Avian necrotic enteritis (NE) is caused by certain strains of type A
Clostridium perfringens, an anaerobic Gram-positive bacterium. It is of significant economic importance to the poultry industry, estimated to cost approximately US$6 billion worldwide in 2015 [
1]. The necrotic enteritis beta toxin-like (NetB) toxin was recently identified and shown to be a critical virulence factor in NE pathogenesis [
2]. In these studies, a
netB knockout mutant was unable to induce disease in an NE challenge chicken model system, while complementation with
netB restored the virulence of the mutant [
2]. Furthermore, several studies have examined
netB prevalence in a wide variety of
C. perfringens strains and found a strong correlation between the presence of
netB and host disease status [
3‐
6]. Recently, we took the approach of comparative genomics and identified a genetic “signature” of NE disease in chickens [
7]. We found that
netB resides on a 42 kb pathogenicity locus, NELoc-1, located on a plasmid (pNetB) and associated specifically with virulent strains. This locus contains 36 genes in addition to
netB, several of which are predicted to play a role in virulence. Two other loci (NELoc-2 and -3) associated with NE-producing strains were also identified: NELoc-2 consists of 11 genes and is chromosomally-encoded, whereas NELoc-3 consists of five genes and is located on a plasmid (pCpb2) distinct from that harboring NELoc-1. The complete sequences of pNetB and pCpb2 from virulent strains originating in Canada and Australia have been reported and contain sequences with >99% identity to NELoc-1 and NELoc-3, respectively [
8,
9]. The strict conservation of these loci suggests that they may encode genes important in the pathogenesis of NE. However, functional confirmation of these putative additional virulence factors has yet to be provided. The extrachromosomal location of most of the NE-associated genes (
i.e. NELoc-1 and NELoc-3) presents an opportunity to remove these genes
en masse, through plasmid loss, and thereby assess their collective role in virulence in a common genetic background. Furthermore, putative virulence genes may be reintroduced into the strains cured of their virulence plasmids, either individually or in combination, to assess each gene for phenotypic effects and possible interactions.
To extend our previous findings from the comparative genomic analyses, we have characterized a virulent C. perfringens strain (CP1) that has spontaneously lost its netB-carrying plasmid (pCP1netB). The virulence of the mutant, a netB-complemented derivative, and the wild-type strain has also been assessed both in vitro and in vivo. The results are reported herein.