Virus Titer Determination and Quality Evaluation of the Commercial Marek’s Disease Vaccines Sold in 2020—2021

doi:10.15933/j.cnki.1004-3268.2022.06.015

Journal of Henan Agricultural Sciences ›› 2022, Vol. 51 ›› Issue (6): 134-143.DOI: 10.15933/j.cnki.1004-3268.2022.06.015

Special Issue: 动物新型疫苗专题

• Animal Science Veterinary Medicine • Previous Articles Next Articles

Virus Titer Determination and Quality Evaluation of the Commercial Marek’s Disease Vaccines Sold in 2020—2021

ZHENG Luping^1,2，TENG Man^1,2，LIU Jinling^1,2，LUO Qin^1,2,3，CHU Yushu^1,2，WANG Weidong^1,2,4，ZHANG Wenkai^1,2,5，LUO Jun^1,2,5

（1.Key Laboratory of Animal Immunology，Henan Academy of Agricultural Sciences/Ministry of Agriculture and Rural Affairs of China & Henan Provincial Key Laboratory of Animal Immunology，Zhengzhou 450002，China；2.UK⁃China Centre of Excellence for Research on Avian Diseases，Henan Academy of Agricultural Sciences，Zhengzhou 450002，China；3.College of Veterinary Medicine，Henan University of Animal Husbandry and Economy，Zhengzhou 450046，China；4.College of Public Health，Zhengzhou University，Zhengzhou 450001，China；5.College of Animal Science and Technology，Henan University of Science and Technology，Luoyang 471003，China）

Received:2021-12-15 Published:2022-06-15 Online:2022-08-17

2020—2021 年市售马立克病疫苗的效价测定及质量评估

郑鹿平^1,2，滕蔓^1,2，刘金玲^1,2，罗琴^1,2,3，楚钰淑^1,2，王伟东^1,2,4，张文凯^1,2,5，罗俊^1,2,5

（1.河南省农业科学院动物免疫学重点实验室/农业农村部动物免疫学重点实验室/河南省动物免疫学重点实验室，河南郑州 450002；2.河南省农业科学院中英禽病国际研究中心，河南郑州 450002；3.河南牧业经济学院动物医药学院，河南郑州 450046；4.郑州大学公共卫生学院，河南郑州 450001；5.河南科技大学动物科技学院，河南洛阳 471003）

通讯作者: 罗俊（1978-），男，河南罗山人，研究员，博士，主要从事动物病毒学研究。E-mail：luojun593@aliyun.com
作者简介:郑鹿平（1984-），女，河南扶沟人，助理研究员，硕士，主要从事家禽免疫抑制病与肿瘤病研究。E-mail：1064033209@qq.com
基金资助:
国家自然科学基金项目（U21A20260）；河南省重点研发与推广专项（212102110097）；河南省自然科学基金（212300410359）；河南省农业科学院自主创新项目（2022ZC65）

Abstract

Abstract: The effective prevention and control of Marek’s disease（MD）mainly depends on vaccine immunization. In order to determine and evaluate the quality of present MD vaccines，virus titers of 11 commercial MD vaccine products provided by 9 distinct companies during 2020—2021 were determined and analyzed by indirect immunofluorescence assay(IFA).Then the potential contamination of avian leukosis virus（ALV）and reticuloendotheliosis virus（REV）was detected using commercial ELISA kits，colloidal gold test strip or RT⁃PCR.The results showed that for all the tested liquid nitrogen vaccines，no significant difference was observed among the three batches of imported or domestic CVI988 vaccines，while significant differences between the two 814 vaccines（P<0.05） and two bivalent MD vaccines(CVI988+HVT or 814+HVT)(P<0. 01)were found. The virus titers of the tested liquid nitrogen MD vaccine products were all above 5 500 PFU/bird，which were more than two folds of value specified in the national standard.However，the virus titers of four freeze⁃dried HVT vaccine products were only about 83.90—109.50 PFU/bird，much lower than 2 000 PFU/bird as indicated in product introductions.Contamination of ALV or REV was not detected in any of the tested MD vaccines，indicating that these vaccines were clean.

Key words: Marek’s disease, Vaccine, Titration, IFA, ALV, REV

摘要： 为了解和评估现有鸡马立克病（MD）疫苗的质量，采用间接免疫荧光试验（IFA）对2020—2021年市场销售的9家不同企业生产的11个批次MD疫苗产品进行病毒效价测定和对比分析，同时用ELISA试剂盒、胶体金试纸条或RT-PCR 方法分别对禽白血病病毒（ALV）和禽网状内皮组织增生症病毒（REV）的污染状况进行检测。结果表明，在所检测的MD液氮苗中，3批进口或国产的CVI988单价液氮苗之间病毒效价无显著差异，2批不同品牌814单价液氮苗效价差异显著(P<0.05)，2批不同品牌双价液氮苗（CVI988+HVT或814+HVT）病毒效价差异极显著(P<0.01)，但上述全部类型液氮苗产品的病毒效价均大于5 500 PFU/羽份，达到国标规定值2倍以上。然而，4批不同品牌HVT冻干苗的病毒效价均远低于产品说明书标注的2 000 PFU/羽份，仅为83.90~109.50 PFU/羽份。全部MD 疫苗产品的ALV和REV检测结果均为阴性，表明这些产品洁净无污染。

关键词: 马立克病, 疫苗, 效价测定, 间接免疫荧光试验, 禽白血病病毒, 禽网状内皮组织增生症病毒

CLC Number:

S859.79+7

ZHENG Luping, TENG Man, LIU Jinling, LUO Qin, CHU Yushu, WANG Weidong, ZHANG Wenkai, LUO Jun. Virus Titer Determination and Quality Evaluation of the Commercial Marek’s Disease Vaccines Sold in 2020—2021[J]. Journal of Henan Agricultural Sciences, 2022, 51(6): 134-143.

郑鹿平, 滕蔓, 刘金玲, 罗琴, 楚钰淑, 王伟东, 张文凯, 罗俊. 2020—2021 年市售马立克病疫苗的效价测定及质量评估[J]. 河南农业科学, 2022, 51(6): 134-143.

Figures/Tables 6

References

［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，2012：129⁃151.
SAIF Y M. Diseases of poultry［M］.SU J L，GAO F，SUO X，trans.12th ed. Beijing：China Agriculture Press，2012：129⁃151.
［2］WITTER R L.Increased virulence of Marek’s disease virus field isolates［J］.Avian Diseases，1997，41（1）：149⁃163.
［3］WITTER R L，CALNEK B W，BUSCAGLIA C，et al.Classification of Marek’s disease viruses according to pathotype：Philosophy and methodology［J］.Avian Pathology，2005，34（2）：75⁃90.
［4］CHURCHILL A E，PAYNE L N，CHUBB R C.Immunization against Marek’s disease using a live attenuated virus［J］.Nature，1969，221：744⁃747.
［5］SCHAT K A，CALNEK B W.Protection against Marek’s disease⁃derived tumor transplants by the nononcogenic SB⁃1 strain of Marek’s disease virus［J］.Infection and Immunity，1978，22（1）：225⁃232.
［6］RISOENS B H，VAN V H，MASTENBROEK N，et al.Control of Marek’s disease in the Netherlands：Field trials on vaccination with an avirulent strain（CVI988）of Marek’s disease virus［J］.Avian Diseases，1972，16（1）：108⁃125.
［7］童昆周，林英华，徐宜为，等.鸡马立克氏病（MD）免疫的研究：MD病毒弱毒疫苗株的培育和免疫试验［J］.畜牧兽医学报，1984，15（2）：34⁃41.
TONG K Z，LIN Y H，XU Y W.Study on the immunization of chicken against Marek’s disease：Report on a naturally avirulent vaccine strain of Marek’s disease herpesvirus［J］.Acta Veterinaria et Zootechnica Sinica，1984，15（2）：34⁃41.
［8］NAIR V，SCHUDEL A，LOMBARD M，et al.Successful control of Marek’ s disease by vaccination［J］.Developmental Biology，2004，119：147⁃154.
［9］YU Z H， TENG M， LUO J， et al.Molecular characteristics and evolutionary analysis of field Marek’s disease virus prevalent in vaccinated chicken flocks in recent years in China［J］. Virus Genes，2013，47（2）：282⁃291.
［10］ZHUANG X Y，ZOU H T，SHI H Y，et al.Outbreak of Marek’s disease in a vaccinated broiler breeding flock during its peak egg⁃laying period in China［J］.BMC Veterinary Research，2015，11：157.
［11］马圣明，滕蔓，李会珍，等.河南土鸡群马立克病病毒流行毒株的分离鉴定及分子进化分析［J］.中国兽医学报，2018，38（4）：637⁃644.
MA S M，TENG M，LI H Z，et al.Isolation，identification and phylogenetic analysis of field Marek’s disease virus
isolated from the local chicken breeds in Henan Province［J］.Chinese Journal of Veterinary Science，2018，38（4）：637⁃644.
［12］SHI M，LI M，WANG W W，et al.The emergence of a vv+MDV can break through the protections provided by the current vaccines［J］. Viruses，2020，12：1048.
［13］李慧姣.禽用病毒性活疫苗外源病毒污染现状［J］.中国家禽，2010，32（22）：37⁃38.
LI H J.Present situation of exogenous virus contamination of live poultry viral vaccines［J］.China Poultry，2010，32（22）：37⁃38.
［14］ZAVALA G，CHENG S.Detection and characterization of avian leukosis virus in Marek’s disease vaccines［J］.Avian Diseases，2006，50（2）：209⁃215.
［15］SILVA R F，FADLY A M，TAYLOR S P，et al.Development of a polymerase chain reaction to differentiate avian leukosis virus（ALV） subgroups：Detection of an ALV contaminant in commercial Marek’s disease vaccines［J］.Avian Diseases，2007，51（3）：663⁃667.
［16］BARBOSA T，ZAVALA G，CHENS S，et al.Molecular characterization of three recombinant isolates of avian leukosis virus obtained from contaminated Marek’s disease vaccines［J］.Avian Diseases，2008，52（2）：245⁃252.
［17］KAWAMURA H，WAKABAYASHI T，YAMAGUCHI S，et al.Inoculation experiment of Marek’s disease vaccine contaminated with a reticuloendotheliosis virus［J］.National Institute of Animal Health Quarterly，1976，16（4）：135⁃140.
［18］JACKSON C A，DUNN S E，SMITH D I，et al.Proventriculitis，“nakanuke” and reticuloendotheliosis in chickens following vaccination with herpesvirus of turkeys（HVT）［J］.Australian Veterinary Journal，1977，53（9）：457⁃459.
［19］WEI K，SUN Z，ZHU S，et al.Probable congenital transmission of reticuloendotheliosis virus caused by vaccination with contaminated vaccines ［J］.PLoS One，2012，7（8）：e43422.

［20］宿靖伟，滕蔓，罗俊. 等.马立克病病毒与禽网状内皮增生病病毒的混合感染及在传代过程中的重组［J］.畜牧兽医学报，2016，47（1）：128⁃134.

SU J W，TENG M，LUO J，et al.Co⁃infection of field Marek’s disease virus with reticuloendotheliosis virus prevalent in vaccinated chicken flocks［J］.Acta Veterinaria et Zootechnica Sinica，2016，47（1）：128⁃134.
［21］中国兽药典委员会. 中华人民共和国兽药典［M］北京：中国农业出版社，2020.
Chinese Veterinary Pharmacopoeia Committee.Chinese veterinary pharmacopoeia［M］.Beijing：China Agriculture
Press，2020.
［22］刘英楠，孟凡峰，李阳，等.3株马立克氏病病毒的分离鉴定及其主要致病基因分析［J］.中国兽医学报，2017，37（8）：1479⁃1484，1500.
LIU Y N，MENG F F，LI Y，et al.Isolation and identification of three Marek’s disease virus strains and sequence comparison of genes involved in pathogenesis［J］.Chinese Journal of Veterinary Science，2017，37（8）：1479⁃1484，1500.
［23］王友，尤永君，徐兆强，等.马立克病毒二价活疫苗效价检测实验［J］.中国动物保健，2013，15（8）：16⁃18，20.
WANG Y，YOU Y J，XU Z Q，et al.The test of the CVI988/Rispens+FC126 bivalent live vaccine potency［J］.China Animal Health，2013，15（8）：16⁃18，20.
［24］白雪雁，张小荣，张成成，等.一株超强马立克病病毒的分离鉴定与致病性试验［J］.动物医学进展，2020，41（11）：41⁃47.
BAI X Y，ZHANG X R，ZHANG C C，et al.Isolation，identification and pathogenicity test if a very virulent
Marek’s disease virus strain［J］.Progress in Veterinary Medicine，2020，41（11）：41⁃47.
［25］汪洁.四个厂家MD商品活疫苗病毒含量的评价及其免疫雏鸡后抗体水平的比较研究［D］.杨凌：西北农林科技大学，2014.
WANG J.Comparison of virus plaque measurement and antibody induction of four Marek’s disease commercial
live vaccines ［D］.Yangling：Northwest A&F University，2014.
［26］石梦雅，李海娟，王威威，等.2017年广西及周边地区地方品种鸡三种肿瘤病的流行病学调查［J］.中国家禽，2019，41（3）：73⁃76.
SHI M Y，LI H J，WANG W W，et al.Epidemiological investigation of three neoplasms in chickens of local breeds in Guangxi and its surrounding areas in 2017［J］.China Poultry，2019，41（3）：73⁃76.
［27］孙嘉.地方品种JS鸡多发肿瘤病的病原学探究［D］.泰安：山东农业大学，2017.
SUN J.Etiological study on mu ltiple tumor disease of local JS Chicken［D］.Taian：Shandong Agricultural University，2017.
［28］徐天鹏，吴思乐，温健，等.慢羽鸡成纤维细胞内源性白血病病毒ev21基因敲除［J］.南方农业学报，2021，52（8）：2259⁃2266.
XU T P，WU S L，WEN J，et al.Knockout of endogenous leukemia gene ev21 in slow feathering chicken［J］.Journal of Southern Agriculture，2021，52（8）：2259⁃2266.
［29］李会珍，滕蔓，党露，等.马立克氏病病毒编码的miR-M12-5p对鸡HVCN1基因表达的靶向调控［J］.河南农业科学，2016，45（6）：121⁃125，129.
LI H Z，TENG M，DANG L，et al.Targeted regulation of host gene HVCN1 by viral microRNA miR⁃M12⁃5p encoded by Marek’s disease virus［J］.Journal of Henan Agricultural Sciences，2016，45（6）：121⁃125，129.
［30］李海娟.过去五年10株ALV-J分离株全基因序列分析及接种ALV-J污染的活疫苗对三黄鸡的致病性研究［D］.南宁：广西大学，2019.
LI H J.Full⁃length genome sequence analysis of ten subgroup J avian leukosis virus(ALV⁃J)strains isolated in the past five years and the study on pathogenicity of three⁃yellow chickens infected with the ALV⁃J contaminated live vaccines［D］.Nanning：Guangxi University，2019.
［31］吕艳艳.禽白血病病毒通过弱毒疫苗污染途径对鸡免疫机能及生产性能的影响［D］.泰安：山东农业大学，2017.
LÜ Y Y.Effect of avian leukosis virus on chicken immune function and production performance through the attenuated vaccine contamination［D］.Taian：Shandong Agricultural University，2017.
［32］马馨，刘一飞，王志宇，等.晋北散养边鸡群ALV-p27携带率及ALV-A/B与ALV-J亚群的ELISA检测［J］.山西农业科学，2020，48（9）：1523⁃1525.
MA X，LIU Y F，WANG Z Y，et al.ELISA detection of ALV⁃p27 carrying rate，ALV⁃A/B and ALV⁃J subgroups
of free⁃range Bian chickens in northern Shanxi［J］.Journal of Shanxi Agricultural Sciences，2020，48（9）：1523⁃1525.
［33］许曾焜，李凯，刘永振，等.表达禽网状内皮组织增生病病毒gp90蛋白的重组马立克氏病病毒的构建及生物学特性分析［J］.中国预防兽医学报，2020，42（9）：867⁃871.

XU Z K，LI K，LIU Y，et al.Construction and characterization of a recombinant Marek’s disease virus expressing gp90 protein of reticuloendotheliosis virus［J］.Chinese Journal of Preventive Veterinary Medicine，2020，42（9）：867⁃871.
［34］陈瑞爱，罗琼，郭凯，等.整合REV LTR序列的MDV弱毒株的分离鉴定与生物学特性［J］.微生物学报，2014，54（6）：670⁃678.
CHEN R A，LUO Q，GUO K，ea al.Isolation and characterization of a non⁃pathogenic Marek’s disease virus with a naturally inserted REV LTR sequence［J］.Acta Microbiologica Sinica，2014，54（6）：670⁃678.
［35］SU S，CUI N，CUI Z Z，et al.Complete genome sequence of a recombinant Marek’s disease virus field strain with one reticuloendotheliosis virus long terminal repeat insert［J］.Journal of Virology，2012，86（24）：13818⁃13819.
［36］滕丽琼，韦平，宋中宝，等.一株整合有禽反转录病毒长末端重复序列的马立克氏病病毒野毒株的致病性研究［J］.病毒学报，2009，25（5）：376⁃381.
TENG L Q，WEI P，SONG Z B，et al.Evaluation of the pathogenicity of a field isolate of Marek’s disease virus integrated with retroviral long terminal repeat sequence［J］.Chinese Journal of Virology，2009，25（5）：376⁃381.
［37］许晓云，孙爱军，崔言顺，等.带有REV-LTR片段的马立克氏病病毒重组野毒株与超强毒株致病性和横向传播性比较［J］.微生物学报，2009，49（4）：540⁃543.
XU X Y，SUN A J，CUI Y S，et al.Comparison of pathogenicity and horizontal transmission ability between recombinant Marek’s disease virus field strain with REV⁃LTR and a very virulent reference strain［J］.Acta Microbiologica Sinica，2009，49（4）：540⁃543.
［38］李启红，杨承槐，刘丹，等.鸡马立克病活疫苗中污染禽网状内皮组织增生症病毒检测方法的建立［J］.中国兽医杂志，2014，50（11）：81⁃84.
LI Q H，YANG C H，LIU D，et al.Establishment of indirect immunofluorescence assay method for detection of avian reticuloendotheliosis virus contamination in Marek’s disease live vaccine［J］.Chinese Journal of Veterinary Medicine，2014，50（11）：81⁃84.
［39］毕玉彧.活疫苗污染外源性ALV的检测及泛素介导的ALV-J多基因共表达DNA疫苗的构建［D］.南宁：广西大学，2015.
BI Y Y.The detections of exogenous ALVs from the live vaccines and the constructions of the ubiquitin⁃mediated
ALV⁃J multigenes co⁃expressed DNA vaccines［D］.Nanning：Guangxi University，2015.
［40］刘丹，侯力丹，李启红，等.禽用活疫苗外源病毒污染情况的调查研究［J］.中国兽药杂志，2018，52（4）：19⁃25.
LIU D，HOU L，LI Q H，et al.Investigation about the extraneous virus contaminations of the avian live vaccines［J］.Chinese Journal of Veterinary Drug，2018，52（4）：19⁃25.
［41］汪宏才，汪最，罗玲，等.湖北地方鸡种鸡场弱毒疫苗ALV的分离与鉴定［J］.湖北农业科学，2019，58（24）：167⁃169．
WANG H C，WANG Z，LUO L，et al.Isolation and identification of attenuated vaccine ALV from Hubei local chicken breeding farm［J］.Hubei Agricultural Sciences，2019，58（24）：167⁃169.

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Virus Titer Determination and Quality Evaluation of the Commercial Marek’s Disease Vaccines Sold in 2020—2021

2020—2021 年市售马立克病疫苗的效价测定及质量评估

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