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For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by  Clostridium perfringens . However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a  C. perfringens  strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from  C. perfringens  (38% identity) and alpha-toxin from  Staphylococcus aureus  (31% identity). NetB was only identified in  C. perfringens  type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a  netB  mutant of a virulent  C. perfringens  chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The  netB  mutant was unable to cause disease whereas the wild-type parent strain and the  netB  mutant complemented with a wild-type  netB  gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of  C. perfringens  to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian  C. perfringens  strains capable of causing NE. Furthermore, the  netB  mutant is the first rationally attenuated strain obtained in an NE-causing isolate of  C. perfringens ; as such it has considerable vaccine potential.

NetB, a New Toxin That Is Associated with Avian Necrotic Enteritis Caused by Clostridium perfringens