Gastrointestinal Candidiasis in an Aldabra Giant Tortoise (Geochelone Gigantea)

Home , Aldabra giant tortoise, Giant tortoise, Greek tortoise, Megalochelys, Tortoise

NOTE Pathology

Gastrointestinal Candidiasis in an Aldabra Giant Tortoise (Geochelone gigantea)

Vetnizah JUNIANTITO1), Takeshi IZAWA1), Mitsuru KUWAMURA1), Masao YONEZAWA2), Shu ITO2) and Jyoji YAMATE1)

1)Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Rinkuu Ourai Kita 1–58, Izumisano-shi, Osaka, 598–8531 and 2)Adventureworld AWS Co. Ltd, Nishimurogun, Shirahama-cho, Katada, Wakayama 649–2201, Japan

(Received 25 April 2009/Accepted 30 May 2009)

ABSTRACT. An adult female Aldabra giant tortoise (Geochelone gigantea) was found dead with a 2 month-history of decreased appetite. At necropsy, multiple ulcers were seen in the mucosa of stomach and colon. Histopathologically, the ulcers were characterized by extensive necrosis, hemorrhage, and marked edema, along with numerous chlamidospores and aseptate fungal hyphae. Fungal structures infiltrated into the lamina propria, submucosa and muscle layer; their invasion into blood vessels formed thrombosis. Immunohistochem- ically, the fungal structures were strongly positive with an antibody against Candida spp. This case is the first report on gastrointestinal candidiasis in an Aldabra giant tortoise. KEY WORDS: Aldabra giant tortoise, candidiasis, gastrointestinal ulcer. J. Vet. Med. Sci. 71(9): 1269–1272, 2009

Aldabra giant tortoises (Geochelone gigantea) living in death. The animal was not treated with any antimicrobial the islands of Aldabra Atoll in the Seychelles have a long therapy. Organs fixed in 10% neutral buffered formalin life span, ranging from 25 to 90 years old, and large body were sent to our laboratory for further histopathological size (19–120 kg) are considered as one of the largest tortoise examinations. Afterwards, tissues were embedded in paraf- [21]. This species are selective grazers, but occasionally fin, sectioned at 3–5 m in thickness, and stained with consume carrion, goat feces, and camp debris [3]. Studies hematoxylin-eosin (HE). Selected sections were also on the ecology, environmental conditions, nutrition, health stained with Grocott’s methenamine silver nitrate (GMS) management, and disease investigation should contribute to and by periodic acid-Schiff (PAS) reaction. Because fresh wildlife conservations in their natural habitat and captivity. tissues for cultures could not be taken at necropsy, immuno- Generally, candidiasis (thrush or moniliasis) occurs as an histochemical studies were performed to identify a possible opportunistic infection, particularly in the hosts undergoing causative agent. Sections were incubated with a rabbit poly- immunosupression or reduction of competing normal clonal antibody against C. spp. or Aspergillus spp. (each microflora when antibiotic therapy is prolonged [20]. As dilution, 1:100; provided by Dr. M. Kubo, NIAH, Japan) for compared with candidiasis in the poultry, reptiles, and mam- 14 hr at 4C. Subsequent incubation with the secondary mals, reports on the infectious disease in the chelonian are antibody was carried out using the Histofine kit simple stain much smaller in the number. In the chelonian, to our knowl- rabbit MAX PO method (Nichirei, Tokyo, Japan) and the edge, an infection due to Candida albicans has been positive signals were visualized with 3,3’-diaminobenzidine reported in a Greek tortoise (Tetsudo graeca) [7] and a log- tetrahydrochloride (DAB) (Vector Laboratories Inc., Cali- gerhead sea turtles (Caretta caretta) [17]; the affected che- fornia, U.S.A.). lonian developed severe enteritis or granulomatous Grossly, distended gas-filled intestine was observed. The pneumonia. In an Aldabra giant tortoise, although C. albi- mucosal layer of stomach and colon showed multifocal cans was isolated from a granulomatous lesion in the oral ulcerative lesions, accompanied with pseudomembrane and and gastric mucosa due to Hyalohypomycosis, C. albicans hemorrhage. The diameter of ulcers in the stomach was was not regarded as the pathogen [6]. Here, we describe the smaller (1–2 cm) compared to that in colonic ulcers (1–5 pathological characteristics in gastrointestinal candidiasis cm) (Figs. 1 and 2). Histopathologically, the stomach and encountered in an Aldabra giant tortoise. colonic ulcers were characterized by extensive necrosis, A female Aldabra giant tortoise was brought to Japan 10 hemorrhage, edema, and fibrinous exudate with infiltration years ago and kept in the Adventureworld Park, Wakayama of heterophils, lymphocytes, macrophages and occasional Prefecture, Japan. The animal was found dead in January, giant cells (Fig. 3). In HE-stained sections, numerous lightly 2007; necropsy was performed at the park. Clinically, the stained fungal organisms were seen throughout the ulcer- animal showed decrease in appetite for 2 months prior to ative areas. Both ovoid chlamidospores and aseptate sausage-like twisted pseudophyphae were positively stained *CORRESPONDENCE TO: YAMATE, J., Veterinary Pathology, Graduate School of Life and Environmental Sciences, Osaka Prefecture red by the PAS reaction (upper inset in Fig. 4) and brown- University, Rinkuu Ourai Kita 1–58, Izumisano-shi, Osaka, 598– black with the GMS stain (Fig. 4 and lower inset). Fungal 8531, Japan. organisms were often detected, penetrating into the deep e-mail: [email protected] lamina propria and muscle layer of stomach (Fig. 4). Fungi 1270 V. JUNIANTITO ET AL.

Fig. 1. Mutifocal ulcers seen in the stomach with marked hemorrhage (arrows). Bar=5 cm. Fig. 2. Mutifocal ulcers seen in the colon. Pseudomembrane and hemorrhage in the periphery are characteristically seen in the ulcerative foci (arrows). Bar=5 cm. Fig. 3. Necrotizing colitis characterized by completely denuded epithelia and numerous inflammatory cells. HE. Bar=200 m. Inset: heterophil infiltration within the ulcerated mucosa (arrows). Bar=30 m. Fig. 4. Fungus invasion into the muscular layer of stomach (arrows). GMS. Bar=100 m. Insets: long sausage-like twisted pseudohyphae (arrows) stained with GMS (lower) and PAS (upper) methods. Bar=30 m. Fig. 5. Immunohistochemistry with the antibody to Candida spp. Hyphae and chlamidospores are strongly positive to the antibody (arrows). Counterstained with hematoxylin. Bar=60 m. Fig. 6. Numerous protozoa seen in the superficial part of the ulcerated mucosa of colon (arrows). HE. Bar=50 m. Inset: cluster of GMS-positive protozoa (arrows). Bar=30 m. GASTROINTESTINAL CANDIDIASIS IN A TORTOISE 1271 invaded into the blood vessels, resulting in thrombosis. without forming ulcers [5]. Apparently, the size and shape Immunohistochemically, both fungal chlamidospores and of the present protozoa were similar to those of cryptospo- pseudohyphae were strongly reacted with the antibody ridia, although definite identification should be made by fur- against C. spp. (Fig. 5), but negative with the antibody to A. ther investigations. spp. In the colonic lesions, large numbers of protozoa, In conclusion, a female adult Aldabra giant tortoise, approximately 4 m in diameter and oval or round in shape, found dead, developed a necrotizing gastrointestinal candid- were seen, occasionally forming clusters (Fig. 6). The iasis, presumably due to an opportunistic infection of Can- organisms were distributed only in the superficial part of the dida spp. The death might be caused by dehydration or lesions, but not in the deep areas. The organisms were pos- hypoglycemia as a result of low feed intake due to the gas- itively stained with the GMS stain (inset in Fig. 6), but neg- tric and colonic ulcerations. This is the first report on gas- atively by the PAS reaction. trointestinal candidiasis in the Aldabra giant tortoise. Histopathological findings seen in other organs included hepatocyte degeneration, mild lipofuscin deposition in renal ACKNOWLEDGMENT. The authors would like to thank epithelia, catarrhal enteritis of the small intestines, and Dr. M. Kubo in National Institute of Animal Health (NIAH), white pulp atrophy in the spleen. Japan, who kindly provided the antibodies. Based on morphological and immunohistochemical findings, the present case was diagnosed as gastrointestinal can- REFERENCES didiasis; the reactivity of the fungal chlamisdopora and pseudohyphae for the special stains such as GMS and PAS 1. Cabanes, F. J., Alonso, J. M., Castella, G., Alegre, F., Dom- methods were consistent with those of candidiasis reported ingo, M. and Ponts, S. 1997. Cutaneous hyalohypomycosis in dogs [12, 15]. Additionally, the histopathological find- caused by Fusarium solani in a logger-head sea turtle (Caretta ings were similar to those of intestinal candidiasis reported caretta L.). J. Clin. Microbiol. 35: 3343–3345. 2. Frye, F. L. 1991. Nonhemic Protozoa. pp. 284–292 In: Reptile in a loggerhead sea turtle [17], although the anatomical loca- care. T.F.H. Publications, Neptune, New Jersey. tion differed in that in the present case ulcerative lesions 3. Gibson, C. W. D. and Hamilton, J. 1983. Feeding ecology and were found in the stomach and colon, whereas the lesions seasonal movements of giant tortoises on Aldabra atoll. Oeco- developed only in the duodenum of the sea turtle [17]. C. logia 56: 84–92. albicans and C. tropicalis have been reported to cause gas- 4. González-Cabo, J. F., Espejo-Serrano, F. and Bárcena-Asen- troenteritis in chelonian [24]. To identify the true pathogen, sio, M. C. 1995. Mycotic pulmonary disease by Beauveria culture should be performed. Therefore, the description of bessiana in a captive tortoise. Mycoses.38: 167–169. Candida spp. is more appropriate as the possible causative 5. Graczyk, T. K., Cranfield, M. R., Mann, J. and Strandberg, J. agent for the present case. D. 1998. Intestinal Cryptosporidium sp. infection in the Egyp- Besides candidiasis, Hyalohypomycosis [1, 16], Mucor- tian tortoise, Testudo kleinmanni. Intl. J. Parasitol. 28: 1885– 1888. mycosis [8], and Phaeohypomycosis [11], as well as infec- 6. Heard, D. J., Cantor, G. H., Jacobson, E. R., Purich, B., Ajello, tions due to Beauveria bessiana [4] and Colletotrichum L. and Padhye, A. A. 1986. Hyalohyphomycosis caused by acutatum [13] have been reported in the marine turtles so Paecilomyces lilacinus in an Aldabra tortoise. J. Am. Vet. Med. far. Mycoses in chelonian and reptiles has been considered Assoc. 189: 1143–1145. to be associated with multiple factors involving immunosu- 7. Hernandez-Divers, S. J. 2001. Pulmonary candidiasis caused pression, poor physical condition, prolonged administration by Candida albicans in a Greek tortoise (Testudo graeca) and of antibiotics, poor housing condition, poor water quality, treatment with intrapulmonary amphotericin B. J. Zoo. Wildl. malnutrition, sudden change of temperature, and sequence Med. 32: 352–359. from other organisms invasion such as bacteria and parasites 8. Jacobson, E. R., Calderwood, M. B. and Clubb, S. L. 1980. [9, 10, 18, 22]. Although basic reasons for the present case Mucormycosis in hatchling Florida softshell turtles. J. Am. Vet. Med. Assoc. 177: 835–837. were not decided, the presence of atrophy of lymphoid folli- 9. Jacobson, E. R., Clubb, S. L. and Greiner, E. 1983. Amebiasis cles in the spleen might indicate decreased immune in red-footed tortoises. J. Am. Vet. Med. Assoc. 183: 1192– response. 1194. The protozoa seen in the colonic lesions were considered 10. Jacobson, E. R., Cheatwood, J. L. and Maxwell, L. K. 2000. to be secondary to the ulceration, because the protozoa were Mycotic diseases of reptiles. Semin. Avian. Exotic. Pet. Med. 9: present only in the superficial of ulcers without deep pene- 94–101. tration into the lamina propria or muscular layer. The proto- 11. Joyner, P. H., Shreve, A. A., Spahr, J., Fountain, A. L. and zoa were different from amoeba species that have been Sleeman, J. M. 2006. Phaeohyphomycosis in a free-living east- reported in colonic ulcers in tortoise as the pathogen [18, ern box turtle (Terrapene carolina carolina). J. Wildl. Dis. 42: 19], in that the present protozoa were smaller in size and 883–888. 12. Kuwamura, M., Ide, M., Yamate, J., Shiraishi, Y. and Kotani, negative by the PAS reaction. Tortoise and turtles are T. 2006. Systemic candidiasis in a dog, developing spondylitis. served as carriers for Entamoeba [2], Coccidia [14] and J. Vet. Med. Sci. 68: 1117–1119. Cryptosporidium species [23]. The cyptosporidia has been 13. Manire, C. A., Rhinehart, H. L., Sutton, D. A. Thompson, E. reported in the Egyptian tortoise, however the organism was H., Rinaldi, M. G., Buck, J. D. and Jacobson, E. 2002. Dissem- distributed only in the apical part of the intestinal epithelia inated mycotic infection caused by Colletotrichum acutatum in 1272 V. JUNIANTITO ET AL.

a Kemp’s ridley sea turtle (Lepidochelys kempi). J. Clin. 158: 567–569. Microbiol. 40: 4273–4280. 20. Ruiz-Sánchez, D., Calderón-Romero, L., Sánchez-Vega, J. T. 14. Mc Allister, C. T. and Upton, S. J. 1989. The coccidia (Api- and Tay, J. 2002. Intestinal candidiasis. A clinical report and complexa: Eimeridae) of the testudines, with descriptions of comments about this opportunistic pathology. Mycopathologia three new species. Can. J. Zool. 67: 2459–2467. 156: 9–11. 15. Ochiai, K., Valentine, B. A. and Altschul, M. 2000. Intestinal 21. Swingland, I. R. 1977. Reproductive effort and life history candidiasis in a dog. Vet. Rec. 146: 228–229. strategy of the Aldabran giant tortoise. Nature 269: 402–404. 16. Orós, J., Ramírez, A. S., Poveda, J. B., Rodríguez, J. L. and 22. Tangredi, B. P. and Evans, R. H. 1997. Organochlorine pesti- Fernández, A. 1996. Systemic mycosis caused by Penicillium cides associated with ocular, nasal, or otic infection in the east- griseofulvum in a Seychelles giant tortoise (Megalochelys ern box turtle (Terrapene carolina carolina). J. Zoo. Wildl. gigantea). Vet. Rec. 139: 295–296. Med. 28: 97–100. 17. Orós, J., Arencibia, A., Fernández, L. and Jensen, H. E. 2004. 23. Traversa, D., Iorio, R., Otranto, D., Modrý, D. and Slapeta, J. Intestinal candidiasis in a loggerhead sea turtle (Caretta 2008. Cryptosporidium from tortoises: genetic characteriza- caretta): an immunohistochemical studies. Vet. J. 167: 202– tion, phylogeny, and zoonotic implication. Mol. Cell. Probes. 207. 22: 122–128. 18. Ozaki, K., Matsuo, K., Tanaka, O. and Narama, I. 2000. Amoe- 24. Zwart, P. and Buitelaar, M. 1981. Candida tropicalis: enteric bosis in the Flat-shelled spider tortoise (Acinixys planicauda). infections and their treatment in chelonians. pp. 58–59. In: Pro- J. Comp. Pathol. 123: 299–301. ceedings of the American Associations of Zoo Veterinarians, 19. Philbey, A. W. 2006. Amoebic enterocolitis and acute myone- 1980, Washington, DC. crosis in leopard tortoises (Geochelone pardalis). Vet. Rec.