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<p>This project is organised around three modules that take a comprehensive approach to improving diagnosis of tuberculosis in badgers. The first module is broken down into two separate objectives. The second and third modules each constitute a distinct objective. Each objective within the project is comprised of a series of work-packages that outline the scientific approach to be taken.</p>
<p>The first module focuses on evaluation and development of improved serodiagnostic tests for TB in badgers. We have been involved in the development and evaluation of a large number of serological tests for TB in badgers over the last 15 years and published widely on the subject (reviewed in Chambers et al. 2009). Serodiagnosis of badger TB offers the prospect of simple, rapid, and affordable testing but as outlined above, has consistently lacked sensitivity at the individual animal level unless the animal is heavily infected with M. bovis. The inclusion of multiple antigenic targets has given modest improvement to sensitivity but the repertoire of conventional protein antigens recognised by infected badgers appears rather limited (Greenwald et al. 2003). Whilst accepting that badgers at an early stage of infection may simply have very little circulating antibody directed to M. bovis, or that antibody may be present but with low affinity or avidity, the greatest promise for improved serodiagnosis lies with improved technology and more accurate testing platforms.</p>
<p>In this proposal we focus on two technologies and collaborations for which we already have some working experience and proof of principle data (IDEXX and Vantix). In both cases the ultimate aim of the work is a robust, affordable and field-able test of enhanced sensitivity. Additionally, we intend to evaluate a commercially available lateral flow device (the dual path platform, or DPP) which is currently planned by the manufacturers to replace the Brock TB Stat-Pak.</p>
<p>The second module moves to a novel PCR-based approach for detection of TB infection in badgers. Despite the reservations expressed by an expert panel assembled by Defra in July 2010 to consider bovine TB and the use of PCR, they concluded that “more research is needed to improve infected sett identification using PCR. However, it cannot be guaranteed that such research will result in a practical test or one that is low cost”. In recognition of the fact that conventional approaches for TB diagnosis using PCR rely on amplification of a relatively large genomic target that is predicated on the organism being present in the diagnostic sample itself, we wish to explore a promising PCR-based approach: the detection of transrenal mycobacterial DNA in urine. We already have proof of principle that this approach can be used to detect TB infection in badgers even when M. bovis could not be cultured from the urine itself. In this project we shall optimise and evaluate the method further to determine if it could be used diagnostically.</p>
<p>The first two modules require diagnostic samples obtained from live, trapped badgers. These include serum, whole-blood and urine as those most practicably obtained from animals without recourse to anaesthesia. It is intended that this project will run in parallel with another AHVLA-led project funded through Defra’s Bovine TB Research Requirements Call 2011-2012 - R4: Developing a Minimally Invasive Method of Obtaining Blood from an Unanaesthetised Trapped Badger (SE3273). Throughout this proposal, emphasis is given to those approaches most likely to be field-able; that is, practical and cost-effective for deployment in the field.</p>
<p>The third module is focused on a novel method for detecting infection remotely; that is, without requiring badgers to be caught and sampled. This approach relies on the testing of faecal samples obtained from badger latrines. However, unlike previous attempts to develop a PCR-based approach to detect M. bovis genomic DNA in the sample, we wish to build on proof of principle data generated in collaboration with Professor David Minnikin (University of Birmingham) that mycobacterial lipid can be detected in faeces from tuberculous badgers, even when the organism itself cannot be isolated by culture.</p>
<p>The Department for Environment, Food and Rural Affairs (Defra) has emphasised the need to identify practical tools with which to detect bovine tuberculosis (bTB) infection in free-living badgers, at both the individual and group levels. The diagnosis of tuberculosis in live badgers represents a considerable scientific and methodological challenge, and to date no technique has been identified which can be applied to a cage-trapped wild animal without the need for anaesthesia. The work proposed in the current project will evaluate a range of novel diagnostic approaches for bTB in wild badgers, with the emphasis on assays which may be readily transferred to an in-field test.
Currently, the only methods available for the rapid diagnosis of bTB in badgers, which could potentially be applied in-field, are based on the detection of antibody responses to infection. However, these methods lack sufficient sensitivity to detect an adequate proportion of infected individuals. Diagnostic assays based on the detection of earlier stages of the immune response to infection are more sensitive but currently require specialised laboratory facilities and technical expertise. Thus the development of more sensitive methods for detecting an antibody responses to infection is crucial. The current project will evaluate two novel serological assays, which have shown potential for the diagnosis of bTB in wildlife. Furthermore, both of these assays have the potential to be transferred to portable in-field platforms, and evaluation of these technologies is included within this project.</p>
<p>An alternative approach to the identification of bTB infection in badgers, which we propose to assess within the current project, is to attempt to identify products from the breakdown of Mycobacterium bovis (the causative agent of bTB) by the host, using urine samples. We propose to assess the sensitivity of a nucleic acid amplification based test for the presence of short fragments of M. bovis DNA, which we have demonstrated to be present in the urine of infected badgers even when the bacterium itself cannot be cultured. This approach would potentially permit evaluation of the infection status of badgers using samples which might be obtained from live animals. Finally, we propose the development of a novel technique to detect TB on the basis of the detection of mycobacterial lipids in faeces from experimental and wild badgers and to develop a simplified protocol for it's use in the field. In contrast to the other methods to be developed within this project, this approach would not require trapping of wild badgers, and would thus enable the identification of infection at the sett or social group level.</p>
<p>Each of these approaches forms a distinct objective within the proposed project. Work will be undertaken in collaboration with relevant experts from both academic and commercial organisations. The research is underpinned by on-going projects at both AHVLA and FERA, providing access to unique sample sets which permit the evaluation of the diagnostic potential of each approach. This provides significant cost savings within the project, as a minimal amount of additional work is required to provide access to diagnostic samples.</p>