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5 January 2013 Relationship Between Levels of Very Virulent MDV in Poultry Dust and in Feather Tips from Vaccinated Chickens
Susan J. Baigent, Lydia B. Kgosana, Ahmed A. Gamawa, Lorraine P. Smith, Andrew F. Read, Venugopal K. Nair
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To assess the effect of various vaccine strains on replication and shedding of virulent Marek's disease virus from experimentally infected chickens, quantitative PCR (q-PCR) methods were developed to accurately quantify viral DNA in infected chickens and in the environment in which they were housed. Four groups of 10 chickens, kept in poultry isolators, were vaccinated at 1 day old with one of four vaccines covering each of the three vaccine serotypes, then challenged with very virulent MDV strain Md5 at 8 days of age. At regular time-points, feather tips were collected from each chicken and poultry dust was collected from the air-extract prefilter of each isolator. DNA was extracted from feather and dust samples and subjected to real-time q-PCR, targeting the US2 gene of MDV-1, in order to measure Md5 level per 104 feather tip cells or per microgram of dust. Accuracy of DNA extraction from dust and real-time q-PCR were validated by comparing either q-PCR cycle threshold values or the calculated MDV genome level; for use in q-PCR, DNA was extracted from serial dilutions of MDV-infected dust diluted with noninfected dust, or DNA from MDV-infected dust was diluted with DNA from noninfected dust. The results confirmed the accuracy and sensitivity of dust DNA extraction and subsequent q-PCR and showed that differences in virus levels between dust samples truly reflect differences in shedding. Vaccination delayed both replication of Md5 in feather tips and shedding of Md5. First detection of Md5 in feather tips always preceded or coincided with first detection in dust in each group. pCVI988 and HVT SB-1 were the most efficient vaccines in reducing both replication and shedding of Md5. There was close correlation between mean virus level in feathers of each group and mean virus level in the dust shed by that group. This relationship was similar in each of the vaccinated groups, demonstrating that measurement of the virus in dust can be used to monitor accurately both the infection status of the chickens and environmental contamination by MDV.

Relación entre los niveles del virus de Marek muy virulento en el polvo de instalaciones avícolas y en cañones de las plumas en pollos vacunados.

Para evaluar el efecto de diferentes cepas de vacunas sobre la replicación y diseminación del virus muy virulento de la enfermedad de Marek en pollos infectados experimentalmente, se desarrollaron métodos cuantitativos de PCR (q-PCR) para determinar con precisión el ADN viral en los pollos infectados y en el entorno en el que se alojaron. Se alojaron cuatro grupos de 10 pollos por unidad de aislamiento, estas aves fueron vacunadas al primer día de edad con una de cuatro vacunas que incluyen los tres serotipos vacunales. Posteriormente fueron desafiados a los ocho días de edad con la cepa muy virulenta del virus de la enfermedad de Marek, Md5. En intervalos de tiempo regulares, se recolectaron cañones de plumas de cada pollo y también se recolectó el polvo del prefiltro del extractor de cada unidad de aislamiento. Se extrajo el ADN de los cañones de las plumas y de las muestras de polvo y se analizaron por PCR en tiempo real, para el gene US2 del virus de Marek 1, con el fin de medir el nivel de virus Md5 por 104 células de plumas o por microgramo de polvo. Se validó la precisión de la extracción de ADN del polvo y el método de PCR en tiempo real mediante la comparación de los ciclos umbrales del método de PCR cuantitativo o por los valores del genoma del virus de Marek. Para su uso en método de PCR cuantitativo, el ADN fue extraído de diluciones seriadas de polvo contaminado con el virus de Marek, diluido con polvo no contaminado, o también, el ADN extraído de polvo contaminado con el virus de Marek se diluyó con ADN de polvo no contaminado. Los resultados confirmaron la precisión y la sensibilidad de la extracción de ADN del polvo y el método de

American Association of Avian Pathologists
Susan J. Baigent, Lydia B. Kgosana, Ahmed A. Gamawa, Lorraine P. Smith, Andrew F. Read, and Venugopal K. Nair "Relationship Between Levels of Very Virulent MDV in Poultry Dust and in Feather Tips from Vaccinated Chickens," Avian Diseases 57(2s1), 440-447, (5 January 2013).
Received: 21 September 2012; Accepted: 1 December 2012; Published: 5 January 2013
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