Indicator organisms are used to assess public health risk in recreational waters, to highlight periods of challenge to drinking water treatment plants, and to determine the effectiveness of treatment and the quality of distributed water. However, many have questioned their efficacy for indicating

Male-specific (F؉) coliphages have been investigated as viral indicators of fecal contamination that may provide source-specific information for impacted environmental waters. This study examined the presence and proportions of the different subgroups of F؉ coliphages in a variety of fecal wastes and surface waters with well-defined potential waste impacts. Municipal wastewater samples had high proportions of F؉ DNA and group II and III F؉ RNA coliphages. Bovine wastewaters also contained a high proportion of F؉ DNA coliphages, but group I and IV F؉ RNA coliphages predominated. Swine wastewaters contained approximately equal proportions of F؉ DNA and RNA coliphages, and group I and III F؉ RNA coliphages were most common. Waterfowl (gull and goose) feces contained almost exclusively F؉ RNA coliphages of groups I and IV. No F؉ coliphages were isolated from the feces of the other species examined. F؉ coliphage recovery from surface waters was influenced by precipitation events and animal or human land use. There were no significant differences in coliphage density among land use categories. Significant seasonal variation was observed in the proportions of F؉ DNA and RNA coliphages. Group I F؉ RNA coliphages were the vast majority (90%) of those recovered from surface waters. The percentage of group I F؉ RNA coliphages detected was greatest at background sites, and the percentage of group II F؉ RNA coliphages was highest at human-impacted sites. Monitoring of F؉ coliphage groups can indicate the presence and major sources of microbial inputs to surface waters, but environmental effects on the relative occurrence of different groups need to be considered.In the last decade, increased attention has been given to the physical and biological integrity of our nation's water bodies. The 2000 National Water Quality Inventory reported that 39% of the rivers and streams evaluated were polluted, and pathogens are among the top three causes of impairment (http: //www.epa.gov/305b/2000report/). In addition, the leading source of impairment was reported to be non-point-source pollution from urban and agricultural lands during periods of precipitation and runoff. Factors contributing to water impairment include increased land use and rural development which place receiving water bodies at high risk of contamination (25) and alterations in the hydrologic cycle, associated with global climate change, that are increasing the magnitude and frequency of runoff events (18,25). States are addressing compromised water quality by identifying impaired water bodies and establishing total maximum daily load (TMDL) programs designed to limit pollution and restore water quality. An important part of a regional TMDL program is assessment of the relative microbial impacts of various potential waste inputs in a watershed, and a source-specific microbial indicator system would be an important component of such a program.Enteric bacteria have traditionally been used as indicators of fecal contamination in source, drinking, and recreational waters. ...

In recent years, there has been increased interest in the use of male-specific or F؉ coliphages as indicators of microbial inputs to source waters. Sero-or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F؉ RNA (FRNA) viruses are well studied, while little is known about the F؉ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Q␤, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F؉ coliphages as a useful tool for large-scale MST studies.

The survival of seven F+RNA phages (MS2 Group I ATCC type strain, two Group I environmental isolates, a Group II environmental isolate, a Group III environmental isolate, and two Group IV environmental isolates) and six F+DNA phages (M13, fd, f1, and ZJ/2 ATCC type strains, and two environmental isolates) were examined in microcosms using a surface drinking water source. Phages were spiked into replicate aliquots of a source water at about 20,000 pfu/ml. Replicate spikes were incubated at 4 and 20 degrees C and monitored for 110 days. At 4 degrees C, Groups I and II F+ RNA phages were detectable through 110 days, with reductions of about 1 and 3 log10, respectively. The Group III F+RNA phage demonstrated 5 log10 reduction after 3 weeks, and the Group IV F+RNA phages were reduced to detection limits (5 log10 reduction) within 10 days. Of the F+DNA phages, all four type strains were detectable with about 2.5 log10 reduction after 110 days at 4 degrees C. The F+DNA environmental isolates were detectable with about a 4 log10 reduction after 110 days at 4 degrees C. All phages demonstrated faster decay at 20 degrees C. These results suggest that differences in F+ phage survival may influence their prevalence in environmental waters and the ability to attribute their prevalence to specific human and animal sources of faecal contamination.

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