AFIP Wednesday Slide Conference - No. 28
April 28, 1999

Conference Moderator: Dr. Don Nichols, Diplomate, ACVP
Department of Pathology
National Zoological Park
Washington DC 20008
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Case I - 97-214-3 (AFIP 2655235)

one electron micrograph photo transparency
Signalment: Adult, male, hairy-footed hamster (Phodopus sungorus).
History: This hamster was noted to have lameness or ataxia of the left rear leg and impacted cheek pouches. It was presented to the Department of Animal Health in a moribund condition and was euthanized.
Gross Pathology: The carcass was in poor nutritional condition with little to no subcutaneous and cavitary fat. The right maxillary incisor was absent, and the mandibular incisors were slightly overgrown and deviated to the right. Other findings were unremarkable.
Laboratory Results: None.
Contributor's Diagnosis and Comments: Brain, meningoencephalitis, suppurative to pyogranulomatous, multifocal, severe.

Etiologic Diagnosis: Algal (protothecal) encephalitis

Etiology: Prototheca sp.
The lesions in the brain were associated with the presence of round, 5-10 mm endosporulating eukaryotic organisms. The walls of these organisms stained with the periodic acid-Schiff reaction, GMS, and Gridley fungal stain, but the organisms did not stain with acid-fast or Gram stains. Ultrastructural examination revealed that they had a thick outer cell wall and contained up to 3 endospores which had numerous, variably-sized, electron-dense granules and electron-lucent granules in their cytoplasm. The light and electron microscopic features of the organisms were compatible with a species of Prototheca.

Prototheca are colorless, unicellular saprophytic microorganisms that are believed to be achlorophyllous algae closely related to green algae in the genus Chlorella. Opportunistic infections have occasionally been reported in humans, cattle, dogs, cats, and other species. These infections are often localized to the skin or subcutis, although disseminated infection can occur. In cattle, protothecosis typically occurs as chronic mastitis.
In this hamster, protothecal infection was only detected in the brain. This animal had spent its entire life inside the Small Mammal House at the National Zoo. The source and route of infection were not determined.
AFIP Diagnosis: Cerebrum: Meningoencephalitis, necrotizing, pyogranulomatous, multifocal, moderate, with algae, hairy-footed hamster (Phodopus sungorus), rodent.
Conference Note: Prototheca are generally regarded as opportunistic infectious organisms of low pathogenicity, and dysfunction of the host immune system is usually required before organisms can invade and act as pathogens. In humans, protothecosis occurs primarily in two clinical forms: cutaneous infection and olecranon bursitis. Cutaneous infection, accounting for most cases, is often progressive and tends to occur in individuals who are immunosuppressed or have underlying debilitating disease. Olecranon bursitis accounts for one-third of human cases, and occurs in healthy individuals, usually associated with trauma. While Prototheca sp. typically cause mastitis in cattle, in dogs protothecosis is frequently a systemic disease, and the organisms have a predilection for the brain and eyes. Cutaneous disease has been described in cats.
Three species of Prototheca are recognized: P. stagnora, P. zopfii, and P. wickerhamii. The latter two are pathogenic and have similar morphologic characteristics in tissue sections, although they differ somewhat in size. P. zopfii are 10 to 25 mm in diameter, whereas P. wickerhamii are 1.5 to 11 mm. Also, the endospores of P. zopfii are larger than those of P. wickerhamii.
The differential diagnosis considered by conference participants included Prototheca and Chlorella. Chlorella sp. are green, chlorophyll-containing algae with otherwise similar morphological features to those of Prototheca. The chlorophyll is contained within cytoplasmic chloroplasts that also contain abundant starch. The starch granules stain intensely with the periodic acid-Schiff reaction and are diastase sensitive. By electron microscopy, the chloroplasts consist of a highly organized, twisted or lamellar component associated with amorphous, electron dense, vacuolated material representing starch. Prototheca may contain similar, smaller starch granules in the form of storage plastids. Ultrastructurally, the storage plastids are homogenous, electron dense, and are not associated with the lamellar component found in true chloroplasts. Participants did not identify chloroplasts in the electron micrograph provided by the contributor, and thus favored the diagnosis of protothecosis.
Contributor: Department of Pathology, National Zoological Park, Smithsonian Institution, Washington, DC 20008.
1. Chandler FW, Watts JC: Protothecosis and infections caused by green algae. In: Pathologic Diagnosis of Fungal Infections, Chandler FW, Watts JC, eds., pp. 43-53, ASCP Press, Chicago, IL, 1987.
2. Dillberger JE, Homer B, Daubert D, Altman NH: Protothecosis in two cats. J Amer Vet Med Assoc 192:1557-1559, 1988.
3. Jones TC, Hunt RD, King NW: Diseases caused by fungi. In: Veterinary Pathology, Jones TC, Hunt RD, King NW, eds., 6th ed., pp. 534-535, Williams & Wilkins, Baltimore, MD, 1997.
4. Taniyama H, et al: Disseminated protothecosis caused by Prototheca zopfii in a cow. Vet Pathol 31:123-125,1994.

Case II - Fac. Med. Vet. da USP (AFIP 2600309)

Signalment: Juvenile male and female Tracaja turtles (Podocnemis unifilis).
History: An environmental project aimed at raising Tracaja turtles, an endangered species in South America, is based in the Goias State of Brazil. Sixty of these animals were sent to San Paulo for a research study. These animals were sacrificed during the research protocol. Ninety percent of these contained protozoans within the kidneys.
Gross Pathology: The kidneys were markedly swollen and pale.
Laboratory Results: Urine examination revealed developmental stages of a protozoan. The largest of these organisms contained spore-like bodies.
Contributor's Diagnosis and Comments: Chronic interstitial nephritis due to a myxosporidian of undetermined generic status.
Note: The hyaline, refractive spore wall was present rarely in the large pansporoblasts within the lumen of the tubules. When stained with Giemsa (enclosed), the polar capsules were a reddish-purple and were distinguishable from the blue of the developing spores. Also note the cytoplasm and nuclei of the mature spore pushed to one side of the spore.
In the majority of the cases, chronic interstitial nephritis extending into the parenchyma was present. Heterophils were common. Tubules were found to contain cellular debris and sloughed cells. In other cases, only a moderate inflammatory infiltrate was seen. In infected turtles, sporonts and pansporoblasts were present within epithelial cells. As the pansporoblasts matured, they enlarged, ruptured the epithelial cell, and were found within the lumen of the tubules. Immature spores were formed and gave rise to mature spores.
For years, it was thought that myxosporean parasites used only one host in their life cycle. From research in the last ten years, it is now known that an intermediate host is required to complete the life cycle (we refer those interested to references 3, 4, 5). In those organisms for which life cycle studies have been conducted, the intermediate host has been found to be an Oligochaeta or Polychaete worm. Life cycles have been elucidated in both fresh water and marine habitats.
In the turtles of this case, trophozoites were found in the epithelial cells and lumens of renal tubules. Spores developed in disporoblastic pseudoplasmodium in lumens of the renal tubules. These pseudoplasmodia are very large and contain the hyaline spores. Spores are found in the urine of infected turtles. Ongoing studies are attempting to elucidate the intermediate host. Additional studies are underway to determine the genus of the myxosporean in question. Myxosporean parasites have been found in the kidneys of fish, such as sockeye salmon from Canada and red drum from Florida.
AFIP Diagnoses:
1. Kidney: Tubular ectasia and tubular epithelial hyperplasia, multifocal, moderate, with numerous intracellular and extracellular myxosporidia, Tracaja turtle (Podocnemis unifilis), chelonian.
2. Kidney: Nephritis, interstitial, lymphoplasmacytic and heterophilic, multifocal, moderate.
Conference Note: The class Myxosporea are protozoan parasites belonging to the phylum Myxozoa. Myxozoans primarily parasitize invertebrates (mostly annelids) and poikilothermic vertebrates, the vast majority of which are fish. Thus, most published reports of disease have been in fish. The myxozoans that infect fish are all members of the class Myxosporea.
Myxosporea are transmitted by means of spores to susceptible hosts. The spores, which have a characteristic morphology that is the basis for taxonomic classification, are multicellular spheroid structures that in fish measure approximately 10 mm in diameter. Spores have one binucleate or two uninucleate sporoplasms, one to six (usually two) polar capsules, and a shell. One of the key characteristics of myxosporidia is that all stages, except during sexual reproduction, are multinucleated forms that have enveloping (primary) cells that contain enveloped (secondary) cells.
Most myxosporean infections of fish are relatively harmless, with only mild host reaction. Heavy infections can be pathogenic, however, causing mechanical damage due to pseudocysts, or tissue necrosis and inflammation due to trophozoite feeding. Some of the diseases in fish caused by myxosporidians include whirling disease of young salmonids caused by Myxobolus cerebralis; renal and blood sphaerosporosis in cyprinids caused by Sphaerospora renicola; and proliferative kidney disease of salmonids caused by Myxidium lieberkuhni.
One of the first Myxosporean infections described was Hoferellus (Mitraspora) cyprini, the cause of kidney enlargement disease in young cyprinids. The descriptions of the microscopic lesions associated with H. cyprini bear some resemblance to the lesions observed in the turtles. Trophozoites are associated with the epithelium of renal tubules (or ureters), and undergo endodyogeny, becoming large plasmodia. Trophozoites initially congregate on the apical surface of the epithelium, but become progressively incorporated into the cell layer as epithelial hyperplasia and papillary growth occurs. In the terminal stages of infection, the tubules are markedly enlarged, displacing the swimbladder, and distending the abdomen.
Contributor: Departamento de Patologia, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Brazil.
1. Gardiner CH, Fayer R, Dubey JP: Myxozoa. In: An Atlas of Protozoan Parasites in Animal Tissues, Agriculture Handbook No. 651, pp. 14-15, U.S. Department of Agriculture, Washington DC, 1988.
2. Kreier JP: Myxosporidia. In: Parasitic Protozoa, Kreier JP, ed., vol. IV, pp. 115-154, Academic Press, New York, NY, 1977.
3. Markiw ME, Wolf K: Myxosoma cerebralis (Myxozoa: Myxosporea): Etiological agent of salmonid whirling disease requires tubificid worm (Annelida: Oligochaeta) in its life cycle. J Protozool 30:561-564, 1983.
4. Wolf K, Markiw ME: Biology contravenes taxonomy in the Myxozoa: New discoveries show alteration of invertebrate and vertebrate hosts. Science 225:1449-1452, 1984.
5. Ruidisch S, El-Matbouli M, Hoffman RW: The role of tubificid worms as an intermediate host in the life cycle of Myxobolus pavlovskii (Akhmerov, 1954). Parasitol Res 77:663-667, 1991.
6. Kent ML, Whitaker DJ, Dawe SC: Parvicapcula minibicornis (Myxozoa, Myxosporea) from the kidney of sockeye salmon (Oncorhynchus nerka) from British Columbia, Canada. J Parasitol 83:1153-1156, 1997.
7. Landsberg JH: Kidney myxosporean parasites in red drum Sciaenops ocellatus (Sciaenidae) from Florida, USA, with a description of Parvicapsula renalis. Diseases of Aquatic Organisms 17:9-16, 1993.
8. Garden O: The myxosporea of fish: A review. British Vet J 148:223-239, 1992.
9. Noga EJ: Myxozoan infections: General features. In: Fish Disease: Diagnosis and Treatment, Noga EJ, ed., pp. 173-178, Mosby-Year Book, St., Louis, MO, 1996.

Case III - 97-3720 (AFIP 2641212)

Signalment: Adult slender tree frog (Litoria adelensis).
History: Slender tree frog tadpoles had been introduced into a large pond built to reproduce the natural wetland habitat of an endangered species, the Western Swamp Tortoise in the Perth Zoological Gardens. Whilst intended as a feed source, some tadpoles had survived, and a colony had developed within the pond environs. Mortalities were first observed in slender tree frogs within the pond, initially with no premonitory clinical signs noted by the zoo attendants. As the outbreak progressed, several moribund and depressed frogs that appeared reluctant to move when approached were seen, and mortalities in tadpoles were noted.
Gross Pathology: There was an increased volume of clear coelomic fluid and multiple raised pale foci, 0.5 mm to 1.0 mm in diameter which were randomly distributed over the surface and extended into the parenchyma of the kidney, liver and spleen. One frog had a unilateral opacity of the cornea with mild exophthalmia, and another had an irregular pale area at the apex of the heart in addition to pale foci within the liver and kidney.
Laboratory Results: Mucor amphibiorum was cultured from kidney, liver and skin.
Contributor's Diagnosis and Comments: Liver: Hepatitis, necrotizing and granulomatous within intralesional Mucor amphibiorum sphaerules.
Histopathologically, frogs showed a multisystemic, multifocal and coalescing granulomatous inflammatory reaction centered about Mucor amphibiorum sphaerules. The sphaerules ranged from 10 µm in diameter in those with no internal structures, to larger than 30 µm diameter mother sphaerules which contained between four and eight daughter sphaerules.2,3 The eosinophilic refractile capsule of the sphaerules stained strongly by the periodic acid-Schiff reaction.
Mucor amphibiorum is a fungus which replicates in soil and in culture as aerial mycelia and sporangiospores typical of other members of the Mucor genera. In tissues, it occurs solely as unique circular structures or sphaerules which contain between 2 and 11 daughter sphaerules. Mucor amphibiorum was first isolated in 1972 from an Australian green tree frog (Litoria caerulea) held in a collection in Germany.1 It was believed that L. caerulea may have been infected prior to importation from Australia. However, the collection also contained frogs imported from South America, and the exact source of the infection could not be determined.
Infection by Mucor amphibiorum has been reported in Australia in skin lesions in the platypus (Ornithorhynchus anatinus) in Tasmania, and as a multisystemic infection in cane toads (Bufo marinus) and a green tree frog (Litoria caerulea) in Queensland.2,3 The detection of Mucor amphibiorum in Western Australia indicates the fungus is more widely distributed than previously believed and raises concerns over the susceptibility of other Australian anuran species to this fungus.
AFIP Diagnosis: Liver: Hepatitis, granulomatous, necrotizing, diffuse, severe, with numerous fungal spherules, slender tree frog (Litoria adelensis), amphibian.
Conference Note: Mucor amphibiorum is unusual among the Zygomycetes in that it is a dimorphic fungus, occurring in tissues in a spherical form, while hyphae and reproductive structures are found only in the external environment. The fungus was classified as Mucor sp. by Schipper in 1978 based on morphology. Within tissues, the organism reproduces by forming numerous daughter spherules that are released upon degeneration of the wall of the mother spherule.
Mucor amphibiorum is a primary pathogen in anurans, and several routes of infection are suspected or described, depending upon the species affected. In infected cane toads from Australia, ingestion of contaminated soil was suspected as the route of infection, with dissemination to the liver via the hepatic portal system and subsequent hematogenous spread to multiple organs. Infected toads can excrete fungus in their feces. In a single case report of Mucor amphibiorum in a free-ranging green tree frog from Australia, a respiratory route of infection was suspected; dissemination to multiple organs was also present. In platypuses, animals appear to be infected percutaneously and the infection remains localized.
The infectivity and dissemination of M. amphibiorum are probably related to its temperature tolerance. The maximum temperature at which the fungus grows in vitro is 36°C. In the platypus, whose body temperature is 32°C, lesions occur primarily in the dermal tissues of the body and on the extremities. In poikilothermic anurans, however, organisms become disseminated and are able to grow in various organs.
In tissue sections, organisms that occur as spherical forms similar to M. amphibiorum include Prototheca sp. and Coccidiodes immitis. The mother spherules of M. amphibiorum are larger than the cells of Prototheca, although the smaller spherules may be similar in size to the algae. In culture, Prototheca retain their spherical form, whereas M. amphibiorum forms a mycelial phase. Coccidiodes immitis can be distinguished from M. amphibiorum by the endospores, which are smaller and more numerous than spherules.
Contributor: Agriculture Western Australia- Division of Veterinary and Biomedical Sciences, Murdoch University, South Street, Murdoch, Western Australia 6150.
1. Obendorf DL, Peel BF, Munday BL: Mucor amphibiorum infection in platypus (Ornithorhynchus anatinus). J Wildl Dis 29:485-487, 1993.
2. Berger L, Speare R, Humphrey J: Mucormycosis in a free-ranging green tree frog from Australia. J Wildl Dis 33:903-907, 1997.
3. Speare R, Berger L, O'Shea P, Ladds PW, Thomas AD: Pathology of mucormycosis of cane toads in Australia. J Wildl Dis 33:105-111, 1997.
4. Speare R, Thomas AD, O'Shea P, Shipton WA: Mucor amphibiorum in the toad, Bufo marinus, in Australia. J Wildl Dis 30:399-407, 1994.

Case IV - 97-263 (AFIP 2635662)

Signalment: Adult, male freshwater turtle (Chrysemas picta).
History: The turtle was one of several purchased from a commercial vendor for research purposes, which included in vitro intracellular recording of the cerebellum using a patch-clamp technique. Several of the turtles died during the quarantine period, and the investigator indicated that a small parasitic worm was found moving within one of the brain preparations.
Gross Pathology: There was focal necrosis within the pancreas. There were white spots within the liver and spleen.
Laboratory Results: None.
Contributor's Diagnoses and Comments:
1. Granulomatous pancreatitis - trematode ova.
2. Embolized trematode ova - small intestine and pancreas.
The lesions are compatible with Spirorchid blood fluke infection. There are 16 genera within the family Spirorchidae, all of which affect turtles. Although monogenetic and digenetic fluke infections are common in freshwater turtles and considered relatively nonpathogenic, the Spirorchid blood flukes are an exception. Spirorchis scriptae was first described in 1923. Three species of blood flukes have recently been associated with mortalities in stranded green turtles. The adult flukes generally live in the heart and larger arteries where they produce numerous ova that are widely distributed to body organs. The adult flukes have been associated with focal intimal villus proliferation and thromboembolic disease. The pathogenicity of the flukes varies somewhat between the species, and is also related to widespread deposition of the ova which form granulomas. Spirorchis parvus are capable of invading various tissues including the pancreas and the central nervous system.
AFIP Diagnoses:
1. Pancreas: Pancreatitis, granulomatous, with abundant necrotic debris, freshwater turtle (Chrysemas picta), chelonian.
2. Small intestine and pancreas: Granulomas, multiple, with trematode eggs.
Conference Note: In the pancreas, and within the serosa, tunica muscularis and submucosa of the small intestine, there are numerous trematode eggs that lack an operculum, have golden-brown shells that often appear triangular in histologic section, and occasionally contain multiple, small, round basophilic structures (intact miracidia). Some eggs are not associated with any host response, while others are surrounded by immature fibrous connective tissue and/or a few multinucleate giant cells. Adult schistosomes are present in vessels of the small intestine and pancreas in some sections.
In most slides, at least one of the serial sections of the pancreas is focally replaced by extensive granulomatous inflammation with abundant eosinophilic, necrotic material; sections of a degenerate larval nematode may also be present in this area on some slides. In some sections, bacilli are found at the periphery of areas of necrosis and granulomatous inflammation, within the surrounding pancreatic mesentery, and occasionally within mesenteric vessels. These were found to be Gram-negative with tissue Gram stains performed at the AFIP. An acid-fast stain and a GMS did not demonstrate acid-fast bacteria or mycotic organisms.
The schistosomes are the only trematodes that do not have operculated eggs. The schistosomes are also the only group of trematodes that have separate sexes; other trematodes are hermaphroditic. Important aspects of the pathogenicity of spirorchid infections in turtles include dissemination of trematode eggs to many organs and tissues, occlusion of vessels by the eggs, and migration of eggs through vessel walls inciting an initial acute inflammatory response followed by granuloma formation. Passage of eggs from vessels in the intestine through the epithelium and into the lumen allows invasion of bacteria, and secondary bacterial infections are likely responsible for the deaths of some turtles infected by spirorchid flukes. The Gram-negative bacteria present in this turtle were likely secondary to spirorchid infection, and may have contributed to the lesions and inflammatory response in the pancreas. The presence of larval nematodes in some sections may have also contributed to pancreatic lesions.
Contributor: Department of Comparative Medicine, M.S. Hershey Medical Center, Penn State University, 500 University Drive, H054, Hershey, PA 17033.
1. Gordon AN, Kelly WR, Cribb TH: Lesions caused by cardiovascular flukes (Digenea: Spirorchidae) in stranded green turtles (Chelonia mydas). Vet Pathol 35:21-30, 1998.
2. Holliman RB, Fischer JE, Parker JC: Studies on Spirorchis parvus (Stunkard, 1923) and its pathological effects on Chrysemas picta picta. J Parasitol 57:71-77, 1971.
3. Johnson CA, et al.: Fatal trematodiasis in research turtles. Lab Anim Sci 48:340-343, 1998.
Course Coordinator:
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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