J亚群禽白血病病毒与粪肠球菌混合感染的诊断及相关基因分析

doi:10.16431/j.cnki.1671-7236.2021.12.035

摘要/Abstract

摘要： 为了解贵阳市某养殖场J亚群禽白血病病毒(Avian leukosis virus,ALV)gp85基因的变异情况及粪肠球菌(Enterococcus faecalis,EF)毒力基因的携带情况,本研究对该养殖场ALV与粪肠球菌混合感染的病例展开了病原学调查分析,采集病料进行病毒核酸检测、gp85基因克隆测序分析、核苷酸序列相似性分析、细菌分离鉴定、毒力基因检测等。结果显示,成功克隆出ALV gp85基因并分离鉴定出1株粪肠球菌,分别命名为GZM52021株和GZEF2021株。ALV核酸检测仅在约422 bp处出现特异性扩增条带,说明存在ALV-J感染;成功克隆ALV gp85基因,且GZM52021株与J亚群参考毒株gp85基因(GenBank登录号为KM655820、KF796654、JN378888和Z46390)的相似性较高,在97.9%~98.2%之间;gp85基因核苷酸序列遗传进化树构建结果与相似性结果一致。GZEF2021株在LB 固体平板上长出圆形凸起、不透明的乳白色菌落,在鲜血琼脂平板上有明显的溶血现象,革兰氏染色镜检为球形或卵圆形阳性链状球菌;16S rRNA测序结果显示,GZEF2021株与粪肠球菌(GenBank登录号为KJ626240、MT611693、KY399248、MH919370、MN379663和KU937389)的相似性均在99%以上;药敏试验结果显示,GZEF2021株对头孢哌酮、青霉素、哌拉西林和羧苄西林4种药物敏感,对复方新诺明、头孢拉定、头孢唑啉3种药物中度敏感,对新霉素、丁胺卡那、多西环素和米诺环素4种药物耐药;毒力基因检测显示,GZEF2021株携带有ace、gelE、asa1、EF3314和efaA 5个毒力基因,说明从鸡中分离出的粪肠球菌可能具有一定的致病性。该病死鸡存在J亚群ALV与粪肠球菌共同感染的情况,本试验结果可为了解J亚群ALV和粪肠球菌在贵州的流行趋势及其混合感染的诊治提供一定的参考资料。

关键词: 禽白血病病毒(ALV); 粪肠球菌; 混合感染; 毒力基因

Abstract: In order to understand the variation of gp85 gene of Avian leukemia virus (ALV) subgroup J and the carrying of virulence gene of Enterococcus faecalis (EF) in a farm in Guiyang city, etiological investigation and analysis were carried out on the cases of co-infection of ALV and EF in this farm.Samples were collected for virus nucleic acid detection, gp85 gene cloning and sequencing analysis, nucleotide sequence similarity analysis, bacterial isolation and identification, virulence gene detection, etc.The results showed that one ALV strain and one Enterococcus faecalis strain were successfully isolated and identified, which were named GZM52021 and GZEF2021 respectively.The specific amplification band of ALV only appeared at about 422 bp, indicating the existence of ALV-J infection.ALV gp85 gene was successfully cloned, and the similarity between GZM52021 strain and gp85 gene of subgroup J reference strain (GenBank accession No.:KM655820, KF796654, JN378888 and Z46390) was high, ranging from 97.9% to 98.2%.The construction results of genetic evolutionary tree of nucleotide sequence of gp85 gene were consistent with the similarity results.GZEF2021 strain grew round convex and opaque milky white colonies on LB solid plate, and there was obvious hemolysis on blood agar plate.Gram staining microscopy showed spherical or oval positive Streptococcus.16S rRNA sequencing showed that the similarity between GZEF2021 strain and Enterococcus faecalis (GenBank accession No.:KJ626240, MT611693, KY399248, MH919370, MN379663 and KU937389) was more than 99%.The results of drug sensitivity test showed that GZEF2021 strain was sensitive to cefoperazone, penicillin, piperacillin and carbenicillin, moderately sensitive to compound sulfamethoxazole, cefradine and cefazolin and resistant to neomycin, amikacin, doxycycline and minocycline.The detection of virulence genes showed that GZEF2021 strain carried ace, gelE, asa1, EF3314 and efaA five virulence genes, indicating that the Enterococcus faecalis isolated from chicken might be pathogenic.The co-infection of ALV subgroup J and Enterococcus faecalis existed in the dead chicken.The results of this experiment could provide some references for understanding the epidemic trend of ALV subgroup J and Enterococcus faecalis and the diagnosis and treatment of mixed infection in Guizhou.

Key words: Avian leukosis virus (ALV); Enterococcus faecalis; mixed infection; virulence gene

中图分类号:

S852.65⁺7

刘蔓蔓, 温贵兰, 文明, 张小波, 胡璇, 孙芸, 张秀莉. J亚群禽白血病病毒与粪肠球菌混合感染的诊断及相关基因分析[J]. 中国畜牧兽医, 2021, 48(12): 4671-4680.

LIU Manman, WEN Guilan, WEN Ming, ZHANG Xiaobo, HU Xuan, SUN Yun, ZHANG Xiuli. Diagnosis and Related Gene Analysis of Co-infection of Avian Leukemia Virus Subgroup J and Enterococcus faecalis[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4671-4680.

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