Development, Application, and Results of Routine Monitoring of Marek's Disease Virus in Broiler House Dust Using Real-Time Quantitative PCR

Abstract

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/microl 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.

An observational study of the temporal and spatial patterns of Marek's-disease-associated leukosis condemnation of young chickens in the United States of America

“…The results of our STL analysis showed that MD dynamics fluctuated within years, with relatively high rates in the winter/fall and relatively low rates in the spring/summer, consistent with previous studies (Witter 1996; Walkden-Brown et al 2013). Preliminary analysis also revealed that these fluctuations closely matched a sine curve.…”

Modeling Marek's disease virus transmission: A framework for evaluating the impact of farming practices and evolution

“…Yet the ecology of MDV is poorly understood. This is perhaps most clearly exemplified by the conventional wisdom that the virus is ubiquitously found on industrialized poultry farms (Office International des-Epizooties, 2010; Dunn, 2013), despite recent surveillance data suggesting that the virus may not be present on a large fraction of farms (Groves et al, 2008; Wajid et al, 2013; Walkden-Brown et al, 2013; Bettridge et al, 2014; Kennedy et al, 2015b, 2017; Ralapanawe et al, 2015). …”

“…The concentration of MDV in dust can vary several orders of magnitude between farms and within farms over time (Walkden-Brown et al, 2013; Kennedy et al, 2017). The underlying cause of this variation is unknown.…”

Real-time PCR quantification of infectious laryngotracheitis virus in chicken tissues, faeces, isolator-dust and bedding material over 28 days following infection reveals high levels in faeces and dust

“…Another avian herpesvirus, gallid herpesvirus 2 (GaHV-2) or Marek's disease virus (MDV), is the causative agent of Marek's disease and is also transmitted by inhalation of infective material. For this virus, routine monitoring of large flocks based on quantitative PCR (qPCR) determination of MDV viral load in poultry house dust samples has been developed as a useful disease monitoring tool (Walkden-Brown et al, 2013). This method may be useful for other avian viral pathogens such as ILTV.…”