No abstract

No abstract

Canada's National Agri-Environmental Standards Initiative sought to develop an environmental benchmark for low-level waterborne pathogen occurrence in agricultural watersheds. A field study collected 902 water samples from 27 sites in four intensive agricultural watersheds across Canada from 2005 to 2007. Four of the sites were selected as reference sites away from livestock and human fecal pollution sources in each watershed. Water samples were analyzed for Campylobacter spp., Salmonella spp., Escherichia coli O157:H7, Cryptosporidium spp., Giardia spp., and the water quality indicator E. coli. The annual mean number of pathogen species was higher at agricultural sites (1.54 ± 0.07 species per water sample) than at reference sites (0.75 ± 0.14 species per water sample). The annual mean concentration of E. coli was also higher at agricultural sites (491 ± 96 colony-forming units [cfu] 100 mL(-1)) than at reference sites (53 ± 18 cfu 100 mL(-1)). The feasibility of adopting existing E. coli water quality guideline values as an environmental benchmark was assessed, but waterborne pathogens were detected at agricultural sites in 80% of water samples with low E. coli concentrations (<100 cfu 100 mL(-1)). Instead, an approach was developed based on using the natural background occurrence of pathogens at reference sites in agricultural watersheds to derive provisional environmental benchmarks for pathogens at agricultural sites. The environmental benchmarks that were derived were found to represent E. coli values lower than geometric mean values typically found in recreational water quality guidelines. Additional research is needed to investigate environmental benchmarks for waterborne pathogens within the context of the "One World, One Health" perspective for protecting human, domestic animal, and wildlife health.

A single-tube PCR method was developed for efficient identification of nontuberculous mycobacteria (NTM) and their environmental isolates in about 3 h without conventional DNA isolation. The following three steps were optimized or developed: (i) a simple, 6-min direct cell lysis protocol as a PCR prestep for generation of DNA-template, (ii) an improved Mycobacterium-specific PCR amplification protocol with a broader species specificity using newly designed primers targeting a 228-bp region of the 65-kDa heat shock protein (hsp) gene and optimal PCR amplification conditions, and (iii) a genus-specific restriction analysis of the PCR product for conclusive identification of the unknown NTM isolates.Nontuberculous mycobacteria (NTM) are important causes of nosocomial infections and occupational illnesses. These organisms are commonly associated with natural ecosystems such as water supplies, aerosols, food, and soil (3,4,6,7,17). NTM that cause nosocomial infections are frequently associated with hospital water supplies and washing equipment. From an occupational health standpoint, NTM are considered causal agents for hypersensitivity pneumonitis, asthma, and bronchitis in machine workers exposed to metalworking fluids (MWF) and their aerosols, which are used in metalworking industries for cooling and lubrication (8,10,12,14,18). A method for early and reliable detection of mycobacteria from these environments might help minimize these illnesses. The existing practice of identification of mycobacteria from clinical and environmental sources includes isolation using enrichment and selective agar media, a method which often results in the collection of a large number of putative isolates for subsequent screening and confirmation by morphological, culture, and biochemical methods (20). Molecular methods such as PCR offer a significant alternative for rapid screening and identification of these bacteria (11). In practical terms, the major limiting steps of the PCR approach for the rapid screening are the extraction of amplifiable-quality genomic DNA and the availability of genus-specific primers with broad specificity for different species. Due to the complexity and rigidity of the cell walls of these acid-fast bacteria, several efforts have been reported for the rapid isolation of amplifiable genomic DNA by using physical, chemical, or enzymatic strategies or combinations thereof (1,5,9,13,16,22). However, these cumbersome and/or time-consuming protocols failed to yield effective cell lysis, thereby preventing their use in routine screening and diagnosis of diverse environmental species of Mycobacterium. The two existing genus-specific PCR-protocols, one based on a 16S rRNA gene (15) and the other based on Mycobacteriumspecific 65-kDa heat shock protein (hsp), are applied for identification of mycobacteria (16). The latter protocol has received wider acceptance. However, this protocol fails to reliably amplify the target hsp sequence in some environmental species such as Mycobacterium immunogenum, which is an importan...