Molecular Detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in Soil

Abstract

PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 ؋ 10 3 to 3.6 ؋ 10 3 gene copies g of soil ؊1 , depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37°C with moist soil (؊20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.Environmental and wildlife reservoirs of Mycobacterium bovis are of significance due to the increasing number of bovine tuberculosis breakdowns in cattle herds in both the United Kingdom and Ireland. In Europe, the badger (Meles meles) has been implicated, but it is unclear how the disease is transmitted to cattle from badgers. Few studies have considered dissemination and persistence of environmental M. bovis. Tanner and Michel (12) reported survival of up to 6 weeks for M. bovis cells inoculated into soil and feces, detected by traditional selective cultivation methods. However, cultivation techniques for monitoring M. bovis and other mycobacteria in soil are impeded by the slow growth rates of M. bovis and the need for prolonged incubation of highly selective agars. Pretreatment or decontamination of samples is required involving the addition of 1 to 5% NaOH, often followed by further treatments with H 2

Is Mycobacterium bovis in the environment important for the persistence of bovine tuberculosis?

“…Notwithstanding, our findings strongly indicate that the mycobacterial DNA detected in this study was contained within intact and viable cells: detection of 16S rRNA by RT-PCR is a sensitive indication of active cell metabolism, and extracellular mycobacterial DNA fails to survive for more than a few days in soil (Young et al 2005). Growth of the organism on culture following direct extraction from soil and faeces by MTBC specific immunomagnetic capture (Sweeney et al submitted) is further evidence of its viability.…”

“…Detection of rRNA in only 6 of 22 PCR positive samples may be due to the poorer stability of RNA and the likely low number of ribosomes within cells in soil. In a study of M. bovis persistence in Ireland, little difference was detected in semi-quantitative PCR signal strength in samples from setts tested at 4 and 15 months after badger and (Young et al 2005), highlighting the known ability of the organism to viably persist in soil and faeces for months (Maddock 1933). Critically, historical studies of M. bovis showed contaminated soil, badger and cattle faecal inoculates to be infectious in in vivo guinea-pig models (Williams & Hoy 1930;Maddock 1933;Wilesmith et al 1986).…”

“…(b) Molecular detection Mycobacterium DNA was detected by a PCR assay targeted at the MPB70 antigen gene specific to the MTBC (Young et al 2005). Positive samples from the Woodchester and Wytham badger populations were subsequently confirmed as M. bovis using a second PCR targeting Rv1510 (RD4), present in all members of the complex except M. bovis (Huard et al 2003).…”

“…Positive samples from the Woodchester and Wytham badger populations were subsequently confirmed as M. bovis using a second PCR targeting Rv1510 (RD4), present in all members of the complex except M. bovis (Huard et al 2003). RT-PCR for 16S rRNA was performed (Young et al 2005) (d) Statistical analyses Data generated from the badger populations were analysed by maximum-likelihood logistic regression. The outcome variable was the proportion of environmental replicate samples that tested PCR positive per territory.…”

“…Potential obstacles to bTB control are the existence of the badger (Meles meles) as a wildlife reservoir (Griffin et al 2005), and the presence of M. bovis in the environment where the organism can survive for months (Maddock 1933;Young et al 2005) and may remain infectious (Williams & Hoy 1930;Maddock 1933;Wilesmith et al 1986). Badgers form social groups that use communal underground setts where conditions are likely to facilitate transmission and provide one focus of environmental contamination.…”