茶花鸡MHC单倍型及其对HPAI免疫应答水平影响的测定
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摘要
为筛选茶花鸡H5N1高致病性禽流感(HPAI)抗性MHC单倍型,本实验对293只脱温茶花鸡先后两次接种同一HPAI灭活疫苗(0.4 m L/只),二次免疫前、后采集抗凝外周血,分别应用病毒血凝抑制(HI)试验和夹心酶联免疫吸附试验(ELISA)测定血浆特异性抗体效价和IFN-γ水平;通过PCR扩增分析微卫星LEI0258和MCW0371,根据扩增产物的长度多态性确定个体MHC单倍型及基因型。选取频数不少于5的MHC单倍型或基因型,对其与抗体效价和IFN-γ水平的关联性分别做单因素方差分析。结果显示,应用PCR扩增LEI0258获得50种长度多态性片段,包括已有命名的20种和新发现的30种;二免后茶花鸡实验群体特异性抗体效价和IFN-γ水平均极显著高于二次免疫前(p<0.01); 249 bp、307 bp、319 bp携带组和B17单倍型纯合子组二次免疫后抗体效价和IFN-γ水平均显著高于其它组(p<0.05)。本实验初步判定249 bp、307 bp、319 bp携带者和B17单倍型纯合子具有较高的HPAI免疫应答水平,提示其可以作为进一步鉴定HPAI抗性的候选MHC单倍型,有望为建立HPAI控制新策略提供思路。
In order to screening MHC haplotypes of H5N1 highly pathogenic avian influenza(HPAI)resistant from Chahua chickens,a total of 293 birds were inoculated twice with 0.4mL dose of the same inactivated HPAI vaccine,Peripheral blood with anticoagulant was collected before and after the 2nd immunization and the specific antibody titers and IFN-γlevels in the plasma were examined by viral hemagglutination inhibition(HI)assay and ELISA,respectively.Microsatellite LEI0258 and MCW0371were analyzed by PCR,individual MHC haplotypes and genotypes were determined according to the length polymorphism of microsatellite.Finally,those MHC haplotypes and genotypes with a frequency no fewer than 5 were selected and subjected to one-way analysis of variance for possible association with antibody titers or IFN-γlevels.Consequently,the PCR amplification of LEI0258 resulted in 50 length polymorphism fragments,of which 20 had been given nomenclature and the other 30 were newly discovered.Both specific antibody titer and IFN-γlevel were significantly higher post booster immunization than those prior to it(p<0.01)in the bird population.One-way ANOVA revealed that chicken groups bearing 249bp,307bp,319bp and B17homozygous produced significantly higher antibody titers and IFN-γlevel than the other groups after 2nd immunization(p<0.05).These findings preliminarily demonstrate that 249bp,307bp,319bp carriers and B17 homozygotes had stronger acquired immune response to HPAI,indicating their role as candidates for identification of HPAI-resistant MHC haplotypes in further,it is expected to provide ideas for establishment of a new strategy for HPAI control.
In order to screening MHC haplotypes of H5N1 highly pathogenic avian influenza(HPAI)resistant from Chahua chickens,a total of 293 birds were inoculated twice with 0.4mL dose of the same inactivated HPAI vaccine,Peripheral blood with anticoagulant was collected before and after the 2nd immunization and the specific antibody titers and IFN-γlevels in the plasma were examined by viral hemagglutination inhibition(HI)assay and ELISA,respectively.Microsatellite LEI0258 and MCW0371were analyzed by PCR,individual MHC haplotypes and genotypes were determined according to the length polymorphism of microsatellite.Finally,those MHC haplotypes and genotypes with a frequency no fewer than 5 were selected and subjected to one-way analysis of variance for possible association with antibody titers or IFN-γlevels.Consequently,the PCR amplification of LEI0258 resulted in 50 length polymorphism fragments,of which 20 had been given nomenclature and the other 30 were newly discovered.Both specific antibody titer and IFN-γlevel were significantly higher post booster immunization than those prior to it(p<0.01)in the bird population.One-way ANOVA revealed that chicken groups bearing 249bp,307bp,319bp and B17homozygous produced significantly higher antibody titers and IFN-γlevel than the other groups after 2nd immunization(p<0.05).These findings preliminarily demonstrate that 249bp,307bp,319bp carriers and B17 homozygotes had stronger acquired immune response to HPAI,indicating their role as candidates for identification of HPAI-resistant MHC haplotypes in further,it is expected to provide ideas for establishment of a new strategy for HPAI control.
引文
[1]焦同路,王靖飞,赵双成,等.表达H5亚型禽流感病毒HA蛋白的重组禽痘病毒的构建[J].中国预防兽医学报,2012,34(5):341-344.
[2]Desvaux S,Marx N,Ong S,et al.Highly pathogenic avian influenza virus(H5N1)outbreak in captive wild birds and cats,Cambodia[J].Emerg Infect Dis,2009,15(3):475-478.
[3]张立凡,连林生,闭兴明,等.茶花鸡种群遗传结构分析及其保种措施的探讨[J].畜牧与兽医,2006,38(11):10-13.
[4]叶朗惠,苗永旺,霍金龙,等.茶花鸡群体遗传多样性[J].动物学杂志,2006,41(2):37-42.
[5]包文斌,陈国宏,李碧春,等.红色原鸡和中国家鸡遗传多样性及亲缘关系[J].中国科学C辑:生命科学,2008,38(1):43-51.
[6]Blankert J J,Albers G A,Briles W E,et al.The effect of serologically defined major histocompatibility complex haplotypes on Marek's disease resistance in commercially bred White Leghorn chickens[J].Avian Dis,1990,34(4):818-823.
[7]Miller T K,Bowman D D,Schat K A.Inhibition of the in vitro development of Eimeria tenella in chick kidney cells by immune chickens plenocytes[J].Avian Dis,1994,38(3):418-427.
[8]李启军,宋超,徐超,等.我国家养雉鸡MHC B-F基因表达量测定与分析[J].吉林农业大学学报,2017,10.13327/j.jjlau.2017.3924
[9]Bacon L D.Influence of the major histocompatibility complex on disease resistance and productivity[J].Poult Sci,1987,66(5):802-811.
[10]Abd-Ellatieff H A,Abou R A A,Ellakany H F,et al.Molecular characterization and phylogenetic analysis of a virulent Marek's disease virus field strain in broiler chickens in Japan[J].Avian Pathol,2017,47(1):47-57.
[11]Fulton J E,Young E E,Bacon L D.Chicken MHC alloantiserum cross-reactivity analysis by hemagglutination and flow cytometry[J].Immunogenetics,1996,43:277-288.
[12]Miller M M,Abplanalp H,Goto R.Genotyping chickens for the B-G,subregion of the major histocompatibility complex using restriction fragment length polymorphisms[J].Immunogenetics, 1988,28:374-379.
[13]Goto R M,Afanassieff M,Ha J,et al.Single-strand conformation polymorphism(SSCP)assays for major histocompatibility complex B genotyping in chickens[J].Poult Sci,2002,81:1832-1841.
[14]Nikbakht G,Esmailnejad A,Barjesteh N.LEI0258 microsatellite variability in Khorasan,Marandi,and Arian chickens[J].Biochem Genet,2013,51:341-349.
[15]Han Bo,Lian Ling,Qu Lu-jiang,et al.Abundant polymorphisms at the microsatellite locus LEI0258in indigenous chickens[J].Poultry Sci,2013,92:3113-3119.
[16]Nikbakht G,Esmailnejad A.Chicken major histocompatibility complex polymorphism and its association with production traits[J].Immunogenetics,2015,67(4):247-252.
[17]徐日福.中国部分地方鸡种MHC B-LBⅡ、B-G基因变异及其群体遗传结构研究[D].武汉:华中农业大学,2005.
[18]Gillingham M A,Béchet A,Courtiol A,et al.Very high MHCClass IIB diversity without spatial differentiation in the mediterranean population of greater Flamingos[J].Bmc Evol Biol,2017,17(1):56.
[19]张富寿.鸡主要组织相容性复合体(MHC)研究进展[J].中兽医学杂志,2017,(05):100-101.
[2]Desvaux S,Marx N,Ong S,et al.Highly pathogenic avian influenza virus(H5N1)outbreak in captive wild birds and cats,Cambodia[J].Emerg Infect Dis,2009,15(3):475-478.
[3]张立凡,连林生,闭兴明,等.茶花鸡种群遗传结构分析及其保种措施的探讨[J].畜牧与兽医,2006,38(11):10-13.
[4]叶朗惠,苗永旺,霍金龙,等.茶花鸡群体遗传多样性[J].动物学杂志,2006,41(2):37-42.
[5]包文斌,陈国宏,李碧春,等.红色原鸡和中国家鸡遗传多样性及亲缘关系[J].中国科学C辑:生命科学,2008,38(1):43-51.
[6]Blankert J J,Albers G A,Briles W E,et al.The effect of serologically defined major histocompatibility complex haplotypes on Marek's disease resistance in commercially bred White Leghorn chickens[J].Avian Dis,1990,34(4):818-823.
[7]Miller T K,Bowman D D,Schat K A.Inhibition of the in vitro development of Eimeria tenella in chick kidney cells by immune chickens plenocytes[J].Avian Dis,1994,38(3):418-427.
[8]李启军,宋超,徐超,等.我国家养雉鸡MHC B-F基因表达量测定与分析[J].吉林农业大学学报,2017,10.13327/j.jjlau.2017.3924
[9]Bacon L D.Influence of the major histocompatibility complex on disease resistance and productivity[J].Poult Sci,1987,66(5):802-811.
[10]Abd-Ellatieff H A,Abou R A A,Ellakany H F,et al.Molecular characterization and phylogenetic analysis of a virulent Marek's disease virus field strain in broiler chickens in Japan[J].Avian Pathol,2017,47(1):47-57.
[11]Fulton J E,Young E E,Bacon L D.Chicken MHC alloantiserum cross-reactivity analysis by hemagglutination and flow cytometry[J].Immunogenetics,1996,43:277-288.
[12]Miller M M,Abplanalp H,Goto R.Genotyping chickens for the B-G,subregion of the major histocompatibility complex using restriction fragment length polymorphisms[J].Immunogenetics, 1988,28:374-379.
[13]Goto R M,Afanassieff M,Ha J,et al.Single-strand conformation polymorphism(SSCP)assays for major histocompatibility complex B genotyping in chickens[J].Poult Sci,2002,81:1832-1841.
[14]Nikbakht G,Esmailnejad A,Barjesteh N.LEI0258 microsatellite variability in Khorasan,Marandi,and Arian chickens[J].Biochem Genet,2013,51:341-349.
[15]Han Bo,Lian Ling,Qu Lu-jiang,et al.Abundant polymorphisms at the microsatellite locus LEI0258in indigenous chickens[J].Poultry Sci,2013,92:3113-3119.
[16]Nikbakht G,Esmailnejad A.Chicken major histocompatibility complex polymorphism and its association with production traits[J].Immunogenetics,2015,67(4):247-252.
[17]徐日福.中国部分地方鸡种MHC B-LBⅡ、B-G基因变异及其群体遗传结构研究[D].武汉:华中农业大学,2005.
[18]Gillingham M A,Béchet A,Courtiol A,et al.Very high MHCClass IIB diversity without spatial differentiation in the mediterranean population of greater Flamingos[J].Bmc Evol Biol,2017,17(1):56.
[19]张富寿.鸡主要组织相容性复合体(MHC)研究进展[J].中兽医学杂志,2017,(05):100-101.