【作者】 邹宁；

【导师】 刘思当；

【作者基本信息】 山东农业大学 ， 兽医（专业学位）， 2011， 硕士

【摘要】 猪繁殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome, PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的猪的一种高度接触性传染病。自1987年首次在美国暴发以来,目前已遍布全球各主要养猪国家。2006年夏季,我国南方部分地区在高温高湿季节发生了以感染猪繁殖与呼吸综合征病毒为主要病原同时伴发多种病原引起的猪“高热病”,随后猪“高热病”在山东及全国发生流行,给养猪业造成前所未有的损失。本研究对高致病性蓝耳病山东分离株SD0612在抗体选择压下传代致弱毒株回归猪的感染状况、病理变化、免疫反应及对PRRSV强毒人工感染的保护作用进行了系统的研究。为了比较SD0612原始株及其在抗体选择压下突变株在回归猪体后的抗体动态变化,初步探讨其作为疫苗候选毒株免疫猪后的安全性及对强毒的抵抗能力。我们先后进行了两次动物试验。在第一次试验中,将15头健康猪分为五组,分别为SD0612原毒感染组、有抗体条件下第一次传40代突变株F40感染组及有抗体条件下再次传40代突变株e40感染组、无抗体条件下再次传40代突变株a40感染组及阴性对照组,分别于接毒后1w、2w、3w、4w、5w采血,收集血清以ELISA试剂盒测定其抗体水平。剖检死亡猪及到期剖杀猪,取病料固定,石蜡切片,HE染色,镜检。结果显示：F40在接种猪后引起的ELISA抗体较原始株迟缓,约滞后一周左右。而e40和a40诱发的抗体和F40未见明显区别。四个毒株比较后发现,SD0612原始株感染猪后引发的ELISA抗体始终是最高的,显示其在体内的复制能力最强和抗原性最好。SD0612原始株接种组迅速出现高致病性蓝耳病的典型病症,于接种10-13天后死亡,剖检及病理组织学病变也完全符合高致病性蓝耳病的典型病变。而三株突变株除一过性发烧外,未见其它症状,剖检也未见明显的眼观病变,但镜检1/3的猪见肺泡壁轻度增厚,少量淋巴细胞浸润,肝细胞轻度颗粒变性及水泡变性,各毒株间未见明显的差异,表明突变株的致病力显著下降,已成“非致病性”弱毒株,但安全性可能还存在一定的问题。为了比较F40、e40及a40接种猪体后对强毒攻击的免疫保护作用,进行了第二次试验,将20头猪分为四组,分别为F40接种组、e40接种组、a40接种组及阴性对照组,在第40天时接种强毒,于攻毒后1w、2w、3w、4w、5w采血,收集血清以ELISA试剂盒测定其抗体水平变化,测量体温,观察其发病表现,并到期剖杀试验猪,采取组织病料做石蜡切片,光镜下观察不同组织器官特别是肺脏、淋巴组织及大脑的组织病理学变化。结果显示：F40接种猪经强毒攻击后引起的ELISA抗体升高幅度较e40组迟缓,升高的幅度也低于e40组。尽管个别猪出现了一过性的发热现象,肺有极轻微的炎症反应,但F40诱发的免疫力足以经受强毒的攻击,显示F40虽然在某些位点发生了一些氨基酸的变化,但其抗原性与原始株仍在较近的范围内。a40接种猪经强毒攻击后引起的ELISA抗体变化与F40接种组基本相同,本组强毒攻击后的症状表现及病理变化与F40组也基本相同,同样具有良好的免疫保护作用,说明其抗原性未再发生明显变化。而e40接种猪经强毒攻击后ELISA抗体升高快且显著高于F40、a40接种组；e40接种组虽然未有死亡,但在3-10天期间均出现了高热症状,且表现严重的精神沉郁、食欲不振等,e40诱发的免疫未能有效保护实验猪经受强毒的攻击,这从一个侧面反映了e40较F40特别是原始株抗原性发生了更大的变化。三个毒株比较后发现,a40或F40能够保护免疫猪经受强毒的攻击,安全性高,是极具开发价值的候选疫苗毒株。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is a highly contagious disease in pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV). Since the first outbreak in the United States1987, it has been spread to the the major pig-raising countries around the world. In the summer of2006, pigs "high fever" disease characterized by high fever took place in south of China in hot and humid season. The study showes it was caused mainly by porcine reproductive and respiratory syndrome virus as the main pathogen associated with a variety of pathogenic infection and secondary infection of other pathogen at the same time. Pig "high fever" epidemic in Shandong Province and across all the country, causing unprecedented damage to the pig industry. In this study, it shows infection, pathology, immune response and artificial infection of virulent PRRSV protective effect of HPRRSV Shandong isolate SD0612antibody selection pressure passage-attenuated strain returning to swine. To compare the dynamic of response in pigs between SD0612original strain and it’s mutants in the selective pressure, and study its immunization safety and virulent resistance after the inoculation to swine as a vaccine candidate strain,two animal experiments were conducted in this study. In the first experiment,15healthy pigs were divided into five groups as the SD0612original strain infection group, the first passage mutant F40infection group and the second passage mutant e40infection group, the second passage mutant e40infection group without antibody and negative control group, antibody was determined by ELISA kit in1w,2w,3w,4w,5w after challenge. Then dead pigs were dissected, and specimens were immediately fixed by formalin and imbedded by paraffin. Microscopic section was examined by microscopy after HE staining. The results showed that:Antibodyof the F40infection group is one week later than the original strain group, and it has no significant difference with the e40and a40group. It is found after the comparison of four strains that the antibody of SD0612original strain infection group is always the highest, indicating its highest graphetic level and the best antigenicity.SD0612original strain infection group quickly appeared the typical symptoms of PRRS, then died after vaccination10-13days. Dissected checks and pathological changes also fully comply with the typical symptoms. And except for one reason mutations of the fever, the other three mutant strains did not see other symptoms and obvious pathological changes, but Alveolar walls mild thickening, a few lymphocytic infiltratesand liver cells mild granulovacuolar degeneration and vacuslation were found by microscopy in one-third of the pigs.Each strain has not the obvious difference, suggesting the pathogenicity of mutant strains decreased significantly,and it has become a "nonpathogenic " weak strain, but there may be still some problems in security.To compare the immune protective effect after challenge among e40, F40and a40,20pigs were divided into four groups in the second experiment as the F40vaccinated group, e40vaccinated group, the a40vaccinated group and control group. At the40th day all groups were inoculated with virulent PRRSV,antibody was determined by ELISA kit in1w,2w,3w,4w,5w after challenge.Temperature was measured and incidence of performance was observed daily.Then pigs were dissected regularly,and specimens were immediately fixed by formalin and imbedded by paraffin. Pathological changes of different organs especially lung, lymphoid tissue and the brain were examined by microscopy after HE staining. The results showed that: Antibody rise of the F40vaccinated group is slower than the control group, and there is no significant difference. The immunity F40induced could still be able to protect the immune pig from strong virus attack, indicating some amino acids of F40has changed in some sites, but its antigenicity still in the near range of the original strain. Antibody changes of the vaccinated group have no significant difference with the F40vaccinated group.,and their symptoms and pathological changes were also similiar with the F40vaccinated group,showing good immune protective effect and antigenicity not changing greatly.But antibody of the e40vaccinated group was obviously higer than the F40and a40vaccinated group’s.The immunity e40induced could not be able to protect the immune pig from strong virus attack,it shows from one side the antigenicity of e40has changed greater than F40especially the original strain.After comparing three strains, F40and a40with high safety will be able to protect the immune pig from strong virus attack, and is the candidate vaccine strain with high development value.更多还原