Molecular Detection and Phylogenetic Analysis of the cpb2 Gene in Clostridium perfringens Isolates Recovered from Poultry with Suspected Necrotic Enteritis

Abstract

Necrotic enteritis (NE) is an economically important enteric disease of poultry associated with pathogenic Clostridium perfringens strains. The beta2 toxin gene, cpb2, has been detected in isolates from several host species, but its                      contribution to avian NE remains uncertain. This descriptive study evaluated the occurrence of cpb2 among C. perfringens isolates recovered from poultry with clinical or pathological findings suggestive of NE and characterised two partial cpb2 sequences. A total of 62 samples, comprising 54 intestinal samples and eight cloacal swabs, were collected from affected poultry in Maharashtra, India. C. perfringens was isolated using conventional anaerobic bacteriological methods and confirmed by species-specific polymerase chain reaction (PCR) targeting the 16S rRNA gene. Confirmed isolates were screened for cpb2 by PCR. Amplicons from two cpb2-positive isolates were sequenced, aligned with selected GenBank reference sequences, and evaluated by Maximum Parsimony phylogenetic analysis. C. perfringens was recovered from 57 of 62 samples (91.9%), including 52 of 54 intestinal samples (96.3%) and five of eight cloacal swabs (62.5%). All 57 culture-positive isolates yielded the expected 481-bp 16S rRNA amplicon. Five of the 57 confirmed isolates (8.8%) yielded the expected 548-bp cpb2 amplicon. The two partial cpb2 sequences showed relatedness to previously reported reference sequences. In the phylogenetic tree, PZ547701 grouped with the poultry sequence AY884035 with 77% bootstrap support, whereas PZ547700 grouped with the cattle sequence DQ525205 with 62% support and joined AJ537534 within a broader node supported at 89%. These findings document cpb2 carriage in a minority of the C. perfringens isolates examined and indicate that the two partial sequences fall within previously reported cpb2 diversity. Further studies involving unaffected control groups, comprehensive toxinotyping, analysis of toxin expression, and appropriate comparison groups are warranted to further establish the causal role of CPB2 in the development of necrotic enteritis (NE).