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CASE 11 - SLIDE # 59350 - 9 CONTRIBUTORS/ INSTITUTION: Teresa Southard and Richard J. Montali, Department of Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore MD Melody Roelke, Catoctin Wildlife Reserve and Zoo, Thurmont MD SIGNALMENT: Adult male carpet python (Morelia spilota) HISTORY: The snake was given to the Catoctin Mountain Zoo in the summer of 2006. It was initially individually housed and then added to a group of snakes in April of 2007. The snake stopped feeding and was found dead on June 17, 2007. GROSS FINDINGS: Distal to the gall bladder, the small intestinal mucosa was hyperemic and covered by a fibrinonecrotic pseudomembrane. The mucosal changes extended to the distal colon, where there was a 7 x 3.5 x 3 cm firm mass in the mucosa, with multifocal areas of necrosis. HISTOPATHOLOGIC FINDINGS: Colon: The section of colon is characterized by mucosal necrosis and transmural inflammation. The surface epithelium is denuded and the lamina propria and segments of the muscularis mucosa are necrotic and contain mixed bacterial colonies, fibrin and cellular debris. This zone of necrosis extends in to the superficial submucosa. Small vessels in the submucosa often contain fibrin thrombi and larger vessels in the submucosa and serosa are dilated and surrounded by infiltrates of lymphocytes and macrophages, with fewer plasma cells and heterophils. Mixed with the inflammatory cells and necrotic debris in the mucosa and submucosa are multiple round amoeba trophozoites, 11-15 um in diameter, with granular amphophilic cytoplasm and small, round, basophilic nuclei. MORPHOLOGIC/ETIOLOGIC DIAGNOSIS: Colon: colitis, necrotizing, segmental, severe, mucosa and submucosa, with intralesional amoebae, bacterial colonies and thrombosis, etiology consistent with Entamoeba invadens. DISCUSSION: Entamoeba invadens is one of the most common causes of gastrointestinal disease in snakes (Kojimoto et al., 2001). E. invadens can also cause disease in other reptiles, although chelonians, and possibly crocodilians, are more likely to be asymptomatic carriers. Snakes in zoo settings are often infected by E. invadens when they are exposed to or co-housed with chelonians (Donaldson et al., 1975). Transmission of E. invadens is fecal-oral, and the infectious form is an 11-20 um cyst with four nuclei. Once ingested, the cyst passes through the stomach and excystment occurs in the small intestine. The four nuclei replicate resulting in a form with 8 nuclei, which divides to form 8 small amebulae. The amebulae grow into mature 10-15 um trophozoites, which have a single nucleus and a central endosome with a ring of peripheral granules beneath the nuclear membrane. Trophozoites multiply in the surface mucus layer of the colon, and either invade the mucosa or encyst and pass out in the feces. Trophozoites can also migrate outside of the gastrointestinal tract and cause disease in the liver and other organs. The chemical environment in the colonic lumen, including concentration of short chained fatty acids (Byers et al., 2005), calcium ions (Makioka et al., 2002), and catecholamines (Coppi et al., 2002) is thought to regulate the processes by which the amoeba excyst and encyst. Entamoeba spp. are also pathogens of primates, canids and swine (E. histolytica), horses (E. equi), ducks (E. anatis) and amphibians (E. ranarum). E. invadens is the only one of these species which can be induced to encyst and excyst in culture, and therefore is used as a model to study human amebiasis. Although the morphology of the protozoa identified in the colon of this snake is consistent with Entamoeba invadens, a definitive diagnosis requires immunohistochemistry. In a study of 51 snakes with diagnosed or suspected amebiasis, less than half of the diagnoses were confirmed with an antibody to E. invadens. The protozoa in the remaining cases were identified as flagellates using electron microscopy (Jakob and Wesemeier, 1995). This snake may have been predisposed to colitis and protozoal invasion because of a large mass in the distal colon which was likely obstructing the lumen. The tumor was composed of nests of round cells containing abundant PAS- and mucicarmine-positive foamy eosinophilic material, separated by a fine fibrovascular stroma. Special stains are pending to further define this mucusproducing neoplasm. REFERENCES: Byers J, Faigle W, Eichinger D. 2005. Colonic short-chain fatty acids inhibit encystation of Entamoeba invadens. Cell Microbiol. Feb;7(2):269-79. Coppi A, Merali S, Eichinger D. 2002. The enteric parasite Entamoeba uses an autocrine catecholamine system during differentiation into the infectious cyst stage. J Biol Chem. 2002 Mar 8;277(10):8083-90 Donaldson M, Heyneman D, Dempster R, Garcia L. 1975. Epizootic of fatal amebiasis among exhibited snakes: epidemiologic, pathologic, and chemotherapeutic considerations. Am J Vet Res. 36(6):807-17. Jakob W, Wesemeier HH. 1995. Intestinal inflammation associated with flagellates in snakes. J Comp Pathol. 112(4):417-21. Kojimoto A, Uchida K, Horii Y, Okumura S, Yamaguch R, Tateyama S. 2001. Amebiasis in four ball pythons, Python reginus. J Vet Med Sci. 63(12):1365-8. Makioka A, Kumagai M, Kobayashi S, Takeuchi T. 2002. Possible role of calcium ions, calcium channels and calmodulin in excystation and metacystic development of Entamoeba invadens Parasitol Res. 2002 Sep;88(9):837-43. ACKNOWLEDGMENTS: This work supported by NIH training grant RR07002. Thanks to Patricia Wilcox who provided histology services for this case.