Development of a high-throughput platform for evaluation of chicken immune responses

The poultry industry has successfully applied breeding and production programmes to meet growing consumer demands for chicken meat and eggs. Over the last four decades, poultry breeders have selected birds not only for productivity, but also for improved health, welfare, fitness and environmental robustness. Intensive production settings contribute to faster spread of diseases and greater losses in production due to increased morbidity and mortality of the flock. Traditional methods of disease treatment and prevention have played a critical role in control of disease. However, growing resistance of pathogens to therapeutic measures and consumer concerns led to the withdrawal of antibiotics as growth promoting additives in chicken feed. In addition, some vaccines have been overcome by increasing variation and virulence of pathogens and are no longer successful in disease prevention. The emergence of virulent and drug resistant pathogens have emphasised the need to focus on other solutions to disease, particularly natural genetic resistance. Genetic loci or gene expression patterns associated with the differential resistance of lines to specific pathogens have been identified, providing valuable markers for selective breeding. However, to date relatively few of these have been successfully incorporated into commercial lines. An ability to suppress or resist multiple pathogens, by selection for improved innate immune robustness has also been studied but it has not been introduced in commercial production, partly as the phenotype is ill-defined. Previous studies that focused on pro-inflammatory cytokines and their mRNA levels expressed by innate immune effector cells (heterophils and macrophages) identified differences between resistant and susceptible chicken lines, with the former producing stronger responses, supporting efforts to select poultry with an efficient early innate response. Here, small-scale qPCR screening and cellular techniques were evaluated with the conclusion that a more rapid, cheaper and reproducible method needs to be applied. The main objective of this project was therefore to design and validate a diagnostic tool that could be used to phenotype the immune responses of chickens at the level of innate immunity. For this purpose, a panel of 89 genes was selected based on previously published infection studies and on RNA-seq results obtained from stimulation of heterophils, macrophages and dendritic cells with lipopolysaccharide (LPS). Target genes were cloned and sequenced to optimise the design of qPCR reactions and primers. A multiplex qPCR platform, the Fluidigm 96.96 Dynamic Array, was selected as the tool of choice with the capacity to measure transcription of 96 genes of interest in 96 samples simultaneously. The preamplification reaction was optimised and the platform validated using a commercial line of chickens housed in clean or pathogen-challenged environments. Lymphoid tissues, including bursa of Fabricius, spleen, ileum with Peyer’s patches, caecal tonsils, and blood leukocytes were isolated and transcript levels for immune-related genes defined between organs, birds and farms. For qPCR analysis, a panel of reference genes was normalised and TBP, ACTB and GAPDH genes were selected and validated as the most stable. The high-throughput qPCR analysis identified peripheral blood leukocytes as a potentially reliable indicator of immune responses among all the tissues tested with the highest number of genes significantly differentially expressed between birds housed in varying hygienic environments. The research described here could potentially aid the selection of poultry for improved immune robustness. The technical optimisation and validation of a new tool to simultaneously quantify expression of tens of relevant immune-related genes will prime research in many areas of avian biology, especially to define baseline immune gene expression for selection, the basis of differential resistance, and host responses to infection, vaccination or immuno-modulatory substances.