青藏高原牦牛流产衣原体分离株免疫原性和免疫保护实验

发布时间：2018-05-18 20:02

  本文选题：衣原体 + 牦牛　； 参考：《甘肃农业大学》2017年博士论文

【摘要】：衣原体是严格胞内寄生的革兰氏阴性细菌,具有广泛的致病性,能引起动物和人的多种疾病。感染牦牛的衣原体主要有三种,即流产衣原体(C.abortus)、鹦鹉热衣原体(C.psittaci)、以及反刍动物衣原体(C.pecorum),其中流产衣原体感染可引起牦牛流产和生殖障碍性疾病。衣原体病导致牦牛的流产严重阻碍了牦牛相关产业的发展,给牧区人民带来了巨大的经济损失。因此,对于牦牛衣原体病的防控具有重要的公共卫生及经济意义。本研究组织开展了牦牛衣原体病流行病学调查,采集了青海省多个县的牦牛血清和组织样品,通过间接血凝试验(IHA)和PCR检测,结果显示:2014年间接血凝试验阳性率为24.02%,PCR检测阳性率为23.8%;2015年间接血凝试验阳性率为55.76%,PCR检测阳性率为41.67%。表明青藏高原相关区域牦牛感染衣原体的形势非常严峻。本研究对牦牛流产组织样本进行了分离鉴定,采用鸡胚卵黄囊接种培养法从贵南县(GN)和海晏县(HY)共分离到6株衣原体,经PCR-RFLP鉴定,GN-1、GN-4、GN-6以及HY-1与流产衣原体标准株SX5的特征一致,为流产衣原体。同时,有两株流产衣原体毒株连续传代至第18代,繁殖滴度为10-10,且稳定。本研究以分离株GN-6作为制苗菌株,进行了牦牛流产衣原体灭活苗的研究,以BALB/c小鼠为实验动物进行免疫试验。以接种鸡胚卵黄囊培养分离得到的牦牛流产衣原体作为抗原,经甲醛灭活,以206为佐剂制成牦牛流产衣原体灭活疫苗。经过小鼠免疫试验证明:免疫小鼠的抗体亚型测定结果为以κ链轻链为主IgG1型、血清与淋巴细胞培养上清中的细胞因子IFN-γ含量明显升高、DTH检测结果显示足垫明显增厚、淋巴细胞增殖系数升高。结果表明免疫小鼠体内产生了Th1型细胞免疫应答,对衣原体感染后的有效清除起到一定作用。抗体中和试验结果显示,免疫小鼠血清中形成了能够中和衣原体的保护性抗体。攻毒试验结果表明,免疫组小鼠能够有效抵抗衣原体的感染。以上结果表明小鼠在接种了该疫苗以后,诱导机体产生特异性细胞免疫应答和体液免疫应答。本研究对研制的牦牛流产衣原体疫苗进行了本动物试验。牦牛流产衣原体灭活疫苗安全试验结果表明这种衣原体灭活疫苗安全可靠;通过最小免疫剂量试验和免疫效力检测证明,免疫剂量为牦牛犊1ml、成年牦牛2ml,即可达到良好的免疫效果。疫苗的免疫持续期检测结果显示,本研究所研制的牦牛流产衣原体疫苗免疫后210天时平均免疫抗体滴度保持在1:256以上。此外,疫苗的保存期试验目前正在进行中,将疫苗在4℃条件下保存一年,已于2016年8月进行免疫,免疫后抗体效价较好,达到了预期的免疫效果。本研究在青藏高原地区开展了牦牛衣原体的流行病学调查,成功分离鉴定到4株流产衣原体,制备了具有良好免疫效果的流产衣原体疫苗,为牦牛衣原体病的防控奠定了基础。
[Abstract]:Chlamydia chlamydia is a strictly cytosolic gram-negative bacteria with a wide range of pathogenicity and can cause a variety of animal and human diseases. There are mainly three kinds of chlamydia infection in yak, I. E. C. abortus, C. psittacius, and C. pecorumus, among which the infection of chlamydia miscarriage can cause abortion and reproductive disorders in yak. The abortion of yak caused by chlamydia has seriously hindered the development of yak related industries and brought huge economic losses to the people in pastoral areas. Therefore, the prevention and control of Chlamydia yak chlamydia has important public health and economic significance. In this study, the epidemiological investigation of yak chlamydia was carried out, and yak serum and tissue samples were collected from several counties of Qinghai Province, and detected by indirect hemagglutination test (IHA) and PCR. The results showed that the positive rate of indirect hemagglutination test in 2014 was 24.02% and that of indirect hemagglutination test in 2015 was 55.76% and 41.67% respectively. The results showed that the situation of Yak infected with Chlamydia was very serious in Qinghai-Tibet Plateau. In this study, six strains of Chlamydia chlamydia were isolated from chicken embryo yolk sac inoculation and culture from Guinan County (GNN) and Haiyan County (HYY). The characteristics of GN-1GN-4GN-6 and HY-1 were consistent with that of Chlamydia abortus standard strain SX5. For chlamydia miscarriage. At the same time, two strains of chlamydia abortus were subcultured to the 18th generation, the reproductive titer was 10-10 and stable. In this study, the inactivated Chlamydia abortus vaccine of yak was studied with GN-6 as the vaccine preparation strain, and the BALB/c mice were used as experimental animals to carry out the immune test. Using Chlamydia abortus isolated from yak embryo yolk sac culture as antigen, Chlamydia abortus inactivated vaccine was prepared by formaldehyde inactivation and 206 as adjuvant. The antibody subtype of immunized mice was mainly 魏 chain light chain IgG1, and the level of cytokine IFN- 纬 in the supernatant of serum and lymphocyte culture was significantly increased. Lymphocyte proliferation coefficient increased. The results showed that the immune response of Th1 type cells was produced in the immunized mice, which played a certain role in the effective clearance of chlamydia infection. Antibody neutralization test showed that protective antibodies against chlamydia were formed in serum of immunized mice. The results showed that the immunized mice could resist chlamydia infection effectively. The above results suggest that the specific cellular and humoral immune responses were induced after the mice were inoculated with the vaccine. In this study, the Yak Chlamydia abortus vaccine was tested. The safety test of the inactivated Chlamydia chlamydia vaccine in yak showed that the vaccine was safe and reliable. The immune dose was 1 ml for yak calf and 2 ml for adult yak. The results of immunization duration test showed that the average antibody titer of Chlamydia abortus vaccine was above 1: 256 at 210 days after immunization. In addition, the preservation period of the vaccine is currently under way. The vaccine was immunized at 4 鈩，

本文编号：1907013