Occurrence of Clostridium perfringens β2-toxin amongst animals, determined using genotyping and subtyping PCR assays

Abstract

Clostridium perfringens isolates are currently classified into one of five biotypes on the basis of the differential production of alpha-, beta-, epsilon- and iota-toxins. Different biotypes are associated with different diseases of man and animals. In this study a multiple PCR assay was developed to detect the genes encoding these toxins. In addition, detection of the genes encoding the C. perfringens enterotoxin and beta2-toxin allowed subtyping of the bacteria. C. perfringens isolates taken from a variety of animals, including foals, piglets or lambs, were genotyped using this assay. Most of the isolates were found to be genotype A and the gene encoding beta2-toxin [corrected] was present in 50% of the isolates genotyped. A significant association between C. perfringens possessing the beta2-toxin gene and diarrhoea in piglets was identified, suggesting that beta2-toxin may play a key role in the pathogenesis of the disease.

Detection of Enterotoxigenic Clostridium perfringens Type A Isolates in American Retail Foods

“…Multiplex PCR is now routinely used to assign C. perfringens isolates to one of five types (A to E), based upon whether an isolate carries the genes encoding alpha-, beta-, epsilon-, or iota-toxin (8). Recent versions of the multiplex PCR toxin genotyping assay can also determine whether a C. perfringens isolate possesses the cpe and cpb2 genes encoding CPE and beta2-toxin, respectively (8).…”

“…Multiplex PCR is now routinely used to assign C. perfringens isolates to one of five types (A to E), based upon whether an isolate carries the genes encoding alpha-, beta-, epsilon-, or iota-toxin (8). Recent versions of the multiplex PCR toxin genotyping assay can also determine whether a C. perfringens isolate possesses the cpe and cpb2 genes encoding CPE and beta2-toxin, respectively (8). Determining the toxin genotypes of C. perfringens food isolates is epidemiologically significant, since C. perfringens type A food poisoning is nearly always caused by CPE-positive type A isolates (13).…”

“…The multiplex PCR assay used in our study has been described previously (8) and can detect the presence of genes encoding alpha-toxin (cpa), beta-toxin (cpb), beta2-toxin (cpb2), epsilon-toxin (etx), CPE (cpe), or the A component of iota-toxin (iap). The sequences of the six primer pairs used in this multiplex PCR were published previously (8).…”

“…The sequences of the six primer pairs used in this multiplex PCR were published previously (8). Each PCR had a total volume of 50 l, which contained 5 l of the primer pair mixture (final primer concentration, 0.34 M each cpe oligonucleotide, 0.36 M each cpb oligonucleotide, 0.36 M each cpb2 oligonucleotide, 0.44 M each etx oligonucleotide, 0.50 M each cpa oligonucleotide, and 0.52 M each iap oligonucleotide), 40 l of Taq Complete 1.1ϫ Master mix (GENE CHOICE), and 5 l of a cell lysate (prepared as described above) as a template.…”

Multilocus Sequence Typing Subtypes of Poultry Clostridium perfringens Isolates Demonstrate Disease Niche Partitioning

“…All isolates used in the MLST scheme were toxinotyped (Table 1) using PCR assays for the ␣, ␤, ε, , and ␤2 toxin genes and the C. perfringens enterotoxin (CPE) gene (9,22,44). Isolates were also assayed for the presence of netB by using a previously published PCR assay (17).…”