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<OL> <LI> Establish the in vitro and in vivo patterns of temporal gene expression by massively parallel signature sequencing and macro- and microarray analysis of Salmonella enterica Typhimurium, Brucella abortus, Cryptosporidium parvum, and Mycobacterium avium subspecies paratuberculosis infected target tissue culture cells and intact bovine ileum. <LI> Apply bioinformatics and computational biology to compare the temporal gene profiles for common and unique expression patterns for Salmonella enterica Typhimurium, Brucella abortus, Cryptosporidium parvum, and Mycobacterium avium subspecies paratuberculosis. <LI> Confirm selected common and uniquely expressed genes by real-time PCR, Northern analysis, RNAse protection and/or RNA interference assays and define the roles of candidate genes in major cell physiological pathways critical to survival of the pathogen or the host. <LI> Sequence host genes essential for survival of each pathogen in nuclear families of cattle segregating differential response to pathogens and identify single nucleotide polymorphisms (SNPs).
NON-TECHNICAL SUMMARY: This will provide deterrents to biological warfare through innate resistant cattle, biosignatures for diagnostics, and improved vaccines. The outcomes from these projects have application for improving public health, producing safer foods and minimizing agricultural and human bioterrorism.
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projects as described below. Ruminant Ileal Loop Core Laboratory: For the in vivo genomic comparative analysis of ruminant host responses to the selected intracellular pathogens, the initial focal point of all investigators is at the level of the host:pathogen entry interface and subsequent interactions in Peyers patches. Samples for bacteriologic culture, histopathology and ultrastructural studies are collected as required for the specific pathogens, for example at 5, 15, and 30 minutes and 1, 2, 3, 4, 5, 6, 8, 10, and 12 hours. For gene expression analysis, total RNA is extracted immediately after dissection of the mucosa from samples obtained at specified time intervals post-infection. Microarray & Informatics Core Laboratory: We will use a commercial bovine microarray to study the differential expression of bovine genes in response to the microbial infections, bacterial and protozoal pathogens will be inoculated into bovine ligated ileal loops or appropriate cell lines. Total RNA will be isolated from infected and control bovine tissues or cells using appropriate RNA isolation kits and mRNA may be further purified from the total RNA to increase the ratio of under-representative transcripts that include many regulatory or defensive genes critical to the host-pathogen interaction. Hybridization is detected using cDNA probes synthesized with one of two color fluorescent deoxyribonucleotide precursors. The intensities of hybridization in microarrays will be quantified and analyzed using a GeneSight software. Using microarray techniques and ratio analysis, we expect to identify bovine genes whose expression levels are strongly regulated by the invasion of microbial pathogens for future confirmation by real-time PCR. Analysis of Genes in Radiation Hybrid Cell Lines: The development of a 5000 rad radiation hybrid (RH) panel for cattle genome mapping ushered in high-resolution comparative mapping of the cattle genome relative to the genomes of human and mice. Two-point linkage and multipoint map construction will be carried out with the RHMAPPER package. The most likely order will be subjected to a multipoint maximum likelihood algorithm to determine breakage frequency and cR distances.