An experiment was conducted to develop and optimize a chicken bioassay to detect the presence of infective viral pathogens in poultry litter from a variety of sources. The experiment also aimed to determine the effect of type of chickens and age of exposure to litter on the level of viral infectivity. The bioassay detected chicken anaemia virus, infectious bursal disease virus and fowl adenovirus from chicken litters. SPF chickens showed higher sensitivity than commercial broiler chickens without any effect of age of exposure, however, the assay was more sensitive in broiler chickens when exposed at day 8. We conclude that the bioassay based on exposure of day-old SPF chickens is a viable assay of viral infectivity of poultry litter.

Objective: To develop a chicken bioassay to detect infective viral pathogens in poultry litter and to determine the effects of type of chicken and age of exposure, as well as the effect of simulated litter transportation, on the level of viral infectivity detected. Design: A 5 x 2 x 2 factorial design, plus negative controls. Five chicken litters, including two with deliberate contamination (one transported and one not), two chicken types (specific-pathogen-free (SPF) Leghorns and Cobb broilers) and two ages at initial exposure (days 1 and 8). Two replicates of each treatment combination. Methods: The 10 chickens in each of 22 isolators were either exposed (20 isolators) or not (2 isolators) to 8 L of previously used or deliberately contaminated poultry litter in two deep scratch trays. At day 35 post-exposure, sera were assayed for antibodies against chicken anaemia virus (CAV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV) and fowl adenovirus (FAV). Spleen samples were tested for Marek's disease virus (MDV) using real-time polymerase chain reaction. Results: The bioassay detected CAV, IBDV and FAV, but not NDV, IBV or MDV, in chickens exposed to infected litters. Infection in SPF chickens was detected with greater sensitivity than in the broiler chickens. Sensitivity increased with age at exposure in broiler but not SPF chickens. Simulated transportation for 24 h had little effect on pathogen detection. Conclusion: A bioassay based on the exposure of day-old SPF chickens to poultry litter and measurement of seroconversion at day 35 post-exposure is a useful semi-quantitative assay for viral infectivity in poultry litter, with overnight transportation of litter having little effect on the level of viral infectivity detected. This bioassay has applications in research on litter treatment protocols.

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/ml 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.

Marek's disease (MD) is a common disease of chickens worldwide, including Australia, caused by the Marek's disease virus (MDV), a cell-associated alphaherpesvirus. The virus is lymphotrophic, but also infects cells of the feather follicle epithelium in which it replicates rapidly and is shed in feather dander, being transmitted laterally via inhalation of this material. The disease as first described by Josef Marek in 1907 was characterised by paralysis and lymphocytic infiltration of peripheral nerves in older chickens. This form of the disease, known as "classical" MD was superseded in the 1950s and 60s by "acute" MD characterised primarily by lymphomas in multiple organs in younger chickens. This development, associated with the intensification of the poultry industry, was a major threat to the industry until the concurrent discovery of the causative agent and development of live vaccines in the UK and USA in 1969. Since that time, there has been an ongoing evolution of virulence of MDV in countries such as the USA, associated with sequential vaccine failure and greater virulence in unvaccinated chickens with or without maternal antibody directed against MDV. Marek's disease vaccines are "imperfect" in that they protect against MD but not infection and shedding of virus, and as such are implicated in the evolution of virulence of the virus. Recent modelling work, based on our Australian research has shown that both vaccination and the reduced host lifespan that has occurred with the intensification of the chicken meat industry are implicated as causes of the observed increase in virulence.

Objective: To pathotype Australian isolates of Marek's disease virus (MDV) in commercial broiler chickens using standard methods and to evaluate early markers of pathotype. Methods: A complete 3 x 4 factorial experiment with two replicates was conducted using 648 Cobb broiler chickens in 24 isolators. The experimental factors were vaccination (unvaccinated, herpesvirus of turkeys (HVT), bivalent (HVT + SB1 strain of serotype 2 MDV) and MDV challenge (unchallenged or 500 plaque-forming units of isolates MFP57, 02LAR or FT158). Mortality, body weight, immune-organ weights and viral load were measured to 56 days post challenge (dpc). Vaccinal protective index (PI) and virulence rank (VR) were calculated based on gross Marek's disease (MD) pathology. Results: The PIs provided by the HVT and bivalent vaccines against challenge with MPF57, 02LAR, and FT158 were 84.6% 56%, 61.4% and 82.2%, 60.8%, 57.7%, respectively, leading to putative pathotypes of virulent MDV for MPF57 and very virulent MDV for 02LAR and FT158. Significantly more of the unvaccinated chickens (85.7%) had MD lesions than chickens vaccinated with either the HVT (26.8%) or bivalent vaccine (27.6%). Strong linear relationships were observed between the incidence of MD at 56 dpc and MDV load in the spleen at 7 dpc (R² = 0.71) and MDV load in the isolator exhaust dust at 14 dpc (R² = 0.57) and 21 dpc (R² = 0.51). Immune organ weights had a weaker association with subsequent MD incidence. Conclusion: Pathotyping results in broiler chickens with maternal antibody broadly agreed with those in specific-pathogen-free chickens in other studies, with some important differences. MDV load in the spleen at 7 dpc and in isolator dust at both 14 and 21 dpc was a powerful early predictor of subsequent MD incidence.

Since 2004 a major Australian poultry company in has used routine monitoring for MDV serotype 1 (MDV1) in poultry dust using serotype-specific real-time quantitative PCR (qPCR) as part of their MD management strategy, particularly in one production region. Dust samples are collected from one house for each farm batch at approximately 6 weeks of age. Combined with standard PCR monitoring of in ovo HVT vaccination in spleen samples, the monitoring program allows assessment of broiler vaccination value, assessment of the seasonal pattern of MDV1 presence and evaluation of MDV1 challenge levels over time. In the one region monitored consistently over the period MDV levels in dust have fallen to very low levels in the last two years. Monitoring has also allowed a rapid comparison of MDV1 status in new sections of the operation. Between 2005 and 2011 1487 shed dust samples were analysed for MDV1 by qPCR with 22.5% being positive for MDV overall. The positive rate ranged from a low of 6% in 2007 to 40% in 2010. Mean (±SD) MDV copy number per mg of dust in positive samples was 4.51±3.2 x 105 on the untransformed scale or 3.96±1.14 on the log10 transformed scale. The range in annual averages on the two scales was 0.091 to 13.2 x 105 with peak levels in 2009 reflecting samples from a new region with very high counts. The experience has been that routine monitoring of MDV by qPCR is a useful tool in managing cost effective control of MD in broiler operations.

Vaccination with herpesvirus of turkey (HVT) vaccine provides protection against clinical Marek's disease (MD) but does not preclude infection with wild-type MD virus (MDV). The quantity of MDV detected in circulating lymphocytes during the early period after infection may be a useful predictor of subsequent clinical MD later in the life. A study was designed to quantify MDV and HVT copy number in peripheral blood lymphocytes (PBL) using real-time polymerase chain reaction between days 5 and 35 post-challenge and to relate this to subsequent development of gross MD lesions. Female commercial broiler chickens were vaccinated with HVT or were sham-vaccinated at hatch, then challenged with MDV strain MPF-57 at day 2 post-vaccination and reared in positive-pressure isolators up to 56 days post-challenge, when all survivors were euthanized. All dead and euthanized chickens were examined post mortem for gross MD lesions. Birds were scored for MD lesions and mortality. MDV and HVT genome copy numbers were determined for each PBL sample. There was an increase in HVT load in PBL between days 7 and 37 post-vaccination, with marked increases between days 7 and 16 and again between days 30 and 37. There was a steady increase in MDV load to 35 days post-challenge. The mean MDV copy number (log10) was greater in chickens subsequently exhibiting gross MD lesions (5.05±0.21) than in those that did not (2.88±0.223), with the largest difference at 14 and 21 days post-challenge (P<0.001). Quantification of MDV during early infection is therefore a potential tool for monitoring MD in broiler flocks.