Dairy Cow Mastitis Related Innate Immune Genes Screening And Function Study

In recent 20 years,milk production of dairy cow has been increased dramatically through genetic improvement,that render it more susceptible to various pathogens.Dairy industry has suffered immensely by diseases such as mastitis and that of uterine which results in lower reproductive performance and shorter lifespan.Innate immunity is the first defending line to eliminate many kinds of pathogens.The genetic manipulation of immune related genes will be of great importance in providing disease resistance to dairy cow.Genome-wide differential gene expression technologies help us understand the innate immune response mechanism of dairy cow.SNP arrays and genome-wide association study(GWAS)will be useful to identify associated markers and genes for genetic improvements of the innate immune traits of dairy cows.In this study,the differential gene expression file from intra-mammary infection of dairy cow by different pathogens(from GEO database)and GWAS study of dairy cow population from different countries were combined and used to screen innate immune related candidate genes and signaling pathways on mastitis,and then we preliminarily studied the important innate immune candidate genes function and their genetic variation effect at cellular and molecular level.1.Dairy cow mastitis related innate immune genes screeningIngenuity pathway analyse technologe(by IPA software)was used to do combine analysis of the public data to screen innate immune candidate genes which shows promising character are to be used in disease resistance improvement of dairy cow(1)505 common differential expressed genes in a similar direction were appeared when dairy cow suffered intra-mammary infection with either E.coli or S.uberis.Functional cluster,signaling pathway and gene network was analyzed to these 505 genes.It was shown that pathogen infection can improve proliferation and inhibit apoptosis of mammary gland cells.Leukocyte trafficking was also significant activated in the infected tissue.IL-6 play foundational role to amplify inflammatory signal.IL-10 and glucocorticoid receptor signaling pathways play fundamental role to control excessive inflammatory response.Leukocyte was guided to infection site depending on 4 signaling pathways(Granulocyte Adhesion and Diapedesis,Agranulocyte Adhesion and Diapedesis,Leukocyte Extravasation Signaling,fMLP Signaling in Neutrophils).Gene network indicated genes which mediate cell survival and movement are linked.(2)Ninety-three SCS significantly associated SNP markers were obtained from 7 reported GWAS studies on dairy cow and 1635 protein-coding genes were located within 2 Mb of the 93 significant SNPs.Of these 1635 genes,182 genes were significantly related to 32 signaling pathways,most of them are immune and inflammatory pathways.Of the 182 genes,9 genes(XCL1,PPP3 CA,NFATC2,PRKAA1,HSF1,CSTF1,UGT2A3,CYP4F2,MAOA)were located closest to the candidate SNP.These 9 genes take part in 6 signaling pathways which are related to immune response or health in dairy cow.(3)Of the 1635 genes,48 genes expression changed when dairy cow mammary gland suffered E.coli or S.uberis(in the 505 genes).11 of the 48 genes shows significance in 5 signaling pathway including Granulocyte Adhesion and Diapedesi,Agranulocyte Adhesion and Diapedesis,Role of IL-17 A in Psoriasis,Ephrin Receptor Signaling,Stearate Biosynthesis I(Animals).Network showed 16 genes of 48 genes which can be toned for organismal survival and cellular movement and was significantly linked.(4)Combined analysis of functional cluster,signaling pathway and gene network,as well as distance between candidate genes and significant SNPs reported,a final 17 genes(SELP,SELL,CXCL2,CXCL6,CXCL8,CXL1,ACTR3,BCAR1,FABP4,FASN,GNAS,SORBS1,HSF1,PPP3 CA,NFATC2,PRKAA1,CYP4F2)which take part in leukocyte diapedesis,inflammation regulation and mammary gland health were sort out for further study.2.LPS stimulated bovine aortic endothelial cell(BoAEC)and peripheral blood leucocyte(PBL)was used as a model and studied for the role of SELP and SELL play when inflammation happens in dairy cow and also the inflammatory signals mediated leukocyte apoptosis inhibition.(1)Both bovine SELP and SELL play role in the adhesion,LPS stimulated fast adhesion enhancement between Bo AEC and PBL depends on SELP.SELL can play as a receptor for downstream signaling to activate PBL and strengthen PBL adhesion ability.(2)LPS stimulation improved some important adhesion genes(SELP,SELE,ICAM1,VCAM1)expression in BoAEC but inhibit adhesion genes(PSGL-1,CXCR2)expression in PBL while no effect on SELL gene expression.(3)LPS stimulation inhibited spontaneous apoptosis of granulocyte while no effect on agranulocyte.Some genes BCL2,BCL2A1,SGK1 encoding proteins that inhibit apoptosis in PBL was improved by LPS stimuli,which may decrease spontaneous apoptosis of granulocyte inhibition.3.Polymorphisms in coding exons and flanking introns of candidate genes SELP,SELL with another selectin family gene SELE were indentified and associated study was performed with traits of dairy cow including disease,fertility,milk production and longevity.(1)Nine SNPs were identified in exons of SELP,three of them lead missense mutation(amino acid change).Three SNPs were found in gene SELL and all of them are synonymous mutation(no amino acid change).Only one synonymous SNP in SELE was found.(2)Five SNPs(SELPE4,SELPE5,SELPE6,SELPE8,SELPE13)in SELP gene?two SNP(SELLE3,SELLE4)in SELL gene and one in SELE gene(SELEE14)were genotyped.Linkage disequilibrium analysis was done for these 8 SNPs in selectin genes and another SNP rs41579632.3 SNPs SELPE13,SELLE4 and SELEE14 in gene SELP,SELL and SELE respectively represented linkage disequilibrium(LD)(0.31? r2 ?0.80).SLEPE4 and SELPE5 in exons 4 and 5 of SELP gene were in complete LD with each other(r2 = 1).SLEPE4 and SELPE5 were also in LD with SELPE6.No SNP in selection genes was found in LD with rs41579632.(3)Among the nine SNPs genotyped,only one(SELLE3)was found associated with uterine diseases(metritis or endometritis)risk during first 100 days in the second lactation.(4)Association study was undertaken between 9 SNP genotyped and 7 reproductive traits including age at First service?age at first conception,days to first service from calving,days to conception after calving,age at calving,service times needed for conception,calving interval.SNP SELPE6 was significantly associated with many traits including service times needed for first conception,age at first and second calving,calving interval at first lactation,dairy cow with genotype GG performed better.SNP SELPE8 was significantly associated with traits including age at first service,age at first conception,age at first and second calving,dairy cow with genotype GG performed better.Haplotype combination of SELPE13,SELLE4 and SELEE14 was significantly associated with service times needed for second conception.(5)Association study was done between 9 SNP genotyped and 4 milk production traits including days of milking,305 days milk yield,milk fat percentage,milk protein percentage.SELPE6 was significantly associated with days of milking in first lactation and 305 days milk yield in second lactation,cows with AG genotype is better.SELPE13 was significantly associated with milk fat percentage in first lactation,cows with AT genotype is better.SELEE14 was significantly associated with 305 days milk yield in second lactation,cows with CG genotype is better.rs41579632 was significantly associated with milk fat percentage in first lactation,cows with AA and AG genotype performs better.(6)Production longevity of dairy cow with different genotype in each of 9 SNPs was analyzed.SELPE5 was found associated with production longevity,whereas cows with AC and CC genotype were observed to be better.4.Polymorphisms in coding exons and flanking introns of important innate immune genes RelA and TREM1 indetified and association study was done with traits of dairy cow including disease,fertility,milk production and longevity.(1)No SNP was found in exons of RelA and only one got in intron 5(RelA-intron5).2 SNP TREM-SP and TREM-3'UTR which were located at splicing area and 3' untranslated area respectively.(2)RelA-intron5 was significantly associated with many traits,cows with CC genotype is better at production longevity,milk fat and protein percentage in first lactation,and have shorter calving interval but are of older age at first calving.No association was found between TREM-SP and any trait used in this study.TREM-3'UTR was only found associated with longevity.