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Title:   Author  item
                                            Thacker, Tyleritem
                                            Palmer, Mitchellitem
                                            Waters, Wade
  Submitted to: Meeting Abstract
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 10/11/2011
                                    Publication  Date: 10/11/2011
                                    Citation: Thacker, T.C., Palmer, M.V., Waters, W.R. 2011. Transcriptome analysis of stimulated PBMC from Mycobacterium bovis infected cattle [abstract]. 4th International Symposium on Animal Functional Genomics. p. 28.Interpretive Summary:
                                Technical Abstract: Immunological responses of cattle to Mycobacterium bovis  (M. bovis) infection are of interest in terms of understanding the biology of M. bovis infection and for the development of improved diagnostic techniques. Although considerable time and resources have been invested in understanding immune responses to M. bovis infection, the protective response has yet to be identified. To begin to elucidate the immune response of cattle to M. bovis infection, 6 outbreed Holstein calves were infected via aerosol with 8 x 10**3 CFU/ml M. bovis. Peripheral blood was collected by venipuncture prior to infection and at 30 and 60 days post infection. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated with purified protein derivative (PPD) of M. bovis for 16 hours. Total RNA was isolated using a Qiagen RNeasy Mini column. The quality of the RNA was evaluated using an Agilent 2100 Bioanalyzer. Samples were randomly assigned to one of two pools for each time point. Each sample pool was prepared for RNA-Seq and sequenced (75 base run) on the Illumina Genome Analyzer II, one pool per channel. Sequences were trimmed then aligned against the Btau_4.0 assembly using myrna. Utilizing the count data, differential gene expression was determined using EdgeR. When post infection was compared to pre infection, 97 genes were differentially expressed at both time points after infection. Differentially expressed genes were primarily composed of cytokine/T cell specific genes including: IFN-gamma, LT-alpha, IL-22, CXCL-10, CXCL-9, IL-17a, IL-17F, Granzyme A and Granzyme B. Expression was confirmed by real-time quantitative PCR using RNA from individual animals. Differential splicing and unannotated exon expression was explored.


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