Exploring the biology of the potentially zoonotic pathogen Mycobacterium avium subsp. paratuberculosis to inform detection methods

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) causes Paratuberculosis (Johne’s disease) in mammals. This is a chronic, untreatable and ultimately fatal wasting disease that primarily affects ruminants. There are also some concerns about the zoonotic potential of MAP, as MAP infection has been linked to Crohn’s disease and various other autoimmune disorders. Many countries have introduced MAP control programmes, both to improve the health of their livestock and to limit human exposure to MAP. However, these programmes have had varied success, in part due to the lack of sensitive and specific MAP detection methods. Typically, MAP is detected by ELISA, PCR or culture, but none of these tests are completely sensitive and specific for MAP. Therefore, the aim of this PhD research was to investigate the biology of MAP in order to improve current MAP detection methods. Particular emphasis was placed on MAP culture, as this is the current ‘gold standard’ detection method for viable MAP. This means that novel MAP detection methods must be validated against culture, despite its lack of sensitivity. Other areas investigated included: (1) using MAP-specific monoclonal antibody and peptide binders to identify epitopes of MAP antigens with the potential to be used for the development of immune-based MAP diagnostic detection assays, and (2) characterisation of recently isolated mycobacteriophages with potential to be used in place of mycobacteriophage D29 in bacteriophage-based detection methods for MAP.

MAP may be cultured in liquid or solid media. Culture in broth is faster, but culture on agar is arguably more useful as single colonies are obtained which can be isolated and stored for further studies. For this reason, two novel MAP solid culture methods were developed during this study that have the potential to reduce the time until MAP colonies become visible on agar. The first was an optimised solid culture medium consisting of Middlebrook 7H9 broth with 1.0% Tween 80, 0.019% casitone, 1.4% bacteriological agar, 10% egg yolk, 10% ADC and 1.65 µg/ml Mycobactin J. MAP growth became visible two weeks earlier on this new QUB medium than on Herrold’s Egg Yolk Medium (HEYM). The second novel culture method was designed to take advantage of speed of culture in broth while still enabling single colonies to be obtained. The ‘2-in-1’ system consists of a tissue culture flask or universal tube containing a layer of agar along with a small volume of broth, enabling samples to be enriched in broth prior to inoculation of the agar surface. The ‘2-in-1’ culture system significantly improved the incubation periods and final colony numbers of MAP cultured on HEYM compared to culture on solid media without an enrichment period in broth. A novel antimicrobial cocktail was also developed to reduce contamination of cultures when isolating MAP from raw milk samples. The new cocktail consisted of 8 µg ampicillin, 16 µg nalidixic acid and 50 µg amphotericin B per ml medium. The new cocktail inhibited non-target microorganisms from raw milk samples more effectively than the standard 2.5% PANTA but was also detrimental to MAP viability.Further optimisation will be required before it could be used for MAP culture.

With a view to potentially being able to replace MAP whole cell antigens used in current ELISA assays with peptide mimics of key MAP cell surface epitopes, during Covid lockdown time was spent on computational analysis of several 12-mer peptide mimics identified previously through phage display biopanning of the MAP-specific antibodies MAb 6G11 and MAb 15D10, and a 12-mer peptide Biotin-EEA402. The most promising peptides identified were peptide sequence TAKYLPMRPGPL for MAb 6G11, peptide sequence HNDNNTDQTWWW for MAb 15D10, and peptide sequence VYSPCGPCRRFL for BiotinEEA402. However, there was evidence that the peptides may bind to multiple Mycobacterium spp. rather than MAP specifically. These results will need to be confirmed experimentally with ELISAs.

Finally, because phage-based MAP detection methods show promise for detecting viable MAP quickly, four recently isolated mycobacteriophages were characterised to determine if they might substitute or complement the currently employed D29 mycobacteriophage. Phage DNA sequencing and subsequent bioinformatics indicated that all four isolates were identical members of the H1 subcluster of lytic Siphoviridae phages. On the basis of the limited amount of laboratory work possible, the ability of the novel phage isolate to infect and amplify within MAP is suspected, but has yet to be conclusively demonstrated.

Thesis is embargoed until 31 July 2025.

Date of Award	Jul 2023
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Irene R. Grant (Supervisor) & Linda Stewart (Supervisor)