Bacteroidesfragilis has been associated with causation of diarrheal disease in livestock and humans. To date, conventional tissue culture and animal assays used to detect the biologic activity of bacterial enterotoxins have failed with enterotoxigenic B. fragilis. Although enterotoxigenic B. fragilis stimulates intestinal secretion in lamb and calf ligated intestinal loops, infant rabbits, and adult rabbits with ligated ceca, these animal systems are costly and complicated, which limits their usefulness for identification of enterotoxigenic B. fragilis strains. Using the cloned human colonic-epithelial-cell line HT29/Cl, we have developed an in vitro assay that is 89% sensitive and 100% specific in detecting enterotoxigenic B. fragilis strains as defined by the lamb ligatedintestinal-loop assay. Subconfluent HT29/Cl cells treated with concentrated bacterium-free culture supernatants of enterotoxigenic B. fragilis strains develop specific and striking morphologic changes including loss of cell-to-cell attachments, rounding, swelling, and, in some cases, pyknosis. These morphologic changes are initially visible at 1 h after treatment and progress over at least the first 24 h. This tissue culture assay should prove useful in epidemiologic studies of enterotoxigenic B. fragilis and may facilitate basic studies to identify the B. fragilis toxin(s) and its mechanism of action.
Escherichia coli may produce a heat-labile enterotoxin (LT) or two heat-stable enterotoxins (STa, STb). STb consistently causes secretion in vivo in 5-hr weaned-pig intestinal loops (P less than .0001). In Ussing chambers in vitro, crude culture filtrates of STb initiate a prompt increase in short circuit current (SCC; P less than or equal to .0001) and potential difference (P less than or equal to .0001) when compared with nontoxigenic culture filtrates. In bidirectional in vitro studies of ion flux (22Na and 36Cl), STb did not alter 22Na or 36Cl unidirectional or net fluxes. The calculated residual ion flux increased significantly (P less than .03), however, in tissues treated with STb and fully accounted for the STb-induced increase in SCC. Measurement of the electrolyte content of ligated intestinal segments in vivo further suggested that STb stimulated bicarbonate secretion. Relative to controls, significant accumulation of Na and Cl also occurred intraluminally in vivo. These data indicate that STb is a unique enterotoxin that causes net secretion in pig jejunum in vivo. In vitro and in vivo studies show that STb stimulates active secretion of nonchloride anion. We postulate that STb causes active bicarbonate secretion in weaned-piglet jejunum.
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