Molecular characterization of sheep ruminal enrichments that detoxify pyrrolizidine alkaloids by denaturing gradient gel electrophoresis and cloning.

An enrichment of strictly anaerobic bacteria from ovine rumen fluid, which has previously been named L4M2, is known to detoxify animal hepatotoxins from the pyrrolizidine alkaloid family. These toxins are present in the tansy ragwort plant (Senecio jacobaea). These plants have been described in livestock animals' range forages in regions of the world such as the Northwest United States and South Africa. The bacterial enrichment was characterized by molecular cloning techniques and by the molecular fingerprinting technique of denaturing gradient gel electrophoresis (DGGE). Phylogenetic analysis of the enrichment revealed that the consortium is composed of no more than five putative bacterial species which associated to the Anaerovibrio, Desulfovibrio, Megasphaera, Prevotella, and Synergistes generas. These are all known to exist in the upper gastrointestinal tract of ruminant animals. This work improved upon previous attempts to characterize the consortium by obtaining nearly full-length ribosomal 16S rDNA sequences through cloning. The DGGE results were directly compared to the cloning data by polymerase chain reaction (PCR) amplifying eight phylogenetically representative clones and analyzing them by DGGE. Direct DGGE analysis of the enrichment displayed greater 16S diversity than the clone library used in this study, suggesting that at least one of the organisms present in the enrichment comprises less than 1% of the total cell population. These data will be used to further refine the enrichment in hopes of future use as a probiotic, which could be administered to animals challenged by the presence of tansy ragwort in their forage.