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 ability of antisera to lipid A, induced in rabbits by immunization with lipid A complexed to various carriers, to protect mice against gram-negative infection and to inhibit the fluid loss caused by an enteropathogenic strain of Escherichia coli in the piglet ligated gut was investigated. No significant protection was obtained in either case, although passive hemolysis and quantitative precipitation tests showed the presence of antilipid A antibodies in the sera. Fluorescent antibody studies suggest that the lipid A is in a cryptic position on the surface of smooth strains of gram-negative bacteria.

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.

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