Development, application, and results of routine monitoring of Marek's disease virus in broiler house dust using real-time quantitative PCR.

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.

Horizontal transmission dynamics of a glycoprotein G deficient candidate vaccine strain of infectious laryngotracheitis virus and the effect of vaccination on transmission of virulent virus.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens worldwide. The virus is horizontally transmitted and causes large outbreaks of disease. Recent studies have shown that a glycoprotein G deficient candidate vaccine strain of ILTV (ΔgG ILTV) is safe and protects birds from disease following challenge with virulent virus. This study examined the transmission dynamics of this candidate vaccine and of ILTV in field and experimental settings. The reproduction ratio (R0, average number of secondary infectious cases from a typical infectious case) was calculated from the growth rate of disease epidemics in broiler flocks. Assuming a latent period of 2 days and an infectious period of 4 days R0 was estimated to be 2.43 (95% CI 2.25-2.69). In experimental settings the transmission characteristics of ΔgG ILTV were similar to those of wildtype virus, and importantly ΔgG ILTV remained safe following one in vivo passage and subsequent infection via contact-exposure. There was minimal transmission of wildtype virus in vaccinated birds. The findings from this study further demonstrate the suitability of ΔgG ILTV for use as a live attenuated vaccine. Knowledge of the basic reproduction ratio of ILTV will be valuable for future studies that aim to improve disease control using vaccination programs.