Date of Award
Masters of Science (Research)
Prof. Jim O'Mahony
Prof. Aidan Coffey
The incidences of Johne’s disease (JD) in farms worldwide have increased significantly over the last 80 years. The increase in JD is owing to the lack of diagnostic tests available and the long incubation period of the disease causing pathogen, Mycobacterium avium paratuberculosis (MAP). In addition, treatment of JD with antibiotics has proven difficult due to the emergence of antibiotic resistance MAP strains and the presence of the thick lipid rich mycobacterial cell wall. An alternative diagnostic and treatment method for JD involves the use of mycobacteriophages. As part of this research, individual mycobacteriophages (TM4) and a mycobacteriophage cocktail (LE1 to LE6) were utilised to develop a mycobacteriophage based diagnostic and viability assay for MAP. The first part of the study was to develop a preliminary optimisation assay utilising the fast growing mycobacterial strain; M.smegmatis mc2 155. The premise of the assay was; (a) if the drug is not effective against killing mycobacteria, mycobacterial cells would remain viable and mycobacteriophage would infect these cells causing an increase in plaque forming units over a four hour time period and (b) If the drug is effective against mycobacteria, then the mycobacterial cells would no longer be viable, therefore the mycobacteriophage have no cells to infect and plaque forming units will remain equivalent over time. The use of the mycobacteriophage TM4 proved to be effective in determining drug susceptibilities as M.smegmatis mc2 155 was determined to be sensitive to Isonazid, Rifampicin, Pyranzaimide and Ethambutol (plaque numbers increased) and resistant to Ampicillin, Erythromycin, Amoxicillin, Streptomycin as plaque numbers remained static. The second part of the study was aimed to utilise the optimisation conditions devised from the first part and implement them against six Irish MAP field isolates in order to assess their drug susceptibility profiles. The six MAP strains all yielded resistance to Ampicillin, Streptomycin, Erythromycin and Amakacin as plaque numbers increased over 4 hours. The six MAP strains also yielded resistance to Rifampicin, Isoniazid and Clindamycin as plaque numbers remained static over 4 hours. Resistance levels for three MAP strains against Vancomycin, Ciproflaxin, Clofazimine and Cefdinir were moderate and for the remaining MAP strains drug sensitivity was high against Trimethoprim, Tetracycline and Doxycycline. A mycobacteriophage cocktail was applied for comparison against an individual mycobacteriophage to ensure that the addition of extra phage didn’t interfere with the fidelity of the assay. Drug susceptibility profiles were identical to the profiles obtained in utilising one mycobacteriophage type (TM4).
Crowley, Gillian Catherine, "The use of mycobacteriophages as a novel therapeutic option to control and treat Johne's disease" (2020). Theses [online].
Available at: https://sword.cit.ie/allthe/21
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