JPC SYSTEMIC PATHOLOGY
DIGESTIVE SYSTEM
October 2021
D-P03

 

SLIDE A: Signalment (JPC #2317341):  Pilot black snake 

 

HISTORY:  This snake was found dead after a brief period of inactivity.

 

HISTOPATHOLOGIC DESCRIPTION:  1.  Liver:  Affecting 70% of the section are random, multifocal to coalescing areas of coagulative necrosis (characterized by loss of differential staining with retention of tissue architecture), lytic necrosis (characterized by loss of tissue architecture with replacement by karyorrhectic and cellular debris, hemorrhage, few heterophils, macrophages, and lymphocytes), and hepatocyte degeneration (characterized by cytoplasmic swelling and vacuolization). There are multifocal areas of hepatocellular loss and Kupffer cell hyperplasia.  Multifocally necrotic areas contain many extracellular, intravascular, or intrahistiocytic 10-20um diameter amoebic trophozoites with a thin cell wall, abundant granular to vacuolated basophilic cytoplasm with rare phagocytized necrotic debris, and a 5-7um round to oval nucleus with marginated chromatin and a lightly basophilic karyosome.  There are scattered colonies of 1 x 2um coccobacilli.  Multifocally the tunica media and tunica adventitia of blood vessels are expanded and replaced by heterophils and eosinophilic and karyorrhectic cellular debris (vascular necrosis), and contain few previously described amoebic trophozoites and inflammatory cells.

 

2. Mesentery: The fibroadipose tissue is moderately expanded by clear space and dilated lymphatics (edema), fibrin, hemorrhage, few degenerate heterophils, and karyorrhectic debris (lytic necrosis).  Multifocally, there are many scattered colonies of coccobacilli.

 

3. Kidney with spermatic ductules: Multifocally separating and surrounding tubules are variably sized areas of hemorrhage, fibrin, and edema.  There are multifocal areas of vascular necrosis of intermediate and large vessels, and rare ameobic trophozoites within or adjacent to affected vessels.  Diffusely, tubular epithelium is sloughed or elevated off the basement membrane (autolysis).

 

MORPHOLOGIC DIAGNOSIS:  1.  Liver:  Hepatitis, necrotizing, random, acute, multifocal to coalescing, moderate, with vascular necrosis, and extracellular and intrahistiocytic amoebic trophozoites and coccobacilli, pilot black snake (Elaphe obsoleta), ophidian.

2. Mesentery: Steatitis, necrotizing, acute, diffuse, moderate, with hemorrhage.
3. Kidney: Hemorrhage, multifocal, moderate, with vascular necrosis and rare amoebic trophozoites.

 

ETIOLOGIC DIAGNOSIS: Amoebic hepatitis

 

CAUSE: Entamoeba invadens

 

SLIDE B: Signalment (JPC #1296472):  Snake

 

HISTORY:  None.

 

HISTOPATHOLOGIC DESCRIPTION:  Colon:  There is diffuse loss of mucosal architecture with replacement by a coagulum of fibrin, hemorrhage, and eosinophilic and karyorrhectic cellular debris (lytic necrosis), admixed with  few macrophages, heterophils, lymphocytes, and plasma cells, as well as moderate numbers of 10-20um diameter amoebic trophozoites with a thin cell wall, abundant granular to vacuolated basophilic cytoplasm, and a 5-7um round to oval nucleus with marginated chromatin and a lightly basophilic karyosome.  Necrosis, inflammation, and amoebic trophozoites extend into the submucosa, tunical muscularis and serosa.  Transmurally, the colon is expanded by clear space and dilated lymphatics (edema).  Multifocally blood vessel walls within necrotic areas are discontinuous and replaced with necrotic debris, fibrin, and edema (vascular necrosis) and often contain small fibrin thrombi and/or amoebic trophozoites.  There is diffuse mesenteric fat atrophy.    

 

MORPHOLOGIC DIAGNOSIS:  Colon:  Colitis, necrohemorrhagic, acute, diffuse, severe, with many extracellular and intravascular amoebic trophozoites, edema, and mesenteric fat atrophy, snake, ophidian.

 

ETIOLOGIC DIAGNOSIS:  Amoebic colitis

 

CAUSE:  Entamoeba invadens

 

Signalment (JPC #2317380):  Slide C & D:  Golden lion tamarin (Leontopithecus rosalia)

 

HISTORY:  None.

 

SLIDE C: HISTOPATHOLOGIC DESCRIPTION:  1.  Colon:  Approximately 70% of the mucosa is characterized by loss of mucosal architecture with replacement by abundant eosinophilic and karyorrhectic cellular debris and fibrin (lytic necrosis) admixed with neutrophils, lymphocytes, macrophages, hemorrhage, edema, and numerous colonies of 1 x 2um basophilic bacilli.  Necrosis and inflammation multifocally extends through the muscularis mucosa into the submucosa.  Within necrotic areas, there are few 10-25um diameter amoebic trophozoites with clumped to globular basophilic cytoplasm and an eccentric, 3-4um diameter nucleus with a karyosome.  Colonic crypts are variably necrotic with epithelial cells that are sloughed or shrunken with hypereosinophilic cytoplasm and pyknotic nuclei, or ectatic and filled by abundant eosinophilic cellular and karyorrhectic luminal debris (crypt abscesses) admixed with numerous filamentous bacteria.  There is diffuse mesenteric fat atrophy.

 

2. Duodenum: Focally within an ectatic submucosal lymphatic, there is a cross section of a larval nematode surrounded by few degenerate inflammatory cells.  The nematode is 40um in diameter and has a 1-2um cuticle, paired lateral alae, coelomyarian-polymyarian musculature, an indistinct digestive tract, and lacks a reproductive tract. 

 

3. Multiple sections throughout the esophagus, stomach, and small intestine: No significant lesions.

 

SLIDE D:  PAS:  Colon:  There are multiple PAS-positive amoebic trophozoites within the necrotic mucosa. 

 

MORPHOLOGIC DIAGNOSIS:  1. Colon:  Colitis, necrotizing, subacute, multifocal, moderate, with crypt necrosis, crypt abscesses, few PAS-positive amoebic trophozoites  and multifocal colonies of bacilli, golden lion tamarin (Leontopithecus rosalia), nonhuman primate.

2. Duodenum, lymphatic: Larval nematode, focal.

 

ETIOLOGIC DIAGNOSIS:  Amoebic colitis

 

CAUSE:  Entamoeba histolytica

 

CONDITION:  Amoebiasis

 

GENERAL DISCUSSION:

• Entamoeba are obligate protozoan parasites with direct life cycle Entamoeba invadens

• Causes ulcerative colitis and hepatitis with high morbidity and mortality in snakes and lizards
• Clinically affects many snake families (Boidae, Pythonidae, Colubridae, Elaphidae, Hydrophidae, and Viperidae); giant tortoises; leopard tortoises
• Garter snakes, northern black racers, and box turtles are common carriers; rarely clinically affected
• Most turtles and crocodiles are resistant; serve as reservoirs Entamoeba histolytica

• Causes amebic dysentery in humans and nonhuman primates (especially Old World), and rarely infects other species (dogs, cats, pigs, cattle)
• Many infections are asymptomatic
• Zoonotic in human beings, nonhuman primates, dogs, cats, and other animals

 

PATHOGENESIS:

• Amoebae are usually nonpathogenic inhabitants of the large bowel lumen
• Diet, immune status, and parasite species and virulence influence pathogenicity
• Contact of amoeba and host cells likely mediated by glycoproteins, carbohydrate chains, and adhesins; soluble factors produced by amoebae mediate pathogenicity:
  • Cysteine proteases result in epithelial cell damage and inflammation
  • Amoebic contact with host cells leads to cytolysis
• Ingestion > amoebae attach to GI epithelium > trogocytosis of epithelial cell by amoebae> elevates intracellular calcium > epithelial cell death > invade crypts of colonic glands > burrow into lamina propria > invade submucosa > (in reptiles > portal vein > liver)
• The trophozoite first adheres to the intestinal mucus and epithelial cells by a Gal/GalNAc-specific lectin
• Pore-forming polypeptides called amoebapores (channel forming proteins) are released by the trophozoite
• Pathogenic amoebae are erythrophagocytic
• Trogocytosis allows amoebae to acquire/display host cell membrane proteins which confers protection from lysis by complement; leads to invasive disease in humans

 

LIFE CYCLE:

• Direct; rapid spread of infection
• Fecal-oral transmission via contaminated food or water, arthropod vectors (flies, roaches)
• Ingestion of infective cysts > develop into motile trophozoites in the intestine > trophozoites multiply by binary fission > invade mucosa or are transformed into cysts and pass in the feces
• Trophozoites may disseminate to brain, liver, and other organs through blood vessels and lymphatics and produce large, lytic lesions (amoebic abscesses)

 

TYPICAL CLINICAL FINDINGS:

• Bloody and mucoid diarrhea
• Anorexia, dehydration, lethargy, abdominal pain
• Neurological signs such as seizures and ataxia if CNS involvement

 

TYPICAL GROSS FINDINGS:

• Necrohemorrhagic and ulcerative colitis frequently with a fibrinonecrotic membrane, +/- enteritis, gastritis
• Thickened and friable intestinal wall, covered by a necrotic membrane
• Lumen filled with blood, necrotic debris, and mucus
• Necrosis and abscesses in brain, liver, lungs, and kidneys

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Diffuse necrohemorrhagic colitis; erosion and ulceration; necrosis down to the muscularis mucosa
• Flask-shaped ulcers in colon, with a narrow neck through the mucosa and a broad base in the submucosa
• Amoebae commonly present in small clusters in mucus on colonic surface, in necrotic exudate, and in adjacent viable tissue
• histolytica: Spherical to irregular surrounded by a clear halo; 10-50 um in diameter; nucleus with a central dense karyosome and chromatin plaques at the periphery; light staining, granular cytoplasm with remnants of erythrocytes and glycogen (PAS positive)
• invadens: Similar to E. histolytica, but 10-35 um with darker cytoplasm and agranular at one pole

 

ADDITIONAL DIAGNOSTIC TESTS:

• Demonstration of cysts and/or trophozoites in fecal smears or flotation
• Histochemical stains: PAS, GMS 
• PCR
• Culture
• Immunohistochemistry
• Indirect immunofluorescence

 

DIFFERENTIAL DIAGNOSIS:

Other causes of colitis in primates:

• Shigella flexneri, S. sonnei: Necrohemorrhagic colitis; differentiate with culture
• Salmonella enteritidis, S. typhimurium: Necrotizing, suppurative enterocolitis, (paratyphoid nodules) and possibly  septicemia; pyogranulomas in other organs
• Campylobacter jejuni, coli: Lesions usually less severe; affects both small and large intestine; spiral bacteria evident with silver stains; colonic mucosa sometimes hyperplastic
• Yersinia enterocolitica, Y. pseudotuberculosis: Necroulcerative enterocolitis; large colonies of gram-negative bacteria in necrotic centers nearly always diagnostic
• Balantidium coli: Ulcerative colitis; ciliated trophozoites 40-60um in diameter; kidney-shaped macronucleus

Other causes of gastroenteritis in snakes:

• Salmonella
• Proteus
• Coccidia (Eimeria, Isospora spp., Caryospora spp.)

 

COMPARATIVE PATHOLOGY:

Entamoeba sp. in other veterinary species:

• Dogs and cats: Clinical disease rare, signs and lesions similar to those in primates; concomitant infection with Trichuris or Ancylostoma makes disease more severe; dissemination rare, usually with immunosuppression (i.e. distemper infection); usually infected via cysts in human feces; dogs and cats rarely pass cysts in feces
• Gastric amoebiasis due to histolytica reported in wallaroos, red kangaroos; one case in wallaby; one case of meningoencephalitis in captive juvenile kangaroo
• Gastric amoebiasis due to histolytica also occurs in leaf-eating primates (colobus monkey, silver-leafed monkey) due to higher stomach pH (more alkaline) that is conducive to survival of amoebae; erosive and ulcerative gastritis
• Amphibians: Several species susceptible to Entamoeba ranarum; signs and lesions similar to those in invadens in snakes; one case documented in ball python housed with toads (Michaely, J Comp Pathol. 2020); novel species documented in wild cane toad (Shilton, Vet Pathol. 2020)
• Entamoeba bovis: Nonsuppurative ileitis and typhlitis; necrotizing and pyogranulomatous lymphadenitis in pronghorn antelope fawns

Other pathogenic amoebae:

• Amoebic gill disease (Neoparamoeba): Proliferative condition affecting the gills characterized by hyperplasia and fusion of gill lamellae and filaments; most often of farmed fish, primarily salmonids
• Infections by free-living amoebae are relatively rare, but increasingly reported; usually not pathogenic; may cause fatal disease, especially in immunosuppressed patients:
• Naegleria fowleri: Acute fatal necrohemorrhagic meningoencephalitis; usually invades olfactory mucosa and migrates to brain via olfactory nerves; reported in humans (primary amebic meningoencephalitis or PAM), cattle, and a South American tapir; most human cases occur in healthy young individuals
• Acanthamoeba sp.: Granulomatous amoebic encephalitis (GAE) and pneumonia, and keratitis; in humans; usually invades cribriform plate; reported in sheep, cows, dogs, birds
• Balamuthia mandrillaris: Granulomatous amoebic encephalitis (GAE) and respiratory infections in humans, a baboon, orangutan, and sheep; acute and necrotizing meningoencephalitis in gorillas and other Old World primates; granulomatous nephritis and meningoencephalitis in a dog
• Sappinia diploidea: Necrohemorrhagic amoebic meningoencephalitis in humans
• Hartmanella : Gangrenous pneumonia in a bull; cervical lymphadenitis in sheep
• Willaertia : Reported in a dog with gastric ulcers

 

REFERENCES:

1. Carrera-Jativa PD, Morgan ER, Barrows M, Wronski T. Gastrointestinal parasites in captive and free-ranging birds and potential cross-transmission in a zoo environment. J Zoo Wildl Med. 2018; 49(1):116-128.
2. Gardiner CH, Fayer R, Dubey JP. An Atlas of Protozoan Parasites in Animal Tissues. 2nd ed. Washington DC: Armed Forces Institute of Pathology; 1998: 10-11, 16-17.
3. Gelberg HB. Alimentary system and the peritoneum, omentum, mesentery, and peritoneal cavity. In: McGavin MD, Zachary JF, eds. Pathologic Basis of Veterinary Disease. 6th ed. St Louis, MO: Elsevier; 2017:381.
4. Greiner EC, Mader DR. Parasitology. In: Mader DJ, ed. Reptile Medicine and Surgery. 2^nd Philadelphia, PA: WB Saunders; 2006:347.
5. Ilha MRS, Coarsey MD. Gastric and Caecal Amoebiasis in a Red Kangaroo (Macropus rufus) with Disseminated Toxoplasmosis. J Comp Pathol. 2019;172: 17-21.
6. Michaely LM, von Dornberg K, Molnar V, et al. Entamoeba ranarum Infection in a Ball Python (Python regius). J Comp Pathol. 2020;179: 74-78.
7. Miller HW, Suleiman RL, Ralston KS. Trogocytosis by Entamoeba histolytica Mediates Acquisition and Display of Human Cell Membrane Proteins and Evasion of Lysis by Human Serum. ASM Journals / mBio.  2019:10(2), 1-16.
8. Roberts, RJ. The parasitology of teleosts. In: Roberts RJ, ed. Fish Pathology. 4th ed. Philadelphia, PA: Elsevier Science Limited; 2012:306-307.
9. Shilton CM, Slapeta J, Shine R, Brown GP. Pathology associated with an outbreak of entamoebiasis in wild cane toads in tropical Australia. Vet Pathol. 2020;56: 921-931.
10. Strait K, Else JG, Eberhard ML. Parasitic diseases of non-human primates. In: Bennet BT, Abee CR, Henrickson R, eds. Nonhuman Primates in Biomedical Research. 2. 2nd ed. San Diego, CA: Elsevier; 2012:206-209.
11. Uzal FA, Plattner BL, Hostetter JM. Alimentary system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer's Pathology of Domestic Animals.  Vol 2. 6th ed.  Louis, MO: Elsevier; 2016: 98-99, 242.

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