In the industrialised world, where zoonotic human tuberculosis incidence is low, the main impact of the disease is economic with losses in agricultural productivity. In the UK, despite an ongoing test and slaughter programme for cattle and periods of statutory badger culling, there has been an average 18% increase in the annual number of new confirmed cattle herd breakdowns since the mid-1980s with an estimated cost of approximately �� 108 million in 2008�C2009. In parts of the developing world where there are few animal control measures in place, Citiolone infection in cattle can also have a significant impact on human health. The WHO has recently designated bovine tuberculosis as a neglected zoonosis, with particular reference to the developed world. A further issue for concern is the transmission of M. bovis from livestock into wildlife reservoirs in free-ranging ecosystems. M. bovis has become established in the African buffalo populations within South Africa��s Kruger National Park and been observed in a number of other species, from primates to predators, including lions. White-tailed deer are now considered to be the primary reservoir and maintenance host of bTB in Michigan, USA. M. bovis transmission between wildlife, livestock and humans is expected to be primarily via aerosol routes of contact, however there is growing evidence to suggest that the environment may be a potentially important reservoir of the organism. Furthermore, current methods for M. bovis detection in wildlife involve invasive trapping and sampling, a time-consuming and expensive process. The development of a non-invasive and sensitive tool to detect M. bovis in animals and their immediate environment would make a valuable contribution to bTB surveillance and epidemiological studies. Monitoring excretion, rather than infection, is of particular relevance because excreting animals are responsible for transmission. Molecular detection methods have been recently developed for quantification of M. bovis by real time PCR in environmental samples and further optimised with particular regard to DNA extraction methodology. An inhibition control assay has also been developed. This study validates this molecular assay through rigorous testing in three independent laboratories aimed to assess concordance, reliability and sensitivity. We collected and tested badger faeces from Moexipril HCl latrines in areas of high and low bTB incidence in cattle. In addition, we spiked a sub-sample of the faeces collected from a low incidence area with known titres of M. bovis BCG. In parallel, an inhibition assay was applied to all samples to assess presence of PCR inhibitors and thus to limit false negative results. The sample panel comprised of 24 spiked faecal samples and 300 field samples taken from 30 badger latrines and the Badger Trust. The bTB breakdown incidence was calculated using the VetNet TB in Cattle system data which provides national data on farm level bTB skin tests.