Development of Novel Diagnostic Strategies for the Ante-Mortem Immunodiagnosis of Bovine Tuberculosis and Johne's Disease

<p>Mycobacterial infections of livestock such as bovine tuberculosis (bTB) or Johne\'s disease (JD) exact an enormous cost on European agriculture. bTB and JD are chronic inflammatory diseases caused by Mycobacterium bovis (MB) and M. avium paratuberculosis (MAP), respectively. Detection and slaughter of MB infected animals is required under EU law but JD control relies on voluntary cooperation. Both diseases can affect multiple domestic animals and wildlife. Current diagnostic tests are based on immune responses to bovine, avian and johnin tuberculin (aka PPD), reagents which have specificity, sensitivity and standardisation constraints. Due to cross reactivity, use of PPD reagents in MB skin testing elicit immune responses that may confound subsequent immunological detection of both diseases. Furthermore, the demonstration that JD infection interferes with bTB diagnosis in dually infected herds further increases concerns about the performance of PPD-based diagnostic tests. In addition, the absence of adequate diagnostic tools for early detection of latent MAP and MB infected livestock severely hampers disease control.</p>
<p>We propose an integrated approach to the development of improved diagnostic tests based on assay platforms (skin testing, serology, and defined MB and MAP antigens) that can be applied across both diseases and adapted to multiple host species whilst exploiting differences in the immunobiology of bTB and JD. Our aim is to improve the diagnosis of bTB and JD per se and to generate tools that are not compromised in sensitivity or specificity by co-infection or testing regimes. Rather than working in isolation on either bTB or JD, as has been common in the past, our consortium bridges both fields in a multi-disciplinary approach. Together, we have experience in the biology of the diseases; experimental MB, MAP and MB/MAP infection models (cattle and goats), cellular immunology, bioinformatics, antigen mining, lipid and protein biochemistry, test development and exploitation. The consortium will generate sera collections from MB, MAP infected and MAP vaccinated animals and will have access to samples from live animals to validate and develop antigens and tests. Ours is a multi-pronged approach; validating prioritised MB and MAP antigens and platforms, platform development and antigen discovery combined with a basic research arm.
Work packages. WP1: To validate performances in relation to sensitivity and specificity of already available MB and MAP peptides, proteins, and lipid antigens on existing platforms (skin test, IFN-gamma release assay (IGRA), serological assays). WP2: Development of multiplex assays to diagnose TB and JD. The aim is to develop (i) serology multiplex systems using Luminex platform in conjunction with MB and MAP antigens tested in WP1; (ii) to develop a combined serology and cytokine multiplex based on IFNgamma and other cytokines. The latter approach will also encompass the use of non-peptide glycolipid and lipopeptide ligands. WP3: Antigen discovovery. Additional potential MB or MAP specific antigens will be identified by the following approaches: (i) Antigen mining using bioinformatics to predict MB and MAP peptides recognised by CD4 T cells, with promising peptides then chemically synthesised and tested; (ii) Preparation of MB and MAP glycolipids, (ii) MAP protein antigens by comparative proteomics with candidates to be expressed as recombinant proteins. Antigens from all three approaches to be tested (serology and IGRA). WP4: Improved understanding of T cell immunity. (i) Characterisation of T cells recognising non-proteinaceous antigens. We will functionally characterise T cells that recognise glycolipid and lipopeptide antigens by defining the T cell subset(s) recognising these antigens, their restriction elements, cytokine profiles and ability to kill or lyse MB or MAP infected target cells; (ii) Identify host biomarkers of latency using a caprine sub-clinical MAP infection model: expression of modulatory cytokines, eicosanoid, local immunity or effector/memory T cell balance.</p>
<p>Outputs: (i) Diagnostic tests that improve the performance of TB and JD diagnosis; (ii) development of
novel serological and cytokine multiplexes; (iii) discovery of novel MB and MAP antigens; (iv) characterisation of non-conventional T cell populations; (v) biomarkers associated with latency to detect animals at different disease stages to faciliate a risk-based approach to disease control.</p>