| Mastitis is the most frequent and important disease in the dairy industry worldwide with great economic losses to the dairy industry. Most researchers have managed to prevent and control mastitis in cow through genetic and breeding technology, and veterinary technology besides the improvement of milking technology and management in nutrition and sanitation.In the study, mainly causing pathogens subclinical mastitis, including Staph ylococcus, Escherichia coli and Streptococcus, were isolated and identified among4dairy cattle herds in Yangzhou, Jiangsu province. The mRNA levels of five genes, IL8, CXCR1, TLR4, LYZ and LF, were detected by real-time PCR technique in the mammary cell lines of dairy cow, after infected by main pathogenic bacteria. To explore the associations between these five genes and milking traits and somatic cell score (SCS), polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing were performed to screen single nucleotide polymorphisms (SNPs) in610Chinese Holstein cows from30bull families. MDR and Logistic regression methods were used to identify the synergic interaction of five SNP loci of CXCR1and IL8and their impacts on the mastitis resistance. The mRNA levels of CXCR1and IL8gene were detected by real-time PCR technique. The main results were as follows:1. In order to investigate the incidence of mastitis in Yangzhou area, the total310 Holstein cows were sampled from four dairy cattle farms for subclinical mastitis test with the method of BMT (Beijing Mastitis Test). The results showed that139dairy cows with208mammary areas among totally1163mammary areas were suffering with subclinical mastitis, the incidence of subclinical mastitis for cow numbers and mammary zones were44.84%and17.88%, respectively.322strains of bacteria were isolated from the above208mastitis positive milk samples, and identified as mainly Staphylococcus, Escherichia coli and Streptococcus.71strains among them were Staphylococcus (22.05%),95strains were Escherichia coli (29.50%),156strains were Streptococcus (48.45%). Among the139mastitis positive cows, the infection rate of single pathogen was43.16%, within which the infection rate of Streptococcus was31.65%. The infection rate of dual and more pathogens was54.68%, including mainly Escherichia coli and Streptococcus.2. Mammary epithelial cell line was cultured, and challenged by Staphylococcus, Escherichia coli and Streptococcus. The five genes, IL8, CXCR1, TLR4, LYZ and LF, were detected by real-time PCR to explore their reaction against these pathogenic bacteria. The results showed that the expressions of the five genes exhibited with different mRNA values in different phases, and the five genes reacted to all three pathogens. The expression values of IL8and CXCR1genes increased gradually in the Staphylococcus challenge, while the expression values of TLR4gene under E.coli challenge were significantly higher than any other pathogen. The expression values of LF gene increased gradually, while LYZ gene showed a very low expression level under these three pathogens, however, with a tendency of rise.3. In the polymorphic research,11SNPs were detected in IL8, CXCR1, TLR4, LYZ and LF genes:IL8-233(G>A), IL82789(A>G)&2862(T>C), CXCR1-1830(A>G), CXCR1-1768(T>A), CXCR1-344(T>C), CXCR1783(C>A), LYZ115(T>G), TLR4-226(G>C), TLR4exon31760(C>T), LFexonl133(G>C) and LF-3727(C>G)&-3717(A>G)), The associations between these11SNPs loci and milking traits and somatic cell score (SCS) were analyzed, the results showed:Significant association of the SNP, IL8-233(G>A), with somatic cell score (SCS)(P<0.01) was indentified, GG genotype had very significantly lower (P<0.01) SCS than GA or AA. The very significant association of the SNPs, IL82789(A>G) and2862(T>C) with somatic cell score (SCS), test day milk yield,305-day corrected milk yield (P<0.01) were indentified. The results showed that KK genotype had higher test day milk yield,305-day corrected milk yield than AA or KA genotype (P<0.01), Least squares mean of SCS of KK was significantly lower than that of AA or KA genotype (P<0.01). AA genotype was significant lower in tested day milk protein percentage than KK or KA genotype (P<0.05).The very significant association of SNP CXCR1-1830(A>G) with somatic cell score (SCS), test day milk yield, test day milk fat percentage,305-day corrected milk yield (P<0.01) were identified. The results showed that the least squares mean of SCS in AA genotype was significantly lower than that in AG (P<0.01), AG was very significantly higher in test day milk yield than AA or AG (P<0.01), AA was significantly higher in test day milk fat percentage than AG (P<0.01), AG was very significantly higher in305-day corrected milk yield than AA (P<0.01), and significantly higher than GG (P<0.05).The very significant association of SNP CXCR1-1830(A>G) with somatic cell score (SCS), test day milk yield,305-day corrected milk yield (P<0.01) were indentified. The results showed the least squares mean of SCS in TT genotype was significantly lower than that in TA (P<0.01), TA was very significantly higher in test day milk yield than AA or TT (P<0.01), TA was very significantly higher in305-day corrected milk yield than AA or TT (P<0.01).The very significant associations of SNP CXCR1-344(T>C) with somatic cell score (SCS), test day milk yield, test day milk protein percentage (P<0.01) were indentified. The results showed that the least squares mean of SCS in TT genotype was significantly lower than that in TC or CC (P<0.01), TT was very significantly higher in test day milk yield than CC (P<0.01), and significantly higher than TC (P<0.05), CC was very significantly lower in test day milk protein percentage than TT or CC (P<0.01).The very significant association of SNP TLR4-226(G>C) with305-day corrected milk yield (P<0.01) and significantly association with test day milk yield (P<0.05) were indentified. The results showed that CC genotype was very significantly higher in305-day corrected milk yield than GC or GG (P<0.01), CC was very significantly higher in test day milk yield than GC or GG (P<0.01).The very significant association of SNP TLR4exon31760(C>T) with somatic cell score (SCS), test day milk protein percentage, test day milk yield (P<0.01) and significant association with test day fat percentage (P<0.05) were indentified. The results showed that the least square mean of SCS in CC genotype was highly significantly lower than that in TC or TT (P<0.01), and CT was very significantly higher in test day milk yield than TT (P<0.01), and significantly higher than CC (P<0.05). TT was very significantly higher in test protein percentage than TC (P<0.01), CC was significantly higher in test day protein percentage than TC (P<0.05), TT was very significantly higher in test day fat percentage than TC or CC (P<0.01), CC was significantly higher in test day fat percentage than TC (P<0.05).The very significant association of SNP LYZ115(T>G) with somatic cell score (SCS), test day milk protein fat percentage,305-day corrected milk yield (P<0.01) were indentified. The results showed that the least square mean of SCS in GG genotype was highly significantly lower than that in TT (P<0.01), and significantly lower than TG (P<0.05). The least square mean of305-day corrected milk yield in GG was greatly significantly higher than that in TT (P<0.01), and significantly higher than in TG (P<0.05).The very significant association of SNP LF-3727(C>G) and-3717(A>G) with somatic cell score (SCS) and significant association with test day milk yield (P<0.05) were indentified. The results showed that the least square mean of SCS in TT was highly significantly lower than that in AA or TA (P<0.01), and TT or TA was very significantly higher in test day milk yield than AA (P<0.01).The very significant association of SNP LF exonl133(G>C) with test day milk protein percentage, test day milk yield, test day milk fat percentage,305-day corrected milk yield (P<0.01) and significant association with somatic cell score (SCS)(P<0.05) were indentified. The results showed that GG was very significantly higher in test day milk yield than CC (P<0.01), and GC was significantly higher in SCS than GG or CC (P<0.05).4. Identify the interaction of CXCR1and IL8and their impacts on the risk of mastitis susceptibility using MDR, MPVA and Logistic regression. The main effects of polymorphism of5SNPs of CXCR1and IL8gene showed no significant impacts on the mastitis susceptibility of cow based on the single factor Logistic regression. The interactions of CXCR1(-1768)-IL8, CXCR1(-1768)-(783)-IL8, and CXCR1(-1768)-(-344)-(783)-IL8showed significant impacts on the mastitis susceptibility of cow using MDR model, and the cross-validation consistency were maximum (10/10). The interactions of CXCR1(-1768)-IL8and CXCR1(-1768)-(783)-IL8showed high testing balance accuracy (0.5457and0.5485respectively). The interaction of CXCR1(-1768)-IL8was high degree synergy for mastitis susceptibility from the interaction dendrogram of multi-genotypes of CXCR1and IL8. CXCR1(-1830)-(-1768)-IL8were the best combination from MPVA method. In the meanwhile, the interactions of CXCR1(-1768)-IL8showed significant impacts on the mastitis susceptibility of cow using Logistic regression model including the main effect of5SNPs and the interaction effect of CXCR1(-1768)-IL8and CXCR1(-1768)-(783)-IL8(P<0.05).5. The variations of mRNA expression of CXCR1and IL8were detected by real-time PCR technique for different genotypes of CXCR1and IL8based on SNPs, CXCR1-1768(T>A), IL8-233(G>A), IL82789(A>G) and IL82862(T>C), in peripheral blood of Holstein. The results showed the expression levels of TT or TA were significantly higher than AA based on SNP CXCR1-1768(T>A) site. The expression level of GG genotype was very significantly higher than that of AA or AG (P<0.01) based on SNP IL8-233(G>A) site. The expression level of KK genotype was highly significantly higher than that of KA (P<0.01), and significantly higher than that of AA (P<0.05). |
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