A preliminary analysis of existing data to provide evidence of a genetic basis for resistance to infection with M.bovis

Porcine Reproductive and Respiratory Syndrome which is caused by a viral pathogen (PRRSv) is an endemic disease with major effects on pig production. We will investigate the host's response to PRRSv infection in cultured alveolar macrophages and whole animals. We will identify genes involved in the host's response to PRRSv infection by expression profiling using microarray technology. We will identify loci determining the host's response to PRRSv through a genome scan exploiting linkage disequilibrium to single nucleotide polymorphisms. This project represents a paradigm for exploiting genomic and post-genomic knowledge and technology to identify the genes involved in the host's response to infectious disease in pigs. The knowledge acquired from this project will underpin improvements in healthier animal production. (Joint with BBSRC grants 104/EGA16309 and 8/EGA16308).

More than 20 years after its emergence, porcine reproductive and respiratory syndrome (PRRS) still has major impacts on pig health and welfare. PRRS results in reproductive failure and respiratory disorders. The pathogen responsible is the PRRS virus which has a tropism for alveolar macrophages (immune cells found in lungs). The virus establishes a persistent infection through suppression of the host immune response and a high mutation rate during viral replication. Vaccination and management strategies have had a limited impact on the spread of the disease. PRRS remains a challenge to sustainable pig production, especially with the emergence of highly pathogenic PRRSV strains. The aim of this project was to look for evidence of genetic variation in the response of pigs to infection with PRRSV.

Understanding the genetic control of host response to infectious disease in domestic animals is of fundamental scientific interest and strategically important. Insights gained will shape future livestock disease control strategies, and genetic markers associated with differences in susceptibility will enable breeders to select animals better able to cope with the disease. Knowledge of the genetic control of host responses to infection may also impact on vaccine strategies as variation in vaccine efficacy may reflect genetic variation in host responses.

Initially, resources were developed to facilitate pig genetics and genomics research. Libraries of genes expressed in a range of tissues both uninfected and infected with PRRSV were constructed. About 15,000 genes from these libraries were partially sequenced and the so-called Expressed Sequence Tags (ESTs) were deposited in public DNA sequence databases. The ESTs were used to design DNA chips for monitoring gene expression. The ends of over 70,000 BAC clones containing large fragments of the pig genome were sequenced. These sequences contributed to the development of a physical map of the pig genome. Twenty-two pigs were scanned for variation in ca. 7,000 of these sequences and over 9,000 single nucleotide polymorphisms (SNPs) were discovered and a DNA chip developed for typing pigs for ca. 6,500 SNPs simultaneously.

Changes in host gene expression in response to PRRSV infection were examined in vitro and in vivo. Alveolar macrophages were recovered from pig from four different genetic lines. The macrophages were infected with PRRSV and changes in gene expression monitored using microarrays (DNA chips). The macrophages from the different lines responded differently to infection. We found evidence that one line appeared to suppress the initial replication of the virus and thus is potentially more resistant.

These differences between lines informed the design of the animal challenge experiments. First growing pigs from two lines (one more and one less susceptible) were infected with PRRSV and samples collected to monitor changes in gene expression. Similarly, pregnant gilts from the more and less susceptible lines were infected with PRRSV and samples collected to monitor changes in gene expression. Outcomes from these animal challenge experiments confirmed the predictions from the in vitro experiments.

Using the DNA SNP chip, the genome was scanned for loci associated with genetic differences in PRRS tolerance. The data comprised 1,545 sows, most of which had reproductive performance data measured both before and during a PRRS outbreak. Six highly significant SNP markers were identified for reproductive failure traits measured during the PRRS outbreak. These significant effects disappeared when PRRS was absent, indicating that they are associated with the pathogenic effects of PRRS.

The results from the genome scan and profiling of changes in gene expression in response to infection in vivo and in vitro confirmed that there is exploitable genetic variation in host responses to PRRSV infection. Further research is required to identify the molecular basis of this genetic variation.

We identified differences in host responses to infection with PRRSV between genetic lines at the level of changes in gene expression, both in vitro (alveolar macrophages) and in vivo. These expression profiling experiments revealed an apparent ability of one genotype to suppress the initial replication of PRRSV. Outcomes from in vivo challenge experiments were consistent with this genetic difference in susceptibility.

We completed the first whole genome scan in pigs using a linkage disequilibrium / whole genome association study approach for a major endemic disease – PRRS. We identified loci with significant effects on PRRS tolerance, as quantified by changes in female reproductive performance during a PRRS outbreak.

The results from the expression profiling and genome scan experiments jointly confirm that there is exploitable genetic variation in host responses to infection with PRRSV.

Important resources for genetics and genomics research in pigs were developed within the project (EST, SNPs and BAC end sequences). These represent valuable contributions to international efforts to develop platforms for expression profiling and SNP genotyping, and to establish the physical map of the pig genome that underpins the current pig genome sequencing project.