猪支原体肺炎活疫苗RM48株气雾免疫技术的研究

发布时间：2018-01-01 12:35

本文关键词：猪支原体肺炎活疫苗RM48株气雾免疫技术的研究　出处：《山东农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：猪支原体肺炎(Mycoplasmal pneumonia of swine,MPS)即猪的喘气病,由猪肺炎支原体(Mycoplasma hyopneumoniae,MHP)引起的一种常见的慢性呼吸道疾病,一旦感染MHP后常常会继发感染别的呼吸道疾病很难被治愈,给养猪业造成很大的经济损失。对于该病的根治理论上是完全可行的,但是在实际的操作过程中基本无法实现。目前对该病的控制措施最有效的方法是接种疫苗。目前我国市面上的疫苗主要是灭活苗和弱毒苗,灭活苗多为进口疫苗价格高昂成为了其在各大猪场部署的障碍,而我国国产的弱毒疫苗多为肺内注射,操作复杂且不被大多数养殖户所接受同样影响了其在个大养殖场的部署。鉴于此,本课题以猪肺炎支原体RM48疫苗株为研究对象,并对弱毒苗的佐剂进行筛选,旨在验证MPS的气雾免疫的可行性以及筛选出合适的疫苗佐剂。具体的实验分为以下的三个步骤实施:1.雾化对RM48疫苗株的影响及雾化器的筛选分别使用购买的超声雾化器和压缩空气雾化器在无菌实验室中对RM48疫苗株进行雾化,无菌保存备用。使用CCU法对收集到的疫苗进行支原体计数,并设立未雾化对照组。在生化培养箱中培养12d后最后一瓶不再变色。结果显示超声和压缩空气雾化组的活菌数均和对照组无差别,说明雾化对RM48疫苗株并无太大损害。通过比对两种雾化器的雾化速度、雾化后气雾的粒径以及喷射距离后选择了压缩空气雾化器作为喷雾免疫的雾化器。2.利用家兔模型评价各种佐剂对猪支原体肺炎活疫苗的免疫增强作用以及活疫苗喷雾免疫的可行性佐剂的初步筛选及用量确定:将事先准备并配制好的黄芪多糖、天蚕素、板蓝根+乌梅黄酮、CPG、天冬提取物佐剂按一定的浓度与RM48活疫苗混合稀释,分别室温下放置30min、1h和2h进行CCU活性计数看佐剂对活疫苗的毒性。结果显示板蓝根+乌梅黄酮对疫苗的活性影响较大弃用。其余佐剂对活疫苗基本无毒性作用。天蚕素和CPG的用量由实验室提供分别为20μg/头和10μg/头。通过活性计数实验确定黄芪多糖和天冬提取物的用量为1mg/头和5mg/头。家兔免疫后体内疫苗株的PCR检测:在免疫后第7d和14d每组分别剖杀一只兔子取肺脏、脾脏和肝。研磨处理后提取DNA然后进行PCR实验检测体内疫苗株分布。结果显示在第7d肺内的PCR结果为阳性,其他组织均为阴性,第14d只有一组的肺呈弱阳性。免疫后体内细胞因子及抗体的检测:于首次免疫后第7d、14d、21、28d分别取兔鼻拭子和血液,处理后用事先买好的试剂盒分别测定IL-4、IFN-γ和SIgA,结果显示各佐剂组均能诱导兔子产生高水平的细胞免疫、体液免疫以及黏膜免疫水平,其中尤其以CPG佐剂组最高。3.用喷雾的方式对仔猪进行免疫在本动物试验研究中,实验分为天冬提取物喷雾免疫组、CPG喷雾免疫组、黄芪多糖喷雾免疫组、天冬提取物喷雾免疫组、肺内注射无佐剂组、无佐剂喷雾免疫组和对照组。分别在免疫前测定每组四只初生仔猪的体重,并在免疫后7d、14d、21d和28d采鼻拭子并测体重。用试剂盒测定鼻拭子中的SIgA和IFN-γ。结果显示除对照组外,各组SIgA和IFN-γ水平均明显升高,其中以CPG组最高与兔子实验相符。各免疫组间猪的体重均无太大差异。综上所述,本试验结果显示了猪肺炎支原体气雾免疫的可行性,为以后猪肺炎支原体气雾免疫的大规模推广提供了基础使人们更加关注这一疫苗预防接种的新方式。
[Abstract]:Mycoplasma hyopneumoniae (Mycoplasmal pneumonia of swine, MPS) swine enzootic pneumonia, mycoplasmal pneumonia of swine (Mycoplasma hyopneumoniae MHP) is a common chronic respiratory disease caused by MHP infection, once after secondary infection of other respiratory diseases are often difficult to be cured, which caused great economic losses to the pig industry for this. The radical theory of disease is feasible, but in the actual operation of the process can not be achieved. The basic method of control measures for this disease is currently the most effective vaccination. At present on the market the vaccine is mainly inactivated vaccine and attenuated vaccine, inactivated vaccine for the high import price has become a vaccine the obstacle is deployed in all large farms, and China's domestic attenuated vaccine for pulmonary injection, complex operation and will not be accepted by most farmers also affected the large farms in this deployment, This subject to Mycoplasma hyopneumoniae vaccine strain RM48 as the research object, and adjuvant on the attenuated vaccine were selected to verify the feasibility of MPS aerosol immunization and screened vaccine adjuvant appropriate. The implementation of specific experiments can be divided into three steps: ultrasonic atomizer screening 1. atomization on the effect of RM48 vaccine strain and atomizer are used to buy and compressed air atomizer atomization of RM48 vaccine strains in sterile laboratory, aseptic preservation reserve. CCU method is used to collect the vaccine for Mycoplasma counts, and the establishment of non atomized control group. After 12D culture, the last bottle of no discoloration in biochemical incubator. The results showed that ultrasonic and compressed air atomization the number of live bacteria group and control group were no difference, indicating atomization of the RM48 vaccine strain is not much damage. Two kinds of velocity ratio by atomizing atomizer, atomization spray particle size and After the selection of jet distance immune compressed air atomizer as a spray atomizer using.2. immune rabbit model to evaluate various adjuvant of swine mycoplasma pneumonia vaccine to determine the enhancement effect and preliminary selection of vaccine immune adjuvant feasibility and spray dosage: advance preparation and preparation of Astragalus polysaccharide, cecropin, Radix Isatidis + ebony flavonoids, CPG, aspartate extract concentration and RM48 adjuvant according to certain vaccine dilution, 30min were placed at room temperature, 1H and 2H activity of CCU vaccine adjuvant on counter toxicity. The result shows that the influence of ebony flavonoids on activity of Radix Isatidis + vaccine adjuvant on large abandoned. The remaining live vaccine has no toxicity effect of cecropin. And the amount of CPG provided by the laboratory were 20 g/ and 10 g/ head head. Through the activity counting experiment to determine the astragalus polysaccharide and aspartate extract dosage of 1mg/ and 5m head The head of the g/. In vivo detection of vaccine strain PCR in rabbits after immunization: after immunization of 7D and 14d in each group were killed a rabbit from the lung, spleen and liver. Mechanicalattrition DNA extraction followed by PCR assay in vivo vaccine strain distribution. The results showed that in the 7d lung PCR were positive, which he organized the 14d was negative, only one set of lungs was weakly positive in vivo detection of cytokines and antibodies after immunization in the first immunization: after 7d, 14d, 21,28d were obtained from rabbit nasal swabs and blood after treatment with kit bought in advance were measured by IL-4, IFN- and SIgA. The results showed that the gamma. The adjuvant group can produce high levels of immune cells induced by rabbit, humoral and mucosal immune level, especially with CPG adjuvant group had the highest.3. spray way of piglets were immunized in the animal experimental study, the experiment was divided into aspartate extract spray immune group, CPG injection The fog immune group, astragalus polysaccharide extract spray spray immune group, ASP immune group, pulmonary injection without adjuvant group, no spray adjuvant immune group and control group respectively. Four rats in each group were measured in newborn piglets before immunization and post immunization in weight, 7d, 14d, 21d and 28d in nasal swabs and weight. Determination of nasal swabs of SIgA and IFN- gamma kit. The results showed that except the control group, SIgA group and IFN- gamma were significantly increased, especially in CPG group and the rabbit experimental results. There was no much difference in all immunized groups between the weight of the pigs. In conclusion, the experimental results show the feasibility of pneumonia of swine Mycoplasma aerosol immunization, for future large-scale promotion of Mycoplasma hyopneumoniae aerosol immunization provides the basis to make people pay more attention to this new way of vaccination.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.28

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