2012 Annual Report 1a.Objectives (from AD-416): To determine gene expression changes in monocytes/neutrophils isolated during the progression of a mammary gland infection in dairy cattle. 1b.Approach (from AD-416): Lactating dairy cows will be infected with S. uberis. At various time points cells will be isolated from milk and sorted by cell type. Monocytes and neutrophils will be isolated and stored for subsequent RNA analysis. RNA from cells will be isolated and used to perform large-scale RNA sequencing. This data would be analyzed using current bioinformatics software designed by our collaborator. This data will become a part of a public database modeling bovine innate immunity during infections. 3.Progress Report: Cows were infected with S. uberis and at various time points milk was collected and CD14+ monocytes were isolated from milk and other cells. Monocytes were processed to purifiy DNA, RNA, and proteins and each was separated and stored for various assays. The DNA was sent to a researcher at the Beltsville Agricultural Research Center (BARC). The RNA, both mRNA and miRNA, was sent to the Iowa State University Sequencing Facility. Sequencing of mRNA was completed and data was shipped to researchers at Teagasc for analysis. The sequencing facility is still working on the miRNA. To date, protein levels in the samples have been insufficient for proteomic analysis due to the method to protein isolation that was used that combined nucleotide and protein isolation.