History

An 18-year-old sexually intact male ball python (Python regius) housed at a local Indiana zoo was found moribund without prior clinical signs and was transported to the Animal Disease Diagnostic Laboratory at Purdue University. On arrival at the diagnostic laboratory, the snake was alive, yet extremely lethargic; it was placed in a carbon dioxide–filled chamber until completely immobile and unresponsive followed by decapitation prior to necropsy.

Clinical and Gross Findings

Caretakers of the snake did not note any clinical signs prior to the sudden onset of lethargy. At necropsy, the snake was lean with atrophied fat bodies; the gastrointestinal tract was devoid of ingesta. A 7-cm-long segment of the distal portion of the colon was transmurally and circumferentially thickened and firm with a red to dark purple serosal surface; petechiae were scattered in the adjacent serosa overlying fat bodies (Figure 1). The affected colonic mucosa was friable and covered by a layer of dark brown to tan material. The liver was mottled tan to red.

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Photographs of the coelomic cavity (A) and opened colon (B) of an 18-year-old lean ball python (Python regius) that was found moribund without prior clinical signs and was euthanized. The head is to the left. In panel A, the colon is segmentally thickened, firm, and dark red (arrow); petechiae are scattered in the adjacent serosa overlying fat bodies. The liver is mottled tan to red (arrowheads). T = Trachea. In panel B, the colonic mucosa is covered by a layer of dark brown to tan material.

Citation: Journal of the American Veterinary Medical Association 245, 5; 10.2460/javma.245.5.501

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Histopathologic, Microbiological, and Immunofluorescence Test Findings

Samples of the colon, liver, heart, small intestine, stomach, lung, trachea, kidneys, and brain were routinely processed for histologic examination. No lesions were noted in the examined sections of heart, stomach, trachea, and brain tissue. The colonic wall was transmurally effaced and distended by a coagulum of eosinophilic necrotic cellular debris, fibrin, edema, and scattered amoebic trophozoites; colonies of coccobacilli; and few erythrocytes (Figure 2). Colonic inflammation was mild and predominantly histiocytic with fewer lymphocytes, plasma cells, and heterophils. Amoebic trophozoites were round (20 μm in diameter) with a nucleus, karyosome, and abundant vacuolated cytoplasm. Colonic trophozoites were present in both intravascular and extravascular locations. In all layers of the colonic wall, multiple blood vessel walls were expanded by eosinophilic fibrillar material (fibrinoid change) or were encircled by fibrin and some vascular lumina were occluded by fibrin thrombi.

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Photomicrographs of a section of colon from the ball python in Figure 1. A—The colonic wall is transmurally effaced and distended by a coagulum of eosinophilic necrotic cellular debris, fibrin, edema, and a mild, predominantly histiocytic inflammatory cell infiltrate. Multifocally, inflammatory cell infiltrate, fibrin, and edema coat the eroded mucosal surface and extend into the colonic lumen. There is fibrinoid change in the walls of multiple submucosal vessels (arrow). H&E stain; bar = 1 mm. Lower left inset—At higher magnification, details of vascular changes and inflammatory cell infiltrate are evident. H&E stain; bar = 14 μm. Upper right inset—Notice that colonies of coccobacilli separate adjacent collagen fibers in the submucosa. H&E stain; bar = 28.5 μm. B—A colonic blood vessel contains multiple intraluminal, round (approx 20-μm-diameter) amoebic trophozoites (arrows) with vacuolated cytoplasm and a basophilic karyosome. The vessel wall (asterisk) and lumen (solid circle) are indicated. H&E stain; bar = 28.5 μm. Inset—On the basis of immunofluorescence staining, the amoebic trophozoites are identified as Entamoeba invadens. Fluorescein isothiocyanate–labeled anti–E invadens antibody; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 245, 5; 10.2460/javma.245.5.501

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Microscopic examination of liver tissue revealed randomly distributed multifocal to submassive coagulative hepatocellular necrosis. Hemorrhage mixed with edema, fibrin, hemosiderophages, and coccobacilli surrounded necrotic hepatocytes. Fibrinoid change was present in central veins and portal blood vessels in areas of hepatocellular necrosis. Amoebic trophozoites were scattered in hepatic sinusoids and blood vessels. Multiple sinusoids were distended by lipogranulomas composed of macrophages with abundant foamy light brown cytoplasm.

Amoebic trophozoites in liver and colon samples reacted positively with Giemsa, Gomori methenamine silver, and periodic acid–Schiff stains. Paraffin sections of these tissues underwent fluorescent antibody testing with antibodies targeting Acanthamoeba castellanii, Balamuthia mandrillaris, Entamoeba histolytica, and Entamoeba invadens; these tests were performed by the Amoeba Laboratory of the CDC Waterborne Disease Prevention Branch. Amoebae in the colon and liver only reacted with fluorescein isothiocyanate–labeled antibody specific for E invadens. Salmonella enterica subsp arizonae was cultured from tissue samples of the small and large intestines, liver, kidneys, and lungs. Microscopically, rare small intestinal erosions were coated by coccobacilli that infiltrated into an underlying hemorrhagic, fibrinous, and edematous lamina propria. There was no histologic evidence of Salmonella-associated renal or pulmonary lesions in the examined sections.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: severe, transmural, fibrinonecrotizing colitis and submassive necrotizing hepatitis with amoebic trophozoites and coccobacilli.

Case summary: E invadens infection in a ball python.

Comments

For the snake of the present report, a presumptive diagnosis of infection with E invadens was made on the basis of the animal species affected, the gross and histologic lesions, and the presence of intralesional amoebic trophozoites; however, a definitive diagnosis relies on results from an immunofluorescence or PCR assay because nonpathogenic commensal amoeba can be present in reptiles.¹ Amoebic trophozoites in liver and colon from this snake were definitively identified as E invadens by immunofluorescence assay.

Entamoeba invadens infections in snakes are often pathogenic; however, low environmental temperatures can result in a carrier state.² Snakes are infected by ingesting amoebic cysts present in the environment; once cysts are in the gastrointestinal tract, they develop into trophozoites, which can invade colonic mucosa and hematogenously spread to the liver and other viscera.^1,3 Anorexia, regurgitation, dehydration, lethargy, and death with no prior clinical signs have all been described for snakes with amebiasis.^4,5 Affected snakes often develop fibrinonecrotizing to hemorrhagic colitis and necrotizing hepatitis with intralesional amoebic trophozoites.^3,4 Entamoeba invadens has also caused disease in lizards, chelonians, and crocodiles; however, chelonians and crocodiles can act as subclinical carriers of this organism.⁶ Gross and microscopic lesions in these reptile species have included necrotic colitis, enteritis, and hepatitis⁶; there is a single report⁷ of myositis in a common water monitor lizard.

Although E invadens was the cause of the intestinal disease in the snake of this report, other differential diagnoses for the gross lesions in the colon included Salmonella infection, lymphoma, or adenocarcinoma. Salmonella spp can also cause bacteremia with subsequent necrotizing hepatitis, or snakes can serve as subclinical carriers of this bacterium.⁸ Cryptosporidium serpentis, the cause of proliferative gastritis, or boid inclusion body disease, which is associated with regurgitation and neurologic signs, can also cause gastrointestinal signs in snakes.^9,10

For the ball python of the present report, the gastrointestinal tract was considered the portal of entry for the amoebae, which then likely spread to the liver via portal circulation. Amoebic trophozoites can severely damage colonic mucosa and allow secondary bacterial infections to develop.³ In the snake described in this report, S enterica subsp arizonae was cultured from samples of multiple visceral organs, including the colon and liver. Salmonella enterica subsp arizonae likely contributed to lesion development as a secondary invader and may have facilitated systemic distribution of the amoeba.

Reports of infections with E invadens often involve outbreaks affecting multiple snakes in captivity, particularly pythons and boas, and these protozoal parasites are often suspected to originate from unaffected carriers such as turtles or alligators in the same, or nearby, enclosures.^4,11 The origin of E invadens infection in the ball python of the present report could not be determined. The snake was not known to be exposed to turtles or alligators, and an additional snake housed with the affected ball python and other snakes and lizards in nearby enclosures did not develop entamebiasis.