绿壳蛋鸡MDV与J亚群外源性ALV混合感染病原的鉴定及主要基因序列分析

Pathogen identification and main gene sequence analysis of mixed infection of MDV and exogenous ALV J subgroup in green-shell laying hens

为了确定贵州省某养殖场病死绿壳蛋鸡的发病原因,试验对送检的病死鸡进行剖检观察,无菌采集病变组织提取核酸并进行基因克隆及测序,分析同源性并构建遗传进化树。结果表明:3只病死鸡剖检可见脾脏肿大、质脆;肝脏肿大,有肿瘤结节;腺胃肿大。马立克病病毒(Marek’s disease virus,MDV)核酸检测仅MDV meq基因出现与预期片段大小相符的特异性扩增条带;禽白血病病毒(Avian leukosis virus,ALV)分离鉴定仅ALV J gp85基因出现与预期片段大小相符的特异性扩增条带;将目的片段克隆至pMD19-T载体上,经测序获得3个MDV meq基因片段和3个ALV J gp85基因片段,核苷酸序列大小分别约为1020,930 bp;获得的3株MDV毒株和3株ALV毒株与GenBank中相应参考毒株的核苷酸序列同源性分别为98.6%~100%、46.1%~99.6%,分别与参考毒株G2株和HPRS103株在同一分支上,亲缘关系较近。说明贵州省某养殖场鸡只的死亡原因是MDV与J亚群外源性ALV混合感染所致。

In order to determine the cause of the disease and death of green-shell laying hens in a farm in Guizhou Province, the experiment carried out necropsy and observation on the diseased and dead hens submitted for inspection, aseptically collected the diseased tissues to extract nucleic acids for gene cloning and sequencing, analyzed homology and constructed genetic evolution tree. The results showed that the necropsy of 3 sick and dead hens showed enlarged and fragile spleens, enlarged liver with tumor nodules, and enlarged glandular stomach. Only the meq gene of Marek’s disease virus(MDV) showed specific amplified bands that corresponded to the expected fragment size, and the isolation and identification of Avian leukemia virus(ALV) only showed the presence of ALV J gp85 gene which showed specific amplified bands that corresponded to the expected fragment size. The target fragments were cloned into the pMD19-T vector, and 3 MDV meq gene fragments and 3 ALV J gp85 gene fragments were obtained after sequencing. The nucleotide sequence sizes were about 1 020 and 930 bp, respectively. The nucleotide sequence homology of the 3 MDV strains and 3 ALV strains obtained with the corresponding reference strains in GenBank were 98.6%-100% and 46.1%-99.6% respectively, which were on the same branch with the reference strains G2 and HPRS103 respectively, and showed close genetic relationship. The results suggested that the cause of death of hens in a farm in Guizhou Province was caused by mixed infection of MDV and exogenous ALV J subgroup.