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A genomics approach to increasing disease resistance in dairy cows through improvements in innate immunity

Final Report Summary - INNATELYBETTERCOWS (A genomics approach to increasing disease resistance in dairy cows through improvements in innate immunity)


Background:
The EU dairy industry should produce a safe food supply for consumers in an economically viable fashion without compromising the health and welfare of the cows. Dairy cows are susceptible to pathogens commonly present on farms which can cause infections, particularly involving the mammary gland and uterus. Poor health reduces longevity and encourages a high reliance on antibiotic usage. We proposed that the situation could be improved by identifying key genes in signalling pathways involving innate immune function which are activated by a wide range of pathogens. It would then be possible to increase disease resistance by selectively breeding cows using marker assisted selection to improve their innate immune response. The project built on a previously established collaboration between Huazhong Agricultural University, China (HZAU) and the Royal Veterinary College (RVC, London, UK), giving access to two distinct populations of Holstein dairy cows. The project had six Research Objectives and also offered training and networking opportunities to the researcher, Professor Shujun Zhang, during her 2 year stay in the UK.

Research Objectives:
• Perform genome wide association studies in cows to identify regions associated with health traits.
• Use comparative genomics to identify candidate genes associated with the innate immune response.
• Select one key candidate gene to determine how alterations in its level of expression affected the cellular response to bacterial lipopolysaccharide (LPS).
• Undertake gene sequencing of selected candidate genes in different cattle populations to discover novel single nucleotide polymorphism (SNPs).
• Validate the relevance of these genes in association studies using disease traits in two new populations of UK and Chinese dairy cows.
• Perform a functional validation study on peripheral blood cell responses to selected bacterial pathogens in cows with differing genotypes for innate immune genes.

Work performed and main results to date:
Work on the first four of these objectives is nearly complete whereas that on the last two will continue into Year 3 when Professor Zhang returns to China.
Genome wide association studies were performed using data obtained from 496 UK and Chinese Holstein-Friesian cows previously genotyped using the Illumina 50K SNP chip. Genotypes were analysed with respect to somatic cell count (SCC) as an indicator trait for mastitis in both populations and against general health data (incidence of lameness, uterine disease, mastitic events, displaced abomasa, milk fever and others) available from the UK cows over the first two lactations. These results highlighted regions of association of SCC with SNP in Chromosomes 1, 4 and 21. The region in Chromosome 4 had previously been reported to show an association with SCC and clinical mastitis in a population of Norwegian dairy cows. These regions were not, however the same as those we identified as being important in the response of the innate immune system to mastitis from an extensive review of published literature and available data bases. This approach instead identified a number of promising candidate genes relating to the pathways “Granulocyte adhesion and diapedesis signaling” and “Ephrin receptor signaling”. An important role for lipid signalling, specifically long chain fatty acids (LCFA), was also suggested. These shortlisted genes were located mainly on Chromosomes 14 and 16. A region on Chromosome 6 including the chemokines CXCL2, CXCL6 and CXCL8 which are key molecules to attract immune cells into affected tissues was also highlighted. Treatment of cultured uterine endometrial cells with bacterial LPS showed increased gene expression of a number of vascular adhesion factors and cell-to-cell adhesion molecules which included the selectins SELL and SELP. These genes also have important roles in leucocyte migration during inflammatory processes.
We therefore selected SELP (P-selectin) as our chosen molecule of interest. We investigated the time course of SELP expression in bovine endothelium using qPCR and western blotting. SELP mRNA expression showed a peak at 3 h post LPS stimulation whereas the protein level was elevated from 10 min to 3 h post treatment. The initial, more rapid, increase in protein probably reflects release of pre-formed protein within the cell to the cell membrane. This time course correlated well with results from an adhesion assay measuring attachment of bovine leukocytes to monolayers of bovine endothelial cells. This also showed two peaks of attachment at 10 min and again at 3-4 h after LPS treatment. The adhesion following LPS stimulation was inhibited by pre-treating the endothelial cells with SELP antibody.
Fifteen candidate genes were sequenced confirming the presence of 52 SNP in our populations of British and Chinese Holstein cows. DNA from the population of UK cows was genotyped with respect to 12 of these SNP and the associations between genotype and SCC, health and survival data were analysed. This showed that several of the SNP were indeed associated with both disease and survival time within the herd.
Responses of macrophages derived from blood and milk to in vitro challenges representative of gram negative and gram positive bacteria and fungal infections were compared between two breeds of dairy cow, Brown Swiss and Holstein which were shown to differ in their susceptibility to mastitis. These tests revealed functional breed differences involving several key pathways of the innate immune system which altered the ability of the cells to kill bacteria.
Further DNA samples are currently being obtained from both UK and Chinese Holstein cow populations to perform a validation study to test the relevance of our candidate genes in disease incidence. More in vitro functional validation tests are also planned to compare biological responses of leukocytes and macrophages from cows with differing genotypes or gene expression levels for genes of interest which are important for innate immunity.

Summary and Impact:
The data obtained support the initial hypothesis that differences in genotype for candidate genes with important roles in the innate immune system do indeed influence both the health and survival of Holstein dairy cows through altered susceptibility to a variety of diseases. Key pathways identified involve both the migration of immune cells into affected tissues and their ability to kill bacteria on arrival. Following further validation studies, this information can be used to improve selection of animals for breeding. This will benefit future generations of cows through improved health and at the same time has the potential to reduce antibiotic use in the dairy industry and to minimise the numbers of animals required to maintain milk production, so also reducing the impact of dairy farming on the environment.