Date of Award

2010

Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Jim O' Mahony

Abstract

MAP is a proven animal pathogen causing Johne's disease, one of the most widespread and economically important diseases of ruminants. Since 1992 and the opening of the European market, the prevalence of MAP in Ireland has increased considerably. Improvements in diagnostic strategies for Ireland and elsewhere are urgently required. MAP infects a wide host range and has emerged as a potential threat to human health and has been associated with Crohn's disease. The control of paratuberculosis infection requires a better understanding of the genetic diversity of the pathogen. The objective of this study was to isolate and characterize this enteric pathogen in Ireland using sophisticated molecular typing methods.

In total, 290 cattle collected from seven selected Irish herds over 2 years were screened for MAP by a conventional culture method and two PCR assays. MAP was isolated and cultured from 23 faecal samples (7.9%) on solid medium. From a molecular perspective, 105 faecal samples (36%) were PCR positive for MAP specific DNA. The cultivation of MAP was characterized by poor recovery but high specificity whereas the PCR produced faster results with improved sensitivity. In total a bank of 38 MAP strains (15 pre-existing and 23 novel isolates) was characterized using 11 multi locus short sequence repeat (MLSSR) loci and 8 variable number of tandem repeat (VNTR) loci. Three subtypes were detected by the VNTR method with a discrimination index (DI) of 0.540. In contrast, the MLSSR method differentiated the 38 MAP isolates into 18 types with Dl of 0.921. Among these, 6 types have not been recorded previously.

This thesis also describes the development of a new diagnostic PCR assay and the evaluation of a novel method to differentiate MAP strains using High Resolution Melting PCR. Based on analysis of a strategically chosen genomic region, 37 genes were screened by in silica analysis to evaluate their potential as a diagnostic target. Five genes were predicted to be unique to MAP and were then tested by PCR. Among them the MAP2179 gene was identified as a specific and sensitive novel target for MAP diagnosis. As previously mentioned, this study also described the application of an optimized High Resolution Melting technique capable of clearly differentiating MAP "C" type from MAP "S" type. This broad genotyping technique is simple, inexpensive and provides results in less than 20 minutes after amplification using a fluorescent dye.

Access Level

info:eu-repo/semantics/openAccess

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