鸭肝炎病毒的分离鉴定及ELISA检测抗原、抗体方法的建立
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摘要
鸭病毒性肝炎(Duck virus hepatitis，DVH)是6周龄以下雏鸭的一种急性、接触性、高度致死性的烈性传染病，病原为鸭肝炎病毒(Duck hepatitis virus，DHV)。该病在国内雏鸭群中频频发生，给养禽业生产造成了极大的损失。本实验从一群发病鸭的病料中分离到一株鸭肝炎病毒，并建立了检测鸭肝炎病毒抗原和血清抗体的ELISA快速检测方法，取得了如下结果：
1．鸭病毒性肝炎病毒株的分离鉴定及部分理化特性的研究
对湖北省内某疑似鸭病毒性肝炎感染鸭病料的收集，通过细菌分离、病毒分离、中和试验、动物回归试验、雏鸭保护试验以及病毒的电镜观察，分离到一株鸭病毒性肝炎Ⅰ型病毒株，对分离毒株的部分生物学特性进行了初步研究。
2．鸭病毒性肝炎血清抗体ELISA快速检测方法的建立和初步应用
将通过鸭胚传代的病毒经过纯化后，作为包被抗原，建立了一种检测血清中DHV抗体的间接ELISA方法。通过对ELISA反应条件的系列测定，确定了各组分的最适工作条件：抗原包被浓度为3.75μg，血清稀释约1∶160时，阳性血清OD450值(P)接近1.0，阴性血清值(N)为0.125，P／N值最大．阴性和阳性分界明显；通过对40份阴性鸭血清的检测结果，确定阴阳性临界点为0.2。根据建立的ELISA方法及最佳反应条件，检测了一批雏鸭携带母源抗体及其消长情况以及一批免疫雏鸭的抗体消长情况。结果表明，本试验建立的间接-ELISA法可用于鸭血清中鸭肝炎病毒抗体水平的检测以及未免疫鸭群鸭病毒性肝炎感染情况的检测。
3．鸭病毒性肝炎病毒双抗体夹心ELISA快速检测方法的建立和初步应用
以纯化的鸭抗DHV IgG为包被抗体，兔抗DHV IgG为第二抗体，通过ELISA反应条件的优化选择，建立了检测DHV抗原的夹心ELISA法。结果表明，鸭抗DHV IgG的最佳包被浓度为约2μg／ml，兔抗DHV IgG的最适工作浓度约为3.4μg／ml；与已知鸭病毒性肝炎标准病毒呈强阳性反应，与已知的阴性样品及其它禽类病毒(NDV、IBV、DP、MDPV、IBDV、MDV)无交叉反应；用建立的ELISA方法对97份疑为鸭肝炎的病料进行了检测，结果有73份呈阳性。经过比较测定，夹心ELISA法要比琼脂免疫扩散方法要敏感64倍，说明本方法在检测鸭病毒性肝炎病毒抗原方面具有特异性强、敏感性高的特点。
Duck hepatitis virus (DHV) is the causative agent of duck viral hepatitis,an acute and fatal disease of young ducklings.Virus isolates was obtained through duck embryo allantoic sac inoculation from duckling liver samples from HuBei provinces in china. All these ducklings were diagnosed clinically dying from duck virus hepatitis.and then, development of ELISA for detecting DHV antigent and antibody.and gain some results as follow.
Virus isolates was obtained through embryo allantoic sac inoculation from duckling liver samples from HuBei provinces in china. All these ducklings were diagnosed clinically dying from duck virus hepatitis.Through neutralization test、animal recurrence infectation、young duckling protection experiment and observe virus particle by electron microscope. A strain DHV-Ⅰwas obtainted and identified.
Using the purified virus as coating antigen, an indirect ELISA method was developed.Optimized conditions were as following.The concentration of virus for plate coating was 3.75μg per well.P/N ration reached maximumunder the condition of serum dilution at 1:160,and the demarcation of negative and positive was most obvious at the same dilution.the threshold value of ELISA was 0.2 with OD_(450).It was found use this method to monitor the antibody's level in the young ducks,this assay is sensisive and specific as well.
A sandwich ELISA for detection of DHV antigen was developed by using purified duck anti-DHV IgG as the first antibody coated on the ELISA plate and rabbit anti-DHV IgG regarded as the second antibody. The results showed that the optimum working concentration of duck anti-DHV IgG and rabbit anti-DHV IgG were 2μg/ml and 3.4μg/ml, respectively. The dilution titers of positive DHV sample by ELISA was 32 times higher than that by Agar gel Immunodiffusion (AGID), and other duck hepatitis virus type-Ⅰcan also be detected by this method. No cross reaction was observed with Newcastle disease virus (NDV), infectious bronchitis virus (IBV), duck plague (DP), Muscovy duckling parvovirus(MDPV), infectious bursal disease virus (IBDV), muscovy duck reovirus(MDRV). 73 of 97 suspicious duckling liver samples showing positive detected by this method.
1．鸭病毒性肝炎病毒株的分离鉴定及部分理化特性的研究
对湖北省内某疑似鸭病毒性肝炎感染鸭病料的收集，通过细菌分离、病毒分离、中和试验、动物回归试验、雏鸭保护试验以及病毒的电镜观察，分离到一株鸭病毒性肝炎Ⅰ型病毒株，对分离毒株的部分生物学特性进行了初步研究。
2．鸭病毒性肝炎血清抗体ELISA快速检测方法的建立和初步应用
将通过鸭胚传代的病毒经过纯化后，作为包被抗原，建立了一种检测血清中DHV抗体的间接ELISA方法。通过对ELISA反应条件的系列测定，确定了各组分的最适工作条件：抗原包被浓度为3.75μg，血清稀释约1∶160时，阳性血清OD450值(P)接近1.0，阴性血清值(N)为0.125，P／N值最大．阴性和阳性分界明显；通过对40份阴性鸭血清的检测结果，确定阴阳性临界点为0.2。根据建立的ELISA方法及最佳反应条件，检测了一批雏鸭携带母源抗体及其消长情况以及一批免疫雏鸭的抗体消长情况。结果表明，本试验建立的间接-ELISA法可用于鸭血清中鸭肝炎病毒抗体水平的检测以及未免疫鸭群鸭病毒性肝炎感染情况的检测。
3．鸭病毒性肝炎病毒双抗体夹心ELISA快速检测方法的建立和初步应用
以纯化的鸭抗DHV IgG为包被抗体，兔抗DHV IgG为第二抗体，通过ELISA反应条件的优化选择，建立了检测DHV抗原的夹心ELISA法。结果表明，鸭抗DHV IgG的最佳包被浓度为约2μg／ml，兔抗DHV IgG的最适工作浓度约为3.4μg／ml；与已知鸭病毒性肝炎标准病毒呈强阳性反应，与已知的阴性样品及其它禽类病毒(NDV、IBV、DP、MDPV、IBDV、MDV)无交叉反应；用建立的ELISA方法对97份疑为鸭肝炎的病料进行了检测，结果有73份呈阳性。经过比较测定，夹心ELISA法要比琼脂免疫扩散方法要敏感64倍，说明本方法在检测鸭病毒性肝炎病毒抗原方面具有特异性强、敏感性高的特点。
Duck hepatitis virus (DHV) is the causative agent of duck viral hepatitis,an acute and fatal disease of young ducklings.Virus isolates was obtained through duck embryo allantoic sac inoculation from duckling liver samples from HuBei provinces in china. All these ducklings were diagnosed clinically dying from duck virus hepatitis.and then, development of ELISA for detecting DHV antigent and antibody.and gain some results as follow.
Virus isolates was obtained through embryo allantoic sac inoculation from duckling liver samples from HuBei provinces in china. All these ducklings were diagnosed clinically dying from duck virus hepatitis.Through neutralization test、animal recurrence infectation、young duckling protection experiment and observe virus particle by electron microscope. A strain DHV-Ⅰwas obtainted and identified.
Using the purified virus as coating antigen, an indirect ELISA method was developed.Optimized conditions were as following.The concentration of virus for plate coating was 3.75μg per well.P/N ration reached maximumunder the condition of serum dilution at 1:160,and the demarcation of negative and positive was most obvious at the same dilution.the threshold value of ELISA was 0.2 with OD_(450).It was found use this method to monitor the antibody's level in the young ducks,this assay is sensisive and specific as well.
A sandwich ELISA for detection of DHV antigen was developed by using purified duck anti-DHV IgG as the first antibody coated on the ELISA plate and rabbit anti-DHV IgG regarded as the second antibody. The results showed that the optimum working concentration of duck anti-DHV IgG and rabbit anti-DHV IgG were 2μg/ml and 3.4μg/ml, respectively. The dilution titers of positive DHV sample by ELISA was 32 times higher than that by Agar gel Immunodiffusion (AGID), and other duck hepatitis virus type-Ⅰcan also be detected by this method. No cross reaction was observed with Newcastle disease virus (NDV), infectious bronchitis virus (IBV), duck plague (DP), Muscovy duckling parvovirus(MDPV), infectious bursal disease virus (IBDV), muscovy duck reovirus(MDRV). 73 of 97 suspicious duckling liver samples showing positive detected by this method.
引文
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78. McNulty, M.S., Picornaviridae. In: Jordan, F., Pattison, M., Alexander,D., Faragher, T. (Eds.), Poultry Diseases, fifth ed. W.B. Saunders, London, 2001, pp. 305-318.
79. Murty D K,Hanson IE. A modified microgel diffusion method and its application in the study of the virus of duck hepatitis. Am J Vet Res, 1961. 22:274—278.
80. Park,N.Y. Occurrence of duck virus hepatitis in Korea.Korea J Vet Res, 1985, 25:171-174
81. Rao,S.B.V.,and B.R.Gupta. Studies on a filterable agent causing hepatitis in ducklings,and biliaru cirrhosis and blood dyscrasia in adults.Indian J Poult Sci, 1967, 2:18-30
82. Sandhu T S, Calneck B W, Zenman L. Pathologic and serologic characterization of a variant of duck hepatitis typel virus. Avain Dis, 1992, 36(4): 932—936.
83. Sonja Kothlow,Nina K.Mannes,Beatrice Schaerer,Dierk E.Rebeski,Bernd Kaspers,Ursula Schultz.Characterization of duck leucocytes by monoclonal antibodies.developmental and comparative Immunology.2005,(29):733-748
84. Taylor P. L. and Hanson L. E. Indirect hemagglutination with duck hepatitis virus. Avian Dis, 1967,11(4): 586-592
85. Tempel,E.and J.Beer. Die virus hepatitis derentenJn H.Rohrer(ed.) Handbuch der Virussinfektionen bei Tieren.Gustav Fischer,Jena,Germany. 1968, 1019—1032.
86. Toth T E, Norcross N L. Humoral immune response of the duck to duck hepatitis virus:Virus-neutralizing vs virus-precipitating antibodies. Avian Dis, 1981, 25:17—28.
87. Toth T E. Duck virus hepatitis. Isolation and Identification of Avian Pathogens, In. Hitchner S B, Domermuth C H, Purchase HG, et al(eds. ). 1975:192—196.
88. Vashhenko V. The use of indirect hemagglutination reaction for the diagnosis of virus hepatitis of ducklings. Eksp Inf Inst Ptits, 1982, 109:32-34
89. Wachendorfer,G. Das Agar-prazipitationsverfahren bei derEnten-hepatitis,der Newcastle-Krankheit und Grenzen in der Virusdiagnostik.Zentralbl 1965, Veterinaermed.l2:55 -56.
90. Woolcock PR. An assay for duck hepatitis virus type I in duck embryo liver cells and a comparison with other assays. Avian Pathol, 1986.15:75-82
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79. Murty D K,Hanson IE. A modified microgel diffusion method and its application in the study of the virus of duck hepatitis. Am J Vet Res, 1961. 22:274—278.
80. Park,N.Y. Occurrence of duck virus hepatitis in Korea.Korea J Vet Res, 1985, 25:171-174
81. Rao,S.B.V.,and B.R.Gupta. Studies on a filterable agent causing hepatitis in ducklings,and biliaru cirrhosis and blood dyscrasia in adults.Indian J Poult Sci, 1967, 2:18-30
82. Sandhu T S, Calneck B W, Zenman L. Pathologic and serologic characterization of a variant of duck hepatitis typel virus. Avain Dis, 1992, 36(4): 932—936.
83. Sonja Kothlow,Nina K.Mannes,Beatrice Schaerer,Dierk E.Rebeski,Bernd Kaspers,Ursula Schultz.Characterization of duck leucocytes by monoclonal antibodies.developmental and comparative Immunology.2005,(29):733-748
84. Taylor P. L. and Hanson L. E. Indirect hemagglutination with duck hepatitis virus. Avian Dis, 1967,11(4): 586-592
85. Tempel,E.and J.Beer. Die virus hepatitis derentenJn H.Rohrer(ed.) Handbuch der Virussinfektionen bei Tieren.Gustav Fischer,Jena,Germany. 1968, 1019—1032.
86. Toth T E, Norcross N L. Humoral immune response of the duck to duck hepatitis virus:Virus-neutralizing vs virus-precipitating antibodies. Avian Dis, 1981, 25:17—28.
87. Toth T E. Duck virus hepatitis. Isolation and Identification of Avian Pathogens, In. Hitchner S B, Domermuth C H, Purchase HG, et al(eds. ). 1975:192—196.
88. Vashhenko V. The use of indirect hemagglutination reaction for the diagnosis of virus hepatitis of ducklings. Eksp Inf Inst Ptits, 1982, 109:32-34
89. Wachendorfer,G. Das Agar-prazipitationsverfahren bei derEnten-hepatitis,der Newcastle-Krankheit und Grenzen in der Virusdiagnostik.Zentralbl 1965, Veterinaermed.l2:55 -56.
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91. Woolcock, P.R., 2003. Duck hepatitis. In: Saif, Y.M., Barnes, H.J., Glisson, J.R.,Fadly, A.M., McDougald, L.R., Swayne, D.E. (Eds.), Diseases of Poultry,llth ed. Iowa State Press, Ames, IA, pp. 343-354
92. Zhao XL, PhilliPs RM , Li G D. Studies on the detect ion of antibody to duck hepatitis virus by enzyme—linked immunosorbent assay.Avain Dis, 1991,35(4): 778-782