【作者】 李留安；

【导师】 王艳玲； 李宏基；

【作者基本信息】 河南农业大学 ， 基础兽医学， 2004， 硕士

【摘要】 本试验以能引起肠道疾病的大肠杆菌﹑痢疾志贺氏菌和伤寒沙门氏菌为免疫原，对距预产期60天的奶牛进行高度免疫，定期采集血样和乳样，用微量凝集反应检测其抗体效价，摸索其消长规律，为抗肠道杆菌免疫乳及其它功能免疫乳的研究开发提供理论依据。将购自中国医学细菌保藏管理中心的标准菌株大肠杆菌44210﹑痢疾志贺氏菌51105﹑伤寒沙门氏菌50089经过活化﹑传代﹑增菌培养﹑灭活﹑浓缩﹑加入佐剂等一系列操作后分别制备铝胶苗和油乳剂苗，并进行菌苗的安全性检验。同时将活化的菌液用棉花棒涂抹于营养琼脂平板上，经恒温培养﹑刮下菌苔﹑高压灭活﹑离心洗涤﹑分光光度法统一浓度等一系列操作后制备诊断抗原，4℃保存备用。将各抗原和阳性血清分别按一定比例稀释作微量凝集方阵实验确定各抗原的最适工作浓度，各抗原最适工作浓度结果如下：大肠杆菌为4×109cfu/ml，痢疾志贺氏菌为3×109cfu /ml，伤寒沙门氏菌4×109cfu /ml。在河南农业大学奶牛场选择年龄﹑体重﹑胎次等相近的距预产期60天的健康妊娠奶牛25头，随机分为5组，每组5头，其中A﹑B﹑C﹑D为4个试验组，E组为对照组，A组以大肠杆菌为免疫原，B组以痢疾志贺氏菌为免疫原，C组以伤寒沙门氏菌为免疫原，D组以三种细菌混合制备的三联苗为免疫原，试验组共进行3次免疫：在分娩前60天左右进行第1次免疫，分别肌肉注射灭活水剂苗；10天后第2次免疫，分别肌肉注射灭活水剂苗；分娩前35天左右进行第3次免疫，分别肌肉注射灭活油乳剂苗。采血时间定为每次免疫之前及产后0﹑3﹑7﹑14﹑21﹑28﹑35﹑42﹑49﹑56﹑63﹑70﹑77﹑84天等，分离血清，56℃30分钟灭能，4<WP=8>℃保存备用。采乳时间于分娩后与采血时间同步进行，分离乳清，4℃保存备用。用微量凝集反应检测各特异性抗体效价结果如下：1） 以大肠杆菌为免疫原给妊娠后期乳牛进行免疫，在分娩当天，试验组和对照组血和乳中抗体效价均达到最高峰，试验血为211.6，试验乳为29.8，对照血为28.4，对照乳为27，试验组可以得到持续近80天的含大肠杆菌高免抗体的免疫乳，与对照组差异极显著（P＜0.01），同时在整个试验期间试验血比试验乳高2个滴度左右, 这与国内学者王春风等报道的基本一致。2） 以痢疾志贺氏菌为免疫原给妊娠后期乳牛进行免疫，在分娩当天，试验组和对照组血和乳中抗体效价均达到最高峰，试验血为212.2，试验乳为210.4，对照血为26.4，对照乳为24.6，试验组可以得到持续80多天的含痢疾志贺氏菌高免抗体的免疫乳，且与对照组差异极显著（P＜0.01），同时发现在整个试验期间试验血比试验乳高2个滴度左右。3） 以伤寒沙门氏菌为免疫原给妊娠后期乳牛进行免疫，在分娩当天，试验组和对照组血和乳中抗体效价均达到最高峰，试验血为210，试验乳为28.2，对照血为27.2，对照乳为25.2，试验组可以得到持续50天的含伤寒沙门氏菌高免抗体的免疫乳，且与对照组差异极显著（P＜0.01），同时发现在整个试验期间试验乳比试验血低将近2个滴度。4） 以三联苗为免疫原给妊娠后期乳牛进行免疫，在分娩当天，试验组和对照组血和乳中大肠杆菌抗体效价均达到最高峰，试验血为210.4，试验乳为28.2，对照血为28。4，对照乳为27，试验组可以得到持续56天的含大肠杆菌高免抗体的免疫乳，且与对照组差异极显著（P<WP=9>＜0.01），在整个试验期间试验血比试验乳高2个滴度左右, 这与国内学者王春风等报道的基本一致；在分娩当天，试验组和对照组血和乳中痢疾志贺氏菌抗体效价均达到最高峰，试验血为211.4，试验乳为29.6，对照血为26.4，对照乳为24.6，试验组可以得到持续70天的含痢疾志贺氏菌高免抗体的免疫乳，且与对照组差异极显著（P＜0.01），同时发现在整个试验期间试验血比试验乳高近2个滴度；在分娩当天，试验组和对照组血和乳中伤寒沙门氏菌抗体效价均达到最高峰，试验血为29.6，试验乳为27.8，对照血为27.2，对照乳为25.2，试验组可以得到持续35天的含伤寒沙门氏菌高免抗体的免疫乳，且与对照组差异极显著（P＜0.01），同时发现在整个试验期间试验血比试验乳高近2个滴度。更多还原

【Abstract】 25 pregnant cows were highly immunized with the germs of E.coli﹑S.dysenteriae﹑S.typhi and triple vaccines . serum and milk were collected at different experimental time, the antibody levels of antigens in serum and milk were detected by micro agglutination , the dynamical changes were described in order to supply the theoretical basis for the further research and development of immune milk. Standard bacteria E.coli44210﹑S.dysenteriae51105﹑S.typhi50089 from China center of medical bacteria collection were dealed with activation ﹑cultivation ﹑inactivation﹑concentration and supplement etc. At last obtained alumina gel vaccine and oil emulsion vaccine. Activated bacterial liquid was scribbled to the cultural utensil with nutrient agar, after some operations, got antigens stored at 4℃ and prepared for diagnosis.Every antigen and positive serum were diluted by certain proportion and through micro agglutination in order to ascertain the suitable concentration. The results were showed as follows: 40 hundred million bacteria every millimetre for E.coli﹑30 hundred million bacteria every millimetre for S.dysenteriae and 40 hundred million bacteria every millimetre for S.typhi.In the experiment, 25 pregnant cows were divided into 5 groups randomly, 5 cows every groups and the experimental groups of A﹑B﹑C﹑D were respectively immunized with E.coli﹑S.dysenteriae﹑S.typhi and triple vaccines, the group of E as the control. All the experimental groups were immunized at different times : the first immunization was disposed with inactivated alumina gel vaccine via intramuscular injection at the day 60 before parturition, after 10 days, the second immunization was disposed as the first ways, the third immunization was disposed with inactivated oil emulsion vaccine via intramuscular injection at the day 35 before parturition.The time of collect samples of blood was fixed at the juncture before every immunization and in the 0d﹑3d﹑7d﹑14d﹑21d﹑28d﹑35d﹑42d﹑49d﹑56d﹑63d﹑70d﹑77d﹑84d after parturition, serum was obtained by the centrifugation and stored at 4℃ after making its energy deprived in 56℃ for 30 minutes. The time of collect samples of milk was the same as samples of blood after parturition, whey was obtained by the centrifugation and stored at 4℃ for the further observation. the specific antibody levels were detected by micro agglutination . <WP=55>For cows immunized with E.coli ，the results were showed as follows: on the parturition, antibody levels of serum and whey were simultaneously attained to the climax in the experimental group and control group, the experimental serum and whey were 211.6 and 29.8 respectively, the control serum and whey were 28.4 and 27 respectively. hyper antibody levels of whey in the experimental group can be detected for about 80 days significantly（P＜0.01）, at the same time, the antibody in whey was less 2 titers than in serum during the whole experimental time, this is consisted with the report from Wang chun-feng. For cows immunized with S.dysenteriae，the results were showed as follows: on the parturition, antibody levels of serum and whey were simultaneously attained to the climax in the experimental group and control group, the experimental serum and whey were 212.2 and 210.4 respectively, the control serum and whey were 26.4 and 24.6 respectively, hyper antibody levels of whey in the experimental group can be detected more than 80 days significantly（P＜0.01）, at the same time, the antibody in whey was less 2 titers than in serum during the whole experimental time.For cows immunized with S.typhi ，the results were showed as follows: on the parturition, antibody levels of serum and whey were simultaneously attained to the climax in the experimental group and control group, the experimental serum and whey were 210 and 28.2 respectively, the control serum and whey were 27.2 and 25.2 respectively, hyper antibody levels of whey in the experimental group can be detected more than 50 days significantly（P＜0.01）, at the same time, the antibody in whey was less a更多还原