Escherichia coli P16 was shown to produce two heat-stable toxins '(ST) with differing biological activity. The toxins were separated by methanol extraction, and the first, STa, was methanol soluble, partially heat stable, active in neonatal piglets (1 to 3 days old) and infant mice, but inactive in weaned pigs (7 to 9 weeks old); the second, STb, was methanol insoluble, active in weaned pigs and rabbit ligated loops, but inactive in infant mice. It is therefore suggested that use of suckling mice as indicators of ST production will fail to identify certain STproducing strains.

While studying the involvement of cyclic adenosine 3',5 '-monophosphate (cAMP) in the fluid secretion caused by heat-stable enterotoxin (ST) from Escherichia coli P16 in infant mice, it was noted that the culture filtrate containing ST also contained large amounts of cAMP. The present paper details attempts to obtain a cAMP-free ST preparation. The organisms were grown in a defined medium, and the heated culture filtrate was concentrated by reverse osmosis. After methanol extraction of the filtrate, which removed 80% of the nonactive solids, the methanol-soluble ST was further purified by gel filtration through a Sephadex G-10 column. The first fraction recovered after gel chromatography contained ST with a negligible amount of cAMP. Treatment with methanol did not adversely affect the enterotoxic activity. Certain parameters of the infant mouse model have been investigated, and using our ST preparation it has been found that animals remain responsive up to 15 days of age with an optimum assay time of 2 h after toxin challenge.

The mucosal surface pH of rat small intestine was measured in vivo . The surface pH in the normal jejunum was 6.20 ± 0.02 (67) and 7.00 ± 0.05 (5) in the ileum. Escherichia coli STa toxin induced a rapid and reversible alkalinization of both jejunal and ileal mucosae to a pH of 6.91 ± 0.08 (10) and 7.67 ± 0.06 (5) respectively. The synthetic ST analogue, STh-(6-19), had an effect identical to native STa toxin on jejunal surface pH. Theophylline (20 mM) maintained the STa-elevated jejunal surface pH after toxin removal but had no effect on untreated tissue. 8-Bromo cyclic GMP resembled STa by causing similar mucosal alkalinization in the jejunum; 8-bromo cyclic AMP, forskolin and cholera toxin individually had considerably smaller effects on surface pH, although combining forskolin or cholera toxin with theophylline resulted in alkalinization of the jejunal mucosa to a pH of 6.92 ± 0.03 (5) and 6.76 ± 0.04 (4). These results indicate that cyclic-GMP-dependent secretory processes are more capable of inducing surface pH changes than those dependent on cyclic AMP. The ability of STa to alter mucosal surface pH makes it a useful tool to investigate the microclimate hypothesis for weak electrolyte absorption.

Partially purified heat-stable enterotoxin obtained from Escherichia coli strain F11/P155 caused an accumulation of cyclic GMP in the intestines of 8-day-old mice.