The potential of reed bed technology – the use of constructed wetlands for wastewater treatment – was first realized in the 1960s in the Netherlands (Brix & Schierup, 1989). Reed beds have since been used worldwide for many purposes, including removal of parasitic helminth eggs from wastewaters in Egypt (Stott et al., 1999), reduction of pathogenic bacteria levels in dairy wastewater (Karpiscak et al., 2001), removal of viral pathogens from wastewater (Jackson & Jackson, 2008), and in the treatment of human sewage in many countries (Kadlec & Knight, 1996). Wetlands act as biofilters through a combination of physical, chemical, and biological processes (Brix, 1993). Physical factors may include mechanical filtration by vegetation, adsorption to organic matter, and sedimentation (Wood & McAtamney, 1994). The chemical processes of oxidation and exposure to biocides excreted by some hydrophytes act to reduce bacterial loads (Brix, 1997). Predation by nematodes and protozoa was found to be an important factor in the removal of bacteria from wastewaters in subsurface flow wetlands by Green et al. (1997). Attack by lytic bacteria and viruses, and natural die-off in the reed bed are other biological mechanisms thought to play a role in the removal of pathogenic bacteria (Gersberg et al., 1989, cited by Rivera et al., 1995). Several studies have shown the potential of reed bed technology in removing pathogenic bacteria from wastewater (Rivera et al., 1995; Green et al., 1997; Ottova et al., 1997; Karpiscak et al., 2001; Stenstrom & Carlander, 2001). Constructed wetlands typically remove >90% of coliforms [reportedly up to 99.999% in one study (Soto et al., 1999)] and >80% of faecal streptococci (Kadlec & Knight, 1996). Such research has focused primarily on the removal of common faecal bacteria (reviewed in Edwards et al., 2005). Consequently, little is known of reed bed filtration efficacy with regard to mycobacteria.
Mycobacteria are ubiquitous environmental saprophytes, found in marshes, ponds, and rivers at the interface of air and water, and in soil, particularly that which is rich in organic matter (Grange, 1987). Several species of mycobacteria cause disease in birds, with Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium genavense implicated most frequently (Tell et al., 2001). Avian tuberculosis (ATB) is endemic within captive wildfowl populations at several Wildfowl & Wetlands Trust (WWT) sites in the United Kingdom (Cromie et al., 1991; Painter, 1997; Evans, 2001; Zsivanovits et al., 2004). This is hampering a range of WWT's conservation programmes, and is the single greatest cause of death of adult birds at WWT Slimbridge (Thorpe, 2000). In captive wildfowl in WWT collections, ATB is caused principally, but not exclusively, by M. avium serotype 1 (Cromie et al., 1991; Painter, 1997). Evidence that the water flowing through the captive wildfowl pens is the source of infection comes from isolation of M. avium from ‘soil, mud or muddy water’ at WWT Slimbridge (Schaefer et al., 1973), an epidemiological study of disease spread progressively downstream from the initial case of infection (Cromie, 1991) and studies showing that the pathology of affected birds indicates oral infection (Brown & Cromie, 1996). Attempts have been made to control ATB in WWT collections using a range of approaches including development of diagnostic tests (Cromie et al., 1993), vaccination (Cromie et al., 2000), management of the bird collection (Thorpe, 2000) including rotation according to age (R.L. Cromie, unpublished data) and through environmental control (Evans, 2001). Reed beds have been used at WWT sites for several years (Billington, 2000; MacKenzie et al., 2004) but thorough investigations into their effectiveness in removing mycobacteria have until now been lacking.
Although culture is a definitive means of confirming mycobacterial presence, the technique has several practical limitations. Mycobacteria require special culture media and many species grow exceedingly slowly: 2–4 weeks may be required for visible colonies to form on culture media, and some strains of M. avium require up to 6 months before colonies become identifiable (Matthews et al., 1978). PCR holds several potential advantages over culture of mycobacteria. Not only is PCR a rapid technique, it can detect very low numbers of organisms and distinguish accurately between species of mycobacteria (Aranaz et al., 1997). Christopher-Hennings et al. (2003) showed nested PCR (nPCR) to be similarly sensitive to culture for the identification of M. avium spp. paratuberculosis from bovine faeces; nPCR can thus be considered a valid alternative to culture. Techniques for the recovery of mycobacterial DNA from soil samples have been described (Zhou et al., 1996). Mendum et al. (2000) successfully used PCR to amplify sequences of mycobacterial nucleic acids extracted from environmental samples.
The aim of this study was to investigate the fate of environmental mycobacteria, with special reference to M. avium, in a constructed reed bed that filters effluent from a large captive wildfowl collection, in an effort to clarify the potential role of reed beds in the environmental control of ATB. This was achieved through the application of single-stage PCR and nPCR on samples of water, sediment, and vegetation taken from before, within, and after the reed bed. A comparison was made between areas of the reed bed planted with common reed (Phragmites australis) and greater reedmace (Typha latifolia) as well as between samples taken at the water's surface, from the submerged stems and from the root systems of the reed bed vegetation.