Project Number: 3625-32000-093-08
Project Type: Trust

Start Date: Oct 01, 2008
End Date: Sep 30, 2010

Objective:
To determine the host (bovine) response to Mycobacterium avium ssp. paratuberculosis, and the response of the bacterium, in tissues most affected by Johne's disease in order to better target vaccine and therapeutic countermeasures against disease.

Approach:
This project addresses the critical relationship between Mycobacterium avium subsp. Paratuberculosis; MAP) and its host at a transcriptional level during the course of an infection. RNA isolated from ileum and draining mesenteric lymph node specimens from mature dairy cows clinically symptomatic (3), asymptomatic (sub-clinical) (3) naturally infected with MAP and uninfected (1) will be interrogated using the AffymetrixTM bovine GeneChip® Array in a high-throughput comparative analysis of AffymetrixTM array data and whole transcriptome shotgun deep resequencing (DRS) to provide suitable lead profiles for eventual vaccine development. Transcriptional profiles will be generated using the bovine GeneChip® Array and the results compared to and integrated with digital transcriptome expression (DTE) and digital gene expression profiles (DGE) developed from ~ 15 GB of transcriptome sequence generated by very high-throughput DRS (Illumina GA2) from identical RNA samples. This depth of sequencing will deliver 6-10x coverage of the bovine genome and 40-50x coverage of the NCBI unigene transcriptome. We will use AlpheusTM, GMAPTM and SAS JMP-genomicsTM software to compare sequencing results to bovine unigene assemblies, the extant bovine genome sequence, the human and MAP genome sequences to identify whether any synonymous and non-synonymous variants (SNPs, indels and alternative splicing) are present within host genes and if mutations arise in the MAP during a persistent infection. We expect to document the dynamic relationship between host and MAP gene expression as a first step toward 1) uncovering the basis for the persistent infection, and 2) provide entry portals for vaccine development. As we uncover new genes, and more importantly, coordinately-expressed gene profiles, we will integrate array and sequencing results through software developed at NCGR and make an annotated view of derived data available to BRDC clients on a secure server.