犊牛大肠杆菌K99、F41二价灭活油剂菌苗的研制及免疫效果测定

发布时间：2018-06-17 09:55

  本文选题：犊牛腹泻 + 大肠杆菌　； 参考：《石河子大学》2015年硕士论文

【摘要】：犊牛腹泻是造成规模化奶牛场内犊牛死亡的主要疾病之一,肠致病性大肠杆菌(EPEC)及产肠毒素大肠杆菌(ETEC)是引起初生犊牛腹泻和败血症的主要病原,其中以K99、F41菌毛致病菌株的致病几率最高。免疫预防是控制该病最有效的方法,但目前国内外尚无牛用的商品化疫苗。为此本研究采用自本地区致病犊牛腹泻病料分离并鉴定的O8(K99)、O78(F41)菌株,采用菌毛抗原和全菌体抗原分别制成油佐剂灭活苗,通过免疫小鼠攻毒保护试验,间接ELISA方法测定免疫母牛初乳及所产犊牛的血清抗体,观察记录犊牛腹泻自然发病率的情况确定其免疫效果,结果如下:1.将O8、O78菌株分别接种LB肉汤及改良Minca肉汤,经扩大培养后灭活,离心菌体在60℃水浴振荡下提取菌毛,饱和硫酸铵盐析法提取菌毛蛋白,加入ISA206双相油佐剂分别制备了犊牛腹泻大肠杆菌K99-F41菌毛油佐剂苗和全菌体疫苗。2.疫苗检验:对两种菌苗的抗原含量进行测定、物理性状检查,无菌检验,安全检测,免疫小鼠攻毒实验,结果显示,菌毛苗蛋白含量为0.5~1.0 mg/m L,全菌体苗细菌数为40~150亿/m L。免疫小鼠攻毒实验结果中菌毛疫苗和全菌体苗的保护率分别是100%和93.3%,表明两种疫苗达到无菌,安全,性状稳定,免疫原性良好指标。3.利用O8、O78菌株所提取的K99-F41菌毛抗原作为包被抗原,通过棋盘滴定法建立了K99-F41菌毛疫苗免疫抗体的间接ELISA检测方法,并测定阴性血清的OD450值计算出阴性临界值,结果表明,抗原包被稀释倍数为1:80,被检血清及乳清稀释倍数为1:100为最佳条件。4.将两种疫苗分别按不同剂量给产前2-4周的怀孕母牛免疫接种,通过已建立的间接ELISA方法检测免疫组母牛产后3 h、6 h、12 h、24 h、36 h、72 h的初乳及所产犊牛灌服初乳1 d、3 d、5 d、7 d血清中相应大肠杆菌抗体滴度,来比较两种大肠杆菌油佐剂疫苗不同注射剂量对妊娠母牛及所产犊牛免疫抗体滴度的差异。结果表明,全菌体疫苗剂量为5 m L组的母牛初乳中抗体滴度最高;菌毛疫苗5 m L组犊牛血清中的抗体滴度最高;两组疫苗免疫剂量5 m L组的初乳及犊牛血清中相应大肠杆菌抗体滴度均显著高于3 m L组;未免疫母牛初乳及所产犊牛的大肠杆菌抗体显著低于免疫组,初生犊牛腹泻自然发病率明显高于免疫组。
[Abstract]:Calf diarrhea is one of the major causes of calf death in a large scale dairy, and enteropathogenic Escherichia coli EPECand enterotoxigenic Escherichia coli ETEC) are the main pathogens causing diarrhea and septicemia in newborn calves. Among them, the pathogenic strain K 99 F 41 had the highest pathogenicity. Immunization prevention is the most effective method to control the disease, but there is no commercial vaccine for cattle at home and abroad. In this study, oil adjuvant inactivated vaccine (O8K99O78F41) isolated and identified from disease-causing calves in this area was made into oil adjuvant inactivated vaccine using fimbriae antigen and whole cell antigen respectively. Indirect Elisa method was used to detect the serum antibody of immune mother bovine colostrum and calves, and to observe and record the natural incidence of diarrhea in calves to determine the immune effect. The results were as follows: 1. Strain O8 O78 was inoculated with LB broth and modified Minca broth, and then inactivated after expanded culture. The fimbriae were extracted by centrifugal bacteria in water bath at 60 鈩，

本文编号：2030625