陕西省部分地区猪病毒性腹泻流行病学调查及病原学分析

发布时间：2018-04-26 08:08

本文选题：猪流行性腹泻 + 猪传染性性胃肠炎　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)和猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)是猪病毒性腹泻的主要病原,不同年龄段的猪(尤其是仔猪)均易感,猪感染后以腹泻、脱水、呕吐为主要临床特征,PEDV和TGEV已成为降低仔猪成活率的重要原因之一,对世界各国的养猪业造成了非常严重的经济损失。为了解陕西省PEDV和TGEV的流行病学及病原学特征,明确陕西省的流行毒株与疫苗株、经典毒株和国内外流行毒株的亲缘关系及变异情况,进而为陕西省综合防控PEDV和TGEV及研发更加合理有效的疫苗提供理论依据及参考数据,本研究拟对陕西省部分地区进行PEDV和TGEV的流行病学调查,并对这两种病毒的遗传变异情况进行分析。研究取得如下结果。1.利用PCR法检测了714份腹泻病料中的PEDV。结果显示,检测出PEDV阳性病料288份,PEDV的阳性率为40.33%。在西安、咸阳、渭南、宝鸡地区病料中均检测到了PEDV,其中西安地区病料中PEDV阳性率最高,为46.94%。不同季节采集的病料中均检测到了PEDV,其中冬季病料中PEDV阳性率最高,为57.96%。采集不同生长时期猪群的病料中也均检测出了PEDV,其中哺乳仔猪的PEDV阳性率最高,为70.42%。2.利用PCR法检测714份腹泻病料中的TGEV。结果显示,检测出TGEV阳性病料69份,TGEV阳性率为9.97%,其中PEDV与TGEV混合感染的阳性病料20份,PEDV与TGEV混合感染阳性率为2.81%。在西安、咸阳、渭南、宝鸡地区病料中均检测到了TGEV,其中咸阳地区病料中TGEV的阳性率最高,为14.34%。不同季节采集的病料中均检测出了TGEV,其中冬季病料中TGEV的阳性率最高,为17.05%。采集哺乳仔猪、断奶仔猪、育肥猪、经产母猪的病料中均检测出了TGEV,阳性率分别为15.88%、8.52%、4.45%、7.29%,而后备母猪和种公猪中未检测到TGEV。3.将处理好的PEDV阳性病料接种Vero细胞进行病毒的分离,通过细胞病变观察、PCR、电镜观察等方法鉴定病毒。结果表明,该病毒能使细胞变圆、皱缩,继而出现细胞破碎、脱落、拉丝等病变,经PCR和电镜观察鉴定为PEDV,并命名为PEDV-SX株。以疫苗株CV777的基因组序列为模板,利用Primer 5.0设计了15对相互重叠的引物,进行全基因组的扩增及测序。利用DNA star软件中的SeqMan软件对序列进行拼接,并用MegAlign软件对目的基因进行同源性的比较和遗传进化分析。结果表明,PEDV-SX株与疫苗株的同源性为98%,与经典毒株的同源性为97.8%,与国内外流行株的同源性为97.2%~99.9%。PEDV-SX株与2012中国山东分离的SD-M株和2008年中国江苏分离的JS2008株处在系统发育树的同一个分支上。4.通过对TGEV-SX株的S、M、N、E基因进行分子克隆和测序,成功获得了S、M、N、E基因序列。利用DNA star软件中的MegAlign软件对目的基因进行同源性比较和遗传进化分析。结果表明,TGEV-SX株的S、M、N、E基因与8株参考毒株的同源性分别为98.0%~99.5%、98.1%~100%、97.9%~99.8%、96.8%~99.2%。TGEVSX株的S基因与SC-Y株、SHXB株、virulent Purdue株、Purdue P115株同属一个大的分支,而TGEV-SX株的M、N、E基因与Miller M6株、Miller M60株、H16株、TS株同属一个大的分支。本研究发现在陕西省部分地区猪群中均有PEDV与TGEV感染,PEDV的感染率较高,且主要感染哺乳仔猪,并存在二者混合感染。同源性分析及遗传进化分析发现,PEDV-SX株与2012中国山东分离的SD-M株和2008年中国江苏分离的JS2008株的亲缘关系较近,而与疫苗株CV777亲缘关系较远。同时也发现TGEV-SX株S基因与SC-Y株、SHXB株、virulent Purdue株、Purdue P115株亲缘关系较近,而TGEVSX株的M、N、E基因与H16、TS株、Miller株亲缘关系较近。
[Abstract]:Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV) and swine infectious gastroenteritis virus (transmissible gastroenteritis virus, TGEV) are the main pathogens of porcine viral diarrhea. Pigs of different ages (especially piglets) are susceptible to diarrhea, dehydration and vomiting as the main clinical characteristics after swine infection, PEDV and TGEV have become descending. One of the important reasons for the survival rate of low piglets has caused very serious economic loss to the pig industry of all countries in the world. To understand the epidemiological and pathogenic characteristics of PEDV and TGEV in Shaanxi Province, the relationship and variation of the epidemic strains and vaccine strains in Shaanxi Province, the classic strains and the epidemic strains at home and abroad, and the variation of the epidemic strains in Shaanxi province are also made. The prevention and control of PEDV and TGEV and the development of more reasonable and effective vaccines provide theoretical basis and reference data. This study intends to carry out an epidemiological survey of PEDV and TGEV in parts of Shaanxi Province, and analyze the genetic variation of these two viruses. The following results have been obtained by.1. and PCR method for detecting PEDV. in 714 diarrhoeal diseases. The results showed that 288 PEDV positive diseases were detected. The positive rate of PEDV was 40.33%. in Xi'an, Xianyang, Weinan and Baoji, and PEDV was detected in the disease materials in Baoji, among which the positive rate of PEDV was highest in Xi'an, and PEDV was detected in all the diseases collected in the different seasons of 46.94%.. The positive rate of PEDV in winter disease was the highest, which was 57.96%. mining. PEDV was also detected in the pigs of different growth periods, among which the positive rate of PEDV was the highest for suckling piglets. The TGEV. results in 714 diarrhoea samples were detected by 70.42%.2. using PCR method. The positive rate of TGEV positive was detected, the positive rate of TGEV was 9.97%, among which 20 were PEDV and TGEV mixed infection, PEDV and TGEV mixed feeling. The positive rate of 2.81%. was TGEV in Xi'an, Xianyang, Weinan and Baoji, among which the positive rate of TGEV was highest in Xianyang area, and TGEV was detected in all the diseases collected in different seasons. The positive rate of TGEV in winter disease was the highest, and 17.05%. collected suckling piglets, weanling piglets, fattening pigs, and production. TGEV was detected in the sows, and the positive rates were 15.88%, 8.52%, 4.45%, 7.29% respectively. In the back-up sow and the boar, the virus was isolated by the Vero cells that were treated with the PEDV positive disease, and the virus was identified by the pathological observation, PCR and electron microscopy. The results showed that the virus could make the cells change. The circle, crinkle, and then cell breakage, shedding, drawing and other lesions were identified as PEDV by PCR and electron microscopy, and named PEDV-SX strain. The genome sequence of the vaccine strain CV777 was used as a template, and Primer 5 was used to design 15 pairs of overlapping primers for whole genome expansion and sequencing. The sequence of SeqMan software in DNA Star software was used to sequence the sequence. The homologous comparison and genetic evolution analysis of the target genes were carried out with MegAlign software. The results showed that the homology of PEDV-SX strain and vaccine strain was 98%, the homology with the classic strains was 97.8%, and the homology of the domestic and foreign epidemic strains was 97.2%~99.9%.PEDV-SX strains isolated from 2012 China Shandong and Jiangsu of China in 2008. The isolated JS2008 strain was located on the same branch of the phylogenetic tree and.4. was sequenced by molecular cloning and sequencing of the S, M, N and E genes of the TGEV-SX strain. The sequence of S, M, N, E gene was successfully obtained. The homology of 8 strains of reference strain was 98.0%~99.5%, 98.1%~100%, 97.9%~99.8%, and 96.8%~99.2%.TGEVSX strain of S and SC-Y strain, SHXB strain, virulent Purdue strain and Purdue P115 strain. PEDV and TGEV infection were found among the pigs in some regions of western province, and the infection rate of PEDV was high, and the main infection was lactation piglets, and there were two mixed infections. Homology analysis and genetic evolution analysis found that the relationship between PEDV-SX strain and 2012 SD-M isolates from Shandong in China and JS2008 strains isolated from China in 2008 was close, and the vaccine strain CV77 was similar to the vaccine strain CV77. At the same time, it was found that the S gene of TGEV-SX strain was close to SC-Y, SHXB, virulent Purdue and Purdue P115, while M, N, and E genes were close.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.28

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