Production of cholera-like enterotoxin by a Vibrio cholerae non-O1 strain isolated from the environment

Abstract

Vibrio cholerae non-O1 strain E8498, isolated in 1978 from fresh water in Louisiana, produced a vascular permeability factor when cultured in shallow resting cultures of Casamino Acids-yeast extract-glucose medium for 24 h at 30 degrees C. Undiluted resting culture filtrates contained heat-labile permeability factor activity which was only partially neutralized by cholera antitoxin and GM1 ganglioside. Supernatants concentrated with PM-10 membranes caused hemorrhage and necrosis in rabbits within 1 h after intracutaneous injection, whereas appropriate dilutions of both filtrates and concentrates demonstrated delayed permeability factor activity, without hemorrhage or necrosis, which was indistinguishable in appearance from that caused by purified cholera enterotoxin produced by V. cholerae O1 Inaba strain 569B. Crude E8498 filtrates contained the biological equivalent of about 5 ng/ml of purified enterotoxin. Permeability factor activity in the fraction obtained by 20 to 50% saturation of filtrate concentrate with ammonium sulfate could be completely neutralized by reference standard cholera antitoxin prepared against purified 569 B enterotoxin. Hemorrhagic activity was unaffected by cholera antitoxin. A 5,000-fold concentrate of the culture supernatant yielded a line of identity with purified cholera enterotoxin in an agar gel double-diffusion test against cholera antitoxin purified by affinity column chromatography with BrCN-activated Sepharose 4B-linked purified cholera enterotoxin as the adsorbent. These findings indicate that V. cholerae non-O1 E8498 produces a permeability factor which is immunologically and biologically indistinguishable from that produced by a strain of V. cholerae O1 classical biotype.