Intra- and extra-cellular mechanisms affecting the persistence of Mycobacterium bovis in the environment: towards molecular surveillance of bovine TB

The incidence of bovine tuberculosis, caused by the bacterium Mycobacterium bovis is increasing in the UK at a cost of £70 million per annum. There is convincing evidence that badgers are important in maintaining the disease in cattle herds, and infected badgers can excrete M. bovis into the environment in their saliva, faeces and urine, which may then be a source of infection to livestock, wildlife and humans. The likelihood that M. bovis excreted into the environment is infectious will depend partially on the activity of the cells and the environmental conditions that favour their survival. Little work has been conducted on M. bovis in the environment as it is difficult to isolate these cells. We have developed technology that allows us to detect and count M. bovis cells using a DNA-based technique known as PCR, and to extract these cells from soil without the need for growing them. A technique known as flow cytometery combined with staining and microscopy to determine if cells are alive or dead is a powerful way in which to study the characteristics of the organism in the soil. It is not known what changes the cells undergo to enable them to survive harsh environmental conditions, therefore we also aim to determine the activity level of these cells and relate this to variations in microclimate and the structure of the soil including the presence of bacterial predators such as amoeba which may play a significant role in enhancing M. bovis survival. The development of sampling methods to enable screening of farms for hotspots of M. bovis contamination is important to aid in the management of bovine TB. We have shown that the level of contamination of soil at badger setts detected by PCR is related to the rates that badgers that live within the setts excrete the organism, suggesting that the PCR may be useful as a non-invasive way of screening for TB contamination and infection. The reliability of the environmental test to identify infected badger social groups requires post-mortem diagnosis of infection, since infectious animals excrete the bacteria intermittently. A unique set of experiments to be conducted in the Republic of Ireland by collaborators at University College Dublin (UCD) provide us with the opportunity to generate the required data with which to optimise the performance of the PCR to detect TB in badger social groups by sampling their local environment. Under local UCD department licence, badgers will be intensively trapped and humanely culled, and diagnosed at post-mortem. From the data provided to us by UCD, we can relate the detection of M. bovis at setts to the prevalence of disease in the resident badgers and determine the spatial scale of sampling that gives the greatest reliability. We will also test the reliability of the PCR when compared to standard methods for post-mortem diagnosis of TB infection in individual badgers using samples of organs and tissues. Another of the Irish experiments is to trial an oral BCG vaccine in wild naturally infected, and captive, badgers. Preliminary studies of oral BCG in captive badgers have shown them to excrete the vaccine in their faeces for at least 17 days after vaccination. This has implications for the interpretation of the current routine diagnostic skin test in cattle, since exposure of cattle to BCG vaccine excreted into the environment by badgers may cause them to give a positive result to the skin test even though they are not infected with a pathogenic M. bovis strain. Using a PCR that will specifically detect BCG, we aim to determine the timing and duration of vaccine excretion in individual captive badgers, and to measure the quantity, spatial distribution and survival of BCG in the latrines of a wild population of vaccinated badgers. This will help us to assess the potential magnitude of any negative impacts that oral BCG vaccination of wild badgers may have on the tuberculin skin test and slaughter policy.

Persistent bovine TB is thought to be partially associated with the maintenance of Mycobacterium bovis by badger populations. Badgers can excrete the organism into the environment. The probability of transmission from environmental sources is expected to relate to intra- and extra-cellular conditions that trigger differential gene expression affecting the abundance, survival and infectivity of these cells. These include their physiological status, soil microclimate, and soil physical and biological composition. We have developed M. bovis specific real-time PCR primers, and immunomagnetic capture (IMC) technology to identify and isolate M. bovis cells from environmental substrates, circumventing the need for problematic cultivation which is likely to alter their gene expression profiles. The degree of environmental contamination at badger setts measured by PCR was shown to be related to the excreting rates of the resident badgers indicating the potential of PCR-based non-invasive surveillance tool. This study proposes to quantify the temporal abundance of live/dead stained and culturable environmental cells relative to soil microclimate and biotic composition, and to identify differential gene expression profiles of environmental vs cultured cells. Methods will include amplification of mRNA using RAP and T7 polymerase approaches and RT real-time PCR, live cell sorting using fluorescence activated cell sorting technology (FACS), cultivation following IMC and FACS, and quantification using real-time PCR with M. bovis-specific RD4 flanking primers. Sampling protocols will be developed to optimise the PCR for its use as a non-invasive surveillance tool by comparing detection probabilities and bacterial cell counts at badger setts to post-mortem diagnostics of the resident badgers. The test will be applied to monitor the distribution and survival of environmental contamination with BCG during an imminent randomised oral BCG badger vaccine efficacy trial.