Elizabeth Glass

Variation matters: TLR structure and species-specific pathogen recognition

Toll-like receptors (TLRs) are a family of pattern recognition receptors that are an important link between innate and adaptive immunity. Many vaccines incorporate ligands for TLRs as an adjuvant and are developed in rodent models, with the resulting data transferred to other species. Vaccine features can be improved markedly by emphasizing the biological relevance when evaluating other animal models for host-pathogen interaction and by taking greater advantage of the unique experimental opportunities that are offered by large animal, non-rodent models. Here, we aim to summarize our current knowledge of species-specific TLR responses and briefly discuss that vaccine efficacy in relevant host species might be improved by considering the species-specific TLR responses.

Comparative organization and function of the major histocompatibility complex of domesticated cattle

Summary: This review focuses on recent advances in research on the bovine major histocompatibility complex (BoLA), with specific reference to the genetic organization, polymorphism and function of the class II genes. The BoLA region is unlike the MHC of humans and mice in that a large inversion has moved several class II genes, including the TAP/LMP cluster, close to the centromere of bovine chromosome 23. Therefore, dose linkage of MHC genes and other genes associated with the MHC in humans and mice does not appear to be required for normal immunological function. In cattle, polymorphism in the class IIa genes influences both the magnitude and the epitope specificity of antigen‐specific T‐cell responses to foot‐and‐mouth disease virus peptides. Disease association studies have demonstrated that BoLA alleles affect the subclinical progression of bovine leukemia virus (BLV) infection. This association is strongly correlated with the presence of specific amino acid motifs within the DRB3 antigen‐binding domain. In addition to the practical significance of these findings, the association between BoLA and BLV provides a unique model to study host resistance to retrovirus infection in a non‐inbred species. These studies contribute to our understanding of the evolution of the MHC in mammals, to the development of broadly effective vaccines, and to breeding strategies aimed at improving resistance to infectious diseases.

Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system

Mastitis caused by Escherichia coli and Staphylococcus aureus is a major pathology of dairy cows. To better understand the differential response of the mammary gland to these two pathogens, we stimulated bovine mammary epithelial cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus culture supernatant (SaS) to compare the transcriptomic profiles of the initial bMEC response. By using HEK 293 reporter cells for pattern recognition receptors, the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that lipoteichoic acid, protein A and α-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling revealed a core innate immune response partly shared by LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greater magnitude than those induced by SaS. Microarray data analysis suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. The higher upregulation of chemokines (Cxcl10, Ccl2, Ccl5 and Ccl20) that target mononuclear leucocytes by LPS than by SaS is likely to be related to the differential activation of the type I IFN pathway, and could induce a different profile of the initial recruitment of leucocytes. The MEC responses to the two stimuli were different, as LPS was associated with NF-κB and Fas signaling pathways, whereas SaS was associated with AP-1 and IL-17A signaling pathways. It is noteworthy that at the protein level secretion of TNF-α and IL-1β was not induced by either stimulus. These results suggest that the response of MEC to diffusible stimuli from E. coli and S. aureus contributes to the onset of the response with differential leucocyte recruitment and distinct inflammatory and innate immune reactions of the mammary gland to infection.

Bovine tuberculosis: the genetic basis of host susceptibility

The prevalence of bovine tuberculosis (BTB) in the UK remains a significant economic burden and problem for the agri-food industry. Much effort has been directed towards improving diagnostics, finding vaccine candidates and assessing the usefulness of badger culling. The contribution that host genotype makes to disease outcome has, until recently, been overlooked; yet, it is biologically untenable that genetic variation does not play a role. In this review, we highlight the evidence, past and present, for a role of host genetics in determining susceptibility to BTB in livestock. We then address some of the major issues surrounding the design of future studies tasked with finding the exact causative genetic variation underpinning the TB susceptibility phenotype. Finally, we discuss some of the potential future benefits, and problems, that a knowledge of the genetic component to BTB resistance/susceptibility may bring to the agricultural industries and the wider scientific community.

The protozoan parasite, Theileria annulata, induces a distinct acute phase protein response in cattle that is associated with pathology.

Acute phase proteins (APP) are synthesised in the liver in response to the systemic presence of high levels of pro-inflammatory cytokines. Bacteria are considered to be strong inducers of APP whereas viruses are weak or non-inducers of APP. Very few reports have been published on APP induction by parasites. Here, we report that the tick-borne protozoan parasite of cattle, Theileria annulata, induced an atypical acute phase response in cattle. Following experimental infection, serum amyloid A (SAA) appeared first, followed by a rise in alpha(1) acid glycoprotein (alpha(1)AGP) in all animals, whereas haptoglobin, which is a major APP in cattle, only appeared in some of the animals, and generally at a low level. All three APP only became elevated around or after the appearance of schizonts in draining lymph nodes and after the first observed temperature rise. Increased alpha(1)AGP levels coincided with the appearance of piroplasms. The production of SAA and alpha(1)AGP correlated strongly with each other, and also with some clinical measures of disease severity including the time to fever, development of leucopaenia, parasitaemia and mortality. These results are consistent with the hypothesis that T. annulata causes severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines.

In-silico identification of chicken immune-related genes.

In order to increase the resources available in chicken, a large-scale expressed sequence tag (EST) project was recently undertaken, resulting in the addition of more than 330,000 sequences to the databases. With the sequencing of further EST collections, there are now more than 460,000 chicken EST sequences publicly available (http://www.ncbi.nlm.nih.gov/). Previous analyses of the EST data estimate that the chicken genome may contain up to 35,000 genes. However, human data indicate that there may only be around 25,000, although there may be many more transcripts than actual genes. Here we describe how we used a bioinformatics approach with this large EST collection in order to identify immune-related genes, many of which were previously unreported in the chicken. The ESTs include cytokines, chemokines, antigens, cell surface proteins, receptors and MHC-associated genes. The identification of these kinds of genes will allow further study of avian immunology and will pave the way for large-scale immune-related microarray experiments, giving new insight into functional and evolutionary studies.

Molecular evolution of bovine Toll-like receptor 2 suggests substitutions of functional relevance

BackgroundThere is accumulating evidence that polymorphism in Toll-like receptor (TLR) genes might be associated with disease resistance or susceptibility traits in livestock. Polymorphic sites affecting TLR function should exhibit signatures of positive selection, identified as a high ratio of non-synonymous to synonymous nucleotide substitutions (ω). Phylogeny based models of codon substitution based on estimates of ω for each amino acid position can therefore offer a valuable tool to predict sites of functional relevance. We have used this approach to identify such polymorphic sites within the bovine TLR2 genes from ten Bos indicus and Bos taurus cattle breeds. By analysing TLR2 gene phylogeny in a set of mammalian species and a subset of ruminant species we have estimated the selective pressure on individual sites and domains and identified polymorphisms at sites of putative functional importance.ResultsThe ω were highest in the mammalian TLR2 domains thought to be responsible for ligand binding and lowest in regions responsible for heterodimerisation with other TLR-related molecules. Several positively-selected sites were detected in or around ligand-binding domains. However a comparison of the ruminant subset of TLR2 sequences with the whole mammalian set of sequences revealed that there has been less selective pressure among ruminants than in mammals as a whole. This suggests that there have been functional changes during ruminant evolution. Twenty newly-discovered non-synonymous polymorphic sites were identified in cattle. Three of them were localised at positions shaped by positive selection in the ruminant dataset (Leu227Phe, His305Pro, His326Gln) and in domains involved in the recognition of ligands. His326Gln is of particular interest as it consists of an exchange of differentially-charged amino acids at a position which has previously been shown to be crucial for ligand binding in human TLR2.ConclusionWithin bovine TLR2, polymorphisms at amino acid positions 227, 305 and 326 map to functionally important sites of TLR2 and should be considered as candidate SNPs for immune related traits in cattle. A final proof of their functional relevance requires further studies to determine their functional effect on the immune response after stimulation with relevant ligands and/or their association with immune related traits in animals.

Duplicated DQ Haplotypes Increase the Complexity of Restriction Element Usage in Cattle

The MHC of cattle encodes two distinct isotypes of class II molecules, DR and DQ. Unlike humans, cattle lack the DP locus and about half the common haplotypes express duplicated DQ genes. The number and frequency of DQA and DQB alleles means that most cattle are heterozygous. If inter- and/or intrahaplotype pairing of DQA and DQB molecules occurs, cattle carrying DQ-duplicated haplotypes may express more restriction elements than would be predicted by the number of expressed alleles. We are investigating whether duplicated haplotypes cause differences in immune response, particularly in terms of generating protective immunity. We have analyzed the Ag-presenting function of DQ molecules in two heterozygous animals, one of which carries a duplicated haplotype. We compared the class II isotype specificity of T cell clones recognizing a putative vaccinal peptide from foot-and-mouth disease virus (FMDV15). We show for the first time that bovine T cells can recognize Ag in the context of DQ molecules. We also present evidence that interhaplotype pairings of DQA and DQB molecules form functional restriction elements. Both animals showed distinct biases to usage of particular restriction elements. Mainly DQ-restricted clones were derived from the animal with duplicated DQ genes, whereas the majority of clones from the animal with a single DQ gene pair were DR restricted. Furthermore, haplotype bias was observed with both animals. These experiments show that understanding of class II chain pairing in addition to knowledge of the genotype may be important in vaccine design where effective epitope selection is essential.

Innate and Adaptive Immune Responses Co-operate to Protect Cattle against Theileria annulata

For many years it was assumed that Theileria annulata resembled T. parva, parasitizing lymphocytes and causing lymphoproliferative disease, with the two species being controlled by similar protective immune responses. Patricia Preston et al. here review the evidence that has led to a different view of T. annulata. It is now thought that the schizonts of T. annulata inhabit macrophages and B cells, and that tropical theileriosis is not a lymphoproliferative disease. Both innate and adaptive responses contribute to recovery from infection and resistance to challenge and cytokines produced by infected and uninfected cells influence the outcome of infection. Partial protection has been stimulated recently by defined recombinant antigens; efficacy depended upon the delivery system.

Genome-wide association study identifies novel loci associated with resistance to bovine tuberculosis

Tuberculosis (TB) caused by Mycobacterium bovis is a re-emerging disease of livestock that is of major economic importance worldwide, as well as being a zoonotic risk. There is significant heritability for host resistance to bovine TB (bTB) in dairy cattle. To identify resistance loci for bTB, we undertook a genome-wide association study in female Holstein–Friesian cattle with 592 cases and 559 age-matched controls from case herds. Cases and controls were categorised into distinct phenotypes: skin test and lesion positive vs skin test negative on multiple occasions, respectively. These animals were genotyped with the Illumina BovineHD 700K BeadChip. Genome-wide rapid association using linear and logistic mixed models and regression (GRAMMAR), regional heritability mapping (RHM) and haplotype-sharing analysis identified two novel resistance loci that attained chromosome-wise significance, protein tyrosine phosphatase receptor T (PTPRT; P=4.8 × 10−7) and myosin IIIB (MYO3B; P=5.4 × 10−6). We estimated that 21% of the phenotypic variance in TB resistance could be explained by all of the informative single-nucleotide polymorphisms, of which the region encompassing the PTPRT gene accounted for 6.2% of the variance and a further 3.6% was associated with a putative copy number variant in MYO3B. The results from this study add to our understanding of variation in host control of infection and suggest that genetic marker-based selection for resistance to bTB has the potential to make a significant contribution to bTB control.