Isolation and purification of Aerornonas sobria cytotonic enterotoxin and p- haemolysin

Summary. Aeromonas sp., grown in tryptone soya broth supplemented with yeast extract, 06 %, pH 7.5, and incubated with agitation at 100 oscillations/min for 15 h at 37°C produced optimal amounts of p-haemolysin and cytotonic enterotoxin. More prolonged incubation resulted in the loss of enterotoxic activity and anion exchange chromatographic analysis indicated the presence of a moiety capable of breaking down the toxin. Anion exchange fast protein liquid chromatography resulted in a single peak of haemolytic activity and two peaks with enterotoxic activitiy. The cytotonic enterotoxin was purified from the fraction most active in the infant mouse assay ; the second peak, which did not cross-react immunologically, may represent a second cytotonic enterotoxin. Neither peak was observed in the chromatographic fractions of filtrates from strains devoid of activity in the infant mouse assay. Purified enterotoxin, estimated to have a mol. wt of 15 kDa by SDS-PAGE, caused fluid accumulation in the infant mouse assay, was non-haemolytic to rabbit erythrocytes, caused an increase in CAMP activity in tissue culture cells and did not cross-react immunologically with components of cholera toxin or the whole toxin. Purified P-haemolysin had an estimated mol. wt of 55 kDa, lysed rabbit erythrocytes and did not cause fluid accumulation in the infant mouse test.