Development of lateral flow assays for improved detection of tuberculosis in cattle

Abstract

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), is a globally prevalent, economically devastating disease. M. bovis is an established zoonotic threat in low-and-middle income countries. Although used statutorily in the UK, a test and slaughter strategy to control bTB is not practical for many regions of the world. The tuberculin skin test (TST) and the ancillary interferon gamma release assay (IGRA) are the main diagnostic tests utilised for bTB control. However, these tests present challenges of low field sensitivity (Se) and specificity (Sp) respectively. Moreover, these tests do not have DIVA capability i.e., ability to differentiate infected from vaccinated (with Bacille Calmette-Guérin, BCG) animals. This makes research for improved diagnostics extremely important. The development of sensitive, specific, DIVA diagnostics is a major goal for TB research. Multibiomarker Lateral Flow Assays utilising novel Upconverting reporter Particles (UCP-LFAs) detecting multiple cytokines/antibodies have recently been developed for human diseases.

The overall aim of this thesis was to develop lateral flow assays for improved bTB diagnosis using this UCP technology. The first step was to identify the host immune biomarkers that could be discriminatory for bTB. A panel of nine host proteins was selected on the basis of gene expression differences in M. bovis infected animals compared to controls that had been reported in the literature and for which anti-bovine antibodies were available. This allowed quantification using enzyme linked immunosorbent assays (ELISAs). The data showed that bovine tuberculin (PPDb) specific levels of IL-2, CCL4, CXCL9 and IP-10, in addition to IFN-γ, have promising biomarker potential to identify experimental M. bovis infection and additionally as DIVA markers, at 12 weeks post challenge or vaccination. As an alternative to lab-based ELISA, user friendly, individual UCP-LFAs were then developed for six promising bTB protein biomarkers, which have shown high robustness in detecting respective recombinant bovine proteins; and enabling differentiation of animals based on their M. bovis infection or BCG vaccination status.

In order to improve diagnostic assays in particular for bTB, new monoclonal antibodies (mAbs) for bovine IP-10 (bovIP-10), a key infection biomarker, were developed and characterised. The utility of one selected mAb was explored in three immunological applications. While ELISA and flow cytometric results indicated successful detection of bovIP-10 from blood samples of M. bovis infected cattle and intra-cellular IP-10 expression, respectively; IP-10 could not be detected in the UCP-LFA format, and the assay requires further optimisation. The UCP-LFA analysis was extended to samples from field M. bovis infected animals, and from animals with Johne’s disease, a major confounder for bTB diagnostics, caused by infection with M. avium subsp. paratuberculosis (MAP). PPDb specific IFN-γ and IL-2 levels, detected by UCP-LFAs, displayed significant potential to discriminate bTB infected animals from those that were M. bovis naïve or MAP infected. Further exploratory biomarker identification experiments using UCP-LFAs developed for detection of human TB protein biomarkers were performed using bovine samples.

These results re-affirmed the importance of antibody selection for species-specific assay development. A research use only lateral flow format from a commercial company, detecting humoral immune responses against M. bovis proteins was tested using serum samples from M. bovis infected, naïve and MAP infected animals. The results with the tested cohorts suggest limited use of this platform for bTB diagnosis.

These results show promise of measuring PPDb specific IL-2 and IP-10 in addition to IFN-γ as additional bTB protein biomarkers, and importantly provide proof of concept for UCP-LFAs to act as a potential alternative to conventional ELISAs. The novel mAbs developed will be instrumental for studying dynamics of bovIP-10 in health and disease. Future work should include further optimisation and multiplexing of these user-friendly UCP-LFAs; and validation of the findings in longitudinal and larger cohorts of naturally and experimentally M. bovis infected, M. bovis naïve and MAP co-infected animals. This will provide foundation to develop a multi-biomarker user-friendly assay for bTB with enhanced diagnostic Se and Sp, suitable for applicability in low resourced settings.