A Novel Pore-Forming Toxin in Type A <i>Clostridium perfringens</i> Is Associated with Both Fatal Canine Hemorrhagic Gastroenteritis and Fatal Foal Necrotizing Enterocolitis

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