Evaluation of a Fecal Shedding Test To Detect Badger Social Groups Infected with Mycobacterium bovis.

Bovine tuberculosis (bTB) is an economically important disease affecting the cattle industry in England and Wales. bTB, caused by Mycobacterium bovis, also causes disease in the Eurasian badger (Meles meles), a secondary maintenance host. Disease transmission between these two species is bidirectional. Infected badgers shed M. bovis in their feces. The Animal and Plant Health Agency (APHA) of the United Kingdom organized a comparative trial to determine the performance of tests in detecting M. bovis in badger feces for the Department for Environment, Food, and Rural Affairs (DEFRA). Here, we assessed the performance of the existing Warwick Fast24-qPCR test and its modified version based on a high-throughput DNA extraction method (Fast96-qPCR). We found Fast24-qPCR to have a sensitivity of 96.7% (95% confidence interval [CI], 94.5 to 99%; n = 244) and a specificity of 99% (95% CI, 97.8 to 100%; n = 292). Fast96-qPCR requires further optimization. Determining the disease status of badger social groups requires multiple tests per group. Therefore, to increase specificity further, we independently repeated the Fast24-qPCR test on positive samples, increasing stringency by requiring a second positive result. Fast24-qPCR with repeat testing had a sensitivity of 87.3% (95% CI, 83.1 to 91.5%; n = 244), and a specificity of 100% (95% CI, 100 to 100; n = 201) on an individual-sample level. At the social-group level, this repeat testing gives Fast24-qPCR high herd specificity, while testing multiple samples per group provides high herd sensitivity. With Fast24-qPCR, we provide a social-group-level test with sufficient specificity and sensitivity to monitor shedding in badgers via latrine sampling, delivering a potentially valuable tool to measure the impacts of bTB control measures.

Prospecting environmental mycobacteria: combined molecular approaches reveal unprecedented diversity.

BACKGROUND: Environmental mycobacteria (EM) include species commonly found in various terrestrial and aquatic environments, encompassing animal and human pathogens in addition to saprophytes. Approximately 150 EM species can be separated into fast and slow growers based on sequence and copy number differences of their 16S rRNA genes. Cultivation methods are not appropriate for diversity studies; few studies have investigated EM diversity in soil despite their importance as potential reservoirs of pathogens and their hypothesized role in masking or blocking M. bovis BCG vaccine. METHODS: We report here the development, optimization and validation of molecular assays targeting the 16S rRNA gene to assess diversity and prevalence of fast and slow growing EM in representative soils from semi tropical and temperate areas. New primer sets were designed also to target uniquely slow growing mycobacteria and used with PCR-DGGE, tag-encoded Titanium amplicon pyrosequencing and quantitative PCR. RESULTS: PCR-DGGE and pyrosequencing provided a consensus of EM diversity; for example, a high abundance of pyrosequencing reads and DGGE bands corresponded to M. moriokaense, M. colombiense and M. riyadhense. As expected pyrosequencing provided more comprehensive information; additional prevalent species included M. chlorophenolicum, M. neglectum, M. gordonae, M. aemonae. Prevalence of the total Mycobacterium genus in the soil samples ranged from 2.3×10(7) to 2.7×10(8) gene targets g(-1); slow growers prevalence from 2.9×10(5) to 1.2×10(7) cells g(-1). CONCLUSIONS: This combined molecular approach enabled an unprecedented qualitative and quantitative assessment of EM across soil samples. Good concordance was found between methods and the bioinformatics analysis was validated by random resampling. Sequences from most pathogenic groups associated with slow growth were identified in extenso in all soils tested with a specific assay, allowing to unmask them from the Mycobacterium whole genus, in which, as minority members, they would have remained undetected.

An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis.

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 10(5) 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.

Short term exposure to elevated trinitrotoluene concentrations induced structural and functional changes in the soil bacterial community.

We investigated the acute impact of trinitrotoluene (TNT) contamination of soil on the aerobic bacterial community composition and function. The contamination of the environment with explosive residues presents a serious problem at sites across the world, with the highly toxic compound TNT being the most widespread explosive contaminant. We investigated the acute impact of trinitrotoluene (TNT) contamination of soil on the aerobic bacterial community composition and function. Soil microcosms were amended with a range of concentrations of TNT for 30 days. A polyphasic approach encompassing culture-independent molecular analysis by DGGE, community-level physiological profiling (CLPP) and cell enumeration revealed that the amendment of soils with TNT resulted in a shift from slower growing k-strategists towards faster growing r-strategists. Pseudomonads became prevalent at high concentrations of TNT. Pollution induced community tolerance (PICT) was observed as TNT concentrations increased. Chemical analyses revealed that TNT was reduced to its amino derivatives, products of reductive microbial transformation. The transformation to amino derivatives decreased at high concentrations of TNT, indicative of inhibition of microbial TNT transformation.

Microbial and plant ecology of a long-term TNT-contaminated site.

The contamination of the environment with explosive residues presents a serious ecological problem at sites across the world, with the highly toxic compound trinitrotoluene (TNT) the most widespread contaminant. This study examines the soil microbial community composition across a long-term TNT-contaminated site. It also investigates the extent of nitroaromatic contamination and its effect on vegetation. Concentrations of TNT and its metabolites varied across the site and this was observed to dramatically impact on the extent and diversity of the vegetation, with the most heavily contaminated area completely devoid of vegetation. Bryophytes were seen to be particularly sensitive to TNT contamination. The microbial population experienced both a reduction in culturable bacterial numbers and a shift in composition at the high concentrations of TNT. DGGE and community-level physiological profiling (CLPP) revealed a clear change in both the genetic and functional diversity of the soil when soil was contaminated with TNT.

Impact of transgenic tobacco on trinitrotoluene (TNT) contaminated soil community.

Environmental contamination with recalcitrant toxic chemicals presents a serious and widespread problem to the functional capacity of soil. Soil bacteria play an essential role in ecosystem processes, such as nutrient cycling and decomposition; thus a decrease in their biomass and community diversity, resulting from exposure to toxic chemicals, negatively affects the functioning of soil. Plants provide the primary energy source to soil microorganisms and affect the size and composition of microbial communities, which in turn have an effect on vegetation dynamics. We have found that transgenic tobacco plants overexpressing a bacterial nitroreductase gene detoxify soil contaminated with the high explosive 2,4,6-trinitrotoluene (TNT), with a significantly increased microbial community biomass and metabolic activity in the rhizosphere of transgenic plants compared with wild type plants. This is the first report to demonstrate that transgenic plants engineered for the phytoremediation of organic pollutants can increase the functional and genetic diversity of the rhizosphere bacterial community in acutely polluted soil compared to wild type plants.

Cloning, sequencing, and characterization of the hexahydro-1,3,5-Trinitro-1,3,5-triazine degradation gene cluster from Rhodococcus rhodochrous.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.