The Development and Application of an Indirect ELISA Test for the Detection of Chicken Anaemia Virus (CAV) by VP1 in Chicken Flock Serum


Chicken anaemia virus (CAV) causes a viral disease in chickens worldwide and thus has economic importance. The main aim of this study was to develop a rapid, sensitive and specific VP1-CAVI indirect ELISA for the detection of CAV infection. The CAV-VP1, was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV-VP1 protein was then coated as an antigen on an ELISA plates to evaluate its reactivity against chicken sera. The resulting indirect ELISA was then compared with a commercial ELISA. The specificity and sensitivity of the indirect ELISA were measured as 93.3% and 100%, respectively. A t-test produced a t-value of 15.805 for the indirect ELISA and revealed a significant difference between CAV-positive serum and CAV-negative serum (p-value of 0.001). For the second variable (i.e., a commercial ELISA), the t-test yielded a t-value of 5.063, which revealed a significant difference between CAV-positive serum and CAV-negative serum (p-value of 0.015). This intervention produces statistically significant improvements in both variables (p-values < 0.05). The correlation coefficient for the indirect ELISA was r = 0.93. Therefore, this work can be considered as a new achievement in diagnosis for Chicken anaemia virus in chicken flocks.

Share and Cite:

Mahgoub, E. (2014) The Development and Application of an Indirect ELISA Test for the Detection of Chicken Anaemia Virus (CAV) by VP1 in Chicken Flock Serum. Open Journal of Genetics, 4, 316-331. doi: 10.4236/ojgen.2014.44029.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Yuasa, N., Taniguchi, T., Imada, T. and Hihara, H. (1983) Distribution of Chicken Anemia Agent (CAA) and Detection of Neutralizing Antibody in Chicks Experimentally Inoculated with CAA. National Institute of Animal Health Quarterly (Tokyo), 23, 78-81.
[2] Engstrom, B.E. (1988) Blue Wing Disease of Chickens: Isolation of Avian Reovirus and Chicken Anaemia Agent. Avian Pathology, 17, 23-32.
[3] Lurkert, P., Boer, D. and McNulty, M.S. (1995) Virus Taxonomy-Classification and Nomenclature of Viruses: Sixth Report of International Committee on Taxonomy of Viruses. Springer-Verlag, Vienna, 25, 166-168.
[4] McNulty, M.S., Curran, W.L., Todd, D. and Mackie, D.P. (1990) Chicken Anemia Agent: An Electron Microscopic Study. Avian Diseases, 34, 736-743.
[5] Todd, D., Mawhinney, K.A. and McNulty, M.S. (1990) Detection and Differentiation of Chicken Anemia Virus Isolates by Using the Polymerase Chain Reaction. Journal of Clinical Microbiology, 30, 1661-1666.
[6] Noteborn, M.H.M., de Boer, G.F., van Roozelaar, D.J., Karreman, C., Kranenburg, O., Vos, J.G., Jeurissen, S.H.M., Hoeben, R.C., Zantema, A., Koch, G., van Ormondt, H. and van der Eb, A.J. (1991) Characterization of Cloned Chicken Anemia Virus DNA that Contains All Elements for the Infectious Replication cycle. Journal of Virology, 65, 3131-3139.
[7] Cardona, C.J. and Schat, W.B. (2000) Distribution of Chicken Anaemia Virus in Reproductive Tissues of Specific Pathogen Free Chicken. Journal of General Virology, 45, 2067-2072.
[8] Koch, G., Van Roozelaar, D.J., Verschueren, C.A.J., Vander, E.A.J. and Neteborne, M.H.M. (1995) Immunogenic and Protective Properties of Chicken Anaemia Virus Protein Expressed by Baculovirus. Vaccine, 13, 763-770.
[9] Novak, R. and Ragland, W.L. (2001) Competitive DNA Hybridization in Microtitre Plates for Chicken Anaemia Virus. Molecular and Cellular Probes, 15, 1-11.
[10] Mcllory, S.G., McNulty, M.S., Bruce, D.W., Smyth, J.A., Goodall, E.A. and Alcorn, M.J. (1992) Economic Effect of Clinical Chicken Anaemia Agent Infection on Profitable Broiler Production. Avian Diseases, 36, 566-574.
[11] McNulty, M.S., Connor, T.J., McNeilly, F. and Spackman, D. (1989) Chicken Anemia Agent in the United States: Isolation of the Virus and Detection of Antibody in Broiler Breeder Flocks. Avian Diseases, 33, 691-694.
[12] Kurian, A.J. and Lackner, C. (2011) Effects of Blood Sample Mishandling on ELISA Results for Infectious Bronchitis Virus, Avian Encephalomyelitis Virus and Chicken Anaemia Virus. The Veterinary Journal, 192, 378-381.
[13] Brentano, L., Lazzarin, S., Bassi, S.S., Klein, T.A.P. and Schat, K.A. (2005) Detection of Chicken Anemia Virus in the Gonads and in the Progeny of Broiler Breeder Hens with High Neutralizing Antibody Titers. Veterinary Microbiology, 105, 65-72.
[14] Todd, D., Creelan, J.L. and McNulty, M.S. (1991) Dot Blot Hybridization Assay for Chicken Anemia Agent Using a Cloned DNA Probe. Journal of Clinical Microbiology, 29, 933-939.
[15] Lien, Y.-Y., Huang, C.-H., Sun, F.-C., Sheu, S.-C., Lu, T.-C., Lee, M.-S., Hsueh, S.-C., Chen, H.-J. and Lee, M.-S. (2012) Development and Characterization of a Potential Diagnostic Monoclonal Antibody against Capsid Protein VP1 of the Chicken Anemia Virus. Journal of Veterinary Science, 13, 73-79.
[16] Sun, S.Q., Guo, H.C., Sun, D.H., Yin, S.H., Shang, Y.J., Cai, X.P. and Liu, X.T. (2010) Development and Validation of an ELISA Using a Protein Encoded by ORF2 Antigenic Domain of Porcine Circovirus Type 2. Virology Journal, 7, 274-280.
[17] Kessler, E.H., Matthes, E. and Schein, M.W. (2003) Detection of Antibodies in Sera of Weaned Pigs after Contact Infection with Sarcoptes scabiei var. suis and after Treatment with an Antiparasitic Agent by Three Different Indirect ELISAs. Veterinary Parasitology, 114, 63-73.
[18] Schlosser, U., Lackner, K.J., Scheckenhofer, C. and Schmitz, G. (1996) Calpastatin Autoantibodies: Detection, Epitope Mapping, and Development of a Specific Peptide ELISA. Clinical Chemistry, 42, 1250-1256.
[19] Köhler, G. and Milstein, C. (1976) Derivation of Specific Antibody Producing Tissue Culture and Tumor Lines by Cell Fusion. European Journal of Immunology, 6, 511-519.
[20] Anon (1998) Rules and Guidelines of the Australian Johne’s Disease Market Assurance Program for Cattle. Australian Animal Health Council Ltd and Veterinary Committee, 14, 54.
[21] Iwata, N., Fujino, M., Tuchiya, K., Iwata, A., Otaki, Y. and Ueda, S. (1998) Development of an Enzyme-Linked Immunosorbent Assay Using Recombinant Chicken Anemia Virus Proteins Expressed in a Baculovirus Vector System. Journal of Veterinary Medical Science, 60, 175-180.
[22] Pallister, J., Fahey, K.J. and Sheppard, M. (1994) Cloning and Sequencing of the Chicken Anaemia Virus (CAV) ORF-3 Gene, and the Development of an ELISA for the Detection of Serum Antibody to CAV. Veterinary Microbiology, 39, 167-178.
[23] Huang, C.H., Lai G.H., Lee M.S., Lin, W.H., Lien, Y.Y., Hsueh, S.C., Kao, J.Y., Chang, W.T., Lu, T.C., Lin, W.N., Chen, H.J. and Lee, M.S. (2010) Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Chicken Anaemia Virus. Journal of Applied Microbiology, 108, 917-924.
[24] Lee, M.S., Lien, Y.Y., Feng, S.H., Huang, R.L., Tsai, M.C., Chang, W.T. and Chen, H.J. (2009) Production of Chicken Anemia Virus (CAV) VP1 and VP2 Protein Expressed by Recombinant Escherichia coli. Process Biochemistry, 44, 390-395.
[25] Rourke, D., Michalski, W.P. and Bagust, T.J. (1994) Chicken Anaemia Virus Antibody ELISA Reactions Practical Experiences and Problems in High-Security SPF Flocks. Avian Diseases, 12, 456-464.
[26] Daum, I., Finsterbusch, T., Härtle, S., Göbel, T.W., Mankertz, A., Korbel, R. and Grund, C. (2009) Cloning and Expression of a Truncated Pigeon Circovirus Capsid Protein Suitable for Antibody Detection in Infected Pigeons. Avian Pathology, 38, 135-141.
[27] Lou, Z., Li, X., Li, Z., Yin, X., Li, B., Lan, X., Yang, B., Zhang, Y. and Liu, J. (2011) Expression and Antigenicity Characterization for Truncated Capsid Protein of Porcine Circovirus Type 2. Canadian Journal of Veterinary Research, 75, 61-64.
[28] Shapira, R., Paster, N., Menasherov, M., Eyal, O., Mett, A., Meiron, T., Kuttin, E. and Salomon, R. (1997) Development of Polyclonal Antibodies for Detection of Aflatoxigenic Molds Involving Culture Filtrate and Chimeric Proteins Expressed in Escherichia Coli. Applied and Environmental Microbiology, 15, 990-995.
[29] Doumit, S.M., Calpastatin, M.E., Lonergan, J.R., Killefer, A.J. and Ohmaraie, M. (1996) Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Quantification of Skeletal Muscle. Journal of Animal Science, 74, 2679-2686.
[30] Gillette, K.G. (1983) Enzyme-Linked Immunosorbent Assay Used to Monitor Serum Antibodies to Bovine Respiratory Syncytial Virus: Comparison with Complement Fixation and Neutralization Tests. American Journal of Veterinary Research, 44, 2251-2255.
[31] Kovarcik, K. (2001) The Development and Application of an Indirect ELISA Test for the Detection of Antibodies to Bovine Respiratory Syncytial Virus in Blood Serum. Veterinary Medicine Czech, 46, 29-34.
[32] Kimman, T.G., Westebrink, F., Schreuder, B.E.C. and Straver, P.J. (1987) Local and Systematic Antibody Response to Bovine Respiratory Syncytial Virus Infection and Reinfection in Calves with and without Maternal Antibodies. Journal of Clinical Microbiology, 2, 1097-1106.
[33] Florent, G. and Wiseman, A. (1990) An IgM specific ELISA for the Serodiagnostics of Viral Bovine Infections. Comparative Immunology, Microbiology and Infectious Diseases, 13, 203-208.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.