Non-toxic perfringolysin O and α-toxin derivatives as potential vaccine candidates against bovine necrohaemorrhagic enteritis.

Bovine necrohaemorrhagic enteritis is a fatal Clostridium perfringens type A-induced disease that is characterised by sudden death. Recently the involvement of perfringolysin O and α-toxin in the development of necrohaemorrhagic lesions in the gut of calves was suggested, and thus derivatives of these toxins are potentially suitable as vaccine antigens. In the current study, the perfringolysin O derivative PFO, alone or in combination with α-toxin derivative GST-cpa, was evaluated as possible vaccine candidate, using in vitro assays. PFO showed no haemolytic effect on horse red blood cells and no cytotoxic effect on bovine endothelial cells. Furthermore, calves immunised with PFO raised antibodies against perfringolysin O that could inhibit the perfringolysin O-associated haemolytic activity on horse red blood cells. Antisera from calves immunised with PFO had a significantly higher neutralising capacity against the cytotoxic effect of C. perfringens culture supernatant to bovine endothelial cells than serum from control calves (P <0.05). Immunisation of calves with PFO in combination with GST-cpa elicited antibodies against perfringolysin O and α-toxin and consequently inhibited both the perfringolysin O-associated haemolytic activity and the α-toxin-associated lecithinase activity in vitro. Additionally, the neutralising ability of these antisera on the cytotoxic effect of C. perfringens culture supernatant to bovine endothelial cells was significantly higher than that from calves immunised with PFO (P <0.001). In conclusion, perfringolysin O derivative PFO is an immunogenic antigen that can potentially be used to produce vaccine against bovine necrohaemorrhagic enteritis. Including α-toxin derivative GST-cpa has an additional protective effect and therefore vaccination of calves with a combination of both antigens seems even more promising.