Abstract
Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 105 cells g−1 and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.
Highlights
Mycobacterium bovis is the causative agent of bovine tuberculosis which affects cattle and a wide range of other mammals, including humans
Inhibition was seen to be low with none of the extractions required to be re-extracted based on the inhibition control values: all no inhibition control (NIC) were within the acceptable range and produced a Delta Ct (DCt) considerably less than 1.5 indicating negligible impurities in the DNA
As with the spiked samples no inhibition was observed, all NICs were within the acceptable range and a DCt of considerably less than 1.5 observed
Summary
Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) which affects cattle and a wide range of other mammals, including humans. M. bovis has been shown to persist in the environment for several months to years [1,2,3,4,5], raising questions about the role of environmental reservoirs in the chronic persistence of bTB in some cattle herds and wildlife populations [2,6,7]. Reservoirs of infection have been reported in wildlife populations in parts of the United Kingdom, Republic of Ireland, North America, Africa and New Zealand [8]. In the United Kingdom and the Republic of Ireland the Eurasian badger (Meles meles) is implicated in the persistence of M. bovis in cattle [9,10]. In parts of the developing world where there are few animal control measures in place, infection in cattle can have a significant impact on human health [13]. A further issue for concern is the transmission of M
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