Evaluation of F RNA and DNA Coliphages as Source-Specific ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2003, p. 6507–6514 0099-2240/03/$08.00⫹0 DOI: 10.1128/AEM.69.11.6507–6514.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 69, No. 11

Evaluation of F⫹ RNA and DNA Coliphages as Source-Specific Indicators of Fecal Contamination in Surface Waters Dana Cole,1* Sharon C. Long,2 and Mark D. Sobsey1 Department of Environmental Sciences and Engineering, The University of North Carolina School of Public Health, Chapel Hill, North Carolina 27599,1 and Department of Civil and Environmental Engineering, University of Massachusetts, Amherst, Massachusetts 352052 Received 28 March 2003/Accepted 8 August 2003

Male-specific (Fⴙ) 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 Fⴙ coliphages in a variety of fecal wastes and surface waters with well-defined potential waste impacts. Municipal wastewater samples had high proportions of Fⴙ DNA and group II and III Fⴙ RNA coliphages. Bovine wastewaters also contained a high proportion of Fⴙ DNA coliphages, but group I and IV Fⴙ RNA coliphages predominated. Swine wastewaters contained approximately equal proportions of Fⴙ DNA and RNA coliphages, and group I and III Fⴙ RNA coliphages were most common. Waterfowl (gull and goose) feces contained almost exclusively Fⴙ RNA coliphages of groups I and IV. No Fⴙ coliphages were isolated from the feces of the other species examined. Fⴙ 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 Fⴙ DNA and RNA coliphages. Group I Fⴙ RNA coliphages were the vast majority (90%) of those recovered from surface waters. The percentage of group I Fⴙ RNA coliphages detected was greatest at background sites, and the percentage of group II Fⴙ RNA coliphages was highest at human-impacted sites. Monitoring of Fⴙ 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. cator organisms for these pathogens has been widely recognized (12, 20; R. Karlin, R. Fayer, M Arrowood, C. Noss, and D. Schoenen, Proc. Am. Water Works Assoc. Source Water Prot. Symp.: Focus on Waterborne Pathogens, 1998 [on CDROM]). Coliphages, viruses that infect Escherichia coli bacteria, have been proposed by the U.S. Environmental Protection Agency as a viral indicator of fecal contamination of groundwater (35), and male-specific (F⫹) coliphages may provide the additional benefit of distinguishing human and animal fecal sources of pollution. Coliphages infect coliform bacteria, are nonpathogenic to humans, and are more similar to enteric viruses with respect to physical characteristics, environmental persistence, and resistance to treatment processes than are indicator bacteria (5, 14, 20, 27, 32, 36). Coliphages are consistently present in domestic raw and treated sewage and have been reported to occur in concentrations ranging from 103 to 107 PFU/liter, depending on the level of sewage treatment (2, 5, 7, 11). In addition, a variety of domestic and feral animals also shed coliphages in their feces (2, 4, 8, 11, 22). The coliphages can be divided into six major morphological groups, two of which infect only F⫹ male hosts through the F sex pilus (30). The two F⫹ coliphage families are the Leviviridae (small, icosahedral, single-stranded RNA phages) and the Inoviridae (filamentous, single-stranded DNA phages). F⫹ RNA coliphages have been highly correlated with virus concentrations in raw and treated wastewater, raw and partially treated drinking water, and surface and recreational waters (14). Other studies have found that F⫹ RNA coliphages behave similarly to enteric viruses in environmental waters and therefore may be used to indicate the viral safety of source

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. However, research has demonstrated that these bacteria may not be appropriate indicators of pathogenic viruses and protozoa (21, 23, 24), and the need for reliable index or indi* Corresponding author. Present address: Department of Large Animal Medicine, The University of Georgia College of Veterinary Medicine, Athens, GA 30602. Phone: (706) 542-0177. Fax: (706) 542-8833. E-mail: [email protected]. 6507

6508

COLE ET AL.

waters (4, 9, 31). In addition, grouping of F⫹ RNA coliphages by serotyping or DNA oligoprobing may be used to identify and distinguish between human and nonhuman fecal contamination sources of F⫹ RNA coliphages (4, 11, 15). Griffin et al. (10) and Brion et al. (1) reported the use of F⫹ RNA coliphage analysis to confirm putative animal and human waste impacts on environmental waters. In contrast to the F⫹ RNA coliphages, little is known about the ecology of F⫹ DNA coliphages (8). Research conducted in Massachusetts (19) and in other geographic areas by Sobsey and colleagues (personal communication) found F⫹ DNA coliphages in a variety of water sources impacted by human and animal fecal waste sources. Furthermore, the F⫹ DNA coliphages were commonly detected during warmer months, when F⫹ RNA coliphages were absent from surface water samples (19). Seasonal differences in the proportions of F⫹ DNA and RNA coliphage occurrence may reflect differential survival characteristics, changes in coliphage excretion patterns of hosts, or changes in land use in impacted watersheds. This research investigated the presence, prevalence, and densities of F⫹ RNA and DNA coliphages in animal feces, animal wastewater from agricultural activities, and municipal wastewater. F⫹ RNA coliphage typing was used to distinguish fecal contamination sources. In addition, the densities and relative proportions of F⫹ RNA and DNA coliphages in surface waters were evaluated at well-defined sampling sites to determine their usefulness as a water quality surveillance tool with which to identify the most significant viral input sources (human versus animal). MATERIALS AND METHODS Study sites and sample collection. Human and animal wastewater, freshly voided animal fecal samples, and surface waters potentially impacted by waste discharges or runoff from a variety of well-defined land uses were collected and analyzed by The University of North Carolina (UNC) Department of Environmental Sciences and Engineering and the University of Massachusetts (UMass) Department of Civil and Environmental Engineering. Samples were collected from surface water sites monthly for 40 months. A subset of surface water samples was also collected during or just following precipitation events (storm samples). At UNC, storm samples were collected with ISCO (Lincoln, Nebr.) automatic samplers that were triggered when the stream height increased by 0.5 in. Analyzed storm samples represented a composite of the storm hydrograph. Storm samples were collected manually at UMass within 24 h of a precipitation event that exceeded 0.1 in. Freshly voided feces (50 g) and liquid wastewater samples (500 ml) were collected aseptically from a variety of feral and domestic animals, from cattle and swine waste lagoons, and from human wastewater treatment plants (WWTP). In addition, surface waters (2-liter samples) were collected from sites identified as being potentially impacted by urban or rural human land use (municipal sewage effluents or septic systems, respectively) or agricultural land use (swine or cattle farms). For each surface water study site, an upstream or background station was identified and sampled on the same day. All samples were collected and transported to the laboratory in sterile, wide-mouth, high-density polyethylene bottles on ice or commercial freezer packs and analyzed within 24 h (WWTP, waste lagoon, and surface water samples) or 72 h (solid wastes) of sample collection. Data recorded at the time of analysis included the sampling site, animal species, or waste source associated with the sample or sampling site, the date of collection, and whether the sample was collected during a storm event. Fⴙ coliphage isolation and serotyping. F⫹ coliphages were enumerated by direct plating of serial dilutions (wastes and wastewater) or cellulose membrane filter adsorption-elution concentration (surface water), followed by double or single agar layer plaque assay methods (U.S. Environmental Protection Agency method 1602) (29, 30). When available, up to 10 coliphage isolates were removed from the sample agar, suspended in phosphate-buffered saline (PBS) containing 20% glycerol, and stored at ⫺80°C until further analysis. F⫹ RNA and DNA coliphages were distinguished by spotting (5 ␮l) and incubation of serial dilutions

APPL. ENVIRON. MICROBIOL. (10⫺2, 10⫺4, and 10⫺6) of the isolated F⫹ coliphage suspended in PBS on nutrient agar-host (E. coli Famp) plates (control) or nutrient agar-host plates containing RNase (experimental) for 12 to 16 h. Phage growth on both the control and experimental plates at all dilutions was indicative of F⫹ DNA phage. A type strain group I coliphage, MS2 (previously molecularly characterized), was used as a positive F⫹ RNA control, and PBS was used as a negative control. F⫹ RNA phage isolates were serotyped to group phages into the following categories: group I (MS2), group II (GA), group III (Q␤), and group IV (SP). Briefly, serial dilutions of the field F⫹ RNA isolate were plated in 5-␮l spots on nutrient agar-E. coli Famp host plates containing neutralizing antisera to MS2, GA, Q␤, or SP coliphages. Failure to propagate at all dilutions in the presence of an antiserum was recorded as a positive serogroup identification. Statistical analysis. The results of all isolate evaluations were entered into a database and examined for bivariate associations with recorded sample data. When stream data were entered into the database, zero was entered when F⫹ coliphages were below the detection limit. When the density of coliphages detected exceeded the countable range, no density entry was made in the database; however, a second variable was assigned that recorded whether phages were detected or not. The distribution of the stream coliphage density data (PFU per liter) was evaluated for log normalcy with a Kolmogorov Smirnov test prior to statistical testing. This distribution was used to estimate the density of coliphages among samples that exceeded the countable range. Paired t tests were used to compare the log10 geometric means of the density data grouped by land use impact. A chi-square or Fisher exact test was used to evaluate potentially significant associations between frequencies of coliphage detection and proportions of coliphage serogroups among land use categories. All statistical tests were evaluated at the 95% confidence level.

RESULTS Presence and proportions of Fⴙ coliphages in waste materials. Overall, wastes from 14 different species were analyzed by the two study facilities (Table 1). However, F⫹ phages were isolated from only five species. F⫹ coliphages were most abundant in swine and WWTP (human) wastewaters and in gull feces. F⫹ DNA phages were most prevalent among the bovine (82% of coliphages evaluated) and human (77%) wastewater isolates. The prevalence of F⫹ DNA phage isolates among swine (50%) and gull (4%) feces was significantly lower (P ⬍ 0.0001) than in these wastewaters. Serotyping of the F⫹ RNA phages revealed significantly different proportions of the four serogroups between WWTP and animal isolates (Fig. 1). Group I isolates were significantly more prevalent among the animal isolates, with the exception of gull wastes. Group I isolates made up only 3.7% of the total F⫹ RNA serogroups in the WWTP wastes and 4.2% of the total in gull wastes, compared to 52.6% of the total in swine wastewaters (P ⬍ 0.001) and 33% of the total in bovine wastewaters (P ⬍ 0.05). Conversely, WWTP wastes had the highest proportion of group II isolates, making up 51.9% of the total F⫹ RNA groups, compared to 5.3% of the total found in swine samples (P ⬍ 0.0001) and 16.67% of that in bovine samples (P ⬍ 0.05). Bird (goose and gull) wastes did not contain either group II or group III serogroups. Group III isolates were only found in swine and human wastes, and there was no significant difference in prevalence between the two species. Group IV isolates were found in cow, swine, and gull wastes but not among the goose samples. There was no significant difference in the prevalence of group IV isolates in swine and bovine wastewaters. Gull wastes had the highest proportion of group IV phages (96%; P ⬍ 0.05) of all of the species evaluated. Cross-reactivity between serogroups (reaction of an F⫹ RNA coliphage isolate with more than one antiserum) was most common among the human isolates (Table 1). Of these, cross-reactivity between serogroups II and III was most fre-

VOL. 69, 2003

WATERSHED SOURCE TRACKING WITH F⫹ COLIPHAGES

6509

FIG. 1. Percentages of F⫹ RNA coliphages isolated from fecal wastes of various animal species.

quently observed. When serogroups II and III were combined and these cross-reactive groups were included (Fig. 2), human wastes had a significantly higher proportion of these two groups (93%) than all of the other species, including swine (30%; P ⬍ 0.0001). Frequency and density of Fⴙ coliphages in environmental water samples. The results of environmental water sampling are shown in Table 2. Seventy-three sets of stream samples were collected, 23 of which represented storm event sampling. Under baseflow (nonstorm) conditions, samples were collected from swine-impacted sites (14% of baseflow samples), cattleimpacted sites (18%), human-impacted sites (32%), and back-

ground sites (36%). Only one storm sample was collected from swine-impacted sites. Overall, F⫹ coliphages were detected in 60% of the samples collected. Differences in the frequency of F⫹ coliphage isolation between the two geographic research facilities were evaluated. Under baseflow conditions, F⫹ coliphages were detected less frequently at UNC background sites (25%) than at UMass background sites (78%; P ⫽ 0.004). For both geographic locations, F⫹ coliphages were detected more frequently in human- or animal-impacted waters than in background waters under baseflow conditions. When the data from both facilities were pooled, the difference between impacted

TABLE 1. Fecal wastes evaluated for the presence of F⫹ coliphages Source

Cow wastes Horse Gull Goose Buffalo Cat Cormorant Rooster Dog Llama Donkey Pig Swine wastewater WWTP a

No. positive/ no. evaluated

14/17 0/4 2/2 3/5 0/2 0/1 0/1 0/1 0/1 0/2 0/1 0/2 11/11 7/7

F⫹ phage density phages (PFU/g or liter) a

Total no. of phages evaluated

% RNA

65

18

RNA no. and phage groups

% DNA

Median

Range

30

25 3

96 100

8.0 ⫻ 104

⬍3–3,267 ⬍3 200–1.5 ⫻ 105 ⬍3–20 ⬍3 ⬍3 ⬍3 ⬍3 ⬍3 ⬍3 ⬍3 ⬍3 2,500–5.5 ⫻ 106

113

50

30 I, 3 II, 14 III, 10 IV

50

3.2 ⫻ 106

600–1.3 ⫻ 108

115

23

1 I, 14 II, 5 III, 1 I/II, 5 II/III, 1 III/IV

77

7.5 ⫻ 104 3

When values were below detection limits, 0.5 times the level of detection was used for the missing value.

4 I, 2 II, 1 II/III, 4 IV 1 I, 23 IV 3I

82 4 0

6510

COLE ET AL.

APPL. ENVIRON. MICROBIOL.

FIG. 2. Proportion of serogroup II and III F⫹ RNA coliphages when cross-reactivity between groups is included.

and background sites was statistically significant (P ⫽ 0.04). In addition, the frequency of F⫹ coliphage detection was greater following storm events (88%) than under baseflow conditions (50%) at UNC (P ⫽ 0.0029), but not at UMass. There were no statistically significant seasonal differences in the frequency of F⫹ coliphage detection. The coliphage density data were consistent with a log normal distribution, so geometric mean data were used to statistically compare coliphage densities between study sites and season and land use categories. Under baseflow conditions, the F⫹ coliphage geometric mean densities measured at human-impacted and background sites were lower and showed greater variability at UNC than at UMass, but this difference was not statistically significant. The geometric mean densities of F⫹ coliphages were higher at human-impacted sites than at back-

ground sites at both research facilities (UNC and UMass), but these differences were not statistically significant for either study site or in pooled-data analysis. Swine-impacted sites had significantly lower coliphage densities than any other land use classifications (P ⫽ 0.010 to 0.026). No other significant differences were observed among season and land use classifications under baseflow conditions. Coliphage density data collected during storm events followed different trends at the two research facilities. At UNC, the geometric mean coliphage density was significantly higher (P ⬍ 0.0001) during storm events than under baseflow conditions. In contrast, at UMass, the geometric mean coliphage density was significantly lower in samples collected during storm events (P ⫽ 0.002) than under baseflow conditions. This difference most likely reflects the different storm sampling

TABLE 2. Frequency and density of F⫹ DNA and RNA coliphages in surface water samples Condition

No. of samples collected

Human

Baseflow Storm

Bovine

No. of samples collected

45 10

Fall Winter Spring Summer

14 6 13 22

UNC Storm UMass Storm

19/32 2/2 11/13 6/8

Baseflow Storm

25 9

Fall Winter Spring Summer

5 6 13 10

Baseflow Storm

Background

Baseflow Storm

50 23

Fall Winter Spring Summer

13 11 19 30

Swine

Baseflow Storm

20 1

Fall Winter Spring Summer

7 0 6 8

a

Values are density in numbers of PFU per gram or liter.

Location or condition

Frequency of detection (no. of samples positive/total)

Season

Land usage

Location

Log10 geometric meana (SD) Baseflow

Storm

UNC UMass

0.55 (1.01) 1.22 (0.88)

2.06 (0.58) 0.38 (0.90)

16/25 8/9

UNC

0.74 (0.93)

3.23 (1.47)

UNC Storm UMass Storm

8/32 4/5 14/18 10/18

UNC UMass

0.48 (1.24) 1.18 (0.79)

2.60 (0.28) 0.36 (0.57)

Baseflow Storm

11/20 1/1

UNC

0.097 (0.55)

1.89

VOL. 69, 2003

WATERSHED SOURCE TRACKING WITH F⫹ COLIPHAGES

6511

TABLE 3. F⫹ coliphage types and subgroups isolated from surface waters No. of phage isolated

Total no. of F⫹ DNA phage

Total no. of F⫹ RNA phage

Group I

Group II

Group III

Group IV

Other

621

369

252

209

17

2

3

9 group I/II, 1 group I/IV, 11 untypeable

Swine Baseflow Storm

17

14 14 NTa

3 3 NT

0

1

0

0

2 untypeable

Bovine Baseflow Storm

61

41 12 29

20 19 1

14

2

1

246

141

105

76

12

0

2

92 49

87 18

9 group I/II, 1 group I/IV, 5 untypeable

173 128 45

124 75 49

119

2

1

1

1 untypeable

Land use impact and condition

Total stream

Human Baseflow Storm Background Baseflow Storm a

297

No. of F⫹ RNA phage isolated

3 untypeable

NT, none typeable.

schemes used at the two study sites: UNC used automatic samplers that sampled during several phases of the hydrograph, whereas UMass collected grab samples within 24 h of a rain event, usually in the falling limb of the hydrograph. It is possible that the late hydrograph samples represent conditions of better water quality due to prior flushing of contaminants and greater dilution by precipitation. Factors influencing Fⴙ DNA and RNA phage groups isolated from environmental waters. Table 3 presents the results of F⫹ DNA and RNA classification. The proportion of F⫹ DNA phages isolated during storm events (64%) was somewhat higher than under baseflow (57%) conditions (P ⫽ 0.086). However, when stratified by land use classification,

impacted sites and background sites did not follow a consistent trend. Cattle-impacted sites exhibited the greatest increase in the proportion of F⫹ DNA phages isolated during storm events (97%) compared to baseflow conditions (37%; P ⬍ 0.0001), followed by human-impacted sites (73 versus 51%; P ⫽ 0.002). At background sites, the proportion of F⫹ DNA phages isolated during storm events was lower (48%) than under baseflow (63%; P ⫽ 0.014) conditions. Seasonal variation in the percentage of F⫹ DNA phage isolation was evaluated among surface water samples collected under baseflow conditions (Fig. 3). The highest proportions of F⫹ DNA phages were isolated during the warmer months of summer (June to August) and fall (September to November).

FIG. 3. Percentage of F⫹ DNA (relative to F⫹ RNA) coliphage isolation.

6512

COLE ET AL.

APPL. ENVIRON. MICROBIOL.

The seasonal trends were the same for both geographic regions and among all of the land use categories, so the data were pooled for statistical analysis. There were no significant differences between the percentages of F⫹ DNA phage isolations during winter (4% F⫹ DNA) and spring (5% F⫹ DNA). However, the percentage of F⫹ DNA phage isolations (19% F⫹ DNA) during fall months (September to November) was significantly higher than in those two seasons (P ⱕ 0.023), and the percentage of F⫹ DNA phage isolation during summer months (83% F⫹ DNA) was significantly higher than during all of the other seasons (P ⬍ 0.0001). The majority of F⫹ RNA coliphages (Table 3) were isolated from human-impacted (43%) or background (46%) sites, which probably reflects the sampling frequency bias for these sites. The most frequently isolated F⫹ RNA coliphage among surface water samples was group I (90%), followed by group II (7%). Comparison of F⫹ RNA coliphage percentages between impacted and background surface water sites revealed significant differences between the land use categories. Background sites had the greatest diversity of coliphage subgroups recovered, but the percentage of group I coliphages recovered at these sites (97%) was significantly higher than that recovered at either bovine-impacted (82%; P ⫽ 0.035) or human-impacted (75%; P ⬍ 0.0001) sites. In contrast, the percentage of group II coliphages recovered from human land use sites (12%) was significantly greater than that recovered from background sites (2%; P ⫽ 0.001). A higher percentage of group II coliphages was also recovered from bovine-impacted sites than from background sites, but the difference was not statistically significant. Swine land use sites were not evaluated because of the low number of coliphages recovered. DISCUSSION A source-specific indicator organism that can be incorporated into water quality management and protection plans is desirable for identification of important sources of fecal contamination within a watershed and establishment of TMDLs. In this and past studies, F⫹ coliphages have shown some promise in this regard. F⫹ coliphages can be isolated consistently from WWTP wastewater, swine and cattle waste, animal waste lagoon liquids, waterfowl feces, and occasionally other domestic and feral animal wastes (2, 6, 10–14, 17, 26). Furthermore, differences in the relative percentages of F⫹ RNA coliphage groups found in various waste and water samples may be useful for source tracking of specific inputs. Consistent with most previous work, this study found significant differences in the proportions of F⫹ DNA and RNA phages among various wastes sources. These differences could aid in distinguishing contamination sources in impacted environmental waters. WWTP samples were characterized by high proportions of F⫹ DNA coliphages, as well as serogroup II and III F⫹ RNA coliphages. Cross-reactivity of F⫹ RNA coliphage groups II and III was noted frequently among human wastewaters, but this did not compromise the interpretation of the coliphage source, since both groups are associated with human wastes. Mixed reactions among isolates were also noted when genotyping was used by Schaper et al. (26) at rates of about 7% for animal slurries, 3% for slaughterhouse wastewater, and up to 9.5% for domestic wastewaters. Bovine waste-

water was similar to human wastewater in containing a high proportion of F⫹ DNA coliphages. Unlike human wastewater, however, serogroups I and IV were the most common F⫹ RNA coliphages isolated from bovine samples. Swine wastewaters had equal proportions of F⫹ DNA and RNA coliphages and exhibited a greater diversity of F⫹ RNA phage serogroups than did the other animal species evaluated. This finding may be related to the fact that human and porcine gut physiology and perhaps their microbial communities are similar (33, 34). High proportions of serogroup I and IV F⫹ RNA coliphages generally distinguished animal source wastes from municipal WWTP wastes. While the variability in the observed densities of F⫹ coliphages among the various species could potentially limit the use of F⫹ coliphages in environmental water monitoring, Calci et al. (2) estimated that the volume of sewage input was the most important factor determining coliphage density in impacted environmental waters. Consequently, an analysis of the proportion of F⫹ DNA coliphages and predominant F⫹ RNA serogroups present in environmental water samples could provide valuable information regarding the most important local or regional sources impacting water quality within a watershed if environmental factors did not significantly alter these proportions. In two previous studies (1, 16), F⫹ coliphages were recovered more frequently from sites downstream from human land use and following storm events than from background sites during baseflow. Both research facilities in this study found the frequencies of F⫹ coliphage occurrence to be significantly greater in human-impacted waters than in background waters. However, in contrast to findings reported by Brion et al. (1), this study found that land used by cattle was also associated with an increased frequency of F⫹ coliphage recovery. There were significant differences between the two research facilities contributing to this study in the frequency of F⫹ coliphage isolation in background surface waters and in the relative coliphage densities in samples associated with precipitation events. This may reflect watershed, climate, and sample collection factors that were dissimilar between the two study regions. At UNC, background surface water sites were located in rural watersheds with very low population densities. At UMass, the background sites included one highly protected site (a water supply aqueduct intake) that did not directly receive inputs from the land but was potentially influenced by roosting gulls. The other UMass background sites were located within a relatively undeveloped subwatershed to an unfiltered source water reservoir. The climates are also different in Massachusetts and North Carolina, with generally colder weather in the former and warmer weather in the latter. In North Carolina, water temperatures often are above 15°C. The generally higher water temperatures in North Carolina may result in faster rates of F⫹ coliphage inactivation and their more frequent absence from the water column (3, 28). In addition, automatic samplers were used at UNC study sites to collect stream waters during the storm hydrograph whereas at UMass grab samples were collected within 24 h of the main precipitation event. These differences limit the direct comparability of coliphage occurrence in precipitation samples from the two study sites, suggesting the need for caution when comparing recovery and density data in other similar but not identical environmental studies.

VOL. 69, 2003

WATERSHED SOURCE TRACKING WITH F⫹ COLIPHAGES

There was a tendency for surface waters from bovine- or human-impacted sites to have higher geometric mean F⫹ coliphage densities than background sites under baseflow sample collection conditions, but these associations were not statistically significant. All of the sampling sites studied were downstream from non-point-source impacts, so contamination was probably relatively diffuse and influenced by local conditions. Therefore, it is not surprising that the temporal variability in measured coliphage densities in surface water samples was too great to observe statistically significant differences in coliphage densities among the land use impact categories during the study period. Nonetheless, there was evidence that land use impacts affected the distribution of F⫹ coliphages in surface waters, although this was influenced by environmental conditions. The proportion of F⫹ DNA coliphages isolated from environmental waters was significantly influenced by season, and stratified analysis suggested that this was not related to research facility or land use impact. The percentage of F⫹ DNA coliphages isolated from surface waters was lowest in the spring and highest in the summer. This finding may be influenced by the effect of water temperature on the relative proportions of F⫹ RNA and DNA coliphages in a sample, with higher F⫹ RNA coliphage inactivation rates in warmer months and a greater likelihood of having F⫹ DNA coliphage-positive samples. However, human- and animal-impacted waters exhibited a more gradual decline in F⫹ DNA isolation than did background sites at both research facilities, suggesting that these land uses may have contributed to the occurrence and concentrations of F⫹ DNA coliphages in adjacent surface waters. This is supported by the fact that the proportion of F⫹ DNA coliphages isolated from surface waters downgradient from animal and human land use sites during storm events was significantly greater than under baseflow conditions, and this is in contrast to background sites where storm events resulted in decreased F⫹ DNA coliphage isolation. Considering that the highest percentages of F⫹ DNA coliphages isolated from fecal wastes were from bovine and human sources, the observed increase in the percentages of F⫹ DNA coliphages isolated from surface waters during storm events may indicate runoff or other sources of coliphage delivery to surface waters from these sources. On this basis, it is plausible that land use impacts on surface waters influence the percentages F⫹ DNA coliphages isolated. The classification of F⫹ RNA coliphage into subgroups to determine sources of fecal wastes in surface waters may be a useful tool for TMDL programs, but the data need to be carefully interpreted. Differential survival of the various F⫹ RNA subgroups may change the distribution of recovered coliphage groups from an impacted surface water site. For example, the overwhelming majority of F⫹ RNA coliphages isolated from stream waters in this study was group I. Previously published work has also reported a high frequency of group I F⫹ RNA coliphage recovery from environmental surface waters (1, 10), and this observation was attributed to the specific fecal impacts in the study region. However, S. C. Long and M. D. Sobsey (101st Gen. Meet. Am. Soc. Microbiol. 2001, abstr. Q-323, p. 649, 2001) found that group I phages survive longer in lake water at both 4 and 20°C than do the other subgroups. In addition, both Schaper et al. (26) and Brion et al.

6513

(1) found increased survival of environmentally derived group I and II coliphages at 25 to 37°C than of other F⫹ RNA serogroups. Furuse (8) described temperature effects on F⫹ RNA coliphage survival indicating that group II coliphages survive preferentially at lower temperatures than do the other three groups. Woody and Oliver (37) described optimal survival of group III coliphages at higher temperatures. Consequently, it is very likely that the presence of F⫹ RNA coliphage serogroups in environmental surface waters is influenced by differences in their environmental survival. Accordingly, a preponderance of group I coliphages in this and previous studies may reflect environmental persistence rather than a high proportion or load of animal source inputs. This differential survival of F⫹ RNA coliphage groups may limit the usefulness of F⫹ RNA coliphage grouping for tracking of fecal sources of contamination in a watershed with poorly characterized or multiple impacts. Nonetheless, surface waters impacted by human wastes in this study did have a significantly higher proportion of type II F⫹ RNA coliphages compared to background sites, suggesting that human land use was affecting the F⫹ RNA coliphage group distribution in downstream waters. The presence and relative percentages of F⫹ RNA and DNA coliphages in waste sources demonstrate that wastes associated with municipal wastewater and high-density agricultural (cattle and swine) activities contain both of these indicators more frequently than do other sources (waterfowl and companion animals). Wildlife and companion animal species contain either predominantly F⫹ RNA coliphages or no coliphages at all, suggesting that F⫹ coliphages might be useful for the surveillance of major sources having regional impacts on microbial water quality. The volume of waste associated with these centralized waste management facilities, combined with the high coliphage density observed in the wastes, minimizes the possible impacts of feral or nonlivestock animal species, confounding the interpretation of F⫹ RNA serogrouping. This is further supported by the fact that densities of F⫹ coliphages increased in surface waters during precipitation events and were higher in waters impacted by human and cattle agriculture land use activities. Therefore, group classification of coliphage isolates appears to be useful in identifying major sources of water quality impairment if regional impacts and differential environmental survival of the F⫹ DNA and RNA coliphage subgroups are taken into account. Ambient water quality standards using fecal indicators are based on quantitative data for microbial densities that are considered indicative of health-related exposure risks from enteric pathogens. Therefore, studies are still needed to determine the quantitative relationships between F⫹ coliphage occurrence, densities, and groups and those of enteric viral pathogens in surface waters if coliphages are to be reliably used for health-related water quality criteria and the development of indicator density standards. ACKNOWLEDGMENTS This material is based on work supported in part by a POWRE grant from the National Science Foundation (BES-9973545), ISA SC MDC 8000 UMS 33 from the Metropolitan District Commission, U.S. Department of Agriculture NRI grant 99-35102-8178, and NIEHS training grant ES07018. Assistance with sample collection and laboratory analyses were provided by Ed Brank of the Metropolitan District Commission; Erin

6514

COLE ET AL.

APPL. ENVIRON. MICROBIOL.

Shafe, graduate assistant at UMass; and Didi Utin at UNC, Chapel Hill. Any opinions, findings and conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the funding agencies. REFERENCES 1. Brion, G. M., J. S. Meschke, and M. D. Sobsey. 2002. F-specific RNA coliphages: occurrence, types, and survival in natural waters. Water Res. 36:2419–2425. 2. Calci, K. R., W. Burkhardt III, W. D. Watkins, and S. R. Rippey. 1998. Occurrence of male-specific bacteriophage in feral and domestic animal wastes, human feces, and human-associated wastewaters. Appl. Environ. Microbiol. 64:5027–5029. 3. Callahan, K. M., D. J. Taylor, and M. D. Sobsey. 1995. Comparative survival of hepatitis A virus, poliovirus and indicator viruses in geographically diverse seawaters. Water Sci. Technol. 31(5–6):189–193. 4. Chung, H. 1993. F-specific coliphages and their serogroups, and Bacterioides fragilis phages as indicators of estuarine water and shellfish quality (fecal contamination). Ph.D. thesis. University of North Carolina, Chapel Hill. 5. Chung, H., L. A. Jaykus, G. Lovelace, and M. D. Sobsey. 1998. Bacteriophages and bacteria as indicators of enteric viruses in oysters and their harvest waters. Water Sci. Technol. 38:37–44. 6. Furuse, K., A. Ando, S. Osawa, and I. Watanabe. 1981. Distribution of ribonucleic acid coliphages in raw sewage from treatment plants in Japan. Appl. Environ. Microbiol. 41:1139–1143. 7. Furuse, K., S. Osawa, J. Kawashiro, R. Tanaka, Z. Osawa, S. Sawamura, Y. Yanageawa, T. Nagao, and I. Watanabe. 1983. Bacteriophage distribution in human faeces: continuous survey of healthy subjects and patients with internal leukemia diseases. J. Gen. Virol. 64:2039–2043. 8. Furuse, K. 1987. Distribution of coliphages in the environment: general considerations, p. 87–124. In S. M. Goyal, C. P. Gerba, and B. Bitton (ed.), Phage ecology. John Wiley & Sons, Inc., New York, N.Y. 9. Geldenhuys, J. C., and P. D. Pretorius. 1989. The occurrence of enteric viruses in polluted water, correlation to indicator organisms and factors influencing their numbers. Water Sci. Technol. 21:105–109. 10. Griffin, D. W., R. Stokes, J. B. Rose, and J. H. Paul III. 2000. Bacterial indicator occurrence and the use of an F⫹ specific RNA coliphage assay to identify fecal sources in Homosassa Springs, Florida. Microb. Ecol. 39:56–64. 11. Havelaar, A. H., K. Furuse, and W. M. Hogeboom. 1986. Bacteriophages and indicator bacteria in human and animal faeces. J. Appl. Bacteriol. 60:255– 262. 12. Havelaar, A. H. 1987. Virus, bacteriophages and water purification. Vet. Q. 9:356–360. 13. Havelaar, A. H., W. M. Pot-Hogeboom, L. Furuse, R. Pot, and M. P. Hormann. 1990. F-specific RNA bacteriophages and sensitive host strains in faeces and wastewater of human and animal origin. J. Appl. Bacteriol. 69:30–37. 14. Havelaar, A. H., M. van Olphen, and Y. C. Drost. 1993. F-specific RNA bacteriophages are adequate model organisms for enteric viruses in fresh water. Appl. Environ. Microbiol. 59:2956–2962. 15. Hsu, F.-C., Y.-S. Shieh, J. van Duin, M. J. Beekwilder, and M. D. Sobsey. 1995. Genotyping male-specific RNA coliphages by hybridization with oligonucleotide probes. Appl. Environ. Microbiol. 61:3960–3966. 16. Jagals, P., W. O. K. Grabow, and J. C. de Villiers. 1995. Evaluation of indicators for assessment of human and animal faecal pollution of surface run-off. Water Sci. Technol. 31:235–241. 17. Lewis, G. D. 1995. F-specfic Bacteriophage as an indicator of human viruses in natural waters and sewage effluents in Northern New Zealand. Water Sci. Technol. 31:231–234.

18. Lipp, E. K., N. Schmidt, M. Luther, and J. B. Rose. 2001. Determining the effects of El Nin ˜o-southern oscillation events on coastal water quality. Estuaries 24:491–497. 19. Long, S. C. 1998. Project report: development of methods to differentiate microorganisms in MDC reservoir watersheds. University of Massachusetts, Amherst. 20. Nasser, A. M., Y. Tchorch, and B. Fattal. 1993. Comparative survival of E. coli, F⫹ bacteriophages, HAV and poliovirus 1 in wastewater and groundwater. Water Sci. Technol. 27:401–407. 21. Nwachuku, N., F. C. Craun, and R. L. Calderon. 2002. How effective is the TCR in assessing outbreak vulnerability. J. Am. Water Works Assoc. 94:88– 96. 22. Osawa, S. K., K. Furuse, and I. Watanabe. 1981. Distribution of ribonucleic acid coliphages in animals. Appl. Environ. Microbiol. 41:164–168. 23. Payment, P., M. Trudel, and R. Plante. 1985. Elimination of virus and indicator bacteria at each step of treatment during preparation of drinking water at seven water treatment plants. Appl. Environ. Microbiol. 49:1418– 1428. 24. Rose, J. B., C. P. Gerba, S. N. Singh, G. A. Toranzos, and B. Keswick. 1986. Isolation of entero- and rotaviruses from a drinking water facility. J. Am. Water Works Assoc. 78:56–61. 25. Rose, J. B., P. R. Epstein, E. K. Lipp, B. H. Sherman, et al. 2001. Climate variability and change in the United States: potential impacts on water- and foodborne diseases caused by microbiologic agents. Environ. Health Perspect. 109(Suppl. 2):211–221. 26. Schaper, M., J. Jofre, M. Uys, and W. O. K. Grabow. 2002. Distribution of genotypes of F-specific RNA bacteriophages in human and non-human sources of faecal pollution in South Africa and Spain. J. Appl. Microbiol. 92:657–667. 27. Simkova, A., and J. Cervenka. 1981. Coliphages as ecological indicators of enteroviruses in various water systems. Bull. W. H. O. 59:611–618. 28. Sinton, L. W., R. K. Finlay, and P. A. Lynch. 1999. Sunlight inactivation of fecal bacteriophages and bacteria in sewage-polluted seawater. Appl. Environ. Microbiol. 65:3605–3613. 29. Sobsey, M. D., K. J. Schwab, and T. R. Handzel. 1990. A simple membrane filter method to concentrate and enumerate male-specific RNA coliphages. J. Am. Water Works Assoc. 82:52–59. 30. Sobsey, M. D., D. A. Battigelli, T. R. Handzel, and K. J. Schwab. 1995. Male-specific coliphages as indicators of viral contamination of drinking water. American Water Works Association Research Foundation, Denver, Colo. 31. Springthorpe, V. S., C. L. Loh, W. J. Robertson, and S. A. Sattar. 1993. In situ survival of indicator bacteria, MS-2 phage and human pathogenic viruses in river water. Water Sci. Technol. 27:413–420. 32. Stetler, R. E. 1984. Coliphages as indicators of enteroviruses. Appl. Environ. Microbiol. 48:668–670. 33. Swindle, M. M., and A. C. Smith. 2000. Comparative anatomy and physiology of the pig. Animal Welfare Information Resource Series, no. 11. [Online.] Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Md. http://www.nal.usda.gov/awic/pubs/swine/swine.htm#art. 34. Tannock, G.W. 2001. Molecular assessment of intestinal microflora. Am. J. Clin. Nutr. 73(Suppl.):410S–414S. 35. U.S. Environmental Protection Agency. 2000. Proposed ground water rule EPA 815-F-00–003. Office of Ground Water, Environmental Protection Agency, Washington, D.C. 36. Wentsel, R. S., P. E. O’Neil, and J. F. Kitchens. 1982. Evaluation of coliphage detection as a rapid indicator of water quality. Appl. Environ. Microbiol. 43:403–434. 37. Woody, M. A., and D. O. Oliver. 1995. Effects of temperature and host cell growth phase on replication of F-specific RNA coliphage Q␤. Appl. Environ. Microbiol. 61:1520–1526.