High concordance of bovine single nucleotide polymorphism genotypes generated using two independent genotyping strategies

Single nucleotide polymorphisms (SNPs) represent the most common form of DNA sequence variation in mammalian livestock genomes. While the past decade has witnessed major advances in SNP genotyping technologies, genotyping errors caused, in part, by the biochemistry underlying the genotyping platform used, can occur. These errors can distort project results and conclusions and can result in incorrect decisions in animal management and breeding programs; hence, SNP genotype calls must be accurate and reliable. In this study, 263 Bos spp. samples were genotyped commercially for a total of 16 SNPs. Of the total possible 4,208 SNP genotypes, 4,179 SNP genotypes were generated, yielding a genotype call rate of 99.31% (standard deviation?±?0.93%). Between 110 and 263 samples were subsequently re-genotyped by us for all 16 markers using a custom-designed SNP genotyping platform, and of the possible 3,819 genotypes a total of 3,768 genotypes were generated (98.70% genotype call rate, SD?±?1.89%). A total of 3,744 duplicate genotypes were generated for both genotyping platforms, and comparison of the genotype calls for both methods revealed 3,741 concordant SNP genotype call rates (99.92% SNP genotype concordance rate). These data indicate that both genotyping methods used can provide livestock geneticists with reliable, reproducible SNP genotypic data for in-depth statistical analysis.     

Receptor‐mediated recognition of mycobacterial pathogens

Mycobacteria are a genus of bacteria that range from the non‐pathogenic Mycobacterium smegmatis to Mycobacterium tuberculosis, the causative agent of tuberculosis in humans. Mycobacteria primarily infect host tissues through inhalation or ingestion. They are phagocytosed by host macrophages and dendritic cells. Here, conserved pathogen‐associated molecular patterns (PAMPs) on the surface of mycobacteria are recognized by phagocytic pattern recognition receptors (PRRs). Several families of PRRs have been shown to non‐opsonically recognize mycobacterial PAMPs, including membrane‐bound C‐type lectin receptors, membrane‐bound and cytosolic Toll‐like receptors and cytosolic NOD‐like receptors. Recently, a possible role for intracellular cytosolic PRRs in the recognition of mycobacterial pathogens has been proposed. Here, we discuss currentideas on receptor‐mediated recognition of mycobacterial pathogens by macrophages and dendritic cells.     

Global gene expression and systems biology analysis of bovine monocyte-derived macrophages in response to in vitro challenge with Mycobacterium bovis

Background

Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cell types to encounter M. bovis following exposure and the response elicited by these cells is pivotal in determining the outcome of infection. Here, a functional genomics approach was undertaken to investigate global gene expression profiles in bovine monocyte-derived macrophages (MDM) purified from seven age-matched non-related females, in response to in vitro challenge with M. bovis (multiplicity of infection 2∶1). Total cellular RNA was extracted from non-challenged control and M. bovis-challenged MDM for all animals at intervals of 2 hours, 6 hours and 24 hours post-challenge and prepared for global gene expression analysis using the Affymetrix® GeneChip® Bovine Genome Array.

Results

Comparison of M. bovis-challenged MDM gene expression profiles with those from the non-challenged MDM controls at each time point identified 3,064 differentially expressed genes 2 hours post-challenge, with 4,451 and 5,267 differentially expressed genes detected at the 6 hour and 24 hour time points, respectively (adjusted P-value threshold ≤0.05). Notably, the number of downregulated genes exceeded the number of upregulated genes in the M. bovis-challenged MDM across all time points; however, the fold-change in expression for the upregulated genes was markedly higher than that for the downregulated genes. Systems analysis revealed enrichment for genes involved in: (1) the inflammatory response; (2) cell signalling pathways, including Toll-like receptors and intracellular pathogen recognition receptors; and (3) apoptosis.

Conclusions

The increased number of downregulated genes is consistent with previous studies showing that M. bovis infection is associated with the repression of host gene expression. The results also support roles for MyD88-independent signalling and intracellular PRRs in mediating the host response to M. bovis.     

Admixture and diversity in West African cattle populations

We present a population genetic analysis of microsatellite variation in 16 West African cattle populations. West Africa represents a unique juxtaposition of different climatic and ecological zones in a relatively small geographical area. While more humid coastal regions are inhabited by the tsetse fly, a vector which spreads trypanosomiasis among cattle, the disease is not transmitted in the drier areas outside this zone. This is the most thorough study of genetic diversity in cattle within this area, which contains genetically important trypanotolerant Bos taurus breeds. Genetic relationships among the many breeds are examined and levels of diversity are assessed. Admixture levels were determined using a variety of methods. Ancestry informative or population-associated alleles (PAAs) were selected using populations from India, the Near East and Europe. Multivariate analysis, the admix program and model-based Bayesian admixture analysis approaches were also employed. These analyses reveal the direct impact of ecological factors and the profound effect of admixture on the cattle of this region. They also highlight the importance of efforts to prevent further dilution of African taurine breeds by B. indicus cattle.     

A microsatellite survey of cattle from a centre of origin: the Near East

Eight humpless cattle breeds from the Near East, three from Europe, one from West Africa and two zebu breeds from India were screened with 20 microsatellite loci. Breeds from the Near East revealed considerable levels of introgression from zebu cattle, which was apparent most in populations from the East and which declined in populations further West. This nonrandom pattern is suggestive of the introduction of zebu cattle from the East. Notwithstanding the overlay of zebu alleles, it was possible to demonstrate that Near Eastern cattle exhibited significantly higher levels of allelic diversity than breeds from other regions, which is consistent with the view that this region represents a primary domestication centre for Bos taurus cattle. The hypothesis that B. taurus and B. indicus cattle have separate domestic origins is also supported by the survey, a large genetic divergence being apparent between the nonhybrid taurine and zebu groups.     

Y-specific microsatellite polymorphisms in a range of bovid species

At least five dinucleotide (CA)n microsatellite repeat arrays have been assigned to the bovine Y-chromosome, with one marker (INRA124) shown to be polymorphic. We describe here the assessment of a panel of four Y-specific microsatellite markers for polymorphism in a range of cattle and related species. It was possible to amplify all the markers in the animals sampled and all showed variation. Three of the microsatellite loci (INRA124, INRA189 and BM861) displayed putative taurine- and zebu-specific alleles which can be useful indicators of male-mediated gene flow in hybrid populations. In the future these microsatellites, in combination with other Y-specific markers should provide a high-resolution Y haplotype system for evolutionary studies in both domesticated cattle and other related species.     

MHC class I bound to an immunodominant Theileria parva epitope demonstrates unconventional presentation to T cell receptors

T cell receptor (TCR) recognition of peptide-MHC class I (pMHC) complexes is a crucial event in the adaptive immune response to pathogens. Peptide epitopes often display a strong dominance hierarchy, resulting in focusing of the response on a limited number of the most dominant epitopes. Such T cell responses may be additionally restricted by particular MHC alleles in preference to others. We have studied this poorly understood phenomenon using Theileria parva, a protozoan parasite that causes an often fatal lymphoproliferative disease in cattle. Despite its antigenic complexity, CD8+ T cell responses induced by infection with the parasite show profound immunodominance, as exemplified by the Tp1(214-224) epitope presented by the common and functionally important MHC class I allele N*01301. We present a high-resolution crystal structure of this pMHC complex, demonstrating that the peptide is presented in a distinctive raised conformation. Functional studies using CD8+ T cell clones show that this impacts significantly on TCR recognition. The unconventional structure is generated by a hydrophobic ridge within the MHC peptide binding groove, found in a set of cattle MHC alleles. Extremely rare in all other species, this feature is seen in a small group of mouse MHC class I molecules. The data generated in this analysis contribute to our understanding of the structural basis for T cell-dependent immune responses, providing insight into what determines a highly immunogenic p-MHC complex, and hence can be of value in prediction of antigenic epitopes and vaccine design.     

Transmission ratio distortion at the growth hormone gene (GH1) in bovine preimplantation embryos: An in vitro culture‐induced phenomenon?

The growth hormone gene (GH1) and its polypeptide product (GH) have a crucial role in reproduction, embryogenesis and general development. A polymorphism present in the fifth exon of the bovine GH1 gene (GH1 p.Leu127Val) has been associated with GH release and milk production in cattle. The objective of the present study was to examine the genotype frequencies of the GH1 p.Leu127Val polymorphism in bovine blastocysts produced in vitro and in vivo to determine if allelic variation of the GH1 gene affects embryo development and survival. A heterozygous (p.Leu127/Val127) sire was used for in vitro fertilization of oocytes of unknown maternal genotype (n = 104) and known maternal genotype (n = 115). PCR amplification and genotyping of the GH1 gene from Day 8 blastocysts derived from these fertilized oocytes demonstrated that there was significant over-representation from the expected Mendelian ratio of GH1 p.Leu127/Leu127 homozygotes from oocytes of known maternal genotype (P = 0.006). Contrary to this, analysis of in vivo-produced bovine blastocysts of known parental GH1 genotype (n = 69) did not reveal an overrepresentation of GH1 p.Leu127/Leu127 homozygotes. These results suggest that developing in vitro-produced embryos are exposed to a selection process, probably due to a less favorable culture environment, that acts to increase the number of GH1 p.Leu127/Leu127 homozygotes, thereby giving rise to the observed transmission ratio distortion (TRD) of GH1 genotypes when compared to in vivo produced embryos.     

Escape from CD8+ T cell response by natural variants of an immunodominant epitope from Theileria parva is predominantly due to loss of TCR recognition

Polymorphism of immunodominant CD8(+) T cell epitopes can facilitate escape from immune recognition of pathogens, leading to strain-specific immunity. In this study, we examined the TCR β-chain (TRB) diversity of the CD8(+) T cell responses of cattle against two immunodominant epitopes from Theileria parva (Tp1(214-224) and Tp2(49-59)) and investigated the role of TCR recognition and MHC binding in determining differential recognition of a series of natural variants of the highly polymorphic Tp2(49-59) epitope by CD8(+) T cell clones of defined TRB genotype. Our results show that both Tp1(214-224) and Tp2(49-59) elicited CD8(+) T cell responses using diverse TRB repertoires that showed a high level of stability following repeated pathogenic challenge over a 3-y period. Analysis of single-alanine substituted versions of the Tp2(49-59) peptide demonstrated that Tp2(49-59)-specific clonotypes had a broad range of fine specificities for the epitope. Despite this diversity, all natural variants exhibited partial or total escape from immune recognition, which was predominantly due to abrogation of TCR recognition, with mutation resulting in loss of the lysine residue at P8, playing a particularly dominant role in escape. The levels of heterozygosity in individual Tp2(49-59) residues correlated closely with loss of immune recognition, suggesting that immune selection has contributed to epitope polymorphism.