【作者】 王春霞；

【导师】 高丰；

【作者基本信息】 吉林大学 ， 兽医（专业学位）， 2015， 硕士

【摘要】 小反刍兽疫（Peste des petits ruminants，PPR）是由小反刍兽疫病毒（Peste despetits ruminants virus，PPRV）引起绵羊、山羊等小反刍动物的一种急性、烈性传染病，是严重威胁和危害养羊业的一种重要的病毒性疾病，该病目前已被我国农业部列为一类动物疫病。该病传染性强，临床上感染动物主要以出现高热、口炎、肺炎、肠炎为特征，具有较高的发病率和死亡率。PPR以往主要在撒哈拉以南的非洲地区、印度和中东国家流行，呈地方流行性，然而近年来该病呈现蔓延的趋势，在周边多个国家均有该病的发生和流行。自2007年我国西藏阿里地区首次发生小反刍兽疫疫情以来，我国新疆、甘肃、内蒙古等多个省份均有该疫情的发生。2014年4月，辽宁省锦州市北镇某养羊户饲养的57只山羊中，先后有18只山羊出现体温升高（升高至41℃左右）、咳嗽、流鼻涕，且眼、鼻周围可见脓性分泌物以及腹泻等临床症状，一些严重病例很快发生死亡。根据该病的流行特点及临床症状，初步诊断为羊小反刍兽疫。为了进一步证实，本研究利用大体病理剖检、病理组织学观察、细菌学检查、电镜负染观察、RT-PCR等方法从病理学和病原学角度对该病的发生原因进行系统深入的研究。具体研究内容如下：1PPR的病理学诊断对送检病羊进行大体病理剖检，肺脏可见有充血、水肿，肺尖叶呈肉样变；胃壁黏膜发生脱落。随后，采集病羊心、肝、脾、肺、肾、肠等多个组织脏器，制作石蜡切片并进行H.E.染色，病理组织学观察可见，肺脏肺泡隔增宽，呈现典型的间质性肺炎变化；肺泡壁毛细血管充血、淤血，肺泡壁上皮细胞增生；支气管黏膜上皮细胞增生，部分细胞坏死、甚至脱落到管腔中；肝脏发生轻度脂肪变性；肾小管周围毛细血管充血淤血较为严重。2PPR的病原学诊断将肺脏、脾脏等病料进行研磨后提取RNA，利用PPRV特异性引物进行RT-PCR扩增，扩增获得与预期目的基因片段大小一致的条带。将经感作处理后的病料研磨上清液接种单层Vero细胞进行病原分离，在接种病料72h后出现明显的CPE。收集细胞病变液，反复冻融3次后进行电镜负染观察，可见椭圆形，有囊膜，直径约为300nm的病毒粒子，与PPRV形态特征一致；此外，细菌学检测排除了细菌混合感染的可能性。综合分析以上结果，从病原学角度证实该疫情发生的原因为PPRV感染所致。之后，在分离获得PPRV毒株的基础上，对该分离株的M和F基因进行扩增、克隆及测序，并通过绘制遗传进化树对其M和F基因的分子生物学特性进行初步研究，遗传进化分析结果显示，该毒株与在我国发生流行的毒株亲缘性较高，属于Ⅳ系。该研究不仅为针对PPRV疫苗的研制提供重要的物质基础，而且将为制定针对PPR切实有效的防控措施提供一定的理论依据。更多还原

【Abstract】 Peste des petits ruminants (PPR) is an acute and highly infectious diseases ofsmall ruminants including sheep and goats, which is caused by Peste des petitsruminants virus (PPRV). The disease is one of the most important viral diseases,which can threat sheep industry seriously. At present, PPR has been listed as A diseasein China. The clinical symptoms of the infectious disease includes fever, stomatitis,diarrhea, pneumonia and enteritis. The morbidity and mortality are high. Thesub-Saharan Africa, India and the Middle East countries are the major epidemic areasof PPR in the past. However, PPR spreads to surrounding countries gradually inrecent years. Since the first occurrence in Ali area of Tibet in2007, the disease isprevalent in many provinces such as Xinjiang, Gansu and Inner Mongolia.The outbreak of the PPR among goats cccurred in a farm with57goats in theLiaoning province of China during April2014. Among them,18goats appearedfever (the temperature up to41degrees), cough, runny nose, purulent secretion aroundthe nasal and eyes, diarrhea and other symptoms. The deaths occurred in several cases.According to the epidemiology features and the clinical symptoms, the disease wasdiagnosed with PPR. The gross pathological examination, histopathologicexamination, bacteriological analysis, negative staining electronmicroscope observation and RT-PCR were applied in order to further confirm. Themain studies are as follows:1The pathological diagnosis of PPRAt first, the gross pathological examinations were performed in sick goats. Thecongestion and edema were observed in the lungs. The gastric mucosa appearedshedding. The heart, liver, spleen, lung, kidney, intestine and other tissues werecollected, which were used for preparation of pathological sections. The interalveolarseptum widen by histopathologic examination. The lungs showed typical interstitialpneumonia. The congestion of the capillary in the alveolar wall were observed. Theproliferation was observed in the alveolar epithelial cells and the bronchial epithelial cells. A part of bronchial epithelial cells occurred necrosis, even falling off into thelumen. The mild fatty degeneration was observed in the liver. In addtion, thecongestion was observed in capillary among kidney tubules.2The etiology diagnosis of PPRThe total RNA was extracted from the lung and spleen of the sick goat. TheRT-PCR was performed using PPRV specitific primers. The target gene fragmentswere amplified. The virus isolation was performed by inoculating the supernatant ofthe tissues into Vero cells. The typical CPE were observed at72h after inoculation.Then the cells having cytopathic effect were collected by freeze-thawing three times.The typical PPR virus particles having envelope were observed by negative stainingelectron microscope observation. The length of the virus particles was about300nm.In addition, the mixed infection of bacteria was excluded by bacteriological detection.According to the above results, the outbreak of the disease was confirmed due toPPRV infection. The M gene and F gene of the PPRV isolate were amplified andsequenced. The phylogenetic trees based on the M gene and F gene were reapectivelyconstructed. The results showed that the isolate belongs to lineage Ⅳ and hadhighhomology with theprevalence strain in China.The study not only provides an important material foundation for thedevelopment of PPRV vaccines, but also provides a theoretical basis for thedevelopment of effective prevention and control measures of PPR.更多还原