Extensive differential DNA methylation between tuberculosis skin test positive and skin test negative cattle.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), represents a significant problem for the agriculture industry as well as posing a risk for human health. Current diagnostic tests for bTB target the cell-mediated immune (CMI) response to infection with M. bovis, primarily through screening of animals with the tuberculin skin test. Epigenetic modifications have been shown to alter the course of the immune response and differentially methylated regions (DMRs) might also influence the outcome of the skin test in cattle. Whole Genome Bisulphite Sequencing (WGBS) was used to profile DNA methylation levels from peripheral blood of a group of cattle identified as test positive for M. bovis (positive for the single intradermal comparative tuberculin test (SICTT) and/or the interferon-γ release assay compared to a test negative control group [n = 8/group, total of 16 WGBS libraries]. Although global methylation profiles were similar for both groups across the genome, 223 DMRs and 159 Differentially Promoter Methylated Genes (DPMGs) were identified between groups with an excess of hypermethylated sites in SICTT positive cattle (threshold > 15% differential methylation). Genes located within these DMRs included the Interleukin 1 receptor (IL1R1) and MHC related genes (BOLA and BOLA-DQB). KEGG pathway analysis identified enrichment of genes involved in Calcium and MAPK signalling, as well as metabolism pathways. Analysis of DMRs in a subset of SICTT negative cattle that were IFN-γ positive showed differential methylation of genes including Interleukin 10 Receptor, alpha (IL10RA), Interleukin 17 F (IL17F) and host defence peptides (DEFB and BDEF109). This study has identified a number of immune gene loci at which differential methylation is associated with SICTT test results and the degree of methylation could influence effective host immune responses.

Identification and validation of quantitative trait loci for ascites syndrome in broiler chickens using whole genome resequencing.

BACKGROUND: Ascites syndrome is a hypertensive, multifactorial, multigene trait affecting meat-type chickens imposing significant economic losses on the broiler industry. A region containing the CPQ gene has been previously identified as significantly affecting ascites phenotype. The region was discovered through whole genome resequencing focused on chicken chromosome 2. The association was confirmed through further genotyping in multiple broiler populations. RESULTS: The whole genome resequencing analyses have now been extended to the current chicken genome assembly. DNA samples were pooled according to gender and phenotype and the pools subjected to next generation sequencing. Loci were identified as clusters of single nucleotide polymorphisms where frequencies of the polymorphisms differed between resistant and susceptible chickens. The chickens are an unselected line descended from a commercial elite broiler line. Regions identified were specific to one or both genders. The data identify a total of 28 regions as potential quantitative trait loci for ascites. The genes from these regions have been associated with hypertensive-related traits in human association studies. One region on chicken chromosome 28 contains the LRRTM4 gene. Additional genotyping for the LRRTM4 region demonstrates an epistatic interaction with the CPQ region for ascites phenotype. CONCLUSIONS: The 28 regions identified were not previously identified in a multi-generational genome wide association study using 60k Single Nucleotide Polymorphism panels. This work demonstrates the utility of whole genome resequencing as a cost effective, direct, and efficient method for identifying specific gene regions affecting complex traits. The approach is applicable to any organism with a genome assembly and requires no a priori assumptions.

Embryo lethality assay as a tool for assessing virulence of isolates from bacterial chondronecrosis with osteomyelitis in broilers.

We used an embryo lethality assay (ELA) to assess virulence for different isolates from cases of bacterial chondronecrosis with osteomyelitis (BCO) in broilers. Lameness is among the most significant animal welfare issues in the poultry industry. Bacterial infections are a major cause of lameness and different bacterial species have been obtained from lame broilers. Reliable lab-based assays are required to assess relative virulence of bacteria obtained from lame broilers. ELA has been used to assess lethal dosage of Enterococcus faecalis and Enterococcus cecorum. We hypothesized that ELA could substitute for more laborious and costly assessments of BCO isolate pathogenicity using live birds. We evaluated 2 different levels of bacteria injected into eggs from layer and commercial broiler embryos. Significant findings include 1) Escherichia coli from neighboring farms operated by the same integrator had very different embryo lethality, 2) isolate Staphylococcus agnetis 908 had low virulence in ELA, even though this isolate can induce more than 50% BCO lameness, 3) Enterococcus cecorum 1415 also had low pathogenicity; even though it was recovered from severe bilateral tibial dyschondroplasia, 4) human and chicken BCO isolates of S. aureus had significant pathogenicity, 5) virulence for some isolates was highly variable possibly corresponding with quality of the embryos/fertile eggs used, and 6) ELA pathogenicity was much lower for our BCO isolates than previous reports which may reflect maternal environment. Overall, ELA virulence and BCO virulence are not always concordant indicating that ELA may not be an effective measure for assessing virulence with respect to BCO.

Whole genome resequencing identifies the CPQ gene as a determinant of ascites syndrome in broilers.

BACKGROUND: Ascites syndrome is the most severe manifestation of pulmonary hypertension in fast-growing broilers. The disease can be attributed to increased body weights of birds, where the higher metabolic load is not matched by sufficient oxygen supply to the cells and tissues. Although there are environmental components, the disease exhibits moderate to high heritability. The current study uses high throughput whole genome resequencing (WGR) to identify genes and chromosomal regions associated with ascites. RESULTS: The WGR data identified the CPQ gene on chromosome 2. The association was confirmed by genotyping a large collection of DNAs from phenotyped birds from three distinct broiler lines using SNPs in intron 6 and exon 8 of the CPQ gene. By combining the genotype data for these two SNP loci, we identified three different alleles segregating in the three broiler lines. Particular genotypes could be associated with resistance to ascites. We further determined that particular genotypes most associated with resistance overexpress CPQ mRNA in three tissues which might explain the role of these alleles in contributing to resistance. CONCLUSIONS: Our findings indicate CPQ is an important determinant of pulmonary hypertension syndrome leading to ascites in broilers. We identified particular SNPs that can be used for marker-assisted selection of broilers for resistance to the disease. Our findings validate WGR as a highly efficient approach to map determinants contributing to complex phenotypic or disease-related traits. The CPQ gene has been associated with pulmonary hypertension in genome-wide association studies in humans. Therefore, ascites investigations in broilers are likely to provide insights into some forms of hypertension in humans.