Turk J Vet Anim Sci 29 (2005) 847-851 © TÜB‹TAK Research Article Isolation of Clostridium perfringens from Chickens and Detection of the Alpha Toxin Gene by Polymerase Chain Reaction (PCR) Hakan KALENDER Veterinary Control and Research Institute, 23100, Elaz›¤ - TURKEY E-mail: hakgg@yahoo.com Hasan Basri ERTAfi Department of Microbiology, Faculty of Veterinary Medicine, F›rat University, 23119, Elaz›¤ - TURKEY Received: 24.03.2004 Abstract: This study was carried out to isolate Clostridium perfringens from chickens and to detect the gene encoding the alpha toxin produced by all types of C. perfringens by polymerase chain reaction (PCR). Intestinal contents of 160 slaughtered chickens from 8 different farms in Elaz›¤ province were analyzed. C. perfringens was isolated from 8 (5%) of the samples. DNA samples extracted from suspected isolates grown on selective agar were amplified by PCR using a pair of primers derived from the alpha toxin gene. All of 8 suspected isolates were found to be C. perfringens by conventional methods and PCR. Isolates were typed by the toxin neutralization test. Of the 8 isolates, 6 were type A and 2 could not be typed. Since C. perfringens type A is associated with food poisoning in humans, the isolation of C. perfringens type A from chickens in this study is important for public health. Key Words: Chicken, Clostridium perfringens, isolation, PCR, alpha toxin Tavuklardan Clostridium perfringens’in ‹zolasyonu ve Alfa Toksin Geninin Polimeraz Zincir Reaksiyonu (PZR) ile Saptanmas› Özet: Bu çal›flma tavuklardan Clostridium perfringens’in izolasyonu ve C. perfringens’in tüm tipleri taraf›ndan üretilen alfa toksinini kodlayan genin Polimeraz Zincir Reaksiyonu (PZR) ile saptanmas› amac›yla yap›ld›. Çal›flmada Elaz›¤ ilinde mezbahada kesilen, 8 iflletmeye ait 160 adet tavu¤un ba¤›rsak içeri¤i incelendi ve örneklerin 8’inden (% 5) C. perfringens izole edildi. Selektif agarda üreyen flüpheli kolonilerden DNA izolasyonu gerçeklefltirildi. ‹zole edilen DNA’lar alfa toksin geninden türetilen bir çift primer kullan›larak PZR’de ço¤alt›ld›. fiüpheli 8 izolat›n tümünün hem klasik yöntemlerle hem de PZR ile C. perfringens oldu¤u saptand›. Toksin nötralizasyon testi ile yap›lan tiplendirmede; 6 sufl tip A olarak tiplendirildi ve 2 sufl tiplendirilemedi. C. perfringens tip A insanlarda g›da kaynakl› enfeksiyonlara neden oldu¤undan, bu çal›flmada tavuklardan C. perfringens tip A’n›n izolasyonu halk sa¤l›¤› aç›s›ndan önem tafl›maktad›r. Anahtar Sözcükler: Tavuk, Clostridium perfringens, izolasyon, PZR, alfa toksin Introduction Clostridium perfringens is a Gram positive, spored and anaerobic bacteria that causes serious infections in humans and animals by toxins. C. perfringens is divided into five types, A, B, C, D and E, based on the synthesis of four major lethal toxins, alpha, beta, epsilon and iota. Alpha toxin is produced by all types. Alpha toxin has lecithinase activity and hydrolyzes lecithin into phosphorylcholine and diglyceride and causes pathologic changes in tissues (1-3). C. perfringens types A, C and D have been shown to cause necrotic enteritis in chickens (4-6). It is reported that coccidiosis plays a role in the occurrence of necrotic enteritis (7,8). C. perfringens is a member of normal intestinal flora that reproduces at high rates and produces toxins (9). C. perfringens type A, which causes infection 847 Isolation of Clostridium perfringens from Chickens and Detection of the Alpha Toxin Gene by Polymerase Chain Reaction (PCR) in chickens, has been reported to cause food poisoning in humans as well (10). Routine typing procedure consumes a lot of antisera and experimental animals. Additionally it is labor. In order to avoid high cost and the use of experimental animals, several researchers have been working on a PCR method for detection of genes encoding C. perfringens toxins (2,3,11-13). There have been a large number of studies conducted to isolate C. perfringens from chickens. Tschirdewahn et al. (14) have reported that they isolated C. perfringens in 80% of feces samples of chickens. Latinovic (6) examined the intestinal contents of 312 chickens with necrotic enteritis and isolated C. perfringens type A in 12, type C in 3 and type D in 3. Awad et al. (4) analyzed samples of 245 diseased and 232 healthy chickens and isolated C. perfringens type A in 138, type C in 41 and type D in 1. Long (15) reported that 855 of 11,076 (7.7%) broiler chickens had necrotic enteritis and isolated C. perfringens in 80 samples and found that 50% of 80 isolates were C. perfringens type A. In another study, intestinal contents of 100 slaughtered broilers were analyzed and C. perfringens was isolated in 41 samples and 21% of isolates were found to be type A (5). Craven et al. (16) reported that C. perfringens was recovered from broiler carcasses after chilling in 13 (81%) of 16 flocks. Among studies conducted in Turkey, Demirözü (9) isolated C. perfringens type A in the intestinal contents of 58 of 500 (11.60%) chickens that were obtained from a slaughterhouse. Nadas et al. (17) looked at sera samples of 101 broiler and 103 laying chickens for the presence of alpha toxin and found 31 and 29 positive sera respectively. Alp (18) examined 127 feed and feed ingredient and isolated C. perfringens in 47, 27 of which were type A, and the rest could not be typed. The aim of this study was to isolate C. perfringens from chickens and to detect the presence of the alpha toxin gene in the isolates. Materials and Methods Samples Intestinal contents were taken from slaughtered apparently healthy broiler chickens aged 45 days from 8 farms at a local abattoir in Elaz›¤, Turkey. The samples were obtained from animals that consumed feed containing antibiotic and coccidiostat. 848 Isolation and Identification of C. perfringens Samples were cultured on Perfringens agar (TSC Agar, Oxoid) plates and incubated in an anaerobic chamber at 37 °C for 24 h. Black colonies, presumed to be C. perfringens, were tested for Gram staining. The suspected isolates were identified by biochemical tests and semi-antitoxin petri method using C. perfringens alpha toxin antisera and agar containing egg yolk (18,19). Typing Typing of C. perfringens was performed by the mouse neutralization test using antisera of alpha, beta and epsilon toxin (1). PCR Primers A pair of primers (5’-TGCTAATGTTACTGCCGTTGAT AG-3’ and 5’- ATAATCCCAATCATCCCAACTATG-3’) was used for detection of the gene for C. perfringens alpha toxin. Procedure A few colonies from suspected isolates grown on selective agar were suspended in 300 µl of distilled water, and the mixture was incubated at 56 °C for 30 min. The samples were treated with 300 µl of TNES buffer (20 mM Tris pH 8.0, 150 mM NaCl, 10 mM EDTA, 0.2% SDS) and proteinase K (200 µg/ml). After incubation at 37 °C for 2 h the mixture was boiled for 10 min. To that suspension, the same volume of phenol (saturated with Tris-HCl) was added, the suspension was shaken vigorously by hand and centrifuged at 11,600 g for 10 min. The upper phase was transferred into another tube and sodium acetate (0.1 volume) and ethanol (2.5 volume) were added. The suspension was kept at -20 °C for 1.5 h and then centrifuged at 11,600 g for 10 min. The pellet was washed with 95% and 70% ethanol, each step followed by 5 min centrifugation. Finally the pellet was dried and resuspended in 50 µl of distilled water (20). The PCR was performed in a touchdown thermocycler (Hybaid) in a total reaction volume of 50 µl containing 5 µl of 10xPCR buffer (10mM Tris-HCl, pH 9.0, 50mM KCl, 0.1% Triton X-100), 5 µl of 25 mM MgCl2, 250 µM of each deoxynucleotide triphosphate, 2 U of Taq DNA polymerase, 1 µM of each primer and 5 µl of template DNA. Amplification was obtained with 30 cycles following an initial denaturating step at 94 °C for 5 min. Each cycle H. KALENDER, H. B. ERTAfi involved denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, and synthesis at 72 °C for 2 min. Then 10 µl of the amplified product was electrophoresed in a 1.5% agarose gel and stained with ethidium bromide. Amplified bands were visualized and photographed under UV illumination (20). Results In this study, 160 intestinal contents were analyzed and C. perfringens was isolated from 8 (5%) samples. All of the 8 suspected isolates grown on selective agar were PCR positive for the alpha toxin gene of C. perfringens. Typing procedures revealed that 6 of these isolates were type A and 2 could not be typed. PCR products for the alpha toxin gene (247 bp) of C. perfringens are shown in the Figure. Discussion Different types of C. perfringens cause enteric infections in chickens. Although generally types C and D produce necrotic infections, type A is also reported to be a causative agent of the infection too (9,18). The isolation rate (5%) of C. perfringens in this study is lower than the rate previously reported in Turkey (9) Figure. or in some other countries (4-6,14,15). This low rate could be due to the use of antibiotic and coccidiostatic drugs in broiler farms. Antibiotic additives can change the intestinal flora of animals. Coccidia are known to have a role in necrotic enteritis, which causes an increase in the number of C. perfringens in the gut (7,8). PCR has been widely used in identifying the toxin genes of C. perfringens because of its high sensitivity. In this study, a pair of primers derived from the alpha toxin gene, which is present in all the strains of C. perfringens, was used. The alpha toxin gene was detected in all suspected isolates. In the present study, we have shown that PCR can be used for identification of C. perfringens. It has previously been shown that C. perfringens could be identified by PCR in feces samples after enrichment (21,22). It is always possible to isolate C. perfringens from intestinal contents as it is a normal habitant of the intestine. Therefore, the detection of toxin produced by bacteria is a more convenient method (1). A routine diagnosis method for C. perfringens infection is the toxin neutralization test applied in mice in which intestinal content is centrifuged and supernatant is injected to mice with antitoxins and the type of toxin is evaluated by the protection of mice from death by antitoxin. Due to easy Agarose gel electrophoresis of PCR products of C. perfringens isolates. M: Marker, N: Negative control, P: Positive control, 1-6: Suspected isolates. 849 Isolation of Clostridium perfringens from Chickens and Detection of the Alpha Toxin Gene by Polymerase Chain Reaction (PCR) inactivation of the toxin, this test might not give true results in nonfresh samples. The toxin neutralization test is also used for typing C. perfringens strains. Some strains may not be able to produce toxin enough to kill mice under laboratory conditions and this causes an obstacle for typing by the test. In two different studies (18,23) conducted in Turkey 42% and 37%, respectively, of strains could not be typed. Similarly, in the present study 6 isolates (75%) were found to be type A and 2 (25%) could not be typed. Several studies reported that the most predominant type in chickens is type A (4,6,9,15,24). The enterotoxins of type A have been reported to cause food-born infections in humans (21,25). In conclusion, isolation of C. perfringens type A in chickens slaughtered for human consumption has a crucial impact on public health. Cross contamination with C. perfringens occurring during slaughter and meat processing and unsuitable storage conditions could be an important threat to public health. As classical identification methods are expensive and time consuming and also because of their low sensitivity, PCR can be used to determine the presence of toxin genes and for typing C. perfringens. This technique gives the opportunity to type isolates that could not be typed by toxin neutralization test. Detailed epidemiological studies are needed to give a certain idea on chicken enterotoxemia. Acknowledgments The authors wish to thank the laboratory workers of the Veterinary Control and Research Institute, Elaz›¤, for their help. References 1. Arda, M., Minbay, A., Lelo¤lu, N., Ayd›n, N., Kahraman, M., Akay, Ö., Ilgaz, A., ‹zgür, M., Diker, K.S.: Özel Mikrobiyoloji. 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