'In vitro' and 'in vivo' characterization of selected Australian isolates of Marek's disease virus

Abstract

Chapter 1 reviews the literature on Marek's disease relevant to the work carried out in this thesis. Methods for TaqMan® quantitative real-time PCR (qPCR) assays to detect the three serotypes of Marek's disease virus (MDV) are already available, and absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in Chapter 2. Using plasmid DNA, the lower detection limit of the MDV2 assay was determined to be 10 copies of the viral genome. Three independent assay runs showed highly reproducible Ct values and calculated copy numbers, with mean intra- and interassay coefficients of variation of less than 3 % for Ct and 21.5 % for calculated copy number. Absolute quantification of MDV2 was performed successfully on dust samples collected from poultry farms across Australia, material from infectious spleens and feather tips from chickens vaccinated with an avirulent strain of MDV2. Thus, it is now possible to use qPCR assays for absolute quantification of all three serotypes of MDV in a sample. The sequencing of the 'meq' gene of 5 Australian isolatres of MDV1 isolated between 1992 and 2004 which had been pathotyped in an experiment using unvaccinated and HVT-vaccinated specific pathogen free (SPF) chickens is reported in Chapter 3. The sequencing results were compared with a range of MDV1 meq sequences published in GenBank® and associations with virulence examined.