Date of Award

2017

Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Prof. Jim O'Mahony

Second Advisor

Prof. Aidan Coffey

Abstract

The slow-growing intracellular pathogen Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease in domesticated and wild ruminants. Johne’s disease inflicts severe annual economic losses to the dairy industry worldwide. This thesis is divided into five chapters that consist of a literature review' chapter and four experimental chapters focusing on MAP strain genotyping and the development of novel molecular MAP detection tools using real-time PCR. Chapter 1 describes published information regarding MAP characteristics, Johne’s disease, MAP genomes, available MAP detection methods, MAP epidemiology and genotyping.

To efficiently control and monitor MAP infection, molecular t>ping of MAP strains provides extensive knowledge regarding the source and genetic diversity of MAP within a geographic location. Knowledge regarding MAP genotypes within Ireland is limited and therefore, a large collection of MAP isolates from 53 herds across the Republic of Ireland were genotyped using the MIRU-VNTR technique and described in chapter 2. Rapid detection of MAP from faeces using real-time PCR is crucial to halt the infectious spread within herds. MAP-specific multi-copy elements are attractive and sensitive molecular targets for real-time PCR assays to detect MAP from faeces. The multi-copy IS_MAP04 has never previously been evaluated as a potential novel target in MAP diagnostics. This evaluation is described in chapter 3.

A further MAP-specific molecular target has been investigated and described in chapter 4. The truncation present in the mycobactin cluster within MAP was used as a novel MAP-specific target in real-time PCR assays to detect and confirm MAP from faeces. The elimination of false-negative detection results is another crucial aspect in molecular MAP diagnostics. Chapter 5 describes the development of two novel MAP DNA extraction and real-time PCR internal controls using Mycobacterium smegmatis as a model mycobacterial strain. These developed internal controls could aide in the elimination of false-negative MAP detection results from faeces.

Access Level

info:eu-repo/semantics/openAccess

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