中国荷斯坦牛乳房炎相关基因遗传效应、IL8和CXCR1基因聚合效应及表达规律的研究
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摘要
乳房炎是奶牛最常见和最复杂的疾病之一,给世界奶牛业造成了巨大的经济损失。除改善母牛营养、加强环境卫生管理和改进挤奶技术外,研究者正试图通过遗传育种和兽医生物技术手段来预防和控制乳房炎。
本研究对扬州周边4个牧场奶样中的主要病原菌(大肠杆菌、葡萄球菌和链球菌)进行了分离鉴定；运用荧光定量PCR技术检测了IL8、CXCR1、TLR4、LYZ和LF5个相关基因在主要病原菌攻毒下在奶牛乳腺细胞中的表达规律；同时以来自30个公牛家系的610头中国荷斯坦牛为研究对象,通过PCR-SSCP、PCR-RFLP和测序相结合的方法,对以上5个基因进行了单核苷酸多态性(SNPs)扫描并分析其与泌乳性状和体细胞评分(SCS)的关系；采用Logistic回归模型和多因子降维法(multifactor dimensionality reduction,MDR)法对IL8和CXCR1基因的5个SNPs进行聚合效应分析；最后通过荧光定量PCR技术对奶牛血液中IL8和CXCR1基因mRNA的含量进行了分析。主要研究结果如下：
1.采用BMT法整群抽样检测了4个奶牛场310头无临床症状的荷斯坦奶牛隐性乳房炎,其中有139头患有隐性乳房炎,发生率44.84%,在1163个乳区中,患病乳区有208个,乳区患病率为17.88%。在208份奶样中共分离到葡萄球菌、大肠杆菌、链球菌3种主要病原菌322株,其中葡萄球菌71株,占分离菌株的22.05%：大肠杆菌95株,占分离株数的29.50%；链球菌156株,占分离菌株的48.45%；分析感染情况表明,病原菌单独感染率为43.16%,其中链球菌的单独感染率达31.65%；病原菌混合感染率占54.68%,主要为大肠杆菌和链球菌混合感染。
2.对奶牛的乳腺细胞进行培养,采用大肠杆菌、葡萄球菌和链球菌对乳腺细胞进行了攻毒实验,检测奶牛乳房炎抗性候选基因(IL8、CXCR1、TLR4、LYZ和LF基因)在不同菌种刺激下在乳腺细胞的表达规律。结果发现在刺激后的不同阶段均检测到5个基因的mRNA,在不同的阶段表达量不同,说明在主要病原菌刺激下,5个基因的均显示出抗菌反应。IL8基因和CXCR1基因在葡萄球菌的刺激下随着时间的延长表达量逐渐提高；TLR4基因在大肠杆菌的刺激下表达量显著高于其它两种菌种；LF基因在三种菌种刺激下,随着时间的延长,表达量逐渐提高；LYZ基因在三种菌的刺激下,表达量较低,但随着时间的延长都有提高的趋势。
3.对IL8、CXCR1、TLR4、LYZ和LF基因的多态性检测发现11SNPs位点(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)、LF exonl133(G>C)和LF-3727(C>G)&-3717(A>G)),将突变位点基因型与泌乳性状以及体细胞评分进行了关联分析,主要结果如下：
IL8-233(G>A)位点对体细胞评分达到极显著水平(P<0.01),GG基因型体细胞评分极显著小于GA和AA基因型(P<0.01); IL82789(A>G)&2862(T>C)位点对体细胞评分、测定日产奶量和305d校正产奶量的影响达到极显著水平(P<0.01),结果显示：KK基因型测定日产奶量、305d校正产奶量极显著高于AA和KA基因型型(P<0.01)。KK基因型的体细胞评分(SCS)极显著低于KA、AA基因型(P<0.01)。对于测定的乳蛋白率AA基因型显著低于KA、KK型(P<0.05)：
CXCR1-1830(A>G)位点对体细胞评分、测定日产奶量、测定日乳脂率和305d校正产奶量的影响达到极显著水平(P<0.01),结果显示：在-1830(A>G)位点AA基因型个体的体细胞评分最小二乘均值与AG基因型相比达到极显著水平(P<0.01)；AG基因型的测定日产奶量显著高于AA和GG基因型(P<0.01)；AA基因型的测定日乳脂率显著高于AG基因型(P<0.01)；AG基因型的305天校正产奶量极显著高于AA基因型(P<0.01),显著高于GG基因型(P<0.05)；
CXCR1-1768(T>A)位点对体细胞评分、测定日产奶量和305d校正产奶量的影响达到极显著水平(P<0.01),结果显示：TT基因型个体的体细胞评分最小二乘均值显著小于TA基因型(P<0.01)；TA基因型的测定日产奶量显著高于TT和AA基因型(P<0.01)；TA基因型的305d校正产奶量极显著高于AA和TT基因型(P<0.01); CXCR1-344(T>C)位点对体细胞评分、测定日产奶量和测定日蛋白率的影响达到极显著水(P<0.01),结果显示：TT基因型个体的体细胞评分最小二乘均值显著小于TC和CC基因型(P<0.01)；TT基因型的测定日产奶量极显著高于CC基因型(P<0.01)；显著高于TC基因型(P<0.05)。CC基因型的测定日蛋白率极显著小于TT和TC基因型(P<0.01)；
TLR4-226(G>C)位点对305d校正产奶量影响达到极显著水平(P<0.01),对测定日产奶量到显著水平(P<0.05),结果显示：CC基因型305d校正产奶量显著高于GC和GG基因型(P<0.01),CC基因型的测定日产奶量显著高于GC和GG基因型(P<0.05)；TLR4基因exon31760(C>T)位点对体细胞评分、测定日产奶量和测定乳蛋白率影响达到极显著水平(P<0.01),测定乳脂率达到显著水平(P<0.05),结果显示：CC基因型的体细胞评分显著低于CT和TT基因型；CT基因型的测定日产奶量极显著高于TT,显著高于CC; TT基因型测定日乳蛋白率极显著高于TC, CC显著高于TC；TT基因型的测定日乳脂率极显著高于TC和CC基因型,CC基因型显著高于TC基因型；
LYZ115(T>G)位点对SCS、乳脂率和305d产奶量有极显著影响(P<0.01),对乳蛋白率有显著影响(P<0.05),GG基因型个体的SCS极显著低于TT基因型(P<0.01),显著低于TG基因型(P<0.05)；GG基因型的305d产奶量极显著高于TT基因(P<0.01),显著高于TG基因型,乳脂率和乳蛋白率基因型之间差异不显著；
LF-3727(C>G)&-3717(A>G)位点对体细胞评分影响达到极显著水平(P<0.01),对测定日产奶量的影响达到显著水平(P<0.05),结果显示：TT基因型体细胞评分极显著低于AA和TA基因型(P<0.01)。TT和TA基因型的测定日产奶量显著高于AA基因型(P<0.01)。LF基因exon1133(G>C)突变位点对测定日产奶量、测定日乳脂率、测定日乳蛋白率和305d校正产奶量影响达到极显著水平(P<0.01),对体细胞评分影响显著(P<0.05)。结果显示：GG基因型的测定日产奶量极显著高于CC(P<0.01),GC基因型的体细胞评分显著高于GG和CC基因型(P<0.05)。
4.MDR法、MPVA法和Logistic回归分析CXCR1和IL8基因各位点突变及其基因型组合对奶牛乳房炎易感性的影响,单因素Logistic回归分析结果表明：CXCR1基因和IL8基因五个SNP位点变异对该牛场奶牛乳房炎易感性均没有达到显著水平,但MDR分析发现CXCR1(-1768)-IL8两位点、CXCR1(-1768)-(783)-IL8三位点及CXCR1(-1768)-(-344)-(783)-IL8四位点交互作用均达到显著水平,且交叉验证一致性均为最大(10/10)。CXCR1(-1768)-(783)-IL8三位点互作模型和CXCR1(-1768)-IL8二位点互作模型检验样本准确度较高(分别为0.5457和0.5485),但四个位点联合效应分级图表明CXCR1(-1768)-IL8两易感基因型为强正交互作用,同时将CXCR1(-1768)-(783)-IL8三位点互作效应和CXCR1(-1768)-IL8二位点互作效应；MPVA法得出CXCR1(-1830)-(-1768)-IL8为最佳组合。综合分析得出：CXCR1(-1768)-IL8二位点互作效应对该牛场奶牛乳房炎易感性达到显著水平。
5.通过荧光定量PCR技术检测CXCR1-1768(T>A)、IL8-233(G>A)和IL82789(A>G)&2862(T>C)的突变位点不同基因型在奶牛血液中的表达变化。结果表明,对于CXCR1-1768(T>A)位点产生的三种基因型,TT和TA基因型的表达量显著高于AA基因型,TT的表达量最高；对于IL8-233(G>A)位点产生的基因型,GG基因型的表达量极显著高于AA和AG两种基因型(P<0.01)；对于IL82789(A>G)&2862(T>C)位点,KK基因型的相对表达量极显著高于KA基因型(P<0.01),显著高于AA基因型(P<0.05)。
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).
本研究对扬州周边4个牧场奶样中的主要病原菌(大肠杆菌、葡萄球菌和链球菌)进行了分离鉴定；运用荧光定量PCR技术检测了IL8、CXCR1、TLR4、LYZ和LF5个相关基因在主要病原菌攻毒下在奶牛乳腺细胞中的表达规律；同时以来自30个公牛家系的610头中国荷斯坦牛为研究对象,通过PCR-SSCP、PCR-RFLP和测序相结合的方法,对以上5个基因进行了单核苷酸多态性(SNPs)扫描并分析其与泌乳性状和体细胞评分(SCS)的关系；采用Logistic回归模型和多因子降维法(multifactor dimensionality reduction,MDR)法对IL8和CXCR1基因的5个SNPs进行聚合效应分析；最后通过荧光定量PCR技术对奶牛血液中IL8和CXCR1基因mRNA的含量进行了分析。主要研究结果如下：
1.采用BMT法整群抽样检测了4个奶牛场310头无临床症状的荷斯坦奶牛隐性乳房炎,其中有139头患有隐性乳房炎,发生率44.84%,在1163个乳区中,患病乳区有208个,乳区患病率为17.88%。在208份奶样中共分离到葡萄球菌、大肠杆菌、链球菌3种主要病原菌322株,其中葡萄球菌71株,占分离菌株的22.05%：大肠杆菌95株,占分离株数的29.50%；链球菌156株,占分离菌株的48.45%；分析感染情况表明,病原菌单独感染率为43.16%,其中链球菌的单独感染率达31.65%；病原菌混合感染率占54.68%,主要为大肠杆菌和链球菌混合感染。
2.对奶牛的乳腺细胞进行培养,采用大肠杆菌、葡萄球菌和链球菌对乳腺细胞进行了攻毒实验,检测奶牛乳房炎抗性候选基因(IL8、CXCR1、TLR4、LYZ和LF基因)在不同菌种刺激下在乳腺细胞的表达规律。结果发现在刺激后的不同阶段均检测到5个基因的mRNA,在不同的阶段表达量不同,说明在主要病原菌刺激下,5个基因的均显示出抗菌反应。IL8基因和CXCR1基因在葡萄球菌的刺激下随着时间的延长表达量逐渐提高；TLR4基因在大肠杆菌的刺激下表达量显著高于其它两种菌种；LF基因在三种菌种刺激下,随着时间的延长,表达量逐渐提高；LYZ基因在三种菌的刺激下,表达量较低,但随着时间的延长都有提高的趋势。
3.对IL8、CXCR1、TLR4、LYZ和LF基因的多态性检测发现11SNPs位点(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)、LF exonl133(G>C)和LF-3727(C>G)&-3717(A>G)),将突变位点基因型与泌乳性状以及体细胞评分进行了关联分析,主要结果如下：
IL8-233(G>A)位点对体细胞评分达到极显著水平(P<0.01),GG基因型体细胞评分极显著小于GA和AA基因型(P<0.01); IL82789(A>G)&2862(T>C)位点对体细胞评分、测定日产奶量和305d校正产奶量的影响达到极显著水平(P<0.01),结果显示：KK基因型测定日产奶量、305d校正产奶量极显著高于AA和KA基因型型(P<0.01)。KK基因型的体细胞评分(SCS)极显著低于KA、AA基因型(P<0.01)。对于测定的乳蛋白率AA基因型显著低于KA、KK型(P<0.05)：
CXCR1-1830(A>G)位点对体细胞评分、测定日产奶量、测定日乳脂率和305d校正产奶量的影响达到极显著水平(P<0.01),结果显示：在-1830(A>G)位点AA基因型个体的体细胞评分最小二乘均值与AG基因型相比达到极显著水平(P<0.01)；AG基因型的测定日产奶量显著高于AA和GG基因型(P<0.01)；AA基因型的测定日乳脂率显著高于AG基因型(P<0.01)；AG基因型的305天校正产奶量极显著高于AA基因型(P<0.01),显著高于GG基因型(P<0.05)；
CXCR1-1768(T>A)位点对体细胞评分、测定日产奶量和305d校正产奶量的影响达到极显著水平(P<0.01),结果显示：TT基因型个体的体细胞评分最小二乘均值显著小于TA基因型(P<0.01)；TA基因型的测定日产奶量显著高于TT和AA基因型(P<0.01)；TA基因型的305d校正产奶量极显著高于AA和TT基因型(P<0.01); CXCR1-344(T>C)位点对体细胞评分、测定日产奶量和测定日蛋白率的影响达到极显著水(P<0.01),结果显示：TT基因型个体的体细胞评分最小二乘均值显著小于TC和CC基因型(P<0.01)；TT基因型的测定日产奶量极显著高于CC基因型(P<0.01)；显著高于TC基因型(P<0.05)。CC基因型的测定日蛋白率极显著小于TT和TC基因型(P<0.01)；
TLR4-226(G>C)位点对305d校正产奶量影响达到极显著水平(P<0.01),对测定日产奶量到显著水平(P<0.05),结果显示：CC基因型305d校正产奶量显著高于GC和GG基因型(P<0.01),CC基因型的测定日产奶量显著高于GC和GG基因型(P<0.05)；TLR4基因exon31760(C>T)位点对体细胞评分、测定日产奶量和测定乳蛋白率影响达到极显著水平(P<0.01),测定乳脂率达到显著水平(P<0.05),结果显示：CC基因型的体细胞评分显著低于CT和TT基因型；CT基因型的测定日产奶量极显著高于TT,显著高于CC; TT基因型测定日乳蛋白率极显著高于TC, CC显著高于TC；TT基因型的测定日乳脂率极显著高于TC和CC基因型,CC基因型显著高于TC基因型；
LYZ115(T>G)位点对SCS、乳脂率和305d产奶量有极显著影响(P<0.01),对乳蛋白率有显著影响(P<0.05),GG基因型个体的SCS极显著低于TT基因型(P<0.01),显著低于TG基因型(P<0.05)；GG基因型的305d产奶量极显著高于TT基因(P<0.01),显著高于TG基因型,乳脂率和乳蛋白率基因型之间差异不显著；
LF-3727(C>G)&-3717(A>G)位点对体细胞评分影响达到极显著水平(P<0.01),对测定日产奶量的影响达到显著水平(P<0.05),结果显示：TT基因型体细胞评分极显著低于AA和TA基因型(P<0.01)。TT和TA基因型的测定日产奶量显著高于AA基因型(P<0.01)。LF基因exon1133(G>C)突变位点对测定日产奶量、测定日乳脂率、测定日乳蛋白率和305d校正产奶量影响达到极显著水平(P<0.01),对体细胞评分影响显著(P<0.05)。结果显示：GG基因型的测定日产奶量极显著高于CC(P<0.01),GC基因型的体细胞评分显著高于GG和CC基因型(P<0.05)。
4.MDR法、MPVA法和Logistic回归分析CXCR1和IL8基因各位点突变及其基因型组合对奶牛乳房炎易感性的影响,单因素Logistic回归分析结果表明：CXCR1基因和IL8基因五个SNP位点变异对该牛场奶牛乳房炎易感性均没有达到显著水平,但MDR分析发现CXCR1(-1768)-IL8两位点、CXCR1(-1768)-(783)-IL8三位点及CXCR1(-1768)-(-344)-(783)-IL8四位点交互作用均达到显著水平,且交叉验证一致性均为最大(10/10)。CXCR1(-1768)-(783)-IL8三位点互作模型和CXCR1(-1768)-IL8二位点互作模型检验样本准确度较高(分别为0.5457和0.5485),但四个位点联合效应分级图表明CXCR1(-1768)-IL8两易感基因型为强正交互作用,同时将CXCR1(-1768)-(783)-IL8三位点互作效应和CXCR1(-1768)-IL8二位点互作效应；MPVA法得出CXCR1(-1830)-(-1768)-IL8为最佳组合。综合分析得出：CXCR1(-1768)-IL8二位点互作效应对该牛场奶牛乳房炎易感性达到显著水平。
5.通过荧光定量PCR技术检测CXCR1-1768(T>A)、IL8-233(G>A)和IL82789(A>G)&2862(T>C)的突变位点不同基因型在奶牛血液中的表达变化。结果表明,对于CXCR1-1768(T>A)位点产生的三种基因型,TT和TA基因型的表达量显著高于AA基因型,TT的表达量最高；对于IL8-233(G>A)位点产生的基因型,GG基因型的表达量极显著高于AA和AG两种基因型(P<0.01)；对于IL82789(A>G)&2862(T>C)位点,KK基因型的相对表达量极显著高于KA基因型(P<0.01),显著高于AA基因型(P<0.05)。
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).
引文
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