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

Abstract

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.

Clostridium perfringens: a review of the disease in pigs, horses and broiler chickens

“…Based on recent reports describing the association of netb and NE, the detection of this gene might also be interesting for a complete laboratory diagnosis (KEYBURN et al, 2008). Intestinal damage and increases in liver condemnation during slaughterhouse inspections strongly suggest NE.…”

“…One of the most interesting of these is Necrotic Enteritis toxin B (NetB), a poreforming toxin capable of causing lesions typical of NE in experimental models (KEYBURN et al, 2008). In addition, it was demonstrated that the NetB-encoding gene (netb) is common in C. perfringens type A isolates from NE birds, whereas it is rare in healthy birds (KEYBURN et al, 2008).…”

“…One of the most interesting of these is Necrotic Enteritis toxin B (NetB), a poreforming toxin capable of causing lesions typical of NE in experimental models (KEYBURN et al, 2008). In addition, it was demonstrated that the NetB-encoding gene (netb) is common in C. perfringens type A isolates from NE birds, whereas it is rare in healthy birds (KEYBURN et al, 2008). A similar association was recently reported in turkeys: netb was identified in 6.6% of the C. perfringens strains isolated from turkeys with NE but in none of the strains isolated from healthy animals (LYHS et al, 2013).…”

Necrotic enteritis in chickens: an important disease caused by Clostridium perfringens

“…For decades after the definitive description of necrotic enteritis by Parish 11 it was thought that alpha-toxin, a toxin produced by all isolates of C. perfringens, was the main virulence determinant. However, in 2006, we demonstrated that alpha-toxin was not essential for experimental disease induction 12 and then went on to discover and characterise the toxin, necrotic enteritis toxin B-like (NetB), that does play an essential role in virulence 13,14 .…”

Jejunal hemorrhage syndrome in a Zebu cow in Brazil

“…The anaerobe bacteriology were performed (SILVA et al, 2013) and characteristic C. perfringens colonies were subjected to multiplex (VIEIRA et al, 2008) and netB (necrotic enteritis B-like toxin) PCR (KEYBURN et al, 2008).…”