【作者】 初小辉；

【导师】 高丰；

【作者基本信息】 吉林大学 ， 兽医， 2011， 博士

【摘要】 猪伪狂犬病(Pseudorabies, PR)又名奥叶基氏病(Aujeszky’s, AD)、奇痒症等,是由疱疹病毒科、α-疱疹病毒亚科的伪狂犬病毒(Pseudorabies Virus,PRV)感染引起多种家畜和野生动物以发热、奇痒(猪除外)和脑脊髓炎等为主要特征的一种急性、高度接触性传染病,其中以猪的感染最为普遍,其主要引起母猪的繁殖障碍,主要表现为流产、死胎、木乃伊胎等;新生仔猪发生感染时,死亡率可高达100%;此外,育肥猪感染往往致使其生长缓慢,种公猪感染则导致精液品质下降甚至丧失种用价值,因此该病的发生给养猪业带来巨大的经济损失。目前,该病已被世界动物卫生组织(OIE)列为B类动物疫病,我国也将其列为二类动物疫病。近年来,该病的发生呈不断上升的趋势,成为当前严重危害养猪业的重要疫病之一。由于当前绝大多数的猪场主要通过使用gE基因缺失疫苗对该病进行免疫预防,因此配套使用PRV gE-ELISA抗体检测试剂盒能快速、准确地对血清中PRV野毒抗体进行检测,从而能有效监测猪群中PRV野毒感染情况。为了解近年来吉林省各地区猪场中PRV野毒的感染情况,本研究采用PRV gE-ELISA抗体检测试剂盒对2008～2010年间采自吉林省12个市县的152个不同规模的免疫猪场不同批次送检的3849份血清样品进行PRV野毒血清抗体检测,从而为针对感染状况日益严重的猪伪狂犬病科学防控措施的制定提供重要的理论依据。血清学调查结果显示,吉林省各地区的抗体阳性率从9.47%～40.00%不等,抗体平均阳性率为22.66%;在被检测的152个猪场中,PRV抗体阳性的猪场103个,各地区猪场阳性率从37.50%～87.50%不等,这表明吉林省各地区猪场中PRV野毒感染情况仍较为严重。此外,本研究还对不同日龄、不同性别以及不同品种猪PRV野毒感染情况进行了调查与分析,以上血清学调查结果将为吉林省针对PR防治工作的开展提供重要的理论依据。2008年11月,吉林省四平地区某猪场怀孕母猪出现流产、死胎,50多头新生仔猪临床上出现呕吐、腹泻,并伴有肌肉震颤、后肢麻痹、四肢呈游泳状滑动等神经症状,经多种抗生素治疗无效,发病后2～3 d后陆续死亡,死亡率高达100%。剖检送检病死仔猪,主要病理变化表现为脑膜充血、淤血,肺脏充血、水肿,肝脏淤血、表面散在有灰白色的坏死灶,而其他组织脏器无明显的病理变化。根据该病的临床症状及眼观病理变化,我们初步诊断为猪伪狂犬病,由于该病在临床上容易与猪繁殖障碍与呼吸综合征、猪细小病毒病、猪布氏杆菌病等相混淆,因此本研究从病理学和病原学角度对该病的发生原因进行了综合分析。首先,本实验从送检病死仔猪的心、肝、脾、肺、肾以及脑组织等进行病理学切片,从病理组织学角度对仔猪的死亡原因进行分析。病理组织学观察可见,脑组织出现“血管套”、噬神经现象等典型的非化脓性脑炎变化,据此我们推断该病变是由病毒感染所引起,同时,细菌学检测结果也排除了细菌感染的可能。此外,肝脏的部分组织坏死形成局灶性的坏死灶,该病变是PRV感染的一个重要的示病病变。为了进一步对该病进行确诊,本试验利用BHK-21细胞进行了系统的病原分离鉴定,并成功获得了1株PRV野毒,并将其命名为PRV JL/08/SP分离株。为了进一步了解PRV JL/08/SP分离株在BHK-21细胞中的增殖能力以及侵染细胞的动态过程,本研究通过制作感染细胞的超薄切片对PRV JL/08/SP病毒粒子的形态学发生过程和感染细胞超微结构的变化进行观察,观察可见,PRV在吸附到细胞膜的表面后通过膜融合的方式进入到细胞中,在胞核中进行病毒的复制,装配好的病毒以出芽方式离开细胞核进入胞浆;在胞浆内的病毒粒子又利用高尔基体的膜结构合成第二层囊膜,形成完整的病毒粒子;最后包裹有完整病毒粒子的高尔基囊泡与细胞膜发生融合,将病毒粒子释放到细胞外。该增殖过程及释放形式与之前文献报道的基本一致,这表明不同的PRV分离株在细胞中的形态发生学过程非常相似,仅在PRV囊膜获得方式上存在有一定的差异。此外,本研究还利用BHK-21细胞对其毒力进行了测定,结果显示,PRV JL/08/SP分离株在BHK-21细胞的感染滴度为10-7.2/0.1 mL,这表明该分离株具有较强的毒力;为了进一步确定该毒株的致病性,还选用对PRV最为易感的家兔以及本源动物仔猪进行了动物回归试验,均复制出与自然感染相类似的临床症状。以上我们对PRV JL/08/SP分离株进行了系统全面的鉴定,并从病原学角度对其致病性进行了初步的研究分析,据此从而确诊该病的发生是PRV野毒感染所致。由于目前各猪场中PRV野毒感染的情况仍非常普遍,在猪群中多呈隐性感染,因此也是彻底根除该病所面临的一个难题。鉴于此,当前非常有必要建立一种快速、特异、敏感的诊断方法用于PRV野毒株的检测。在控制PR的发生流行过程中,PRV基因缺失疫苗起到了非常重要的作用,其中以PRV gE基因缺失疫苗的使用最为广泛。因此,在此基础上建立一种能有效区分野毒感染和疫苗接种毒株的诊断方法对于野毒感染阳性猪的清除以及猪场的净化起到至关重要的作用。由于目前所使用的PRV gE基因缺失疫苗株均部分甚至全部缺失gE基因,而且gE基因能在野毒株中稳定表达且其本身的遗传变异性较小,因此可将gE基因作为鉴别PRV野毒株和疫苗株的一个标志基因用于PRV分子水平上的诊断。本研究在前期分离获得PRV JL/08/SP株的基础上,根据Genbank中公布的gE基因序列保守区设计特异性引物,利用PCR方法扩增并克隆了PRV JL/08/SP分离株的gE基因并进行了序列测定分析,此外,还对该基因的核苷酸及编码氨基酸同源性进行了分析。分析结果表明,与其他分离株相比,gE基因虽然存在有个别核苷酸的突变位点,但从同源性以及遗传进化分析结果来看,PRV JL/08/SP分离株gE基因仍然高度保守。因此,PRV JL/08/SP分离株的gE基因可作为鉴别PRV野毒株和疫苗株的诊断抗原,我们利用该分离株的gE基因建立用于临床样本快速检测的PCR方法,且该方法具有特异性强、敏感性高以及重复性好等优点。应用该方法对吉林省长春、四平、公主岭、吉林等地猪场送检的68份疑似PRV感染猪的脾脏、肺脏、肝脏以及脑组织等组织样本进行检测,其中样本检出阳性率为27.94%,这与之前的血清流行病学调查结果相符合,这说明猪伪狂犬病在吉林省的发生仍较为普遍。在本研究中,我们利用PRV JL/08/SP分离株gE基因建立的PCR诊断方法不仅能快速、特异地对临床样品进行检测,而且能有效地区分PRV野毒株和疫苗株,从而在很大程度上提高了PRV的检出率和准确程度,此外,该方法对于PRV的早期感染、潜伏感染以及持续感染的诊断均具有十分重要的意义,因此可为猪场中PR的净化及根除提供有力的技术支撑。此外,针对当前该病发生的严峻现状,各猪场非常有必要对该病进行净化、根除。因此,本研究在参照国外净化、根除PR成功经验的基础上,在2009～2010年应用PRV gE基因缺失疫苗并结合PRV gE-ELISA抗体检测试剂盒对吉林省6个不同规模的猪场采取免疫、监测以及淘汰等综合防治技术进行净化、根除,并在上述的6个试验猪场达到了预期的净化效果,这将为吉林省乃至全国范围内PR的净化以及根除工作的开展提供参考依据。更多还原

【Abstract】 Pseudorabies (PR) , also known as Aujeszky’s disease, is an acute and high contagious disease of a variety of domestic and wild animals caused by Pseudorabies virus (PRV). The disease is characterized with fever, extreme itch (except for pig) and encephalomyelitis. The infection in swine is most common. The sow usually display the reproductive disorders such as miscarriage, fetal death and mummy foetus. The mortality rates of newly born piglets are up to 100%. In addition, the infection in growth pigs usually leads to the decrease of semen quality, even the loss of species value. Therefore, the occurrence of the disease will result in enormous economics loss of swine industry. At present, the disease has been listed as class B animal loimia by the world health organization (OIE), and listed as the secondary kind animal loimia in our country.In recent years, the occurrence of the disease showed a rising trend. At present, the disease had became an important loimia which composes a threat to swine industry. Currently, the extreme majority of farms carried on immune prevention by the use of PRV gE gene deletion vaccine, so the use of PRV gE-ELISA antibody detection kit could quickly and accurately detect the antibody of PRV wild isolate, thus could effectively monitor the infection of PRV wild isolate. In order to understand the infection status of PRV wild isolate in the farms of different areaes in Jilin province in recent years, the PRV serum antibodies of 3849 serum samples from different batches collected from 152 different scale immunized farms of Jilin province were detected by PRV gE-ELISA antibody detection kit. The results would provide important theoretical basis for the formulation of scientific prevention and control measures. The serological survey result showed that the antibody positive rates in different areaes of Jilin province ranged from 9.47% to 40.00%, and the average positive rates was 22.66%. There were 103 PRV antibody positive farms in 153 detected farms, and the positive rates of farms ranged from 37.50% to 87.50%. The result showed that the infection of PRV wild isolate remained more serious. In addition, we carried on investigation and analysis for the PRV wild infection status in different ages, sexualities and breeds. The serosurvey result would provide important theoretical basis for the prevention and control work in Jilin province.In November 2008, the PRV natural infection occurred in a farm in Siping area of Jilin province. The pregnant sows showed the symptoms of abortion and dead foetus, and newly born piglets more than 50 showed vomit, diarrhoea and the neurological symptoms such as muscle trembling, hind limb parlysis and four limbs swimming sliding. The treatment had no effects by a variety of antibiotic. The sick pigs began death one after another after 2 to 3 days. The mortality rate was up to 100%. Necropsies were performed on died piglets. The main pathological changes of the samples showed meningeal congestion , pulmonary congestion and edema, liver congestion and canous necrosis focuses observed in the surface, however, no obvious pathological changes were observed in the other organs. According to the clinical symptoms and pathological changes, the disease was preliminarily diagnosed as Pseudorabies. Because the disease was easily confused with porcine reproductive and respiratory syndrome, porcine parvovirus and porcine brucellosis, the disease was aggregate analyzed from the pathology and etiology aspects. First, the histological section were performed on the inner organs of heart, liver, spleen, lungs, kidney, and brain tissue. The dead reason of piglets was analyzed by histological examination. Microscopic lesions in the brain tissue showed the non-suppurative encephalitis changes including perivascular cuffing and neuronophagia. So we concluded that the pathological changes were caused by viral infection. And the bacteriology detection result also exclused the possibility of bacterial infection. In addition, part of liver occurred necrosis and formed the focal cellular necrosis focus. The pathological change was an important directive change of PRV infection. In order to final diagnosis for the disease, the systemic isolation and identification of pathogen was performed on BHK-21 cells, and one PRV isolate was successfully obtained which be named PRV JL/08/SP isolate.In order to further understand the proliferation ability and dynamic infextation process of PRV JL/08/SP isolate on BHK-21 cells, the morphology development process of PRV JL/08/SP isolate and the ultrastructural changes of infected cells were observed by the manufacture of ultrathin sections. After adsorption to the cell membrane surface, PRV entered in the endochylema via membrane fusion way. The viral replication were carried out in the cytoplasm. The assembled virus left nucleus into cytoplasm by budding way. Then, the virions in the cytoplasm synthetized the second peplos using the membrane structure of Golgi’s body and further formed complete virions. Eventually, Golgi vesicles packaging with intact virions occurred fusion with cellular membrane and the virons were released to the ecto-cell. The proliferation process and the release form were basic concordance with the report in previous literature. This indicated that the morphology development process of different PRV isolate were very similar, but exist certain differences only in the gain way of peplos. In addition, the virulence of PRV JL/08/SP isolate was determined using BHK-21 cell. The result showed that the titre of PRV JL/08/SP isolate on BHK-21 cells was 107.2/0.1mL. This indicated the PRV isolate had more stronger virulence. In order to further identify its pathogenicity, animal regression experiments were performed on rabbits and piglets, and duplicated the similar clinical symptoms with the natural infection. Above on, PRV JL/08/SP isolate was systemic identified in the study, and its pathogenicity was preliminary analyzed. Thus, the disease was final diagnosed as PRV wild virus infection.Because PRV wild virus infection especially inapparent infection was still very common in pig farms, the thorough eradication of PR confronted with a hard problem. Considering the reason, it was necessary to eatablish a quick, specific and sensitive diagnostic method for the detection of PRV wild isolate. In the process of controlling PR, PRV gene deleted vaccine had played a very important role, and the use of PRV gE gene deleted vaccine was most wide. Thus, the foundation of diagnostic method which could effectively distinguish from wild virus infection and vaccine strains would play extremely important role for the elimination of positive pigs and the cleaning of farms. At present, all PRV gE gene deleted vaccine strains deleted partial even complete gE gene, and the gE gene could stable expression in the wild virus isolate and its genetic variability was small, so gE gene could as a marker gene of differentiation for PRV wild virus and vaccine strains which used for PRV molecular level diagnosis. On the basis of obtaining PRV JL/08/SP, we designed specific primers according to the gE gene conservative sequences which published in Genbank database. Then, gE gene of PRV JL/08/SP isolate was amplified and cloned by PCR, and the gE gene was performed on sequencing analysis. The analytic result indicated that gE gene had individual nucleotide mutations compared with other isolates, but gE gene of PRV JL/08/SP isolate was still highly conserved from the the results of homology and genetic evolution analysis. So, the gE gene of PRV JL/08/SP isolate could be used as the diagnostic antigens for the identification of PRV wild isolate and vaccine strains. In addition, a rapid PCR detection method was established which used for the detection of clinical samples, and the method had strong specificity, high sensitivity and good reproducibility. A total of 68 samples including spleen, lung, liver and brain tissue collected from suspected PRV pigs which comed from the farms of different areaes in Jilin province were detected by PCR method. The positive rate of the samples was 27.94%, and it was consistent with the seroepidemiological survey result. This indicated that PRV infection was still very common in Jilin province. In the study, the PCR method based on gE gene of PRV JL/08/SP isolate not only could rapidly and efficiently detect the clinical samples, but also efficiently distinguish PRV wild virus and vaccine strains, which largely improved the detection rate and accuracy of PRV. In addition, the method had very important meaning for the diagnosis of earlier period PRV infection and latent infection persistent infection, so could provide a powerful technical support for the cleansing and eradication of PR.In addition, in view of the current severe situation of PR occurrence, it was necessary to performed on cleaning and eradication in different pig farms. Therefore, we applied PRV gE gene deleted vaccine combined with PRV gE-ELISA antibody detection kit to perform on immunization, monitoring and elimination in the six different scale pig farms in Jilin province from 2009 to 2010 years, thus achieved cleaning and eradication, and obtained the expectant cleaning effect. This would provide reference basis for PR cleaning and eradication in Jilin province even whole country.更多还原