绵羊伪狂犬病的病毒分离鉴定及预防控制研究

发布时间：2018-03-07 07:51

  本文选题：绵羊伪狂犬病　切入点：PCR扩增　出处：《甘肃农业大学》2017年博士论文　论文类型：学位论文

【摘要】：本研究以临床发现的1例疑似绵羊伪狂犬病病例为研究对象,进行了病毒分离鉴定和病理组织学观察。将病料接种BHK-21细胞,细胞出现病变,间接免疫荧光染色和荧光定量PCR检测结果证实,所分离的病毒为伪狂犬病病毒,并将其命名为SH1311毒株。在电镜下观察病毒,可见到病毒颗粒呈球形,囊膜表面有大量纤突。病理组织学观察显示,发病羊大脑组织神经元发生广泛性变性、坏死并伴有噬神经现象和胶质细胞增生。对分离株g D基因进行PCR扩增、测序和序列分析,并与国内外伪狂犬病病毒株的同源性进行比较发现,该分离株与上海地区2012年以来分离的猪PRV毒株g D基因的同源性最高,为99.8%~100%,而与2010年上海地区流行毒株相比,同源性只有80.5%。构建的g D基因进化树表明,SH1311分离株与上海地区2012~2015年间分离的7株猪伪狂犬病毒以及国内JS-2012、HNB、HN1201、HLJ8、BJRD、ZJ01毒株属于同一个进化分枝,亲缘关系最近,与欧美以及韩国的流行毒株处于不同的分枝上。为了评估绵羊免疫Bartha-K61疫苗后对SH1311分离株攻毒的免疫保护效力,取6月龄湖羊14只,其中6只接种Bartha-K61疫苗,5只为非免疫组,3只为空白对照组。2周后用SH1311分离株对免疫组和非免疫组湖羊攻毒,对攻毒后试验动物的临床症状、直肠温度、鼻腔排毒及感染抗体变化情况进行观察和检测。非免疫组湖羊攻毒后,其中4只出现典型的瘙痒症状,发病率为80%,病死率为100%。用荧光PCR检测攻毒羊鼻拭子,PRV核酸均为阴性,而攻毒后死亡羊只的内脏及脑组织检测结果均为阳性。免疫组羊只攻毒后均未出现明显的临床症状,鼻拭子检测PRV均为阴性。研究结果表明用致死剂量的SH1311分离株对疫苗免疫组和非免疫组进行攻毒,疫苗免疫组对PRV的攻毒具有良好的保护效果。为了进一步探讨伪狂犬病病毒对绵羊的致病机制,将7只健康湖羊随机分成试验组和对照组,其中试验组5只,对照组2只。用SH1311分离株对5只湖羊进行人工攻毒试验,取发病羊内脏组织用于病理组织学检查和免疫组化分析。湖羊在攻毒后3 d开始出现症状,第4 d症状进一步加剧,表现为局部奇痒、体温升高、精神沉郁、食欲废绝等症状,第5 d出现死亡,最终5只湖羊4只发病死亡,1只存活。剖检主要表现为脑膜充血,肺淤血,轻度实变。病理组织学检查显示:大脑、延髓神经元变性、坏死;肺脏呈间质性肺炎病变;脾脏、肾脏出血。免疫组化染色显示,PRV在肝脏、肺脏、大脑、脾脏等器官组织广泛存在,病毒在肝脏中主要存在于库弗氏细胞中,在肺脏中主要存在于Ⅱ型肺泡细胞中,在脾脏中主要存在于巨噬细胞中,而在脑组织中,神经元呈现较强的信号,说明病毒主要存在于神经细胞中。结果证实:PRV对神经组织的亲嗜性以及在神经细胞中复制和传播导致神经元变性、坏死,同时PRV也能损伤肺脏和肾脏组织、导致间质性肺炎病变和肾脏出血。
[Abstract]:In this study, a case of suspected sheep pseudorabies was studied. Virus isolation and histopathological observation were carried out. The disease material was inoculated with BHK-21 cells, and the cells showed pathological changes. The results of indirect immunofluorescence staining and fluorescence quantitative PCR showed that the virus was pseudorabies virus, and it was named as SH1311 strain. There were a large number of fibrils on the surface of the capsule. Histopathological observation showed that the neurons in the brain tissue of the infected sheep showed extensive degeneration, necrosis and glial cell proliferation. The G D gene of the isolated strain was amplified by PCR. Sequencing and sequence analysis showed that this isolate had the highest homology with porcine PRV strain g D gene isolated from Shanghai since 2012, compared with pseudorabies virus strains at home and abroad. There was only 80.5 homology compared with the prevalent strain in Shanghai in 2010. The constructed GD-gene phylogenetic tree showed that the isolated strain SH1311 belonged to the same evolutionary branch as the 7 strains of pseudorabies virus isolated from Shanghai during the period of 20122015 and the domestic strain JS-2012HNBBHN1HN1HN1HN1HN1201HLJRDDZJ01, which belonged to the same evolutionary branch. In order to evaluate the protective effect of sheep Bartha-K61 vaccine against SH1311 isolates, 14 6 months old Hu sheep were selected. Six of them were inoculated with Bartha-K61 vaccine, 5 were non-immunized group, 3 were blank control group. 2 weeks later, Hu sheep were attacked with SH1311 isolate, and the clinical symptoms and rectal temperature of experimental animals after inoculation were analyzed. The changes of nasal cavity detoxification and infection antibody were observed and detected. Four of the non-immunized Hu sheep showed typical pruritus symptoms, the incidence rate was 800.The mortality rate was 1000.The detection of PRV nucleic acid in sheep nasal swabs with fluorescent PCR was negative. The visceral and brain tissues of the dead sheep were positive after attack, and no obvious clinical symptoms were found in the immunized sheep. Nasal swabs were negative for PRV. The results showed that lethal doses of SH1311 isolates were used to attack vaccine and non-immune groups. In order to further study the pathogenic mechanism of pseudorabies virus on sheep, seven healthy Hu sheep were randomly divided into experimental group and control group. The visceral tissues of the infected sheep were used for histopathological examination and immunohistochemical analysis. The symptoms of Hu sheep began to appear 3 days after the attack, and the symptoms became worse on the 4th day. The symptoms were local itching, elevated body temperature, depression of spirit, loss of appetite, death on the 5th day, and death of 1 in 4 Hu sheep. The main manifestations were meningeal hyperemia and pulmonary congestion. Mild consolidation. Histopathological examination showed that the brain, medulla oblongata neurons degeneration, necrosis, pulmonary interstitial pneumonia lesion, spleen, kidney hemorrhage. Immunohistochemical staining showed that PRV in the liver, lung, brain, The spleen and other organ tissues are widespread, the virus mainly exists in the liver, mainly in the type 鈪，

本文编号：1578540