Immune Biomarkers to Identify Resilient and Susceptible Johne's Disease Phenotypes in Red Deer (Cervus elaphus)

Studies have identified breeds of farmed red deer (Cervus elaphus) that express polarised phenotypes for Resilience (R) or Susceptibility (S) to Johne’s disease, following natural infection with Mycobacterium avium subspecies paratuberculosis (MAP). Animals with R phenotype contain or clear the infection while animals with an S phenotype usually succumb to clinical disease. Previous studies showed that macrophages from animals with a susceptible genotype had increased expression of certain innate immune genes, compared to resilient animals. As these traits appeared to be moderately heritable, successive generations of deer were bred over 3 years using artificial insemination with semen obtained from stags with known polarised R or S genotypes and challenged with MAP. These experimental infection studies were carried out to identify immune biomarkers produced by peripheral blood leukocytes cultured in vitro designed to provide immune signatures for either phenotype. The first experiment duplicated an earlier study on innate immune biomarkers found in Monocyte Derived Macrophage (MDM) cultures, stimulated in vitro with MAP. . Quantitative PCR (qPCR) was used to measure gene expression. In the initial screen more than 60 genes were selected as likely candidates, either from prior published literature or from previous transcriptomics findings. Following evaluation of these genes a smaller subset of gene targets (14) were selected for further study in greater depth. The results from the macrophage cultures showed that a small group of genes were expressed at markedly different levels in either R or S animals. The genes IL1A, NOS2, IL12A, CXCL5 and PKLR were expressed at higher levels in macrophages obtained from susceptible animals compared to resilient animals. The remaining genes CCL2, STAT3, SOCS3, SOCS4, MAPK8, ISG15, ISG20, TLR2 and TLR9 did not show any difference in their expression levels between the two groups. Subsequent infection studies targeted genes linked with adaptive immune responses. This involved the culture of Peripheral Blood Mononuclear Cells (PBMCs) stimulated with the superantigen Staphylococcal Enterotoxin B (SEB). PBMC cultures were simple to carry out and provided informative markers that involved a small set of candidate genes capable of discriminating between animals with a R or S phenotype. The genes IL1A, IL1B, IL2, IL17 and IFNG were expressed at significantly higher levels in resilient animals compared to susceptible animals. The genes IL4, IL6, IL8, ISG15 and ISG20 displayed a similar pattern of elevated expression in R animals, but not consistently at all time points measured during the study. The proliferative ability of the PBMCs was also investigated, with PBMCs from resilient animals showing significantly higher proliferative ability than those from susceptible animals. These findings appear to affirm a protective role for the adaptive immune system in Johne’s disease. The data identify a novel immune phenotype in resilient animals that may provide insights into pathways of protective immunity that could in future be targeted by vaccination. The separation of the two phenotypes prefaces technology that could also be applied diagnostically to identify resilient or susceptible stud animals as part of breeding programmes to select for R animals