Understanding resistance and differential vaccine responses to Eimeria in the chicken - novel biomarkers and genetic control.

One of the main underpinning factors for a profitable large-scale poultry industry is the fact that the disease coccidiosis, caused by species of the protozoan parasite Eimeria, is controlled primarily through the use of drugs, or coccidiostats. Vaccines do exist, but these are currently primarily produced by passage of Eimeria through birds and therefore not a cheap nor practical solution to replace coccidostats. Reliance on a single main control measure is not ideal, particularly with political pressure in some parts of the world to ban the use of coccidiostats.

Resistance to Eimeria infection has long been known in inbred lines of chickens, but attempts to map this have been largely unsuccessful. Chromosomes associated with resistance have been identified, but we have yet to identify causative genes, or better still causative mutations. Differential responses to vaccines have also been described, presumably due to similar mechanisms, although this has yet to be formally proven. Resistance has always been described as oocyst output - the fewer oocysts excreted, the more resistant the bird. However, resistance is associated with a stronger innate and adaptive immune response in these birds, and it is unclear, particularly in broilers, if this stronger immune response compromises other parameters, such as feed conversion efficiency (FCE). In other words, do birds that produce fewer oocysts, due to a stronger gut immune response, perform better than birds that produce more oocysts, or is the obverse the case?

We plan to revisit mapping disease resistance, and differential responses to vaccines, using modern techniques, in particular the newly available 600K SNP chip. Similarly to funded work on Campylobacter resistance, we will use both inbred birds in a backcross design, and commercial birds in a genome-wide association study, or GWAS, experiment. We will then assess FCE in resistant versus susceptible birds.

The adaptive immune response is that which clears the pathogen causing the infection, and delivers immunological memory against reinfection. For many years, the adaptive immune response has been split into two arms, each involving a different subset of CD4+ Thelper cells. Th1 responses control infection with intracellular pathogens, such as viruses. Th2 responses control infections with extracellular pathogens such as worms. Adaptive responses are now known to be more complicated, with more subsets of CD4+ T cells involved.

It is well established that infection with Eimeria, an obligate intracellular pathogen, requires a strong inflammatory, Th1 response to control it. However, little has been done recently to investigate the role, if any, of other T cell subsets which have only recently become known in mammals and for which reagents have only recently become available in the chicken - i.e. Th17, Th9 and Treg. We will investigate these arms of the adaptive immune response in both the response to primary infection and to vaccination, with the expectation that this will lead to novel tools to defining disease biomarkers and phenotypes.

Our overarching hypothesis is that QTL controlling resistance to Eimeria are segregating in modern commercial chickens, and that some of these will be in common with those controlling differential vaccine responses. Further, we hypothesise that the sequence variations may allow us to explain the molecular basis of differential resistance and differential vaccine responsiveness. Finally, we hypothesise that innate immune responses and early adaptive immune responses, such as Th17 responses, play a role in driving differential resistance.

We will also perform whole genome association/genomic selection (WGA/GS) for Eimeria resistance in commercial lines and then independently validate the WGA/GS results. We will also determine Eimeria resistance QTL in inbred lines, identify candidate genes and nucleotide changes and analyse the functional consequences of this sequence variation. This dual approach is required as we cannot apply a simple candidate SNP polymorphism approach in the commercial lines, based on the inbred line data, as we have no evidence that there is the same underlying linkage disequilibrium (LD) between the causal mutation and the SNP in the commercial lines. We will then determine QTLs for differential vaccine responsiveness in the inbred lines and determine if these are in common with the QTLs identified for resistance.

The overall aim is to identify markers (SNPs), candidate genes and ultimately causative mutations for resistance/susceptibility to infection with Eimeria in chickens. The resistance-associated genotypes will inform commercial breeding programmes to reduce the incidence of Eimeria infection of poultry but also improve vaccine responses in the same birds. Greater understaning of immune responses to Eimeria infection has the potential to identify novel biomarkers associated with resistance.

The work proposed has direct relevance to the three ARC aims and three of the four prioritised themes within the overarching objective of 'improving farmed animal resistance to pest and disease organisms'. Outputs will include the identification of regions of the chicken genome associated with resistance/susceptibility to infection by one or more Eimeria species parasites and the ability to raise cross-protective immune responses, supplemented by characterisation of specific immune mechanisms underlying these traits to yield a panel of informative biomarkers. Outcomes will assist in increasing UK competitiveness in the global animal production market, improving animal welfare and helping to guarantee a secure supply of safe, healthy food. The following stakeholders have been identified as beneficiaries of this work:

1. The UK poultry production industry
Eimeria parasites cost the UK poultry industry in excess of £800M per annum including disease-induced losses and the cost of control. Addressing the theme 'Understanding the basis of resistance/resilience to pests and diseases in farmed animal species', identification of genomic regions associated with resistance to eimerian disease will facilitate selection for inherently resistant poultry with no loss of productivity. The structure of the UK poultry industry is such that collaboration with the major breeding companies will provide a cascade of breeding developments, 'ensuring exchange of knowledge between the science base and industry through effective networking'.

2. The UK poultry breeding industry
Mapping quantitative trait loci associated with resistance to Eimeria and immune responses correlated with relevant immuno-competence will provide a panel of genetic and phenotypic biomarkers which may be developed as accurate, affordable tools to estimate disease susceptibility and inform breeding strategy ('Developing novel tools for defining disease biomarkers and phenotypes to inform breeding strategies for subclinical infections and increased disease resistance').

3. The UK animal health industry
The UK currently leads the world in the production of live attenuated anticoccidial vaccines, although a major cost is the requirement for two strains of Eimeria maxima. The identification of genetic markers or genes associated with the ability to generate cross-strain immune responses is likely to support selection of chicken genotypes receptive to streamlined live parasite vaccines and impact on the future development of vectored sub-unit vaccines ('Understanding variation in vaccine responsiveness, immuno-competence at different developmental stages and disease outcomes').

4. Animal welfare
The effective reduction of disease as a result of improved breeding supports the Five Freedoms implicit to animal welfare as set out by the Farm Animal Welfare Council.

5. General public and the environment
Increased efficiency in poultry production will raise poultry product availability at a lower cost for the consumer, contributing to improved food security. Consequences of improved disease resistance include a reduction in the requirement for prophylactic chemotherapy, reducing drug consumption and the risk of contamination to the food chain and the environment.

6. Skills, knowledge and training
The multidisciplinary nature of this project will provide opportunities for broad training to all staff, in addition to other members and students of each host institution ('strengthen the research community in the areas of disease and pest resistance of farmed animals through interdisciplinary research and the provision of training').

7. International development
Eimerian parasites impose serious costs on animal production in developing counties. Translating 'high quality, innovative, strategic research within UK universities and institutes to improve the resistance of farmed animals to pest and disease organisms' can improve economic income and alleviate poverty.