【作者】 张坤；

【导师】 刁有祥；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2012， 硕士

【摘要】 鸭瘟(DuckPlague，DP)，是由鸭瘟病毒(Duckplaguevirus，DPV)引起的鸭、鹅和天鹅等水禽的一种急性接触性传染病，其特征是流行广泛、传播迅速、发病率和死亡率高，严重威胁养鸭业的发展。鸭瘟于1923年由Baudet首次在荷兰报道，之后在许多国家均有流行。1957年黄引贤在我国广州报道了本病。至今，全国各养鸭地区均有本病流行的报道，为了更有效的防治DPV，有必要发展简单、快速的检测方法和良好的病原检测及定位手段，并对其致病机理进行深入研究。本研究内容包括四部分：一、鸭瘟病毒SDWF株的分离鉴定通过鸭胚尿囊膜接种，从疑似鸭瘟病例中分离到1株病毒，采用血清中和试验、动物回归试验和PCR方法对分离毒株进行了鉴定，并对毒株的生物学特性进行了研究。致病性试验结果表明，分离毒株人工感染SPF鸭的症状及剖检变化与自然感染病例相同；分离株无血凝活性；鸭胚半数致死量ELD50为10-4.33/0.2mL；对氯仿、乙醚敏感；不耐酸碱、不耐热。分离毒株PCR鉴定结果显示，PCR产物与预期大小一致，其产物序列与鸭瘟病毒标准强毒株相应片段核苷酸的同源性为99.9%。上述结果表明，分离毒株为DPV，且为强毒株，命名为SDWF株。二、鸭瘟病毒环介导等温扩增（LAMP）快速检测方法的建立与应用为建立一种简便、快速、灵敏、特异的DPV检测方法，本研究根据GenBank中登录的DPV基因组序列，设计3对特异性的环介导等温扩增(LAMP)引物，经优化反应体系，建立了LAMP快速检测方法。结果表明，LAMP方法能够在63℃恒温下，1h内实现目的核酸的大量扩增。结果判定时只需要在扩增产物中加入SYBRGreenⅠ染料，就可以直接在可见光或紫外光下观察颜色变化。该方法敏感性可达0.245pg/μL，比普通PCR灵敏性高10倍；对Ι型鸭肝炎病毒、H9N2亚型禽流感病毒、鸭新城疫病毒、鸭源偏肺病毒等的核酸无交叉反应；利用建立的LAMP检测方法对40份临床样品的阳性检出率为30%。该方法简便快捷，省时省力，是一种适用于基层实验室快速、准确检测DPV的方法。三、鸭瘟病毒强毒株感染SPF鸭动态病理组织学研究将纯化的DPV人工感染2月龄SPF鸭，定期剖杀试验组和对照组鸭，对各组织器官的病理组织学变化进行观察，并进行血常规和血液生化指标检测。结果显示：人工感染后24h，感染SPF鸭中枢免疫器官胸腺、法氏囊淋巴细胞数量减少，组织间隙增大；肝脏、脾脏组织病变较为严重，其余组织器官均出现程度较轻的病理变化。感染后48～96h，中枢免疫器官的淋巴细胞极度减少，网状细胞增生，组织器官结构模糊不清，严重充血、出血；其余组织器官出现细胞变性、出血等病理变化。感染后120h，组织细胞变性、坏死，出现大片坏死区。点眼滴鼻组SPF鸭感染DPV后组织学变化与皮下注射组相似，只是发生的时间推后约24～48h。对照组SPF鸭病理组织学观察未见损伤。WBC、HGB、AST、ALT等发生显著变化。该结果表明，接种DPV强毒感染SPF鸭的组织器官严重受损，特别是免疫器官，甚至会引起免疫抑制。四、间接免疫荧光染色（IFA）检测鸭瘟病毒及在鸭体内的抗原定位将纯化的DPV免疫清洁级新西兰白兔，高免血清经辛酸-硫酸铵法和葡聚糖G200柱纯化，制得第一抗体。以FITC标记的山羊抗兔IgG为第二抗体，通过反应条件的优化，建立了DPV间接免疫荧光（IFA）快速诊断方法。试验结果表明：一抗的最佳稀释度为1:100，感作时间为4℃过夜；二抗的最佳工作浓度为1:200，感作时间为37℃1h，DPV特异性黄绿色荧光最清晰。以IFA对DPV人工感染鸭的各组织器官进行检测，在死亡鸭的脾脏、胸腺、法氏囊、肝脏、食道、泄殖腔、肠道、肾脏、肺脏及气管中均检测到DPV抗原，以肝脏、脾脏等组织中荧光最强。表明这些器官是DPV感染后的主要靶器官。更多还原

【Abstract】 Duck plague(DP) is an acute septic infectious disease that caused by duck plague virus (DPV), and characterized by its high prevalence, morbidity and mortality. It is going to serious threat to the development of duck industry.Since the first report of DPV in the Netherlands by Baudet in1923, it was spread rapidly and widely in many countries. In1957, Yinxian Huang reported duck plague in Guangzhou, China. So far, it had been reported in all the country areas raising ducks. In order to efficiently prevent and control DPV, it’s essential to establish simple, rapid and sensitive methods for diagnosis and detection of DPV, and further study of its pathogenesis. Four parts are contained in this present research:Part1:Isolation and identification of duck plague virus SDWF strainA strain virus (SDWF) isolated from a suspected case of duck plague through duck embryo allantoic membrane was identified as duck plague virus (DPV) by serum neutralization test, animal experimental infection and PCR identification. The isolated virus has the same serum type as the standard virulent virus. SPF ducks infected artificially showed the same or similar clinical symptoms and pathological changes as the natural infections in the pathogenicity test. The virus does not agglutinate red blood cells of other animals and it is sensitive to chloroform and ether. It also can not tolerate acid,alkali and heat. Toxicity tests showed that ELD50of SDWF-strain is10-433/0.2mL. It was identified by PCR, and the amplification products were sequenced. It revealed that the amplification result is right, compared with the standard virulent strain, the gene of DPV-SDWF has the homology99.9%in nucleic acids level.Part2:Development and application of a loop-mediated isothermal amplification assay for the rapid detection of duck plague virusTo develop a rapid and sensitive method for detecting duck plague virus(DPV), a loop-mediated isothermal amplification(LAMP) assay was established using three pairs of specific primers. The assay was optimized to amplify DPV DNA by incubation at63℃for1 hour. The results visualized directly or under the UV light with added SYBR Green I dye. The diagnostic method was sensitive and specific, for the amplification results of duck hepatitis virus, H9N2avian influenza virus, duck paramyxovirus and duck avian metapneumovirus were negative, and the lowest detection limit of0.245pg/μL, which was10-fold higher than the conventional PCR. The positive rate of forty duck samples suspected duck plague was30%. Therefore the LAMP assay developed in this study provided a rapid and practical method for DPV detection.Part3:The dynamic study of histopathology of SPF duck infected with duck plague virus virulent strainTwo-month-old SPF ducks were inoculated with duck plague virus(DPV), slaughtered in different period and observed the histopathology changes of tissues and organs. At the same time the blood were collected to be carried out the routine blood test and the detection of blood parameters. Results revealed that24hours later, the number of lymphocyte of experimental ducks’central immune organs, including thymus and bursa, decreased and tissue space enlarged. While the histopathology of liver and spleen was serious, other organs was slight.48-96hours later, the number of lymphocyte decreased severely in central immune organs, hyperplasia of lymphocytic netted cell, blurry configuration, serious hyperemia and hemorrhage, some irreversible pathological changes, cell degeneration and hemorrhage, were observed.120hours later, cell degeneration and focal necrosis of tissue appeared. Histological changes of ducks infected by conjunctival and intraocular-nasal vaccination were similar to those subcutaneous injection appeared with a delay of24-48hours. Histopathology of control group was not observed. WBC,HGB,AST and ALT showed obvious changes. These results suggested tissues and organs, especially the immune organs, of SPF ducks inoculated DPV severely damaged, which even resulted in immunosuppression.Part4:Application of indirect immunofluorescence assay(IFA) for detection and antigen location of duck plague virus virulent strain in paraffin sectionsTo obtain the primary antibody, two clean New Zealand rabbits were vaccinated with oil-emulsion vaccine of DPV. The rabbit anti-DPV specific IgG was extracted by caprylic-ammonium sulfate law method, purified by Sephadex G200chromatography after dialyzing. An IFA test was developed to the rapid diagnosis of DPV by using the anti-DPV IgG as the primary antibody and the fluorescein isothiocyanate(FITC) conjugate goat anti-rabbit IgG as the second antibody. The research suggested that the dilution of primary antibody was1:100and sections were incubated overnight at4℃. Then sections incubated at37℃for1h with diluted FITC-labeled-secondary antibody (1:200). The IFA was applied to detect the DPV antigen in different organs of the artificially infected SPF ducks. The IFA was applied in detecting the virulent DPV antigen in different organs of the artificially infected adult ducks, and the viral antigen was detected in the spleen, thymus, bursa, liver, esophagus, duodenal, renal, lung and trachea of dead infected ducks. The fluorescence intensity of liver and spleen is the strongest, which suggested that the target organs that DPV mainly attacked are liver and spleen.更多还原