Abstract
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 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 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 hemorrhagic 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; some canine and equine netF-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 C. perfringens in specific enteric disease of animals.
Highlights
C. perfringens is an important Gram-positive anaerobic pathogen of humans and animals that is found ubiquitously in soil and the gastrointestinal tract of vertebrates
The present study describes a novel pore-forming toxin toxigenic C. perfringens associated with fatal hemorrhagic gastroenteritis of dogs and fatal necrotizing enterocolitis of neonatal foals
Two PVL open reading frames (ORF) were located in scaffold00006 (GenBank KP739975), while the third was in scaffold00012 (GenBank KP739976), both surrounded by transposon-related sequences
Summary
C. perfringens is an important Gram-positive anaerobic pathogen of humans and animals that is found ubiquitously in soil and the gastrointestinal tract of vertebrates. A novel toxin, NetB, was shown to be produced by the majority of type A isolates recovered from chickens with necrotic enteritis (NE), an important disease in broiler chicken production, and to play a critical role in NE pathogenesis [2]. This important advance raises the possibility that type A strains in a number of other poorly understood but clinically and pathologically distinct enteric diseases of different animal species [1] might contain other as-yet-undescribed necrotizing toxin genes [3]. Clonality has been described for the majority of bovine type E isolates, for porcine type C isolates, and for isolates from chickens with NE [6,7,8,9]
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