Characterization of toxin genes and quantitative analysis of netB in necrotic enteritis (NE)-producing and non-NE-producing Clostridium perfringens isolated from chickens.

Necrotic enteritis (NE) in chickens, a Clostridium perfringens infection, has re-emerged due to the removal of antibiotic growth promoters in feeds in recent years, thus contributing to significant economic losses for the industry. Toxins produced by C. perfringens in conjunction with predisposing factors are responsible for the onset and development of NE. Recently, several lines of evidence indicated the potential role of plasmid-encoded toxins in the virulence of NE, particularly necrotic enteritis B-like (NetB) toxin. However, the association of NetB, beta2 toxin (CPB2), and C. perfringens large cytotoxin (TpeL) in clinical NE isolates are not well-established. Therefore, we characterized the toxinotype and the presence of netB, cpb2, and tpeL genes in 15 NE-producing and 15 non-NE-producing C. perfringens isolates using conventional PCR and quantified netB among those isolates by quantitative PCR (qPCR). All isolates were characterized as toxinotype A and were negative for cpe, which is associated with human food poisoning. The netB was detected in 6.7% and 70% of NE-producing isolates by PCR and qPCR, respectively. In 15 non-NE-producing isolates, netB was not detected by conventional PCR, but was detected in 60% of isolates by qPCR. The presence of and the copy number of netB were not significantly different between NE- and non-NE-producing isolates (p >0.05). No difference was observed between NE- and non-NE-producing isolates in the presence of cpb2 or tpeL (p >0.05). These results suggest that the presence of netB, cpb2, and tpeL, as well as the copy number of netB in C. perfringens is not correlated with clinical NE. In addition, we suggest that qPCR, but not conventional PCR, be used to detect netB.