In cooperation with the 

National Park Service, Department of the Interior 

Presumptive Sources of Fecal Contamination in Four 

Tributaries to the New River Gorge National River, West 

Virginia, 2004 

By Melvin V. Mathes, Tara L. O’Brien, Kriston M. Strickler, Joshua J. Hardy, William B. Schill, Jerzy Lukasik, 

Troy M. Scott, David E. Bailey, and Terry W. Fenger 

Open-File Report 2007–1107 

U.S. Department of the Interior 

U.S. Geological Survey 
U.S. Department of the Interior 

DIRK A. KEMPTHORNE, Secretary 

U.S. Geological Survey 

Mark D. Myers, Director 

U.S. Geological Survey, Reston, Virginia 2007 

For product and ordering information: 

World Wide Web: http://www.usgs.gov/pubprod

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its natural and living resources, natural hazards, and the environment: 

World Wide Web: http://www.usgs.gov 

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Suggested citation: 

Mathes, M.V., O’Brien, T.L., Strickler, K.M., Hardy, J.J., Schill, W.B., Lukasik, J., Scott, T.M., Bailey, D.E., and 

Fenger, T.L., 2007, Presumptive sources of fecal contamination in four tributaries to the New River Gorge 

National River, West Virginia, 2004: U.S. Geological Survey Open-File Report 2007-1107, 28 p. Online at URL 

http://pubs.water.usgs.gov/ofr2007-1107/. 

Any use of trade, product, or firm names is for descriptive purposes only and does not imply 

endorsement by the U.S. Government. 

Although this report is in the public domain, permission must be secured from the individual 

copyright owners to reproduce any copyrighted material contained within this report. 

ii
Contents 

Abstract................................................................................................................................................................................1

Introduction .........................................................................................................................................................................2

Purpose and Scope ........................................................................................................................................................3

Description of Study Area.............................................................................................................................................3

Sample Collection...............................................................................................................................................................6

Fecal Samples .................................................................................................................................................................6

Water Samples................................................................................................................................................................6

Quality-Control Samples................................................................................................................................................7

Laboratory Methods and Procedures.............................................................................................................................9

Pulsed-Field Gel Electrophoresis Method (PFGE) ....................................................................................................9

Human Host-Specific Enterococcus Marker Method .............................................................................................9

Human-Urine Marker Method....................................................................................................................................10

Bacteroidetes Method.................................................................................................................................................10

Host DNA Markers Method ........................................................................................................................................11

Wastewater Compounds Procedure.........................................................................................................................12

Detection of Contaminants, by Analytical Method.....................................................................................................12

Field and Laboratory Determinations of Fecal Indicator Bacteria ......................................................................12

Pulsed-Field Gel Electrophoresis...............................................................................................................................13

Human Host-Specific Enterococcus Marker ..........................................................................................................14

Human-Urine Marker...................................................................................................................................................14

Bacteroidetes................................................................................................................................................................14

Host DNA Markers .......................................................................................................................................................15

Wastewater Compounds.............................................................................................................................................19

Presumptive Sources of Fecal Contamination, by Tributary ....................................................................................20

Arbuckle Creek..............................................................................................................................................................20

Dunloup Creek...............................................................................................................................................................23

Keeney Creek ................................................................................................................................................................23

Wolf Creek .....................................................................................................................................................................24

Summary and Conclusions .............................................................................................................................................24

Acknowledgments............................................................................................................................................................25

References Cited ..............................................................................................................................................................25

iii
Figures 

1. Map showing location of the New River Gorge National River, West Virginia, with park 

area, towns, major roads, tributaries, and sampling sites ............................................................................4 

2-5. Graphs showing: 

2. Relative concentrations of fecal Bacteroidetes in eight water samples collected from four 

tributaries to the New River Gorge National River, West Virginia, 2004 ..................................................17 

3. Relative concentrations of human-specific Bacteroidetes marker in eight water samples 

collected from four tributaries to the New River Gorge National River, West Virginia, 2004...............17 

4. Relative concentrations of filter-bound human DNA in eight water samples collected from 

four tributaries to the New River Gorge National River, West Virginia, 2004 ..........................................19 

5. Relative concentrations of dissolved human DNA in eight water samples collected from 

four tributaries to the New River Gorge National River, West Virginia, 2004 ..........................................19 

Tables

1. Description of the 579-known-source Escherichia coli library with animal sources of fecal samples 

collected from the vicinity of the New River Gorge National River, West Virginia, 2004...............................7

2. Water-quality characteristics of, and bacteria counts in, eight water samples collected from four 

tributaries to the New River Gorge National River, West Virginia, 2004 ...........................................................8

3. Summary of 373 Escherichia coli isolates from water samples collected from four tributaries 

to the New River Gorge National River, West Virginia, 2004, that were compared with the 579-isolate 

known-source library................................................................................................................................................15

4. Summary of 373 Escherichia coli isolates from water samples collected from four tributaries 

to the New River Gorge National River, West Virginia, 2004, that were compared with the expanded 

8,043-isolate known-source library ........................................................................................................................16

5. Host mitochondrial DNA analysis of quality-control samples expressed as sensitivity and 

specificity ....................................................................................................................................................................18

6. Host mitochondrial DNA markers for species other than human detected, but not quantified, 

in eight water samples collected from four tributaries to the New River Gorge National River, 

West Virginia, 2004 ....................................................................................................................................................20

7. Wastewater compounds detected in eight water samples collected from four tributaries to 

the New River Gorge National River, West Virginia, 2004 .................................................................................21

8. Descriptions of findings, by method, for determining animal sources of fecal matter detected in 

eight water samples collected from four tributaries to the New River Gorge National River, 

West Virginia, 2004 ................................................................................................................................................... 22

9. Summary of human-specific markers present in eight water samples collected from four 

tributaries to the New River Gorge National River, West Virginia, 2004. ........................................................23

iv
Conversion Factors, Abbreviations, and Datums 

Inch/Pound to SI 

Multiply By To obtain

Length 

foot (ft) 0.3048 Meter (m) 

mile (mi) 1.609 Kilometer (km)

Area 

square mile (mi

2

) 259.0 Hectare (ha)

square mile (mi

2

) 2.590 square kilometer (km2) 

Volume 

ounce, fluid (fl. Oz) 0.02957 liter (L) 

pint (pt) 0.4732 liter (L) 

quart (qt) 0.9464 liter (L) 

gallon (gal) 3.785 liter (L) 

Flow rate 

cubic foot per second (ft

3

/s) 0.02832 cubic meter per second (m3/s) 

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows: 

 °F=(1.8× °C)+32

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as follows: 

°C=(°F-32)/1.8 

Specific conductance is given in microsiemens per centimeter at 25 degrees Celsius ( μS/cm at 25 °C). 

Concentrations of chemical constituents in water are given either in milligrams per liter (mg/L) or micrograms per liter (μg/L). 

Vertical coordinate information is referenced to the National Geodetic Vertical Datum of 1929 (NGVD 29). 

Horizontal coordinate information is referenced to the North American Datum of 1983 (NAD 83). 

Altitude, as used in this report, refers to distance above the vertical datum. 

v
Presumptive Sources of Fecal Contamination in Four 

Tributaries to the New River Gorge National River, 

West Virginia, 2004 

By Melvin V. Mathes1, Tara L. O’Brien2, Kriston M. Strickler3, Joshua J. Hardy3, William B. Schill1, 

Jerzy Lukasik

4

, Troy M. Scott4, David E. Bailey2, and Terry L. Fenger2

Abstract 

Several methods were used to determine the sources of fecal contamination in water 

samples collected during September and October 2004 from four tributaries to the New River 

Gorge National River -- Arbuckle Creek, Dunloup Creek, Keeney Creek, and Wolf Creek. All four 

tributaries historically have had elevated levels of fecal coliform bacteria. The source-tracking 

methods used yielded various results, possibly because one or more methods failed. Sourcing 

methods used in this study included the detection of several human-specific and animal-specific 

biological or molecular markers, and library-dependent pulsed-field gel electrophoresis analysis 

that attempted to associate Escherichia coli bacteria obtained from water samples with animal 

sources by matching DNA-fragment banding patterns. Evaluation of the results of quality-control 

analysis indicated that pulsed-field gel electrophoresis analysis was unable to identify knownsource bacteria isolates. Increasing the size of the known-source library did not improve the results 

for quality-control samples. A number of emerging methods, using markers in Enterococcus, 

human urine, Bacteroidetes, and host mitochondrial DNA, demonstrated some potential in 

associating fecal contamination with human or animal sources in a limited analysis of qualitycontrol samples. All four of the human-specific markers were detected in water samples from 

Keeney Creek, a watershed with no centralized municipal wastewater-treatment facilities, thus

indicating human sources of fecal contamination. The human-specific Bacteroidetes and host 

mitochondrial DNA markers were detected in water samples from Dunloup Creek, Wolf Creek, and 

to a lesser degree Arbuckle Creek. Results of analysis for wastewater compounds indicate that the 

September 27 sample from Arbuckle Creek contained numerous human tracer compounds likely 

from sewage. Dog, horse, chicken, and pig host mitochondrial DNA were detected in some of the 

water samples with the exception of the October 5 sample from Dunloup Creek. Cow, white-tailed 

deer, and Canada goose DNA were not detected in any of the samples collected from the four 

tributaries, despite the presence of these animals in the watersheds. Future studies with more 

rigorous quality-control analyses are needed to investigate the potential applicability and use of 

these emerging methods. Because many of the detections for the various methods could vary over 

time and with flow conditions, repeated sampling during both base flow and storm events would be 

necessary to more definitively determine the sources of fecal contamination for each watershed. 

 1

 U.S. Geological Survey 2

 Marshall University 3 West Virginia Department of Agriculture 4

 Biological Consulting Services of North Florida, Inc. 

1
Introduction 

More than 200,000 visitors annually raft the whitewater rapids of the New River within the 

confines of the New River Gorge National River in West Virginia (Jesse Purvis, National Park 

Service, Glen Jean, W.Va., oral commun., 1999). Visitors also enjoy swimming, kayaking, 

canoeing, and fishing in the river. Many of these visitors come into direct contact with the waters 

of the New River during these activities. 

Concentrations of fecal coliform bacteria have been monitored by the National Park Service 

and the West Virginia Department of Environmental Protection since 1985 at several mainstem 

sites and tributaries to the New River. During these monitoring activities, fairly high concentrations

of fecal coliform bacteria—with some concentrations in excess of 100,000 colonies per 100 

milliliters (mL) of water—were documented in many tributary inflows to the New River (Purvis 

and others, 2002; National Park Service, 1996). The standard set by the State of West Virginia for 

waters suitable for primary contact recreation is a concentration of fecal coliform bacteria colonies

not to exceed 200 colonies per 100 mL of sample, based on a geometric mean of five samples per 

month. Alternatively, fecal coliform bacteria concentrations should not exceed 400 colonies per 

100 mL of sample in more than 10 percent of the samples collected within one month (West 

Virginia Water Resources Board, 1998). The presence of high concentrations of fecal coliform 

bacteria indicates the possible presence of associated pathogens including bacteria such as Shigella

and Salmonella, protozoans such as Cryptosporidium and Giardia lamblia, and more than 100 

viruses associated with human feces (Britton and Greeson, 1988). All of these pathogens can be 

transmitted to humans through ingestion of contaminated water, and some can be transmitted by 

contact with contaminated water (Benenson, 1995). The presence of Giardia lamblia in Dunloup 

Creek, the tributary to the New River that flows past park headquarters for New River Gorge 

National River, has been reported previously (Messinger, 2002). 

Suspected sources of the fecal bacteria found in tributaries to the New River Gorge National 

River include, but are not limited, to sewage from straight pipes from communities with no sewage 

treatment facilities; partially treated sewage from communities with insufficient sewage treatment, 

especially during storms with excessive amounts of surface runoff; leaking sewer lines; faulty 

septic systems; and runoff from farmlands, pasturelands, and forested areas. Identification of the 

sources of the high bacteria concentrations found in these tributaries could aid the National Park 

Service in findings ways to improve the water quality of the tributaries to the New River Gorge 

area in order to protect park visitors from waterborne diseases.

Bacterial contamination sources commonly are nonpoint sources that are difficult to track 

and identify. Management of nonpoint sources is one way to control and reduce contaminant loads 

to streams. Microbial source tracking and other source tracking methods (hereafter referred to as

sourcing) are tools that can be used to help identify nonpoint sources of bacterial contamination 

(Stoeckel, 2005). Sourcing methods were selected for a study by the U.S. Geological Survey, in 

cooperation with the National Park Service, to attempt to determine, for the time of sample 

collection, sources of contamination for four tributaries –Arbuckle Creek, Dunloup Creek, Keeney

Creek, and Wolf Creek. 

In a previous study, Stoeckel and others (2004) compared the ability of selected microbial 

source tracking methods to associate a challenge set (blinded with respect to animal source) of

Escherichia coli (E. coli) bacteria isolates to their correct animal sources. Sourcing methods 

compared included pulsed-field gel electrophoresis (PFGE) of macrorestriction digests using a 

NotI-based protocol, ribotyping using the restriction enzyme HindIII, ribotyping using the 

restriction enzymes EcoRI and PvuII, repetitive-element DNA polymerase chain reaction (PCR) 

with REP primers, repetitive-element DNA PCR with BOX primers, carbon utilization profiling 

2
using Biolog GN-2 microplates, and antibiotic resistance analysis (ARA). PFGE was the only 

method able to correctly classify 100 percent of a set of blinded replicate E. coli isolates (isolates 

pulled from the known-source library) into the correct species-level categories (animal sources) that 

included human (Homo sapiens), dog (Canis familiaris), white-tailed deer (Odocoileus 

virginianus), Canada goose (Branta canadensis), cow (Bos taurus), swine (Sus scrofa), horse 

(Equus caballus), and chicken (Gallus gallus). For this reason and because of the existence of

large local source libraries of additional PFGE bacteria isolates, PFGE was selected for inclusion 

as a sourcing tool for this study. Several emerging sourcing methods were included in the study. 

These methods included a human-host associated molecular marker found in Enterococcus faecium

(Scott and others, 2005); a urine-carried virus marker associated with humans (McQuaig and 

others, 2006); a general fecal Bacteroidetes marker (Dick and Field, 2004); a human-associated 

Bacteroidetes marker (Seurinck and others, 2005); species-specific host mitochondrial DNA 

markers independently derived but similar to those from Martellini and others (2005) for humans 

and several animals including dog, cow, chicken, pig, horse, white-tailed deer, and Canada goose; 

and analysis for wastewater compounds such as caffeine (Zaugg and others, 2002). 

Purpose and Scope 

This report identifies and describes the inferred sources of fecal contamination at the time of

sample collection, September to October 2004, in four tributaries to the New River Gorge National 

River in West Virginia, as determined by the use of several microbial and related sourcing 

methods. To accomplish this task, 396 fecal samples from humans, Canada geese, white-tailed 

deer, dogs, cows, horses, chickens, coyotes, and turkeys were collected, and two sets of water 

samples were collected from each of four tributary streams - - Arbuckle Creek, Dunloup Creek, 

Keeney Creek, and Wolf Creek. This report also (1) describes the effect of increased source-library 

size on the ability of library-dependent methods such as PFGE to associate fecal bacteria with the 

animal source and (2) describes the potential for several emerging library-independent sourcing 

methods to differentiate between human and nonhuman fecal contamination sources. 

Description of Study Area 

The study area consists of several tributary watersheds (fig. 1) that flow to the New River 

Gorge National River. Four of these tributaries, Arbuckle Creek, Dunloup Creek, Keeney Creek, 

and Wolf Creek, were chosen in consultation with National Park Service scientists from among 

watersheds that had been monitored for fecal bacteria. These tributaries were chosen on the basis 

of accessibility, location near high-usage areas of the river, the availability of several years of 

water-quality and microbiological data, and documented repeated or periodic fecal contamination.

The New River Gorge, into which these four tributaries flow, constricts the river channel of the 

New River, causing it to be narrower, deeper, and steeper in the gorge than at other points along the 

river. This produces the world-class whitewater rafting for which this reach of the gorge is noted. 

The gorge walls rise to a maximum of 1,000 feet (ft) above the river channel, and the channel 

narrows from 1,000 ft upstream from the gorge to a minimum of 200 ft inside the gorge (Wilson 

and Purvis, 2003). The land in the study area and within the four selected watersheds is 

predominantly forest covered (90 percent). About 5 percent of the area consists of agricultural 

pastures, and only about 2 percent of the area consists of developed lands (Purvis and others, 2002). 

3
81 ̊11'0"W 81 ̊4'0"W 80 ̊57'0"W 80 ̊50'0"W 80 ̊43'0"W

38 ̊6'0"N

37 ̊59'0"N

37 ̊52'0"N

37 ̊45'0"N

0 2.5 5 10

0 2.5 5 10 Miles

Kilometers

Stream lines are from U.S. Geological Survey Digital Data, 1:24,000 (USGS, 2002); park boundaries are from National Park Service Digital Data, 1:100,000

(NPS, 1999); towns are from West Virginia Department of Environmental Protection, 1996.

FAYETTEVILLE

WINONA EXPLANATION

New River Gorge

National River

TOWNS

Streams

Sampling sites

Wastewater treatment

plants

Keeney Creek

Wolf Creek

Arbuckle

Creek

HINTON

BECKLEY

THURMOND

OAK HILL

MOUNT

HOPE

MEADOW

BRIDGE

Dunloup

Creek GLEN

JEAN

PRINCE

Piney Creek

Glade Creek

Madam Creek

New River

New River

OH

KY

WV

PA

VA

MD

Study

area

Kanawha

River

Gauley

River

New River

MINDEN

Arbuckle PSD WWTP

White Oak

PSD WWTP

Oak Hill WWTP

Mount

Hope

WWTP

Figure 1. Location of the New River Gorge National River, West Virginia, with park area, towns, 

tributaries, and sampling sites. 

The New River and its tributaries in the study area lie entirely within the Appalachian 

Plateaus Physiographic Province (Fenneman and Johnson, 1946). Rock units in the study area 

consist primarily of interbedded sandstone, shale, clay, and coal layers of Pennsylvanian age that

4
are flat lying or gently folded. The Kanawha and New River Formations are the major geologic 

units that outcrop in the four selected tributary watersheds (Purvis and others, 2002). Most of the 

sedimentary rock beds in the area are fractured in a manner consistent with stress-relief fracturing 

as described in Wyrick and Borchers (1981). Topography and stress-relief fracturing limit groundwater recharge of rivers and streams to the immediate area adjacent to the stream or river extending 

from the ridge on one valley side to the opposite ridge; therefore, each river gorge and tributary 

valley acts as a distinct recharge unit. Thus the tributary inflows to the New River are the major 

pathways for input of fecal contamination to the New River in the gorge. 

Arbuckle Creek is a small tributary that drains 8.7 square miles (mi2

) and flows 

southeasterly through the communities of Oak Hill and Minden and into the western side of the 

New River at the town of Thurmond, 25.1 miles (mi) (Mathes and others, 1982) upstream from the 

confluence of the New and Gauley Rivers. Concentrations of fecal coliform bacteria in 12 water 

samples collected over a wide range of streamflow in Arbuckle Creek by the National Park Service 

from 1998 to 2000 ranged from 30 to 250,000 colonies per 100 mL of streamwater with a median 

concentration of 500 colonies per 100 mL (Wilson and Purvis, 2003). The Oak Hill and Arbuckle 

Public Service District wastewater-treatment plants discharge untreated or partially treated 

wastewater into Arbuckle Creek, and lift stations in the watershed overflow even during dry 

periods, according to Wilson and Purvis (2003). 

Dunloup Creek drains 48.5 mi2

 and flows northeasterly through the communities of Mount 

Hope and Glen Jean and into the western side of the New River just upstream from the town of 

Thurmond, and 25.8 mi (Mathes and others, 1982) upstream from the confluence of the New and 

Gauley Rivers. Concentrations of fecal coliform bacteria in 13 water samples collected over a wide

range of streamflow in Dunloup Creek by the National Park Service from 1998 to 2000 ranged 

from 13 to 1,600 colonies of bacteria per 100 mL of streamwater with a median concentration of 

170 colonies per 100 mL (Wilson and Purvis, 2003). The Mount Hope sewage treatment plant lift 

station and the White Oak Public Service District wastewater-treatment plant are subject to 

overflows during storms and may discharge partially treated wastewater to Dunloup Creek, 

according to Wilson and Purvis (2003). 

Keeney Creek drains 8.9 mi2 and rises southeast of the community of Winona and flows to 

the west through the community of Winona and into the eastern side of the New River 16.0 mi 

(Mathes and others, 1982) upstream from the confluence of the New and Gauley Rivers. 

Concentrations of fecal coliform bacteria in 11 water samples collected over a wide range of 

streamflow in Keeney Creek by the National Park Service from 1998 to 2000 ranged from 430 to 

9,100 bacteria colonies per 100 mL of streamwater with a median concentration of 3,000 colonies 

per 100 mL (Wilson and Purvis, 2003). The communities in the watershed do not have any 

centralized municipal wastewater-treatment facilities, according to Wilson and Purvis (2003). 

Wolf Creek drains 17.4 mi2

 and flows northerly into the western side of the New River just 

upstream from the New River Gorge Bridge and 12.0 mi (Mathes and others, 1982) upstream from 

the confluence of the New and Gauley Rivers. Concentrations of fecal coliform bacteria in 11 

water samples collected over a wide range of streamflow in Wolf Creek by the National Park 

Service from 1998 to 2000 ranged from 1 to 2,600 colonies per 100 mL of streamwater with a 

median concentration of 28 colonies per 100 mL, according to Wilson and Purvis (2003). Wilson

and Purvis (2003) list houses without sewer or adequate septic systems, a malfunctioning sewer 

collection system, pastureland, and an overloaded sewer system lift station on a minor tributary as 

potential sources of bacteria for Wolf Creek. 

5
Sample Collection 

Fecal samples from animal and human sources were collected from the New River Gorge 

and nearby locations and were used to build a regional library of known-source E. coli bacteria 

isolates for use in the analysis by PFGE. Water samples for PFGE analysis were collected from the 

four selected tributaries to the New River Gorge National River for the enumeration of fecal 

coliform bacteria. Water samples also were examined for the presence of genetic markers 

associated with fecal sources and for the presence of a suite of wastewater compounds to aid in the 

determination of sources of fecal contamination. Quality-control samples for the library-dependent 

method PFGE were generated by cultivating E. coli bacteria from fecal samples. Quality-control 

samples for library-independent methods were generated by spiking sterile phosphate buffered 

saline (PBS) water with trace amounts of feces. Quality-control samples were submitted blind to 

the analytical laboratories. 

Fecal Samples 

A total of 396 fecal samples were collected from November 2004 to July 2005 and were 

transported to Marshall University (Huntington, W.Va.) for cultivation of E. coli bacteria isolates 

that were compiled into a library of 579 known-source E. coli. Fecal samples were collected either 

by use of sterile toothpicks and stored in sterile plastic vials, or by use of Starswabs II bacteriology

culture collection and transport system. Samples collected in sterile vials were chilled upon 

collection and during transport to the University laboratory. Fecal samples collected using Starswab 

II were stored at room temperature after collection and during transport. White-tailed deer, Canada

goose, dog, cow, horse, cat, coyote, and wild turkey fecal samples were collected from field 

locations in the four tributary watersheds, in the New River Gorge, and in locations close to the 

tributaries and New River. Human-origin samples were solicited from volunteers in these same 

geographical areas and from New River Gorge National River employees, most of whom lived in 

or near the study area. Fecal samples were streaked onto eosin methylene blue (EMB) media at the 

Marshall University laboratory to isolate E. coli. Attempts were made to isolate three E. coli

isolates from each fecal sample that exhibited E. coli growth. The number of fecal samples 

collected and the number of isolates cultured and added to the known-source library for each 

animal category are listed in table 1.

Water Samples 

Water samples were collected twice in 2004 from Arbuckle Creek (September 27 and 

October 6), Dunloup Creek (September 22 and October 5), Keeney Creek (September 23 and 30), 

and Wolf Creek (September 29 and October 12). Water samples were collected for each site visit 

as follows: one 2-liter (L) sterile bottle for analysis for Bacteroidetes, for host DNA, and for other 

tests at the USGS Leetown Science Center (Leetown, W. Va.); two 1-L sterile bottles for analysis 

for human host-specific Enterococcus marker and human-urine marker by Biological Consulting 

Services of North Florida, Inc.(Gainesville, Fla.); one 1-L bottle for determination of wastewater 

compounds by the USGS National Water-Quality Laboratory (Denver, Colo.), and one or more 

250-mL sterile bottles for membrane filtration of fecal coliform and fecal streptococcus bacteria 

onsite. All water samples were chilled and shipped by overnight mail. Measurements of 

6
Table 1. Description of the 579-known-source Escherichia coli library with animal sources of fecal 

samples collected from the vicinity of the New River Gorge National River, West Virginia, 2004. 

Animal source of

fecal samples 

Number of fecal

samplescollected 

Number of fecal

samples with E. coli

growth

Number of fecal samples 

from which usable data

were collected

Number of E. coli

isolates added to

source library 

Canada goose 179 59 57 160 

White-tailed deer 70 43 43 126 

Dog 51 37 36 103

Human 41 39 36 103

Cat 17 13 13 34

Cow 13 13 8 23

Horse 20 7 7 21

Coyote 3 3 3 9

Wild turkey 2 0 0 0

Totals 396 214 203 579

streamflow, water temperature, pH, specific conductance, and dissolved oxygen were made at each 

site (table 2). Water samples were collected by depth-width integrated methods into sterile bottles 

according to USGS protocols outlined in the National Field Manual (Wilde and others, 1999). 

Water samples were analyzed in the field for fecal-indicator bacteria using a membrane

filtration technique followed by cultivation on fecal coliform media (mFC) and fecal streptococcus

media (KF) (Myers and Wilde, 1997) for fecal coliform and fecal streptococci analysis (table 2), 

respectively. Several different volumes of sample were filtered and plated at each site. For qualitycontrol purposes, an equipment blank and a procedure blank were prepared by filtering sterile 

buffered water before and after each set of water samples. No colonies were observed to grow on 

any of the quality-control blanks. After incubation, the plates containing fecal streptococcus 

colonies were counted and destroyed. The plates containing fecal coliforms were counted, and the 

plates were chilled and shipped by overnight mail to the West Virginia State Agriculture 

Laboratory (Moorefield, W. Va.) for culturing and confirmation of E. coli bacteria isolates for 

analysis by PFGE. 

Total coliforms, enterococci, and E. coli were assayed by standard filtration methodology 

for all water and quality-control samples received by the USGS Leetown Science Center 

laboratory. Total coliforms and E. coli were enumerated from growth on m-ColiBlue24, and 

enterococci were enumerated from growth on m-Enterococcus agar. Digital photographs of plates 

were made, and counts (table 2) were made using image analysis techniques. 

The four sampled streams were generally well mixed and easily wadeable. Stream mixing was 

verified by water-quality cross-sectional measurements of water temperature, pH, specific 

conductance, and dissolved oxygen where possible. Values for these water-quality indicators 

varied only slightly across the width of the stream channel for Dunloup Creek, Wolf Creek, and 

Arbuckle Creek. Depths were too shallow in Keeney Creek to obtain water-quality cross-sectional 

data during both sampling visits, but conditions at this site indicated good mixing. 

Quality-Control Samples 

Quality-control samples were generated to challenge analyses by PFGE, for the human hostspecific Enterococcus marker, for the human-urine marker, and for host DNA markers. A stock 

solution of sterile PBS water was prepared by adding one phosphate buffered saline tablet to each 

7
Table 2. Water-quality characteristics of, and bacteria counts in, eight water samples collected from 

four tributaries to the New River Gorge National River, West Virginia, 2004 

[ft3/s, cubic feet per second; mm, millimeters; mg/L, milligrams per liter; μS/cm, microsiemens per centimeter; °C, 

degrees Celsius; mL, milliliters; E, estimated] 

Sampling site Sampling

date 

Flow 

(ft3

/s) 

Air pressure,

field (mm of

mercury)

Dissolved 

oxygen 

(mg/L) 

Dissolved 

oxygen 

(percent of

saturation) 

pH, field

(standard 

units) 

Specific

conductance, field 

(μS/cm

@25C) 

Arbuckle Creek 09-27-04 1.4 719 10.1 113 8.0 375

Arbuckle Creek 10-06-04 2.5 728 10.4 105 7.8 368

Dunloup Creek 09-22-04 23 740 9.5 96 8.3 485 

Dunloup Creek 10-05-04 22 739 9.7 97 8.3 498 

Keeney Creek 09-23-04 1.3 724 9.4 97 7.6 155 

Keeney Creek 09-30-04 5.2 720 9.3 97 7.5 122 

Wolf Creek 09-29-04 15 736 9.2 97 8.0 274 

Wolf Creek 10-12-04 3.0 731 10.3 103 8.6 631

Sampling site Sampling

date 

Water

temperature 

(°C) 

Fecal 

coliforms, 

field

(colonies 

per 100mL) 

Fecal 

streptococcus, 

field 

(colonies 

per 100 

mL)

Total

coliform 

(colonies 

per 100 mL) 

E. coli

(colonies 

per 100 mL) 

Enterococcus

(colonies 

per 100 mL)

Arbuckle Creek 09-27-04 17.7 160 290 2,200 24 30

Arbuckle Creek 10-06-04 13.8 150 39 2,400 90 74

Dunloup Creek 09-22-04 14.5 87 36 1,000 14 15

Dunloup Creek 10-05-04 13.7 E17 E7 2,900 41 35

Keeney Creek 09-23-04 14.2 E44 E74 11,000 53 98

Keeney Creek 09-30-04 14.6 280 160 3,900 92 88

Wolf Creek 09-29-04 16.4 3,800 1,900 1,600 44 390

Wolf Creek 10-12-04 13.3 E3 E5 1,100 3 5

200 mL of distilled water and autoclaving the resultant solution at 121 degrees Celsius (°C) for 15

minutes. Small unmeasured quantities of fresh feces from known sources (1-3 individuals per 

source) were obtained by use of sterile toothpicks and were added to sterile bottles of this stock 

solution to challenge these sourcing methods. A 1-L sample containing all human feces 

(designated sample Unknown A) and a 1-L sample containing no human feces (designated sample 

Unknown B and containing white-tailed deer and Canada goose feces) were shipped for analysis 

for the human host-specific Enterococcus marker and the human-urine marker, and two 2-L 

samples (Unknowns A and B, same composition as in 1-L bottles) were shipped for analysis for 

host DNA. Because no bacterial growth resulted from the 2-L sample of Unknown B, a 

replacement sample (designated Unknown C and containing white-tailed deer and Canada goose 

feces) was requested by and sent to the lab performing the host DNA analyses. Four additional 

samples were generated by adding white-tailed deer feces to one bottle of PBS stock solution, 

Canada goose feces to a second bottle, human feces to a third bottle, and dog feces to a fourth 

bottle. Fecal coliform bacteria were obtained from these samples by the membrane filtration 

method (Myers and Wilde, 1997). Selected plates of the fecal coliform bacteria were chilled and 

shipped by overnight mail to the West Virginia Agriculture Laboratory from which 37 E. coli

bacteria isolates were cultured, confirmed, and analyzed by PFGE to produce DNA-fragment 

banding patterns. The DNA-fragment banding patterns for these 37 blinded quality-control isolates

8
were sent to the Marshall University Laboratory for computer matching with the DNA-fragment 

banding patterns for the 579 known-source isolates to determine the animal sources of the isolates 

from the blinded quality-control samples. 

Laboratory Methods and Procedures 

Several methods were used to determine the source of fecal contamination in New River 

Gorge National River tributaries. These sourcing methods included analysis by pulsed-field gel 

electrophoresis (PFGE) and analyses for a human host-specific Enterococcus marker, for a humanurine marker, for human-specific and general Bacteroidetes markers, for several host DNA 

markers, and for wastewater compounds. PFGE is a library-dependent method that attempts to 

associate fecal bacteria from water samples with animal or human sources by matching DNAfragment banding patterns generated from bacteria in the water samples to DNA-fragment banding 

patterns generated from fecal bacteria obtained from feces of known animal source. Thus this 

method depends on the existence of a library of known-source bacteria isolates with DNA-fragment 

banding patterns. The other methods with the exception of wastewater compounds are libraryindependent methods that attempt to detect the presence or absence of biological or molecular 

markers that are specific to human or animal source. Most of these biological markers actually 

contain genetic sequences of bacteria or viruses that are thought to be specifically associated with

humans or animals. An experimental method also was tested. This experimental method is based

on directly detecting human or animal DNA that is carried into receiving waters by cells shed from 

the gut of the source, rather than depending on the indirect association of microbes with hosts. 

Specific amplification (an increase in the number of copies of a specific DNA fragment) of sourcespecific sequences by quantitative polymerase chain reaction (qPCR) was used to detect source 

DNA and to measure its relative concentration. 

Pulsed-Field Gel Electrophoresis Method (PFGE) 

PFGE of macrorestriction digests using a NotI-based protocol was applied to the 579 

isolates in the known-source library by researchers at Marshall University and on the 37 isolates 

from the quality-control samples and the 373 isolates from water samples from the four tributaries 

to the New River Gorge National River by researchers at the West Virginia Agriculture Laboratory. 

This PFGE protocol generates DNA-fragment banding patterns that result from whole-genome 

digestion and that are based on variability anywhere in the genome (Stoeckel and others, 2004). 

PFGE generally is considered to be an extremely sensitive method of detecting genetic differences 

between strains (Myoda and others, 2003; Tenover and others, 1997). The protocol for PFGE 

applied in this study uses the same restriction enzyme as that used by Simmons and others (2000). 

DNA extraction and digestion by PFGE were performed by streaking E. coli bacteria isolates from 

the 579-isolate known-source library, the 37 quality-control isolates, and the 373 water-sample 

isolates for confluent growth by a method similar to that reported in Stoeckel and others (2004). 

The DNA-fragment banding patterns generated for bacteria cultured from water samples were 

matched to DNA-fragment banding patterns generated from bacteria isolates cultured from known 

sources of feces in an attempt to associate bacteria in water samples with their correct animal or 

human source. 

Human Host-Specific Enterococcus Marker Method 

Scott and others (2005) proposed the use of a molecular marker in Enterococcus faecium to 

identify human sources of contamination in the environment. The human fecal contamination 

9
marker that they used targets a putative Enterococcus virulence factor, the enterococcal surface 

protein (esp). A validation study conducted by Scott and others (2005) detected this gene in 97 

percent of sewage and septic samples analyzed, but did not detect the gene in samples collected 

from livestock waste lagoons, birds, or from other nonhuman fecal samples. 

Water samples were analyzed using this method by isolating enterococci from the samples 

by membrane filtration and by performing PCR on composite DNA samples from the cultivated 

enterococci. Filters were incubated for 18 to 24 hours (hr) on mE agar (Difco) supplemented with 

indoxyl substrate (Sigma, Inc.). The filters are next lifted, suspended in azide dextrose broth 

(Difco), vortexed vigorously, and incubated for 3 hr at 41°C to wash bacteria from the filters and 

enrich the bacteria. DNA extraction is performed on the resulting culture by use of a QIAamp 

DNA extraction kit (Qaigen, Inc.). Primers specific to the esp gene in Enterococcus faecium were 

developed after DNA sequence alignment of the two available variants of the gene (GenBank 

Accession Numbers AF444000 and AF443999). PCR was performed in a 50-microliter (μL)

reaction mixture containing 1X PCR buffer, 1.5 millimolar (mM) magnesiumchloride, 200 

micromolar (μM) of each of the four deoxyribonucleotides, 0.3 μM of each primer, 2.5 units (U) of

HotStarTaq DNA polymerase (Qiagen), and 5μL of template DNA. Amplification was performed 

initially at 95°C for 15 minutes to activate Taq polymerase, followed by 35 cycles at 94°C for 1 

minute, 58°C for 1 minute, and 72°C for 1 minute. PCR products were separated on a 1.5 percent 

agarose gel stained with GelStar nucleic acid stain (BioWhittaker) and were viewed under 

ultraviolet light. The resultant PCR product is 680 base pairs in length. Positive and negative 

controls were run with each set of reactions. The Enterococcus faecium strain C68, which 

contains the esp gene, was used as the positive control, and sterile water was used as a negative 

control. All DNA extracts were spiked with positive-control DNA to assess whether samples 

contained PCR inhibitors. Samples in which the positive control failed to amplify were 

reprocessed under more stringent conditions designed to remove PCR inhibitors and were 

reanalyzed by PCR. 

Human-Urine Marker Method 

Researchers in Florida (McQuaig and others, 2006) used a method to concentrate and 

extract DNA of human polyomaviruses from water samples. Polyomaviruses have a demonstrated 

prevalence in the worldwide human population (Del Valle and others, 2004). Serological studies 

have shown that 60 to 90 percent of human adults harbor antibodies against polyomaviruses 

(Hirsch and Steiger, 2003). These adults can shed as many as one million viral particles of

polyomaviruses in 1 mL of urine (Bofill-Mas and others, 2000). Individuals with suppressed 

immune systems and individuals with healthy immune systems can shed these viruses (Bofill-Mas 

and others, 2000; Behzad-Behbahani and others, 2004). The human-urine marker uses primers 

specific to the conserved t-antigen of the JC virus and BK virus, both of which are polyomaviruses, 

to detect the polyomaviruses in a nested PCR. The method can detect as little as one μL of raw 

sewage (which contains urine in addition to fecal material and other constituents) added to 100 mL

of water (McQuaig and others, 2006). 

Bacteroidetes Method 

Water samples collected from the four tributaries were analyzed at the USGS Leetown 

Science Center to detect potential fecal contamination and its nature. To accomplish this, the 

methods developed by Dick and Field (2004) and Seurinck and others (2005) were employed to 

detect both general and human-specific Bacteroidetes markers. SYBR Green detection was used to 

10
detect the general Bacteroidetes marker rather than the 5-prime nuclease (TaqMan) detection 

method originally described. 

Water samples (1,000 mL) were filtered (47 mm, 0.45 micron cellulose acetate) through 

sterile filtration devices to collect and concentrate bacteria and particulate-bound DNA. 

Sterilization and decontamination of filtration setups was accomplished by soaking the apparatus 

(without filters) in 10 percent household bleach for 30 minutes, rinsing thoroughly with sterile 

distilled water, and autoclaving for 20 minutes. Just before use and after installation of the 

membrane filter, the device was irradiated with ultraviolet light to further ensure against laboratory 

contamination. For quality control, 100 mL of distilled water was passed through the filter 

apparatus to detect any possible laboratory contamination from filter units and 

extraction/purification reagents. Filters were frozen for later processing. DNA from bacteria and 

particulates retained by filtration were obtained from the frozen filters using a Water Kit (MoBio, 

Carlsbad, Calif.) and following the manufacturer’s instructions. The filtered water was processed

to concentrate and purify dissolved DNA (see “Host DNA Markers Method”). 

All purified and concentrated DNA samples were tested for possible matrix interferences by 

performing PCR on samples composed of 50 percent purified sample DNA and 50 percent known, 

unrelated DNA, then verifying that amplification kinetics were identical to those of the controls 

containing 50 percent known, unrelated DNA and 50 percent distilled water. 

Purified DNA was tested for the presence of general and human-specific Bacteroidetes

markers using a SYBR Green Jumpstart for Quantitive PCR kit (Sigma, Chicago, Ill.). Reactions 

(25 μL) were conducted in a Corbett RotorGene 3000 thermocycler (Corbett Life Sciences, Sydney, 

Australia) using thermocycling conditions as originally described. Data analysis was performed 

using the instrument-associated software. Because no absolute standards were available, qualitycontrol sample Unknown A (human fecal sample) was used as a surrogate after being determined to 

be a human or primarily human fecal sample by host DNA analysis. Decimal dilutions (dilutions 

of one part substance to be diluted added to nine parts water) of Unknown A were used to generate 

standard curves and determine relative concentrations of general and human-specific Bacteroidetes

markers in the water samples collected from the four tributaries. 

Host DNA Markers Method 

The eight water samples collected from the four tributaries to the New River Gorge 

National River were tested for the presence of mitochondrial cytochrome b sequences in both filterbound and dissolved fractions. DNA was measured in both fractions separately (rather than as a 

composite sample) because it is generally thought that sediment-bound DNA is protected somewhat 

from degradation and may reflect more historic DNA contributions to the environment than the 

dissolved fraction, whereas DNA in the dissolved fraction is more subject to degradation than 

filter-bound DNA and, therefore, should have a shorter half-life and be more reflective of recent 

DNA contributions to the environment. DNA in the dissolved fraction was obtained from the 

filtrate of the samples and was concentrated and purified according to the method of Matsui and 

others (2004) with minor modifications. Specifically, 0.25 molar ethylenediaminetetraacetic acid 

(EDTA) was used to redissolve precipitated DNA complexes and to chelate calcium and 

magnesium rather than the TE buffer (a combination of Tris and EDTA) used in the original 

procedure. Recovered DNA was purified further by extraction with a Water DNA Kit (Epicentre, 

Madison, Wis.) following the manufacturer’s instructions and further concentrated by ultrafiltration 

using an Amicon Ultra-4 30,000 MW spin concentration device (Millipore, Billerica, Mass.). 

PCR was used to detect the host DNA sequences, specifically the presence of mitochondrial 

cytochrome b sequences from human, dog, cow, chicken, horse, white-tailed deer, Canada goose 

11
and pig using recently developed methods. (Primer sequences and cycling parameters are not 

discussed here). These methods are similar in approach, but developed independently, from that of 

Martellini and others (2005). Differences that should increase the sensitivity of detection are that 

targeted sequences were smaller in this study (approximately 100-200 base pairs) as opposed to 

those published by Martellini (400-934 base pairs). Targeting smaller sequences should yield 

increased sensitivity in detecting the DNA that is expected to be degraded in the environment. 

Martellini and others (2005) analyzed for DNA that was retained by the filters or sedimented by 

centrifugation, whereas in this study, techniques were adapted for concentrating and purifying 

dissolved DNA as described above. Moreover, this study used real-time PCR that is inherently 

more sensitive than the gel electrophoresis used in the previous study. 

Reactions (25 μL) were conducted using a Corbett RotorGene instrument using SYBR 

Green detection as described in the section “Bacteroidetes Method.” Absolute standards for 

mitochondrial sequences were not available, so human placental DNA (Sigma, Chicago, Ill.) was 

used as a surrogate. Decimal dilutions of the human DNA were used to generate a standard curve 

that was, in turn, used to estimate the amounts of human DNA in dissolved and filter-bound 

fractions. 

Wastewater Compounds Procedure 

Zaugg and others (2002) developed techniques for the analytical determination of more than 

60 wastewater compounds in natural waters by solid-phase extraction and capillary-column gas 

chromatography and mass spectrometry. These techniques were developed in response to concerns

regarding the effect of endocrine-disrupting chemicals in wastewater on aquatic organisms, but the 

method may be useful in evaluating the effect of combined sanitary and storm-sewer overflow on 

the water quality of urban streams. The method focuses on the determination of compounds that 

are indicators of wastewater or are potential endocrine disruptors. Unfiltered water samples were

collected in 1-L amber glass bottles for each of the four tributaries and were chilled and shipped by

overnight mail to the USGS National Water Quality Laboratory (Denver, Colo.) for these analytical 

determinations. 

Detection of Contaminants, by Analytical Method 

PFGE analysis of E. coli bacteria isolates cultured from water samples and fecal samples 

collected in the New River Gorge area, the detection of molecular and biological markers, and 

wastewater compounds were used to analyze potential sources of fecal contamination in Arbuckle 

Creek, Dunloup Creek, Keeney Creek, and Wolf Creek tributaries to the New River Gorge National 

River. Discussion and individual results of each sourcing method follow. Future studies with more 

rigorous quality-control analyses are needed to investigate the potential applicability and use of 

these emerging methods.

Field and Laboratory Determinations of Fecal Indicator Bacteria 

Bacteria counts (table 2) revealed no particular trends or abnormalities and were generally 

what might be expected from surface-water samples; however, field and laboratory counts often 

differed, probably reflecting die-off or regrowth of bacteria during overnight transit. The Wolf 

Creek sample collected September 29, 2004, was delayed in shipment by the contracted carrier. 

12
Pulsed-Field Gel Electrophoresis 

Computers were used to match DNA-fragment banding patterns generated from E coli

bacteria isolates cultured from water samples to DNA-fragment banding patterns generated from E. 

coli cultured from fecal samples of known animal or human source. A curve-based matching 

procedure was used by researchers at Marshall University to classify the 37 blinded quality-control 

isolates and the 373 watershed isolates of unknown source to source categories with the aid of 

BioNumerics image analysis software using the following parameters: 2.0 optimization, 5.0 

percent tolerance, 2.5 percent end of pattern change, and unweighted pair-group method using 

arithmetic averages (UPGMA) clustering. In the previous microbial sourcing methods comparison

study by Stoeckel and others (2004), the same research laboratory assigned patterns that did not 

match known database patterns with better than 85 percent similarity to a “not identified” category. 

Using the above criteria, only 1 of the 37 quality-control isolates was matched against the 

579-isolate known-source library to its correct animal source (a white-tailed deer). Similarities of 

the 37 pattern matches ranged from 66 to 92 percent with only 4 of 37 matches having similarities 

equal to or greater than the 85 percent identification threshold. Curve-based matching of the same 

37 quality-control isolates to an expanded known-source library of 1,266 isolates (an additional 687 

isolates from the New River area were collected independently by Marshall University 

researchers), and a known-source library of 8,043 isolates (all available known-sources isolates 

from West Virginia) again correctly associated only 1 of 37 isolates to its correct animal source (a 

white-tailed deer). Although increasing the size of the known-source library increased the average 

similarity of the matches from 78 percent for the 579-isolate known-source library to 84 percent for 

the 8,043-isolate known-source library, it did not change the number (one white-tailed deer) of 

correct animal-source associations. 

The use of different selective-growth media in the various steps of culturing of E coli

bacteria isolates in this study could partially explain the errors in attempted association of qualitycontrol isolates to animal source. Not all E. coli isolates will grow on each of the available 

selective-growth media for E. coli (Don Stoeckel, U.S. Geological Survey, written commun., 

2006). Thus, it is possible that the subset of E. coli quality-control isolates that were originally 

obtained from fecal coliform colonies grown on mFC media was different from the subset of E. 

coli isolates in the known-source library that were obtained from fecal samples of known source 

that were streaked onto EMB media for E. coli growth. It is also possible that the population of 

DNA-fragment banding patterns derived from E. coli in human and animal feces is too diverse and 

variable to allow accurate associations of watershed bacteria isolates to animal sources by use of 

PFGE analysis. Results of curve-based matching of the 373 watershed isolates to known-source 

libraries are shown (tables 3 and 4) in tabular form, but no interpretations were made from these 

data because of the failure of the PFGE method in this specific project to identify correctly no more 

than 1 of 37 quality-control isolates. Curve-based matching of the 373 watershed isolates to the 

579-isolate known-source library generated 53 of 373 pattern matches (table 3) with a similarity of 

85 percent or greater. Sources represented in this library include human, dog, white-tailed deer, 

Canada goose, cow, horse, cat, and coyote (table1). Curve-based matching of the 373 watershed 

isolates to an expanded library of all 8,043 available E. coli known-source isolates for West 

Virginia generated 167 of 373 pattern matches (table 4) with similarities equal to or greater than 85 

percent. This expanded known-source library includes the 579 isolates in this project; 687 isolates 

collected independently in the New River area by Marshall University; 1,872 isolates collected in 

Berkeley County, West Virginia, to include the isolates collected by Stoeckel and others (2004); 

and 1,902 isolates collected in the Huntington, West Virginia, area. Available animal sources to be 

13
matched in the expanded library included those for the 579-isolate library plus swine, chicken, and 

raccoon. 

Human Host-Specific Enterococcus Marker 

Two water samples were collected in sterile bottles from each of the four tributaries to the 

New River Gorge National River and were submitted for detection analysis for the human 

molecular marker in Enterococcus faecium. Two quality-control samples, each containing minute 

amounts of feces in a sterile PBS solution, also were submitted for detection. Quality-control 

sample Unknown A contained strictly human feces, and quality-control sample Unknown B 

contained Canada goose and white-tailed deer feces. The method correctly detected the human 

marker in sample Unknown A and did not detect the marker in sample Unknown B. The human 

marker was detected in one of the two water samples from Keeney Creek (September 23), but was 

not detected in any of the water samples from Arbuckle Creek, Dunloup Creek, or Wolf Creek. 

The number of enterococci colonies exceeded 200 and were too numerous to count in the 

enrichment samples for both quality-control samples and for all water samples collected from the

four tributary streams, thus reducing the chance of false negative results for the marker in any of 

the analyzed samples. 

Human-Urine Marker 

Two water samples were collected in sterile bottles from each of the four tributaries to the 

New River and were submitted for detection analysis for the human-urine marker for the JC and 

BK polyomaviruses. Two negative quality-control samples, each containing minute amounts of 

feces in a sterile PBS solution, also were submitted for detection. No positive-control samples 

were submitted. Quality-control sample Unknown A contained human feces, but no human urine, 

and quality-control sample Unknown B contained Canada goose and white-tailed deer feces, but no

human urine. The method correctly did not detect the human-urine marker in sample Unknown A 

and sample Unknown B. The human-urine marker was detected in both of the water samples from 

Keeney Creek, but was not detected in any of the water samples from Arbuckle Creek, Dunloup 

Creek, or Wolf Creek. 

Bacteroidetes 

PCR was used to detect bacterial sequences for Bacteroidetes markers. The two markers 

detected are a fecal Bacteroidetes marker for general contamination (Dick and Field, 2004) and a 

human-specific Bacteroidetes marker (Seurinck and others, 2005). Relative fluorescence of the 

SYBR Green I dye as obtained from real-time PCR analysis for each marker indicated the presence 

of the marker and was used to calculate the concentrations of these markers (figs. 2 and 3) relative 

to the surrogate standard (quality-control sample Unknown A). Decimal dilutions of qualitycontrol sample Unknown A were used to generate standard curves by determination of the linear 

regression of the logarithm of standard concentration against Ct (threshold cycle). Ct is defined as 

the fractional cycle number at which relative fluorescence exceeds a set threshold that is in the 

exponential part of the sample growth curves and significantly above background noise levels. 

Equations describing the standard curves for total and human-specific Bacteroidetes concentrations 

were calculated to be Concentration = 10(-0.380*Ct + 6.540), R2

 = 0.99396 and Concentration = 10(-0.312*Ct + 

10.819), R2

 = 0.99025, respectively. Surprisingly, the distribution patterns of these two markers were 

similar, although the concentrations were different. The authors observed that in the 

14
Table 3. Summary of 373 Escherichia coli isolates from water samples collected from four tributaries 

to the New River Gorge National River, West Virginia, 2004, that were compared with the 579-isolate 

known-source library 

[Data are presented according to sample, followed by totals across all samples. Results for two samples are not shown 

because no E. coli bacteria were isolated from these two samples. Patterns with similarities greater than or equal to 85

percent were accepted. RESULTS IN THIS TABLE ARE EXPECTED TO BE INCORRECT, BASED ON 

QUALITY-CONTROL EVALUATION] 

Animal source

Arbuckle 

Creek 

sample 

1 

(9/27/04) 

Arbuckle 

Creek 

sample 

2 

(10/6/04) 

Dunloup 

Creek 

sample 

1 

(9/22/04) 

Dunloup 

Creek 

sample 

 2 

(10/5/04) 

Keeney 

Creek 

sample 

2 

(9/30/04) 

Wolf 

Creek 

sample 

1 

(9/29/04) 

Total

matches by 

source 

Human 0 0 2 3 2 0 7

White-tailed deer 4 1 0 1 2 13 21

Canada goose 0 0 1 1 4 1 7

Dog 1 4 2 0 1 4 12

Cat 2 0 0 0 1 0 3

Coyote 1 0 0 0 0 0 1

Cow 0 1 0 0 0 0 1

Horse 0 0 0 1 0 0 1

Total matches 8 6 5 6 10 18 53

Matches not accepted 39 52 16 45 93 75 320

Total isolate patterns 47 58 21 51 103 93 373

environmental samples, the human-specific marker was measured to be 100 to 1,000 times the 

general marker. This appears to be an artifact of using quality-control sample Unknown A that was 

compounded from only three individuals as a surrogate standard. The environmental samples have 

(presumably) more contributors and thus are more likely to have a higher frequency of the marker. 

Therefore, the concentration of this marker would be expected to be elevated when compared with

samples (including Unknown A) containing feces from low numbers of contributors. Both the 

general-contamination and the human-specific Bacteroidetes markers were detected in all eight 

water samples, but were comparatively elevated in the Dunloup Creek samples and in the second 

Keeney Creek sample (September 30). The concentration of the general-contamination marker was 

elevated (fig. 2) in the first Wolf Creek sample (September 29) in comparison with the second 

sample (October 12). The human-specific marker was most pronounced (fig. 3) in the Keeney 

Creek sample of September 30 and to a lesser degree in the Dunloup Creek sample of October 5.

Host DNA Markers 

Results of the analysis of quality-control samples using the host DNA method are shown in table 5. 

Each of the quality-control samples (Unknowns A, B, and C) were examined for each of the nine

markers specific to the following animal sources: human, Canada goose, white-tailed deer, cow, 

pig, chicken, horse, dog, and sheep. Average sensitivity for the three unknowns was 1.000, and 

average specificity was 0.911. That is, the test correctly identified the presence of host DNAs 

15
Table 4. Summary of 373 Escherichia coli isolates from water samples collected from 

four tributaries to the New River Gorge National River, West Virginia, 2004, that were 

compared with the expanded 8,043-isolate known-source library 

[Data are presented according to sample, followed by totals across all samples. Results for two samples 

are not shown because no E. coli bacteria were isolated from these two samples. Patterns with similarities 

greater than or equal to 85 percent were accepted. RESULTS IN THIS TABLE ARE 

EXPECTED TO BE INCORRECT, BASED ONQUALITY-CONTROL EVALUATION.]

Animal source

Arbuckle 

Creek 

sample 1 

(9/27/04) 

Arbuckle 

Creek 

sample 2 

(10/6/04) 

Dunloup 

Creek 

sample 

1 

(9/22/04) 

Dunloup 

Creek 

sample 

2 

(10/5/04) 

Keeney 

Creek 

sample 

2 

(9/30/04) 

Wolf 

Creek 

sample 

1 

(9/29/04) 

Total

matches by 

source 

Human 2 0 4 4 7 4 21

White-tailed deer 2 4 2 4 8 17 37

Canada goose 3 5 0 2 11 4 25

Dog 0 4 0 0 0 0 4 

Cat 0 0 1 0 0 0 1 

Coyote 0 0 0 2 0 0 2 

Cow 2 4 0 2 3 7 18

Horse 2 0 0 0 0 0 2 

Swine 4 13 2 3 8 1 31 

Chicken 4 1 3 2 4 1 15

Raccoon 0 2 0 0 4 5 11 

Total matches 19 33 12 19 45 39 167

Matches not 

accepted 28 25 9 32 58 54 206

Total isolate 

patterns 47 58 21 51 103 93 373

that were in the quality-control samples 100 percent of the time, but incorrectly identified as 

present host DNAs that were not in the quality-control samples 8.9 percent of the time. Qualitycontrol sample Unknown A (containing human feces) was identified as human and cow. Qualitycontrol sample Unknown B (containing white-tailed deer and Canada goose feces) was identified as 

Canada goose, white-tailed deer, and human. Because no bacterial growth was obtained from this 

sample, a replacement quality-control sample Unknown C (containing white-tailed deer and 

Canada goose) was requested and was correctly identified as white-tailed deer and Canada goose. 

Because quality-control samples were not prepared quantitatively, it is impossible to accurately 

determine the actual mixture composition of the unknown samples. 

A standard curve was established using decimal dilutions of human placental DNA similar 

to those of the Bacteroidetes markers previously described; the equation describing this standard 

curve is Concentration = 10(-0.284*Ct + 7.332), R2 = 0.99972. Results of analyses of the eight water samples 

collected from the four tributaries to the New River indicate detections of human DNA in all eight 

samples and in relatively high amounts in several of the samples (figs. 4 and 5). 

Relative fluorescence of the SYBR Green I dye as obtained from real-time PCR analysis for 

each marker indicates the presence of these markers and is used to calculate the concentration of 

these markers relative to the surrogate standard. In the second Keeney Creek sample (September 

30), the relative concentration of human DNA was particularly elevated in both the dissolved and 

filter-bound fractions. In both Wolf Creek samples, the relative concentration of human DNA was 

16
Arbuckle

Creek

9/27/04 10/06/04 10/05/04 9/22/04 9/23/04 9/30/04 10/12/04 9/29/04

Dunloup

Creek

Keeney

Creek

Wolf

Creek

0.00

0.01

0.02

0.03

0.04

0.05

FECAL BACTERIODES

RELATIVE CONCENTRATION

Figure 2. Relative concentrations of fecal Bacteroidetes in eight water samples collected from four 

tributaries to the New River Gorge National River, West Virginia, 2004. (From Dick and Field, 2004) 

(Concentration is relative to quality-control sample Unknown A, where the concentration of this 

marker in a 1:1000 dilution of the standard equals 1000.) 

Arbuckle

Creek

9/27/04 10/06/04 9/22/04 9/23/04 9/30/04 10/12/04 10/05/04 9/29/04

Dunloup

Creek

Keeney

Creek

Wolf

Creek

0

10

20

30

40

50

HUMAN-SPECIFIC BACTERIODES

RELATIVE CONCENTRATION

Figure 3. Relative concentrations of human-specific Bacteroidetes marker in eight water samples 

collected from four tributaries to the New River Gorge National River, West Virginia, 2004. (From 

Seurinck and others, 2005) (Concentrations are relative to quality-control sample Unknown A, where 

the concentration of this marker in a 1:1000 dilution of the standard equals 1000.) 

17
elevated in both fractions. The relative concentration of filter-bound DNA (fig. 4) was slightly 

higher in the Wolf Creek sample of September 29 than in the Keeney Creek sample of September 

30, but the dissolved human DNA (fig. 5) was much higher in the Keeney Creek sample of 

September 30 than in any other sample. Human DNA was detected in elevated amounts for both 

filter-bound and dissolved DNA for the second Keeney Creek sample (September 30) and for both 

Wolf Creek samples, and for the filter-bound DNA for the second Dunloup Creek sample (October 

5). Animal DNA for dog, chicken, horse, and pig only (table 6) was detected in smaller amounts 

than human DNA, but was not quantified. Animal DNA was detected in all eight water samples 

with the exception of the October 5 sample from Dunloup Creek. Dog DNA was detected in both 

samples from Arbuckle Creek and in the September 22 sample from Dunloup Creek. Chicken 

DNA was detected in both samples from Keeney Creek and in the October 12 sample from Wolf 

Creek. Horse DNA was detected in both samples from Wolf Creek, in the September 23 sample 

from Keeney Creek, and in the September 22 sample from Dunloup Creek. Pig DNA was detected 

only in the September 27 sample from Arbuckle Creek. White-tail deer, Canada goose, and cow 

DNA were not detected in any of the eight water samples from the four tributaries, despite the fact 

that deer and cows were observed in the study area, and geese were observed near the study area. 

Table 5. Host mitochondrial DNA analysis of quality-control samples expressed as sensitivity and 

specificity 

Quality-control

Unknown 

Sample

composition Species detected Species not detected Sensitivity Specificity

Unknown A Human Human Horse 1.000 0.875

Cow Dog

White-tailed deer

Pig

Chicken

Sheep

Canada goose

Unknown B White-tailed deer White-tailed deer Horse 1.000 0.857 

Canada goose Canada goose Dog 

Human Pig

Chicken

Sheep

Cow

Unknown C White-tailed deer White-tailed deer Horse 1.000 1.000 

Canada goose Canada goose Dog 

Pig

Chicken

Sheep

Cow

Human

Sample average 1.000 0.911

18
Arbuckle

Creek

9/27/04 10/06/04 9/22/04 10/05/04 9/23/04 9/30/04 9/29/04 10/12/04

Dunloup

Creek

Keeney

Creek

Wolf

Creek

0.000

0.002

0.004

0.006

0.008

0.010

FILTER BOUND HUMAN DEOXYRIBONUCLEIC

ACID (DNA), RELATIVE CONCENTRATION

Figure 4. Relative concentrations of filter-bound human DNA in eight water samples collected from 

four tributaries to the New River Gorge National River, West Virginia, 2004. (Concentrations are 

relative to a human placental DNA standard that had a concentration of 18.5 nanograms per 

microliter. A 1:1000 dilution of this standard is equal to 1000 on this scale.) 

Arbuckle

Creek

9/27/04 10/06/04 9/22/04 10/05/04 9/23/04 9/30/04 9/29/04 10/12/04

Dunloup

Creek

Keeney

Creek

Wolf

Creek

0.0

0.1

0.2

0.3

0.4

RELATIVE CONCENTRATION OF DISSOLVED

HUMAN DEOXYRIBONUCLEIC ACID (DNA)

Figure 5. Relative concentrations of dissolved human DNA in eight water samples collected from 

four tributaries to the New River Gorge National River, West Virginia, 2004. (Concentrations are 

relative to a human placental DNA standard that had a concentration of 18.5 nanograms per 

microliter. A 1:1000 dilution of this standard is equal to 1000 on this scale.) 

Wastewater Compounds 

The analyses for 62 wastewater compounds yielded detections (table 7) for 24 of the 

compounds (excluding surrogates); only phenol was detected at concentrations that exceeded the 

reporting limit. Although detections for 23 of the wastewater compounds were either not 

19
Table 6. Host mitochondrial DNA markers for species other than human detected, but not quantified, 

in eight water samples collected from four tributaries to the New River Gorge National River, West 

Virginia, 2004 

[X, indicates that the detection was near, but higher than background values; ---, indicates no detection]

Stream Date Dog Pig Cow

White-tailed

deer

Canada

goose Horse Chicken

Arbuckle Creek 9-27-04 X X --- --- --- --- ---

Arbuckle Creek 10-06-04 X --- --- --- --- --- --- 

Dunloup Creek 9-22-04 X --- --- --- --- X 

Dunloup Creek 10-05-04 --- --- --- --- --- --- ---

Keeney Creek 9-23-04 --- --- --- --- --- X X

Keeney Creek 9-30-04 --- --- --- --- --- --- X

Wolf Creek 9-29-04 --- --- --- --- --- X ---

Wolf Creek 10-12-04 --- --- --- --- --- X X

quantified or were estimated detections that were less than the instrument reporting level, these 

detections, especially the estimated detections, are still considered to be significant (Stephen 

Zaugg, U.S. Geological Survey, written commun., 2006). Analysis of the water sample collected 

from Arbuckle Creek on September 27 yielded detections for 22 of 62 of the wastewater 

compounds. The compounds 1,4-dichlorobenzene, AHTN, cotinine, and triclosan that were 

detected in this sample are considered to be good indicators of wastewater, especially sewage. 

Thus, the Arbuckle Creek sample of September 27 likely was affected by human tracer compounds 

with sewage as the likely source of these compounds. These four compounds were not found in 

any of the other water samples except for detections of AHTN in the Arbuckle Creek sample of

October 6 and the Keeney Creek sample of September 30. Caffeine indicates wastewater, but 

historically has been detected in about 50 percent of all field blanks. Phenol is ubiquitous in 

surface waters at the levels detected in this study (Stephen Zaugg, U.S. Geological Survey, written 

commun., 2006). 

Presumptive Sources of Fecal Contamination, by Tributary 

Descriptions of the findings regarding animal sources of fecal matter in the water samples 

from the four tributaries are summarized, by method, in Table 8 and by sample in Table 9. Field 

and laboratory determinations of bacteria concentrations in the eight water samples are listed in 

table 2. A discussion of the presumptive sources of contamination for each of the four tributaries to 

the New River follows. Any inferences of possible sources of fecal contamination are limited to 

the specific dates and times of sample collection. Because many of the detections for the various 

methods could vary over time and flow conditions, repeated sampling during both base flow and 

storm events would be necessary to more definitively determine the sources of fecal contamination 

for each tributary. 

Arbuckle Creek 

Arbuckle Creek was sampled September 27 and October 6, 2004; streamflows at the times of 

sample collection were 1.4 and 2.5 ft3/s, respectively (table 2). Concentrations of fecal coliform 

20
Table 7. Wastewater compounds detected in eight water samples collected from four tributaries to 

the New River Gorge National River, West Virginia, 2004 

[μg/L, micrograms per liter; <, less than; E, estimated; M, presence verified, not quantified] 

Sampling date 1,4-Dichlorobenzene (μg/L)

AHTN 

(μg/L)

Benzophenone 

(μg/L)

Caffeine 

(μg/L)

Camphor 

(μg/L)

Cholesterol

(μg/L)

09-27-04 M E.1 M E.1 M M

10-06-04 <.5 E.2 <.5 E.2 <.5 <2

09-22-04 <.5 <.5 <.5 M <.5 <2

10-05-04 <.5 <.5 <.5 <.5 <.5 <2

09-23-04 <.5 <.5 <.5 <.5 <.5 <2

09-30-04 <.5 M <.5 M <.5 <2

09-29-04 <.5 <.5 <.5 M <.5 M

10-12-04 <.5 <.5 <.5 <.5 <.5 <2

Sampling date

Cotinine 

(μg/L)

DEET 

(μg/L)

Fluoranthene 

(μg/L)

HHCB 

(μg/L)

Isophorone

(μg/L)

Methyl 

salicylate 

(μg/L)

09-27-04 E.2 E.2 M M M <.5

10-06-04 <1.00 <.5 <.5 <.5 <.5 <.5

09-22-04 <1.00 <.5 M <.5 <.5 <.5

10-05-04 <1.00 <.5 <.5 <.5 <.5 <.5

09-23-04 <1.00 <.5 <.5 <.5 <.5 <.5

09-30-04 <1.00 <.5 M <.5 <.5 M

09-29-04 <1.00 <.5 <.5 <.5 <.5 <.5

10-12-04 <1.00 <.5 <.5 <.5 <.5 <.5

Sampling date p-Cresol 

(μg/L)

Phenanthrene 

(μg/L)

Phenol 

(μg/L)

Prometon 

(μg/L)

Pyrene 

(μg/L)

Tetrachloro

-ethene 

(μg/L)

09-27-04 M M 1.1 E.1 M <.5

10-06-04 <1 <.5 <.5 <.5 <.5 <.5

09-22-04 <1 <.5 <.5 <.5 M M

10-05-04 <1 <.5 E1.0 <.5 <.5 <.5

09-23-04 <1 <.5 <.5 <.5 <.5 <.5

09-30-04 <1 <.5 2.9 <.5 M <.5

09-29-04 <1 <.5 <.5 <.5 <.5 <.5

10-12-04 <1 <.5 0.8 <.5 <.5 <.5

Sampling date

Tributyl phosphate 

(μg/L)

Triclosan 

(μg/L)

Triethyl citrate 

(μg/L)

Tris (2-butoxyethyl)

phosphate 

(μg/L)

FYROL CEF 

(μg/L)

FYROL PCF 

(μg/L)

09-27-04 E.1 M E.1 E.2 E.2 E.1

10-06-04 <.5 <1 <.5 <.5 <.5 <.5

09-22-04 <.5 <1 <.5 <.5 <.5 <.5

10-05-04 <.5 <1 <.5 <.5 <.5 <.5

09-23-04 <.5 <1 <.5 <.5 <.5 <.5

09-30-04 <.5 <1 <.5 <.5 <.5 <.5

09-29-04 <.5 <1 <.5 <.5 <.5 <.5

10-12-04 <.5 <1 <.5 <.5 <.5 <.5

21
Table 8. Descriptions of findings, by method, for determining animal sources of fecal 

matter detected in eight water samples collected from four tributaries to the New River 

Gorge National River, West Virginia, 2004 

Method Finding

Pulsed-field gel 

electrophoresis 

Because PFGE correctly identified the source of only one of 37 quality-control 

bacteria isolates, no source inferences as to the source were based on the PFGE

analysis. 

Human host-specific 

Enterococcus marker 

This marker, indicating a human source of contamination, was found only in one

sample (September 23) for Keeney Creek. The marker was detected in one 

positive-control sample and was not detected in one negative-control sample. 

Human-urine marker This marker, indicating a human source of contamination, was found only in

both Keeney Creek samples. The marker was not detected in either of two 

negative-control blind samples, but a positive-control blind sample was not

submitted.

Wastewater compounds At least one wastewater compound was found in all samples, except for the 

Keeney Creek sample of September 23. Detections were all low; the greatest 

number of detections occurred in the Arbuckle Creek sample of September 27. 

Detections in this sample indicate the presence of human tracer compounds 

likely derived from sewage. These analytes were not tested in quality-control

samples. 

Bacteroidetes humanspecific markerQuantitative detection of this marker, indicating human sources of

contamination, was positive in all samples. In general, Keeney Creek contained

the highest concentration of this marker, followed by Dunloup Creek. 

Detections in Arbuckle and Wolf Creeks were near the limit of detection. This 

marker was not tested against control samples. 

Host-specific DNA Detections of host-specific DNA were predominantly of human DNA for all four

tributaries. Concentrations of filter-bound human DNA were slightly higher in 

the Wolf Creek sample of September 29 than in the Keeney Creek sample of 

September 30. Concentrations of dissolved human DNA were much higher in 

the Keeney Creek sample of September 30 than in any other sample. Trace 

amounts of dog, pig, horse, and chicken DNA were detected in some of the 

samples. The human marker was detected in one positive-control sample and in

one of two negative-control samples (white-tailed deer and Canada goose). 

White-tailed deer and Canada goose DNA markers were detected in both tests 

against the positive-control sample. Markers for the other non-human hosts 

were not tested against positive-control samples. 

and fecal streptococcus bacteria measured in the field were low to moderate and were 160 (Sept. 

27) and 150 (Oct. 6) colonies of fecal coliform per 100 mL of sample and 290 (Sept. 27) and 39 

(Oct. 6) colonies of fecal streptococcus per 100 mL of sample, respectively (table 2). Several 

wastewater compounds were detected at low concentrations for both samples (table 7). The 

detections of AHTN, cotinine, triclosan, and 1,4-dichlorobezene in the September 27 sample 

indicate that this sample contains human tracer compounds likely derived from sewage. Potential 

sources of these wastewater compounds are two wastewater-treatment plants that discharge sewage 

to Arbuckle Creek during high streamflow caused by storm runoff and lift stations that have been 

observed to overflow even during dry periods. Also straight pipe discharges, leaking sewer lines, 

22
Table 9. Summary of human-specific markers present in eight water samples collected from four 

tributaries to the New River Gorge National River, West Virginia, 2004. 

[Yes, detected; No, not detected; numbers following “Yes” indicate rank of detection level of samples with especially 

high levels of detection.] 

Sampling site Sampling 

date 

Enterococcus 

marker 

Urine 

marker 

Bacteroidetes 

marker 

Dissolved 

mitochondrial 

DNA 

Filter-bound 

mitochondrial 

DNA 

Dunloup Creek 9/22/2004 No No Yes -3 Yes Yes 

Dunloup Creek 10/5/2004 No No Yes -2 Yes Yes -4

Arbuckle Creek 9/27/2004 No No Yes Yes Yes 

Arbuckle Creek 10/6/2004 No No Yes Yes Yes 

Keeney Creek 9/23/2004 Yes Yes Yes Yes Yes 

Keeney Creek 9/30/2004 No Yes Yes -1 Yes -1 Yes -2 

Wolf Creek 9/29/2004 No No Yes Yes -2 Yes -1 

Wolf Creek 10/12/2004 No No Yes Yes -3 Yes -3 

and faulty septic systems are possible sources. The human-specific Enterococcus marker and the 

human-urine marker were not detected in either Arbuckle Creek sample. The human-specific 

Bacteroidetes (fig. 3) marker and the human-specific mitochondrial DNA marker (figs. 4 and 5) 

were detected in both samples from Arbuckle Creek, but were not especially elevated in either 

sample. Dog DNA was detected in both Arbuckle Creek samples, and pig DNA was detected in 

the sample of September 27 (table 6). 

Dunloup Creek 

Dunloup Creek was sampled September 22 and October 15, 2004; streamflows at the times 

of sample collection were 23 and 22 ft3

/s (cubic feet per second), respectively (table 2). 

Concentrations of fecal coliform and fecal streptococcus measured in the field were relatively low 

at 87 and 17 (estimated) colonies of fecal coliform per 100 mL of sample, respectively, and 36 and 

7 (estimated) colonies of fecal streptococcus per 100 mL of sample, respectively (table 2). The 

human-specific Enterococcus marker and the human-urine marker were not detected in either

Dunloup Creek sample. The human-specific Bacteroidetes (fig.3) marker was detected in relatively 

elevated amounts in both water samples collected from Dunloup Creek. The human-specific 

marker for host mitochondrial DNA (figs. 4 and 5) also was detected in both water samples 

collected from Dunloup Creek Filter-bound mitochondrial DNA (fig. 4) was elevated in the second 

Dunloup Creek sample (October 5). As previously stated, two wastewater-treatment plants in the 

Dunloup Creek watershed could discharge partially treated sewage to Dunloup Creek during high 

streamflow caused by storm runoff. This was obviously not a factor in the base-flow samples 

collected from Dunloup Creek in this study. The fact that human-specific markers for 

Bacteroidetes and host mitochondrial DNA were detected at base flow seems to indicate that 

human waste was reaching Dunloup Creek during the base-flow conditions at the time of sampling. 

Possible sources of the human-specific markers indicating human contamination sources are human 

waste from straight pipe discharges to the stream, from leaking sewer lines, or from faulty septic 

systems. Dog and horse DNA (table 6) were detected in the September 22 sample. 

Keeney Creek 

Keeney Creek was sampled September 23 and 30, 2004; streamflows at the times of sample 

collection were 1.3 and 5.2 ft3

/s, respectively (table 2). Fecal coliform and fecal streptococcus 

23
bacteria concentrations measured in the field were 44 (estimated) (Sept. 23) and 280 (Sept. 30) 

colonies of fecal coliform per 100 mL of sample and 74 (estimated) (Sept. 23) and 160 (Sept. 30) 

colonies of fecal streptococcus per 100 mL of sample, respectively (table 2). The human-specific 

Enterococcus marker was detected in the first but not the second sample, and the human-urine 

marker was detected in both samples (table 9). These two human-specific markers were not 

detected in samples from the other three tributaries. The human-specific Bacteroidetes marker (fig. 

3) and the human-specific host mitochondrial DNA markers (figs. 4 and 5) were detected in both 

samples and were especially elevated in the second sample of September 30. Three of the four 

human-specific marker detections were for the second Keeney Creek sample which had higher 

fecal coliform bacteria concentrations than the first sample. Curiously, the human-specific marker 

for Enterococcus was detected in the first Keeney Creek sample that contained lower 

concentrations of fecal coliform bacteria. The presence of multiple human-specific markers in the

Keeney Creek samples indicates a human source of fecal waste for these samples. The Keeney 

Creek watershed has no centralized municipal wastewater-treatment facilities. Thus, possible 

sources for the human-specific markers during base-flow conditions are straight pipe discharges 

and faulty septic systems. Chicken DNA was detected in both water samples, and horse DNA was 

detected in the September 23 sample (table 6). 

Wolf Creek 

Wolf Creek was sampled September 29 and October 12, 2004; streamflows at times of 

sample collection were 15 and 3.0 ft3

/s, respectively (table 2). The second sample was collected 

during base flow, but the first sample could have contained some surface runoff. Concentrations of

fecal coliform and fecal streptococcus bacteria measured in the field were 3,800 (Sept. 29) and 3 

(estimated) (Oct. 12) colonies of fecal coliform per 100 mL of sample and 1,900 (Sept. 29) and 5 

(estimated) (Oct. 12) colonies of fecal streptococcus per 100 mL of sample, respectively (table 2). 

The first sample collected from Wolf Creek contained bacteria concentrations an order of 

magnitude higher than bacteria concentrations for the remaining seven samples. The humanspecific Enterococcus and human-urine markers were not detected in either Wolf Creek sample. 

The human-specific Bacteroidetes marker (fig. 3) and the human-specific host mitochondrial DNA 

marker (figs. 4 and 5) were detected in both samples. The human-specific DNA marker was 

especially elevated in both samples. Possible sources of the human-specific markers are straight

pipe discharges, leaking sewer lines, and faulty septic systems. The Wolf Creek watershed has a 

sewage distribution system that carries sewage to a sewage-treatment plant located in an adjacent 

watershed. Thus, municipal sewage outfalls or overflows are not possible sources of sewage in 

Wolf Creek. Horse DNA was detected in both samples, and chicken DNA was detected in the 

October 12 sample (table 6). 

Summary and Conclusions 

A study was conducted in the vicinity of the New River Gorge National River to determine 

the source of fecal contamination (sourcing) in four tributaries to the river. Inferences to possible 

sources of fecal contamination are limited to dates and times of sample collection from the 

tributaries. The sourcing methods used in this study to attempt to associate fecal contamination in

water samples collected from the four tributaries to the New River with animal sources yielded 

various results. 

Evaluation of results for quality-control samples indicated that PFGE could only correctly 

identify 1 of 37 known bacteria isolates. It could not identify any human, dog, or Canada goose 

quality-control bacteria isolates and correctly identified only one white-tailed deer quality-control 

24
bacteria isolate. Increasing the size of the PFGE library of known-source isolates did not improve 

quality-control results. The use of different growth media in the various steps of culturing of E coli

bacteria isolates in this study could partially explain the errors in attempted association of qualitycontrol isolates to animal source. E. coli isolates for the water samples and quality-control samples 

were obtained from fecal coliform colonies grown on mFC media, and E. coli isolates were 

obtained from known sources of feces by streaking the fecal samples onto EMB media. It is also 

possible that the population of DNA-fragment banding patterns derived from E. coli in human and 

animal feces is too diverse and variable to allow accurate associations of bacteria isolates in water 

samples from the four tributaries with animal sources by use of PFGE analysis. Due to the poor 

results for the quality-control bacteria isolates, PFGE results for water samples were not used for 

sourcing. 

Results of quality-control tests for emerging sourcing methods that use human-specific and 

animal-specific biological or molecular markers indicate that some of these methods may have 

potential for identifying the source of fecal waste in natural waters. A human-specific 

Enterococcus marker was correctly detected in a positive control sample and was not detected in a

negative control sample. A human-specific urine marker was not detected in two negative control 

samples. A human-specific host mitochondrial DNA marker was correctly detected in a positive 

control sample, but was detected in one of two negative control samples. Canada goose-specific 

and white-tailed deer-specific host mitochondrial DNA markers were correctly detected in two 

positive control samples and not detected in one negative control sample. A cow-specific marker 

was detected in one of three negative control samples. Horse-, pig-, sheep-, dog-, and chickenspecific markers were not detected in any of three negative-control samples. Future studies will 

need rigorous quality-control components on the potential applicability and use of these emerging

sourcing methods. 

The highest relative concentrations of four human-specific markers for Enterococcus and 

human urine (both markers only detected in Keeney Creek), Bacteroidetes, and host mitochondrial 

DNA were detected in water samples from Keeney Creek, a watershed with no centralized 

municipal wastewater-treatment facilities, thus indicating human sources of fecal contamination. 

The human-specific Bacteroidetes and host mitochondrial DNA markers also were detected in 

water samples from Dunloup Creek, Wolf Creek, and to a lesser degree Arbuckle Creek. The 

results of analysis for wastewater compounds indicate that one sample from Arbuckle Creek 

contained numerous human tracer compounds, likely from sewage. Lesser amounts of dog, horse, 

chicken, and pig host mitochondrial DNA were detected in some of the water samples; no animal 

mitochondrial DNA was detected in one water sample from Dunloup Creek. Cow, white-tailed 

deer, and Canada goose DNA were not detected in any of the samples collected from the four 

tributaries, although these animals are present in the watersheds. 

Acknowledgments 

The authors are grateful to the many landowners, facilities, and individuals who either 

allowed access to private and public lands for the collection of fecal samples or who donated fecal 

samples. 

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28