ISRAEL JOURNAL OF VETERINARY MEDICINE AN EPIZOOTIC OF TYZZER'S DISEASE IN RABBITS IN ISRAEL. A review of the literature Waner T.,1, Cohen O.,1, Anug A. M.,2 and Rosner A.1 1. Israel Institute for Biological Research, P.O. Box 19, Ness Ziona 70400, Israel. 2. PathoVet, Shikun Banim 137, Kfar Bilu 76965, Israel. Abstract An outbreak of Tyzzer’s disease (Clostridium piliforme) is described in rabbits in Israel. The outbreak occurred in young rabbits 12-13 weeks of age. The main clinical signs observed were diarrhea, anorexia and death. Major post-mortem findings were diarrhea, severe and extensive miliary hepatitis, hemorrhagic colitis and lymphomegaly of the mesenteric lymph nodes. The diagnosis was based on clinical signs, gross post-mortem findings, typical histological findings and presence of intracellular bacteria in the hepatocytes. To the best knowledge of the authors, the present report is the second account of Tyzzer’s in Israel and the first reported in rabbits. Introduction Tyzzer’s disease is caused by the bacteria Clostridium piliforme (formally Bacillus piliformis)(1). The bacteria are obligatory intracellular microbes and were discovered by Ernest Tyzzer in 1917 after observing an epizootic in Japanese waltzing mice(2). One previous report in Israel has described Tyzzer’s disease in mice(3). To the best of the authors’ knowledge, this is the first report of Tyzzer’s disease among rabbits in Israel. Materials and Methods Fourteen female New Zealand White rabbits of 12-13 weeks weighing between 1.5 to 2.0 kilograms were purchased from a commercial breeder. Within 24 hours of arrival, one rabbit was found dead in its cage. Over the next few days two rabbits were found dead with signs of diarrhea. Four rabbits were anorexic and appeared depressed. These were sacrificed and underwent post-mortem examination. Organs were collected and fixed in 10% formalin buffered saline and submitted for histological examination and evaluation. The tissues were embedded in paraffin, sectioned at about 4µm, and stained with hematoxylin and eosin (H&E). Liver sections were also stained with the Warthin-Starry silver stain and methylene blue. Results Four rabbits were necropsied. Isolation of the bacteria was attempted in one rabbit, however without success. The most prominent macroscopic signs were: Diarrhea and perianal staining. Severe and extensive miliary hepatitis (Figure 1). Hemorrhagic typhylitis. The area of the cecum most prominently affected was the ampulla caecalis coli (Figure 2). Enlargement of the mesenteric lymph nodes. Figure 1. Liver, rabbit: Small multifocal areas of necrosis. Figure 2. Large intestine, rabbit: Typhylitis of the ampulla caecalis coli. The microscopic examination revealed the following findings: Liver: Random multifocal areas of necrosis with abundant pyknosis and virtually no leukocytic infiltrate (Figure 3). Bordering the foci of necrosis, within the cytoplasm of heptatocytes, thin clumped filamentous organisms (15-20µm long) were demonstrated in the liver sections stained with the silver stain and methylene blue. Figure 3. Liver, rabbit: Random multifocal areas of necrosis Small intestine: There were marked differences between sections examined. There were several normal sections with some superficial mucosal autolysis. There were other sections with severe villus atrophy and extensive necrosis of the lamina propria. In these sections few crypts remained and there was abundant pyknosis but virtually no leukocyte infiltration. Colon: Sections examined showed extensive superficial mucosal necrosis with abundant pyknosis and no leukocyte infiltration. The intestinal lumen contained numerous bacterial rods and cocci. Rare filamentous basophilic organisms were observed. Some sections had marked proprial basophilic edema and extensive necrosis. A diagnosis of Tyzzer’s disease was made based on the history, clinical signs, postmortem findings and the identification of the intracellular bacteria within the hepatocytes. Discussion As was the case in this report, young animals are frequently involved in outbreaks of C. piliforme. The severity of Tyzzer’s disease in animals varies from subclinical to acute disease, generally with a high mortality(4). Until 1965 Tyzzer’s disease was thought to be restricted to mice only, when Allen et al. described two enzootics which had clinical and pathological features similar to Tyzzer’s disease in mice (5) In fact clinical Tyzzer’s disease is most often seen in laboratory animals such as rabbits(6-9), gerbils(10-13), hamsters(4, 14-16) and guinea pigs(17-19), whereas infection in mice and rats is oftensubclinical(20-27). The disease has also been reported in horses(2831), cows(32,33), dogs(34-40) and cats(41,42). The bacteria can also cause disease in rhesus monkeys(43) and there has been one documented case in humans(44). In the latter case the individual was severely immunocompromised and presented with skin lesions. Other animals in which Tyzzer’s disease has been reported includes: Red panda(45), serval (Felis capensis)(46), cockatiel(47), Australian marsupials including (possum, koala,wombat and dasyurid)(48,49), raccoon(50), snow leopards(51) and a gray fox(52). C. piliforme is a pleomorphic, spore-forming and mobile bacterium. The organism is gram negative and stains weakly with hematoxylin and eosin. Giemsa stains and silver impregnation methods are used to demonstrate the organism. The vegetative form of C. piliforme is large (8-40µm in length). Young rabbits are most susceptible with entire litters sometimes affected(1). The disease appears acutely. The most prominent signs in rabbits are watery diarrhea and fecal staining of the perineum. Affected rabbits are listless, anorexic and dehydrated. Ninety percent of the animals die within 1-2 days after the onset of symptoms. Surviving rabbits may develop chronic disease with signs of progressive weight loss. The target organs in animal infections are the intestine, liver and less often the heart. The diagnosis of Tyzzer’s disease is usually based on the clinical signs, characteristic gross post mortem findings, light microscopic findings and the demonstration of distinctive intracellular bacteria in affected tissues(1). The diagnosis in this report was based on clinical signs, gross post mortem findings, typical histological findings and the intracellular location of the bacteria in the hepatocytes. The gross liver lesions may not always be present, but in this case the liver pathology and the presence of C. piliforme bacteria in the hepatocytes were considered diagnostic of Tyzzer’s disease. Liver sections were stained with a silver stain and methylene blue in order to visualize the location and structure of the bacterium. The colitis also added evidence to the diagnosis. No myocardial lesions were detected in the animals from this outbreak. A previous report of Tyzzer’s disease in mice in Israel described the disease in the strain of BALB/c mice imported from abroad(3). The disease appeared 4 weeks after arrival. The only organ affected was the liver and the bacteria were demonstrated in the hepatocytes. Diagnosis was based on the post mortem findings and typical histological changes. To the best knowledge of the authors, the present report is the second account of Tyzzer’s in Israel and the first reported in rabbits. Transmission occurs mainly by ingestion of spores in contaminated feces. The vegetative form is labile and probably does not play a role in natural infection. The spores on the other hand may remain infectious for a long period of time. Source of C. piliforme include bedding, feed and water. The exact source of the infection in a barrier breeding facility from which these animals were purchased remains unknown. There is no known treatment for rabbits infected with C. piliforme. Antibiotic therapy gives poor results probably due to the intracellular location of the bacteria(1). No vaccine is available for Tyzzer’s disease, so prevention must depend on good husbandry practices. References 1. DeLong, D. and Manning, P.L., Bacterial Diseases, in The Biology of the Laboratory Rabbit, P.L. Manning, D.H. Ringler,and C.E. Newcomer, Editors. 1994, Academic Press: San Diego. p. 143-146. 2. Tyzzer, E.E.: A fatal disease of the Japanese waltzing mouse caused by a sporebearing bacillus (Bacillus piliformis, n. sp.). J. Med. Res. 37:307-338, 1917. 3. Meshorer, A.: Tyzzer's disease in laboratory mice. Refuah Vet. 31:41-42, 1974. 4. Ganaway, J.R., Allen, A.M. and Moore, T.D.: Tyzzer's disease. Am J Pathol. 64:717-730, 1971. 5. 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