Abstract

Background: Clostridium perfringens is a key pathogen in poultry-associated necrotic enteritis (NE). To date there
are limited Whole Genome Sequencing based studies describing broiler-associated C. perfringens in healthy and
diseased birds. Moreover, changes in the caecal microbiome during NE is currently not well characterised. Thus, the
aim of this present study was to investigate C. perfringens virulence factors linked to health and diseased chickens,
including identifying putative caecal microbiota signatures associated with NE.
Results: We analysed 88 broiler chicken C. perfringens genomes (representing 66 publicly available genomes and 22
newly sequenced genomes) using different phylogenomics approaches and identified a potential hypervirulent and
globally-distributed clone spanning 20-year time-frame (1993–2013). These isolates harbored a greater number of
virulence genes (including toxin and collagen adhesin genes) when compared to other isolates. Further genomic
analysis indicated exclusive and overabundant presence of important NE-linked toxin genes including netB and tpeL
in NE-associated broiler isolates. Secondary virulence genes including pfoA, cpb2, and collagen adhesin genes cna,
cnaA and cnaD were also enriched in the NE-linked C. perfringens genomes. Moreover, an environmental isolate
obtained from farm animal feeds was found to encode netB, suggesting potential reservoirs of NetB-positive C.
perfringens strains (toxinotype G). We also analysed caecal samples from a small sub-set of 11 diseased and healthy
broilers for exploratory microbiome investigation using 16S rRNA amplicon sequencing, which indicated a
significant and positive correlation in genus Clostridium within the wider microbiota of those broilers diagnosed
with NE, alongside reductions in beneficial microbiota members.
Conclusions: These data indicate a positive association of virulence genes including netB, pfoA, cpb2, tpeL and cna
variants linked to NE-linked isolates. Potential global dissemination of specific hypervirulent lineage, coupled with
distinctive microbiome profiles, highlights the need for further investigations, which will require a large worldwide
sample collection from healthy and NE-associated birds.