Introduction and background: A role for type A Clostridium perfringens in acute hemorrhagic and necrotizing gastroenteritis in dogs and in necrotizing enterocolitis of neonatal foals has long been suspected but not characterized.
Basic methodologies: Cytotoxicity testing; DNA sequencing; plasmid analysis; targeted gene mutation and complementation; pulsed-field gel electrophoresis.
Major findings: The supernatant of isolates made from a dog and from a foal that died from these diseases was highly cytotoxic for an equine ovarian (EO) cell line. Partial genome sequencing of the dog isolate revealed three novel putative toxin genes related to the pore-forming Leukocidin/Hemolysin Superfamily; these were designated netE, netF, and netG. netE and netF were located on one large conjugative plasmid, and netG was located with a cpe enterotoxin gene on a second large conjugative plasmid. Mutation and complementation showed that only netF was associated with the cytotoxicity. Although netE and netG were not associated with cytotoxicity, immunoblotting with specific antisera showed these proteins to be expressed in vitro. There was a highly significant association between the presence of netF with type A strains isolated from cases of canine acute haemorrhagic gastroenteritis and foal necrotizing enterocolitis. netE and netF were found in all cytotoxic isolates, as was cpe, but netG was less consistently present. Pulsed-field gel electrophoresis showed that netF-positive isolates belonged to a clonal population. Equine antisera to recombinant Net proteins showed that only antiserum to rNetF had high supernatant cytotoxin neutralizing activity.
Conclusion: The identification of this novel necrotizing toxin is an important advance in understanding the virulence of type A C. perfringens in specific enteric disease of animals.