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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC AND BIOLOGICAL DETERMINANTS OF AVIAN TUMOR VIRUS PATHOGENICITY, TRANSMISSION, AND EVOLUTION

Location: Avian Disease and Oncology Laboratory

Title: Detection and differentiation of CVI988 (Rispens vaccine) from other serotype 1 Marek’s disease viruses

Authors
item Gimeno, Isabel -
item Dunn, John
item Cortes, Aneg -
item El-Gohary, Abd El-Galil -
item Silva, Robert

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 13, 2014
Publication Date: January 13, 2014
Citation: Gimeno, I.M., Dunn, J.R., Cortes, A.L., El-Gohary, A., Silva, R.F. 2014. Detection and differentiation of CVI988 (Rispens vaccine) from other serotype 1 Marek’s disease viruses. Avian Diseases. 58:232-243.

Interpretive Summary: Marek’s disease (MD) is a lymphoproliferative disease of susceptible chickens. Currently, CVI988 is the best vaccine to protect chickens against MD. To monitor vaccination strategies, it is important to be able to detect and differentiate CVI988 from Marek’s disease virus (MDV), the causative agent for MD. However, there are no reliable means to do this. We developed a simple assay that will allow anyone to specifically detect CVI988. We used this assay to detect CVI988 when it was in a mixture of MDVs.

Technical Abstract: The serotype 1 Marek’s disease virus (MDV) is the causative agent for Marek’s disease (MD), a lymphoproliferative disease of chickens of great concern to the poultry industry. CVI988, an attenuated serotype 1 MDV, is currently the most efficacious commercially available vaccine for preventing MD. However, it is difficult to detect and differentiate CVI988 when other serotype 1 MDVs are present. To facilitate the detection of CVI988, we developed two sets of primers for a mismatch amplification mutation assay (MAMA) PCR that targeted the SNP associated with the H19 epitope of the pp38 gene. The PCR was very specific. One primer set (oncogenic primers) amplified DNA from 15 different serotype 1 MDVs except CVI988. The other primer set (CVI988 primers) amplified DNA from CVI988 but not from any of the other 15 serotype 1 MDVs. A real time PCR assay was developed using MAMA primers and specificity and sensitivity was evaluated in vitro and in vivo. Mixtures of plasmids (CVI988 plasmid and oncogenic plasmid) at various concentrations were used to evaluate the sensitivity/specificity of MAMA primers in vitro. Both primer sets were able to amplify as little as one copy of their respective plasmid. Oncogenic primers were highly specific and only amplified CVI988 plasmid when the concentration of oncogenic plasmid was very low (1 x 101) and CVI988 plasmid was very high (1 x 106). Specificity of CVI988 primers was not as high since they could amplify oncogenic plasmids when the concentration of CVI988 plasmid was 1 x 103 and the concentration of oncogenic 1 x 102. Validation of MAMA primers in in vivo samples demonstrated that oncogenic primers can be used for both early diagnosis of MD in feather pulp samples collected at 3 weeks of age and confirmation of MD diagnosis in tumors. CVI988 primers could be used to monitor CVI988 vaccination in samples with low load of oncogenic MDV DNA (latently infected samples or negative) but not in samples with high load of oncogenic MDV DNA (tumors). Our results suggest that monitoring CVI988 vaccination in feather pulp samples collected at one week of age ensures the specificity of the CVI988 primers.

Last Modified: 7/24/2014
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