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

Lead Research Organisation: University of Warwick
Department Name: Biological Sciences


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.

Technical Summary

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.
Description Naturally excreted M. bovis wildtype survives in nature for >12m in contaminated sett soils and badger latrines, as also reflected in long-term survival of wildtype in seeded soil microcosms under all environmental test conditions. Model BCG behaves differently in soil microcosms showing significant sensitivity to temperature and moisture, with rapid biphasic die-off. M. bovis cells in the environment are alive and viable. RT-PCR applied to non-invasive urine and faecal samples is a sensitive tool to measure M. bovis excretion. Analyses of latrine samples collected during a badger BCG vaccine field trial in Republic of Ireland to test vaccine efficacy to reduce M. bovis excretion in faeces (infectivity) can not be released until permission is granted by the Irish Ministry of Agriculture. Unprecedented diversity of mycobacteria was observed using 454 pyrosequencing and analytical techniques in soils collected from cattle farms; differences in mycobacteria communities were observed between control, bTB breakdown and NSI farms. Slow-growing mycobacteria internalised in live foraging amoebae isolated from these soils are thought to facilitate mycobacteria survival.
Exploitation Route The comparative analyses of M. bovis quantitation and prevalence in different tissues of culled badgers in high and low regions of tranmsission, measured by qPCR with a novel internal inhibition control, provides the first insight into the relative sentivities of qPCR in potential invasive versus non-invasive samples in a protected wildlife species. The results suggest that urine (a non-invasive sample) would be one of the samples of choice. However faeces collected from latrines are a more practical non-invasive sample which correlates with M. bovis infection and infectivity (shedding).
Sectors Agriculture, Food and Drink,Education,Environment,Healthcare

Description The work contributed to the development of a potential non-invasive molecular tool to monitor environmental excretion of Mycobacterium bovis by badgers which resulted in later funding by Defra to conduct a ring trial across three international labs to test the assays performance. The project tested the tool as part of an oral BCG vaccine trial in Irish badgers. The latter results can not be published until given authority by the Irish Ministry of Agriculture.
First Year Of Impact 2011
Sector Agriculture, Food and Drink,Environment
Impact Types Societal,Economic,Policy & public services