Molecular detection of Mycobacterium bovis in the environment
Young, Jamie Stuart (2003) Molecular detection of Mycobacterium bovis in the environment. PhD thesis, University of Warwick.
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.