Deciphering The Role Of EDN2 In Mycobacterium avium spp. paratuberculosis Tissue Infection

Johne’s disease is a highly transmissible bacterial disease, caused by Mycobacterium avium subspecies paratuberculosis (Map) infection. We initially found an association with Map tissue infection on BTA3 that was narrowed to a 70 kb region in Holstein cattle. The objective of this study was to identify candidates for the causal variant underlying Map tissue infection in Holstein and Jersey cattle. Thirty Jersey and Holstein cattle were whole genome re-sequenced and SNPs were identified within the 70 kb region. Comparisons of conserved and functional sites across mammalian species within this region were conducted using TRANSFAC 7.06 and ENSEMBLE to select 96 SNPs for the design of a custom Illumina SNP assay for fine mapping in 250 Holstein and 50 Jersey cows. Sixteen putative functional SNPs were associated with Map tissue infection (P <0.01) in the Holstein and Jersey cows. Nuclear extracts from freshly harvested ileo-cecal lymph nodes from Holstein cows were next used to characterize differences in binding affinities of the sequences harboring 3 of the SNPs. Luciferase reporter assays were performed to confirm the transcriptional activity of the EDN2 SNPs that demonstrated mobility shifts. Genotyping of 400 additional cattle for the SNPs associated with putative EDN2 functional variants is ongoing. These results suggest that one or more variants in the promoter of EDN2 may result in susceptibility to Map tissue infection in Holstein and Jersey cattle.