DIAGNOSIS OF CLOSTRIDIUM-PERFRINGENS TYPE-C ENTERITIS IN PIGS USING A DNA AMPLIFICATION TECHNIQUE (PCR)

Clostridium perfringens type C, which produces alpha- and beta-toxin, causes severe haemorrhagic and necrotic enteritis in animals and humans. A polymerase-chain-reaction (PCR) assay was developed for the specific detection of the genes encoding alpha-, beta-, epsilon- and enterotoxin of C. perfringens for rapid typing of C. perfringens strains, and especially for the identification of type C strains. Both the alpha- and beta-toxin genes were detected directly in porcine C. perfringens type C cultures and also in type B and type C collection strains to a sensitivity of 10(3) cells without purification of the DNA. The alpha-toxin gene was detected in all types of C. perfringens. The epsilon-toxin gene was found in type B and type D, and the enterotoxin gene in some type A strains. Nine other species of Clostridium and a variety of intestinal pathogenic bacteria showed no signal for these toxin genes in this PCR assay. The alpha- and beta-toxin genes PCR assay were used to identify C. perfringens strains isolated from intestinal contents of 36 necropsied piglets that had suddenly died or died after premonitory signs of diarrhoea. At necropsy, 20 piglets showed necrotizing enteritis (15 acute and 5 chronic cases) and were suspected to have suffered from a C. perfringens type C infection. All of them had C. perfringens which gave a positive PCR signal for alpha and beta-toxin genes, and, hence, were identified as type C strains. From the 16 other piglets with lesions other than necrotizing enteritis, C. perfringens strains with the alpha-toxin gene, but no beta-toxin gene, were isolated. The necropsy findings and the anamnesis showed a very good correlation with the PCR identification of toxin genes. It was therefore concluded that the PCR-based toxin gene examination is a good alternative to the time-consuming, less specific, and more expensive mouse neutralization test in the routine diagnostic laboratory.