Identification of Equine Cecal Bacteria Producing Amines in an In Vitro Model of Carbohydrate Overload

Abstract

Acute laminitis has been associated with the overgrowth of gram-positive bacteria within the equine hindgut, causing the release of factor(s) leading to ischemia-reperfusion of the digits. The products of fermentation which trigger acute laminitis are, as yet, unknown; however, vasoactive amines are possible candidates. The objectives of this study were to use an in vitro model of carbohydrate overload to study the change in populations of cecal streptococci and lactobacilli and to establish whether certain species of these bacteria were capable of producing vasoactive amines from amino acids. Cecal contents from 10 horses were divided into aliquots and incubated anaerobically with either corn starch or inulin (fructan; both at 1 g/100 ml). Samples were taken at 6-h intervals over a 24-h period for enumeration of streptococci, lactobacilli, and gram-negative anaerobes by a dilution method onto standard selective growth media. The effects of the antibiotic virginiamycin (1 mg/100 ml) and calcium hydrogen phosphate (CaHPO 4 ; 0.3 g/100 ml) were also examined. Fermentation of excess carbohydrate was associated with increases in numbers of streptococci and lactobacilli (2-to 3.5-log unit increases; inhibited by virginiamycin) but numbers of gram-negative anaerobes were not significantly affected. A screening agar technique followed by 16S rRNA gene sequence analysis enabled the identification of 26 different bacterial strains capable of producing one or more vasoactive amines. These included members of the species Streptococcus bovis and five different Lactobacillus spp. These data suggest that certain bacteria, whose overgrowth is associated with carbohydrate fermentation, are capable of producing vasoactive amines which may play a role in the pathogenesis of acute laminitis.Fermentation of carbohydrate in the hindgut has been recognized as one of the primary events associated with acute laminitis in the horse (14). The ability of the equine small intestine to digest starch may be overwhelmed if starch is present in excess (Ͼ0.4% body weight [35]), leading to its metabolism by cecal and colonic bacteria and the production of lactic acid. Fructans, the oligosaccharides present in grass which act as the storage form of carbohydrate, do not appear to be digested by the small intestine (21) and so are also substrates for fermentative bacteria. The variation of fructan levels in grasses under different climatic conditions has been suggested to account for the seasonal incidence of acute laminitis (A. C. Longland, A. J. Cairns, and M. O. Humphreys, Proc. 16th Equine Nutr. Physiol. Soc., p. 258-259, 1999).Gram-positive bacteria have been implicated in the pathogenesis of laminitis in the carbohydrate overload model since they have been shown to overgrow in the cecum of horses given corn starch by stomach tube (16). This overgrowth, particularly of streptococci and lactobacilli, was associated with lactic acid accumulation and the death of gram-negative bacteria leading to endotoxin release, both of which were postulated t...

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Feeding grape seed extract to horses: effects on health, intake and digestion

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