不同类型鸡泄殖腔棉拭子禽白血病病毒p27抗原检测与病毒分离的相关性分析
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摘要
为探讨泄殖腔棉拭子或种蛋中禽白血病病毒(ALV)-p27抗原检测与病毒分离检测之间的相关性,对不同类型鸡编号后分别对应采集泄殖腔棉拭子或蛋清后以ALV-p27抗原检测试剂盒检测,同时无菌采集抗凝血接种DF-1细胞进行ALV的分离鉴定。结果显示,人工感染A亚群ALV的SPF鸡群泄殖腔棉拭子ALV-p27抗原检测与病毒分离有很高的吻合率,相对于病毒分离法,119次泄殖腔棉拭子ALV-p27抗原检测中出现有1例假阳性和3例假阴性;从美国进口的200只祖代肉鸡3次采血DF1细胞分离病毒结果均为阴性,而泄殖腔棉拭子ALV-p27抗原阳性率依次为4.00%(8/200),2.97%(5/168)和2.61%(4/153),假阳性率为3.30%。相对于病毒分离法,正在实施ALV净化的不同遗传背景地方品系鸡泄殖腔棉拭子ALV-p27抗原检测的假阳性率高达93.20%~100.00%,还有一定比例假阴性(1/9,1/4,1/3)。本研究大量对应比较的数据表明,相对于血浆病毒分离法,不同类型鸡的泄殖腔棉拭子ALVp27抗原检测法不仅有很高的假阳性率,还有一定比例的漏检率。在种鸡场的ALV净化过程中考虑到成本,不建议以泄殖腔棉拭子作为检测材料。本试验为我国鸡群禽白血病净化检测和临床鉴别诊断提供参考依据。
To investigate the correlation with the antigen p27 detection of avian leukosis virus(ALV)in cloacal swab or eggs and ALV viral isolation in cell cultivation,different types of chickens were raised separately with wing numbers.cloacal swabs,eggs and sterile anticoagulant were collected according to the number.The p27 antigen in eggs and cloacal swabs were detected by ELISA Kits.The blood samples were inoculated onto DF1 cells,and the supernatant was also detected by ELISA Kits for p27 antigen after cultured 7days.The results showed that the cloacal swab ALV-p27 antigen detection and viral separation of SPF chicken artificially infected subgroup A of ALV in different ways had a high match rate compared with viral isolation,a total of 119 detection also appeared to one false-positive and three undetected cases.The results of the 200 grandparent chicken imported from America showed that in plasma samples collected at 3,18 and 33d of age and inoculated into DF1 cells,no exogenous virus was isolated from all chicken,while the positive rate of the cloacal swab ALV-p27 antigen detection were 4.00%(8/200),2.97%(5/168)and 2.61%(4/153),respectively.The false-positive rate of the cloacal swab ALV-p27 antigen detection was 3.30%.Compared with viral isolation,the detection result of cloacal swab ALV-p27 antigen and viral isolation of local strains chicken of different genetic backgrounds in the process of purification demonstrated that the false-positive rate of cloacal swab ALV-p27 antigen detection was as high as 93.20%-100.00%,and there was a certain proportion of false-negative rate(1/9,1/4,1/3,respectively).A large amount data of one-to-one comparison suggested that compared with viral isolation,the detection of cloacal swab ALV-p27 antigen in different types of chickens not only had a high false-positive rate,but also there was a certain proportion undetection rate.Considering the cost factor in the process of ALV purification in breeder farms,it is not recommended to use cloacal swab for the test material.This experiment will provide a reference for the purification and clinical diagnosis of avian leukosis in china.
To investigate the correlation with the antigen p27 detection of avian leukosis virus(ALV)in cloacal swab or eggs and ALV viral isolation in cell cultivation,different types of chickens were raised separately with wing numbers.cloacal swabs,eggs and sterile anticoagulant were collected according to the number.The p27 antigen in eggs and cloacal swabs were detected by ELISA Kits.The blood samples were inoculated onto DF1 cells,and the supernatant was also detected by ELISA Kits for p27 antigen after cultured 7days.The results showed that the cloacal swab ALV-p27 antigen detection and viral separation of SPF chicken artificially infected subgroup A of ALV in different ways had a high match rate compared with viral isolation,a total of 119 detection also appeared to one false-positive and three undetected cases.The results of the 200 grandparent chicken imported from America showed that in plasma samples collected at 3,18 and 33d of age and inoculated into DF1 cells,no exogenous virus was isolated from all chicken,while the positive rate of the cloacal swab ALV-p27 antigen detection were 4.00%(8/200),2.97%(5/168)and 2.61%(4/153),respectively.The false-positive rate of the cloacal swab ALV-p27 antigen detection was 3.30%.Compared with viral isolation,the detection result of cloacal swab ALV-p27 antigen and viral isolation of local strains chicken of different genetic backgrounds in the process of purification demonstrated that the false-positive rate of cloacal swab ALV-p27 antigen detection was as high as 93.20%-100.00%,and there was a certain proportion of false-negative rate(1/9,1/4,1/3,respectively).A large amount data of one-to-one comparison suggested that compared with viral isolation,the detection of cloacal swab ALV-p27 antigen in different types of chickens not only had a high false-positive rate,but also there was a certain proportion undetection rate.Considering the cost factor in the process of ALV purification in breeder farms,it is not recommended to use cloacal swab for the test material.This experiment will provide a reference for the purification and clinical diagnosis of avian leukosis in china.
引文
[1]SAIF Y M.禽病学[M].苏敬良,高福,索勋,译.12版.北京:中国农业出版社,2011:599-639.
[2]WITTER R L.Avian tumor viruses:persistent and evolving pathogens[J].Acta Vet Hung,1997,45(3):251-266.
[3]SHEN Y W,CAI L M,WANG Y M,et al.Genetic mutations of avian leukosis virus subgroup J strains extended their host range[J].J Gen Virol,2014,95(3):691-699.
[4]张志,崔治中,姜世金.从J亚群禽白血病肿瘤中检测出禽网状内皮组织增生症病毒[J].中国兽医学报,2004,24(1):10-13.
[5]崔治中.免疫抑制性病毒多重感染在鸡群疫病发生和流行中的作用[J].畜牧兽医学报,2003,34(5):417-421.
[6]崔治中,张志,杜岩.我国肉用型鸡群中J亚群白血病流行现状的调查[J].中国预防兽医学报,2002,24(4):292-294.
[7]王建新,崔治中,张纪元,等.J亚群禽白血病病毒与禽网状内皮增生症病毒共感染对肉鸡生长和免疫功能的抑制作用[J].中国兽医学报,2003,23(3):211-213.
[8]陈瑞爱.鸡白血病生物防控措施要点[J].中国家禽,2010,32(7):36.
[9]HIMLY M,FOSTER D N,BOTTOLI I,et al.The df-1chicken fibroblast cell line:transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses[J].Virology,1998,248(2):295-304.
[10]XU B R,DONG W X,YU C M,et al.Occurrence of avian leukosis virus subgroup J in commercial layer flocks in China[J].Avian Pathol,2004,33(1):13-17.
[11]徐镔蕊,董卫星,余春明,等.用ALV-J gp85单克隆抗体证明蛋鸡存在J亚群禽白血病[J].畜牧兽医学报,2005,36(3):269-271.
[12]成子强,张利,刘思当,等.中国麻鸡中发现禽J亚群白血病[J].微生物学报,2005,45(4):584-587.
[13]王辉,崔治中.蛋鸡J亚群白血病病毒的分离鉴定及序列分析[J].病毒学报,2008,24(5):369-375.
[14]何爱飞,徐春志,雷云华,等.蛋鸡血管瘤型禽白血病的诊断[J].动物医学进展,2009,30(1):112-115.
[15]李先桥,史开志,罗明星,等.A、J亚群禽白血病病毒感染肉用种鸡的诊断[J].贵州畜牧兽医,2009,33(4):4-6.
[16]赵冬敏,张青蝉,崔治中.芦花鸡中B亚群禽白血病病毒的分离与鉴定[J].病毒学报,2010,26(1):53-57.
[17]王鑫,齐鹏飞,杜燕,等.一例A亚型禽白血病病毒引起的纤维素肉瘤的病理学和病毒学分析[J].中国动物传染病学报,2011,19(1):11-16.
[18]叶建强,秦爱建.J亚群禽白血病病毒(ALV-J)ELlSA检测方法的建立[J].中国兽医学报,2006,26(3):235-237.
[19]杭柏林,胡建和,李杰,等.J亚群禽白血病病毒检测方法的研究进展[J].中国农学报,2011,27(32):21-24.
[20]ZENG X W,LIU L L,HAO R J,et al.Detection and molecular characterization of J subgroup avian leukosis virus in wild ducks in China[J].PLoS One,2014,9(4):e94980.
[21]沈学怀,张丹俊,潘孝成,等.不同检测材料对ELISA检测禽白血病病毒-p27抗原结果的影响[J].中国畜牧兽医,2015,42(5):1294-1300.
[22]崔治中,孙淑红,赵鹏,等.对种鸡场禽白血病净化方案的建议[J].中国家禽,2014,36(1):3-6.
[23]高娟,姚俊峰,土晓亮,等.采用不同试剂盒检测禽白血病p27抗原阳性率的比较[C]//中国畜牧兽医学会家禽学分会.中国畜牧兽医学会家禽学分会第九次代表会议暨第十六次全国家禽学术讨论会论文集.2013.
[24]王明月,窦雯雯,陈立坤,等.不同组织样品或检测方法对ALV检测结果的影响[J].中国兽医学报,2013,33(5):659-663.
[25]赵鹏,崔治中,马诚太.种蛋中禽白血病病毒p27抗原检出率与鸡群禽白血病发病率的相关性研究[J].畜牧兽医学报,2012,43(10):1618-1622.
[26]CRITTENDEN L B,SMITH E J.A comparision of test material for differentiating avian leukosis virus group-specific antigens of exogenous and endogenous origin[J].Avian Dis,1984,28(4):1057-1071.
[2]WITTER R L.Avian tumor viruses:persistent and evolving pathogens[J].Acta Vet Hung,1997,45(3):251-266.
[3]SHEN Y W,CAI L M,WANG Y M,et al.Genetic mutations of avian leukosis virus subgroup J strains extended their host range[J].J Gen Virol,2014,95(3):691-699.
[4]张志,崔治中,姜世金.从J亚群禽白血病肿瘤中检测出禽网状内皮组织增生症病毒[J].中国兽医学报,2004,24(1):10-13.
[5]崔治中.免疫抑制性病毒多重感染在鸡群疫病发生和流行中的作用[J].畜牧兽医学报,2003,34(5):417-421.
[6]崔治中,张志,杜岩.我国肉用型鸡群中J亚群白血病流行现状的调查[J].中国预防兽医学报,2002,24(4):292-294.
[7]王建新,崔治中,张纪元,等.J亚群禽白血病病毒与禽网状内皮增生症病毒共感染对肉鸡生长和免疫功能的抑制作用[J].中国兽医学报,2003,23(3):211-213.
[8]陈瑞爱.鸡白血病生物防控措施要点[J].中国家禽,2010,32(7):36.
[9]HIMLY M,FOSTER D N,BOTTOLI I,et al.The df-1chicken fibroblast cell line:transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses[J].Virology,1998,248(2):295-304.
[10]XU B R,DONG W X,YU C M,et al.Occurrence of avian leukosis virus subgroup J in commercial layer flocks in China[J].Avian Pathol,2004,33(1):13-17.
[11]徐镔蕊,董卫星,余春明,等.用ALV-J gp85单克隆抗体证明蛋鸡存在J亚群禽白血病[J].畜牧兽医学报,2005,36(3):269-271.
[12]成子强,张利,刘思当,等.中国麻鸡中发现禽J亚群白血病[J].微生物学报,2005,45(4):584-587.
[13]王辉,崔治中.蛋鸡J亚群白血病病毒的分离鉴定及序列分析[J].病毒学报,2008,24(5):369-375.
[14]何爱飞,徐春志,雷云华,等.蛋鸡血管瘤型禽白血病的诊断[J].动物医学进展,2009,30(1):112-115.
[15]李先桥,史开志,罗明星,等.A、J亚群禽白血病病毒感染肉用种鸡的诊断[J].贵州畜牧兽医,2009,33(4):4-6.
[16]赵冬敏,张青蝉,崔治中.芦花鸡中B亚群禽白血病病毒的分离与鉴定[J].病毒学报,2010,26(1):53-57.
[17]王鑫,齐鹏飞,杜燕,等.一例A亚型禽白血病病毒引起的纤维素肉瘤的病理学和病毒学分析[J].中国动物传染病学报,2011,19(1):11-16.
[18]叶建强,秦爱建.J亚群禽白血病病毒(ALV-J)ELlSA检测方法的建立[J].中国兽医学报,2006,26(3):235-237.
[19]杭柏林,胡建和,李杰,等.J亚群禽白血病病毒检测方法的研究进展[J].中国农学报,2011,27(32):21-24.
[20]ZENG X W,LIU L L,HAO R J,et al.Detection and molecular characterization of J subgroup avian leukosis virus in wild ducks in China[J].PLoS One,2014,9(4):e94980.
[21]沈学怀,张丹俊,潘孝成,等.不同检测材料对ELISA检测禽白血病病毒-p27抗原结果的影响[J].中国畜牧兽医,2015,42(5):1294-1300.
[22]崔治中,孙淑红,赵鹏,等.对种鸡场禽白血病净化方案的建议[J].中国家禽,2014,36(1):3-6.
[23]高娟,姚俊峰,土晓亮,等.采用不同试剂盒检测禽白血病p27抗原阳性率的比较[C]//中国畜牧兽医学会家禽学分会.中国畜牧兽医学会家禽学分会第九次代表会议暨第十六次全国家禽学术讨论会论文集.2013.
[24]王明月,窦雯雯,陈立坤,等.不同组织样品或检测方法对ALV检测结果的影响[J].中国兽医学报,2013,33(5):659-663.
[25]赵鹏,崔治中,马诚太.种蛋中禽白血病病毒p27抗原检出率与鸡群禽白血病发病率的相关性研究[J].畜牧兽医学报,2012,43(10):1618-1622.
[26]CRITTENDEN L B,SMITH E J.A comparision of test material for differentiating avian leukosis virus group-specific antigens of exogenous and endogenous origin[J].Avian Dis,1984,28(4):1057-1071.
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