Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in cattle and wildlife. Direct aerosol contact is thought to be the primary route of infection between conspecifics, whereas indirect transmission via an environmental reservoir of M. bovis is generally perceived not to be a significant source for infection. Here, we report on the application of molecular technology (PCR) to quantify the prevalence of M. bovis in the environment and to explore its epidemiological significance. We show that the detectability of viable M. bovis at badger setts and latrines is strongly linked to the frequency of M. bovis excretion by infected badgers, and that putative M. bovis in the environment is prevalent on a large proportion of endemic cattle farms in Britain. These results raise important questions about the role of an environmental reservoir in bTB persistence.

Background Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year.Methodology/Principal FindingsWe report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities.Conclusions M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×105 cells g−1 using Griffiths and 4.25×106 cells g−1 using FastDNA® Spin kit.

Real-time PCR was used to detect and quantify Mycobacterium bovis cells in naturally infected soil and badger feces. Immunomagnetic capture, immunofluorescence, and selective culture confirmed species identification and cell viability. These techniques will prove useful for monitoring M. bovis in the environment and for elucidating transmission routes between wildlife and cattle.Previous studies of Mycobacterium bovis shed into the environment by infected hosts using conventional PCR with primers targeting the MPB70 antigen gene (specific to the M. tuberculosis complex) provided evidence that the organism is likely to persist in the environment for at least 15 months postremoval of the known animal reservoirs (16) and that the probability of detection of M. bovis in soil and badger feces is correlated with the prevalence of excreting badgers (2). For epidemiological studies, M. bovis detection techniques must be 100% species specific with robust and reliable quantification.Real-time PCR has advantages over conventional PCR because it allows absolute quantification by comparison to a standard curve of known target sequence numbers. The complete genome sequence of M. bovis (5) has been used to design primers flanking a region of difference (RD4) between the sequence of M. bovis DNA and that of other M. tuberculosis complex members (1). The presence of M. bovis is confirmed by using a fluorescent (TaqMan) probe which discriminates M. bovis from other M. tuberculosis complex members since it hybridizes with both the 5Ј and 3Ј RD4 deletion flanking sequences, which only occur directly adjacent to each other in M. bovis (1).M. bovis cannot be directly cultured from soil because of the harsh decontamination techniques required to remove competing organisms. This limitation was overcome in a previous study by using immunomagnetic capture (IMC) to extract cells of M. bovis from mixed cell communities with a polyclonal antibody to M. bovis BCG and thus enabling cultivation of M. bovis from soil samples for the first time (13). Greater specificity could be achieved by using a monoclonal antibody, MBS43 (14, 15), which recognizes MPB83, a glycosylated cell wall-associated protein (8), differentiating M. bovis from other members of the M. tuberculosis complex (6).We report here the first use of an M. bovis-specific real-time PCR to detect and quantify M. bovis DNA in environmental samples and confirm the presence of viable cells of M. bovis by using IMC, immunofluorescence, and cultivation.Badgers are an important wildlife reservoir of M. bovis in the United Kingdom, and infected badgers can excrete the organism into the environment (4, 13). Social groups of badgers dig underground tunnel systems known as setts, and they defecate into communal "latrines," which are often located on cattle pasture. Soil was collected from seven badger setts, and feces was collected from five badger latrines during September 2006 on two cattle farms in a region of the United Kingdom where bovine tuberculosis (bTB) is endemic. Replicate samples...

Aims:  To adapt an immunomagnetic capture (IMC) technique to concentrate and cultivate Mycobacterium bovis from environmental samples including soil, faeces and urine. Methods and Results:  Cells of Myco. bovis BCG and wild‐type Myco. bovis were successfully isolated and cultured from seeded and naturally infected materials respectively. The IMC cell recovery estimated by colony forming units (CFUs) counts ranged from 0·10% to 0·16% for spiked media, and 0·15–0·36% for naturally infected soil and faeces. Recovery estimated by cell counts calculated using semi‐quantitative PCR ranged from 80·3% to 88·6% for spiked and 84·1–88·2% for naturally infected material. The differences in the recovery rates estimated by CFUs compared with pixel intensity is likely to be due to clustering of cells on culture plates, thereby underestimating the true cell count. Conclusions:  The IMC techniques can be applied to isolate viable wild type Myco. bovis from naturally contaminated environmental samples. Significance and Impact of Study:  Cultivation of Myco. bovis from environmental samples using traditional methods is extremely problematic. Here, we demonstrate a novel development of IMC techniques that will greatly facilitate the study of the organism in situ in order to assess its epidemiological importance in bovine tuberculosis persistence.

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.

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