40周龄三黄种鸡爆发禽白血病的诊断与病原分析
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摘要
为了解鸡群中禽白血病(Avian leukosis,AL)发生的原因以及感染的禽白血病病毒(Avian leukosis virus,ALV)的亚群,研究对发病鸡进行临床诊断、病理剖检和病理组织学观察,并采集肿瘤样病变组织处理后接种DF-1细胞,培养后分别进行禽白血病病毒p27群特异性抗原ELISA检测、亚群特异性PCR检测以及亚群特异性间接免疫荧光法(IFA)鉴定,确定了40周龄三黄种鸡群感染了J亚群禽白血病病毒(ALV-J,分离株命名为GX16YL02);进一步对分离株env基因进行PCR扩增、克隆和序列测定与比较分析。结果表明:分离株GX16YL02的env基因与ALV-J英国原型株HPRS-103的核苷酸及氨基酸的相似性分别为93.9%和91.9%,与其他8株国内外参考株的核苷酸及氨基酸的相似性分别为90.7%~95.5%和89.4%~95.2%,其中gp85基因与其他参考株的核苷酸和氨基酸相似性相对较低,分别为86.9%~94.9%和85.1%~95.4%,整个env基因有义突变和沉默突变的比例分析显示,与gp37基因相比,gp85基因变异较大,尤其是免疫选择压在高变区hr1区域有一定的作用。
To understand the etiology of Avian Leukosis( AL) and the subgroup of avian leukosis virus in the flocks,in this study,clinical diagnosis,pathologic examination and histopathological observation were performed in diseased chickens. Furthermore,tissue samples with tumor-like lesions were cultured with DF-1 cells and identified by ELISA detection of avian leucosis virus p27 group specific antigen,PCR amplification and indirect immunofluorescence assay( IFA). The results confirmed that the 40-week-old Three-Yellow Chickens were infected with subgroup J ALV( ALV-J) and named as GX16YL02. PCR amplification,cloning and sequencing analysis of env gene of the isolates were further performed and compared. Results revealed that the similarity of nucleotide and amino acid between isolated GX16YL02 env gene and HPRS-103 of the British ALV-J prototype strains were 93. 9% and 91. 9% respectively,and the similarity of nucleotide and amino acid with other 8 strains of domestic and foreign reference strains were 90. 7% ~ 95. 5% and 89. 4% ~ 95. 2%. In addition,the similarity comparison on gp85 gene showed that the isolate had less similarities with the reference strains,which were 86. 9% ~ 94. 9% and 85. 1% ~ 95. 4%,respectively.The proportion of nonsynonymous and synonymous changes in env gene indicated that the mutations in gp85 gene were significantly higher than those in gp37 gene; especially the immune selective pressure which had certain effects on the hyper-variable region of hr1.
To understand the etiology of Avian Leukosis( AL) and the subgroup of avian leukosis virus in the flocks,in this study,clinical diagnosis,pathologic examination and histopathological observation were performed in diseased chickens. Furthermore,tissue samples with tumor-like lesions were cultured with DF-1 cells and identified by ELISA detection of avian leucosis virus p27 group specific antigen,PCR amplification and indirect immunofluorescence assay( IFA). The results confirmed that the 40-week-old Three-Yellow Chickens were infected with subgroup J ALV( ALV-J) and named as GX16YL02. PCR amplification,cloning and sequencing analysis of env gene of the isolates were further performed and compared. Results revealed that the similarity of nucleotide and amino acid between isolated GX16YL02 env gene and HPRS-103 of the British ALV-J prototype strains were 93. 9% and 91. 9% respectively,and the similarity of nucleotide and amino acid with other 8 strains of domestic and foreign reference strains were 90. 7% ~ 95. 5% and 89. 4% ~ 95. 2%. In addition,the similarity comparison on gp85 gene showed that the isolate had less similarities with the reference strains,which were 86. 9% ~ 94. 9% and 85. 1% ~ 95. 4%,respectively.The proportion of nonsynonymous and synonymous changes in env gene indicated that the mutations in gp85 gene were significantly higher than those in gp37 gene; especially the immune selective pressure which had certain effects on the hyper-variable region of hr1.
引文
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[11]安好峰,戴银,潘孝成,等.J亚群禽白血病病毒安徽分离株的鉴定及其gp85基因序列分析[J].中国畜牧兽医,2012,39(12):6-10.
[12]王增福,崔治中.在抗体免疫选择压作用下鸡J亚群白血病病毒gp85基因的变异[J].中国科学:生命科学,2006,36(1):9-16.
[13]韦平,崔治中.地方品种鸡禽白血病和鸡白痢的危害及净化防控的实践[J].中国家禽,2015,37(9):1-4.
[14]毕玉彧,管子言,黄晓姣,等.部分种鸡场常用活疫苗中ALV的PCR检测[J].广西畜牧兽医,2014,30(4):171-174.
[15]杨永立,钟兴福,王培坤,等.2014年广西地方鸡群病毒性肿瘤病例的检测分析[J].中国家禽,2015(17):53-56.
[16]齐岩,张贺楠,刘有昌.禽白血病的危害及防控[J].家禽科学,2010(6):5-9.
[2]PAYNE L N,BROWN S R,BUMSTEAD N,et al.A novel subgroup of exogenous avian leukosis virus in chickens[J].J Gen Virol,1991,72(Pt4):801-807.
[3]王鑫,赵鹏,崔治中.我国地方品种鸡分离到的一个禽白血病病毒新亚群的鉴定[J].病毒学报,2012,28(6):609-614.
[4]石梦霞,王培坤,黄晓姣,等.广西地方品种鸡临床血管瘤型禽白血病的诊断[J].中国家禽,2015,37(2):68-70.
[5]王培坤,毕玉彧,秦丽莉,等.三黄鸡临床血管瘤病例中分离出A亚群与J亚群禽白血病病毒[J].动物医学进展,2015,36(3):13-16.
[6]高跃飞.禽白血病的诊断与防治[J].当代畜禽养殖业,2015(12):32.
[7]SMITH L M,BROWN S R,HOWES K,et al.Development and application of polymerase chain reaction(PCR)tests for the detection of subgroup J avian leukosis virus[J].Virus Res,1998,54(1):87-98.
[8]VENUGOPAL K,SMITH L M,HOWES K,et al.Antigenic variants of J subgroup avian leukosis virus:sequence analysis reveals multiple changes in the env gene[J].J Gen Virol,1998,79(Pt4):757-766.
[9]李德庆,赵鹏,王鑫,等.鸡胚和雏鸡接种ALV-J相关急性纤维肉瘤浸出液的致病性比较[J].畜牧兽医学报,2013,44(2):250-255.
[10]张青婵.A亚群禽白血病病毒不同分离株的基因组和生物学特性比较[D].泰安:山东农业大学,2010.
[11]安好峰,戴银,潘孝成,等.J亚群禽白血病病毒安徽分离株的鉴定及其gp85基因序列分析[J].中国畜牧兽医,2012,39(12):6-10.
[12]王增福,崔治中.在抗体免疫选择压作用下鸡J亚群白血病病毒gp85基因的变异[J].中国科学:生命科学,2006,36(1):9-16.
[13]韦平,崔治中.地方品种鸡禽白血病和鸡白痢的危害及净化防控的实践[J].中国家禽,2015,37(9):1-4.
[14]毕玉彧,管子言,黄晓姣,等.部分种鸡场常用活疫苗中ALV的PCR检测[J].广西畜牧兽医,2014,30(4):171-174.
[15]杨永立,钟兴福,王培坤,等.2014年广西地方鸡群病毒性肿瘤病例的检测分析[J].中国家禽,2015(17):53-56.
[16]齐岩,张贺楠,刘有昌.禽白血病的危害及防控[J].家禽科学,2010(6):5-9.