AFIP Wednesday Slide Conference - No. 11
November 24, 1999

Conference Moderator:
COL Michael J. Topper
Diplomate, ACVP
Division of Pathology
Walter Reed Army Institute of Research
Washington, DC 20307
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Case I - 99-8265 (AFIP 2694722)
Signalment: 2-yr-old, female, domestic shorthaired cat, Felis domesticus.
History: The cat developed dyspnea within 48 hours of being placed in a boarding facility. The owner claimed that the animal exhibited no clinical abnormalities prior to this sudden onset of respiratory distress. Radiographs revealed a diffuse interstitial pattern of pulmonary infiltration. The animal died before further clinical work-up could be pursued.
Gross Pathology: Necropsy was performed by the referring veterinarian. The lungs were described as diffusely red, meaty and heavy. No other abnormalities were noted in the carcass, and the lung was the only organ sampled for microscopic exam.
Contributor's Diagnosis and Comments: Severe, subacute to chronic, diffuse, proliferative interstitial pneumonia with intralesional protozoa

Etiology: Toxoplasma gondii.
Sections have been cut from each of the 3 lung samples submitted, so there is some variability between slides. There is remarkable proliferation of type II pneumocytes, resulting in an adenomatous appearance to the tissue. Many protozoal organisms are visible. These tachyzoites are most prominent in macrophages and multinucleate giant cells but can also be found within respiratory epithelial cells. The alveolar septa and pleura are thickened, and a lymphoplasmacytic infiltrate is evident in this interstitial compartment. Many bronchoalveolar spaces are filled with necrotic cellular debris, which includes tachyzoite-laden cells.
The cat is the definitive host for Toxoplasma gondii, which utilizes an enteroepithelial life cycle to produce oocysts within this host. However, extraintestinal organisms can be found within the cat (and a wide variety of other hosts, including humans) in the form of intracellular tachyzoites and bradyzoites. Cats may be infected transplacentally or orally (through ingestion of food or water contaminated with sporulated oocysts, or by eating tachyzoites and bradyzoites within the tissues of infected intermediate hosts). Immunohistochemical staining of the lung for Toxoplasma antigen was positive (test performed at Cornell Veterinary Diagnostic Lab).
Infection of cats is common, but the clinical outcome is likely influenced by various factors such as the strain and number of infectious organisms ingested, as well as the immune status of the host. Fatal disease in adult cats is uncommon. Dyspnea, as seen in this case, is one of the most common clinical signs of postnatal toxoplasmosis. Toxoplasma-induced lesions are frequently detected in the lung, liver, pancreas and lymph nodes and generally have a necrotizing and granulomatous character. The FIV and FeLV status of this cat is unknown and the possibility of concurrent infection with viral respiratory pathogens such as calicivirus and feline viral rhinotracheitis virus cannot be ruled-out.
AFIP Diagnosis: Lung: Pneumonia, interstitial, proliferative, diffuse, severe, with pleuritis and numerous intracellular protozoa, domestic short hair, feline, etiology consistent with Toxoplasma gondii.
Conference Note: Conference participants agreed that the findings are consistent with Toxoplasma gondii. Other organisms that were considered in the differential diagnosis include: Neospora caninum, Sarcocystis sp., Trypanosoma sp., Leishmania sp., and Histoplasma capsulatum. In general, the most difficult possibility to eliminate is Neospora caninum. Immunohistochemistry and electron microscopy can differentiate the two organisms. Ultrastructurally, Neospora has electron dense rhoptries, while Toxoplasma has honeycomb-like or labyrinthine rhoptries. Also, natural infection of cats with Neospora caninum has not been reported.
Toxoplasma gondii is an obligate intracellular parasite that can infect all warm-blooded animals, but cats are the definitive host (only cats excrete oocysts). The three infectious stages are tachyzoites (rapidly proliferating, infect almost any cell), bradyzoites (slowly proliferating encysted zoites) and sporozoites (four per sporocyst, two sporocysts per sporulated oocyst). Both Toxoplasma and Neospora oocysts require up to 3 days to sporulate after they are shed, but Frenkelia, Sarcocystis and Cryptosporidium are shed as sporulated oocysts.
Contributor: College of Veterinary Medicine, Oregon State University, Magruder Hall, Corvallis, OR 97331
1. Barker IK, Van Dreumel AA, Palmer N: The Alimentary System. In: Pathology of Domestic Animals, 4th ed., vol. 2, eds. KVF Jubb, PC Kennedy, Palmer N, pp. 308-310. Academic Press, San Diego, CA, 1993
2. Dubey JP: Advances in the life cycle of Toxoplasma gondii. Internat J for Parasit 28:1019-1024, 1998
3. Dubey JP, Lappin MR: Toxoplasmosis and Neosporosis. In: Infectious Diseases of the Dog and Cat, ed. CE Greene, 2nd ed., pp. 493-509. WB Saunders, Philadelphia, 1998
4. Dubey JP, Lindsay DS, Speer CA: Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clin Microbiol Rev. 11(2):267-299, 1998
Case II - B99-9032 (AFIP 2693027)
Signalment: Sprague Dawley rat (Rattus norvegicus), young adult male.
History: Rats had surgery for placement of bile duct cannulae 7-10 days prior to sacrifice. These rats were supposed to be used for a metabolism study involving bile excretion. Rats were purchased from a commercial supplier after a surgical procedure to allow sampling of bile outside the body. The bile duct had been cannulated with small bore polyethylene tubing. The tubing was anchored in place via a suture, then exteriorized through the abdominal cavity and skin. The tubing then formed a loose loop outside the body, passed back through the skin into the abdominal cavity, and was anchored to empty into the duodenum. There was a small connector in the tubing loop present outside the skin that could be removed to sample bile. This connector had a small opening in the center which was patent to allow bile to flow freely. This surgical preparation was supposed to allow bile to flow freely through the tubing and eventually return to the duodenum. The exterior connector could be removed to take a sample, then put back in place.
Note: On each slide, a non-cannulated control rat liver is included nearest the label for comparison with the cannulated diseased liver.
Gross Pathology: Livers from cannulated rats were enlarged and mottled. Livers from non-cannulated controls were normal.
Laboratory Results: The following clinical chemistry findings were noted (presented as means for groups of n=5):



Normal range
 ALT (U/L)


 29 - 65
 AST (U/L)


 75 - 118
 SDH (U/L)


 8 - 28
 GGT (U/L)


 0 - 3
 T Bili (mg/dl)


 0.1 - 0.2
 T Bile Acids (umol/L)


 13.7 - 391.2
Contributor's Diagnosis and Comments:
Moderate to severe necrotizing and proliferative cholangiohepatitis
Etiology: Biliary obstruction from surgical cannulation of the bile duct
Multiple rats were used to prepare the submission, so the degree of change present varies somewhat across the slides. In general, there is inflammation centered on portal regions characterized by infiltration of mononuclear cells and neutrophils. Inflammation extends variably into adjacent hepatic parenchyma. Biliary ducts and ductules are dilated and proliferative, with associated edema and dilation of portal lymphatics. Many biliary epithelial cells are hypertrophic and have prominent nuclei. In addition to the biliary change, there is considerable hepatic necrosis in some areas, with associated infiltration by neutrophils and other inflammatory cells. The clinical pathology data is supportive of the histopathology, and provides evidence for both hepatocellular damage as well as cholestatic disease.
Although the surgical cannulation procedure in these rats was supposed to provide free flow of bile out the tubing and back into the duodenum, we speculate that there was too much resistance in the length of tubing present and a functional obstruction resulted. Back-pressure from increased resistance likely produced primary post-hepatic cholestasis and the biliary proliferation. Associated with these events would have been secondary infection, resulting in the inflammation and necrosis.
AFIP Diagnosis:
1. Liver: Cholangiohepatitis, subacute, diffuse, moderate with biliary hyperplasia, Sprague Dawley rat, rodent.
2. Liver: Hepatitis, necrotizing, suppurative, random, multifocal, moderate, with bacilli.
3. Liver: Necrosis, coagulative, multifocal.
Conference Note: The histologic lesions in this case most likely resulted from two processes. The bile duct hyperplasia is attributed to biliary obstruction secondary to cannulation. The hepatitis and necrosis are most likely associated with a secondary ascending bacterial infection and possibly septicemia. Gram stains demonstrated large numbers of Gram negative bacilli within the areas of necrosis.
Clinical pathologic testing for hepatocellular damage is conducted by measuring the soluble cytosolic enzymes that are released into the blood with sublethal cell injury and necrosis. Alanine aminotransferase and sorbitol dehydrogenase are considered to be specific for detecting liver damage in the mouse. In this case both were elevated. Aspartate aminotransferase was also elevated; however, it is not considered to be liver specific. The elevated total bilirubin is attributed to cholestasis.

Contributor: The Procter & Gamble Company, Miami Valley Laboratories, PO Box 398707, Cincinnati, Ohio, 45239-8707.
1. Duncan JR, Prasse KW, Mahaffey EA: Veterinary Laboratory Medicine, Clinical Pathology, 3rd ed., pp. 130-151. Iowa State University Press, Ames, Iowa, 1994
2. Harada T, Enomoto A, Boorman GA, Moronpot RR: Liver and Gallbladder. In: Pathology of the Mouse, eds. Maronpot RR, Boorman GA, Gaul BW, pp. 125-126. Cashe River Press, Vienna, IL, 1999
Case III - S94/99 (AFIP 2681357)
Signalment: Guinea pig (Cavia aperea porcellus), 6-month-old, female.
History: There was high mortality in a breeding colony of a pet shop. No clinical signs were observed the day before this animal's death.
Gross Pathology: Post-mortem examination disclosed focal alopecia, hyperkeratosis, panniculitis and acute congestion of lung, liver and spleen.

Contributor's Diagnosis and Comments: Kidney: focal interstitial nephritis, focal tubular degeneration, intratubular spherical bodies; guinea pig (Cavia aperea porcellus), Cavioidea, Rodentia, Mammalia; Cause: infection with Klossiella cobayae.
Light microscopically, the animal showed a severe focal mononuclear interstitial nephritis, accompanied by lymphohistiocytic perivascular infiltrations and a focal increase of interstitial fibroblasts. Besides, focal tubular degeneration and intratubular spherical bodies of varying size were observed. According to their morphology, the latter were identified as Klossiella cobayae development stages. Additionally, focal interstitial pneumonia was diagnosed.
In general, Klossiella infection, which has up to now been demonstrated in equids, mice, bats, opossums, various rodents, boa constrictors, and guinea pigs, remains asymptomatic. Clinical and macroscopic alterations, represented by minute gray spots in the renal cortex, are only observed in animals exposed to massive stress (e.g., emaciation, transport, primary infections). Klossiella are protozoans belonging to the subclass coccidia. They have a direct life cycle. Ingestion of sporocyst leads to the release of sporozoites, which circulate and parasitize the endothelial cells of small vessels in kidney, lung, spleen, and other organs. Primary schizonts develop predominantly in glomerular endothelial cells. Merozoites invade epithelial cells of the proximal convoluted tubules where secondary schizogony takes place. Released merozoites undergo sexual stages in the loops of Henle. Fertilized gametes develop into sporonts, which bud to form sporoblasts. Each sporoblast undergoes successive division to form sporocysts containing sporozoites, which are shed with the urine.
AFIP Diagnoses:
1. Kidney, tubules: Intraepithelial and intraluminal protozoa, with multifocal mild tubular epithelial degeneration, guinea pig (Cavia aperea porcellus), rodent.
2. Kidney: Nephritis, interstitial, lymphoplasmacytic, multifocal, minimal to mild.
Conference Note: The histologic findings of multiple stages of protozoan parasites within endothelium and tubules in conjunction with the distinctive sporonts rimmed by budding sporoblasts caused conference participants to agree with the diagnosis of Klossiella cobayae. Toxoplasmosis and encephalitozoonosis were discussed, but neither has life stages similar to the sporonts of klossiellosis.

Contributor: Institut für Veterinar-Pathologie, Universität Leipzig, An den Tierkliniken 33, Leipzig, Germany 04103

1. Gardiner CH, Fayer R, Dubey JP: An Atlas of Protozoan Parasites in Animal Tissues, 2nd ed., pp. 61-62. Armed Forces Institute of Pathology, Washington, DC 1998
2. Maxie MG: The kidney. In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N, vol. 2, 4th ed., p. 518. Academic Press, San Diego, CA, 1993
3. Shadduck JA, Pakes SP: Protozoal and Metazoal Diseases. In: Pathology of Laboratory Animals, eds. Benirschke K, Garner FU, Jones TC, vol. II, p. 1609. Springer-Verlag, New York, 1978.
Case IV - 95/351 (AFIP 2698315)

Signalment: 8-year-old, male, Rottweiler dog.
History: In 1994, the dog had episodic weakness and mild, generalized seizures. The first seizure coincided with minor surgery two months before hospitalization. Immediately after surgery a generalized seizure lasting for about three minutes occurred. Diazepam was administered intravenously to alleviate the symptoms. Analysis of a blood sample revealed marked hypoglycemia (1.4 mmol/liter). Nine days later the dog was re-examined. No new seizures had been observed, but the dog was generally depressed and several episodes of weakness and muscle tremor had been noticed. These clinical signs were preceded by periods of excitement.
Blood analysis revealed hypoglycemia (1.7 mmol/liter) and hyperinsulinemia (193 mU/liter). The condition was stabilized and controlled for 46 days by means of prednisolone and a strict diet. Two days after the owner ran out of prednisolone tablets, several seizures occurred. Prednisolone treatment was reinstated and the dose was increased. No new seizures had occurred five days later. The dog was then anaesthetized and a coeliotomy was performed.
No gross abnormalities were identified in the pancreas, but palpation revealed two tiny nodules (diameter 2-3 mm) in the right lobe of the pancreas. The liver and spleen appeared normal. A firm and irregular abdominal mass, thought to be a mesenteric lymph node, was observed. This mass was excised and submitted for histological examination. Since an obvious pancreatic tumor could not be located, intravenous infusion of methylene blue was performed. After 20 to 25 minutes, tiny nodules previously not visible in the right lobe of pancreas became visible and stained reddish-blue. No other color changes in the pancreas or other organs were observed. A partial right lobe pancreatomy was performed and the tissue was sent for histological examination.

Microscopic examination of pancreas and mesenteric lymph node revealed neoplastic tissue in both samples. In the pancreas, a few lobules of tumor cells surrounded by dense fibrous tissue were found. Tumor tissue, located in the mesenteric lymph node, almost completely replaced the lymphoid tissue. Immunocytochemistry showed that some tumor cells stained positively for insulin, and a diagnosis of insulin-secreting carcinoma was made (sections from these biopsies are included, marked 40014/94, nyd 6 and nyd 1). The dog recovered quickly. Fourteen months later, in 1995, the dog suddenly died and an autopsy was performed.
Gross Pathology: The dog was in good body condition. It was dehydrated and had moderate diarrhea. Lungs were heavy, wet and dark red. The left lobe of the pancreas appeared normal. Two mesenteric lymph nodes close to the pancreas were enlarged and contained tumor tissue. The cut surface consisted entirely of grayish-white, lobular tumor tissue. Focal ½ to 1 cm diameter nodules with lobulated cut surface were found in the liver.
Laboratory Results: Immunocytochemistry using an avidin-biotin complex method with an anti-insulin antibody as primary antibody revealed positive staining of a large proportion of tumor cells (see section marked krøsln 1 Insulin ABC).

Contributor's Diagnosis and Comments: Metastatic, insulin-secreting carcinoma, localized to mesenteric lymph nodes (and liver). Other diagnoses were aspiration pneumonia (sections not included) and dehydration.

The biopsies examined in 1994 revealed tumor tissue arranged in irregular cords or in closely packed nests. The neoplastic cells were of two main types: ovoid to polyhedral cells with eosinophilic and finely granular cytoplasm, and cuboidal or columnar cells with a lightly basophilic cytoplasm and frequent small cytoplasmic vacuoles. Immunoreactivity for insulin was found in the polyhedral cells, which resembled normal pancreatic islet cells. Further examination of the insulin negative cell population was not performed. These cells may represent less differentiated B-cells or presence of another cell population. A heterogeneous cell population may be present in canine islet cell tumors, and tumor cells that are immunoreactive for other hormones such as glucagon, somatostatin, pancreatic polypeptide and gastrin have been reported in insulin-secreting tumors.

The submitted sections (material from 1995) of metastatic processes showed tumor tissue composed of a more homogenous population of oval to polyhedral cells, of which many showed immunoreactivity for insulin.
AFIP Diagnosis: Lymph node: Neuroendocrine carcinoma, metastatic, Rottweiler, canine
Conference Note: In adult animals, profound hypoglycemia is most commonly associated with hyperinsulinemia resulting from insulin secreting tumors of the pancreas. An insulin to glucose ration of >30 (insulin mUnits/dl ¸ serum glucose mg/dl) in the presence of <60mg/dl of glucose is indicative of hyperinsulinemia. Other causes of hypoglycemia include:starvation, malabsorption, exertion, systemic disease, liver disease and hypoadrenocorticism. The body utilizes glucose from three sources: ingestion, gluconeogenesis, and glycogenolysis. Glucose in the blood is either used by cells or converted into glycogen.
The histologic pattern of irregularly arranged cords forming a gyriform pattern is consistent with a neuroendocrine tumor. The differential diagnosis considered by conference participants included pancreatic islet cell tumor and other neuroendocrine carcinomas. Immunohistochemistry performed at the AFIP revealed multifocal positivity of the neoplastic cells for insulin, supporting an islet cell tumor. Ultrastructurally, characteristic angular to rhomboid "brick granules" are anticipated in pancreatic islet cell neoplasms.
Contributor: Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway
1. Duncan JR, Prasse KW, Mahaffey EA: Veterinary Laboratory Medicine, Clinical Pathology, 3rd ed., pp. 122-129, 196-197. Iowa State University Press, Ames, Iowa, 1994
2. Fingeroth JM, Smeak DD: Intravenous methylene blue infusion for intraoperative identification of pancreatic islet-cell tumors in dogs. Part II: Clinical trials and results in four dogs. J of the Amer Anim Hosp Assoc 24:175-182, 1988
3. Hawkins KL, Summers BA, Kuhajda FP, Smith CA: Immunocytochemistry of normal pancreatic islets and spontaneous islet cell tumors in dogs. Vet Pathol 24:170-179, 1987
4. O'Brien TD, Hayden DW, O'Leary TP, Caywood DD, Johnson KH. Canine pancreatic endocrine tumors: Immunohistochemical analysis of hormone content and amyloid. Vet Pathol 24:308-314, 1987
5. Thoresen SI, Aleksandersen M, Lønaas L, Bredal WP, Grøndalen J, Berthelsen K: Pancreatic insulin-secreting carcinoma in a dog: Fructosamine for determining persistent hypoglycemia. J Small Animal Pract 36:282-286, 1995.
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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