Vet. Path. 10: 323-329 (1973) Naturally Occurring Gastric Amebiasis in the Wallaroo E. D. ROBERIS, J. C. WILLIAMS and G. PIRIE Department of Veterinary Pathology, School of Veterinary Medicine, and Department of Veterinary Science, Louisiana State University, Baton Rouge, La. ; and Baker Animal Hospital, Baker, La. Abstract. Gastric amebiasis occurred naturally in two wallaroos in the Baton Rouge Zoo. Infection by an Entnmoeba sp. parasite was confined to the forestomach and was characterized by hemorrhage and necrosis of the mucosa; parasitic invasion of the lamina propria, submucosa, and muscularis; and extensive gastric edema. Typical amebic cysts were found in the feces of the animal that died and of those in contact. Gastric amebiasis has not previously been reported in marsupials. E/itan?oeba histolytica is a parasitic ameba of inan and higher primates [ 12, 141 and has been reported in the dog [2, 6, 8, 21 1, cat [ 171, mouse [ 181, goat and pig [19]. The cow, goat and pig have amebic cysts that are morphologically indistinguishable from the cysts of E. I~istolytica [ 161. They are probably not pathogenic for ruminants [16], even though ainebiasis in cattle has been reported [8]. Amebiasis is worldwide and is found within various systems of the animal. Pathogenicity of the amebae is variable and is influenced by various factors such as diet of the host [lo], immunity [20], and secondary bacterial infection [9]. E. histolytica is potentially an invasive protozoan parasite. It may invade the mucosa and lamina propria and localize deep in the niucosal glands and submucosa where extensive tissue necrosis may occur, eventually producing the typical hour-glass type of ulceration [6]. Amebiasis of the kangaroo or wallaroo has not been reported. This paper reports two naturally occurring fatal cases of gastric amebiasis in wallaroos at the Baton Rouge Zoo.
Materials and Methods Eight kangaroos and four wallaroos were housed in a common outside exhibition pen with a shed. They were maintained on a diet of commercial horse and mule feed and fed grass hay adlibifinn. No clinical signs were noted before an IS-month-old female wallaroo was found dead in the pen; about 48 h later the second adult wallaroo was depressed and had diarrhea. It died the following day. Both animals were necropsied (the animal in case 1 about 12 h after death and that in case 2 immediately after death), and gastric contents were collected for mercury, lead and arsenic analysis, routine microbiologic evaluation and parasitological fecal examination (direct saline smear and iodine stained smear). The following tissues were fixed in 10% buffered formalin : heart, liver, lung, spleen, mesenteric lymph node, tongue, pharynx, trachea, brain, eye, stomach (from each of the four major compartments), duodenum, jejunum, ileum, cecum and colon, urinary bladder and kidney. All tissues were embedded in paraffin, cut at 6 pm and stained with heniatoxylin and eosin. Selected slides of the digestive tract were stained with Gomori s methanamine silver nitrate and periodic acid- Schiff (PAS) with hematoxylin counterstain [I 1 ]. Blood was collected in ethylene-dianiinetetraacetic acid (EDTA) tubes from the animal in case 2 for a complete blood count. After Enfanioeba sp. trophozoites were found in sections of the stomach, all remaining animals housed together were examined for amebic cysts. Results Macvoscoj? ic E,uani inat ion The abdomen was greatly distended, and there was evidence of diarrhea before death. The wallaroo has a multicompartmental stomach similar to that of the kangaroo. This entire organ was greatly distended with gas and was congested. The stomach was filled with hemorrhagic, frothy liquid that appeared localized to the gastric area. Excess mucus was throughout the rest of the digestive tract. The liver appeared congested with focal areas of ischemia. The lungs were congested, and all lobes had small areas of pneumonia. Microscopic E,uani ina t ion The forestomach was congested and had focal areas of hemorrhage throughout all levels of the mucosa (fig. 1). Some segments of the mucosa were completely necrotic. Numerous trophozoites (10-20 pm) were seen in necrotic areas of the mucosa where extensive invasion of the submucosa and muscularis had occurred. Lymphatic and capillary necrosis were accompanied by prominent submucosal edema. In most areas the inflammatory infiltrate was slight and was composed of lymphocytes, neutrophils, and focal accumulations of eosinophils.
Gastric Ainebiasis in the Wallaroo 325 Fig. I. Hemorrhage of lamina propria and localization of numerous EntamoeBn sp. (arrows) trophozoites deep in the lamina propria of a wallaroo forestomach. HE. Phagocytosed erythrocytes were occasionally seen in the amebic trophozoiles (fig. 2), and in most instances the cytoplasm of the trophozoites was very foamy. The inflammatory response and trophozoites were prominent throughout all compartments of the forestomaclis. The trophozoites and inflammation stopped abruptly at the true stomach. Desquamation of the surface epithelium with slight lymphocytic and eosinophilic infiltration occurred in the remaining parts of the small and large intestine, but no Enlmnoeba sp. were seen. Gomori s and PAS stains enhanced the observation of the trophozoites. In the lungs there was marked congestion and edema, and the larger bronchi were filled with inhaled ingesta. Inflammatory reaction was slight in the lung. Sinusoidal pooling of blood was noted in the liver. In the kidneys, focal areas of interstitial medullary hemorrhage were present. No change was noted in the skin, adrenal gland, skeletal muscle, myocardium, aorta, spleen, tongue, pharynx, urinary bladder, and brain.
326 ROBERTS/WILLIAMS/PIRIE Fig. 2. Ameba with a phagocytosed erythrocyte (arrow) from a wallaroo forestomach. HE. Entamoeba sp. cysts were seen in feces from the wallaroos and kangaroos (fig. 3). At least three size variants were noted; sizes ranged from 8.3 to 17.9 pm. One to four nuclei were seen with the majority having three to four nuclei. In unstained saline smears, the cysts were small spheres with colorless, finely granular cytoplasm. The nuclei appeared as delicate rings. Results of the analysis of stomach content from the animal in case 1 for mercury, arsenic and lead were negative. The hemogram from the animal in case 2 was 1,500 white cells/mm3; 5.5 x lo6 redcells/mm3; hemato~rit,47.0'~~; and hemoglobin, 16.3 g/100 ml. The differential white cell count was as follows: neutrophils, 15.0':d; band forms, 3 70; lymphocytes, 58%; eosinophils, 25%; and monocytes, 3%. Six smudge cells, three nucleated erythrocytes, and many sickle-type cells were noted. Microbiologic examination did not show pathogenic bacteria.
Gastric Amebiasis in the Wallaroo 321 Fig. 3. Entatnoeba sp. cyst in an iodine-stained direct fecal smear. Note the two nuclei (arrow). Discussion The apparent case of gastric amebiasis is unique. Few animals other than ruminants have a rumen-like organ preceding the true stomach. The abrupt arrest of amebic trophozoite invasion at the true stomach suggests the destructive action or unfavorable environment of the gastric secretion on the trophozoites. The origin of the amebic cysts in feces was not determined, as the small and large intestine were apparently free of amebic invasion. The cysts were probably formed in the forestomach. The red kangaroo has a gastric system morphologically similar to the wallaroo. MYKYTOWYCZ [I 51 in his study of the endoparasites of the red kangaroos determined that the ph of the forestomach varied from 5.6 to 6.5 whilst the true stomach had an average ph of 3.9, thus suggesting a favorable ph environment for development of Entamoeha sp. in the forestomach. The origin of the amebae was not determined as the marsupials had been in contact with various animals. Some ruminants may have harbored an Entamoeba sp. pathogenic for the marsupial species. Several African ruminants have been shown to harbor pathogenic species of amebae [8]. Death of the wallaroos was caused by inhaled vomitus. Amebae alone have been implicated as responsible for typical intestinal lesions, and the process has been mediated by the lytic action of the amebic process. Three stages of invasion, superficial erosion, typical deep ulceration accompanied by cellular infiltration, and chronic undermining ulceration [6]
328 ROBERTS/WILLlAMS/PIRIE have been described. In the wallaroos, the major accumulation of amebae was deep in the inucosal glands and in the submucosa. Marked inucosal necrosis and necrosis of the various vascular structures of the subniucosa were a prominent feature of these wallaroos; similar changes are typical of human and canine ainebiasis [6]. Amebic cysts, although scant, were seen in in-contact aniinals as well as in the two reported animals. Diagnosis of amebiasis by fecal examination is difficult in some animals, especially the dog [5]. In the wallaroos examined cysts were occasionally seen in the feces. Direct examination of an iodinestained fecal smear proved satisfactory for demonstrating cysts. Although the cysts and the pathologic process described are typical of E. histolytica, it is difficult to attribute the process definitively without further studies of the Entarnoeba sp. observed. Hartmannella sp. (Acanthanzoeha), soil amebas, have recently been reported from naturally occurring cases in the dog [I] and ox [13]. Various organs were infected but not the gastrointestinal tract. Acanthamoeba infection in man appears to have a predilection for the brain and meninges [3, 41. In all naturally occurring infections by acanthamoeba, both trophozoites and cysts occur in the tissue phase, in contrast to amebiasis where the extraintestinal infection is by trophozoites only. Cysts were not seen in the tissue phase in the two cases described. The apparent susceptibility of marsupials to gastric amebiasis suggests that in wildlife exhibits they should not be placed in direct contact with wild porcine and African ruminant species, as the Entainoeba sp. in these animals may be pathogenic. A cknowdedgement The authors thank Dr. JOsEPii H. MILLER, Louisiana State University School of Medicine, Department of Tropical Medicine and Medical Parasitology, New Orleans, La., for his assistance in the identification of this protozoan parasite. References I AYERS, K.M.; BILLUPS, L.H., and GARNER, F. M.: Acanthamoebiasis in a dog. Vet. Path. 9: 221-226 (1972). 2 BURROWS, R. B. and LILLIS, W.G.: Intestinal protozoan infection in dogs. J. anier. vet. med. Ass. 150: 880-883 (1967).
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