| Exogenous chicken anemia virus (CAV) has been detected in commercial poultry vaccines in various countries of the world. The presence of unwanted CAV in vaccines not only influences the epidemiology of chicken infectious anemia disease, but may also lead to vaccine failure and confusing results when vaccine responses are monitored. To detect CAV in contaminated vaccines, nucleic acid testing (unlike conventional testing) has a shorter processing time and does not require cell culture or live animals. The aim of the current study was to develop a TaqMan real-time polymerase chain reaction (PCR) assay to detect and quantify CAV in poultry vaccines and investigate CAV contamination in Razi live Newcastle disease vaccines. The TaqMan real-time PCR assay was set up, optimized, and validated in successive experiments. A standard plasmid pUC-VP2 containing viral protein 2 of CAV was constructed and used in the assay to generate a standard curve to quantify CAV genomes. A clear linear correlation was observed between threshold cycle (Ct) values and plasmid copy numbers in the amplification plots of 10-fold serial dilution of the plasmid. Total DNA of three samples of each of four different Razi live Newcastle disease vaccines, namely LaSota, B1, clone.12IR, and thermo-resistant strains, were extracted and subjected to real-time PCR assay. No CAV contamination was detected in the Razi Live Newcastle vaccines. The developed TaqMan real-time PCR assay provides a quick, specific, and sensitive method for use in detecting CAV in quality control vaccine testing and viral load studies. |
- Schat KA, van Santen VL. Chicken Infectious Anemia and Circovirus Infections in Commercial Flocks. In: Swanye DE, Boulianne M, Logue CM, McDougald LR, N. V, D.L. S, editors. Diseases of Poultry. 14 ed. Hoboken, NJ, USA: Wiley-Blackwell; 2020. p. 284-32.
- McNulty MS. Chicken anaemia agent: a review. Avian Pathol: J WVPA. 1991;20(2):187-203.
- Brentano L, Lazzarin S, Bassi SS, Klein TA, Schat KA. Detection of chicken anemia virus in the gonads and in the progeny of broiler breeder hens with high neutralizing antibody titers. Vet Microbiol. 2005;105(1):65-72.
- Cardona C, Lucio B, O'Connell P, Jagne J, Schat KA. Humoral immune responses to chicken infectious anemia virus in three strains of chickens in a closed flock. Avian Dis. 2000;44(3):661-7.
- Cardona CJ, Oswald WB, Schat KA. Distribution of chicken anaemia virus in the reproductive tissues of specific-pathogen-free chickens. J Gen Virol. 2000;81(Pt 8):2067-75.
- Barrios PR, Marín Gómez S, Rios R, Pereira CG, Resende M, Resende JS, et al. A retrospective PCR investigation of avian Orthoreovirus, chicken infectious anemia and fowl Aviadenovirus genomes contamination in commercial poultry vaccines in Brazil. Arq Bras Med Vet Zootec. 2012;64:231-5.
- Li Y, Hu Y, Cui S, Fu J, Wang Y, Cui Z, et al. Molecular characterization of chicken infectious anemia virus from contaminated live-virus vaccines. Poult Sci. 2017;96(5):1045-51.
- Marín Gómez S, Barrios P, Rios R, Resende M, Resende J, Santos B, et al. Molecular Characterization of Contaminating Infectious Anemia Virus of Chickens in Live Commercial Vaccines Produced in the 1990s. Avian Dis. 2013;57:15-21.
- Varela AP, Dos Santos HF, Cibulski SP, Scheffer CM, Schmidt C, Sales Lima FE, et al. Chicken anemia virus and avian gyrovirus 2 as contaminants in poultry vaccines. Biologicals. 2014;42(6):346-50.
- European Pharmacopoeia. Chicken flocks free from specified pathogens for the production and qulaity control of vaccines. 1. Strasbourg Cedex, France: Directorate for the Quality of Medicines & HealthCare, Council of Europe; 2013. p. 579-82.
- Ottiger HP. Development, standardization and assessment of PCR systems for purity testing of avian viral vaccines. Biologicals. 2010;38(3):381-8.
- Kaffashi A, Eshratabadi F, Shoushtari A. Full-length infectious clone of an Iranian isolate of chicken anemia virus. Virus Genes. 2017;53(2):312-6.
- Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870-4.
- Rychlik W. OLIGO 7 primer analysis software. Methods Mol Biol. 2007;402:35-60.
- Kaffashi A, Noormohammadi AH, Allott ML, Browning GF. Viral load in 1-day-old and 6-week-old chickens infected with chicken anaemia virus by the intraocular route. Avian Pathol. 2006;35(6):471-4.
- European Pharmacopoeia. Avian viral vaccines: tests for extraneous agents in seed lots. 1. 8th ed. Strasbourg Cedex, France: Directorate for the Quality of Medicines & HealthCare, Council of Europe; 2013. p. 209-12.
- Li Q, Zhang Y, Meng F, Jiang H, Xu G, Ding J, et al. A New Strategy for the Detection of Chicken Infectious Anemia Virus Contamination in Attenuated Live Vaccine by Droplet Digital PCR. Biomed Res Int. 2019;2019:2750472.
- Su Q, Wang T, Meng F, Cui Z, Chang S, Zhao P. Synergetic pathogenicity of Newcastle disease vaccines LaSota strain and contaminated chicken infectious anemia virus. Poult Sci. 2019;98(5):1985-92.
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