本文選題：綿羊偽狂犬病　切入點(diǎn)：PCR擴(kuò)增　出處：《甘肅農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：本研究以臨床發(fā)現(xiàn)的1例疑似綿羊偽狂犬病病例為研究對象,進(jìn)行了病毒分離鑒定和病理組織學(xué)觀察。將病料接種BHK-21細(xì)胞,細(xì)胞出現(xiàn)病變,間接免疫熒光染色和熒光定量PCR檢測結(jié)果證實(shí),所分離的病毒為偽狂犬病病毒,并將其命名為SH1311毒株。在電鏡下觀察病毒,可見到病毒顆粒呈球形,囊膜表面有大量纖突。病理組織學(xué)觀察顯示,發(fā)病羊大腦組織神經(jīng)元發(fā)生廣泛性變性、壞死并伴有噬神經(jīng)現(xiàn)象和膠質(zhì)細(xì)胞增生。對分離株g D基因進(jìn)行PCR擴(kuò)增、測序和序列分析,并與國內(nèi)外偽狂犬病病毒株的同源性進(jìn)行比較發(fā)現(xiàn),該分離株與上海地區(qū)2012年以來分離的豬PRV毒株g D基因的同源性最高,為99.8%~100%,而與2010年上海地區(qū)流行毒株相比,同源性只有80.5%。構(gòu)建的g D基因進(jìn)化樹表明,SH1311分離株與上海地區(qū)2012~2015年間分離的7株豬偽狂犬病毒以及國內(nèi)JS-2012、HNB、HN1201、HLJ8、BJRD、ZJ01毒株屬于同一個(gè)進(jìn)化分枝,親緣關(guān)系最近,與歐美以及韓國的流行毒株處于不同的分枝上。為了評估綿羊免疫Bartha-K61疫苗后對SH1311分離株攻毒的免疫保護(hù)效力,取6月齡湖羊14只,其中6只接種Bartha-K61疫苗,5只為非免疫組,3只為空白對照組。2周后用SH1311分離株對免疫組和非免疫組湖羊攻毒,對攻毒后試驗(yàn)動(dòng)物的臨床癥狀、直腸溫度、鼻腔排毒及感染抗體變化情況進(jìn)行觀察和檢測。非免疫組湖羊攻毒后,其中4只出現(xiàn)典型的瘙癢癥狀,發(fā)病率為80%,病死率為100%。用熒光PCR檢測攻毒羊鼻拭子,PRV核酸均為陰性,而攻毒后死亡羊只的內(nèi)臟及腦組織檢測結(jié)果均為陽性。免疫組羊只攻毒后均未出現(xiàn)明顯的臨床癥狀,鼻拭子檢測PRV均為陰性。研究結(jié)果表明用致死劑量的SH1311分離株對疫苗免疫組和非免疫組進(jìn)行攻毒,疫苗免疫組對PRV的攻毒具有良好的保護(hù)效果。為了進(jìn)一步探討偽狂犬病病毒對綿羊的致病機(jī)制,將7只健康湖羊隨機(jī)分成試驗(yàn)組和對照組,其中試驗(yàn)組5只,對照組2只。用SH1311分離株對5只湖羊進(jìn)行人工攻毒試驗(yàn),取發(fā)病羊內(nèi)臟組織用于病理組織學(xué)檢查和免疫組化分析。湖羊在攻毒后3 d開始出現(xiàn)癥狀,第4 d癥狀進(jìn)一步加劇,表現(xiàn)為局部奇癢、體溫升高、精神沉郁、食欲廢絕等癥狀,第5 d出現(xiàn)死亡,最終5只湖羊4只發(fā)病死亡,1只存活。剖檢主要表現(xiàn)為腦膜充血,肺淤血,輕度實(shí)變。病理組織學(xué)檢查顯示:大腦、延髓神經(jīng)元變性、壞死;肺臟呈間質(zhì)性肺炎病變;脾臟、腎臟出血。免疫組化染色顯示,PRV在肝臟、肺臟、大腦、脾臟等器官組織廣泛存在,病毒在肝臟中主要存在于庫弗氏細(xì)胞中,在肺臟中主要存在于Ⅱ型肺泡細(xì)胞中,在脾臟中主要存在于巨噬細(xì)胞中,而在腦組織中,神經(jīng)元呈現(xiàn)較強(qiáng)的信號,說明病毒主要存在于神經(jīng)細(xì)胞中。結(jié)果證實(shí):PRV對神經(jīng)組織的親嗜性以及在神經(jīng)細(xì)胞中復(fù)制和傳播導(dǎo)致神經(jīng)元變性、壞死,同時(shí)PRV也能損傷肺臟和腎臟組織、導(dǎo)致間質(zhì)性肺炎病變和腎臟出血。
[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，

本文編號：1578540