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8 January 2013 Development, Application, and Results of Routine Monitoring of Marek's Disease Virus in Broiler House Dust Using Real-Time Quantitative PCR
Stephen W. Walkden-Brown, Aminul Islam, Peter J. Groves, Ambrosio Rubite, Sue M. Sharpe, Susan K. Burgess
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Results are presented from four studies between 2002 and 2011 into the feasibility of routinely monitoring Marek's disease virus serotype 1 (MDV-1) in broiler house dust using real-time quantitative PCR (qPCR) measurement. Study 1 on two farms showed that detection of MDV-1 occurred earlier on average in dust samples tested using qPCR than standard PCR and in spleen samples from five birds per shed assayed for MDV-1 by qPCR or standard PCR. DNA quality following extraction from dust had no effect on detection of MDV-1. Study 2 demonstrated that herpesvirus of turkeys (HVT) and MDV serotype 2 (MDV-2) in addition to MDV-1 could be readily amplified from commercial farm dust samples, often in mixtures. MDV-2 was detected in 11 of 20 samples despite the absence of vaccination with this serotype. Study 3 investigated the reproducibility and sensitivity of the qPCR test and the presence of inhibitors in the samples. Samples extracted and amplified in triplicate showed a high level of reproducibility except at very low levels of virus near the limit of detection. Mixing of samples prior to extraction provided results consistent with the proportions in the mixture. Tests for inhibition showed that if the template contained DNA in the range 0.5–20 ng/µl no inhibition of the reaction was detectable. The sensitivity of the tests in terms of viral copy number (VCN) per milligram of dust was calculated to be in the range 24–600 VCN/mg for MDV-1, 48–1200 VCN/mg for MDV-2, and 182–4560 VCN/mg for HVT. In study 4 the results of 1976 commercial tests carried out for one company were analyzed. Overall 23.1% of samples were positive for MDV-1, 26.1% in unvaccinated and 16.4% in vaccinated chickens. There was marked regional and temporal variation in the proportion of positive samples and the MDV-1 load. The tests were useful in formulating Marek's disease vaccination strategies. The number of samples submitted has increased recently, as has the incidence of positive samples. These studies provide strong evidence that detection and quantitation of MDV-1, HVT, and MDV-2 in poultry house dust using qPCR is robust, sensitive, reproducible, and meaningful, both biologically and commercially. Tactical vaccination based on monitoring of MDV-1 rather than routine vaccination may reduce selection pressure for increased virulence in MDV-1.

Desarrollo, aplicación y resultados de un muestreo rutinario del virus de la enfermedad de Marek en polvo de casetas avícolas mediante PCR cuantitativo en tiempo real.

Se presentan los resultados de cuatro estudios entre los años 2002 y 2011 sobre la viabilidad de un muestreo para la enfermedad de Marek serotipo 1 (MDV-1) llevado a cabo en polvo de casetas avícolas de pollo de engorde mediante PCR cuantitativo en tiempo real (qPCR). El estudio 1 llevado a cabo en dos granjas mostró que la detección de MDV-1 en promedio ocurría antes en las muestras de polvo con el método cuantitativo de PCR en comparación con el convencional y en las muestras de bazo de cinco aves por caseta analizadas para detectar al virus de Marek 1 por PCR en tiempo real o por PCR convencional. La calidad del ADN después de la extracción a partir de polvo no tuvo ningún efecto sobre la detección del virus de Marek 1. El estudio 2 demostró que el herpesvirus de pavos (HVT) y el serotipo 2 del virus de Marek (MDV-2), además del serotipo1 podrían ser fácilmente amplificados a partir de muestras de polvo de granjas comerciales, a menudo en mezclas. El serotipo 2 se detectó en 11 de 20 muestras a pesar de la ausencia de la vacunación con este serotipo. El estudio 3 investigó la reproducibilidad y la sensibilidad de la prueba de PCR cuantitativo y la presencia de factores inhibidores en las muestras. Las muestras extraídas y amplificadas por triplicado mostraron un alto nivel de reproducibilidad, excepto a niveles muy bajos del virus que estaban cerca del límite de detección. El mezclado de mue

American Association of Avian Pathologists
Stephen W. Walkden-Brown, Aminul Islam, Peter J. Groves, Ambrosio Rubite, Sue M. Sharpe, and Susan K. Burgess "Development, Application, and Results of Routine Monitoring of Marek's Disease Virus in Broiler House Dust Using Real-Time Quantitative PCR," Avian Diseases 57(2s1), 544-554, (8 January 2013).
Received: 25 September 2012; Accepted: 1 November 2012; Published: 8 January 2013
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