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

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.

Acute necrotizing colitis with pneumatosis intestinalis in an Amazonian manatee calf

“…SPS agar (Difco) was used to investigate the presence of Clostridium perfringens. After an aerobic incubation for 24 h, 5 colonies with suggestive morphology were individually subjected to previously described PCR protocols for genotyping and investigation of additional virulence factors (Keyburn et al 2008, Silva et al 2014, Gohari et al 2015. The isolated strains were genotyped as C. perfringens type A (positive for alpha toxin-encoding gene) and negative for the following genes: cpb2, cpe, netB, netE, and tpeL.…”

Clostridium perfringens extracellular toxins and enzymes: 20 and counting

“…The most recently characterised toxins are the cytotoxic poreforming toxin NetF and the putative pore-forming toxins NetE and NetG, which were shown to be encoded on plasmids in isolates causing necrotizing gastroenteritis and necrotizing enterocolitis in foals and dogs 28 . There is a significant association between NetFproducing strains and enteric disease in these animals; however, the function of NetE and NetG in disease remains to be investigated 28 .…”

“…There is a significant association between NetFproducing strains and enteric disease in these animals; however, the function of NetE and NetG in disease remains to be investigated 28 .…”

Isolation and genotyping of Clostridium perfringens and Clostridium difficile in Capuchin Monkeys (Sapajus spp.)

“…Furthermore, C. perfringens can produce additional virulence factors such as enterotoxin, which is responsible for diarrhea in humans (LINDSTRÖM et al, 2011), necrotic enteritis toxin B-like (NetB), which is responsible for necrotic enteritis in broiler chickens (KEYBURN et al, 2008), and NetF, which is associated with acute diarrhea in dogs and foals (GOHARI et al, 2015;DINIZ et al, 2016). Despite its importance as an enteropathogen, C. perfringens is commonly reported in the enteric microbiota of healthy animals, thus complicating the laboratory diagnosis of infections caused by this microorganism (SILVA et al, 2015).…”

“…After incubation, at least three characteristic colonies from each dilution were subjected to thermal DNA extraction (BAUMS et al, 2014), and then, a previously described PCR protocol (VIEIRA et al, 2008) was used to detect genes encoding the major C. perfringens toxins (alpha, beta, epsilon and iota), beta-2 toxin (cpb2) and enterotoxin (cpe). For detecting the NetB-, NetE-, NetF and NetG-encoding genes (netB, netE, netF and netG, respectively), PCR protocols described by KEYBURN et al (2008) and GOHARI et al (2015) were applied. Thawed aliquots of stool samples positive for C. perfringens cpe + strains were subjected to enterotoxin detection with a commercial EIA kit (RIDASCREEN ® Clostridium perfringens Enterotoxin -R-Biopharm, Germany).…”