Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations

Abstract

Clostridium perfringens is an important pathogen of animals and humans and is the causative agent of necrotic enteritis (NE) in poultry. This study focuses on the typing of intestinal C. perfringens isolates (n = 61) from outbreaks of NE collected from several areas of Southern Ontario, using a recently developed multilocus sequence typing (MLST) technique. For comparison, C. perfringens isolates from healthy birds were also obtained and typed. An additional locus, the pfoS locus, was included in our analysis, in an attempt to increase the discriminatory ability of the method previously published. Birds were collected from two major poultry processors in Canada, and isolates from processor 2 formed a distinct MLST cluster. Isolates from healthy birds also collected from the outbreak flocks clustered together with isolates from the birds with NE. Although isolates from eight outbreaks clustered together, MLST types were also occasionally different between outbreaks. Strong linkage disequilibrium was observed between loci, suggesting a clonal C. perfringens population structure. Detection assays for toxin genes cpb2 (beta-2 toxin), tpeL, and the newly described netB (NetB toxin) were also performed. netB was almost always found in outbreak isolates, whereas cpb2 was found exclusively in healthy bird isolates. The toxin gene tpeL, which has not been previously identified in C. perfringens type A strains, was also found, but only in the presence of netB. Resistance to bacitracin was found in 34% of isolates from antimicrobial agent-free birds and in 100% of isolates from conventionally raised birds.

FIG. 1.

Dendrogram of concatenated MLST sequences of 61 avian isolates from Southern Ontario generated with pairwise similarities and UPGMA clustering. It includes eight previously published avian C. perfringens MLST sequences (16). Isolates from the study by Jost and collaborators are labeled JGS and shown in gray. CP4 is a virulent NE-associated isolate from another previous study in Ontario (30). The three C. perfringens strains with published genome sequences (strains 13, ATCC 13124, and SM101) were also included, and their MLST types were generated in silico (22, 27). Selected bootstrap values are indicated at nodes. Susceptibility to bacitracin was determined using a 16-μg/ml breakpoint. STs were determined through eBURSTv3 analysis (10), and ST01 and 18 are labeled in boldface type (founders).

FIG. 2.

Linkage disequilibrium matrix of polymorphic sites in 22 STs, generated by Arlequin version 3.11 (9). Darker cells refer to more-significant P values (black cells represent values of ≤0.05 and the lightest-gray cells up to 0.20). Locus names are shown on each axis, in sequential order on the chromosome.