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This study investigated the effect of diet and host on the rumen bacterial microbiome and the impact of an acidotic challenge on its composition. Using parallel pyrosequencing of the V3 hypervariable region of 16S rRNA gene, solid and liquid associated bacterial communities of 8 heifers were profiled. Heifers were exclusively fed forage, before being transitioned to a concentrate diet, subjected to an acidotic challenge and allowed to recover. Samples of rumen digesta were collected when heifers were fed forage, mixed forage, high grain, during challenge (4 h and 12 h) and recovery. A total of 560,994 high-quality bacterial sequences were obtained from the solid and liquid digesta. Using cluster analysis, prominent bacterial populations differed (P≤0.10) in solid and liquid fractions between forage and grain diets. Differences among hosts and diets were not revealed by DGGE, but real time qPCR showed that several bacteria taxon were impacted by changes in diet, with the exception of Streptococcus bovis. Analysis of the core rumen microbiome identified 32 OTU"s representing 10 distinct bacterial taxa including Bacteroidetes (32.8%), Firmicutes (43.2%) and Proteobacteria (14.3%). Diversity of OTUs was highest with forage with 38 unique OTUs identified as compared to only 11 with the high grain diet. Comparison of the microbial profiles of clincial vs. subclinical acidotic heifers found a increases in the relative abundances of Acetitomaculum, Lactobacillus, Prevotella, and Streptococcus. Increases in Streptococcus and Lactobacillus likely reflect the tolerance of these species to low pH and their ability to proliferate on surplus fermentable carbohydrate. The acetogen, Acetitomaculum may thereforeplay a role in the conversion of lactate to acetate in acidotic animals. Further profiling of the bacterial populations associated with subclinical and clinical acidosis could establish a microbial fingerprint for these disorders and provide insight into whether there are causative microbial populations that could potentially be therapeutically manipulated.