AFIP Wednesday Slide Conference - No. 23
March 10, 1999

Conference Moderator:
MAJ (P) Mark Mense
Walter Reed Army Institute of Research
Division of Pathology
Washington, D.C. 20307-5100
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Case I - 12A (AFIP 2658211)

Signalment: 17-year-old, male, Japanese macaque (Macaca fuscata).
History: This monkey was from a large, free-ranging colony located in south Texas (Dilly, Texas). The animal was incontinent, paretic, and paralyzed in the hind limbs. Three of four limbs were edematous. The animal was euthanized for humane reasons.
Gross Pathology: Externally, there was massive subcutaneous edema over the ventral body, legs, and scrotum. Internally, there were marked hydropericardium and ascites. The lungs were multifocally abscessed and infarcted. There were tan to yellow, 2 mm, slightly raised, miliary foci in the lungs, liver, spleen, and kidneys. Gross diagnoses were congestive heart failure, ascites, hydropericardium, and multiple organ abscesses.
Laboratory Results: None.
Contributor's Diagnosis and Comments: Heart: Pericarditis and epicarditis, pyogranulomatous, diffuse, severe, with fungal spherules, Japanese macaque (Macaca fuscata), primate. Etiology consistent with Coccidioides immitis.
In the submitted section of heart, the pericardium is greatly expanded by pyogranulomatous inflammation and necrotic debris that extend through the epicardium. The inflammation is composed of degenerate neutrophils, macrophages, and multinucleated foreign body giant cells, with fewer lymphocytes and plasma cells. Multifocally, these form small, well to poorly organized, discrete granulomas. Within giant cells and free in the necrotic milieu, there are 15 to 45mm diameter spherules with a 4 to 5mm thick, birefringent wall, containing flocculent basophilic granular material (immature stages) or 5 to 7mm diameter endospores (mature endosporulating stages). Other spherules are fragmented and degenerate. Similar yeast organisms were found within pyogranulomatous foci in the lung, spleen, pancreas, lymph nodes, and liver.
Coccidioides immitis is a dimorphic fungus known to infect humans, wild and domestic animals, nonhuman primates, rodents, and exotic animals in the southwestern United States, northern Mexico, and in endemic areas of Central and South America. Inhalation of fungal spores is the only proven mode of natural infection, although direct transmission has been implicated. Fatal coccidioidomycosis has been diagnosed in several nonhuman primate species including the rhesus, bonnet macaque, Japanese macaque, chimpanzee, sooty mangabey, gorilla, and baboons.
Endosporulating mature spherules contain numerous uninucleate endospores and can measure more than 30mm in diameter. Immature spherules contain granular material. Differential diagnosis includes Rhinosporidium seeberi, Chrysosporium parvum var. crescens, Cryptococcus neoformans, Paracoccidioides brasiliensis, Histoplasma capsulatum, Emmonsia parva, and Blastomyces dermatitidis, but usually these can easily be eliminated from diagnostic consideration by histologic features, anatomic location, and host reaction. Complement fixation, immunodiffusion, and fluorescent antibody tests diagnostic for C. immitis are available and may be very useful in difficult cases.
4x obj.
Case 23-1. Heart. The epicardium is markedly thickened by fibrosis and a superficial exudate composed of caseous necrotic debris, a dense cellular infiltrate, with multinucleate giant cells and scattered 50u diameter yeast spherules.
20x obj.
Case 23-1. Epicardial exudate. Immature spherules have a hyaline eosinophilic capsule and contain granular basophilic material. Mature spherules contain abundant 2u diameter endospores. Spherules are surrounded by numerous neutrophils, fewer macrophages, multinucleate foreign body giant cells, lymphocytes and plasma cells.
AFIP Diagnoses:
1. Heart, epicardium: Granuloma, with mature and immature fungal spherules, Japanese macaque (Macaca fuscata), nonhuman primate, etiology consistent with Coccidioides immitis.
2. Heart: Fibrosis, interstitial, multifocal, mild, with multifocal myofiber atrophy and karyomegaly.
Conference Note: Occurrence of coccidioidomycosis in animals and humans is typically associated with arid or semi-arid environments, such as the southwestern United States and parts of Mexico. In humans, the disease is commonly referred to as San Joaquin Valley Fever. Animals most commonly infected are dogs, horses, and feedlot cattle. The infection in cattle is often subclinical and restricted to the lungs.
Interestingly, a case of pulmonary coccidioidomycosis has been recently reported in a stranded bottlenose dolphin from the southern California coast. While coccidioidomycosis has been previously described in marine mammals such as sea otters and California sea lions, the infection in the bottlenose dolphin is unique because it is the first report of coccidioidomycosis in a purely aquatic, free-ranging marine mammal. Coccidiodes immitis can be carried long distances by the wind, and can survive in saline soil and sea water. Bottlenose dolphins venture close to the California shore, and it was speculated that an offshore wind may have carried infectious arthrospores to the dolphin from an endemic area in California.
Mild myocardial fibrosis and karyomegaly of myocardial fibers are common findings in aged macaques and are considered incidental in this case.
Contributor: Southwest Foundation for Biomedical Research, Air Force Research Laboratory, 2509 Kennedy Circle, Brooks Air Force Base, Texas 78235.
1. Bellini S, Hubbard G, Kaufman L: Spontaneous fatal coccidioidomycosis in a native-born hybrid baboon (Papio cynocephalus anubis/Papio cynocephalus cynocephalus). Lab Anim Sci 41:509-511, 1991.
2. Johnson HJ, et al.: Disseminated coccidioidomycosis in a mandrill baboon (Mandrillus sphinx): A case report. J Zoo Wildl Med 29:208-213, 1998.
3. Reidarson TH, Griner LA, Pappagianis D, McBain J: Coccidioidomycosis in a bottlenose dolphin. J Wildl Dis 34:629-631, 1998.
4. Ziemer EL, et al.: Coccidioidomycosis in horses: 15 cases. J Amer Vet Med Assoc 201:910-916, 1992.
5. Samuelson J: Infectious diseases. In: Robbins Pathologic Basis of Disease, Cotran RS, Kumar V, Collins T, eds., 6th ed., page 353, WB Saunders, Philadelphia, PA, 1999.

Case II - NADC MVP-2 (AFIP 2638852)

Signalment: Adult, female, white-tailed deer (Odocoileus virginianus).
History: This deer was from a herd of 700 captive white-tailed deer on a hunting preserve that was depopulated due to Mycobacterium bovis infection. All deer in the herd were in good flesh. No clinical signs had been noted.
Gross Pathology: Medial retropharyngeal lymph nodes were enlarged to 3 to 4 cm in diameter. On cut surface there was caseous necrosis and mineralization surrounded by fibrous connective tissue and more normal lymphoid tissue.
Laboratory Results: Mycobacterium bovis was cultured from samples of the affected lymph nodes. PCR of formalin-fixed, paraffin-embedded sections to detect an insertion sequence specific to M. tuberculosis complex mycobacteria also confirmed the presence of M. bovis.
Contributor's Diagnosis and Comments: Lymph node: Granulomatous lymphadenitis, multifocal to coalescing, with caseous necrosis, mineralization, and peripheral fibrosis.
Mycobacterium bovis, the causative agent of tuberculosis in cattle, is also the cause of tuberculosis in Cervidae. Captive as well as free-ranging Cervidae have been diagnosed with M. bovis infection. A recent outbreak of M. bovis infection in wild white-tailed deer in Michigan represents the first known wild animal reservoir of M. bovis in North America. White-tailed deer come into close contact with cattle and have been seen to share feeding and watering sites. Other wild animal reservoirs for M. bovis include the brushtail possum (Trichosurus vulpecula) in New Zealand and the badger (Meles meles) in the United Kingdom. Wildlife reservoirs of M. bovis represent a serious threat to efforts to eradicate tuberculosis from domestic livestock. Possums and badgers have been documented as a source of infection in domestic cattle herds grazing pastures where infected possums and badgers reside.

Tuberculosis in some species of Cervidae have been found to have lesions morphologically distinct from typical bovine lesions. Elk and red deer (Cervus elaphus) have peripheral mineralization of granulomas rather than the central mineralization often seen in cattle. Lesions in elk and red deer often have more neutrophils and fewer giant cells than lesions seen in cattle. Fallow deer (Dama dama) generally have more giant cells than bovine lesions, but are otherwise indistinguishable from bovine lesions. Sika deer (Cervus nippon) have more giant cells which are larger with more nuclei than giant cells seen in bovine lesions. Lesions in white-tailed deer have been described as typical of lesions seen in cattle. Medial retropharyngeal lymph nodes have been found to be the most common site to contain lesions in white-tailed deer.
4x obj.
Case 23-2. Lymph node. There is a caseating granuloma displacing cortical lymphocytes which contains foci of mineralization and giant cells.
20x obj.
Case 23-2. Lymph node. Partly mineralized caseous necrotic debris is surrounded by multiple foreign body giant cells, epithelioid macrophages, lymphocytes, and plasma cells (tubercle).
AFIP Diagnosis: Lymph node: Granuloma(s), caseo-calcareous, white-tailed deer (Odocoileus virginianus), cervid.
Conference Note: There is marked variability of the examined sections. Some slides contain single to multiple, small, discrete granulomas within clearly identifiable sections of lymph node. In other slides, the lymph node is enlarged and the parenchyma is almost entirely replaced by a single, large, caseo-calcareous granuloma; remnant lymphoid elements are present at the periphery of the lymph node. Staining by Fite's method for acid-fast bacteria performed at the AFIP demonstrated rare, acid-fast bacilli within macrophages.
Mycobacteria are aerobic, nonmotile, non-spore forming bacilli characterized by a waxy coat that retains red dye when subjected to acid in acid-fast stains. The pathogenicity of M. tuberculosis infection has been attributed to several components in the bacterial cell wall that allow the organism to escape killing by macrophages and induce delayed type hypersensitivity. Virulent strains of M. tuberculosis possess cord factor, a glycolipid on the surface of the bacterium. In mice, injection of purified cord factor induces granuloma formation. Lipoarabinomannan, a polysaccharide similar in structure to that of endotoxin in Gram-negative bacteria, inhibits macrophage activation by interferon-g, induces macrophage secretion of TNF-a, causing fever and weight loss, and causes secretion of IL-10, which suppresses mycobacteria induced T-cell proliferation. Complement, activated on the surface of bacilli, serves to opsonize the organisms and facilitate their uptake into macrophages. In addition to the virulence factors associated with the cell wall, mycobacteria reside in phagosomes that fail to become acidified. Lack of acidification of lysosomes has been associated with urease secreted by mycobacteria, and with phagocytosis of bacteria via complement or mannose binding receptors rather than Fc receptors.
Contributor: National Animal Disease Center, 2300 Dayton Road, Ames, Iowa 50010.
1. Rhyan JC, Saari DA: A comparative study of the histopathologic features of bovine tuberculosis in cattle, fallow deer (Dama dama), Sika deer (Cervus nippon), and Red deer (Cervus elaphus). Vet Pathol 32:215-220, 1995.
2. Schmitt SM, Fitzegerald SD, Cooley TM, et al.: Bovine tuberculosis in free-ranging white-tailed deer from Michigan. J Wild Dis 17:749-758, 1997.
3. Miller J, Jenny A, Rhyan J, et al.: Detection of Mycobacterium bovis in formalin-fixed, paraffin-embedded tissues of cattle and elk by PCR amplification of an IS6110 sequence specific for Mycobacterium tuberculosis complex organisms. J Vet Diag Lab Inv 9:244-249, 1997.
4. Krebs JR, Anderson RM, Clutton-Brock T, et al.: Badgers and bovine TB: Conflicts between conservation and health. Science 279:817-818, 1998.
5. Morris RS, Pfeiffer DU: Directions and issues in bovine tuberculosis epidemiology and control in New Zealand. NZ Vet J 43:256-265, 1995.
6. Jackson R, Cooke MM, Coleman JD, et al.: Naturally occurring tuberculosis caused by Mycobacterium bovis in brushtail possums (Trichosurus vulpecula): III. Routes of transmission and excretion. NZ Vet J 43:322-327, 1995.
7. Samuelson J: Infectious diseases. In: Robbins Pathologic Basis of Disease, Cotran RS, Kumar V, Collins T, eds., 6th ed., pp. 349-352, WB Saunders, Philadelphia, 1999.

Case III - 7-78-96 (AFIP 2657523)

Signalment: One-month-old greater rhea (Rhea americana).
History: Three juvenile (one to three-month-old) rheas from a flock of 200 birds died with leg weakness and lethargy.
Gross Pathology: The necropsy was performed by the submitting veterinarian. Within the liver there were disseminated, pale, friable foci up to 3 mm in diameter. The liver was enlarged, pale, and firm.
Laboratory Results:
1. Liver selenium: 3.64 mg/g dry weight.
2. Liver Vit E: 28.92 mg/g dry weight.
3. Liver: Mg 184 ppm, Cu 3.67 ppm, Zn 154 ppm, Mn 1.99 ppm, Cd <0.10 ppm, and Mo 0.523 ppm.
Contributor's Diagnosis and Comments:
1. Liver: Granulomata, heterophilic, multiple.
2. Liver: Amyloidosis, multifocal and coalescing, marked.
A Gram-negative, curved rod was isolated from the liver. The isolate was identified as Campylobacter coli, an enteric pathogen in ratites. Systemic infection with associated hepatic lesions and encephalitis attributed to Campylobacter jejuni has been previously documented in juvenile ostriches. Lesions include focal hepatic necrosis, ascites, hydropericardium, and swollen kidneys. The liver copper level was below normal range in this case, and may have been contributory. Hepatic amyloid deposition is commonly encountered in birds with chronic and active inflammatory processes.
10x obj.
Case 23-3. Liver. A granuloma composed of necrotic debris surrounded by waxy eosinophilic material (amyloid) and degenerate inflammatory cells replaces hepatic parenchyma.
Congo red, 20x obj.
Case 23-3. Liver. Hepatic plates are separated by amorphous deposits of red staining material (amyloid) and low numbers of lymphocytes.
AFIP Diagnosis: Liver: Granulomas, heterophilic, with multifocally extensive amyloidosis, greater rhea (Rhea americana), avian.
Conference Note: Conference participants considered a variety of bacteria as potential causes of the hepatic lesions observed in this greater rhea. Infection with Mycobacterium avium was the primary consideration in the differential diagnosis of most attendees. Escherichia coli, Salmonella sp., and Campylobacter sp. were also mentioned. Ziehl-Neelsen and Fite's acid-fast stains performed at the AFIP did not demonstrate acid-fast bacteria. Additionally, tissue Gram stains, the Warthin-Starry method and Steiner's method did not demonstrate bacteria. A Congo red stain confirmed the presence of amyloid.
A variety of pathogenic bacteria may cause similar histologic lesions in the livers of birds. While conference participants agreed that the hepatic lesions and culture results in this rhea are consistent with campylobacteriosis, some were reluctant to definitively attribute the changes to Campylobacter sp. without other evidence of its presence within the liver. Bacterial culture results are most reliable when interpreted in context with histopathologic findings and observation of bacteria within lesions. Techniques such as in situ hybridization and immunohistochemistry are often very useful in demonstrating infectious agents in lesions that contain few microorganisms.

Contributor: Montana Veterinary Diagnostic Laboratory, PO Box 997, Bozeman, Montana 59771.
1. Jensen J, Johnson, JH, Weiner ST: Husbandry and medical management of ostriches, emus and rheas. Wildlife and Exotic Animal TeleConsultants, 1992.
2. Perelman JB: Campylobacteriosis. In: Proceedings of Third Annual Ostrich Conference: Ostrich Medicine and Surgery for Veterinarians, College of Veterinary Medicine, Texas A&M University, 1991.
3. Post K, Ayers JR, Gilmore WC, Raleigh RH: Campylobacter jejuni isolated from ratites. J Vet Diagn Invest 4:345-437, 1992.
4. Boukraa L, Messier S, Robinson Y: Isolation of Campylobacter from livers of broiler chickens with and without necrotic hepatitis lesions. Avian Dis 35:714-717, 1991.
5. Oyarzabal OA, Conner DE, Hoer FJ: Incidence of campylobacters in the intestine of avian species in Alabama. Avian Dis 39:147-151, 1995.

Case IV - CID (AFIP 2658212)

Signalment: Adult feral pig.
History: Tissue was collected from one of several pigs slaughtered at a local meat processing plant. The pigs were heavily infested with various metazoan parasites, including lungworms, roundworms, tapeworms, and acanthocephalids.
Case 23-4. Liver. Hepatic parenchyma is discolored by numerous 2-3cm white foci. Occasionally thin walled white cysts elevate the hepatic serosa.
Case 23-4. Kidneys, perirenal fat, ureters. Several 1-2cm cysts are scattered throughout the perirenal fat. A ruptured cyst contains a creamy brown exudate.
Gross Pathology: Multifocal and coalescing, firm, nodular areas were observed markedly thickening the loose fibrous connective tissue surrounding the renal hilus and ureters. When incised, the fibrotic nodules were abscessed and contained encysted adult nematodes 2 to 4 cm in length. Multifocal, white, irregularly round to linear areas of fibrosis were present in the subcapsular and interstitial tissue of the liver.
Contributor's Diagnosis and Comments: Perirenal and periureteral connective tissue: Granulomas, eosinophilic, multifocal, with intralesional adult nematodes and eggs, etiology consistent with Stephanurus dentatus.
Infestation with Stephanurus dentatus, the kidney worm of swine, is a common problem of feral swine in most tropical and subtropical climates. Clinical findings in affected animals vary with the magnitude of infection and location of migrating larvae, ranging from stunted growth to emaciation, ascites, rear limb lameness, and posterior paresis. Pigs are initially infected with the 3rd stage larvae via ingestion, skin penetration, or prenatally. Ingested larvae migrate through the portal circulation or lymphatics to the liver, while those acquired cutaneously pass to the lungs prior to reaching the liver via the systemic circulation. Grossly, S. dentatus migration through the liver can often be distinguished from that of Ascaris suum by the severe inflammation and tract-like, irregular pattern of fibrosis produced by the former. Severe hepatitis leading to cirrhosis and ascites can occur. Larvae in the liver are destroyed, encapsulated or eventually break through the hepatic capsule, migrating to the preferred perirenal and mesenteric tissue sites. Other lesions ascribed to aberrant larval migration of S. dentatus in pigs include portal phlebitis, pancreatitis, splenitis, lymphadenitis, myositis, and myelitis leading to paralysis.
The encysted perirenal larvae develop into adults. Perirenal cysts typically contain a pair of worms surrounded by inflammatory cells and a dense fibrous capsule. The cysts communicate with the ureter allowing large numbers of eggs to pass freely in the urine of the host. The eggs develop into the infective L3 larval stage approximately four days later, completing the life cycle. Larvae can survive in the appropriate environment for up to five months. Once infected, up to nine months may be required to establish a patent infection in the host animal.
Features which help to identify the adult worms in these sections as true strongyles include the platymyarian, meromyarian musculature, prominent lateral chords, a pseudocoelom, and a relatively large intestine composed of few, multinucleated cells which display a thick eosinophilic microvillar border ("strongyle gut"). Thin-walled, morulated eggs can be seen in the reproductive tract and free in surrounding tissue in most sections.
2x obj.
Case 23-4. Ureter, perirenal fat. There is a large granuloma within the periureteral fat which contains a cross section of a nematode parasite.
2x obj.
Case 23-4. Parasitic granuloma. Three cross-sections of a nematode parasite contain profiles of ovaries and multiple cross-sections of nematode digestive tract.
AFIP Diagnosis: Fibrous and adipose tissue, periureteral (per contributor): Eosinophilic granulomas, multiple, with globule leukocytes, and adult nematodes and eggs, feral pig, porcine, etiology consistent with Stephanurus dentatus.
Conference Note: The swine kidney worm measures 20-40 mm in length and is found principally in perirenal fat and adjacent tissues. The nematode is especially common in the southern United States. Earthworms can serve as transport hosts. As described by the contributor, extensive migration by the parasite may produce widespread tissue damage and a variety of clinical signs. Stephanurus dentatus has been found in various organs and tissues including the kidneys, lumbar muscles, heart, lungs, pleural cavity, spleen, and spinal canal. Differential diagnosis considered by conference participants included Dioctophyma renale, the giant kidney worm of several animal species including swine, and aberrant migration of Ascaris suum.
Contributor: Wilford Hall Medical Center, 59th MDW/MSR, 1255 Wilford Hall Loop, Lackland Air Force Base, Texas 78236.
1. Blood DC, Henderson JA, Radostits OM: Diseases caused by helminth parasites. In: Veterinary Medicine, 5th ed., pp. 780-782, Lea & Febiger, Philadelphia, 1979.
2. Soulsby EJL: Class: Nematoda. In: Helminths, Arthropods, and Protozoa of Domesticated Animals, 7th ed., pp. 193-195, Lea & Febiger, Philadelphia, 1982.
3. Corwin, DiMarco, McDowell, Pratt: Internal parasites. In: Diseases of Swine, Leman AD, ed., 6th ed., pp. 658-659, Iowa State Univ. Press, Ames, IA, 1986.
4. Maxie GM: The urinary system. In: Pathology of Domestic Animals, Jubb KVF, Kennedy PC, Palmer N, eds., 4th ed., vol. 2, pp. 516-517, Academic Press, 1993.
5. Jones TC, RD Hunt, NW King: Diseases caused by parasitic helminths and arthropods. In: Veterinary Pathology, 6th edition, page 648, Williams and Wilkins, Baltimore, MD, 1997.
Ed Stevens, DVM
Captain, United States Army
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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