Anti-CPB2 monoclonal antibody, clone BIUBGJ-5082

Reasons for performing the studyToxin detection and screening could contribute to knowledge of the transmission patterns, risk factors and epidemiology of Clostridium difficile and Clostridiumperfringens. ObjectiveTo isolate C.difficile and C.perfringens and to detect A/B toxins in faecal samples from diarrhoeic and nondiarrhoeic foals. Study designCross-sectional observational study. MethodsA total of 153 samples from foals were collected: 139 samples from farms and 14 samples from diarrhoeic foals admitted to a veterinary hospital. The A/B toxins were detected by cytotoxicity assay. All suspected colonies of C.perfringens were subjected to polymerase chain reaction for detection of the major toxin genes (, , epsilon and ) and for detection of 2-, NetB- and enterotoxin-encoding genes. Furthermore, C.difficile and C.perfringens isolates were evaluated for in vitro antimicrobial susceptibility. ResultsSeven of 153 (4.6%) samples, all from diarrhoeic foals, were positive for C.difficileA/B toxin. Of these, 5 of 14 (35.7%) were from hospitalised foals, and only 2 of 63 (3.2%) diarrhoeic foal samples were from farms (P = 0.002). Clostridiumperfringens was isolated from 31 (20.3%) foals, of which 21 of 76 (27.6%) were diarrhoeic and 10 of 76 (13.2%) were nondiarrhoeic, demonstrating a difference between these 2 groups (P = 0.045). Only 4 strains were positive for the 2-encoding gene (cpb2). All C.difficile and C.perfringens isolates were susceptible to metronidazole and vancomycin. ConclusionsThe present report highlights the need for laboratory diagnostics to differentiate C.difficile-associated infection in foals from other causes of diarrhoea to facilitate adequate antimicrobial therapy. Potential relevanceMore studies are needed to clarify the role of C.perfringens as a primary agent of diarrhoea in foals.

Enterotoxin-producing Clostridium perfringens type A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carrying cpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lacking cpb2 and IS1151 but carrying cpe and IS1470-like sequences. By completely sequencing these two cpe plasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an similar to 35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon TOM. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the similar to 43.6-kb pCPF5603 variable region contains a functional cpb2 gene and several metabolic genes. Diversity studies indicated that other type A plasmid cpe(+)/IS1151 SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmid cpe(+)/ IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in the cpe plasmids of other type A SPOR/AAD isolates, as well as in representative C. perfringens type B to D isolates carrying other virulence plasmids, possibly suggesting that most or all C. perfringens virulence plasmids transfer conjugatively.