HEMORRHAGIC ENTEROCOLITIS AND DEATH IN TWO FELINES (PANTHERA TIGRIS ALTAICA AND PANTHERA LEO) ASSOCIATED WITH PERFRINGENS TYPE A Author(s): Yanlong Zhang, Ph.D., Zhijun Hou, Ph.D., and Jianzhang Ma Source: Journal of Zoo and Wildlife Medicine, 43(2):394-396. 2012. Published By: American Association of Zoo Veterinarians DOI: http://dx.doi.org/10.1638/2010-0197.1 URL: http://www.bioone.org/doi/full/10.1638/2010-0197.1

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HEMORRHAGIC ENTEROCOLITIS AND DEATH IN TWO FELINES (PANTHERA TIGRIS ALTAICA AND PANTHERA LEO) ASSOCIATED WITH TYPE A

Yanlong Zhang, Ph.D., Zhijun Hou, Ph.D., and Jianzhang Ma

Abstract: Severe hemorrhagic enterocolitis was observed in a Siberian tiger (Panthera tigris altaica) and a lion (Panthera leo). Both animals developed acute depression, anorexia, and bloody diarrhea several days before death. Small and large intestines were diffusely congested, edematous, necrotic, and filled with hemorrhagic fluid, and mesenteric lymph nodes were enlarged and congested. Pure and abundant growth of gram-positive bacilli was obtained in culture under anaerobic conditions from the livers of both felines. Identification of highly virulent Clostridium perfringens Type A was based on pathologic lesions, hemolytic patterns, morphologic structure, and polymerase chain reaction. Animal inoculation assays indicated that C. perfringens Type A played an important role in the pathogenesis of both felines. Key words: Alpha-toxin, a-toxin, Clostridium perfringens, hemorrhagic enterocolitis, lion, Panthera leo, Siberian tiger, Panthera tigris altaica.

BRIEF COMMUNICATION China) staining showed very similar lesions for both animals. Macroscopic lesions in the small Clostridium perfringens is a gram-positive, spore- and large intestines were diffusely congested, forming, anaerobic bacterium responsible edematous, necrotic, and filled with hemorrhagic for a variety of diseases in humans, including gas fluid. The mesenteric lymph nodes were enlarged gangrene, food poisoning, and diarrhea, as well as and congested. Severe multifocal of the enterotoxemia and hemorrhagic gastroenteritis in superficial epithelium was observed in both the many domestic animals and wildlife.2,10 Clostridi- large and small intestines, and the lamina propria um perfringens is considered to be the most had a diffuse infiltration of lymphocytes, plasma important cause of clostridial enteric disease and cells, and neutrophils and a few eosinophils. is classified into five types (A, B, C, D, and E) Liver samples from each animal were also based on the production of four major toxins: collected aseptically and cultured on sheep blood alpha, beta, epsilon, and iota ( , , , and ).7 a b e i agar (nutrient agar, Tianhe Microorganism Re- Two adult felines (a Siberian tiger [Panthera agent Co., with sheep blood) under aerobic and tigris altaica] and a lion [Panthera leo]) among a anaerobic conditions. No were isolated population of more than 100 felines in a Harbin under aerobic conditions. However, anaerobic Zoo in northeastern China died several days after liquid culture (ordinary broth medium, Tianhe developing acute depression, anorexia, and Microorganism Reagent Co.) from both animals bloody diarrhea. Necropsy of both animals was had a dense growth of short bacilli when grown performed following routine procedures. Portions for 24 hr at 378C. Many bubbles were observed of liver, heart, and intestines were collected from under liquid paraffin (liquid paraffin, Kermel each animal and fixed in 10% formalin (10% Chemical Reagent Co.), which were attributed to formalin neutral buffer solution, Kermel Chemi- bacterial growth. Gram staining (Gram staining cal Reagent Co., Ltd., Tianjian, 300350, China) solution, Tianhe Microorganism Reagent Co.) for 24 h. Tissues were then embedded in paraffin revealed short, gram-positive bacilli. A presump- (paraffin, Kermel Chemical Reagent Co.) and tive diagnosis of enterotoxemia caused by C. sectioned at a thickness of 4 lm. Hematoxylin perfringens was made, based on the pathologic and eosin (1% Eosin Y Eosin ethanol solution and lesions, hemolytic patterns, and bacterial mor- Mayer’s hematoxylin solution, Tianhe Microor- phology. ganism Reagent Co. Ltd., Hangzhou, 310008, Ten anaerobic isolates of C. perfringens from liver samples (five isolates from each animal) were randomly selected and tested for the presence of From the College of Wildlife Resources, Northeast Forestry University, Harbin, Heilongjiang 150040, Chi- genes for major toxins, including a (cpa), b (cpb), e na (Zhang, Hou, Ma). Zhijun Hou and Yanlong Zhang (etx), i (itx), b2(cpb2), and C. perfringens entero- contributed equally to this work. Correspondence should toxin (cpe), by multiplex polymerase chain reac- be directed to Dr. Ma ([email protected]). tion (PCR), as described previously.6 Sterile

394 ZHANG ET AL.—HEMORRHAGIC ENTEROCOLITIS IN TWO FELINES 395 conditions were used, and negative (no DNA) animal inoculation assays documented that iso- controls were included in every trial. Reactions lates of C. perfringens from both felines were were run in a TaKaRa thermocycler (TaKaRa highly virulent, indicating that C. perfringens PCR Thermal Cycler DiceTM [Gradient model], played an important role in the pathogenesis of TaKaRa Bio Inc., Shiga 520-2193, Japan] using a enterotoxemia in both felines. step-cycle program. Cycling conditions com- Some isolates of C. perfringens type A can be prised an initial DNA denaturation at 948C for 5 considered nontoxigenic because they do not min; followed by 35 cycles of 948C for 1 min, 558C produce sufficient a-toxin to kill experimental for 1 min, and 728C for 1 min; and a final animals under test conditions.1 However, animal extension at 728C for 10 min. PCR products were inoculation data from this study indicated that visualized on agarose gels (Biowestt Regular this was not the case for the C. perfringens type A AgarosG-10,GeneCo.Ltd.,HongKong isolates from both felines. 202100, China) stained with EB (ethidium bro- Multiplex PCR revealed the presence of the cpa mide, TaKaRa Biotechnology Co., Ltd., Dalian toxin gene, but not other toxin genes, in C. 116600, China). PCR results demonstrated that perfringens isolates from both felines in this study, the strains isolated from the liver were positive for indicating that only the a-toxin was produced by the gene encoding a-toxin, indicating that the the isolates. The a-toxin is a lecithinase that is isolates were C. perfringens type A. Furthermore, thought to be responsible for yellow lamb disease the strains lacked the genes coding for other C. in sheep, a condition characterized by massive perfringens major toxins (cpb, etx, and itx), as well and icterus, as well as red-tinged as the cpb2 and cpe genes. urine,7,10 symptoms not observed in either the Five cats and two rabbits were obtained from Siberian tiger or the lion in this study. Studies The Institute of Oncology of Harbin (license: have reported goats, calves, pigs, bears, Amur SCXK[HEI]:2007–2010) and experimentally in- leopards, and deer infected with C. perfringens type oculated with C. perfringens (under guidelines of A that did not display these symptoms,1–5,7–10 local animal care committees) using cultures from indicating that the symptoms do not always liver samples sporulated in anaerobic broth. All appear in C. perfringens enterotoxemia, or possibly inoculated animals died between 18 hr and 5 days only appear in some species. after inoculation, and all exhibited pathology Although C. perfringens is a normal inhabitant similar to that seen in the felines. of the intestines of most animal species, it Although enterotoxemias are commonly ob- proliferates in large numbers and produces sev- served in some domestic animals and wildlife,1–7,9–10 eral potent toxins when the intestinal microbial description of enterocolitis in big felines caused by environment is altered by diet or other factors. Clostridium is rare in the literature, except one Furthermore, the toxin can act locally, systemi- report of Amur leopards (Panthera pardus orientalis) cally, or both, with devastating effects on the that had been infected by C. perfringens.8 In the host.3,10 Braxy, a clostridial disease caused by present study, C. perfringens type A infection was Clostridium septicum, is most often related to found in a Siberian tiger and a lion. Some authors consumption of frozen food.5 Any injury to the have reported that sudden access to carbohydrate- mucosa of the small intestine might also predis- rich food stimulates C. perfringens growth and pose an individual to changes in the bacterial subsequent pathogenesis in ruminants.9 Lubbs et flora, resulting in overgrowth of . Mild al.4 had reported that C. perfringens populationswere indigestion could also cause the intestines to elevated (P , 0.05) in adult cats fed a high- become static, preventing normal flushing of toxic diet when compared with those fed moderate- substances from the system.1 Neiffer8 thought protein diets. The diet of tigers and lions consists that partially thawed meat in the leopards’ diet primarily of meat,4 suggesting that felines may be at might have precipitated the production of an a higher risk to C. perfringens infection because of endogenous clostridial enterotoxicosis by disrupt- their high protein diet. ing digestive tract flora with resultant clostridial Clostridium perfringens type A is ubiquitous in overgrowth and sporulation.8 The diet of felines the environment and is a normal inhabitant of the in the zoo involved with this study consists of gastrointestinal tract of most healthy warm- frozen beef and chicken, which is stored in a blooded animals. Therefore, controversy exists freezer at À208C and thawed before feeding. regarding its pathogenic role in animals.3,7 Al- Incomplete thawing of the meat might, therefore, though enterotoxemia in the two felines could not have contributed to enterotoxemia of the Siberian be confirmed to be caused by C. perfringens, tiger and lion. 396 JOURNAL OF ZOO AND WILDLIFE MEDICINE

Diagnosis of enterotoxicosis caused by C. ium perfringens in polar bears from Svalbard, Norway. J. perfringens type A is difficult because the bacteri- Wildl. Dis. 44: 55–158. um is ubiquitous in the environment, and healthy 4. Lubbs, D. C., B. M. Vester, N. D. Fastinger, and K. individuals can be carriers. However, evaluation S. Swanson. 2009. Dietary protein concentration affects intestinal microbiota of adult cats: a study using of gross and pathologic lesions, culture morphol- DGGE and qPCR to evaluate differences in microbial ogy, multiplex PCR results, and animal inocula- populations in the feline gastrointestinal tract. J. Anim. tion assays, as well as the absence of other Physiol. Anim. Nutr. (Berl.) 93: 113–121. enterotoxigenic pathogens in both the Siberian 5. Manteca, C., T. Jauniaux, G. Daube, G. Czaplichi, tiger and lion, make C. perfringens type A enter- and J. G. Mainil. 2001. Isolation of Clostridium otoxicosis the most likely diagnosis. perfringens from three neonatal calves with hemorrhagic abomasitis. Rev. Me´d. Ve´t. 152: 637–639. Acknowledgment: This research was supported 6. Meer, R. R., and J. G. Songer. 1997. Multiplex by the China Postdoctoral Science Foundation polymerase chain reaction assay for genotyping Clos- (200974224 LRB11-589). tridium perfringens. Am. J. Vet. Res. 58: 702–705. 7. Miyakawa, M. E. F., J. Saputo, J. S. Leger, B. Puschner, D. J. Fisher, B. A. McClane, and F. A. Uzal. LITERATURE CITED 2007. Necrotizing enterocolitis and death in a goat kid associated with enterotoxin (CPE)-producing Clostrid- 1. Embury-Hyatt, C. K., G. Wobeser, E. Simko, and ium perfringens type A. Can. Vet. J. 48: 1266–1269. M. R. Woodbury. 2005. Investigation of a syndrome of 8. Neiffer, D. L. 2001. Clostridium perfringens enter- sudden death, splenomegaly, and small intestinal otoxicosis in two Amur leopards (Panthera pardus hemorrhage in farmed deer. Can. Vet. J. 46: 702–708. orientalis). J. Zoo. Wildl. Med. 32: 134–135. 2. Greco, G., A. Madio, V. Martella, M. Corrente, D. 9. Tammy, D. 2004. Clostridium perfringens type A Buonavoglia, and C. Buonavoglia. 2005. Enterotox- and b2 toxin associated with enterotoxemia in a 5- emia associated with beta 2 toxin-producing Clostridi- week-old goat. Can. Vet. J. 45: 251–253. um perfringens type A in two Asiatic black bears 10. Uzal, F. A. 2004. Diagnosis of Clostridium (Selenarctos thibetanus). J. Vet. Diagn. Invest. 17: 186– perfringens intestinal infections in sheep and goats. 189. Anaerobe 10: 135–143. 3. Jores, J., A. E. Derocher, C. Staubach, and A. Aschfalk. 2008. Occurrence and prevalence of Clostrid- Received for publication 11 January 2011