Multilocus sequence typing subtypes of poultry Clostridium perfringens isolates demonstrate disease niche partitioning

Abstract

Clostridium perfringens is a ubiquitous and versatile pathogenic bacterium and is implicated in the etiology of the poultry diseases necrotic enteritis (NE) and poultry gangrene (PG). In this study, multilocus sequence typing was used to investigate genotypic relationships among 139 C. perfringens isolates from 74 flocks. These isolates had multiple disease, host, and environmental origins. The results indicated a polymorphic yet highly clonal population, with 79.6% of all isolates partitioning into one of six clonal complexes or two dominant sequence types, ST-9 and ST-31. The most prolific clonal complex, CC-1, contained 27.3% of all isolates and was not clearly associated with one particular disease. The subtypes CC-4 and ST-31 were highly associated with NE and represented 9.4% and 7.2% of the total isolates, respectively. No PG-associated and NE-associated C. perfringens isolates shared the same sequence type or clonal complex. NE-associated subtypes were more clonal and appeared more evolutionarily divergent than PG-associated subtypes, which tended to cluster in the more ancestral lineages alongside isolates from asymptomatic chickens and turkeys. Toxin gene screening identified cpb2 throughout these isolates and correlated the presence of netB with NE pathology. Previous investigations into the genetic basis of C. perfringens pathogenicity have focused on toxins and other variable genetic elements. This study presents the first sequence-based comparison of C. perfringens isolates recovered in clinical cases of PG and NE and demonstrates that niche specialization is observable in the core genomes of poultry-associated C. perfringens isolates, a concept with both epidemiological and evolutionary significance.

Fig. 1.

Sequence type disease partitioning.

Fig. 2.

eBURST representation of relationships between sequence types and clonal complexes. Clonal complexes are designated CC-1 to CC-6. Separate dots represent sequence types, which are designated by sequence type number. The size of each dot correlates with the number of isolates in that sequence type.

Fig. 3.

Neighbor-joining phylogeny of C. perfringens isolates from the current study. The tree was generated on the basis of a maximum composite likelihood model and was condensed where bootstrap support was <50% of replicates. Clonal complexes are designated CC-1 (○), CC-2 (●), CC-3 (□), CC-4 (■), CC-5 (▵), and CC-6 (▴).

Fig. 4.

Neighbor-joining phylogenetic comparison of C. perfringens isolates from the current study (STs followed by “AGTP”) with those from a prior study (STs followed by “Chalmers”) (2). The tree was generated on the basis of a maximum composite likelihood model and was condensed where bootstrap support was <50% of replicates. ST numbers are equivalent for Fig. 3 and 4 but have been condensed in Fig. 4 due to the merging of the typing schemes. Clonal complexes are indicated by symbols: ○, CC-1; ●, CC-2; □, CC-3; ■, CC-4; ▵, CC-5; ▴, CC-6.