Both epsilon-toxin and beta-toxin are important for the lethal properties of Clostridium perfringens type b isolates in the mouse intravenous injection model

Clostridium perfringens is capable of producing up to 15 toxins, including alpha toxin (CPA), beta toxin (CPB), epsilon toxin (ETX), enterotoxin (CPE), beta2 toxin (CPB2), and perfringolysin O (PFO). Type B isolates, which must produce CPA, CPB, and ETX, are associated with animal illnesses characterized by sudden death or acute neurological signs, with or without intestinal damage. Type B pathogenesis in ruminants is poorly understood, with some animals showing lesions and clinical signs similar to either type C or D infections. It is unknown whether host or environmental conditions are dominant for determining the outcome of type B disease or if disease outcomes are determined by variable characteristics of type B isolates. To help clarify this issue, 29 type B isolates were evaluated for toxin production during late log-phase growth via quantitative Western blotting and by biological activity assays. Most type B isolates produced similar CPB levels to type C isolates in vitro, and have the potential to produce genotype C-like disease. The lethality of type B isolate supernatants administered intravenously to mice was evaluated with or without prior trypsin treatment and monoclonal antibody neutralization studies also were performed. Correlation analyses comparing toxin levels in type B supernatants versus lethality and neutralization studies both found that the main contributor to lethality without pre-treatment with trypsin was CPB, whereas neutralization studies indicated that CPB and ETX were both important after trypsin pre-treatment. At least part of the CPB produced by type B isolates remained active after trypsin treatment.