal.

Contributor’s Comment: The current gross and histologic lesions are typical of equine
recurrent exertional rhabdomyolysis (ERER). Polysaccharide storage disease was
ruled out by negative PAS (periodic acid-Schiff) stain. ERER, or myoglobinuria, belongs
to a group of muscle diseases called exertional myopathies. Exertional myopathies also
include capture myopathy and porcine stress syndrome (malignant hyperthermia),
characterized by a common initiating factor of intensive or exhaustive muscle activity.1
ERER is characterized clinically by sudden onset of stiff gait, reluctance to move, and

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swelling of affected muscles groups especially gluteal muscles.1 Diagnosis is based on
clinical signs in association with an increase in serum creatinine kinase (CK), aspartate
amino-transferase (AST), and in severe cases myoglobinuria. The exact etiology and
pathogenesis of ERER are still unknown. Recently, a heritable defect in muscle cell
calcium regulation of muscle excitation-contraction coupling was suggested as the
primary factor for this disease.2 Few reported cases of ERER have an underlying
polysaccharide storage myopathy; however, the extent of PSM in the majority of ERER
is unknown.3

AFIP Diagnosis: Skeletal muscle: Degeneration and necrosis, acute, multifocal,

moderate, Baladi horse, equine.

Conference Comment: Conference attendees discussed the differential diagnosis of

the gross lesion, pale streaks in striated muscle. Differentials include: exertional
rhabdomyolysis, equine polysaccharide storage myopathy (EPSSM), nutritional
myopathy (Vitamin E/Selenium deficiency), ischemic myopathy due to anesthesia, plant
toxicity (Cassia occidentalis, coffee weed), ionophore toxicity (monensin), clostridial
myositis (malignant edema, or botulism), malignant hyperthermia-like syndrome,
protozoal myopathy (Sarcocystis spp.), and Streptococcus-associated myopathy.

In this case, the top three differentials are equine recurrent exertional rhabdomyolysis
(ERER), EPSSM, and nutritional myopathy. EPSSM is an inherited polysaccharide
storage disease of quarter horses, warmbloods, and draft-related breeds. Histologically
this disease can be diagnosed by the accumulation of periodic acid-Schiff (PAS)-
positive and amylase resistant material in affected muscles. ERER is a group of
myopathies which include EPSSM and other often unidentified causes of
rhabdomyolysis. ERER is also known as tying up, azoturia, or Monday morning
disease. There is some evidence to suggest that ERER in Thoroughbreds is due to
abnormal calcium homeostasis within skeletal muscle. Vitamin E and selenium
deficiency most commonly occurs in foals and young adult horses. In foals, the most
severely affected muscles are those that have the highest workload (cervical muscles,
proximal limb muscles, tongue, and masticatory muscles). In young adult horses, the
most severely affected muscles are often the temporal and masseter muscles.
Histologically, the lesions in the affected muscles are those of a multifocal, multiphasic
segmental necrosis.3

Comparatively, wildlife, especially deer, can exhibit capture myopathy, which is identical
to exertional rhabdomyolysis. Cattle, sheep, racing greyhounds, and sled dogs can
exhibit exertional rhabdomyolysis. And pigs, dogs, and humans can have malignant
hyperthermia, which is a hereditary molecular defect in the ryanodine receptor which is
involved with calcium regulation in muscle.3

Contributor: Egyptian Society of Comparative and Clinical Pathology (ESCCP),

Alexandria University, Department of Veterinary Pathology, Alexandria, Egypt.

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References:
1. Hulland TJ: Muscle and tendon. In: Pathology of Domestic Animals, eds. Jubb KV,
Kennedy PC, Palmer N, 4th ed., vol. 1, pp. 239-244. Academic Press, San Diego, CA,
1993
2. Mlekoday JA, Mickelson JR, Valberg SJ, Horton JH, Gallant EM, Thompson LV:
Calcium sensitivity of force production and myofibrillar ATPase activity in muscles from
Thoroughbreds with recurrent exertional rhabdomyolysis. Am J Vet Res 62(10):1647-
52, 2001
3. McGavin MD, Valentine BA: Muscle. In: Thomson's special veterinary pathology,
eds. McGavin MD, Carlton WW, Zachary JF, 3rd ed., pp:477, 480-484, 496. Mosby, St.
Louis, MO, 2001

SLIDE 99
CONFERENCE 25 / CASE II – S 2/04 (AFIP 2942015)

Signalment: 15-years-old male, Lady Amherst’s Pheasant (Chrysolophus amherstiae).

History: This animal was kept in a zoological garden and was found dead in its aviary.

Gross Pathology: The pheasant was emaciated. The cecal wall was multifocally
thickened with numerous, up to 3 mm large, round, subserosal and intramural white
nodules. In the lumen there were several, up to 1.5 cm long, nematodes.

Laboratory Results: Bacteriology revealed Salmonella enteritidis in all parenchyma

and intestine.

Contributor’s Morphologic Diagnosis: Cecum, submucosa: Typhlitis,

granulomatous and fibroplastic with intralesional larval and adult ascarid nematodes,
etiology consistent with Heterakis spp.

Contributor’s Comment: Infections with Heterakis spp. occur worldwide and affect
ducks, chickens, quails, grouse and especially pheasants. Besides H. dispar, H.
gallinarum and H. isolonche are the species most commonly found in pheasants.1
These nematodes are morphologically characterized by prominent cuticular alae, large
lateral chords, polymyarian-coelomyarian musculature, and triradiate intestine with
uninucleate columnar intestinal cells. The exact identification of the species can be
made by measuring the length and estimating the shape of the spicules and
oesophagus.2 H. isolonche has a direct life cycle, lives within the cecal submucosa and
induces nodular granulomas with considerable mesenchymal proliferation or even
leiomyomas.2,3 Defecated non-embryonated eggs gain their infectivity outside the host.
After hatching in the small intestine, the larvae reach the cecal submucosa and develop
into adult worms.

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AFIP Diagnosis: Cecum: Typhlitis, nodular and granulomatous, multifocal, moderate,
with marked mesenchymal infiltration and intralesional adult and larval nematodes,
etiology consistent with Heterakis spp., Lady Amherst’s pheasant (Chrysolophus
amherstiae), avian.

Conference Comment: Conference attendees discussed the histogenesis of the cecal

nodules. Some reports classify them as granulomas, while others describe them as
fibrous hyperplastic tissue or even leiomyomas.1 The spindle cells forming the nodules
in this case are not producing collagen as identified with Masson’s trichrome stain, nor
are they immunohistochemically positive for smooth muscle actin. Therefore, these
cells may be of histiocytic origin; however, further immunohistochemistry would be
necessary to determine the definitive histogenesis.

Heterakis spp. are known to cause nodular typhilitis in a number of avian species,
including chickens, turkeys, ducks, geese, grouse, guinea fowl, partridges, pheasants,
and quail. However, the chief economic importance of Heterakis gallinarum, the cecal
worm, lies in its role as a carrier of Histomonas meleagridis.5

Histomoniasis is a parasitic disorder of the ceca and liver of many gallinaceous birds.
Grossly, the disease is characterized by well-demarcated necrotic foci surrounded by a
raised hyperemic ring in the liver and necroulcerative lesions in the ceca that often lead
to the development of cecal cores composed of necrotic debris. Microscopically,
histomonads are pale, lightly stained, 15-20 _m, oval bodies within lacunae in the
lamina propria and muscularis mucosa and are admixed with lymphocytes,
macrophages, and heterophils. Histomonad invasion with necrosis may extend well into
the muscular tunics, nearly to the serosa. With time, large numbers of giant cells form
nodules that may be seen grossly as granulomas bulging from the serosal aspect of the
cecum.5

The life cycle of H. meleagridis is complex with histomonads being found in the
intestinal epithelial cells of H. gallinarium. Histomonas meleagridis infected Heterakis
gallinarium eggs are passed in the feces of susceptible avian species. The eggs then
embryonate and may either be swallowed by a susceptible host (direct transmission) or
they may be ingested by the earthworm (indirect transmission). In the earthworm, eggs
hatch and larvae may live for months. The earthworm is then eaten by a susceptible
host, resulting in infection with both Heterakis gallinarium and Histomonas meleagridis.5

Contributor: Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal

Health, Boddenblick 5A, 17493 Greifswald – Insel Riems, Germany
www.bfav.de

References:

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1. Menezes RC, Tortelly R, Gomes DC, Pinto RM: Nodular typhlitis associated with the
nematodes Heterakis gallinarum and Heterakis isolonche in pheasants: Frequency and
pathology with evidence of neoplasia. Mem Inst Oswaldo Cruz 98:1011-1016, 2003
2. Permin A, Hansen JW: The Epidemiology, Diagnosis, and Control of Poultry
Parasites, pp. 29-30
(http://www.poultry.kvl.dk/training/The_Epidemiology,_Diagnosis_and_Control_of_Poult
ry_Parasites.pdf)
3. Griner LA, Migaki G, Penner LR, McKee AE Jr: Heterakidosis and nodular
granulomas caused by Heterakis isolonche in the ceca of gallinaceous birds. Vet Pathol
14:582-590, 1977
4. http://www.afip.org/vetpath/WSC/WSC97/97wsc15.htm (case III)
5. McDougald LR: Internal Parasites. In: Disease of Poultry, ed. Saif YM, 11th ed., pp.
947-948, 1001-1004. Iowa State Press, Ames, IA, 2003

SLIDE 100
CONFERENCE 25 / CASE III – NADC MVP-1 2004 (AFIP 2936146)

Signalment: Full term fetus, white-tailed deer (Odocoileus virginianus).

History: Twin full-term fawns were born to a 2.5 year-old white-tailed deer. One fawn
was stillborn while the other appeared healthy.

Gross Pathology: The stillborn fawn had marked abdominal distention due to marked
bilateral nephromegaly. Kidneys maintained their reniform shape but were enlarged
(8.8 x 6.0 cm), pale, tan and smooth. The capsule was thin, tightly adherent and
translucent, through which could be seen numerous fluid-filled cysts. On cut section,
there were numerous 1-5 mm, round to fusiform cysts that contained clear fluid. The
ureters and bladder were grossly normal. On cut surface of the liver, the intrahepatic
bile ducts were variably ectatic with irregular outlines and intraluminal bile. Gross
lesions were not seen in other organs.

Contributor’s Morphologic Diagnosis: Kidney, glomeruli, tubules and collecting

ducts: Ectasia and cysts, diffuse, severe, white-tailed deer (Odocoileus virginianus).

Contributor’s Comment: Microscopically, there is severe dilatation of all renal tubules.

The corticomedullary junction is obscured. Dilated tubules are lined by low cuboidal to
flattened squamous epithelium. Most dilated tubules are empty, but some contained a
flocculent eosinophilic material. The number of glomeruli is greatly reduced; those
present are small and located within a dilated Bowman’s capsule. The liver (not
submitted) is characterized by marked biliary hyperplasia. Bile ducts are dilated to 1 to 5
mm in diameter, irregular in contour and contain intraluminal inspissated bile.

In humans, polycystic kidney disease (PKD) is characterized by progressive

enlargement of the kidneys due to numerous expansile cysts and ultimately leads to

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renal failure. In humans, PKD is heritable and recognized in at least 2 genetically
distinguishable forms; autosomal recessive PKD (ARPKD) or autosomal dominant PKD
(ADPKD). The autosomal dominant form is often associated with a variety of extrarenal
manifestations and usually leads to death from renal failure in late adulthood. The
autosomal recessive form is rare, often diagnosed in early infancy by massive
nephromegaly, and is rapidly progressive.1 Syndromes resembling both the recessive
and dominant forms of human PKD have been recognized in animals including cats,2,3
with Persian cats appearing disproportionately affected,4 dogs,5,6 mice,7 pigs, raccoons8
and ruminants such as cattle,9 goats,10,11 sheep12 and Springbok (Antidorcas
marsupialis).13 PKD has not been reported previously in any member of the family
Cervidae.

In domestic animals, PKD is most often consistent with the human ARPKD in that
disease manifests as stillbirths or death within the first few weeks of life, although
manifestations consistent with the ADPKD have also been described. Reported
extrarenal manifestations of PKD in animals include biliary and pancreatic cysts.3,5,6,10-12

Many humans with ARPKD have been found to have mutations in the gene, polycystic
kidney and hepatic disease 1 (PKHD1). This gene is predicted to code for a protein that
is known as fibrocystin or polyductin.14,15 The protein is expressed on adult and fetal
kidney, liver and pancreas and may be a receptor protein that plays a role in collecting
duct and bile duct differentiation. The basic defect in ARPKD may, therefore, be a
failure of terminal differentiation in collecting and bile ducts.15 PKHD1 gene products
are members of a novel class of proteins that share structural features with hepatocyte
growth factor receptor and plexins, members of a class of proteins involved in the
regulation of cell proliferation, cellular adhesion and repulsion.14,15 Genetic factors may
be involved in congenital PKD of Cairn Terriers, springbok and Persian cats as the
condition has been described in groups of related animals.

AFIP Diagnosis: Kidneys, glomeruli and tubules: Cystic change, diffuse, severe,
white-tailed deer (Odocoileus virginianus), cervid.

Conference Comment: The contributor provides a thorough overview of polycystic

kidney disease in humans and animals. As mentioned by the contributor, animals with
polycystic kidney disease (PKD) often have biliary cysts and pancreatic cysts in addition
to the renal changes. Extrarenal cysts are not found in conditions leading to congenital
or acquired renal cysts.

Congenital renal cysts can occur in cases of renal dysplasia or can occur as a primary
entity. There may be only one cyst or there may be many cysts that often distort the
contour of the kidney. Some cysts may cause no alteration in renal function, and are
therefore considered incidental findings. Cysts may arise anywhere along the nephron
and can be located in either the cortex or the medulla and may range in size from barely
visible to several centimeters in diameter.16

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Acquired renal cysts can occur as a result of renal interstitial fibrosis, as in chronic renal
disease, or they may occur in renal diseases that cause intratubular obstruction. These
cysts are usually small, only 1-2 mm in diameter, and occur primarily in the renal cortex.
In all cases with renal cysts, the cysts must be differentiated from hydronephrosis.16

Contributor: National Animal Disease Center, ARS, USDA/ARS, 2300 Dayton

Avenue, Ames, IA 50010
http://www.nadc.ars.usda.gov/

References:
1. Zerres K, Rudnik-Schoneborn S, Eggermann T, Bergmann C: Autosomal recessive
polycystic kidney disease. J Nephrol 16:453-458, 2003
2. Battershell D, Garcia JP: Polycystic kidney in a cat. J Amer Vet Med Assoc 154: 665-
666, 1969
3. Crowell WA, Hubbell JJ, Riley JC: Polycystic renal disease in related cats. J Amer
Vet Med Assoc 17:286-288, 1979
4. Maxie MG: The urinary system. In: Pathology of domestic animals, eds. Jubb KVF,
Kennedy PC, Palmer N, 4th ed., vol. 2, pp.463-465. Academic Press, San Diego, CA,
1993
5. McKenna SC, Carpenter JL: Polycystic disease of the kidney and liver in the Cairn
Terrier. Vet Pathol 17:436-442, 1980
6. Van Den Ingh TSGAM, Rothuizen J: Congenital cystic disease of the liver in seven
dogs. J Comp Pathol 95:405-414, 1985
7. Sweeney WE, Avner ED: Pathophysiology of renal tubular cyst formation in murine
models of polycystic kidney disease. Contrib Nephrol 97:23-34, 1992
8. Hamir AN, Klein L: Polycystic kidney disease in a raccoon (Procyon lotor). J Wild Dis
32:674-677, 1996
9. Ushigaki K, Uchida K, Murakami T, Yamaguchi R, Tateyama S: Multicystic renal
dysplasia in a Japanese Black bull. J Vet Med Sci 61:839-842, 1999
10. Krotec K, Meyer BS, Freeman W, Hamir AN: Congenital cystic disease of the liver,
pancreas, and kidney in a Nubian goat (Capra hircus). Vet Pathol 33:708-710, 1996
11. Newman SJ, Leichner T, Crisman M, Ramos J: Congenital cystic disease of the
liver and kidney in a pygmy goat. J Vet Diagn Invest 12:374-378, 2000
12. Dennis SM: Urogenital defects in sheep. Vet Rec 105:344-347, 1979
13. Iverson WO, Fetterman GH, Jacobson ER, Olsen JH, Senior DF, Schobert EE:
Polycystic kidney and liver disease in Springbok: I. Morphology of the lesions. Kid
Internat 22:146-155, 1982
14. Onuchic LF, Furu L, Nagasawa Y, Hou X, Eggermann T, Ren Z, Bergmann C,
Senderek J, Esquivel E, Zeltner R, Rudnik-Schoneborn S, Mrug M, Sweeney W, Avner
ED, Zerres K, Guay-Woodford LM, Somlo S, Germino GG: PKHD1, the polycystic
kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple
immunoglobulin-like plexin-transcription factor domains and parallel beta-helix 1
repeats. Am J Hum Genet 70:1305-1317, 2002
15. Ward CJ, Hogan MC, Rossetti S, Walker D, Sneddon T, Wang X, Kubly V,
Cunningham JM, Bacallao R, Ishibashi M, Milliner DS, Torres VE, Harris PC: The gene

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mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-
like protein. Nat Genet 30:259-269, 2002
16. Confer AW, Panciera RJ: The urinary system. In: Thompson’s Special Veterinary
rd
Pathology, eds. McGavin MD, Carlton WW, Zachary JF, 3 ed., pp.240-241. Mosby, St.
Louis, MO, 2001

ELECTRON MICROGRAPH
CONFERENCE 25 / CASE IV – Case #2 (AFIP 2946684)

Signalment: Young female striped skunk (Mephitis mephitis) of unknown age with an
approximate weight of 4 pounds (1.8 Kg).

History: The skunk had a history of seizures and accumulation of ocular/periocular

crusty debris. The skunk was found in a populated area where dogs and cats resided,
and was transferred to a wildlife rehabilitation center where it subsequently died. The
carcass was refrigerated until a necropsy was performed. Selected tissues were
collected and preserved in a 10% buffered neutral formaldehyde solution for
histopathologic examination. Small fragments of lung and gastric mucosa were
collected and preserved in a 3% buffered glutaraldehyde solution for examination by
transmission electron microscopy.

Gross Pathology: The stomach and duodenum had numerous white nematode
parasites that measured approximately 2.5 mm long. The uterus contained six fetuses
each measuring approximately 1.5 cm in length.

Laboratory Results:
1. Canine Distemper Virus (CDV) and Rabies Virus fluorescent antibody tests –
positive CDV antigen immunoreactivity in the lungs, tongue, and brain. Negative for
Rabies Virus antigen immunoreactivity. Viruses were not isolated from the lungs, brain,
or tongue.
2. Histopathology (Tissues not submitted for review) –
a. Lung: Interstitial pneumonia with bronchial epithelial inclusion bodies.
b. Brain: Meningoencephalitis, lymphocytic.
c. Bronchial lymph node: Histiocytosis and follicular lymphoid hyperplasia
d. Liver: Congestion and hepatocellular vacuolar degeneration with biliary
epithelial inclusion bodies.
e. Spleen: Lymphoid depletion
f. Epithelium (multiple sites): Inclusion bodies

Contributor’s Morphologic Diagnosis: Ciliated columnar epithelium (bronchus):

Intracytoplasmic and intranuclear viral inclusions consistent with CDV.

Contributor’s Comment: The transmission electron photomicrograph of bronchial

epithelium contained ciliated columnar epithelial cells with intracytoplasmic and

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intranuclear viral inclusion bodies consistent with CDV. Intracytoplasmic inclusion
bodies were consistent with accumulated viral nucleocapsids and were characterized
most frequently by amorphous aggregates of moderately electron dense granular
material, and less frequently by amorphous aggregates of tubular-like structures. The
nuclei of most cells were of decreased electron density due to dispersion of the
chromatin pattern. One cell had intranuclear inclusions characterized by parallel,
stacked arrays of electron dense, tubular to filamentous material. Other findings in the
bronchial epithelial cells consisted of vacuolar epithelial cell degeneration characterized
by mild dilatation of the smooth endoplasmic reticulum and the perinuclear cisterna, loss
of apical microvilli, low numbers of secondary lysosomes, and cellular debris in the
luminal surface. Examination of fine mitochondrial detail was not possible due to mild
autolysis.

CDV is grouped in the family Paramyxoviridae, genus Morbillivirus, and is related to

Measles Virus, Peste de Petits Ruminants Virus, and Rinderpest Virus.1 It is an RNA
virus that displays spherical to filamentous morphology ultrastructurally. It is reported
that there is a single serotype of CDV; however, more than one genotype is known to
exist. CDV isolates vary in biologic activity and tissue tropism.

CDV is transmitted primarily by contact with respiratory, ocular, or oral fluids.1 CDV may
also be transmitted less frequently by contact with infected shed skin cells, feces, and
urine, and by the transplacental route. Transmission is likely enhanced by increased
density and contact between susceptible animal populations, animal behaviors
conducive to transmission, increasing dose of virus, and immunosuppression.1 Other
factors that influence susceptibility to CDV are age, species of host, virus strain, and
environmental conditions. A strong antibody response to infection by the virus is
reportedly protective, while weak antibody responses are associated with illness.

CDV may infect and cause clinical disease in a wide variety of carnivores including
canids, felids, mustelids, procyonids and others.1 Striped skunks are reportedly less
susceptible to disease caused by CDV, but may suffer from the disease nonetheless.
CDV-infected wild and domestic carnivores present a significant risk to zoological
collections with susceptible species. The incubation period reportedly varies from one
week to > 1 month, and clinical disease may last approximately 1-6 weeks. Infection
may be fatal, particularly in highly susceptible species such as domestic ferrets (Mustela
putorius furo). Clinical signs associated with CDV infection include depression,
mucopurulent oculonasal discharge, fever, cough, anorexia, vomition, diarrhea, and
central nervous system (CNS) dysfunction.1-4 Clinical signs consistent with CNS
dysfunction in CDV-infected animals include seizures, convulsions, paresis, paralysis,
and other clinical signs. CDV-infected mustelids, such as striped skunks, may have
symptoms similar to those described above.1

Macroscopic lesions include oculonasal discharge, diarrhea, hyperkeratosis (in

prolonged infections primarily), poor body condition, and pulmonary changes consistent
with pneumonia.1 Other less frequently seen macroscopic findings may occur and in
some cases lesions may not be evident. Commonly seen microscopic findings include

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interstitial and/or bronchopneumonia, and lymphoid depletion in the spleen, lymph
nodes, and thymus.1 Formation of intracytoplasmic and intranuclear eosinophilic
inclusions are a characteristic microscopic feature and can be seen in epithelial and
neural cells of infected animals. In the present case, inclusions were seen in multiple
epithelial tissues by light microscopy. CDV-induced primary lesions may also be seen
in other tissues including tissues of the CNS, stomach, and intestines.1-4 Concurrent
opportunistic infections may occur in CDV-infected animals secondary to
immunosuppression, and may obscure CDV-induced lesions.1,5 This skunk had
microscopic lesions consistent with CDV.

Laboratory tests, other than light microscopic examination, utilized to diagnose CDV-
infection include transmission electron microscopy, fluorescent antibody testing
(FAT)/immunohistochemistry, PCR assays, nucleic acid hybridization, virus isolation,
determination of antibody titers, and cytologic examination of tissue samples or blood
smears.1,6-15 In the present case the ultrastructural characteristics of the viral particles
and the FAT results were consistent with CDV.

The most important differential diagnosis in a suspected CDV-infected carnivore,

particularly those with CNS dysfunction, is Rabies Virus.1 In the present case Rabies
was considered an important differential diagnosis and specific testing indicated that the
skunk was not infected with Rabies Virus. Infection with other microbiologic agents or
exposure to toxins are differential diagnoses that must also be considered.1

AFIP Diagnosis: Ciliated respiratory epithelium: Degeneration and necrosis, with

intracytoplasmic and intranuclear viral inclusions, striped skunk (Mephitis mephitis),
mustelid.

Conference Comment: The contributor provides a thorough overview of Canine

Distemper Virus (CDV) including species affected, clinical signs, transmission,
microscopic and ultrastructural findings, diagnostic tests, and etiologic differentials.

For most pathologists, describing ultrastructural changes and interpreting electron

micrographs can be challenging. However, when examining electron micrographs one
must only consider three cellular alterations: degeneration, necrosis, and something
added or taken away. Then one must systematically evaluate and describe the cells
present and their organelles in order to identify the cells/tissue and the process. When
describing electron micrographs it is important to start with a brief description of the
normal features which allow one to identify the cells and or tissue. Such features may
include: number and arrangement of cells, plasma membrane, surface decorations,
cellular junctions, cytosol, endoplasmic reticulum (smooth and rough), lysosomes,
mitochondria, nuclei, and other intra- and extracellular features. Below are two helpful
charts to assist in evaluating electron micrographs.16,17

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 Organelle Normal features and changes to note on EM
Plasma membrane Cilia, villi; loss of surface specialization; cytoplasmic blebs; types and locations of
intercellular junctions
Cytosol Rarefaction (swelling); presence of myelin figures; inclusion bodies
ER (smooth and rough) Relative amounts of SER and RER; swelling/dilation; detachment of ribosomes;
SER / RER increased amounts of SER
Mitochondria Relative number and location; low amplitude / high amplitude swelling; matrix
flocculent densities; calcification; vacuolization; rupture
Lysosome Relative number; swelling; rupture
Nucleus Clumped, dispersed, or marginalized chromatin; heterochromatin, euchromatin;
pyknosis, karyorrhexis, karyolysis; viral inclusions
Other Intranuclear or intracytoplasmic inclusions; bacteria; parasites; fungi; algae

Organelle Reversible Changes Irreversible Changes

Plasma membrane Blebbing, blunting, distortion; loosening of Disruption of cellular membranes;
intercellular attachments
Mitochondria Swelling, rarefaction, small amorphous Marked dilation; large amorphous
densities densities
ER Dilation, detachment of ribosomes
Nucleus Chromatin clumping Pyknosis, karyorrhexis, karyolysis
Other Cellular swelling; creation of myelin figures Increased cellular swelling; swelling
and disruption of lysosomes;
increased numbers of myelin figures

Contributor: Eli Lilly and Company, Lilly Research Laboratories, 2001 West Main
Street, Greenfield, IN

References:
1. Williams ES: Morbilliviral Diseases. In: Infectious Diseases of Wild Mammals, eds.
Williams ES, and Barker IK, 3rd ed., pp. 37-76. Iowa State University Press, Ames, Iowa,
2001
2. Lincoln SD, Gorham JR, Davis WC, Ott RL: Studies of old dog encephalitis II:
electron microscopic findings and immunohistologic findings. Vet Path 10:124-129,
1973
3. Lisiak JA, Vanvelde M: Polioencephalomalacia associated with canine distemper
virus infection. Vet Pathol 16:650-660, 1979
4. Machida N, Kiryu K, Oh-ishi K, Kanda E, Izumisawa N, Nakamura T: Pathology and
epidemiology of canine distemper in raccoon dogs (Nyctereutes procyonoides). J Comp
Pathol 108:383-392, 1993
5Reed WM, Turek JJ: Concurrent distemper and disseminated toxoplasmosis in a red
fox. JAVMA 187(11):1264-1265, 1985.
6. Miry C, Ducatelle R, Thoonen H, Hoorens J: Immunoperoxidase study of canine
distemper virus pneumonia. Res Vet Sci 34:145-148, 1983
7. McLaughlin BG, Adams PS, Cornell WD, Elkins AD: Canine distemper viral
inclusions in blood cells of four vaccinated dogs. Can Vet J 26:368-372, 1985
8. Alleman AR, Christopher MM, Steiner DA, Homer BL: Identification of inclusion
bodies in mononuclear cells from the cerebrospinal fluid of a dog with canine distemper.
Vet Pathol 29:84-85, 1992

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9. Baumgartner W, Krakowka S: Fixation procedures for retention of cellular
morphologic features and for preservation of immunoreactivity of canine paramyxovirus
antigens. Am J Vet Res 49(4):477-481, 1988
10. Watson ADJ, Wright RG: The ultrastructure of cytoplasmic inclusions in circulating
lymphocytes in canine distemper. Res Vet Sci 17:188-192, 1974
11. Richter WR, Moize SM: Ultrastructural nature of canine distemper inclusions in the
urinary bladder. Path Vet 7:346-352, 1970
12. Confer AW, Kahn DE, Koestner A, Krakowka S: Comparison of canine distemper
viral strains: an electron microscopic study. Am J Vet Res 36(6):741-748, 1975
13. Narang HK: Ultrastructural study of long-term canine distemper virus infection in
tissue culture cells. Infection and Immunity 36(1):310-319, 1982
14. Oglesbee M: Intranuclear inclusions in paramyxovirus-induced encephalitis:
evidence for altered nuclear body differentiation. Acta Neuropathol 84:407-415, 1992
15. Koestner A, Long JF: Ultrastructure of canine distemper virus explant tissue
cultures of canine cerebellum. Laboratory Investigation 23(2):196-201, 1970
16. Kumar V, Abbas AK, Fausto N: Cellular adaptations, cell injury, and cell death. In:
Robbins and Cotran Pathologic Basis of Disease, 7th ed., pp. 12. Elsevier Saunders,
Philadelphia, PA, 2005
17. Scott DP: Ultrastructural pathology. Notes from 13th Annual Descriptive Veterinary
Pathology Course. Armed Forces Institute of Pathology, 2004

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