Combining Genome Wide Association Studies and Differential Gene Expression Data Analyses Identifies Candidate Genes Affecting Mastitis Caused by Two Different Pathogens in the Dairy Cow

Mastitis is a costly disease which hampers the dairy industry. Inflammation of the mammary gland is commonly caused by bacterial infection, mainly Escherichia coli, Streptococcus uberis and Staphylococcus aureus. As more bacteria become multi-drug resistant, one potential approach to reduce the disease incidence rate is to breed selectively for the most appropriate and potentially protective innate immune response. The genetic contribution to effective disease resistance is, however, difficult to identify due to the complex interactions that occur. In the present study two published datasets were searched for common differentially expressed genes (DEGs) with similar changes in expression in mammary tissue following intra-mammary challenge with either E. coli or S. uberis. Additionally, the results of seven published genome-wide association studies (GWAS) on different dairy cow populations were used to compile a list of SNPs associated with somatic cell count. All genes located within 2 Mbp of significant SNPs were retrieved from the Ensembl database, based on the UMD3.1 assembly. A final list of 48 candidate genes with a role in the innate immune response identified from both the DEG and GWAS studies was further analyzed using Ingenuity Pathway Analysis. The main signalling pathways highlighted in the response of the bovine mammary gland to both bacterial infections were 1) granulocyte adhesion and diapedesis, 2) ephrin receptor signalling, 3) RhoA signalling and 4) LPS/IL1 mediated inhibition of RXR function. These pathways comprised a network regulating the activity of leukocytes, especially neutrophils, during mammary gland inflammation. The timely and properly controlled movement of leukocytes to infection loci seems particularly important in achieving a good balance between pathogen elimination and excessive tissue damage. These results suggest that polymorphisms in key genes in these pathways such as SELP, SELL, BCAR1, ACTR3, CXCL2, CXCL6, CXCL8 and FABP may influence the ability of dairy cows to resist mastitis

Toxoplasma gondii profilin does not stimulate an innate immune response through bovine or human TLR5

Toxoplasma gondii is responsible for one of the most prevalent infections in people. T. gondii profilin (TgPr) is a protein integral to parasite movement and cellular invasion. Murine TLR has been described to bind TgPr. Furthermore, more recently, human TLR5 has been described to recognise recombinant TgPr, as well as bacterial flagellin. In addition to infections in humans, T. gondii infects farm animals, but little information is available about its innate recognition. We aimed to investigate whether, similarly to their human orthologue, bovine and porcine TLR5 could also be stimulated by TgPr by using a combination of reporter cell lines expressing full length TLR5 from each species as well as primary cells. Although human and bovine TLR5-transfected cells responded to flagellin, no response was detected upon stimulation with profilin. Furthermore, TgPr failed to elicit IL-6 secretion in human peripheral blood mononuclear cells and CD14þ monocytes. In contrast, exposure of RAW cells, known to express TLR11 to TgPr, slightly increased the IL-6 response. Our data cast doubts on the possibility that profilin is a specific ligand for human TLR5 and bovine TLR5. This leaves the immunogenic properties of this potential target antigen uncharacterised outside of the murine system

Poultry Coccidiosis: Design and Interpretation of Vaccine Studies

Eimeria infection impacts upon chicken welfare and economic productivity of the poultry sector. Live coccidiosis vaccines for chickens have been available for almost 70 years, but the requirement to formulate blends of oocysts from multiple Eimeria species makes vaccine production costly and logistically demanding. A multivalent vaccine that does not require chickens for its production and can induce protection against multiple Eimeria species is highly desirable. However, despite the identification and testing of many vaccine candidate antigens, no recombinant coccidiosis vaccine has been developed commercially. Currently, assessment of vaccine efficacy against Eimeria, and the disease coccidiosis, can be done only through in vivo vaccination and challenge experiments but the design of such studies has been highly variable. Lack of a “standard” protocol for assessing vaccine efficacy makes comparative evaluations very difficult, complicating vaccine development, and validation. The formulation and schedule of vaccination, the breed of chicken and choice of husbandry system, the species, strain, magnitude, and timing of delivery of the parasite challenge, and the parameters used to assess vaccine efficacy all influence the outcomes of experimental trials. In natural Eimeria infections, the induction of strong cell mediated immune responses are central to the development of protective immunity against coccidiosis. Antibodies are generally regarded to be of lesser importance. Unfortunately, there are no specific immunological assays that can accurately predict how well a vaccine will protect against coccidiosis (i.e., no “correlates of protection”). Thus, experimental vaccine studies rely on assessing a variety of post-challenge parameters, including assessment of pathognomonic lesions, measurements of parasite replication such as oocyst output or quantification of Eimeria genomes, and/or measurements of productivity such as body weight gain and feed conversion rates. Understanding immune responses to primary and secondary infection can inform on the most appropriate immunological assays. The discovery of new antigens for different Eimeria species and the development of new methods of vaccine antigen delivery necessitates a more considered approach to assessment of novel vaccines with robust, repeatable study design. Careful consideration of performance and welfare factors that are genuinely relevant to chicken producers and vaccine manufacturers is essential

The Phylogeography of Rabies in Grenada, West Indies, and Implications for Control

In Grenada, West Indies, rabies is endemic, and is thought to be maintained in a wildlife host, the small Indian mongoose (Herpestes auropunctatus) with occasional spillover into other hosts. Therefore, the present study was undertaken to improve understanding of rabies epidemiology in Grenada and to inform rabies control policy. Mongooses were trapped island-wide between April 2011 and March 2013 and examined for the presence of Rabies virus (RABV) antigen using the direct fluorescent antibody test (dFAT) and PCR, and for serum neutralizing antibodies (SNA) using the fluorescent antibody virus neutralization test (FAVN). An additional cohort of brain samples from clinical rabies suspects submitted between April 2011 and March 2014 were also investigated for the presence of virus. Two of the 171 (1.7%) live-trapped mongooses were RABV positive by FAT and PCR, and 20 (11.7%) had SNAs. Rabies was diagnosed in 31 of the submitted animals with suspicious clinical signs: 16 mongooses, 12 dogs, 2 cats and 1 goat. Our investigation has revealed that rabies infection spread from the northeast to the southwest of Grenada within the study period. Phylogenetic analysis revealed that the viruses from Grenada formed a monophyletic clade within the cosmopolitan lineage with a common ancestor predicted to have occurred recently (6–23 years ago), and are distinct from those found in Cuba and Puerto Rico, where mongoose rabies is also endemic. These data suggest that it is likely that this specific strain of RABV was imported from European regions rather than the Americas. These data contribute essential information for any potential rabies control program in Grenada and demonstrate the importance of a sound evidence base for planning interventions

Short communication:Pegbovigrastim treatment in vivo does not affect granulocyte ability to migrate to endometrial cells and kill bacteria in vitro in healthy cows

In periparturient dairy cows, immune suppression, resulting in decreased neutrophil numbers and function, leads to increased susceptibility to postpartum conditions such as mastitis, retained placenta, and metritis. Administration of polyethylene glycol-conjugated bovine granulocyte colony stimulating factor (pegbovigrastim, Imrestor; Elanco Animal Health, Greenfield, IN) 7 d before and within 24 h of calving, effectively improves granulocyte production and function in vivo as well as in milk. A recently developed coculture assay was adapted for use with endometrial epithelial cells to assess the effects of pegbovigrastim application on directed granulocyte migration and bactericidal activity in vitro on a per-cell basis in endometrial cell cultures. Granulocytes from treated and untreated periparturient cows (6 and 5 per group, respectively) were evaluated for their ability to migrate to and kill bacteria after treatment, in context of the infected endometrium. We hypothesized that in addition to increasing the absolute concentration of circulating neutrophil granulocytes, pegbovigrastim treatment in vivo alters the ability of granulocytes to migrate to endometrial cells in vitro. The results clearly show a marked increase in the total concentration of granulocytes and monocytes between the 2 treatment groups as early as 2 d after the first injection, and this increased between the samples taken 2 d after calving. No migratory or killing differences were identified between granulocytes of both groups, suggesting that pegbovigrastim-induced granulocytes were as effective as non-induced cells. This may also be due to the absence of negative energy balance in the study animals and leads us to conclude that the positive effects seen in vivo are most likely based on the larger number of granulocytes present rather than a direct effect of pegbovigrastim treatment on the functionality of cells for the parameters tested in this study

A Genome-Wide Association Study for Calving Interval in Holstein Dairy Cows Using Weighted Single-Step Genomic BLUP Approach.

The aim of the present study was to identify genomic region(s) associated with the length of the calving interval in primiparous (n = 6866) and multiparous (n = 5071) Holstein cows. The single nucleotide polymorphism (SNP) solutions were estimated using a weighted single-step genomic best linear unbiased prediction (WssGBLUP) approach and imputed high-density panel (777 k) genotypes. The effects of markers and the genomic estimated breeding values (GEBV) of the animals were obtained by five iterations of WssGBLUP. The results showed that the accuracies of GEBVs with WssGBLUP improved by +5.4 to +5.7, (primiparous cows) and +9.4 to +9.7 (multiparous cows) percent points over accuracies from the pedigree-based BLUP. The most accurate genomic evaluation was provided at the second iteration of WssGBLUP, which was used to identify associated genomic regions using a windows-based GWAS procedure. The proportion of additive genetic variance explained by windows of 50 consecutive SNPs (with an average of 165 Kb) was calculated and the region(s) that accounted for equal to or more than 0.20% of the total additive genetic variance were used to search for candidate genes. Three windows of 50 consecutive SNPs (BTA3, BTA6, and BTA7) were identified to be associated with the length of the calving interval in primi- and multiparous cows, while the window with the highest percentage of explained genetic variance was located on BTA3 position 49.42 to 49.52 Mb. There were five genes including , , , , and inside the windows associated with the length of the calving interval. The biological process terms including alanine transport, L-alanine transport, proline transport, and glycine transport were identified as the most important terms enriched by the genes inside the identified windows

Adaptive evolution of Toll-like receptor 5 in domesticated mammals

<p>Abstract</p> <p>Background</p> <p>Previous studies have proposed that mammalian toll like receptors (TLRs) have evolved under diversifying selection due to their role in pathogen detection. To determine if this is the case, we examined the extent of adaptive evolution in the TLR5 gene in both individual species and defined clades of the mammalia.</p> <p>Results</p> <p>In support of previous studies, we find evidence of adaptive evolution of mammalian TLR5. However, we also show that TLR5 genes of domestic livestock have a concentration of single nucleotide polymorphisms suggesting a specific signature of adaptation. Using codon models of evolution we have identified a concentration of rapidly evolving codons within the TLR5 extracellular domain a site of interaction between host and the bacterial surface protein flagellin.</p> <p>Conclusions</p> <p>The results suggest that interactions between pathogen and host may be driving adaptive change in TLR5 by competition between species. In support of this, we have identified single nucleotide polymorphisms (SNP) in sheep and cattle TLR5 genes that are co-localised and co-incident with the predicted adaptive codons suggesting that adaptation in this region of the TLR5 gene is on-going in domestic species.</p

Genome-wide association studies of inflammatory bowel disease in German shepherd dogs

Canine Inflammatory Bowel Disease (IBD) is considered a multifactorial disease caused by complex interactions between the intestinal immune system, intestinal microbiota and environmental factors in genetically susceptible individuals. Although IBD can affect any breed, German shepherd dogs (GSD) in the UK are at increased risk of developing the disease. Based on previous evidence, the aim of the present study was to identify single nucleotide polymorphisms (SNPs), which may confer genetic susceptibility or resistance to IBD using a genome-wide association study (GWAS). Genomic DNA was extracted from EDTA blood or saliva samples of 96 cases and 98 controls. Genotyping of cases and controls was performed on the Canine lllumina HD SNP array and data generated was analyzed using PLINK. Several SNPs and regions on chromosomes 7,9,11 and 13 were detected to be associated with IBD using different SNP-by-SNP association methods and F-ST windows approach. Searching one Mb up-and down-stream of the most significant SNPs, as identified by single SNP analysis as well as 200Kb before and after the start and the end position of the associated regions identified by F-ST windows approach, we identified 63 genes. Using a combination of pathways analysis and a list of genes that have been reported to be involved in human IBD, we identified 16 candidate genes potentially associated with IBD in GSD