AFIP Wednesday Slide Conference - No. 1
8 September 1999

Conference Moderator:
COL William Inskeep II
Diplomate, ACVP
Chairman, Department of Veterinary Pathology
Deputy Director, Armed Forces Institute of Pathology
Washington, DC 20306
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Case I - 96-4834 (AFIP 2597320)
Signalment: Neonatal bovine.
History: Seven of 70 calves had diarrhea.

Laboratory Results: Florescent antibody testing was positive for coronavirus and negative for rotavirus. Coronaviruses were found by electron microscopy on negatively stained preparations of colonic content.

No significant bacteria were isolated. Indirect florescent antibody examination for E. coli K99 pilus antigen was negative. Examination of colonic content smears was negative for Cryptosporidium sp. by volusol AF stain.
Contributor's Diagnoses and Comments:
1. Enteritis, ulcerative, viral, coronavirus.
2. Colitis, ulcerative, viral, coronavirus.
The small intestine has marked shortening of the villi and flattened villous epithelium. Crypt epithelium has prominent regeneration. The colon has concurrent degeneration and regeneration of crypt epithelium.
AFIP Diagnoses:
1. Small intestine: Enteritis, erosive and necrotizing, acute, diffuse, moderate, with blunting and fusion of villi, crypt abscesses, and regeneration. breed unspecified bovine calf.
2. Large intestine: Colitis, erosive and necrotizing, acute, diffuse, moderate, with crypt abscesses and regeneration.
Conference Note: The main infectious causes of neonatal calf diarrhea are rotavirus, coronavirus, enterotoxigenic Escherichia coli, Salmonella species, and Cryptosporidium sp. Clinical differentiation is difficult because the potential pathogens cause similar clinical signs and frequently two or more causative agents are present.
Coronavirus causes both upper respiratory and intestinal infections, with intestinal infections occurring generally between one and two weeks of age. Viral replication begins in the epithelium of the proximal small intestine and spreads throughout the small and large intestines causing necrosis of crypts and villi.

· Clinical signs: Profuse watery diarrhea, dehydration.
· Gross lesions: Mild fibrinous enterocolitis and edema in lymph nodes.
· Pathophysiology: Infection via fecal/oral or nasal/oral route Þ replication in oral mucosa Þ swallowed Þ tips of villi.
· Clinical pathology: Acidosis, hyperkalemia, and hyponatremia.
· Comparative pathology: The same coronavirus is responsible for winter dysentery in older cattle; other coronaviruses cause feline infectious peritonitis, vomiting and wasting disease of swine, transmissible gastroenteritis of swine and hepatitis in mice.

Rotavirus produces enteric infection as early as three days of age and as late as about 3 weeks of age. The lower small intestine is generally affected. Lesions are histologically similar to those seen in coronaviral enteritis. E. coli enterotoxicosis occurs in calves up to 6 days of age. Diarrhea results from the release of thermostabile enteroxin that produces profound hypersecretion from enterocytes without significantly damaging the epithelium. Salmonella sp. generally infect calves between one and seven weeks of age. These bacteria damage the mucosa through invasion and the production of enterotoxins. Infection frequently results in septicemia. Cryptosporidium sp. Infect calves in the first three weeks of life. These extracellular protozoa attach to the epithelium of the small and large intestine and displace the microvilli.
Contributor: California Veterinary Diagnostic Laboratory System, U.C. Davis, 105 West Central Avenue, San Bernardino, CA 92408

1. Jones T C, Hunt R D, King N W: Diseases caused by viruses. In: Veterinary Pathology, 6th Ed, 1997, pp. 281-286.
2. Vermunt JJ: Rearing and management of diarrhoea in calves to weaning. Australian Vet Journ. Vol. 71, No. 2, 33-41, February 1994.
3. Barker, IK, Van Dreumel, AA: The alimentary system. In: Pathology of Domestic Animals, Vol 2. Jubb, KVF, Kennedy, PC, and Palmer, N 4th Ed, 1993, pp 184-192.
5. Janke BH: Protecting calves from viral diarrhea. Vet Med 803-811, August 1989.
Case II - Case 2/BE2D/NIEHS (AFIP 2677917)
Signalment: 8-week-old, female, Fischer 344 rat
History: The rat was treated once daily for 2 days by gavage with an ethylene glycol ether (EGE).
Gross Pathology: No significant gross abnormalities noted.
Laboratory Results: None.

Contributor's Diagnoses and Comments:
1. Nasal cavity, maxillo- and nasoturbinates, submucosal vessels - thrombosis.
2. Nasal cavity, incisor teeth, dental pulp - thrombosis.

In rats exposed for 2 days, disseminated thrombosis was noted in the liver, teeth, heart, and bone marrow (in the tail and femur) and in the lungs. Infarction was noted in the bone and bone marrow. Use of this EGE in rats was associated with a wide range of hematologic and pathological abnormalities (Ghanayem 1996). Blood smears obtained from exposed rats had significant alteration of erythrocyte morphology. These changes included stomatocytosis, spherocytosis, fragmentation of erythrocytes, formation of ghost cells, and vesciculation. It is suggested that chemical exposure induced either primary anemia, leading to anoxic endothelial injury or alternatively induced changes in the erythrocyte morphology, resulting in spherocytosis and contributing to compromised blood flow. Either of these events may have triggered acute disseminated intravascular coagulation (DIC) and eventual bone infarction.
AFIP Diagnosis: Nasal mucosa and dental pulp: Fibrin thrombi, Fischer 344 rat, rodent.
Conference Note: Various mechanisms that may be involved in the pathogenesis of chemically induced thrombosis were discussed, including vascular damage, induction of a hypercoagulable state, and disturbance of blood flow.

Disseminated thrombosis (disseminated intravascular coagulation (DIC) or consumption coagulopathy) is a thrombohemorrhagic disorder that may develop as a complication in a variety of diseases. Two mechanisms for triggering DIC are 1. Release of procoagulant tissue factor(s) into the circulation following injury and 2. Activation of factor XII following widespread endothelial injury and resulting surface contact with collagen. The thrombi may be formed in the general circulation or may be localized to a specific organ or tissue. The thrombi may cause ischemia of more severely affected or more vulnerable organs.

Nyska et al. reported that 2-butoxyethanol (BE) (ethylene glycol monobutyl ether) can produce disseminated thrombosis and bone infarction in female rats. The authors did not know the pathogenesis of BE-induced disseminated thrombosis, but proposed that BE-induced hemolysis (and release of procoagulant factors from damaged erythrocytes) may result in thrombosis via disturbances of blood flow, but direct endothelial damage and other mechanisms were also possible. The primary support for hemolysis being the underlying cause of DIC stems from studies that have shown that female rats are more susceptible to BE-induced hemolysis; thrombosis and bone infarction are observed only in female rats.
Contributor: National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709
1. Ghanayem B: An overview of the hematoxicity of ethylene glycol ethers. Occupat Hyg 2:253-268, 1996
2. Nyska A, et al: Disseminated thrombosis and bone infarction in female rats following inhalation exposure to 2-butoxyethanol. Tox Path 27(3):287-294, 1999
Case III - 98N061 GUH DC 20007 (AFIP 2681360)
Signalment: Two-year-old male, neutered, skunk, Mephitis mephitis
History: This skunk presented with a 2 cm diameter ulcerated lesion on its back, duration unknown. The entire lesion was excised and submitted for histopathology. The skunk is alive and doing well.
Gross Pathology: This two cm diameter lesion was dark brown-black and ulcerated. It extended 1cm down into the dermis.
Laboratory Results: None.
Contributor's Diagnosis and Comments: Low grade leiomyosarcoma
The H and E stained section of the mass shows that it is composed of homogeneous round to spindloid cells arranged in cords and short bundles embedded in a myxomatous to focally hemorrhagic matrix. The cells are characterized by oval nuclei, 1-2 indistinct nucleoli and scant eosinophilic cytoplasm. The mitotic rate is low: 0-1per high power field. The overlying skin is ulcerated with mild to moderate mixed inflammation. With the H and E stained section alone, this neoplasm was diagnosed as a low grade sarcoma, with leiomyosarcoma and neurofibrosarcoma included in the differential diagnosis.
Immunohistochemistry (avidin-biotin immunoperoxidase method) was performed utilizing monoclonal antibodies to desmin, muscle specific actin (MSA), myoglobin, S-100 protein and neuron specific enolase (NSE). The tissue was positive for desmin and MSA, and nonspecific for myoglobin and NSE. There were occasional positive cells for S-100 but these were of uncertain significance. Therefore, this mass was determined to be a leiomyosarcoma. Leiomyosarcomas of the subcutaneous tissue are rare tumors of domestic animals. They are speculated to originate from the smooth muscle of vessel walls or arrrector pili muscle.
AFIP Diagnosis: Haired skin and subcutis: Leiomyosarcoma, skunk (Mephitis mephitis), mustelid.
Conference Note: Cutaneous leiomyosarcomas are rarely reported in animals. These tumors may be underdiagnosed because of their resemblance to more common spindle cell sarcomas. The distinguishing histologic features in this case include cells forming long streams and bundles, end to end rowing of nuclei, blunt-ended and occasionally plicated nuclei, moderate amounts of variably vacuolated cytoplasm, and minimal collagenous stroma.
Immunohistochemistry performed at the AFIP confirmed that the neoplastic cells are positive for smooth muscle actin, and negative for glial fibrillary acidic protein. Unfortunately, immunostains for S-100 protein did not work properly on two attempts. The AFIP's Department of Soft Tissue Pathology reviewed this case and favored a diagnosis of epithelioid leiomyosarcoma. In humans and animals, cutaneous leiomyosarcomas are generally low-grade malignancies.

Contributor: Georgetown University/DCM, 3950 Reservoir Rd. NW, Washington, DC 20007
1. Brunnert SR, Herron AJ, and Altman NH: Leiomyosarcoma in a domestic ferret: morphologic and immuncytochemical diagnosis. Lab Ani Sci, vol 40, no. 2:208-209, 1990.
2. Brunnert SR, Herron AJ, and Altman NH: Leiomyosarcoma in a Peruvian squirrel monkey (Saimiri sciureus). Vet Pathol, vol. 27:126-128, 1990.
3. Gross TL, Ihrke PJ, Walder EJ: Veterinary Dermatopathology, pp. 444-445. Mosby-Year Book Inc, St Louis, Missouri, 1992.
4. Hanzaike Tl, Ito I, Ishikawa T et al: Leiomyosarcoma of soft tissue in a cow. J Comp Path, vol.112:237-242, 1995.
5. Sartin EA, Doran SE, Riddell MG et al: Characterization of naturally occurring cutaneous neurofibromatosis in Holstein cattle. Am J Pathol, vol 145, no. 15:1168-1174, 1994.
Case IV - 99-0002 (H99-0065 D) (AFIP 2681727)
Signalment: Fledgling Nankeen kestrel (Falco cenchroides)
History: A wild Nankeen kestrel fledgling with a history and clinical signs of episodic nervous disease, blindness, head-pressing, intermittent seizures, loss of balance and spontaneous screaming was presented for necropsy examination. Ophthalmological examination demonstrated a swollen and hyperaemic pecten and mild hyphaema.
Gross Pathology: There was congestion of cerebral vessels and multifocal petechial haemorrhages in the leptomeninges and throughout the brain parenchyma.
Laboratory Results: Haematological examination demonstrated a normal total white blood cell count with a moderate relative lymphocytosis and heteropaenia. Occasional rare, large, round, bluish, granular, intracytoplasmic gametocytes and eccentrically displaced sometimes distorted nuclei in circulating leukocytes were observed. Plasma creatine kinase concentration was slightly elevated but other biochemical parameters were normal. Cultures of liver and lung failed to yield bacterial isolates.
Contributor's Diagnoses and Comments: Brain and eye: Severe, subacute vascular endothelial hyperplasia, granulomatous perivasculitis and pectinitis with endothelial parasitic cysts measuring 40 to 60 mm in diameter.
The lesions presented in this case are characteristic of a disease which is seasonally common in young Western Australian falcons (Jaensch and Raidal 1996; Raidal et al 1999). The inflammatory lesions centered on the vessels of the brain and pecten represent the schizogenous phase of a Leucocytozoon sp. Transmission electron microscopy demonstrated marked proliferation of endothelial cells and swollen endothelial cells containing abundant mitochondria. Occasional endothelial cells contained intracytoplasmic parasitophorous vacuoles containing granular material or larger similar vacuoles (10-20 mm in diameter) containing electron-dense granular material, nuclear membranes and electron-dense aggregates. Spherical protozoal merozoites measuring 1 mm in diameter were present within thin-walled endothelial cysts and also free within the lumens of vessels. The zoites contained a spherical, indented nucleus measuring 0.5 mm in diameter and paired, tear-shaped, electron-dense rhoptries and microneme-like electron-densities.

Severely affected kestrels typically have low numbers of circulating leucocytozoon gametocytes in blood smears. Although acute disease can occur rapidly before the completion of gametogony, endothelial schizonts occur consistently in the arterioles of the brain, spinal cord, optic nerve, pecten and kidney and less frequently in arterioles of the lungs, heart, liver, intestines and spleen.

Leucocytozoon are parasites of birds and, in most species, schizogony with the production of small schizonts, and in some species megaloschizonts, occurs in hepatocytes or hepatic sinusoidal endothelial cells although schizogony and gametogony can also occur in other tissues (Steele and Noblet 1992). All affected falcons and kestrels examined so far have had no evidence of hepatic phases of schizogony. The predilection for arterioles in the central nervous system, eye and kidney is unusual for Leucocytozoon species.
Case 1-4. Transmission electron micrograph.
AFIP Diagnoses:
1. Cerebrum and brain stem: Endothelial hyperplasia and hypertrophy, multifocal, marked, with intraendothelial protozoal schizonts, hemorrhage, and perivascular hemosiderophages, Nankeen kestrel (Falco cenchroides), avian.
2. Eye: Endothelial hyperplasia and hypertrophy, multifocal, moderate, with intraendothelial protozoal schizonts, and histiocytic pectinitis.

Conference Note: The differential diagnosis in this case includes Leucocytozoon sp, Hemoproteus sp., Toxoplasma gondii, Sarcocystis sp., Plasmodium sp. and microsporidia. The size and location of schizonts within the cytoplasm of endothelial cells leads to a shortened differential diagnosis of Leucocytozoon sp. and Hemoproteus sp. The presence of intraleukocytic stages in circulating erythrocytes and leukocytes supports the final diagnosis of Leucocytozoon sp.
Contributor: Division of Biochemical Sciences, Murdoch University, South Street Murdoch, Western Australia, 6150

1. Gardiner CH, Fayer R, Dubey JP: An Atlas of Protozoan Parasites in Animal Tissues, 2nd ed., pp. 73-74. Armed Forces Institute of Pathology, Washington, DC, 1998
2. Jaensch SM, Raidal SR: Neurological disease and blindness - two case studies. Annual Proceedings of the Australian Chapter of the Association of Avian Veterinarians., O'Reilly's Rainforest Resort, Lamington National Park, Queensland, pp 151-154, 1996
3. Raidal SR, Jaensch SM, Ende J: Preliminary report of a parasitic infection of the brain and eyes of a Peregrine Falcon Falco peregrinus and Nankeen Kestrels Falco cenchroides in Western Australia, EMU, 99:1-2, 1999
4. Steele EJ, Noblet GP: Schizogonic development of Leucocytozoon smithi. Journ of Parasit, 39:530-536, 1992.
J Scot Estep, DVM
Captain, VC, USA
Registry of Veterinary Pathology*
Department of Veterinary Pathology
Armed Forces Institute of Pathology
(202)782-2615; DSN: 662-2615
* The American Veterinary Medical Association and the American College of Veterinary Pathologists are co-sponsors of the Registry of Veterinary Pathology. The C.L. Davis Foundation also provides substantial support for the Registry.
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