奶牛乳房炎基因工程亚单位疫苗实验免疫研究
摘要
本文从生产实践出发，对金黄色葡萄球菌基因工程亚单位疫苗防治奶牛乳房炎进行了实验免疫研究，探讨了疫苗免疫对奶牛乳房炎发病率及生产性能等方面的影响，建立了检测特异性抗体的ELISA方法，并根据统计学方法建立了血清OD值与效价之间的回归曲线和回归方程，对疫苗免疫后血清中特异性抗体消长规律等方面作了较为详细的研究。
乳房炎是奶牛的一种常见病、多发病，其中又以隐性乳房炎为主。隐性乳房炎由于没有临床明显可见症状，因此一般多应用一些特殊的检查方法进行诊断。奶牛隐性乳房炎的检测方法很多，本试验采用了SMT法、苛性钠法、过氧化氢法及BTB法等四种较为常用的方法，对供试奶牛173头进隐性乳房炎检测。经综合判定，隐性乳房炎阳性牛89头，占51.45％，阳性乳区181个，阳性率26.66％；对181个阳性乳样进行细菌分离鉴定，有156个乳区共分离到细菌225株，细菌检出率为82.54％。分离到的细菌中，以金黄色葡萄球菌、停乳链球菌和无乳链球菌为主，分别占40.44％、19.11％和18.22％，其他还有大肠杆菌、乳房链球菌及表皮葡萄球菌等。根据试验需要，在供试奶牛中，按不同情况挑选60头，作为试验组和对照组。A组为经产隐性乳房炎阳性牛10头，B组为经产健康牛10头，C组为初产健康牛10头，对照组为与各试验组相对应的同期、同舍、同种情况的奶牛各10头。试验组于臀部皮下注射1ml制备的疫苗，连续注射3次，每次间隔15天，并在每次注射疫苗和最后一次注射后1、2、3、4和5个月时分别采血10ml，分离血清，将血清-20℃冷冻保存，以备进行特异性抗体检测。在三免后第四个月时，对各免疫组与其相应的对照组之间的隐性乳房炎发病率、头目均产奶量等进行了统计分析，发现免疫组的隐性乳房炎发病率明显低于对照组，有效率分别为55.6％、66.7％和50.0％，而产奶量明显提高，增加12.84％、10.29％和9.10％，从临床角度证明了疫苗的使用价值。
根据常规的ELISA方法，建立了检测本疫苗的血清特异性抗体的ELISA检测方法，并对条件进行优化，建立检测特异性抗体的ELISA标准程序，应用此ELISA方法检测试验奶牛的特异性抗体的水平，监测免疫后特异性抗体的消长规律，为该疫苗临床应用提供依据。免疫组在二免，血清特异性抗体水平迅速提高，OD值的平均值为1.08，在三免1个月后特异性抗体达到最高值1.80，至三免5个月后，仍然维持较高水平。各免疫组之间特异性抗体效价无差异。为了能更好的用ELISA检测的血清OD值来反应血清特
吉林农业大学硕士学位论文
奶牛乳房炎墓因工程亚单位疫苗实验免疫研究
异性抗体的效价的高低，试验将血清1:1 00倍稀释度下的0D值与对应的血清效价进行
了比较，并作了统计学分析，得出了二者之间存在着一定的线性关系，得出了回归方程
和回归曲线，回归方程为y一1.62卜0.2441092‘(y为血清1:1的稀释时0D值，x为血
清效价的倒数)。并对回归方程的实测值与预测值进行了比较，无显著差异，证明了血
清效价在1:1叨倍稀释时的0D值与抗体效价倒数的以2为底的对数成直线关系。可以
直接应用血清1:100稀释时0D值表示效价，使检测血清效价更简单方便。
通过本试验，建立了检测金黄色葡萄球菌基因工程亚单位疫苗特异性杭体的ELISA
方法，并从临床实践角度出发，验证了该疫苗的安全性、可靠性及实用性，可有效预防
由金黄色葡萄球菌引起的奶牛隐性乳房炎，为该疫苗的推广应用莫定基础。
For the benefit of practical production, we applied the genetic engineering subunit vaccine of Staphylococcus aureus to immune the cow, then observed how it can influence the incidence of mastitis and milk productivity. We have established ELISA method to detect peculiar antibody and established regressive equation and regressive curve base on the serum OD value and serum titers according statistics. We also reasearched the increase and decline regulation of peculiar antibody after the immunity.
Mastitis is an usual and common disease of cow. Subclinical mastitis is the most usual clinical case. Because of it have no obvious clinical symptom, we usually apply some special methods to examine it. There are many methods to examine this disease. We have examined 173 cows through the usual mehtod such as SMT method, NaOH method, H2O2 mehtod and BTB method, through general analysis, there are 89 cows suffered subclinical mastitis, the positive rate is 51.45%. There were 181 positive breast districts and the positive rate was 26.66%. We also isolated 225 strains of bacteria from the positive breast districts, the rate is 82.54%. The bacteria mainly include Staphylococcus aureus, Streptococcus dysgalacitiae and Streptococcus agalactiae, the positive rate was 40.44%, 19.11% and 18.22% respectively. There also were other bacteria such as E.coli, Streptococcus uberis and Staphylococcus epidermidis etc. Base on the need of test, we chose 60 cow from test cow according to different conditions. Then we allocated
them into 2 large groups, namely the test group and control group. A group include cow which suffered subclinical mastitis; B group included 10 healthy 10 post partum; C group included 10 healthy cow that beginning to propagate. The control group included 10 same period, same house and same kind condition cows comparing with test group. We gave the test cow subcutaneous injection with lml vaccine at buttocks. We immunized the cow 3 times and the interval was 15 days. We immediately sampled 10 ml blood after the every injection and 1, 2, 3, 4, 5 months after the final injection. Then we isolated serum from blood and Frozen it at - 20 C for carrying out peculiar antibody detection. At the 4th month after the third immunity, we carried out statistical analysis of the incidence of subclinical mastitis, the daily milk production and so on. The result indicated that the incidence of subclinical mastitis of test group was obviously lower than that of control
group. The effective rate was 55.6%, 66.7% and 50.0% respectively. The milk production was obviously increased. The increasing rate was 12.84%, 10.29% and 9.10% respectively. From the clinical standpoint, we made concluded that the genetic engineering subunit vaccine have practical value.
Base on the conventional ELISA method, we have established ELISA detection method to detect the serum peculiar antibody of vaccine. We also optimized the reaction condition and established standard peculiar ELISA detection program. Then we applied this method to supervise the increasing and declining regulation of peculiar antibody and offered basis theory for clinical application of this vaccine. At the time of second immunity, the average peculiar serum antibody titers increased quickly and the average OD value was 1.08. The peculiar antibody titers reached the highest level and the OD value was 1.80 at 1 month after the third immunity. The peculiar antibody titers still maintained high level at 5 month after the third immunity. There was no discrepancy among the immune groups. For the better of estimating the peculiar antibody titers through the OD value, we diluted serum 1:100 times and compared the OD value with corresponding serum antibody titers, then analyzed the relation by statistics. We found that
the there are certain linear relation between them. Then we worked out the regressive equation and drew out the regressive curve. The regressive equation is y = - 1.621 + 0.244 log2x( y: the OD value of the serum which was diluted times; x: the reciprocal of serum antibody titer
乳房炎是奶牛的一种常见病、多发病，其中又以隐性乳房炎为主。隐性乳房炎由于没有临床明显可见症状，因此一般多应用一些特殊的检查方法进行诊断。奶牛隐性乳房炎的检测方法很多，本试验采用了SMT法、苛性钠法、过氧化氢法及BTB法等四种较为常用的方法，对供试奶牛173头进隐性乳房炎检测。经综合判定，隐性乳房炎阳性牛89头，占51.45％，阳性乳区181个，阳性率26.66％；对181个阳性乳样进行细菌分离鉴定，有156个乳区共分离到细菌225株，细菌检出率为82.54％。分离到的细菌中，以金黄色葡萄球菌、停乳链球菌和无乳链球菌为主，分别占40.44％、19.11％和18.22％，其他还有大肠杆菌、乳房链球菌及表皮葡萄球菌等。根据试验需要，在供试奶牛中，按不同情况挑选60头，作为试验组和对照组。A组为经产隐性乳房炎阳性牛10头，B组为经产健康牛10头，C组为初产健康牛10头，对照组为与各试验组相对应的同期、同舍、同种情况的奶牛各10头。试验组于臀部皮下注射1ml制备的疫苗，连续注射3次，每次间隔15天，并在每次注射疫苗和最后一次注射后1、2、3、4和5个月时分别采血10ml，分离血清，将血清-20℃冷冻保存，以备进行特异性抗体检测。在三免后第四个月时，对各免疫组与其相应的对照组之间的隐性乳房炎发病率、头目均产奶量等进行了统计分析，发现免疫组的隐性乳房炎发病率明显低于对照组，有效率分别为55.6％、66.7％和50.0％，而产奶量明显提高，增加12.84％、10.29％和9.10％，从临床角度证明了疫苗的使用价值。
根据常规的ELISA方法，建立了检测本疫苗的血清特异性抗体的ELISA检测方法，并对条件进行优化，建立检测特异性抗体的ELISA标准程序，应用此ELISA方法检测试验奶牛的特异性抗体的水平，监测免疫后特异性抗体的消长规律，为该疫苗临床应用提供依据。免疫组在二免，血清特异性抗体水平迅速提高，OD值的平均值为1.08，在三免1个月后特异性抗体达到最高值1.80，至三免5个月后，仍然维持较高水平。各免疫组之间特异性抗体效价无差异。为了能更好的用ELISA检测的血清OD值来反应血清特
吉林农业大学硕士学位论文
奶牛乳房炎墓因工程亚单位疫苗实验免疫研究
异性抗体的效价的高低，试验将血清1:1 00倍稀释度下的0D值与对应的血清效价进行
了比较，并作了统计学分析，得出了二者之间存在着一定的线性关系，得出了回归方程
和回归曲线，回归方程为y一1.62卜0.2441092‘(y为血清1:1的稀释时0D值，x为血
清效价的倒数)。并对回归方程的实测值与预测值进行了比较，无显著差异，证明了血
清效价在1:1叨倍稀释时的0D值与抗体效价倒数的以2为底的对数成直线关系。可以
直接应用血清1:100稀释时0D值表示效价，使检测血清效价更简单方便。
通过本试验，建立了检测金黄色葡萄球菌基因工程亚单位疫苗特异性杭体的ELISA
方法，并从临床实践角度出发，验证了该疫苗的安全性、可靠性及实用性，可有效预防
由金黄色葡萄球菌引起的奶牛隐性乳房炎，为该疫苗的推广应用莫定基础。
For the benefit of practical production, we applied the genetic engineering subunit vaccine of Staphylococcus aureus to immune the cow, then observed how it can influence the incidence of mastitis and milk productivity. We have established ELISA method to detect peculiar antibody and established regressive equation and regressive curve base on the serum OD value and serum titers according statistics. We also reasearched the increase and decline regulation of peculiar antibody after the immunity.
Mastitis is an usual and common disease of cow. Subclinical mastitis is the most usual clinical case. Because of it have no obvious clinical symptom, we usually apply some special methods to examine it. There are many methods to examine this disease. We have examined 173 cows through the usual mehtod such as SMT method, NaOH method, H2O2 mehtod and BTB method, through general analysis, there are 89 cows suffered subclinical mastitis, the positive rate is 51.45%. There were 181 positive breast districts and the positive rate was 26.66%. We also isolated 225 strains of bacteria from the positive breast districts, the rate is 82.54%. The bacteria mainly include Staphylococcus aureus, Streptococcus dysgalacitiae and Streptococcus agalactiae, the positive rate was 40.44%, 19.11% and 18.22% respectively. There also were other bacteria such as E.coli, Streptococcus uberis and Staphylococcus epidermidis etc. Base on the need of test, we chose 60 cow from test cow according to different conditions. Then we allocated
them into 2 large groups, namely the test group and control group. A group include cow which suffered subclinical mastitis; B group included 10 healthy 10 post partum; C group included 10 healthy cow that beginning to propagate. The control group included 10 same period, same house and same kind condition cows comparing with test group. We gave the test cow subcutaneous injection with lml vaccine at buttocks. We immunized the cow 3 times and the interval was 15 days. We immediately sampled 10 ml blood after the every injection and 1, 2, 3, 4, 5 months after the final injection. Then we isolated serum from blood and Frozen it at - 20 C for carrying out peculiar antibody detection. At the 4th month after the third immunity, we carried out statistical analysis of the incidence of subclinical mastitis, the daily milk production and so on. The result indicated that the incidence of subclinical mastitis of test group was obviously lower than that of control
group. The effective rate was 55.6%, 66.7% and 50.0% respectively. The milk production was obviously increased. The increasing rate was 12.84%, 10.29% and 9.10% respectively. From the clinical standpoint, we made concluded that the genetic engineering subunit vaccine have practical value.
Base on the conventional ELISA method, we have established ELISA detection method to detect the serum peculiar antibody of vaccine. We also optimized the reaction condition and established standard peculiar ELISA detection program. Then we applied this method to supervise the increasing and declining regulation of peculiar antibody and offered basis theory for clinical application of this vaccine. At the time of second immunity, the average peculiar serum antibody titers increased quickly and the average OD value was 1.08. The peculiar antibody titers reached the highest level and the OD value was 1.80 at 1 month after the third immunity. The peculiar antibody titers still maintained high level at 5 month after the third immunity. There was no discrepancy among the immune groups. For the better of estimating the peculiar antibody titers through the OD value, we diluted serum 1:100 times and compared the OD value with corresponding serum antibody titers, then analyzed the relation by statistics. We found that
the there are certain linear relation between them. Then we worked out the regressive equation and drew out the regressive curve. The regressive equation is y = - 1.621 + 0.244 log2x( y: the OD value of the serum which was diluted times; x: the reciprocal of serum antibody titer
引文
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