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Toxin-associated and other genes in Clostridium perfringens type A isolates from bovine clostridial abomasitis and jejunal hemorrhage syndrome - Benjamin Schlegel Clostridium perfringens, a Gram-positive anaerobic bacterium, is widespread in the environment and in the intestines of warm-blooded animals (1). A classification system for C. perfringens strains divides them into types A through E based on toxin production, and there are several important toxins that fall outside of the traditional typing system. C. perfringens type A is defined as strains producing α toxin (cpa gene), type B as strains producing α, β (cpb gene), and ε (cpe gene) toxins, type C as strains producing α and β toxins, type D as strains producing α and ε toxins, and type E as strains producing α and ι (itx gene) toxins (1). Enterotoxin (cpe gene) is associated with human food poisoning strains and domestic animal diarrheal strains and may be produced by any C. perfringens type (1). β2 toxin (cpb2 gene) positive C. perfringens strains are associated with the occurrence of enteric disease in domestic animals (11). Atypical β2 toxin (atypical cpb2 gene) has 62-80% similarity to consensus β2 toxin and is far more common in non-porcine animal isolates than consensus β2 toxin, although the atypical β2 toxin is often not expressed (11). NetB and TpeL are recently discovered toxins that have been shown to be a part of the pathogenesis of necrotic enteritis in broiler chickens (12,13). In young calves, severe abomasitis associated with C. perfringens type A is characterized by sudden onset of disease, abomasal tympany, abdominal pain, and hemorrhagic diarrhea (2-4). Pathologically, the disease is characterized by acute emphysematous and necrotizing hemorrhagic inflammation of the mucosa of the abomasum, with marked edema in the lamina propria and submucosa (2-4). The disease may produce similar lesions in the rumen, reticulum, and duodenum. In these cases, Gram-positive bacilli typical of C. perfringens are often found on the mucosa and in the submucosa (3), and C. perfringens is invariably isolated. The disease may be very acute; calves with a normal appearance may be found dead 12 hours later with abomasal necrosis and bloat (4). To describe this disease the term bovine clostridial abomasitis (BCA) will be used. Bovine clostridial abomasitis has been reproduced by experimental infection with a C. perfringens BCA isolate in calves (5). This disease of calves can be persistent in a herd, hard to diagnose, and hard to treat (4). Diagnosis of C. perfringens type A enteritis is however complicated by the fact that C. perfringens type A is found in the gastrointestinal tract of healthy animals. C. perfringens type A BCA disease is identified by the presence of characteristic pathological and histopathological changes of hemorrhage and necrosis, including gas production and the involvement of the abomasum, as well as the isolation of C. perfringens in high numbers from the intestinal contents. Traditionally, the diagnosis of enteric disease caused by C. perfringens is based on the detection of specific toxins in the intestinal contents (6), but no specific toxin has been associated with BCA. Laboratory diagnosis would be simpler if the infection was linked to a specific toxin (6) or other C. perfringens “virulence signature” (7), but no such characteristic has been identified to date (8.9). Jejunal hemorrhage syndrome (JHS) is characterized by mechanical obstruction of some length of the jejunum with sloughed mucosa and clotted blood in mature cattle (9). Affected animals have a mean age of 4 years (9,10); it occurs in both male and female beef and dairy cattle, and the syndrome is associated with the winter season (9). C. perfringens type A has been isolated from the intestinal contents of many affected cattle and may have a causative role in the syndrome (9,10). This study examined known or possible virulence-associated genes in C. perfringens type A from cases of BCA and from cases of JHS, and compared these to isolates from calves which were healthy or had undifferentiated diarrheal illness. A real time PCR assay was used to genotype the 218 C. perfringens isolates examined. Isolates were sourced from healthy and diarrheic young and mature cattle (n = 191), from calves with confirmed or suspected BCA (n = 22), and from mature cattle with JHS (n = 5). Two hundred and eight of 216 (96%) isolates were positive for the cpa gene, and 13% (29/218) for atypical cpb2. Three of 8 (37.5%) confirmed BCA isolates, 2 of 13 (15.4%) suspected CA isolates and no JHS isolates tested positive for atypical cpb2. All isolates were negative for cpb, cpb2, cpe, etx, itx, netB, and tpeL, so that the results of the present study do not support a role for these genes in BCA or JHS. A subset of unique genes identified in one bovine clostridial abomasitis isolate (F262), for which a genome sequence is available, was searched for in 8 BCA isolates by PCR. None of the 10 genes were consistently present in all or even in a majority of BCA isolates. Many of these genes were also variably and inconsistently present in type A isolates from calves that did not have BCA. C. perfringens type A has been previously isolated from cases of BCA and JHS and genotyped, however the present study looked at additional C. perfringens toxin genes such as netB, tpeL, and atypical cpb2 that have not been examined before (2-4). Since all BCA and suspected BCA isolates, and all JHS isolates, were negative for cpb, consensus cpb2, cpe, etx, itx, netB, and tpeL genes, the present study does not support a role for these genes in BCA or JHS of cattle. The data from PCR amplification failed to identify novel genes found in the F262 isolate sequence common to the BCA and JHS isolates examined. The unique virulence characteristics, if any, of BCA and JHS isolates therefore remain to be determined. References 1. Songer JG. Clostridial enteric diseases of domestic animals. Clin Microbiol Rev 1996; 9:216234. 2. Morris WE, Venzano AJ, Elizondo A, Vilte DA, Mercado EC and Fernandez-Miyakawa ME. Necrotic enteritis in young calves. J Vet Diagn Invest 2011; 23:254-259. 3. Songer JG and Miskimins DW. Clostridial abomasitis in calves: Case report and review of the literature. Anaerobe 2005;11:290-294. 4. Van Kruiningen HJ, Nyaoke CA, Sidor IF, Fabis JJ, Hinckley LS, and Lindell KA. Clostridial abomasal disease in Connecticut dairy calves. Can Vet J 2009; 50:857-860. 5. Roeder BL, Chengappa MM, Nagaraja TG, Avery TB, Kennedy GA. Experimental induction of abdominal tympany, abomasitis, and abomasal ulceration by intraruminal inoculation of Clostridium perfringens type A in neonatal calves. Am J Vet Res 1988; 49:201-207. 6. Sterne M, Batty I. Pathogenic Clostridia. London, UK: Butterworths, 1975, pp144. 7. Nowell V. Genomic and proteomic analysis of a bovine hemorrhagic abomasitis Type A Clostridium perfringens isolate MSc thesis. Guelph, Ontario: University of Guelph, 2011. 8. Manteca C, Daube G, Jauniaux T, Linden A, Pirson V, et al. A role for the Clostridium perfringens β2 toxin in bovine enterotoxaemia? Vet Microbiol 2002; 86:191-202. 9. Lebrun M, Filée P, Mousset B, Desmecht D, Galleni M, et al. 266 The expression of Clostridium perfringens consensus beta2-toxin is associated with bovine enterotoxaemia syndrome. Vet Microbiol 2007; 120:151-157. 10. Abutarbush SM and Radostits OM. Jejunal haemorrhage syndrome in dairy and beef cattle: 11 cases (2001 to 2003). Can Vet J 2005; 46:711-715. 11. Jost BH, Billington SJ, Trinh HT, Bueschel DM and Songer JG. Atypical cpb2 genes, encoding Beta2-toxin in Clostridium perfringens isolates of nonporcine origin. Infect Immun 2005; 73:652-656. 12. Keyburn AL, Boyce JD, Vaz P, et al. NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathog. 2008 Feb 8; 4: e26. 13. Chalmers G, Bruce HL, Hunter DB, et al. Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations. J Clin Microbiol 2008; 46:3957-3964.