【作者】 杨丰利；

【导师】 何宝祥；

【作者基本信息】 广西大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 奶牛乳腺炎是导致奶牛业经济损失最严重的疾病之一。本文对南宁市郊奶牛乳腺炎的发病情况、致病菌的流行状况、金黄色葡萄球菌毒性基因的分布、隐性乳腺炎乳汁酶类诊断、治疗乳腺炎中草药制剂组方的确定、乳腺炎疫苗的研制和效果评价以及临床乳腺炎对繁殖性能的影响等方面进行了探索和研究,取得结果如下：(1)2005-2009年对南宁市郊某规模化奶牛场全场奶牛进行乳腺炎调查,平均瞎乳头的奶牛占11.5%,平均瞎乳头率3.7%,临床乳腺炎奶牛平均发病率8.7%,乳区平均发病率3.7%,隐性乳腺炎奶牛平均发病率48.8%,乳区平均发病率19%,此外,临床乳腺炎和隐性乳腺炎的发病率随着胎次的增加而升高。临床乳腺炎和隐性乳腺炎乳汁致病菌检出率分别为96%和34%,临床乳腺炎的主要致病菌是金黄色葡萄球菌(29.5%)、大肠杆菌(25.7%)和新型隐球菌(16.2%),隐性乳腺炎的主要致病菌是金黄色葡萄球菌(32.1%)、凝固酶阴性葡萄球菌(19.7%)和无乳链球菌(17.9%),本实验所用的大多数抗生素和乳炎消对致病性细菌都比较敏感,分离的大肠杆菌和金黄色葡萄球菌的毒性最强,致病率分别为61%和54%。(2)对39株细菌的23S rRNA序列进行检测,全部确定为金黄色葡萄球菌,与之前生化鉴定结果完全一致,PCR结果检出了clfA、tsst-1、nuc、fnb、hla、hlb、cap5、cap8和mecA共九种基因,未检测出sea、fnbB基因,金黄色葡萄球菌携带一种以上毒力基因的现象较为普遍,提示菌株间毒力基因连锁流行的情况较普遍,并且毒性基因的流行病学具有一定的地域性。(3)从奶牛产后两周到产后十周采集的124个乳样,同时进行细菌学检测和上海隐性乳腺炎检测(SMT),均为阳性结果的乳汁确定为隐性乳腺炎(SCM)。结果显示,SCM发病率为26.6%,主要致病菌是金黄色葡萄球菌(47%)和凝固酶阴性葡萄球菌(27%),SCM乳汁中的MDA浓度和乳酸脱氢酶(LDH)、碱性磷酸酶(ALP)的活性显著高于正常乳汁,而SCM乳汁中的谷胱甘肽过氧化物酶(GPx)活性显著低于正常乳汁,SCM乳汁中的超氧化物歧化酶(SOD)和天门冬氨酸氨基转移酶(AST)的活性与正常乳汁没有显著差异。因此,测定乳汁中的MDA浓度和LDH、ALP与GPx的活性可以作为奶牛隐性乳腺炎诊断的一种方法,并且在一定程度上为探明奶牛乳腺炎的发病机理提供依据。(4)金黄色葡萄球菌对连翘和黄连极度敏感,大肠杆菌对乌梅高度敏感。诃子、黄连对金黄色葡萄球菌的抑制作用最强,最低抑菌浓度(MIC)达到8 mg/mL,乌梅和黄连对大肠杆菌的抑制作用最强,MIC达到16mg/mL。通过正交设计,连翘、诃子、乌梅和黄连均能明显提高对金黄色葡萄球菌和大肠杆菌的抑制作用,可以作为奶牛乳腺炎中草药组方的主要成分。该组方对金黄色葡萄球菌和大肠杆菌的MIC分别为8 mg/mL和16mg/mL。(5)所研制的两种金黄色葡萄球菌疫苗对小白鼠的攻毒保护率达到60%以上。根据常规间接ELISA方法,通过反应体系的优化,确定了最佳反应条件,初步建立了间接ELISA检测奶牛乳腺炎金黄色葡萄球菌抗体的方法。同时确定了建立的间接ELISA阴阳临界值(OD=0.28)和建立了标准曲线和回归方程,回归方程计算的预测效价和实际效价基本一致。接种疫苗后7-14天,奶牛血清的特异性抗体效价逐渐升高,疫苗1组奶牛于接种后14天达到最高峰(1：2995),随后逐渐缓慢下降；疫苗2组奶牛于接种后21天达到最高峰(1：2062),随后逐渐缓慢下降。直至实验结束(接种疫苗后第150天),两个实验组奶牛血清的特异性抗体效价仍然高于阴阳临界值。(6)对南宁市郊某规模化奶牛场152头2-4胎次的经产奶牛产后进行临床乳腺炎的发病时间和繁殖指标的调查,包括首次配种天数、配种次数、首次配种怀孕率、空怀期、产后70天内怀孕率、流产率。产后至怀孕之间发生临床乳腺炎奶牛的首次配种天数和空怀期均极显著多于对照组奶牛(P<0.01),配种次数显著多于对照组奶牛(P<0.05)；首次配种怀孕率显著低于对照组奶牛(P<0.05),产后70天内怀孕率低于对照组奶牛(P>0.05),流产率高于对照组奶牛(P>0.05)。产后至首次人工授精之间发生临床乳腺炎奶牛的首次配种天数和空怀期极显著多于对照组奶牛(P<0.01),首次配种怀孕率显著低于对照组奶牛(P<0.05)。首次人工授精至怀孕之间发生临床乳腺炎奶牛的首次配种天数和空怀期极显著多于对照组奶牛(P<0.01),配种次数显著多于对照组奶牛(P<0.05),首次配种怀孕率显著低于对照组奶牛(P<0.05)。结论：奶牛产后至怀孕之间发生临床乳腺炎对繁殖性能具有严重的负面影响。更多还原

【Abstract】 Bovine mastitis is one of the most serious diseases which caused economic losses to the dairy industry. In this study, it was explored that the incidence of dairy cows mastitis, the prevalence of pathogens caused mastitis in Nanning region, and the distribution of S. aureus virulence genes, the diagnosis of subclinical mastitis (SCM) by milk enzymes, the determination of Chinese herbal medicine compound which treatment of mastitis, the development and evaluate the effectiveness of mastitis vaccines, and the influence of clinical mastitis (CM) on reproductive performance of dairy cows. The results are as following:ⅰ) We have investigated the prevalence of cows mastitis in a commercial dairy farm which located in Nanning suburb between 2005 and 2009. The average percentages of blind quarter(s) at cow and quarter level were 11.5% and 3.7%, respectively. The incidence of CM at cow and quarter level were 8.7% and 3.7%, respectively, while that of SCM at cow and quarter level were 48.8% and 19%, respectively. In addition, the incidence of CM and SCM were increased with the parity. The positive rate of pathogens in CM and SCM milk was 96% and 34%, respectively. The most common bacterial isolates from CM cases were S. aureus (29.5%), E. coli (25.7%), and C. neoformans (16.2%), however, in SCM they were S. aureus (32.1%), coagulase-negative staphylococci (CNS) (19.7%), and St. agalactiae (17.9%). The antimicrobial sensitivity test indicated that most of the antimicrobial agents we employed and Ruyangxiao showed lower proportion of resistant isolates of all the isolated bacteria. The most toxic pathogens of mastitis were E. coli and S. aureus, the pathogenic rate were 61% and 54%, respectively.ii) A total of 39 bacterial isolates from bovine mastitis were detected the 23 S rRNA sequence, and all identified as S. aureus, they are consistent with the results of biochemical identification. A total of nine genes were detected in the PCR results, they are elf A, tsst-1, nuc, fnbA, hla, hlb, cap5, cap8 and mecA, but sea and fnbB genes were not detected. It was suggesting that more than one virulence genes were exist in a strain of S. aureus, and that the virulence genes chain between strains are more prevalent, and the epidemiology of virulence genes in S. aureus have a certain regionality. ⅲ) A total of 124 milk samples were collected from lactating glands in the period between the 2nd week after calving and the 1 0th week postpartum. Those which were classified by bacterial culture and the California Mastitis Test (CMT) as positive were deemed to have glands with SCM. The results showed that the periodic incidence rate of SCM was 26.6%. The most common bacterial isolates from SCM cases were S. aureus (47%) and CNS (27%). The mean level of MDA and activities of lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) were significantly higher in SCM milk than in normal milk, while the mean activity of glutathione peroxidase (GPx) was significantly lower in SCM milk than in normal milk. There were no differences in the activities of superoxide dismutase (SOD) and aspartate aminotransferase (AST) between normal milk and SCM milk. Therefore, the measurement of milk MDA level and GPx, LDH and ALP activities appears to be a suitable diagnostic method for identifying SCM in dairy cows. And provide the theoretical basis for the pathogenesis of mastitis to some extent.ⅳ) It was shown that S. aureus is significantly sensitive to the forsythia and coptis, and . coli is highly sensitive to the dark plum. Medicine terminalia and coptis have the strongest inhibition on the S. aureus, and the minimum inhibition concentration (MIC) was all 8 mg/mL to S. aureus. Dark plum and coptis have the strongest inhibition on the E. coli, and the MIC was all 16 mg/mL to E. coli. By the method of orthogonal design, Orsythia, medicine terminalia, dark plum, and coptis have the positive inhibition effect on S. aureus and E. coli, they would be used as the main component of Chinese herbal medicine prescription which treatment dairy cows mastitis. The MIC of the herbal prescription are 8 mg/mL and 16 mg/mL to S. aureus and E. coli, respectively.ⅴ) The protection rate of the two developed S. aureus vaccines were more than 60% in mice. According the method of conventional indirect ELISA, we have been optimization the reaction conditions, and initially established the method of indirect ELISA for detection the antibody of S. aureus mastitis in dairy cows. At the same time, we determined the cutoff value (OD= 0.28) and established the standard curve and regression equation of the indirect ELISA. The result of the actual value was consistent with the prediction value by the regression equation. The serum antibody titers of S. aureus were gradually increased after vaccinated 7 to 14 days in dairy cows. The peak of serum antibody titers was shown up in the 14 days after inoculation (1:2995) of the vaccine 1 group cows, and then gradually slows down. While, the peak of serum antibody titers was shown up in the 21 days after inoculation (1:2062) of the vaccine 2 group cows, and then gradually decreased. Until the end of the experiment (150 days after vaccination), the two groups of cows’serum antibody titers are still higher than the cutoff value. ⅵ) We have investigated the timing of cows clinical mastitis occurrence and reproductive performance on 152 Holstein dairy cows which are multiparous in a commercial dairy farm in Nanning suburb. The reproductive indexes including days in milk at first service (DIMFS), services per conception (S/C), conception rate at first postpartum AI, days not pregnant (DNP), estrus rate within 70 days postpartum and abortion rate. First clinical mastitis occurred between calving and pregnancy diagnosis group cows (MG) had greater (P< 0.01) DIMFS and DNP compared with control group cows (CG). Group MG had greater (P<0.05) S/C than CG. Group MG had lower (P<0.05) conception rate at first postpartum AI compared with CG. Group MG had lower (P> 0.05) estrus rate within 70 days postpartum than CG. And group MG had higher (P> 0.05) abortion rate compared with CG. First clinical mastitis occurred between calving and first postpartum artificial insemination (AI) group cows (MG1) had greater (P<0.01) DIMFS and DNP compared with CG. Group MG1 had lower (P<0.05) conception rate at first postpartum AI compared with CG. First clinical mastitis occurred between first postpartum AI and pregnancy diagnosis group cows (MG2) had greater (P<0.01) DIMFS and DNP compared with CG. Group MG2 had greater (P<0.05) S/C than CG. Group MG had lower (P<0.05) conception rate at first postpartum AI compared with CG. In conclusion, clinical mastitis occurred between claving and pregnancy diagnosis had a serious negative impact on reproductive performance of dairy cows.更多还原