POSTER PRESENTATIONS 251 SafePork 2005 ISOLATION OF CLOSTRIDIUM PERFRINGENS FROM SWINE CARCASSES AND FECES Andrea M. Moreno*, Thais S. P. Ferreira, Renata R. Almeida, Luciane T. S. Zucon, Renata Paixão, Débora D. S. Gobbi, Cleise R. Gomes, Antônio J. P. Ferreira Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, n.87, CEP 05508000. São Paulo, SP, Brasil. *E-mail: morenoam@uol.com.br. Abstract Bacterial hazards are the major concern in the production of food of animal origin. Clostridium perfringens is a common cause of food-borne illness. The illness is characterized by profuse diarrhea and acute abdominal pain. At this study 30 carcass swabs from dorsal area and 30 fecal samples from a swine abattoir were analyzed over C. perfringens presence. The frequen- cy of agent in carcasses was 40% and in fecal samples 73.2%. The forty-seven isolated strains were characterized by PCR for the presence of genes codifying the enterotoxin, alpha, beta, epsilon, iota and beta-2 toxins. All strains were positive for presence of alpha and beta 2 toxin gene, and were classified as type A. None were positive to enterotoxin gene through PCR. Single enzyme amplified fragment length polymorphism (SE-AFLP) analyses were used to characterize 47 C. perfringens strains and revealed two common profiles among fecal and carcass strains. Introduction Clostridium perfringens is the etiologic agent of multiple syndromes in domestic animals, some of the most important conditions that producers and veterinary practitioners have to face (Songer, 1998; Klaasen, 1999). The various toxins produced by the bacteria play key roles in the pathogenesis of the disease and are divided into five biotypes, designated A through E, based on the production of alpha- (-), beta- (-), epsilon- (-), and iota- (-) toxins. The -toxin is produced by all types, -toxin is produced by type B and C strains, -toxin is produced by type B and D strains, and -toxin is produced by type E strains (Garmory et al., 2000). Different biotypes of C. perfringens are associated with different diseases. Type A enterotoxigenic strains are a com- mon cause of food poisoning outbreaks worldwide. Type C, is generally considered to be the pri- mary cause of necrotic enteritis in piglets. In addition to the major toxins, other toxins may play a role in disease (Garmony et al., 2000). A novel toxin produced by C. perfringens, named beta 2- (2) toxin, has recently been identified and its encoding gene characterized. The occurrence of C. perfringens in swine carcass at slaughter is not reported in Brazil. The genotyping of these isolates may lead to a better understanding of the potential of this agent to contaminate pork meat and cause outbreaks in humans. Single enzyme AFLP (SE-AFLP) has previously been used for the differentiation of Clostridium perfringens and others agents as Pasteurella multocida to strain level (McLauchlin et al, 2000; Moreno et al, 2003). The objectives of this trial are to isolate C. perfringens from carcass swabs and swine feces at abattoir, characterize the strains in relation to the presence of enterotoxin, alpha, beta, epsilon, iota and beta-2 toxins genes using polymerase chain reaction (PCR) and analyze the strains through the SE-AFLP technique. Materials and Methods A total of 30 carcass swabs from dorsal area and 30 fecal samples from one swine abattoir were analyzed. A sample of 5 g of feces and the carcass swabs were submit- ted to enrichment in 25 ml of liquid thioglycolate medium and cultured overnight under anaerobic condition. After that, the culture was streaked in TSC agar under anaerobic condition. The suspect colonies were identified by the use of Gram stains, lecithinase and lipase reaction in egg-yolk agar and presence of storm clout in Litmus milk. The colonies of C. perfringens were cultured in 10 ml of thioglycolate broth for 24 hr at 37°C. The DNA extraction was conducted with 200 μl of bacterial culture treated with Lysozyme (140μl of 100mg/ml) and Proteinase K (40μl of 20 mg/ml) for 1 hour at 37°C. The bacterial lysates were sub- mitted to DNA purification with the guanidium thiocyanate method described by Pitcher et al. (1989). PCR assays were performed using specific primers the toxin genes (alpha, beta, beta2, epsilon, iota and enterotoxin) as described elsewhere (Moreno, 2003). Reference strains C. per- fringens type A (ATCC 3624), type C (ATCC 3628), type B (ATCC 3626) and type D (ATCC 3629) were kindly supplied by Instituto Biológico de São Paulo, and were used as positive controls. To SE-AFLP technique an aliquot containing 10μg of DNA was digested overnight (16 h) at brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by Digital Repository @ Iowa State University