7/1/2006 - 6/30/2008
- Joel Pedersen, UW-Madison
- Katherine McMahon, UW-Madison
- Sharon Kluender, Wisconsin State Laboratory of Hygiene
Confirming the presence and determining the source(s) of fecal contamination to water is critical to protect human health and environmental quality. This is especially true in large portions of Wisconsin, where fractured and/or karst bedrock is located within a few feet of the ground surface, facilitating rapid transport of fecal pathogens to aquifers. Detection of commonly targeted fecal indicators (e.g., coliform bacteria, caffeine) suggests that water quality has been compromised. However, these indicators do not adequately track or confirm the presence of several enteric pathogens of concern, including viruses. In addition, current indicators do not discriminate between important sources of fecal contamination without labor-intensive and time-consuming investigation. Therefore, the causes of elevated indicator concentrations may be misjudged or remain unidentified.
Adenoviruses (AdV) have been advocated as fecal indicators with potential to both distinguish contamination sources and track environmental transport of enteric viruses. Despite these promising features, the following challenges have prevented efficient and confident use of AdV as source-specific fecal indicators: (1) current polymerase chain reaction (PCR) methods for AdV detection cannot simply or definitively discriminate between AdV of human and livestock origin; (2) filters commonly used to collect/concentrate viruses in 10- to 100-L water samples often exhibit poor virus recovery and are costly; and (3) compounds present in concentrated samples often interfere with DNA amplification by PCR.
The objectives of this study were (1) to develop a quantitative PCR (qPCR) method capable of simply and confidently distinguishing between human and bovine AdV; and (2) to optimize virus recovery from environmental samples by two newly-advocated, competitively priced filters while minimizing concomitant concentration or introduction of PCR-inhibiting compounds.
Oligonucleotides for the detection of HAdV and of BAdV were designed based on alignments of all available non-redundant sequences of the hexon gene (or complete AdV genome) present in the NCBI GenBank database. Primers were evaluated first by conventional PCR, agarose gel electrophoresis, and quantitative PCR. Cationic Nanoceram® filters were evaluated for the retention and recovery of two bacteriophages (MS2 and PRD1) and two AdV (bovine 1 and human 41) by virus adsorption-elution. A variety of eluents were investigated based on their previously demonstrated or hypothesized ability to disrupt virus-filter interactions. Fresenius hollow-fiber hemodialysis ultrafilters were evaluated for the recovery of bacteria (Escherichia coli and Enterococcus faecalis), bacteriophages (MS2 and PRD1), BAdV 1 and HAdV 41. AdV were assayed by qPCR, while bacteriophage were enumerated via plaque-forming-unit (pfu) assay on host bacteria cultures. E. coli and E. faecalis were enumerated using the quantitray application of the Colilert and Enterolert assays (IDEXX, Inc.).