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Molecular detection of Mycobacterium bovis in the environment
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Young, Jamie Stuart (2003) Molecular detection of Mycobacterium bovis in the environment. PhD thesis, University of Warwick.
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WRAP_THESIS_Young_2003.pdf - Submitted Version Download (22Mb) | Preview |
Official URL: http://webcat.warwick.ac.uk/record=b1666036~S1
Abstract
An investigation was carried out to determine the presence and persistence of
Mycobacterium bovis in the environment. Soil samples were taken in April 2000 from a
farm in Ireland which had undergone a bovine tuberculosis outbreak some four months
prior. Total community DNA was extracted from these samples and PCR carried out
targeted to two genes specific for the Mycobacterium tuberculosis complex; mpb64 and
mpb70. These genes were detected in soil samples taken from entrances to two badger
sets and in soil from two sites where the infected cattle grazed. Further analysis of DNA
using oligonucleotide primers specific for the 16S rRNA genes of slow-growing
mycobacteria was carried out. This revealed the presence of 16S rRNA genes relating to
Mycobacterium bovis. RT-PCR was also carried out using these primers on total
community RNA. Sequences relating to M. bovis were found, indicating that the DNA
and RNA came from viable, intact cells in the soil, and that M. bovis persists in soil for
up to four months after contamination of the soil. Sampling was repeated in November
2002 following a second TB outbreak in January 2001. DNA sequences for mpb64 and
mpb7O were only found in the samples from the badger setts, as were 16S rRNA
sequences.
The survival of the vaccine strain M. bovis BCG was also determined, using soil
microcosms experiments in defined environmental conditions. M. bovis BCG was found
to survive for longest at temperatures of 37°C and at soil wetting levels of 30%.
Culturable cells could not be detected after 60 days, however DNA and RNA relating to
M. bovis BCG could be detected up to 18 months after initial inoculation. This suggests
the cells persisted in a viable non-culturable state. Experiments to determine the rate of
persistence of DNA in soil were carried out. DNA was found to persist for no longer than
10 days in soil.
Diversity studies were carried out on the farm samples and on Warwick soil, to determine
the diversity of the mycobacterial population. 16S rRNA analysis was carried out and
showed the presence ofsequences relating to M. bovis, Al. hiberniae, M. avium, Al. fallax,
and M. farcinogenes.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QR Microbiology | ||||
Library of Congress Subject Headings (LCSH): | Mycobacterium bovis -- Detection, Bacteriology, Agricultural, Soil microbiology, Badgers -- Habitations | ||||
Official Date: | September 2003 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Biological Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Wellington, E. M. H. (Elizabeth M. H.), 1954- | ||||
Extent: | xviii, 175 leaves | ||||
Language: | eng |
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