Present status of cholera vaccines.

crease in enterotoxin production. Based on their findings, toxin-enriched culture filtrates for separation studies have been produced by the Centre for Applied Microbiology and Research, Porton Down, in collaboration with this laboratory, using a 20 litre batch fermentation technique with controlled pH 7.5, aeration of 10 litres/min, agitation at 500rev./min and incubation at 36°C for 5-6h. Cells were removed by continuous centrifugation and the enterotoxin extracted from concentrated culture filtrates using narrowrange preparative isoelectric focusing in dextran gel (Turnbull et a[., 19796). The diarrhoeal enterotoxin is a thermolabile antigenic protein produced to some degree by most strains of B. cereus. As a metabolite it is distinct from phospholipase C and the two haemolysins of B. cereus, and constitutes one of the two lethal factors produced by this organism (the other being the thiol-activated haemolysin, cereolysin). The instability of the enterotoxin, its sensitivity to proteolytic enzymes, the partial loss of toxigenicity in strains on repeated sub-culture and the problems encountered in separating it from other metabolites of B. cereus have all contributed to the slow progress in purification of the product. Although complete purification and characterization are still awaited there is now good evidence that this enterotoxin is responsible for the diarrhoeal syndrome of illness, and that the dermonecrotic and intestinonecrotic properties are relevant in the pathogenesis of the various non-gastrointestinal B. cereus infections (Turnbull & Kramer, 1983). The emetic toxin produced by B. cereus has only been partially purified (Melling & Capel, 1978; Melling et al., 1978). Optimal production occurs in a rice culture slurry incubated at 25-30°C during the stationary growth phase of the organism (Fig. 2) and synthesis may be associated with sporulation. This toxin is a non-antigenic polypeptide of low relative molecular mass possessing remarkable properties in its stability to heat, pH extremes and enzymes. Strains of B. cereus that synthesize this toxin are not exceptional with respect to their ability to produce other metabolites including the diarrhoeal toxin, but until a simpler model than monkey feeding is available it is not possible to predict the proportion of strains that might be able to elaborate the toxin. Epidemiological evidence, however, that 75% of strains incriminated in the emetic syndrome outbreaks are serotype 1 suggest that only certain strains may be able to produce this toxin; the spores of serotype 1 strains also exhibit a markedly higher resistance to heat than spores of other serotypes. A detailed review of the principal toxic metabolites produced by B. cereus has been presented by Turnbull (1981).