小鹅瘟病毒PCR检测方法的建立和山东济宁地区小鹅瘟流行病学调查

发布时间：2018-10-21 11:52

【摘要】：小鹅瘟是由鹅细小病毒引起的一种急性传染病,主要侵害30日龄以内的雏鹅和雏番鸭,以发病急,死亡率高,传播快为特点。发病率和死亡率随着日龄的增加而下降,主要侵害7日龄以内的雏鹅和雏番鸭,根据发病情况分为3种类型,最急性型,急性型和亚急性型(殷震and刘景华,1997)。发病鹅主要以肠粘膜脱落,坏死,肠栓,脱水等为症状。1966年,匈牙利学者Derszy用鹅胚也分离到病毒,他是国外第一个用鹅胚分离到病毒的人,并于1967年对本病进行了详尽的描述,但当时他却把该病误认为流感,直到1971年Schettler确定这种病是由细小病毒引起。1978年建议把这种病称为鹅细小病毒感染,1974年世界禽类学会为纪念Derzsy的先期工作而将此病命名为Derzsy氏病(Derzsy D,1975)。1995年Zadori等首次报道了GPV全基因序列,并将其与番鸭细小病毒基因序列进行了同源性比较(Zadori Z,1995)。本病大流行具有一定周期性。在大流行以后当年余下的鹅群都获得了主动免疫,使次年的雏鹅具有天然被动免疫力,故本病不会在同一地区连续两年发生大流行。由此可见,本病的发病率和死亡率一方面和日龄有关,另一方面取决于亲代母鹅的特异性抗体水平。免疫预防是控制小鹅瘟的重要措施。目前已建立了PCR、ELISA、间接免疫荧光技术、微量中和试验等检测小鹅瘟的方法,但是这几种检测方法均需特殊的仪器设备,且耗时长,不适于在基层推广应用。因此,建立一种更加快速、灵敏的病毒检测方法迫在眉睫。本研究建立的聚合酶链式反应,具有操作简单,快速,适合基层推广的特点。近年来小鹅瘟在我国的一些省、市、自治区均有流行,给养漖业造成严重的危害,同时也给饲主带来巨大的经济损失,因此受到国内外学者和工作者的广泛关注。本研究通过工作之便,对所在地区鹅养殖现状和小鹅瘟发病情况做了相应的调查,并对其流行情况做了汇总,以期对养鹅业小鹅瘟疾病的发生做出相关的预防措施。本研究根据GENBANK上发表的小鹅瘟的vp3的序列信息,设计了一对特异性引物,通过优化各种条件,最终扩增出与预期片段大小一致的314bp的核酸片段,在通过相应的特异性实验发现此引物仅能扩增出小鹅瘟的特异片段,能够用来检测小鹅瘟的发生。本研究中对于分离到的10株病毒进行测序分析,结果显示分离株之间,同源性最高达100%,最低也在95.2%以上,说明小鹅瘟病毒的扩增片段核苷酸变异很小,VP3是小鹅瘟的主要抗原,所以本研究对于免疫预防具有指导性的意义。依据本研究中调查的小鹅瘟的发病日龄等基本信息,尤其每个鹅场的免疫预防程序进行了调查,发现小鹅瘟的发病日龄集中在12-2月份之间,根据小鹅瘟的发病情况,对其免疫程序作出了相应的调整,结果显示小鹅瘟的发病概率降低了,说明合适的免疫程序是预防该病的比较有效的措施。
[Abstract]:Gosling plague is an acute infectious disease caused by goose parvovirus, which mainly infects goslings and young muscovy ducks within 30 days of age. It is characterized by rapid onset, high mortality and rapid transmission. The morbidity and mortality decreased with the increase of the age of the day, mainly affecting the goslings and young muscovy ducks within 7 days of age, which were divided into three types according to the incidence, the most acute type, the acute type and the subacute type (Yin Zhen and Liu Jinghua, 1997). In 1966, Hungarian scholar Derszy also isolated the virus from goose embryos. He was the first person in foreign countries to isolate the virus from goose embryos, and described the disease in detail in 1967. But at the time he mistook the disease for the flu, It was not until 1971 that Schettler determined that the disease was caused by parvovirus. In 1978, it was suggested that the disease should be called goose parvovirus infection. In 1974, the World Avian Society named the disease Derzsy's disease (Derzsy Dl1975) to commemorate the advance work of Derzsy. Zadori et al. The entire GPV gene sequence, The gene sequence of muscovy duck parvovirus was compared with that of muscovy duck parvovirus (Zadori ZJ 1995). The epidemic has a certain periodicity. After the pandemic, the remaining geese got active immunity, which made the next goslings have natural passive immunity, so the disease would not occur in the same area for two consecutive years. Therefore, the morbidity and mortality of the disease are related to the age of day, on the other hand, it depends on the specific antibody level of the parent goose. Immune prevention is an important measure to control goose plague. At present, PCR,ELISA, indirect immunofluorescence technique and micro neutralization test have been established to detect Gosling plague. However, these methods all need special instruments and equipment, and take a long time, so they are not suitable for popularization and application in basic level. Therefore, it is urgent to establish a more rapid and sensitive method for virus detection. The polymerase chain reaction established in this study has the characteristics of simple operation, fast operation and suitable for basic level popularization. In recent years, Gosling plague has been prevalent in some provinces, cities and autonomous regions of China, which has caused serious harm to the industry of feeding and feeding, and at the same time has brought huge economic losses to the breeders, so it has been widely concerned by scholars and workers at home and abroad. In this study, the current situation of goose breeding and the incidence of Gosling plague in the region were investigated, and the epidemic situation was summarized, in order to make relevant preventive measures to the occurrence of goose plague in the goose industry. In this study, a pair of specific primers were designed based on the sequence information of vp3 of Gosling plague published on GENBANK. By optimizing various conditions, the nucleic acid fragments of 314bp with the expected size were amplified. It was found that the primer could only amplify the specific fragment of Gosling plague and be used to detect the occurrence of Gosling plague. In this study, 10 isolates were sequenced. The results showed that the highest homology was 100% and the lowest was over 95.2% among the isolates, indicating that the nucleotide variation of the amplified fragments of Gosling plague virus was very small, and VP3 was the main antigen of Gosling plague. Therefore, this study has instructive significance for immune prevention. According to the basic information of Gosling plague in this study, especially the immune prevention program in every goose farm, it was found that the incidence age of Gosling plague was between December and February, and according to the incidence of Gosling plague, The results showed that the incidence probability of Gosling plague had been reduced, which indicated that the proper immunization procedure was an effective measure to prevent the disease.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.33

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