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.
Highlights
Clostridium perfringens is the main causative agent of avian necrotic enteritis (NE), an enteric disease of chickens that was first described in 1961 [1] and has since been found in all poultry producing countries
Until recently alpha-toxin was thought to be the major virulence factor involved in necrotic enteritis
This paper details the identification and characterisation of a novel toxin, NetB, and provides evidence that the protein is an essential factor in causing necrotic enteritis in chickens
Summary
Clostridium perfringens is the main causative agent of avian necrotic enteritis (NE), an enteric disease of chickens that was first described in 1961 [1] and has since been found in all poultry producing countries. NE in chickens manifests as an acute or chronic enterotoxemia [2]. The acute disease results in significant levels of mortality whereas the chronic disease leads to loss of productivity and welfare concerns. It has been estimated that the disease costs the international poultry industry in excess of $US2 billion per year [3,4,5]. Clinical NE is thought to occur when C. perfringens proliferates to high numbers in the small intestine and produces extracellular toxins that damage the gastrointestinal tract
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