Document Type

Theses, Masters

Rights

Available under a Creative Commons Attribution Non-Commercial Share Alike 4.0 International Licence

Disciplines

1.6 BIOLOGICAL SCIENCES

Publication Details

Thesis submitted for award of M.Phil, to the school of Biological Sciences, Technological University Dublin, August 2013, in conjunction with Department of Agriculture Food and the Marine, Celbridge, County Kildare.

Abstract

Common strain typing methods for differentiation of Mycobacterium bovis isolates include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping and more recently, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing. MIRU-VNTR and spoligotyping were evaluated in this study and these typing methods were compared with RFLP typing. A total of 386 M. bovis isolates from cattle, badgers and deer in the Republic of Ireland that had previously been typed by IS6110, polymorphic GC-rich sequence (PGRS) and direct repeat (DR) RFLP were included in the study. An initial panel of VNTR loci was established to analyse the first 60 isolates to determine that six VNTR loci (QUB 11a, QUB 11b, ETR A, 4052, MIRU 26 and 1895) displayed the greatest degree of discrimination between strains. Analysis of these six VNTR loci and spoligotyping was performed on all 386 isolates. RFLP was the method that gave the greatest differentiation of strains with a Hunter Gaston discriminatory index (HGDI) of 0.927, the HGDI recorded for MIRU-VNTR was marginally lower at 0.918 and spoligotyping was the least discriminatory method with a HGDI of 0.7. The HGDI for VNTR (6 loci) and spoligotyping was 0.933, however when 4 VNTR loci were used a comparable HGDI of 0.93 was recorded. Spoligotype SB0140 represented approximately 50% of the isolates. Within the group of isolates represented by SB0140 there was a high level of diversity between RFLP and MIRU-VNTR typescompared to groups represented by other spoligotypes. The most common MIRU-VNTR types within the SB0140 group were widely distributed geographically which limited their usefulness for tracing geographic spread of infection. However, the less common MIRU-VNTR types with the SB0140 group were largely concentrated in defined geographic areas. A combination of spoligotyping and MIRUVNTR typing offered advantages over MIRU-VNTR typing alone. In a combined spoligotyping and MIRU-VNTR typing protocol the number of VNTR loci could be reduced to four (QUB 11a, QUB 11b, ETR A and 4052) while maintaining a high level of strain differentiation.

DOI

https://doi.org/10.21427/D73305

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