牛支原体灭活疫苗的研制与免疫效果研究

发布时间：2018-09-08 08:59

【摘要】：牛支原体(Mycoplasma bovis,M.bovis)是感染牛的一种重要的致病性病原体,主要引起牛的肺炎、乳腺炎和关节炎等多种疾病。美国于1961年首次从患乳腺炎的牛乳中分离得到该病原。国内于2008年首次从患肺炎的犊牛肺脏中分离得到,此后在国内逐步蔓延传播,造成了巨大的经济损失,为研发防控牛支原体病的有效疫苗,在综合前期分析研究的基础上,本文对牛支原体生长滴度测定、抗原菌悬液浓缩、灭活疫苗制备以及犊牛免疫效果评价等疫苗研发关键技术进行了试验研究,主要研究内容及结果如下:1 TTC应用于牛支原体活菌计数研究及牛支原体扩大培养效果测定为探讨氯化三苯基四氮唑(TTC)应用于M.bovis菌落计数的可行性及实验条件,通过平板计数法进行了TTC浓度筛选;采用倾注培养法对M.bovis扩大培养效果进行了菌落形成单位(Colony forming unit,cfu)计数,通过颜色变化单位(Colour change unit,ccu)法进行了浓度检测,并测定了630 nm处OD值及培养物p H的变化。结果:培养基中TTC质量浓度在0.3~0.6 g/L时菌落正常生长且显色效果明显,TTC质量浓度较低时菌落显色效果不明显,较高时则会抑制M.bovis的生长,TTC作用最佳质量浓度为0.4 g/L;CFU法测得菌液浓度为1.8~2.42×108 CFU/m L,CCU法测得牛支原体菌株在96 h时的生长滴度为5.0×108 CCU/m L,两种方法检测结果基本一致;而不同时段牛支原体培养物OD值变化范围在0.024~0.213,在对应时间点OD值与上述两种方法未表现出一致性,OD值测定仅可做为培养效果检测时的辅助方法;培养物p H变化与OD值测定却表现出一定的相关性。结果表明:TTC可应用于M.bovis活菌计数,菌落呈醒目红色且特征明显,可有效、准确地对M.bovis进行活菌计数;CFU法与CCU法均可应用于牛支原体扩大培养效果测定。2牛支原体生长曲线测定及不同培养基生长效果比较通过颜色变化单位法(Colour change unit,ccu)对牛支原体新疆分离株在牛支原体专用液体扩大培养基中不同培养阶段的生长滴度进行了测定并绘制了生长曲线,同时采用活菌计数法对牛支原体菌株在牛支原体专用液体扩大培养基、改良Thiaucourt’s培养基、牛心汤培养基、葡萄糖血清肉汤培养基中的培养效果进行了比较。结果表明:活化培养后的牛支原体菌株在牛支原体专用液体扩大培养基中生长良好,该培养基相比其他三种培养基具有一定的经济优势,可作为制备疫苗时牛支原体抗原获取的最优培养基。3牛支原体灭活疫苗的研制采用透析法、高速离心法及超滤浓缩法对M.bovis培养物进行了浓缩试验对比研究,测定不同处理方法浓缩物蛋白含量,并利用CFU与CCU法测定了超滤浓缩后浓缩液浓度;应用ISA 206双相佐剂制备M.bovis灭活疫苗,先后进行了疫苗无菌检测、物理性状检测、稳定性与保存期试验、动物安全性试验,结果显示超滤浓缩法处理得到的浓缩物蛋白浓度最高,CFU法与CCU法检测结果基本一致;所制备疫苗物理性状良好,4℃可保存180 d、37℃可存放3 d,受试动物接种后未见异常反应,注射部位均正常,未见化脓及红肿现象。结果表明:超滤浓缩法适用于M.bovis培养物的浓缩,可用于疫苗生产时抗原的富集;疫苗安全可靠,为M.bovis灭活疫苗的规模化生产提供了一定的方法与技术支持。4牛支原体灭活疫苗免疫小鼠最小免疫剂量及抗体消长规律初步研究应用牛支原体灭活疫苗对小鼠进行最小免疫剂量及抗体消长规律初步研究。将制备的疫苗分别以0.1、0.2、0.3、0.4 m L/只的不同剂量,经腿部肌肉途径免疫小鼠,结果显示最小免疫剂量为0.2 m L/只;以0.2 m L/只剂量免疫小鼠,免疫3周后,抗体滴度达到最高峰,随后抗体滴度开始下降,但仍保持相对稳定,5周后抗体滴度表现出下降趋势。5牛支原体灭活疫苗免疫犊牛的免疫保护性评价随机选取10-15日龄犊牛20头,免疫组10头于10-15日龄首免,间隔10天进行二免,肌肉注射3 m L/每头,未免疫组10头不注射疫苗,注射等剂量生理盐水。测定两组犊牛二免后14 d抗体水平并在二免后21 d随机选取相同日龄免疫与未免疫犊牛各3头进行攻毒保护试验,每头犊牛经鼻腔黏膜接种(滴注)2 m L、气管注射3 m L牛支原体培养物(1.8×109 CFU/m L),记录犊牛体温,观察临床症状,检测血清抗体水平。攻毒后28 d剖检观察肺脏临床解剖学及病理组织学变化,记录犊牛肺脏病变指数并采集病变肺脏组织进行牛支原体分离培养。结果:免疫组犊牛在二免后14 d血清牛支原体抗体达到0.404(OD450);3头攻毒犊牛均保持良好的精神状态且体温变化不明显,临床症状综合评分为3,肺脏病变指数为2,肺脏解剖学及病理组织学观察无异常且肺脏中未分离回收到牛支原体。未免疫组犊牛同期血清牛支原体抗体为0.142(OD450);3头攻毒犊牛均先后出现体温升高、轻度咳喘、脓性鼻液,明显消瘦等症状,临床症状综合评分为11,肺脏病变指数为17,肺脏尖叶、心叶及部分膈叶均表现明显肝变,两头犊牛表现胸膜与肺脏黏连,一头犊牛整个尖叶广泛分布黄白色蚕豆状大小坏死性结节,与自然感染病例相同,病理组织学观察显示肺泡腔和支气管中浸润有大量中性粒细胞,支气管管壁充血、水肿,肺脏部分坏死灶呈均质红染,为典型的化脓性及坏死性肺炎变化,3头未免疫组犊牛病变肺组织中均分离回收到M.bovis。
[Abstract]:Mycoplasma bovis (M. bovis) is an important pathogen of bovine pneumonia, mastitis, arthritis and other diseases. The pathogen was first isolated from the milk of cows suffering from mastitis in 1961 in the United States. It was first isolated from the lungs of calves suffering from pneumonia in China in 2008 and has been in the country since then. In order to develop an effective vaccine to prevent and control Mycoplasma bovis disease, on the basis of comprehensive preliminary analysis, the key technologies of vaccine development, such as determination of Mycoplasma bovis growth titer, concentration of antigen suspension, preparation of inactivated vaccine and evaluation of calf immune effect, were studied. The main research contents and results are as follows: 1. TTC application in Mycoplasma bovis viable count study and Mycoplasma bovis enlarged culture effect determination to explore the Triphenyltetrazolium chloride (TTC) application in M. bovis colony count feasibility and experimental conditions, through plate counting method for TTC concentration screening; The colony forming unit (cfu) was counted, and the concentration of the colony forming unit (ccu) was measured. The OD value and the change of the culture P H were measured at 630 nm. Results: The colony grew normally and showed obvious colour effect when the concentration of TTC in the medium was 0.3-0.6 g/L. The best mass concentration of TTC was 0.4 g/L; the concentration of CFU was 1.8~2.42 *108 CFU/m L, and the growth titer of Mycoplasma bovis was 5.0 *108 CCU/m L at 96 h by CCU method. The results of the two methods were basically the same; but the best mass concentration of TTC was 0.4 g/L. The OD values of the cultures ranged from 0.024 to 0.213. The OD values were not consistent with the above two methods at the corresponding time points. The determination of OD values could only be used as a supplementary method for the detection of culture effect, but the changes of culture P H showed a certain correlation with the determination of OD values. CFU method and CCU method can be used to determine the effect of Mycoplasma bovis in the enlarged culture. 2 The growth curve of Mycoplasma bovis and the growth effect of different media were compared. Colour change unit (ccu) was used to detect Mycoplasma bovis in Xinjiang isolates of Mycoplasma bovis. The growth titers of Mycoplasma bovis strains in different culture stages were measured and their growth curves were plotted. Meanwhile, the effect of Mycoplasma bovis strains in liquid enlarged culture medium for Mycoplasma bovis, improved Thiaucourt's culture medium, bovine heart soup culture medium and glucose serum broth culture medium were studied by counting live bacteria. The results showed that the strains of Mycoplasma bovis grew well in the special liquid expanded medium for Mycoplasma bovis. Compared with the other three media, this medium had certain economic advantages and could be used as the optimal medium for obtaining Mycoplasma bovis antigen in the preparation of vaccine. 3 The development of inactivated Mycoplasma bovis vaccine was made by dialysis. Methods: The concentration of M. bovis culture was studied by high-speed centrifugation and ultrafiltration. The protein content of M. bovis culture concentrate was determined by different treatment methods, and the concentration of concentrated solution after ultrafiltration was determined by CFU and CCU methods. The results showed that the protein concentration of the concentrate was the highest by ultrafiltration and concentration, and the results of CFU and CCU were basically the same. The prepared vaccine had good physical properties, and could be stored at 4 ~C for 180 days, 37~C for 3 days. No abnormal reaction was observed after inoculation, and the injection site was normal. The results showed that the ultrafiltration method was suitable for the concentration of M. bovis culture and could be used for antigen enrichment during vaccine production; the vaccine was safe and reliable, which provided a certain method and technical support for the large-scale production of M. bovis inactivated vaccine. 4 Minimum immune dose and antibody elimination of mice immunized with bovine mycoplasma inactivated vaccine A preliminary study on the length rule of mice immunized with inactivated Mycoplasma bovis vaccine was carried out. The mice were immunized with the vaccine at different doses of 0.1,0.2,0.3,0.4 m L per mouse through the leg muscle route. The results showed that the minimum immune dose was 0.2 m L/mouse, and 0.2 m L/mouse was immunized with the vaccine at different doses. After 3 weeks, the antibody titer reached its peak, then began to decline, but remained relatively stable. After 5 weeks, the antibody titer showed a downward trend. 5 Immunoprotective evaluation of calves immunized with inactivated Mycoplasma bovis vaccine was randomly selected from 10 to 15 days of age. 10 calves in immunization group were immunized for the first time from 10 to 15 days of age. The levels of antibodies were measured 14 days after the second immunization and randomly selected 3 calves of the same age immunized and 21 days after the second immunization. Each calf was inoculated (dripped) 2 ml through nasal mucosa and 3 ml Mycoplasma bovis by tracheal injection. The body temperature of calves was recorded, clinical symptoms were observed and serum antibody levels were detected. The clinical anatomy and histopathological changes of lungs were observed 28 days after treatment. The pathological index of lungs of calves was recorded and Mycoplasma bovis was isolated and cultured by collecting pathological lung tissues. Mycoplasma antibody was 0.404 (OD450); all the three calves maintained good mental state and had no significant changes in body temperature. The comprehensive score of clinical symptoms was 3, the index of lung lesion was 2, the pulmonary anatomy and histopathology were normal, and the antibody of Mycoplasma bovis was not isolated and recovered from the lungs. The clinical symptoms were 11, pulmonary lesion index was 17, pulmonary apical lobe, heart lobe and part of diaphragm lobe showed obvious liver degeneration, pleura and lung adhesion in two calves, and yellow and white in the whole apical lobe of one calf. Histopathological observation showed that there were a large number of neutrophils infiltrated in alveolar cavity and bronchus, the wall of bronchial tube was congested, edema, and some necrotic lesions of lung were homogeneous red stained, which was a typical change of suppurative and necrotizing pneumonia. The pathological changes in the lung tissues of the three unimmunized calves were all the same. Separation and recovery to M.bovis.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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