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CASE I – Case 1 (AFIP 2741233)
Signalment: Young, adult male, olive baboon (Papio cyanocephalus anubis)
History: Incidental findings in a wild-caught baboon in Kenya.
Gross Pathology: The cecum and colon contained many blue-black, round, raised mucosal and serosal foci that were approximately 4 mm in diameter.
Laboratory Results: None reported.
Contributor’s Diagnosis and Comment: In the colonic submucosa, large hemorrhagic granulomas are composed of hemosiderin-laden macrophages, lymphocytes, plasma cells, multinucleated giant cells and a mixture of neutrophils and eosinophils. The centers of the granulomas contain small areas of mineralization, an eosinophilic granular material and segments of nematode larvae. The parasites have meromyarian-platymyarian muscle and contain few intestinal cells in cross sections with dense microvillar layer.
Within the lamina propria, and occasionally in the submucosa, multiple elongated, ovoid eggs are present. The egg wall is pale yellow, appears to contain a lateral spine, and stains acid fast. Many of the eggs appear to be within slightly dilated capillaries. The cellular reaction is primarily composed of lymphocytes and plasma cells, admixed with occasional multinucleated giant cells. There are small foci of mineralization in the mucosa, and the deepest portion of the lamina propria is infiltrated by hemosiderin-laden macrophages.
Diagnosis: Colitis, granulomatous with intralesional larvae of Oesophagostomum spp. and eggs of Schistosoma mansoni.
This case illustrates two common, naturally occurring, parasitic infestations of baboons in Kenya. In a recent survey of wild-trapped olive baboons, 76% of animals were positive by fecal examination for Oesophagostomum spp. In another survey, 40% of baboons in an endemic area had positive fecal samples for Schistosoma mansoni. In oesophagostomiasis, the characteristic nodular lesions of the large intestine appear as a result of intestinal penetration by larvae.
In contrast to the direct life cycle of the nematode parasite Oesophagostomum, blood flukes such as schistosomes require an intermediate host for their development, typically a species-specific snail. The trematode ovum releases a miracidium that penetrates the snail and matures into infective cercaria. Cutaneous lesions in the final host are due to penetration of the skin by cercarial schistosomes. Schistosomes migrate into the peripheral vasculature via the lung and portal vein and develop into adults in the portal or pelvic venous system. Large numbers of eggs are produced daily which elicit granulomatous and fibrosing lesions in the lungs, liver and gut.
AFIP Diagnoses: 1. Colon, tunica muscularis and submucosa: Granulomas, with hemorrhage, hemosiderin, hematoidin, and few nematode parasites, etiology consistent with Oesophagostomum sp., olive baboon (Papio cyanocephalus anubis), nonhuman primate.
2. Colon, mucosa and submucosa: Colitis, granulomatous, multifocal, mild, with schistosome eggs, etiology consistent with Schistosoma mansoni.
Conference Comment: Infection of monkeys with nematodes in the genus Oesophagostomum, the most common nematode parasite of Old World monkeys and great apes, is usually asymptomatic and goes unrecognized. Lesions seen at necropsy are, most frequently, granulomas on the serosal surface of the colon and cecum and in the mesentery, but can be found elsewhere in the body because of aberrant larval migration. Rupture of the serosal nodules may result in acute or chronic peritonitis. The mild, chronic serositis, present in some sections that were examined in conference, may represent such a reaction.
Schistosoma mansoni has been reported to infect New World and Old World monkeys and the great apes, but again, is usually an incidental finding at necropsy. Pathologic effects are principally due to the presence of eggs within the tissues. The prominent lateral spine on the eggs of S. mansoni is believed to aid in the penetration of eggs from the vasculature into the intestinal or urinary bladder lumen where they may be shed. Hepatic granulomas surrounding schistosome eggs are one of the key lesions of a patent S. mansoni infection. High tissue eosinophilia and multinucleate giant cell formation are characteristic responses of man and the baboon, a proposed animal model, to the presence of eggs. This inflammatory response is thought to be important in bringing about the destruction of schistosome eggs and subsequent resolution of the egg granuloma without fibrosis. Antibody-mediated mechanisms and T-helper 1 and T-helper 2-associated cytokine production are thought to aid in the development of resistance to reinfection.
Contributor: Novartis Pharmaceuticals Corporation, 59 Route 10, Building 406/248, East Hanover, NJ 07901
References: 1. Farah IO, Nyindo M, King CL, Hau J: Hepatic granulomatous response to schistosoma mansoni eggs in BALB/c mice and olive baboons. J Comp Pathol 123(1):7-14, 2000
2. Toft JD, Eberhard ML: Nematodes and trematodes in parasitic diseases. In: Nonhuman Primates in Biomedical Research: Diseases, ed. Bennett BT, Abee CR, Henrickson R, pp. 133-166. Academic Press, New York, 1998
3. Munene E, Otsyula M, Mbaabu DAN, Mutahi WT, Muriuki SMK, Muchemi GM: Helminth and protozoan gastrointestinal tract parasites in captive and wild-trapped African non-human primates. Vet Parasitol 78:195-201, 1998
4. Nyindo M, Farah IO: The baboon as a non-human primate model of human Schistosome infection. Parasitol Today 15:478-482, 1999
CASE II – X6456 (AFIP 2744078)
Signalment: This is a 45-year-old female Asian elephant, Elephas maximus indicus.
History: This elephant was living with at least twelve other retired circus elephants and was euthanized because of intractable foot disease that no longer responded to medication. She was given a lethal dose of M-99. Serum and whole blood collected in EDTA were obtained. This elephant was deemed to be Mycobacterium tuberculosis complex negative from trunk wash specimens obtained four months earlier.
Gross Pathology: 1. Right bronchial lymphadenopathy, rule out large solid granuloma
2. Bronchial and tracheal lymphadenopathy, with multiple granulomas
3. Pulmonary caseous nodules, right lung
4. Marked cystic endometrial hyperplasia
5. Chronic bursitis, olecranon, extensive
Laboratory Results: Lung lesions were acid-fast bacilli positive and M. tuberculosis was isolated by culture.
Contributor’s Diagnosis and Comment: Lung with bronchus, bronchopneumonia, granulomatous, tuberculous.
Etiology: Mycobacterium tuberculosis
This elephant was euthanized because of extensive, chronic, dissecting, and necrotizing lesions of the right front foot that had been progressive for four years despite medical treatment. The disease process became intractable and no longer responded well to surgical and antibiotic therapy and the elephant was no longer responding to pain management. A chronic, organizing bursitis of the left olecranon bursa was also cause for chronic front-end lameness.
At necropsy, a significant finding, although at this stage not life-threatening, was progressive tuberculous lesions in the upper region of the right lung lobe with extension into the bronchi and trachea. There was rather extensive involvement of the bronchial lymph node and lesser changes in the peritracheal nodes. The lesions in the pulmonary parenchyma were caseous with minimal liquefaction and minimal calcareous foci. Spreading, proliferative, epithelioid granulomas extended into the surrounding parenchyma and into the bronchi and trachea indicative of open lesions that were most likely shedding organisms into the upper respiratory tract.
Cultures were submitted to the University of Florida Diagnostic Laboratory for mycobacterial and other bacterial isolation. Lesions were initially confirmed to be smear-positive for acid-fast bacilli. Sparse numbers of acid-fast bacilli were evident in tissue sections of pulmonary tissue, airway lesions and lymph nodes. Mycobacteria isolated from lung and lymph node lesions were sent to the National Veterinary Services Laboratory and confirmed to be M. tuberculosis by standard culture methods.
Spoligotyping, provided by the National Animal Disease Center (NADC), showed evidence of M. tuberculosis, but not M. bovis, in paraffin-embedded tissues. Of the 18 isolates recovered from infected Asian elephants in North America since 1996, there were 4 different restriction fragment length polymorphism (RFLP) types identified. When the same types are identified between two or more cases, it usually suggests a common source of exposure. RFLP testing at NADC showed a pattern that differed from the other 3 strains, indicating another source for this elephant’s infection.
AFIP Diagnosis: Lung: Granulomas, caseous and epithelioid, multifocal, with granulomatous and ulcerative bronchiolitis, Asian elephant (Elephas maximus indicus), Proboscidea.
Conference Comment: Infection with M. tuberculosis or M. bovis has not been reported in non-domesticated Asian or African elephants. Close or prolonged contact with actively infected humans is considered the most likely source of exposure once these animals are brought into captivity. Specific clinical signs of tuberculosis are often absent in elephants until advanced disease exists.
Currently, captive elephants are tested, by means of trunk washes conducted on 3 consecutive days, on an annual basis as part of the Guidelines for the Control of Tuberculosis in Elephants, developed by the National Tuberculosis Working Group for Zoo and Wildlife Species in the USA. Culture of respiratory secretions obtained from these trunk lavages is considered the most reliable test for antemortem diagnosis of tuberculosis in elephants. A nucleic acid amplification technique has been developed as an additional test, displaying moderate sensitivity and high specificity, for use on trunk wash samples.
Spoligotyping is a recently developed form of polymerase chain reaction that has been adapted to differentiate M. tuberculosis from other mycobacteria in formalin-fixed tissues. Acid-fast tubercle bacilli are typically rare in histological sections, but are usually found in central areas of caseation in the lungs.
RFLP typing can lead to important epidemiological information and allow trace back of cases. When the same RFLP types are identified in 2 or more cases, it usually suggests a common source of exposure. The unique RFLP type of this case made identification of the source of exposure more difficult.
Contributor: Smithsonian National Zoological Park, Department of Pathology, 3001 Connecticut Avenue NW, Washington, DC 20008
References: 1. Binkley M: Tuberculosis in captive elephants. In: Proceedings of the American Association of Zoo Veterinarians, pp. 116-119. Houston, TX, 1997
2. Michalak K, Austin C, Diesel S, Bacon JM, Zimmerman P, Maslow JN: Mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants. Emerg Inf Dis 4(2):283-287, 1998
3. Mikota SK, Larsen RS, Montali RJ: Tuberculosis in elephants in North America. Zoobiol [In press, May, 2000]
4. Montali RJ, Mikota SK, Cheng LI: Mycobacterium tuberculosis in zoo and wildlife species. Scien Tech Rev, International Office of Epizootics (OIE), [In press, July, 2000]
CASE III – 00F0168 (AFIP 2741052)
Signalment: Immature (2.5 cm fork length), sex undetermined, goldfish, Carassius auratus
History: This fish was selected from a group of young fantail goldfish, for sale at a local pet shop, as part of a parasitological survey.
Gross Pathology: Externally, the fish exhibited prominent asymmetrical abdominal swelling that was characterized by scoliosis and bulging of the caudal 2/3 of the left body wall. Internally, the caudal or trunk kidney was grossly enlarged and occupied the bulk of the abdominal cavity. It displaced the swim bladder and viscera cranially and to the right. Incision of the kidney revealed multiple fluid-filled cysts up to 1 mm in size.
Laboratory Results: None provided.
Contributor’s Diagnosis and Comment: Kidney (trunk), tubular epithelial hypertrophy and hyperplasia, with papillary cystic dilatation and intracellular protozoal trophozoites.
Gross and microscopic changes were consistent with infection by the myxosporan parasite Hoferellus carassii (Mitraspora cyprini). Hoferellus carassii is a common parasite of goldfish (Carassius auratus) and has been reported in other members of the genus Carassius. Hoferellosis is known by several common names, including kidney bloater, kidney enlargement disease and polycystic kidney disease of goldfish, due to its ability to induce proliferative changes in renal tubular epithelial cells leading to massive polycystic enlargement of the excretory kidney. Changes may be either uni- or bilateral. Described in Japan as “kidney enlargement disease” (KED), the organism was misidentified as Mitraspora cyprini. The genus Mitraspora is now considered a junior synonym of the genus Hoferellus, belonging to the family Sphaerosporidae. The parasite was first reported in the United States in 1984.
The life cycle of H. carassii has only recently been elucidated and has been demonstrated, as with other myxosporan parasites of fish, to include the causative agents of whirling disease (Myxobolus cerebralis) and proliferative gill disease of catfish (Aurantiactinomyxon spp.), to involve a two-host life cycle. The life stage infective to goldfish develops within the intestinal epithelium of a tubificid oligochaete (Phylum: Annelida). Actinosporea, once considered a separate group of parasites, are released from the oligochaete in the form of an aurantiactinomyxon spore. Following infection of the goldfish kidney, trophozoites develop within the cytoplasm of renal tubules and are then released into the tubular lumen. Plasmodia and spores appear to mature preferentially within the lumen of the ureters and bladder. Ultimately, myxosporan spores are released via the urine into the water to infect oligochaetes and complete the life cycle.
Most authors consider the parasite to follow a seasonal pattern of infection and development in the goldfish. Initial infection occurs in the spring and early summer. By fall, essentially every epithelial cell within an infected tubule will contain a trophozoite within its apical cytoplasm. Development of the 6.5–7.2 m m, round trophozoites corresponds to the period of tubular hypertrophy and proliferation. Tubular lumens become progressively dilated and lined by hypertrophic columnar cells. The epithelium itself may eventually develop papillary folds. Inflammation is not a feature of the infection, although granulomas are present in some sections, presumably arising from effete tubules. Interstitial hematopoietic tissues may become compacted, but are otherwise unaffected. At the time of their release into the tubular lumen, trophozoites are composed of four cells having a cell within a cell structure. The primary and secondary cells form the plasmodium and eventually degenerate to release the spores. Spores form within tertiary cells, while sporoblasts develop from quaternary cells. During the winter, multicellular plasmodia are present in the bladder lumen and spore formation is initiated in the spring. A single plasmodium may produce 20 or more spores. Throughout the cycle, additional trophozoites continue to mature within the tubular epithelium.
Myxosporan spores released from plasmodia average 18.56 x 12.21 m m, have a rounded anterior apex, prominent suture line dividing the shell into two symmetrical valves, and longitudinal ridges that extend into brush-like caudal filaments. Two pyriform polar capsules, oriented 45° to the suture line, are present at the anterior end. The aurantiactinomyxon spore, found within the oligochaete and infective to the goldfish, possesses a typical tri-radial morphology formed by three shell valves that form long rounded processes. The bases of the shell valves form a triangular episome with three polar capsules and as many as 22 amoeboid germ cells.
Infections acquired in the spring usually become grossly apparent by summer’s end and most mortality occurs the following spring. Affected fish initially behave and feed normally, but show progressive abdominal enlargement and difficulty swimming. Infected fish may survive for months, but the condition is ultimately fatal. There are no proven effective treatments. Disinfecting ponds and replacing stock with disease free fish may prevent recurrence. Ponds with mud bottoms and excessive buildup of detritus are most conducive to the presence of oligochaete intermediates.
AFIP Diagnosis: Trunk kidney: Tubular hypertrophy and epithelial hyperplasia, diffuse, moderate, with numerous apical cytoplasmic protozoa, and few granulomas, goldfish (Carassius auratus), piscine.
Conference Comment: The contributor provided a detailed discussion of the life cycle and histopathological changes induced by Hoferellus carassii. Early stages of the trophozoites within renal epithelial cells have been shown to induce tubular hypertrophy and epithelial hyperplasia. It is not known, however, whether the cystic transformation and enlargement of the kidney is a direct effect of tubular epithelial proliferation, or if cysts form secondary to mechanical blockage of efferent tubules and obstruction of the outflow of excreted fluid.
Contributor: Louisiana State University, School of Veterinary Medicine, Department of Veterinary Pathology, Baton Rouge, LA 70803
References: 1. Gardiner CH, Fayer R, Dubey JP: In: An Atlas of Protozoan Parasites in Animal Tissues, 2d ed., pp. 53-56, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, DC, 1998
2. Noga E: In: Fish Disease: Diagnosis and Treatment, pp 181-182. Mosby-Year Book, Inc, St. Louis, MO, 2000
3. Trouillier A, El-Matabouli M, Hoffman R: A new look at the life-cycle of Hoferellus carassii in the goldfish (Carassius auratus auratus) and its relation to “kidney enlargement disease” (KED). Folia Parasitologica 43:173-187, 1996
4. Yokoyama H, Ogawa K, Wakabayashi H: Involvement of Branchiura sowerbyi (Oligochaeta: Annelida) in the transmission of Hoferellus carassii (Myxosporea: Myxozoa), the causative agent of kidney enlargement disease (KED) of goldfish Carassius auratus. Fish Pathol 28(3):135-139, 1993
5. El-Matabouli M, Fischer-Scherl T, Hoffman R: Transmission of Hoferellus carassii (Achmerov, 1960) to goldfish Carassius auratus via an aquatic oligochaete. Bull Eur Assoc Fish Pathol 12(2):54-56, 1992
6. Yokoyama H, Ogawa K, Wakabyashi H: Light and electron microscopic studies on the development of Hoferellus carassii (Myxosporea), the causative organism of kidney enlargement disease of goldfish. Fish Pathol 25:149-156, 1990
7. Molnar K, Fischer-Scherl T, Baska F, Hoffman R: Hoferellosis in goldfish Carassius auratus and gibel carp Carassius auratus gibelio. Dis Aquat Org 7:89-95, 1989
CASE IV – 00-1420 (AFIP 2741886)
Signalment: 7-year-old, male Russian tortoise, Testudo horsfieldii, reptile.
History: Several tortoises were bought from a source in the Southern California desert. They became anorectic, developed severe oral infections and showed compromised respiratory signs. Throat cultures isolated numerous mixed Gram positive organisms, moderate growth of Proteus vulgaris and Stenotrophomonas maltophilia, and low numbers of Klebsiella oxytoca. All organisms were sensitive to enrofloxacin and TribrissenÒ . Despite antibiotic therapy, several tortoises died.
Gross Pathology: One tortoise was submitted for necropsy. It had a yellow and white, 1–2 mm thick, friable, diphtheritic membrane lining the oral cavity, pharynx and larynx. Numerous translucent white worms, 1-mm diameter x 10-mm in length, were present within the colon.
Laboratory Results: Culture of the oral cavity yielded heavy growth of Stenotrophomonas maltophilia and Acinetobacter anitratus, with moderate growth of Fusarium spp. Fecal flotation yielded high numbers of oxyurid eggs.
Contributor’s Diagnosis and Comment: Diffuse, severe, ulcerative and proliferative laryngitis, chronic, with epithelial intranuclear eosinophilic inclusion bodies, superficial bacterial colonies, and fungal hyphae.
Etiologic Diagnosis: Herpesviral laryngitis.
The laryngeal mucosa is covered by a thick, fibrinonecrotic membrane containing fibrin, granulocytes, degenerate and necrotic epithelial cells, mucus, proteinaceous fluid, mononuclear cells, bacterial colonies and parallel-walled, septate, 45° -branching fungal hyphae. The mucosa is hyperplastic and multifocally ulcerated, with foci of epithelial cells in the basal layer and stratum spinosum with intracellular edema and undergoing degeneration and necrosis. Many of the reactive epithelial cells have large and multiple eosinophilic nucleoli. Occasional mucosal and desquamated epithelial cells have a nucleus that contains a pale eosinophilic, large, intranuclear inclusion with a peripheral clear halo and a thin rim of marginated chromatin. Multifocally, the superficial submucosa and the mucosal/submucosal interface have an infiltrate of lymphocytes and plasma cells. Some of the skeletal muscle fibers in the deeper aspects of the tissue are swollen, hyalinized and vacuolated.
Herpesvirus, causing necrotizing glossitis and stomatitis, has been reported in several species of tortoises and turtles. Additional lesions include rhinitis, pneumonia, tracheitis, gastritis, esophagitis and encephalitis. Intranuclear inclusion bodies may be found in the liver, spleen, adrenal glands, duodenum, jejunum, colon, pancreas, endothelium of glomeruli, and neurons and glial cells of the medulla oblongata and diencephalon. Syncytial cells also may be formed.
A prior case of chelonian herpesvirus, presented in the AFIP Wednesday Slide Conference (5/17/95, case 2, AFIP 2468749), reported inclusion bodies in epithelium of the ductus deferens. The contributors of that case suggested the possibility of vertical transmission. This tortoise had a mild interstitial pneumonia and vacuolar degeneration of hepatocytes; however, intranuclear inclusion bodies were not observed in these tissues. Horizontal transmission is thought to occur through body secretions.
Diagnosis is achieved by identifying typical inclusion bodies in tissues or cytologic specimens, or by viral isolation. Recently, PCR analysis has been used to amplify herpesviral DNA from oral mucosa and liver of affected tortoises. Differential diagnosis of upper respiratory tract infections includes Mycoplasma agassizii, Pasteurella spp., and iridovirus. Treatment consists of isolation of affected tortoises, antiviral drug therapy, and antimicrobials to prevent secondary infections. The bacteria and fungus isolated in this case were judged to be secondary invaders. Pinworms are commonly present in tortoises and usually thought to be of little pathologic significance. High worm burdens, however, have been associated with morbidity and mortality.
AFIP Diagnosis: Tongue: Glossitis, necrotizing and ulcerative, focally extensive, severe, with epithelial eosinophilic intranuclear inclusion bodies, syncytia, and superficial bacterial colonies and fungal hyphae, Russian tortoise (Testudo horsfieldii), reptile.
Conference Comment: Alphaherpesviral infections of tortoises usually involve the oral cavity, commonly with extension into the pharyngeal and laryngeal regions. The route of disease transmission among chelonians is uncertain, although spread via contaminated saliva, nasal secretions, urine and feces is considered most likely.
Subclinical herpesviral infections are suspected to occur among tortoises and other reptiles. Transport stress, crowding or environmental changes have been associated with the manifestation of disease in these animals or with the infection of other reptiles in the same exhibit.
Contributor: Office of the County Veterinarian, Department of Agriculture, Weights and Measures, 5555 Overland Avenue, Building 4, San Diego, CA 92123
References: 1. Une Y, Uemura K, Nakano Y, Kmiie J, Ishibashi T, Nomura Y: Herpesvirus infection in tortoises (Malacochersus tornieri and Testudo horsfieldii). Vet Pathol 36(6):624-627, 1999
2. Cooper JE, Gschmeissner S, Bone RD: Herpes-like virus particles in necrotic stomatitis of tortoises. Vet Rec 123:554, 1988
3. Jacobson ER, Gaskin JM, Roelke M, Greiner EC, Allen J: Conjunctivitis, tracheitis and pneumonia associated with herpesvirus infection in green sea turtles. J Am Vet Med Assoc 189(9):1020-1023, 1986
4. University of Florida website: http://www.vetmed.ufl.edu/sacs/wildlife/TortoiseHerpesvirus/Pages/THerp.html, Dec 3, 1999
5. Rideout BA, Montali RJ, Phillips LG, Gardiner CH: Mortality
of captive tortoises due to viviparous nematodes of the genus
Proatractis (family Atractidae). J Wildlife Dis 23(1):103-108,
1987
*Sponsored by the American Veterinary Medical Association, the American College of Veterinary Pathologists and the C. L. Davis Foundation.
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