Identification of Novel Pathogenicity Loci in Clostridium perfringens Strains That Cause Avian Necrotic Enteritis

Dion Lepp,Joshua Gong,J Glenn Songer,Pradeep R Marri,Everett L Rosey,Bryan Roxas,Valeria R Parreira,John F Prescott,Gayatri Vedantam,Holger Bruggemann

doi:10.1371/journal.pone.0010795

Abstract

Type A Clostridium perfringens causes poultry necrotic enteritis (NE), an enteric disease of considerable economic importance, yet can also exist as a member of the normal intestinal microbiota. A recently discovered pore-forming toxin, NetB, is associated with pathogenesis in most, but not all, NE isolates. This finding suggested that NE-causing strains may possess other virulence gene(s) not present in commensal type A isolates. We used high-throughput sequencing (HTS) technologies to generate draft genome sequences of seven unrelated C. perfringens poultry NE isolates and one isolate from a healthy bird, and identified additional novel NE-associated genes by comparison with nine publicly available reference genomes. Thirty-one open reading frames (ORFs) were unique to all NE strains and formed the basis for three highly conserved NE-associated loci that we designated NELoc-1 (42 kb), NELoc-2 (11.2 kb) and NELoc-3 (5.6 kb). The largest locus, NELoc-1, consisted of netB and 36 additional genes, including those predicted to encode two leukocidins, an internalin-like protein and a ricin-domain protein. Pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed that the NE strains each carried 2 to 5 large plasmids, and that NELoc-1 and -3 were localized on distinct plasmids of sizes ∼85 and ∼70 kb, respectively. Sequencing of the regions flanking these loci revealed similarity to previously characterized conjugative plasmids of C. perfringens. These results provide significant insight into the pathogenetic basis of poultry NE and are the first to demonstrate that netB resides in a large, plasmid-encoded locus. Our findings strongly suggest that poultry NE is caused by several novel virulence factors, whose genes are clustered on discrete pathogenicity loci, some of which are plasmid-borne.

Highlights

Avian necrotic enteritis (NE) is caused by specific strains of Clostridium perfringens and costs the worldwide poultry industry an estimated $2 billion annually, largely due to costs of antimicrobial prophylaxis and inefficient feed conversion [1,2]
The alpha toxin (CPA) of C. perfringens was long assumed to play a central role in the pathogenesis of NE, but Keyburn and others [3] showed that challenge of broiler chicks with a control methods. The alpha toxin (CPA) mutant yielded lesions comparable to those in birds inoculated with the wildtype strain
The total number of contigs and N50 values associated with these draft genomes were comparable to that of the six publicly available draft C. perfringens genomes generated via a traditional WGS sequencing approach (Table 2)

Summary

Introduction

Avian necrotic enteritis (NE) is caused by specific strains of Clostridium perfringens and costs the worldwide poultry industry an estimated $2 billion annually, largely due to costs of antimicrobial prophylaxis and inefficient feed conversion [1,2]. The alpha toxin (CPA) of C. perfringens was long assumed to play a central role in the pathogenesis of NE, but Keyburn and others [3] showed that challenge of broiler chicks with a CPA mutant yielded lesions comparable to those in birds inoculated with the wildtype strain. Other studies have revealed that immunization of birds with CPA toxoid protects against virulent C. perfringens challenge [4,5]. The intestines of birds challenged with the CPA mutant contained CPA [6], likely produced by the resident C. perfringens strains and contributing to gut lesions. Recent work has largely confirmed the critical association of netB with NE strains [8], netB-negative C. perfringens may sometimes produce NE [9]

Methods

Results

Conclusion