Signalment:  

2-year-old female green anaconda, (Eunectes murinus).The snake was kept in a zoological garden and died suddenly.

Gross Description:  

The serosal surface of posterior half of small intestine was severely congested and the mucosal surface was diffusely covered with fibrinonecrotic exudate. The mucosa of large intestine had severe edema with mild fibrinous exudate on the surface.

Histopathologic Description:

The normal structure of small intestine is largely lost but a portion of the lamina propria and tunica muscularis retains a normal lymphoid follicle. The lumen of small intestine is filled with fibrinonecrotic debris and the intestinal wall is diffusely and transmurally necrotic with moderate hemorrhage and edema. Smooth muscle fibers in the tunica muscularis are separated by edema and hemorrhage. Diffusely, macrophages and heterophils infiltrate the submucosa and extend to the serosal surface. Macrophages often contain hemosiderin and there are scattered multinucleated giant cells. Numerous amoebic trophozoites are admixed with multifocal aggregates of bacteria within the luminal necrotic debris. Amoebic trophozoites are also present within the submucosa and tunica muscularis; they are often vacuolated and rarely contain cellular debris. The trophozoites average 16 μm and range from 10 to 27 μm in diameter. They are positive for PAS stain. Cysts are also occasionally noted in and around the serosa. There is also multifocal vasculitis with thrombi and/or necrotic debris occluding the lumen. Serosal blood vessels are severely congested and occasionally contain amoebic trophozoites. 

Morphologic Diagnosis:  

Small intestine: Enteritis, fibrinonecrotizing, transmural, acute, diffuse, severe, with numerous amoebic trophozoites.

Condition:  

Entamoeba invadens

Contributor Comment:  

Based on the species and histopathology as well as their size and morphology, amoebic trophozoites were tentatively identified as Entamoeba sp., an obligate intracellular parasite with a direct life cycle.(4) E. invadens is one of the most significant protozoal parasites of reptiles, especially captive snakes and lizards.(2) E. invadens trophozoites range from 10 to 35 μm in diameter and the cysts range from 10 to 20 μm, with up to four nuclei.(6) Although the trophozoites and quadrinucleated cysts described above are morphologically indistinguishable from the primate amoeba, E. histolytica, E. invadens prefers a host or culture temperature of less than 31^oC whereas E. histolytica evolves to thrive at 37^oC.(2)

Herbivorous turtles are thought to serve as the natural host for E. invadens. In turtles, the parasite likely lives as a commensal symbiont without any pathogenicity.(2) While in the trophozoite state, these protozoa locomote and feed by forming pseudopodia. The resistant cyst stage is excreted in the feces. Following contamination of the water supply of snakes and lizards, the protozoa are ingested and induce a fulminating enteritis and hepatitis in these species. The clinical signs include regurgitation of undigested food and severe diarrhea, occasionally accompanied by blood- or bile-tinged green mucus, and/or remnants of intestinal mucosa. 

The most characteristic microscopic lesion induced by E. invadens is severe intestinal erosion, ulceration and inflammation, often with formation of fibrinonecrotic pseudomembranes (diphtheritic enteritis).(2,6) Typically, the ileum and colon are the most severely affected intestinal segments and the affected gut wall is severely thickened. The protozoa invade blood vessels, spread systemically and induce necrosis and inflammation in various extra-intestinal tissues, especially the liver. 

This anaconda has typical diphtheritic ileitis with numerous amoebic trophozoites and similar (though less severe) inflammation is observed in the large intestine. Although the protozoa often migrate in the sinusoids of liver and occasionally in the vessels of lung and kidney, there is no apparent damage to these organs in this case. We conferred with a veterinarian in the zoological garden in an attempt to elucidate the mechanism of infection in this case and were unable to reach a definitive conclusion; however, we suspect that the anacondas water supply was contaminated by turtle feces. 

Immunosuppression has been suggested as an important predisposing factor in snake Entamoeba infections. There is a recent report of diphtheritic colitis caused by an amphibian Entamoeba sp. in a Boa constrictor kept in a zoological garden.(6) Histopathological changes in this case were limited to the large intestine, implying that this Entamoeba may have a lower pathogenicity than E. invadens. As the snake suffered simultaneously from endoparasitism and inclusion body disease, the animal was suspected to be in an immunocompromised state. Our case also demonstrated moderate atrophy of lymphoid follicles in spleen, which may be a result of inadequate nutrition; however, the details are unclear.

JPC Diagnosis:  

1. Intestine: Enteritis, fibrinonecrotic, circumferential, diffuse, severe, with numerous amoebic trophozoites. 
2. Mesenteric vessels: Vasculitis, necrotizing, multifocal, moderate.

Conference Comment:  

Entamoeba spp. is a protozoan parasite belonging to the phylum Sarcomastigophora, subphylum Sarcodina (Rhizopoda), order Amoebida, family Entamoebidae, genus Entamoeba.(3) Amoebae are normal, nonpathogenic inhabitants of the large bowel lumen in many species. Disease occurs upon invasion of the intestinal mucosa, and severity is influenced by diet, immune status, and the particular strain and virulence of the organism.(8) E. invadens and E. histolytica are two commonly important species in veterinary medicine, both of these amoebae have similar life cycles with two morphologically distinct stages. The labile trophozoite inhabits the host and is capable of locomotion, while the resistant quadrinucleate cyst form is protected by a cell wall and is able to survive under unfavorable environmental conditions. The cyst is infectious, and releases the motile trophozoite following ingestion by the host.(1,3) Virulence factors of Entamoeba spp. include 1) a Gal/GalNAc-specific lectin, which facilitates trophozoite adhesion to intestinal epithelial cells and may contribute to amoebic resistance to complement, 2) pore-forming polypeptides called amoebapores that insert a channel into the host cell membrane, leading to cell lysis, and 3) a family of cysteine proteases that function to break down the extracellular matrix, allowing amoebic tissue invasion.(1,5)

The contributor provides an excellent summary of Entamoeba invadens in reptiles. Chelonians and crocodilians are considered to be the natural hosts of E. invadens and may serve as a reservoir for infection in snakes and lizards in captivity. As noted by the contributor, transmission is typically feco-oral (probably related to contamination from turtle feces) and the most common presentation of invasive amoebiasis in reptiles is enteritis, often with subsequent hepatitis following hematogenous dissemination via the portal vein. After entering the mesenteric circulation, trophozoites may disseminate to other organs, although this is uncommon. There is a single report of amoebic myositis in a water monitor (Varanus salvator), presumably due to either hematogenous spread or direct invasion via skin wounds.(1)

Entamoeba histolytica is the etiologic agent of amoebic dysentery in humans, with an incidence of up to 500 million clinical cases per year, including up to 100,000 fatalities.(5) E. histolytica is distributed worldwide among humans, but also occurs in a broad range of New and Old World monkeys and apes, and can be transmitted to dogs, cats, cattle and macropods; the pathogenesis is similar to E. invadens.(5,7,8) In Old World monkeys, amoebic dysentery typically induces necrotizing colitis, with occasional dissemination to the liver or (rarely) other tissues. Conversely, in certain species of leaf-eating monkeys, including colobus monkeys, silver leaf monkeys, douc langurs and proboscis monkeys, fibrinonecrotizing gastritis is the principle lesion associated with E. histolytica. It is thought that the normal neutral pH within these gastric compartments provides a favorable environment for excystation of ingested E. histolytica, followed by tissue invasion.(7) E. histolytica can be transmitted to dogs, cats and cattle, where it typically causes colitis;(3) gastric amoebiasis has been reported in a wallaby, a species with a complex, sacculated stomach adapted for fermentation that is similar to that of leaf-eating monkeys.(7)

In addition to severe fibrinonecrotic enteritis, conference participants found the multifocal necrotizing vasculitis and thrombosis within adjacent mesenteric vessels particularly striking. The etiology of this finding is unclear; discussion centered upon the possibility of direct damage from amoebic trophozoites versus the potential of a concomitant gram-negative septicemia (i.e. salmonellosis) with subsequent vasculitis. 

References:

1. Chia MY, Jeng CR, Hsiao SH, Lee AH, Chen CY, Pang VF. Entamoeba invadens myositis in a common water monitor lizard (Varanus salvator). Vet Pathol. 2009;46(4):673-676.

2. Frye F. Reptile Care: An Atlas of Diseases and Treatments. Vol. I. Neptune City, NJ: T.F.H. Publications; 1991:278-285.

3. Gardiner CH, Fayer R, Dubey JP. An Atlas of Protozoal Parasites in Animal Tissues. 2nd ed. Washington, DC: Armed Forces Institute of Pathology; 1998:10-11.

4. Gelberg, HB. Alimentary system and the peritoneum, omentum, mesentery, and peritoneal cavity. In: Zachary JF, McGavin MD, eds. Pathologic Basis of Veterinary Disease. 5th ed. St. Louis: Elsevier; 2012:395.

5. Petri, WA Jr. Intestinal invasion by Entamoeba histolytica. Subcell Biochem. 2008;47:221-232.

6. Richter B, K+�-+bber-Heiss A, Weissenb+�-�ck H. Diphtheroid colitis in a Boa constrictor infected with amphibian Entamoeba sp. Vet. Parasitol. 2008;153:164-167.

7. Stedman NL, Munday JS, Esbeck R, Visvesvara GS. Gastric amebiasis due to Entamoeba histolytica in a Dama Wallaby (Macropus eugenii). Vet Pathol. 2003;40(3):340-342.

8. Strait K, Else JG, Eberhard ML. Parasitic diseases of nonhuman primates. In: Abee CR, Mansfield K, Tardif S, Morris T, eds. Nonhuman Primates in Biomedical Research: Diseases. London, UK: Academic Press; 2012:206-208.

Click the slide to view.

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4-1. Ileum

4-2. Intestine

4-3. Intestine

4-3. Intestine

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