Further analysis of the incidence of Mycobacterium avium Complex (MAC) in drinking water supplies (Including the detection of Helicobacter pylori in water and biofilm samples)
DWI0838

February 2005

INTRODUCTION

Mycobacterium avium subsp. paratuberculosis (MAP) is a putative cause of Crohn’s disease, a chronic inflammatory disease of the gastrointestinal tract of humans. The association is poorly understood, and based largely on clinical similarity between Crohn’s disease and Johne’s disease, a chronic, contagious and lethal disease that effects a wide range of livestock and domestic and wild animals, of which MAP is a known etiological agent. The presence of MAP in the faecal matter of clinically and subclinically infected cattle is well established (Whitlock et al., 1985 and Stabel, 1998). The entry of MAP into the water system is plausible, for example in agricultural run-off from the land into rivers. Given the zoonotic potential of MAP it is important to identify the incidence and extent of MAP in the environment and assess the exposure and risk to humans.

Concern has been raised because of some studies in which MAP DNA has been detected in human biopsy samples taken from Crohn’s patients. For example, MAP DNA was detected in 65-95% of samples taken from the guts of patients with Crohn’s (Sanderson et al., 1992 and Bull et al., 2003). Likewise the direct detection of MAP in samples may be due to the presence of DNA from dead organisms and not necessarily live organisms. It is also possible that components of dead mycobacteria may be relevant in acting as a trigger for Crohn’s.

Recent studies report the presence of both MAP DNA and viable MAP in bulk tank and pasteurised milk (Pillai and Jayarao, 2002, Stabel et al., 2002, Cousins et al., 1999). The reported detection rates of MAP directly from samples (by molecular methods) compared to culture of viable MAP from samples were apparently higher. This has caused debate because it is hard to establish whether or not the low culture rates are a reflection of the extremely slow growth rate and fastidious nutritional requirements of the organism.

In addition to the presence of MAP in milk concern has been expressed at the potential contamination of drinking water. In a recent study (Whale et al., 2004), no MAP was cultured from a range of samples collected from domestic water systems, including mains drinking water. This study demonstrated the problems associated with attempts to isolate MAP from environmental samples, particularly contamination and competition from rapid growing and more abundant mycobacteria species. For these reasons we proposed to use direct molecular detection methods. The most established methodology is detection of the insertion sequence 900 (IS900) by a polymerase chain reaction (PCR)-based assay. IS900 is a 1,451 bp element inserted into the MAP genome at 14-18 conserved loci (Bull et al., 2003). A number of protocols have been developed and commercial kits are available with varying performance (Pilliai and Jayarao, 2002). An optimised and specific nested IS900 PCR (in which the products of a primary PCR reaction serve as the template for a second PCR) as described by Bull et al. (2003) was adopted in this study due to its increased sensitivity over conventional IS900 PCR. The feasibility of alternative approaches for MAP detection such as immunomagnetic separation (IMS) or long term culture prior to PCR analysis was also examined.

This aim of the study was to retrospectively analyse the samples collected in the previous DWI study (listed in appendix) using IS900 analysis. Whilst we have had success using a cheap and rapid crude DNA extraction, others have advocated the importance of mechanical disruption, the use of Proteinase K and DNA purification (Pilliai and Jayarao, 2002, Bull et al., 2003 and Irene grant, personal communication).

Copies of this report may be available as an Acrobat pdf download under the 'Post 2000 Reports' heading on the DWI website.