猪伪狂犬病综合诊断及分离毒株gE和TK基因遗传变异分析

2012年以来,许多使用gE基因缺失活疫苗免疫过的猪场广泛性出现伪狂犬病毒(PRV)感染,gE抗体阳性率不断升高,伪狂犬典型病例不断增加。2018年3月鲁南地区几个种猪场先后发生疑似伪狂犬病疫情,怀孕母猪流产、产死胎和木乃伊胎,仔猪出现神经症状且死亡率高。通过对病死猪及死胚剖检进行初步诊断,取病料进一步进行病理组织学诊断及病毒分离鉴定。结果显示,病死猪均可见病毒性脑炎、肝细胞变性坏死及淋巴组织坏死等病理变化,在病变的神经元、肝细胞、扁桃体隐窝上皮细胞等细胞核内见红染包涵体。对分离到的4株PRV进行了gE和TK基因的序列测定及遗传变异分析发现,4株PRV的gE和TK核苷酸序列的同源性分别为98.8%~99.3%和98.9%~99.6%,与国内流行毒株其核苷酸序列的同源性分别99.1%~99.7%和98.6%~99.8%,与匈牙利和美国等流行毒株核苷酸序列的同源性分别为97.3%~97.8%和98.8%~99.5%,表明4株分离株高度同源,与国内PRV变异株处在同一分支,而与匈牙利和美国等毒株遗传距离较远。传统疫苗对PRV变异毒株不能提供有效保护,给猪场伪狂犬病的防控和净化工作带来了新的挑战。

Comprehensive Diagnosis of Pseudorabies in Pigs and Analysis of Genetic Variation of gE and TK Genes in Isolated Strains

Since 2012,many pig farms immunized with gE gene deleted live vaccines have been extensively infected with PRV(pseudorabies virus),and the positive rate of gE antibody has been increasing,and the typical cases of pseudorabies have been increasing.In March 2018,suspected pseudorabies occurred in several breeding pig farms in Lunan region of Shandong province.Pregnant sows miscarried,stillborn and mummified fetuses,and piglets showed neurological symptoms and high mortality rate.A preliminary diagnosis was carried out on the dead pigs and dead embryos,and the histopathological diagnosis,virus isolation and identification were further carried out.The results showed that pathological changes such as viral encephalitis,degeneration and necrosis of hepatocytes and lymphoid tissue necrosis were all observed in the dead pigs.Red stained inclusion bodies were found in the nucleus of the neurons,hepatocytes and tonsil crypt epithelial cells.The sequence analysis and genetic variation analysis of the gE and TK genes of the four strains of PRV isolated showed that the homology of the gE and TK nucleotide sequences of the four strains of PRV were 98.8%-99.3%and 98.9%-99.6%,respectively.The identity with the nucleotide sequences of the domestic strains were 99.1%-99.7%and 98.6%-99.8%,and the similarity with the nucleotide sequence of Hungarian and American strains were 97.3%-97.8%and 98.8%-99.5%respectively,which indicated that the four isolated strains are highly homologous,and they are in the same branch with the domestic PRV mutants,while they are far away from the Hungarian and American strains in the distance of inheritance.The traditional vaccine cannot provide effective protection for the mutant strains of PRV,which brings new challenges to the prevention and purification of eradication in pig farms.