Evolution of the Bovine TLR Gene Family and Member Associations with Mycobacterium avium Subspecies paratuberculosis Infection

Fisher, Colleen A.; Bhattarai, Eric K.; Osterstock, Jason B.; Dowd, Scot E.; Seabury, Paul M.; Vikram, Meenu; Whitlock, Robert H.; Schukken, Ynte H.; Schnabel, Robert D.; Taylor, Jeremy F.; Womack, James E.; Seabury, Christopher M.

A case-control study was performed to estimate the association between specific TLR genotypes and MAP infection in Holstein cattle. The study population was derived from an established repository [69] that included whole blood samples preserved from adult Holstein cattle in three herds that were characterized on the basis of: 1) MAP bacterial culture of feces; 2) MAP bacterial culture of tissues for harvested cattle; 3) ELISA values for MAPspecific antibody. Cattle from which MAP was cultured in the feces and/or the tissues collected at harvest were selected as cases (n = 68). Herd-matched controls (n = 270) were selected from those cattle in the repository with negative ELISA and bacterial culture data. Cattle with multiple negative tests were preferentially selected to reduce the probability of misclassification relative to infection status due to the low sensitivity of available diagnostic methods for MAP. DNA was extracted from available blood specimens using a commercial kit (MoBio DNA non-spin, Carlsbad, CA) and assessed for quality as well as concentration by standard spectrophotometric methods. Genotypes for validated SNPs and indels in the 59 upstream regions, introns, and those associated with nonsynonymous or putative nonsense mutations in bovine TLR genes recognizing bacterial ligands (TLR1, TLR2, TLR4, TLR5, TLR6, TLR9, TLR10) (see refs [11, 14] ) were evaluated for further analysis. Loci fixed for the major allele in our dairy population were excluded, leaving 35 nonsynonymous and 1 putative nonsense substitution, and 37 other SNP loci within the 59 upstream regions or intragenic introns. For these 73 variable sites, we excluded SNPs and indels with MAFs,0.01 in our infected cases, leaving 32 SNPs and 3 indels for association tests (see Table S1 ).