摘要
2006年5月,我国出现了一种以无名高热为主要特征的猪病,病情迅速蔓延,猪只大批量死亡。7月份,湖南省宁乡县也出现该病病例,为了调查发病情况,探索发病的原因,开展了本次实验研究。
对发病猪场进行实地走访并采集典型发病猪病料,采用挑取单菌落划线的方法分离纯化细菌,并做了相关初步鉴定。结果表明,从高热病发病猪体内可以分离到多种病原菌。
我们将采集的病料进行电子显微镜观察,发现了直径60～100nm,球形,有囊膜的病毒粒子;并通过病毒抗体检测,核酸检测等手段,将病毒锁定在猪流感病毒和蓝耳病病毒。通过对这两种病毒进行的分离鉴定实验,排除了猪流感病毒,最后确定猪蓝耳病病毒在此次疫情中起到了主导作用。
从病料中分离到使MARC-145细胞产生病变的病毒,经过血清学中和试验、RT-PCR测定,确定病料中含有猪繁殖与呼吸道病毒(PRRSV)。然后进行病毒RNA提取,PCR扩增其中的ORF5基因,将回收得到的DNA片段进行测序,最后用DNASTAR软件对比分析我们分离的毒株和Genebank中的PRRSV毒株之间的同源性和进化关系。
结果表明:本试验分离到的两株PRRSV变异毒株,经过血清学实验和序列分析证明为美洲型PRRSV毒株;ORF5基因同源性比较发现,与VR-2332标准株的同源性为89.4%,与其他学者于2006年从其他省份的高热病发病猪体内分离到的PRRSV毒株的同源性为96.8%～99.5%,其中又以江西分离株同源性最高,达到99.5%。经过基因进化树分析,我们分离的两株毒株和国内其他学者分离的高致病性蓝耳病病毒为同一个种。
In May 2006,there was an outbreak of swine loimia,which the main characteristic was high fever.It was quickly spreaded and caused thousands death of swine.In July, this Ioimia occurred in Ningxiang County,Hunan Province.In order to investigate the epidemic and explore the pathogene,we carried out this experimental research.
We visited the pig farm that broke out the disease and acquisited materials from typical swine;isolated,purified and identificated bacterias.The result shows this is a multi-pathogen disease,we can isolate several pathogenic bacterias from the swine catched the disease.
We found some spherical and capsuled virus particles under the electronic microscope,which diameter is 60～100nm;through anti-body detection,nucleotide detection, and some other methods.We inferred that it would be caused by SIV or PRRSV.Through isolate and identity experiment on these two viruses,we precluded the SIV,and certained PRRSV was the main reason in the whole loimia.
We isolated virus which make MARC-145 cells appeared CPE.We certained there was PRRSV in the sample by SN,RT-PCR ect.We extracted RNA,and amplificated gene ORF5 by PCR,sequenced gene ORF5 retrieved and compared with other ORF5 strains which published on Genebank,using DNASTAR to compare the homology and evolutionary relationship.
We confirmed that the PRRSV we isolated are NA type by sequence analysis and serum experiment,the homology of gene ORF5 compared with the VR-2332 was 89.4%, and compared with other PRRSV strains which isolated by other researchers in the the other provinces in 2006 was 96.8%to 99.5%,among which the homology of separation strain in Jiangxi was the highest,reached 99.5%.By gene phylogenetic tree analysis,the two strains we separated and other PRRSV which isolated by other researchers were the same species.
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