牛支原体感染动物模型的建立及其在评估灭活疫苗免疫效力上的应用

Establishment of an Mycoplasma bovis infection model and its application in the evaluation of the inactivated vaccine

为了找到能够成功模拟牛支原体（Mycoplasma bovis,M.bovis）感染的方便可行的替代动物,并为灭活疫苗的初步探索做出贡献,采用原代次、第80代次、第160代次的M.bovis,每代分别设1.0×10^9、1.0×10^10和1.0×10^11CFU/m L 3个浓度对健康家兔进行感染试验,通过临床表征、剖检观察、病理组织切片镜检、分离培养、PCR法进行验证,显示每个代次M.bovis均呈阳性,且浓度越高、代次越低,症状越明显。家兔可成功模拟M.bovis感染情况,并确定M.bovis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,第160代次M.bovis可作为疫苗备选株;将160代次M.bovis灭活后与铝胶盐佐剂1∶1混合制成灭活疫苗对家兔进行免疫试验,在第1次免疫后第14天进行第2次免疫,在第28天时对免疫组和空白对照组家兔进行攻毒试验,结果显示免疫组家兔经血清抗体法检测出体内具有较高水平的抗体,且经上述方法检测免疫组结果均呈阴性,空白对照组检测结果均呈阳性。结果表明：弱毒的M.bovis灭活疫苗也能够起到免疫防御的作用,为M.bovis疫苗回归本动物试验的研究提供了有力的数据支撑。

In order to find out a kind of animal which is convenient and feasible to be tested to effectively simulate Mycoplasma bovis（M.bovis） infection and to be prepared for preliminary exploration of inactivated vaccine of M. bovis,three concentrations of the 1 st,80 thand160 thgenerations of M. bovis at 1. 0×10^9 CFU/m L,1. 0×10^10 CFU/m L and 1. 0×10^11 CFU/m L were used to infect healthy rabbits in a clinical test. The results of clinical symptoms,anatomic observation,microscopic examination of pathological tissue sections,conventional separated culture and polymerase chain reaction showed that the rabbits could successfully simulate the state of infected bovis. Also,as the subculturing progressed,the virulence of the M. bovis strain decreased,but it still had pathogenicity with the 160 thgeneration of the M. bovis strain showing the performance of a candidate strain for vaccine. Then,we inactivated the 160 thgeneration of the M. bovis and mixed it with alhydrogel salt adjuvant in a ratio of 1 ∶ 1,and immunized one group of rabbits as an experimental group with another group as a blank control. 14 days later,the experimental group received a second immunization. 28 days later,both the groups of rabbits were challenged with M.bovis. The results were： the rabbits of the immunized group showed no symptoms of M. bovis infection,and no lesion was seen in their lung tissues,while the rabbits of the control group showed symptoms of hyperpyrexia,fever and apparent pathological changes in lungs. The enzyme-linked immuno sorbent assay showed that the serum of the immunized group had a high level of M. bovis antibodies. The rabbits of the immunized group showed negative reaction to M. bovis challenge and those of the blank control group exhibited positive reaction. Our results proved that inactivated attenuated M. bovis vaccine could function in immune protection for cattle,and offered strong support for test of the vaccine on the intended herd.