Five wild-type mutant strains of serogroup O1 that lacked the CTX virulence cassette, or contained a natural deletion of a virulence gene within the CTX virulence cassette, or possessed an additional virulence gene, along with a prototype toxigenic strain representing the El Tor classical biotypes were examined by in-vivo and in-vitro methods to determine their enterotoxic potential. The ability of whole cells and culture supernates of the strains to cause fluid accumulation in the rabbit ileal loop model revealed a pattern consistent with the presence of the various virulence gene(s), with those possessing the intact CTX virulence cassette being the most secretogenic. Culture supernates of strains without the CTX virulence cassette or the strain with an incomplete cassette were also able to evoke mild to moderate fluid accumulation in the rabbit ileal loop. Of the various media used, AKI and brain heart infusion broth appeared to support the production of a hitherto unknown secretogenic factor, because culture supernates of the non-toxigenic O1 strains showed higher fluid accumulation ratios when grown in these media than in the others. To confirm that the fluid accumulation elicited by these strains in the ileal loop was due to enterotoxin activity, the effect of supernate of the strains was examined in rabbit small intestine mounted on Ussing chambers. Increases in short circuit current and tissue conductance, as compared with the medium control, were observed even with the strains that did not possess the CTX virulence cassette, confirming their ability to disrupt the function of intestinal tissue. From these studies, it was concluded that strains of O1 devoid of the CTX virulence cassette were still able to elicit a secretory response in the ileal loop and displayed enterotoxic activity in an in-vitro experimental model.


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