g you The direct determination of haplotypes from extended regions of genomic DNA

Background: One of the major obstacles to the exploitation of genetic variation in human medicine, veterinary medicine, and animal breeding is the difficulty in defining haplotypes in unrelated individuals. Results: We have developed a Multiplex Double Amplification Refractory Mutation System combined with Solid Phase PCR on Fluorescently labelled beads. The process is inherently amenable to automation. It provides a high degree of internal Quality Control, as each PCR product is represented in duplicate on the bead array, and each SNP is tested against multiple partners. This technique can resolve very complex genotypes into their constituent haplotypes; it defined all the alleles at 60 SNP in exon 2 of the ovine DRB1 MHC locus in a sample of 109 rams. These 60 SNP formed 33 DRB1 exon 2 alleles; two of which had not been previously identified; although both of them have been independently confirmed. Conclusion: This technique has the same resolution as allele specific sequencing. Sequencing has the advantage of identifying novel polymorphic sites but where all SNP sites have been identified this novel procedure can resolve all alleles and haplotypes and identify novel combinations of polymorphisms. This method is similar in price to direct sequencing and provides a low cost system for direct haplotyping of extended DNA sequence

An explicit immunogenetic model of gastrointestinal nematode infection in sheep

Gastrointestinal nematodes are a global cause of disease and death in humans, wildlife and livestock. Livestock infection has historically been controlled with anthelmintic drugs, but the development of resistance means that alternative controls are needed. The most promising alternatives are vaccination, nutritional supplementation and selective breeding, all of which act by enhancing the immune response. Currently, control planning is hampered by reliance on the faecal egg count (FEC), which suffers from low accuracy and a nonlinear and indirect relationship with infection intensity and host immune responses. We address this gap by using extensive parasitological, immunological and genetic data on the sheep–Teladorsagia circumcincta interaction to create an immunologically explicit model of infection dynamics in a sheep flock that links host genetic variation with variation in the two key immune responses to predict the observed parasitological measures. Using our model, we show that the immune responses are highly heritable and by comparing selective breeding based on low FECs versus high plasma IgA responses, we show that the immune markers are a much improved measure of host resistance. In summary, we have created a model of host–parasite infections that explicitly captures the development of the adaptive immune response and show that by integrating genetic, immunological and parasitological understanding we can identify new immune-based markers for diagnosis and control

The transfer of IgA from mucus to plasma and the implications for diagnosis and control of nematode infections

Immunoglobulin A (IgA) activity has been associated with reduced growth and fecundity of Teladorsagia circumcincta. IgA is active at the site of infection in the abomasal mucus. However, while IgA activity in abomasal mucus is not easily measured in live animals without invasive methods, IgA activity can be readily detected in the plasma, making it a potentially valuable tool in diagnosis and control. We used a Bayesian statistical analysis to quantify the relationship between mucosal and plasma IgA in sheep deliberately infected with T. circumcincta. The transfer of IgA depends on mucosal IgA activity as well as its interaction with worm number and size; together these account for over 80% of the variation in plasma IgA activity. By quantifying the impact of mucosal IgA and worm number and size on plasma IgA, we provide a tool that can allow more meaningful interpretation of plasma IgA measurements and aid the development of efficient control programmes

The potential for vaccines against scour worms of small ruminants

This review addresses the research landscape regarding vaccines against scour worms, particularly Trichostrongylus spp. and Teladorsagia circumcincta. The inability of past research to deliver scour-worm vaccines with reliable and reproducible efficacy has been due in part to gaps in knowledge concerning: (i) host-parasite interactions leading to development of type-2 immunity, (ii) definition of an optimal suite of parasite antigens, and (iii) rational formulation and administration to induce protective immunity against gastrointestinal nematodes (GIN) at the site of infestation. Recent ‘omics’ developments enable more systematic analyses. GIN genomes are reaching completion, facilitating “reverse vaccinology” approaches that have been used successfully for the Rhipicephalus australis vaccine for cattle tick, while methods for gene silencing and editing in GIN enable identification and validation of potential vaccine antigens. We envisage that any efficacious scour worm vaccine(s) would be adopted similarly to “Barbervax™” within integrated parasite management schemes. Vaccines would therefore effectively parallel the use of resistant animals, and reduce the frequency of drenching and pasture contamination. These aspects of integration, efficacy and operation require updated models and validation in the field. The conclusion of this review outlines an approach to facilitate an integrated research program

Divergent Allele Advantage provides a quantitative model for maintaining alleles with a wide range of intrinsic merits

The Major Histocompatibility Complex (MHC) is the most genetically diverse region of the genome in most vertebrates. Some form of balancing selection is necessary to account for the extreme diversity, but the precise mechanism of balancing selection is unknown. Due to the way MHC molecules determine immune recognition, overdominance (also referred to as heterozygote advantage) has been suggested as the main driving force behind this unrivalled diversity. However, both theoretical results and simulation models have shown that overdominance in its classical form cannot maintain large numbers of alleles unless all alleles confer unrealistically similar levels of fitness. There is increasing evidence that heterozygotes containing genetically divergent alleles allow for broader antigen presentation to immune cells, providing a selective mechanism for MHC polymorphism. By framing competing models of overdominance within a general framework, we show that a model based on Divergent Allele Advantage (DAA) provides a superior mechanism for maintaining alleles with a wide range of intrinsic merits, as intrinsically less fit MHC alleles that are more divergent can survive under DAA. Specifically, our results demonstrate that a quantitative mechanism built from the Divergent Allele Advantage hypothesis is able to maintain polymorphism in the MHC. Applying such a model to both livestock breeding and conservation could provide a better way of identifying superior heterozygotes and quantifying the advantages of genetic diversity at the MHC

Identification of the amino acids in the major histocompatibility complex class II region of scottish blackface sheep that are associated with resistance to nematode infection

Lambs with the Major Histocompatibility Complex DRB1*1101 allele have been shown to produce fewer nematode eggs following natural and deliberate infection. These sheep also possess fewer adult Teladorsagia circumcincta than sheep with alternative alleles at the DRB1 locus. However, it is unclear if this allele is responsible for the reduced egg counts or merely acts as a marker for a linked gene. This study defined the MHC haplotypes in a population of naturally infected Scottish Blackface sheep by PCR amplification and sequencing, and examined the associations between MHC haplotypes and faecal egg counts by generalised linear mixed modelling. The DRB1*1101 allele occurred predominately on one haplotype and a comparison of haplotypes indicated that the causal mutation or mutations occurred in or around this locus. Additional comparisons with another resistant haplotype indicated that mutations in or around the DQB2*GU191460 allele were also responsible for resistance to nematode infections. Further analyses identified six amino acid substitutions in the antigen binding site of DRB1*1101 that were significantly associated with reductions in the numbers of adult T. circumcincta

Development of a porcine skeletal muscle cDNA microarray: analysis of differential transcript expression in phenotypically distinct muscles

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background Microarray profiling has the potential to illuminate the molecular processes that govern the phenotypic characteristics of porcine skeletal muscles, such as hypertrophy or atrophy, and the expression of specific fibre types. This information is not only important for understanding basic muscle biology but also provides underpinning knowledge for enhancing the efficiency of livestock production. Results We report on the de novo development of a composite skeletal muscle cDNA microarray, comprising 5500 clones from two developmentally distinct cDNA libraries (longissimus dorsi of a 50-day porcine foetus and the gastrocnemius of a 3-day-old pig). Clones selected for the microarray assembly were of low to moderate abundance, as indicated by colony hybridisation. We profiled the differential expression of genes between the psoas (red muscle) and the longissimus dorsi (white muscle), by co-hybridisation of Cy3 and Cy5 labelled cDNA derived from these two muscles. Results from seven microarray slides (replicates) correctly identified genes that were expected to be differentially expressed, as well as a number of novel candidate regulatory genes. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray. Conclusion We have developed a porcine skeletal muscle cDNA microarray and have identified a number of candidate genes that could be involved in muscle phenotype determination, including several members of the casein kinase 2 signalling pathway.Peer Reviewe

Genetic dissection of MHC-associated susceptibility to Lepeophtheirus salmonis in Atlantic salmon

Background: Genetic variation has been shown to play a significant role in determining susceptibility to the salmon louse, Lepeophtheirus salmonis. However, the mechanisms involved in differential response to infection remain poorly understood. Recent findings in Atlantic salmon (Salmo salar) have provided evidence for a potential link between marker variation at the major histocompatibility complex (MHC) and differences in lice abundance among infected siblings, suggesting that MHC genes can modulate susceptibility to the parasite. In this study, we used quantitative trait locus (QTL) analysis to test the effect of genomic regions linked to MHC class I and II on linkage groups (LG) 15 and 6, respectively. Results: Significant QTL effects were detected on both LG 6 and LG 15 in sire-based analysis but the QTL regions remained unresolved due to a lack of recombination between markers. In dam-based analysis, a significant QTL was identified on LG 6, which accounted for 12.9% of within-family variance in lice abundance. However, the QTL was located at the opposite end of DAA, with no significant overlap with the MHC class II region. Interestingly, QTL modelling also revealed evidence of sex-linked differences in lice abundance, indicating that males and females may have different susceptibility to infection. Conclusion: Overall, QTL analysis provided relatively weak support for a proximal effect of classical MHC regions on lice abundance, which can partly be explained by linkage to other genes controlling susceptibility to L. salmonis on the same chromosom

Maternal undernutrition and the ovine acute phase response to vaccination

Background: The acute phase response is the immediate host response to infection, inflammation and trauma and can be monitored by measuring the acute phase proteins (APP) such as haptoglobin ( Hp) or serum amyloid A (SAA). The plane of nutrition during pregnancy is known to affect many mechanisms including the neuroendocrine and neuroimmune systems in neonatal animals but effects on the APP are unknown. To investigate this phenomenon the serum concentration of Hp and SAA was initially determined in non-stimulated lambs from 3 groups (n = 10/group). The dams of the lambs of the respective groups were fed 100% of requirements throughout gestation (High/High; HH); 100% of requirements for the first 65 d of gestation followed by 70% of requirements until 125 d from when they were fed 100% of requirements (High/Low; HL); 65% of liveweight maintenance requirements for the first 65 d gestation followed by 100% of requirements for the remainder of pregnancy ( Low/High; LH). The dynamic APP response in the lambs was estimated by measuring the concentration of Hp and SAA following routine vaccination with a multivalent clostridial vaccine with a Pasteurella component, Heptavac P (TM) following primary and secondary vaccination. Results: The Hp and SAA concentrations were significantly lower at the time of vaccination ( day 8-14) than on the day of birth. Vaccination stimulated the acute phase response in lambs with increases found in both Hp and SAA. Maternal undernutrition led to the SAA response to vaccination being significantly lower in the HL group than in the HH group. The LH group did not differ significantly from either the HH or HL groups. No significant effects of maternal undernutrition were found on the Hp concentrations. A significant reduction was found in all groups in the response of SAA following the second vaccination compared to the response after the primary vaccination but no change occurred in the Hp response. Conclusion: Decreased SAA concentrations, post-vaccination, in lambs born to ewes on the HL diet shows that maternal undernutrition prior to parturition affects the innate immune system of the offspring. The differences in response of Hp and SAA to primary and secondary vaccinations indicate that the cytokine driven APP response mechanisms vary with individual AP