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Title: Novel detection methods for Mycobacterium avium subsp. paratuberculosis : development, optimisation and field validation
Author: O'Brien, Lorna
ISNI:       0000 0004 6060 0884
Awarding Body: Queen's University Belfast
Current Institution: Queen's University Belfast
Date of Award: 2016
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Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a wasting disease that largely affects domestic ruminants such as cattle, sheep and goats. Clinical disease can take up to 5 years to manifest, and therefore identification of subclinically-infected animals (i.e. those which are MAP-infected but are without symptoms) is challenging. Current diagnostic approaches lack detection sensitivity and speed of acquisition of results, so there is an urgent need for the development of a rapid test, which is highly specific and highly sensitive for the detection of viable MAP in naturally-contaminated samples. The peptide-mediated magnetic separation (PMS) phage amplification assay may represent such a test as it allows for the detection of viable MAP cells within 72 hours and has an analytical sensitivity and specificity of >85% and >99%, respectively (Foddai et al., 2010). The primary objective of this research project was to optimise the PMS-phage assay further, through the generation and evaluation of novel MAP binders. Two different MS protocols were successfully developed: (1) a new PMS assay and (2) an immuno-magnetic separation (IMS) assay. Subsequently, the diagnostic potential of each of these MS protocols was evaluated, with the PMS assay successfully combined with a PCR endpoint detection method and the IMS successfully combined with both culture and phage amplification assays for the detection of viable MAP. In order to assess the diagnostic performance of the MS-based assays for the detection of viable MAP from naturally-contaminated bovine milk samples, MS-culture and MS-phage assays were employed. The results generated were compared with those of current diagnostic approaches (faecal culture and serum ELISA). Additionally, an attempt was made to characterise the binding sites of the two novel monoclonal antibodies and the peptide binder by mimotope and computational analysis. Furthermore, an alternative diagnostic application for the novel MAP-specific binders was explored through early-stage experiments to develop a lateral flow immunochromatographic assay for the detection of whole MAP cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available