2012 Annual Report 1a.Objectives (from AD-416): To determine how Rumensin affects the bacterial and archaeal populations within the rumen of lactating dairy cows and to determine the effect of this drug on the bacterial populations within dairy wastewater that is routinely used as a fertilizer for crop plants such as tree nuts. 1b.Approach (from AD-416): In order to determine how different dosages of Rumensin affect the bacterial and archaeal populations within the rumen and waste, rumen fluid and waste samples will be taken from the four dairy cattle receiving either 0, 200, 400 or 600 mg/hd/day. The samples will be assayed by cultural, 16S rRNA gene library analysis and quantitative PCR analysis for total and pathogenic bacteria. 3.Progress Report: Quantitative Polymerase Chain Reaction (PCR) on rumen samples from dairy cattle fed 0, 200, 400 or 600 mg/hd/day rumensin revealed no differences in the levels of methanogenic archaea or protozoan DNA. Sixteen S rRNA gene sequence analysis revealed significant increases in the level of the phylum Firmicutes, with the 200 mg/hd/day group showing the greatest increase and the 600 mg/hd/day group showing the least increase. We also observed a significant decrease in the phylum Bacteroidetes with the 200 mg/hd/day showing the greatest decrease and the 400 mg/hd/day group showing the lowest decrease. We also observed decreases in the ratio of Gram negative / Gram positive bacteria with the greatest decrease in the 200 mg/hd/day group and the lowest decrease in the 600 mg/hd/day group. We observed no significant difference in the levels of E. coli or Salmonella in any of the groups. Taken together these results suggest that rumensin does alter the bacterial populations within the rumen of dairy cattle; however, no deleterious differences were observed. This research is in direct support of objective 1 of the parent project, "Define the critical control points for pathogen contamination during the production stream. Place particular emphasis on agricultural water sources including dairy waste water."