牛支原体流行病学调查及灭活疫苗研制

发布时间：2018-05-11 19:10

本文选题：牛支原体 + 分离培养　；参考：《内蒙古农业大学》2017年硕士论文

【摘要】：牛支原体从发现到现在一直没有引起人们足够的重视,也没有列入重大疫病的名单,但是牛支原体在牛养殖业引起的损失一直都非常巨大,许多的报道已经说明牛支原体病呈世界性分布,牛支原体的致病力非常强,主要导致牛乳房炎、肺炎,这也是人们了解和发现牛支原体的主要原因。同时牛支原体在引起病理变化的过程中可以为其它致病菌大开门户。本课题首先对中国大部分地区分离的菌株进行了鉴定和流行病学调查,然后对纯化得到的牛支原体进行后续试验:在此基础上研发了牛支原体灭活疫苗和建立抗体间接ELISA检测方法。本实验室过去8年时间里对国内12个省30个规模化奶牛场进行了流行病学调查。采用牛支原体间接ELISA、直接培养法和特异性PCR检测,进行了牛支原体感染情况调查及分析,结果显示,血清抗体阳性率为52.31%(441/843);病料阳性率为55%(33/60);乳样阳性率为34.22%(373/1090);并且从中我们收集了具有地方代表性的55株牛支原体菌种,进行了 16SrDNA测序比对,比对结果表明在16SrDNA水平上没有发生变异,这个结果为以后牛支原体免疫预防提供了重要依据。从分离到的55株牛支原体中选取5株牛支原体进行培养和特性研究,我们从牛支原体的生化特征、血清生长抑制、营养及代谢、生长和培养特征、药物敏感等实验,并采用PCR、CCU颜色变化单位、CFU菌落计数单位等方法获取实验数据,并选取优势菌株传代培养,经过传代培养,实现对牛支原体的驯化,传代中牛支原体活菌数可达到10111cfu/mL,并且可以维持稳定,到目前为止,牛支原体共在体外传代200代次。以牛支原体全菌体蛋白的超声波裂解物作为包被抗原,建立检测牛支原体抗体的间接ELISA方法。通过正交筛选确定蛋白包被量为4 μg/mL,包被液为批pH9.6的碳酸盐缓冲液,包被最佳温度和时间为4℃ 16 h,封闭液为1%浓度的脱脂乳,最佳封闭时间和温度为37℃2 h,待检血清最佳稀释度为1:100,反应温度和时间37℃ 1 h,二抗稀释度为1:4000,最佳反应温度和时间为37℃1h。通过实验确定阴性血清的临界值为OD450=0.346,该方法与口蹄疫、副结核、布鲁菌病、牛病毒性腹泻、牛传染性鼻气管炎及结核的阳性血清均无交叉反应,具有极强的特异性。将阳性血清倍比稀释,1:512倍稀释时OD450仍大于临界值,证明该方法具有很好的敏感性。组内和组间变异系数均小于5%,证明该方法重复性较好,利用该方法与加拿大Biovet公司生产的牛支原体间接ELISA试剂盒对150份临床样本进行检测,结果显示两者的符合率为95%。以上结果可以说明我们建立的间接ELISA方法可为该病的快速诊断、流行病学调查及免疫牛只的抗体检测提供一种手段。用分离纯化、传代培养的牛支原体在在家兔上进行毒力鉴定,症状与牛感染牛支原体的症状高度相似;通过病理组织切片、间接免疫荧光、特异性PCR、分离培养等方法验证其为牛支原体感染。确定牛支原体在体外连续传代培养过程中毒力明显减弱但仍具有致病性,可作为疫苗备选株。将第160代次的牛支原体灭活后与铝胶盐佐剂1:1混合做成灭活疫苗对家兔进行免疫试验,第28 d时对疫苗组和对照组家兔进行攻毒试验,疫苗组不发病,解剖后肺脏没有病变,而对照组高热,解剖后肺脏有明显的病变。家兔免疫试验证明牛支原体灭活疫苗在家兔体内可产生较高水平的抗体,并可产生确实的保护作用。在家兔免疫试验的基础上我们制定了牛支原体灭活疫苗免疫程序,并在内蒙古某牧场进行了临床实验,选取了 210头60日龄以内的小公牛进行牛支原体灭活疫苗本动物免疫试验,疫苗中活菌含量为1011cfu/mL,免疫计量为1mL/头。通过牛支原体灭活疫苗免疫后,到第60 d免疫组发病率下降20.7%,分菌率下降35%,PCR阳性率下降50%,对照组发病率上升17%,分菌率上升8.3%,PCR阳性率下降25%。在两次免疫后疫苗所产生的抗体滴度最高可达1:256,80%的小牛抗体滴度达到1:128及以上,到第60 d时仍可检测到抗体,有67%的小牛抗体滴度达到1:64及以上,此次临床实验前后经历一年时间,共进行了三个批次的实验,在不断摸索中最终确定最佳免疫时间、免疫日龄及免疫程序,并初步建立了疫苗免疫后抗体消涨规律。
[Abstract]:Mycoplasma bovine has not been paid enough attention to, and it has not been included in the list of major diseases. But the loss of Mycoplasma in cattle has been very huge. Many reports have shown that the Mycoplasma disease is distributed worldwide and the pathogenicity of bovine mycoplasma is very strong, which is mainly caused by cow milk. Pneumonia, which is also the main reason for people to understand and discover Mycoplasma bovine. At the same time, Mycoplasma bovine can open the door for other pathogenic bacteria in the process of pathological changes. First, this subject has conducted identification and epidemiological investigation of isolated strains in most areas of China, and then carried out follow-up tests on the purified Mycoplasma bovine: On this basis, the bovine mycoplasma inactivated vaccine and the establishment of indirect ELISA for the establishment of antibody were developed. In the past 8 years, the laboratory conducted an epidemiological survey on 30 dairy farms in 12 provinces in China. The infection of Mycoplasma bovine was investigated by indirect ELISA, direct culture and specific PCR detection. The results showed that the positive rate of serum antibody was 52.31% (441/843), the positive rate of the disease was 55% (33/60), the positive rate of milk sample was 34.22% (373/1090), and we collected 55 mycoplasma strains with local representativeness and performed the 16SrDNA sequencing comparison, which showed that there was no variation on the 16SrDNA level compared with the results, and this result was the result. In the future, the immune prevention of Mycoplasma bovine provided an important basis. From the isolation of 55 mycoplasma from 55 strains of mycoplasma, 5 mycoplasma from bovine mycoplasma were selected for culture and characteristics. We studied the biochemical characteristics of Mycoplasma bovine, serum growth inhibition, nutrition and metabolism, growth and culture characteristics, drug sensitivity, and the use of PCR, CCU color change units, and CFU colony. Counting unit and other methods were used to obtain the experimental data, and the dominant strains were selected and cultured to be cultured to domesticate Mycoplasma bovine. The number of Mycoplasma bovine viable bacteria in the passage can reach 10111cfu/mL, and it can maintain stability. So far, Mycoplasma bovine have been passed in vitro for 200 generations. An indirect ELISA method for detecting mycoplasma antibody was established by using the lysate as the envelope antigen. Through orthogonal screening, the protein package was determined to be 4 mu g/mL and the clad liquid was a batch pH9.6 carbonate buffer. The best temperature and time were 4, 16 h, and the closed liquid was 1% concentration of skimmed milk. The optimum closing time and temperature were 37, 2 h, and the serum was tested most. The best dilution was 1:100, the reaction temperature and time were 37 C 1 h, the two anti dilution was 1:4000, the optimum reaction temperature and time was 37 1h., and the critical value of the negative serum was OD450=0.346. The method was not cross against the foot and mouth disease, the accessory tuberculosis, brucellosis, bovine viral diarrhea, bovine infectious rhinotracheitis and the positive serum of tuberculosis. It has very strong specificity. Dilute the positive serum double ratio and OD450 more than the critical value at 1:512 times dilution, which proves that the method has good sensitivity. The coefficient of variation within and between groups is less than 5%, which proves that the method is more repeatable, and 150 copies of the bovine mycoplasma indirect ELISA kit produced by the Canadian Biovet company are used in this method. The clinical samples were tested and the results showed that the coincidence rate of the two was more than 95%.. The results showed that the indirect ELISA method established by us could provide a means for the rapid diagnosis of the disease, the epidemiological investigation and the antibody detection of immunized cattle. The symptoms of Mycoplasma bovine infected cattle were highly similar; the Mycoplasma bovine infection was confirmed by pathological tissue section, indirect immunofluorescence, specific PCR, and isolation and culture. The virulence of Mycoplasma oxabi was obviously weakened but still pathogenicity in the process of continuous subculture in vitro, which could be used as a vaccine candidate. The 160th generation of bovine mycoplasma was used. After the inactivation of the body, the vaccine was mixed with the aluminum gel salt adjuvant 1:1 to make an inactivated vaccine against the rabbit. At twenty-eighth D, the vaccine group and the control group were attacked by the vaccine group. The vaccine group was not sick and the lungs were diseased after dissection. The control group had high fever and the lung had obvious pathological changes after dissection. The rabbit immunization test proved that the bovine mycoplasma inactivated vaccine was in the home. In rabbits, a high level of antibodies can be produced and can produce a certain protective effect. On the basis of the immune test in rabbits, we formulated the immunization program for the inactivated vaccine of Mycoplasma bovine, and carried out a clinical experiment in a pasture in Inner Mongolia, and selected 210 heads of bulls within 60 days of age to be vaccinated with the inactivated vaccine of bovine mycoplasma. The living bacteria content in the vaccine was 1011cfu/mL and the immuno metrology was 1mL/ head. After immunization with the inactivated vaccine of Mycoplasma bovine, the incidence of the sixtieth D immunization group decreased by 20.7%, the rate of bacteria division decreased by 35%, the positive rate of PCR decreased by 50%, the incidence of the control group increased by 17%, the rate of bacteria separation increased by 8.3%, and the PCR positive rate decreased the antibody titer produced by the two post immunization vaccine. The titer of the calf antibody up to 1:256,80% reached 1:128 and above, and the antibody was still detected at sixtieth D, and 67% of the calf antibody titer reached 1:64 and above. After a year, three batch experiments were carried out before and after the clinical trial. The rule of antibody elimination after vaccination was preliminarily established.

【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

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