AFIP Wednesday Slide Conference - No. 29
3 May 2000

Conference Moderator:
Dr. Don Nichols, Diplomate, ACVP
Department of Pathology
National Zoological Park
Washington, DC 20008
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Case I - S-49-98 (AFIP 2681629)
Signalment: Boa constrictor (Boa constrictor constrictor), adult (est. 8 years), unknown sex.
History: Snake presented with a 1 cm2 dorsal mass in November 1993. At surgery, the mass was described as being rubbery and white and did not appear to invade underlying dermis. Biopsy was diagnosed as "granulation tissue". Local recurrence of mass occurred in July 1995, at which time it was again surgically removed.
Gross Pathology: 3 x 2 cm2 white, firm, fibrous dermal mass.
Contributor's Diagnosis and Comments: Morphologic Diagnosis: Iridophoroma, skin, snake.
Sections of skin containing an expansile, nonencapsulated, multilobular, pigmented dermal mass which extends to both lateral and ventral borders in some sections. The mass is composed of whorls, interlacing bundles, nests and fascicles of a uniform population of spindle cells on a fine fibrovascular stroma. The cells are spindle to stellate with indistinct cell borders, moderate amounts of eosinophilic fibrillar cytoplasm with varying amounts of coarse to fine golden brown/olive green pigment granules. The nuclei are ovoid to polyhedral with coarsely clumped chromatin and a single, variably distinct, round, magenta nucleolus. The mitotic index is less than l/HPF. Pigment granules are birefringent on polarized light.
Electron Microscopy: Cells had variable numbers of empty elliptical cytoplasmic membrane lined spaces. The amount of pigmentation seen on light microscopy related to the number of spaces on EM. Membranes ranged in size, shape and arrangement within the cytoplasm. The latticework of empty membranes is characteristic and represents the loss of "reflecting platelets" during routine uranyl acetate processing.
While the histologic features of this mass were suggestive of a melanoma, the presence of pigmented cells in a reptilian skin tumor are not diagnostic of that tumor alone.
In addition to melanophores, snake skin normally contains 3 other classes of pigment producing cells including iridophores, erythrophores and xanthophores. All are derived from neural crest. Neoplasms arising from all four classes have been described in reptiles. Mosaic chromatophoromas with combined features of multiple cell types have also been described.
H&E light microscopic features of these tumors are indistinguishable from those of a melanoma without the use of polarized light. Ultrastructural features, however, are striking and diagnostic and were pursued in this case.

Melanophoromas typically contain electron-dense melanosomes in various stages of development with a characteristic internal structure. Erythrophoromas contain characteristic lamellated pterinosomes. Neither of these structures was identified in this case. Instead, the cytoplasm was packed with a distinctive latticework of empty membranes. These spaces represent the dissolved cytoplasmic reflecting platelets of iridophores which are lost with routine uranyl acetate and lead citrate EM staining.
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Case 29-1. Dermis. Variably pigmented spindle shaped cells contain anisotropic crystalline material when viewed under polarized light.
AFIP Diagnosis: Skin: Iridophoroma, malignant, boa constrictor, reptile.
Conference Note: All conference participants agreed with the contributor's morphologic diagnosis of iridophoroma. The "malignant" designation was added because of infiltrative growth pattern, a mitotic rate that ranged up to 7 per high power field and nuclear atypia.
Note: Communication with the contributor revealed that this neoplasm has not recurred since it was removed in 1995. The original excision from 1993 was reexamined and determined to be the same neoplasm.
Contributor: Department of Pathology, Wildlife Health Center / WCS 185th St. and Southern Blvd., Bronx, New York, NY 10460.
1. Frye FL, Carney JD, Harshbarger JC, Zeigel FF: Malignant chromatophoroma in a western terrestrial garter snake. J Am Vet Med Assoc 167:557-558, 1975
2. Ghadially FN: Diagnostic Electron Microscopy of Tumors, 2nd ed., pp.106-121. Butterworths, London, England, 1985
3. Jacobson ER, Ferris W, Bagnara JT, Iverson WO: Chromatophoromas in a Pine Snake. Pigment Cell Res 2:26-33, 1989
4. Leach MW, Nichols DK, Hartsell W, Torgerson RW: Radiation therapy of a malignant chromatophoroma in a yellow rat snake (Elaphe obsoleta quadrivittata). J Zoo and Wild Med 22(2):241-244, 1991
5. Okihiro MS: Chromatophoromas in two species of Hawaiian butterfly fish, Chaetodon multicinctus and C. miliaris. Vet Pathol 25:422-43 1, 1988
6. Ryan MJ, Hill DL, Whitney GD: Malignant chromatophoroma in a Gopher snake. Vet Pathol 18:827-829, 1981
Case II - 40149 (AFIP 2679508)
Signalment: Adult, female caiman lizard (Dracaena guianensis)
History: This lizard was one of two animals wild-caught in Peru and spent approximately 1 year in at least 2 different reptile dealerships in California. On arrival at the zoo, the animals were in poor body condition and mildly dehydrated. Initial treatment included subcutaneous fluid therapy and tube feeding. Both lizards died suddenly within two weeks of arrival and had similar necropsy findings.
Gross Pathology: At necropsy, the lungs were diffusely hyperemic and exuded copious amounts of clear fluid on section. The lumen of each opened lung contained moderate amounts of a yellow mucoid material. A single adult trematode parasite was attached to the gastric mucosa.
Laboratory Results:
Aerobic bacterial culture of lung yielded 2+ Klebsiella pneumoniae, 3+ Proteus mirabilis, 3+ Pseudomonas sp., 2+ Citrobacter sp., and 2+ Aeromonas sp.
Virus isolation from the lung on viper heart cells demonstrated syncytial cell formation at 5 days post inoculation; negative staining electron microscopy demonstrated virions consistent with a paramyxovirus.
Wet mounts of gastric fluid contained moderate numbers of an unidentified trematode egg.
Contributor's Diagnoses and Comments:
1. Lung: Pneumonia, proliferative and interstitial, diffuse, moderate to marked with syncytial cells and rare intracytoplasmic eosinophilic inclusion bodies.
2. Lung: Encapsulated trematode eggs (may not be present in all sections).
Histologically, there is expansion of faveolar septa by edema and variable infiltrates of predominantly heterophils with fewer mononuclear inflammatory cells. Multifocally, within the lumen there are aggregates of similar inflammatory cells admixed with fewer sloughed epithelial cells, erythrocytes, rare bacteria and cellular debris. Diffusely, there is marked hyperplasia and hypertrophy of faveolar lining epithelial cells (type-2 pneumocytes) with formation of syncytial cells that have 2-3, and occasionally up to 10, nuclei. Occasionally, epithelial cells contain discrete variably sized eosinophilic cytoplasmic inclusion bodies. Rarely, within faveolar septa, there are trematode eggs, which are surrounded by a thin rim of fibrous connective tissue and/or low numbers of macrophages, which are often multinucleate. In addition to virus isolation (noted above), transmission electron microscopy of formalin-fixed lung tissue showed moderate numbers of filamentous and spheroidal virions, morphologically suggestive of paramyxovirus, budding from pneumocyte cell membranes.
The histologically observed proliferative pneumonia with syncytial cell formation and the presence of cytoplasmic inclusion bodies led to a presumptive diagnosis of a viral pneumonia in this case. This was later confirmed by electron microscopy and by isolation of a paramyxovirus from the lung. Although viral pneumonias are well known in snakes, to our knowledge, this is the first time that a viral pneumonia has been documented in a lizard.
Previous reports of paramyxoviruses in lizards have consisted of a serosurvey of wild iguanas and of virus isolation without correlative histopathology. In snakes, paramyxoviral infections have been responsible for epizootics in private and zoological collections and can be associated with high mortality. Lesions in affected snakes are similar to those observed in this caiman lizard and usually include proliferative pneumonia with or without syncytial cell formation and rarely, intracytoplasmic inclusion bodies.
Another frequently reported lesion in affected snakes is pancreatic necrosis with marked ductular hyperplasia and occasionally, syncytial cell formation. Mild pancreatic lesions were also observed in these affected lizards and consisted of mild interstitial edema and ductular syncytial cell formation. In epizootics occurring in snakes at our institution, pancreatic lesions have been more prominent with the classical proliferative pneumonia being observed only infrequently. Secondary bacterial pneumonia and septicemia have been frequently described in snakes with paramyxovirus infections. Consequently, when histologically evaluating what might seem to be a clear-cut bacterial pneumonia in a reptile, care should be taken not to overlook the sometimes-subtle proliferative changes that could be suggestive of an underlying viral infection.
The multiple bacterial isolates obtained from the lung of this lizard were interpreted as representing secondary colonization of the virally compromised respiratory tract. Trematode eggs were observed in multiple tissues, including the lung, with minimal associated host response and consequently were interpreted as incidental findings. Because snails comprise the majority of the diet in caiman lizards, the presence of trematode eggs in tissues was not surprising.
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Case 29-2. Lung. Multifocally air sac epithelium contains syncytial cells.
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Case 29-2. Lung. There are two deformed, collapsed, empty, brown, trematode eggs expanding the interstitium.
AFIP Diagnoses:
1. Lung: Pneumonia, interstitial, proliferative and exudative, subacute, diffuse, moderate, with syncytial cells, caiman lizard (Dracaena guianensis), reptile.
2. Lung, interstitium: Encapsulated trematode eggs (may not be present in all sections).

Conference Note: Caiman lizards (members of the genus Dracaena and the family Teiidae) are found in wet, forested areas of South America. D. guianensis reaches a length of about 122 cm. Caiman lizards should not be confused with caimans, a group which includes several species of Central and South American reptiles related to alligators. The black caiman (Melanosuchus niger) may reach 4.5 m; other species are generally in the 1.2 to 2.1 m range. The moderator, Dr. Nichols, noted that lizard paramyxovirus has now been diagnosed at animal facilities in San Diego, Baltimore, and Florida.
Contributor: Department of Pathology, Zoological Society of San Diego, PO Box 120551, San Diego, CA 92112-0551.
1. Gravendyck M, Ammermann P, Marschang RE, Kaleta EF: Paramyxoviral and reoviral infections of iguanas on Honduran islands. J Wildl Dis 34:33-38, 1998
2. Jacobsen ER, Adams HP, Geisbert TW, Tucker SJ, Hall BJ, Homer BL: Pulmonary lesions in experimental ophidian paramyxovirus pneumonia of Aruba Island rattlesnakes (Crotalus unicolor). Vet Pathol 34:450-459,1997
3. Jacobsen ER, Flanagan JP, Rideout B, Ramsay EC, Morris P: Ophidian paramyxovirus--roundtable discussion. Bull Assoc Rept Amphib Vet 9:15-22, 1999
4. Jacobsen ER, Gaskin JM, Page D, Iverson WO, Johnson JW: Illness associated with paramyxo-like virus infection in a zoologic collection of snakes. JAVMA 179:1227-1230, 1981
5. Jacobsen ER, Gaskin JM, Wells S, Bowler K, Schumacher J: Epizootic of ophidian paramyxovirus in a zoologic collection: pathological, microbiological, and serological findings. J Zoo Wildl Med 23:318-327, 1992
6. Schumacher J: Respiratory diseases of reptiles. Sem Av Exot Pet Med 6 (4):209-215, 1997
Case III - 1906/96 (AFIP 2694783)
Signalment: Adult hawksbill turtle (Eretmochelys imbricata).
History: The turtle had been in a zoo since 1970. Following a shark bite in 1988, the animal had suffered from recurrent episodes of cloacal obstipation and diphtheroid-necrotizing cloacitis. In June 1996 it became anorectic and depressed. Early in July, several well-circumscribed yellow nodules, up to 1 cm in diameter, were noted in the skin of the ventral mandibular and pericloacal area. The animal continued to deteriorate and additional yellow nodules developed on neck and tail. It was found dead one month later.
Case 29-3. Gross tissue. There are multifocal irregularly shaped pale zones (necrosis).
Gross Pathology: Multiple yellowish firm nodules up to 3 cm in diameter were present in the lung, liver, spleen, kidney, esophagus, small and large intestine, pectoral and lumbar muscles, and skin. Skin lesions were located in the mandibular arch, ventral neck, the pericloacal region, and the ventral tail. In addition to those lesions ulcerating the epidermis, several nodules within the dermis were found. The liver revealed large confluent and irregularly marginated areas of necrosis. The serosal surface of liver, lung, spleen, kidney, small and large intestine was ulcerated on multiple locations and covered with patchy fibrinous layers. Furthermore, ulcerations of the intestinal mucosa were present. On cut surface, nodules were friable and contained central caseous material.
Laboratory Results: The blood status showed high levels of urea, uric acid, and CPK, whereas creatinine levels were considered normal.
Contributor's Diagnosis and Comments: Liver: granulomatous hepatitis, multifocal, severe, with intralesional fungal hyphae (Paecilomyces lilacinus)
Paecilomyces spp. belongs to the section of hyaline Hyphomycetes and is closely related to Penicillium spp. They are distinguished from the latter often by their color and by their tapering phialides and divergent conidial chains. P. lilacinus is characterized by its vinaceus color, it's long and rough-walled, usually colored conidiophores and the absence of chlamydospores. Paecilomyces is a saprophytic organism, ubiquitous in soil and decaying material and could be an airborne contaminant.

Several authors have described mycotic diseases in reptiles with constant association of typical, mainly granulomatous lesions to particular species of fungi like Paecilomyces spp., Aspergillus spp. or Beauvaria sp. Most frequently affected organs are the lung and the skin, but systemic disease is also reported. Systemic infection with saprophytic molds is generally associated with impaired immune defense, especially that of cellular immunity.

In our case, a possible site of entry for the fungus is the cloaca or the pericloacal region, as repeated cloacitis was observed in this turtle. Inside the body, the spread of fungi occurred hematogenously and by direct implantation.
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Case 29-3. Liver. Multiple branching septate fungal hyphae are present within the lumen and the wall of a blood vessel.
AFIP Diagnosis: Liver: Hepatitis, necrotizing and granulomatous, multifocally extensive, severe, with necrotizing vasculitis, and fungal hyphae, hawksbill turtle (Eretmochelys imbricata), reptile.
Conference Note: Five species of Paecilomyces (P. javanicus, P. lilacinus, P. marquandii, P. variotii and P. viridis) have been reported to cause disease in humans and animals. In animals, infection is generally characterized by chronic weight loss. Lung is most often affected. The inflammatory reaction is granulomatous. Histologically, budding yeast-like cells, septate hyphae and conidia may be found.
Contributor: Institut für Tierpathologie, Universität Bern, Länggasse 122, CH-3012 Bern.
1. Heard DJ, Cantor GH, Jacobson ER, Purich B, Ajello L, Padhye AA: Hyalohyphomycosis caused by Paecilomyces lilacinus in an Aldabra tortoise. J Am Vet Med Assoc 9:1143-1145, 1986
2. Jacobson ER, Gaskin JM, Shields RP, White FH: Mycotic pneumonia in mariculture-reared green sea turtles. J Am Vet Med Assoc 175:929-933, 1979
3. Posthaus H, Krampe M, Pagan O, Guého E, Suter C, Bacciarini L: Systemic Paecilomycosis in a Hawksbill Turtle (Eretmochelys imbricata). J Mycol Med 7:223-226, 1997
Case IV - 96-735 (AFIP 2701698)
Signalment: 11+ -year-old, female, White's tree frog (Litoria caerulea)
History: This was an aged tree frog that had not been eating well for several weeks. It was found dead in its cage.
Gross Pathology: The carcass was in poor nutritional condition; coelomic fat stores were markedly atrophied.
Contributor's Diagnosis and Comments: Skin, hyperkeratosis and acanthosis, diffuse, moderate, with multifocal epidermal degeneration
Etiologic Diagnosis: Mycotic (chytridiomycotic) dermatosis
Etiology: Batrachochytrium dendrobatidis (Chytridiomycetes fungus)
Until recently, no species of fungi in the Phylum Chytridiomycota was known to be a pathogen of vertebrate animals. However, fatal cutaneous chytridiomycosis has now been reported in a wide variety of wild and captive amphibians and has been associated with declines of wild populations of frogs and toads in Australia, Panama, and the United States.
At the National Zoological Park (NZP), this disease has been identified in more than 40 dead frogs of 4 different species. The causative organism has been isolated in culture from blue poison arrow frogs (Dendrobates azureus), green-and-black poison arrow frogs (Dendrobates auratus), and a White's tree frog at NZP. This organism is unlike any previously described fungal genus or species and has been named Batrachochytrium dendrobatidis.
The most characteristic lesions associated with cutaneous chytridiomycete infection are hyperkeratosis and acanthosis. Focal epidermal cell hypertrophy and/or degeneration are less common. Inflammation is rare and, when it does occur, is usually mild. Typically, low to moderate numbers of chytridiomycetes are located in the superficial epidermis, primarily the keratinized layers. In histologic sections, three stages of the organisms are primarily seen: a uninucleated form, a multinucleated stage containing internal septa, and a cyst-like form (zoosporangium) containing multiple flagellated spores (zoospores). Each spore has a single posterior flagellum that is very difficult to detect in histologic sections.
The entire life cycle of Batrachochytrium dendrobatidis has not been determined. Spores are released through tubular projections on the zoosporangia known as discharge papillae. The motile spores are then thought to swim through water to infect other epidermal cells.
The mechanisms by which chytridiomycosis causes death is not known. The skin of infected animals appears to be the only organ to consistently have lesions. Normal skin functions in amphibians include maintenance of hydration, osmoregulation, thermoregulation, and respiration. The skin lesions probably interfere with these functions and cause fatal metabolic alterations.
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Case 29-4. Skin. Multiple developmental stages of fungal thalli and zoosporangia are present within the epidermis and subepidermal glands.
AFIP Diagnosis: Skin: Epidermal hyperplasia, diffuse, moderate, with hyperkeratosis and numerous intracorneal fungi, White's tree frog (Litoria caerulea), amphibian, etiology consistent with Chytridiomycetes.
Conference Note: Emerging infectious diseases are believed to play important roles in the global declines in amphibian populations. Various factors (host, pathogen, and environmental) may be involved in disease emergence. Infectious diseases may be only the proximate cause of death. Environmental factors such as increased UV-B, chemical pollution, climate change and stress have been hypothesized as possible underlying problems. In addition to Chytridiomycetes, iridoviruses of the Ranavirus genus have been implicated as contributing to the amphibian decline.
Contributor: Department of Pathology, National Zoological Park, Smithsonian Institution, Washington, DC, 20008.
1. Berger L, Speare R, Daszak P, Green DE, Cunningham AA, Goggin CL, Slocombe R, Ragan MA, Hyatt AD, McDonald KR, Hines HB, Lips KR, Marantelli G, Parkes H: Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proceedings of the National Academy of Sciences 95:9031-9036, 1998
2. Longcore JE, Pessier AP, Nichols DK: Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia 91(2): 219-227, 1999
3. Nichols DK, Pessier AP, Longcore JE: Cutaneous chytridiomycosis in amphibians: an emerging disease? Proceedings, Annual Conference of the American Association of Zoo Veterinarians, Omaha, NE, pp. 269-271, 1998
4. Pessier AP, Nichols DK, Longcore JE, Fuller MS: Cutaneous chytridiomycosis in poison dart frogs (Dendrobates spp.) and White's tree frogs (Litoria caerulea). J Vet Diag Invest 11:194-199, 1999
5. Daszak P, Berger L, Cunningham AA, Hyatt AD, Green DE, Speare R: Emerging infectious diseases and amphibian population declines. Emerg Infect Dis 5(6):Nov-Dec,1999
J Scot Estep, 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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