Salivary syndrome in horses: identification of slaframine in red clover hay.

An outbreak of salivary syndrome in horses in North Carolina was investigated. Rhizoctonia leguminicola was the predominant fungus isolated from toxic red clover hay. The fungus was less prevalent in the hay after 10 months of storage, and the hay had also decreased in biological activity after 10 months. Toxic hay caused extreme salivation, piloerection, respiratory distress, and increased frequency of defecation when fed to guinea pigs, and purified extracts of toxic hay and pure slaframine elicited these same responses when injected intraperitoneally into guinea pigs. The freshly acquired hay, based on the biological (slobber-producing) activity in hay and in purified extracts, contained the equivalent to 50 to 100 ppm (50 to 100 microgram/g) of slaframine, but this level had decreased after 10 months by about 10-fold to about 7 ppm. Slaframine and seven synthetic derivates of slaframine were used in presumptive gas-liquid chromatographic identification of this mycotoxin. Slaframine (1-acetoxy-6-amino-octahydroindolizine) was identified in purified extracts of toxic hay by gas-liquid chromatography-mass spectrometry after preparative thin-layer chromatography. This was the first direct identification of slaframine in toxic red clove hay.