猪流行性腹泻病毒YX株的分离鉴定及其灭活疫苗免疫效力试验研究

发布时间：2018-04-19 23:25

本文选题：猪流行性腹泻 + Vero细胞　；参考：《华南农业大学》2016年硕士论文

【摘要】：猪流行性腹泻(PED)是由冠状病毒属的猪流行性腹泻病毒(PEDV)引起的一种以仔猪水样腹泻、呕吐、脱水为主要特征的接触性肠道传染病。猪流行性腹泻是世界上流行最广泛的猪病之一。近年,猪流行性腹泻作为烈性传染病,开始疯狂肆虐我国广大养殖户。我国的广东、广西、江西、湖南、福建、浙江、安徽、江苏、湖北、河北和山东等省(自治区)均有猪流行性腹泻的发现。PED在我国的流行面逐渐扩大,对26个省市自治区的调查表明PED的总死亡率占36种疾病总死亡率的1.74%。该病多发于冬春季节,但就广东地区这个全年高湿度的环境,PED呈高发趋势,全年均可发生,7天龄内的新生仔猪在发生腹泻后3?4天,通常因脱水导致衰竭而死亡,病死率高达100%。目前,在广东粤西地区,猪流行性腹泻呈高发趋势,一旦进入冬春季节,该病都是大规模爆发,病死率高。虽然可以采取疫苗接种方式来控制此病流行,但经典的CV777株疫苗免疫后效果并不理想,接种过疫苗的猪场仍然有此病的流行。本研究主要分为下面三个部分:1、PEDV-YX株的分离鉴定。本研究从广东粤西9个疑似发生腹泻疫情的规模化猪场采集180份病料样本。通过在Vero细胞培养基中添加胰酶的方法孵育、选取1%接毒量接种Vero单层细胞,进行病毒分离纯化,从中分离出9株未知病毒HS1、HS2、HS3、HS4、YC1、YC2、YC3、YC4和KP,经胶体金快速诊断技术、RT-PCR扩增、基因序列比对,确诊均为猪流行性腹泻病毒(PEDV)。通过猪体回归试验,选取临床症状最明显,毒力最强的YC1毒株作为研究对象,命名为PEDV-YX株。在胰酶总浓度为7.5ug/ml的作用下,盲传到第8代,能观察到细胞圆缩、聚集,空泡增多等明显的细胞病变。2、对PEDV-YX株生物学特性、中和试验及其TCID50测定研究。对PEDV-YX株生物学特性、中和试验及其TCID50研究表明,该分离株对5-Brd U不敏感,为具有囊膜的RNA病毒;对乙醚、氯仿敏感;在pH为3.0的环境下易失活;对热敏感,在50℃条件下作用1h,病毒易失活;设计特异性引物,进行RT-PCR扩增,电泳检测得到与预期长度一致的条带;与PEDV抗血清中和效价为120.23;PEDV-YX F10的TCID50为10-5.67/0.1ml。3、PEDV-YX株氢氧化铝胶灭活疫苗制备及不同免疫剂量、不同免疫途径对抗体水平的影响研究。PEDV-YX株F10经0.3%的甲醛溶液灭活,以病毒液样40ml+PBS40ml+20ml铝胶佐剂的培苗方案,配制猪流行性腹泻灭活疫苗。以口服免疫、后海穴注射免疫为免疫途径,2ml、1ml、0.5ml不同免疫剂量,研究该灭活疫苗的抗体消长规律。当免疫剂量相同时,后海穴注射免疫比口服免疫抗体效价更高,且抗体维持时间更长;当免疫途径相同时,免疫2ml的抗体整体水平比免疫1ml的更高,免疫0.5ml的中和抗体水平最低。通过分离广东粤西地区PEDV的流行性毒株,为了解该地区流行性毒株的特性及流行情况,建立PEDV快速准确的诊断方法及新型疫苗的研究提供理论依据,从而为制定有效的防控措施,促进该地区养猪业的稳定发展做好铺垫。
[Abstract]:Porcine epidemic diarrhea (PED) is a kind of contact intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV) of the coronavirus, which is characterized by diarrhea, vomiting and dehydration of piglets. Porcine epidemic diarrhea is one of the most popular swine diseases in the world. In recent years, swine epidemic diarrhea, as a strong infectious disease, began to ravage me wildly. Guangdong, Guangxi, Jiangxi, Hunan, Hunan, Hunan, Fujian, Zhejiang, Anhui, Jiangsu, Hubei, Hebei and Shandong and other provinces (autonomous regions) have been found to have epidemic diarrhea in China. The prevalence of.PED in China is gradually expanded, and the total mortality of PED in 26 provinces and autonomous regions accounts for the incidence of the total mortality of 36 diseases. Yu Dongchun season, but in Guangdong area this year high humidity environment, PED is high trend, can occur throughout the year, 7 days of age of newborn piglets in 3 to 4 days after diarrhea, usually due to dehydration and death, the mortality rate is up to 100%., in the west of Guangdong Province, swine epidemic diarrhea is high trend, once into the winter and spring season, The disease is a large outbreak, with high mortality. Although the vaccine can be taken to control the epidemic, the classic CV777 vaccine is not ideal after immunization. The vaccinated piggery still has the epidemic. This study is divided into three parts: 1, isolation and identification of PEDV-YX strain. This study from Guangdong, Guangdong, 9 180 samples of disease materials were collected in a large-scale pig farm suspected of diarrhea. By incubating the trypsin in the Vero cell culture medium, 1% single cells were inoculated and purified. 9 unknown viruses, HS1, HS2, HS3, HS4, YC1, YC2, YC3, YC4 and KP were isolated from the Vero monolayer. Increase, gene sequence alignment, confirmed as porcine epidemic diarrhea virus (PEDV). Through the pig body regression test, select the most obvious clinical symptoms, the most virulent YC1 strain as the research object, named PEDV-YX strain. Under the effect of total trypsin concentration of 7.5ug/ml, blind to the eighth generation, can observe the cell roundness, aggregation, vacuoles and so on obvious Cytopathic.2, biological characteristics, neutralization test and TCID50 determination of PEDV-YX strain. Biological characteristics of PEDV-YX strain, neutralization test and TCID50 study showed that the isolate was insensitive to 5-Brd U, was a RNA virus with capsule, sensitive to ether, chloroform, and easily inactivated in a pH 3 environment; it was sensitive to heat and acted at 50 degrees centigrade. 1H, the virus was easily inactivated; specific primers were designed for RT-PCR amplification. Electrophoresis was used to detect the expected length of the strip; neutralization titer with PEDV antiserum was 120.23; TCID50 of PEDV-YX F10 was 10-5.67/0.1ml.3, aluminum hydroxide gel inactivated vaccine of PEDV-YX strain and different immunization doses, and the effect of different immune pathways on antibody level .PEDV-YX strain F10 was inactivated by 0.3% Formaldehyde Solution, and the pig epidemic diarrhea inactivated vaccine was prepared by the solution of virus like 40ml+PBS40ml+20ml aluminum gum adjuvant. Immunization was taken orally, the immunization of Houhai acupoint immunization, 2ml, 1ml, 0.5ml in different immune doses to study the law of antibody growth of the inactivated vaccine. When the immune dose was the same, Houhai The titer of acupoint injection is higher than that of oral immunization antibody, and the time of antibody maintenance is longer. When the immune pathway is simultaneously, the overall level of the antibody to the immune 2ml is higher than that of the immune 1ml, and the neutralizing antibody level of the immune 0.5ml is the lowest. By separating the epidemic strains of PEDV in the western Guangdong region, the characteristics and epidemic of the epidemic strains in this area are understood. It provides a theoretical basis for the establishment of a rapid and accurate diagnostic method of PEDV and a new vaccine, so as to lay the groundwork for the formulation of effective prevention and control measures and the promotion of the stable development of the pig industry in this area.

【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.28

【参考文献】