U.S. Department of the Interior

U.S. Geological Survey

Open-File Report 2009–1161

Occurrence of Viable Avian Influenza Viruses in 

Water and Bed Sediments from Selected Water Bodies 

along the Atlantic Flyway, February and May 2006 

and January 2007
Cover. Ardea occidentalis (great white heron), Chinoteague National Wildlife Refuge, 

Accomack County, Virginia (photo by Alan M. Cressler, U.S. Geological Survey).
Occurrence of Viable Avian Influenza Viruses in 

Water and Bed Sediments from Selected Water Bodies 

along the Atlantic Flyway, February and May 2006 

and January 2007

By Melinda S. Dalton, Lisa M. Stewart, and Hon S. Ip

Open-File Report 2009–1161

U.S. Department of the Interior

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

KEN SALAZAR, Secretary

U.S. Geological Survey

Suzette Kimball, Acting Director

U.S. Geological Survey, Reston, Virginia: 2009

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Suggested citation:

Dalton, M.S., Stewart, L.M., Ip, H.S., 2009, Occurrence of viable avian influenza viruses in water and bed sediments 

from selected water bodies along the Atlantic Flyway, February and May 2006 and January 2007: U.S. Geological 

Survey Open-File Report 2009–1161, 12 p. 
iii

Contents

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

Introduction.....................................................................................................................................................1

Previous Studies ...................................................................................................................................2

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

Sample Collection and Analysis..................................................................................................................4

Sampling Method .................................................................................................................................4

Laboratory Analysis..............................................................................................................................5

Occurrence of Viable Avian Influenza Viruses .........................................................................................9

Savannah National Wildlife Refuge...................................................................................................9

Santee National Wildlife Refuge........................................................................................................9

Pee Dee National Wildlife Refuge .....................................................................................................9

Mason Neck and Occoquan Bay National Wildlife Refuges ......................................................10

Blackwater National Wildlife Refuge..............................................................................................10

Chincoteague National Wildlife Refuge..........................................................................................10

Summary .......................................................................................................................................................11

Acknowledgments.......................................................................................................................................11

References ...................................................................................................................................................11

Figures

1. Matrix showing possible avian influenza virus hemagglutinin (H) and 

neuraminidase (N) subtypes.......................................................................................................1

2. Diagram showing possible biotic and abiotic reservoirs and transmission 

pathways for avian influenza viruses........................................................................................2

3. Map showing water and bed-sediment samples collected from selected 

water bodies from National Wildlife Refuges along the Atlantic Flyway for 

analysis of avian influenza viruses, February and May 2006 and January 2007................4

Tables

1. Location of water and bed-sediment samples collected from 

National Wildlife Refuges along the Atlantic Flyway for analysis of 

avian influenza viruses, February and May 2006 and January 2007 ...................................3

2. Sampling-site conditions for water and bed-sediment samples collected 

from National Wildlife Refuges along the Atlantic Flyway for analysis of 

avian influenza viruses, February and May 2006 and January 2007 ....................................6

3. Field measurements of water-quality properties and results of 

matrix RT-PCR and viral isolation tests associated with water and 

bed-sediment samples collected from National Wildlife Refuges 

along the Atlantic Flyway for analysis of avian influenza viruses, 

February and May 2006 and January 2007 ...............................................................................8
iv

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch

meter (m) 3.281 foot (ft) 

meter (m) 1.094 yard (yd) 

Volume

milliliter (mL) 0.03382 once, fluid (fl. oz)

milliliter (mL) 0.000264 gallons (gal)

liter (L) 33.82 ounce, fluid (fl. oz)

liter (L) 2.113 pint (pt)

liter (L) 1.057 quart (qt)

liter (L) 0.2642 gallon (gal)

liter (L) 61.02 cubic inch (in3

) 

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

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

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 in milligrams per liter (mg/L).

Conversion Factors and Horizontal Datum
Occurrence of Viable Avian Influenza Viruses in 

Water and Bed Sediments from Selected Water Bodies 

along the Atlantic Flyway, February and May 2006 

and January 2007

By Melinda S. Dalton, Lisa M. Stewart, and Hon S. Ip

Abstract 

Water and bed-sediment samples were collected 

from selected water bodies along the Atlantic Flyway and 

analyzed for the presence of viable avian influenza viruses. 

Samples were collected during February and May 2006 and 

January 2007 at U.S. Fish and Wildlife Service National 

Wildlife Refuges in Georgia, South Carolina, North Carolina, 

Virginia, and Maryland. Avian influenza viruses were detected 

in samples collected from the Savannah National Wildlife 

Refuge in Georgia during February 2006 and from the Santee 

National Wildlife Refuge in South Carolina and the Pee Dee 

National Wildlife Refuge in North Carolina during January 

2007. Avian influenza virus was detected in water temperatures ranging from 11.8 to 12.7 degrees Celsius when birds 

were either present or had departed at least 10 days prior to 

sampling. Although the literature indicates that avian influenza virus persists in the environment more effectively at 

colder temperature regimes, these detections were made in 

a comparatively warmer climate at a time of the year when 

cooler water temperatures prevail.

Introduction

Three types of influenza viruses occur naturally—A, 

B, and C. Influenza A viruses (avian influenza) can infect 

humans, pigs, horses, and other mammals, in addition to 

birds. Avian influenza viruses (AIV) cause seasonal epidemics 

affecting a small percentage of the human population annually; however, AIV also cause periodic pandemics (1918, 

1957, and 1968) that have resulted in large losses of human 

life. Highly pathogenic AIV is a major cause of substantial 

losses in domestic poultry populations worldwide (World 

Health Organization, 2003, 2006; Zhang and others, 2006). 

AIV subtypes are categorized by the combination of 

proteins (hemagglutinin, “H” and neuraminidase, “N”) found 

on their surfaces and include subtypes with both high and low 

pathogenicity (fig. 1; Karasin and others, 2000; World Health 

Organization, 2006). Highly pathogenic (HP) AIV H5N1 

infected domestic poultry, migratory waterfowl, and humans 

in Asia, Europe, and Africa in 2008. During such widespread 

incidents, mortality rates can be as high as 90 and 100 percent 

for poultry and waterfowl, respectively, and greater than 

H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16

N1

N2

N3

N4

N5

N6

N7

N8

N9

Known subtype combination H5N1–highly pathogenic subtype

AIV subtypes detected (this study)

EXPLANATION

Figure 1. Matrix showing possible avian influenza virus hemagglutinin (H) and neuraminidase (N) subtypes.

No known subtype contamination

Figure 1. Matrix showing possible avian influenza virus hemagglutinin (H) and neuraminidase (N) subtypes.
2 Occurrence of Viable Avian Influenza Viruses in Water and Bed Sediments from Selected Water Bodies

50 percent for infected humans (World Health Organization 

2003, 2006; U.S. Interagency Strategic Plan, 2006; U.S. 

Department of Health and Human Services, 2008). Most cases 

of HPAIV H5N1 infection in humans result from contact with 

infected poultry, domesticated chickens, ducks, and turkeys, 

or from contact with surfaces contaminated with secretions 

or excretions from infected birds (World Health Organization 

2003; U.S. Interagency Strategic Plan, 2006; U.S. Department 

of Health and Human Services, 2008). Because all AIV can 

mutate genetically, world health agencies are concerned that 

in the future, HPAIV H5N1, may mutate and become capable 

of being transmitted efficiently from one human to another, 

which could result in a worldwide influenza pandemic (World 

Health Organization, 2003, 2006; U.S. Interagency Strategic 

Plan, 2006; U.S. Department of Health and Human Services, 

2008). Therefore, surveillance of biotic and abiotic reservoirs 

for AIV (fig. 2) is critical for developing a procedure to 

identify sources of a future influenza pandemic and where 

a future pandemic could emerge. Until recently the study of 

these abiotic reservoirs have focused primarily on water as a 

transmission source (World Health Organization, 2006; Zhang 

and others, 2006); however, it is likely that AIV, once shed, 

would dilute rapidly in any body of water. 

In 2006, the U.S. Geological Survey (USGS) began a 

reconnaissance to determine if AIV was present in the bed 

sediment of selected water bodies along the Atlantic Flyway. 

The movement of AIV between countries and continents 

may be the result of transport by infected migratory birds, 

and bed sediment may be an important substrate for AIV to 

spread between migratory bird populations. Investigating 

the presence of AIV in sediment is important because once 

the virus is shed from an animal it may persist longer in the 

sediment substrate where it is protected from conditions that 

can cause AIV to deteriorate. 

Previous Studies

Investigators have been successful in isolating AIV in surface water when migratory ducks are present and are shedding 

virus in feces (Webster and others, 1978; Hinshaw and others, 

1979; Halvorson and others, 1983; Kelleher and others, 1985; 

Sivanandan and others, 1991). What is less understood is how 

long the virus remains viable in the environment once shed. 

Does the virus persist long enough to infect the next population of migratory birds to use the contaminated water body? 

Does the virus remain in sufficient concentrations to infect the 

next host? Although previous investigators were successful in 

collecting water samples containing AIV, these samples were 

collected in cold-weather climates, such as Canada, Alaska, 

and Siberia (Hinshaw and others, 1979; Ito and others, 1995; 

Zhang and others, 2006); or in areas of concentrated bird 

populations, such as domestic duck production facilities 

(Markwell and Shortridge, 1982; Sivanandan and others, 1991).

Few AIV studies have been conducted to evaluate the 

persistence of the virus in warm-climate waters used by migratory waterfowl such as water bodies in the southeastern United 

States; as a result, little is known about the persistence of AIV 

in these climates. However, it has been established that the persistence of AIV is dependent on the water chemistry and temperature regime of the water body. Several studies have shown 

the infectivity of the virus declined as temperature and salinity 

increased and pH decreased (Stallknecht and others, 1990; 

Ito and others, 1995; and World Health Organization, 2006). 

This study is the first AIV reconnaissance in the southeastern 

United States to attempt to isolate the virus in bed sediments.

Figure 2. Diagram showing possible biotic and abiotic reservoirs 

and transmission pathways for avian influenza viruses (modified 

from Ip and Slota, 2005). 

Biotic

reservoir

Abiotic

reservoir

Known Possible

EXPLANATION

Shorebirds

Waterfowl

Pond Well

Domestic

poultry

Mammals

(primarily swine)

Humans

Humans

 Avian influenza virus transmission pathway

Figure 2. Diagram showing possible biotic and abiotic reservoirs 

and transmission pathways for avian influenza viruses (modified 

from Ip and Slota, 2005).
Introduction 3

Table 1. Location of water and bed-sediment samples collected from National Wildlife Refuges along the 

Atlantic Flyway for analysis of avian influenza viruses, February and May 2006 and January 2007. 

[NWIS, U.S. Geological Survey National Water Information System; NWR, National Wildlife Refuge; °, degrees; ', minutes; 

", seconds; do., ditto; NA, not available; bold text indicates positive results for avian influenza type A viruses by viral isolation tests]

Sample 

number

NWIS site 

identification number Sample date Waterbody Latitude Longitude

Savannah NWR, Georgia and South Carolina

SNWR-01 321128081045201 02/28/2006 King Fisher Pond 32°11'28.1" 81°04'51.7"

SNWR-02 321130081045301 do. do. 32°11'30.4" 81°04'52.9"

SNWR-03 320903081063001 do. Pond #14 32°09'02.6" 81°06'30.4"

SNWR-04 320901081063101 do. do. 32°09'01.3" 81°06'31.0"

SNWR-05 320900081063201 do. do. 32°09'00.2" 81°06'32.5"

SNWR-06 320905081062901 01/16/2007 do. 32°09'04.6" 81°06'28.8"

SNWR-07 320955081064901 do. Pond #10 32°09'55.0" 81°06'49.0"

SNWR-08 320857081062101 do. Pond #14 32°08'57.4" 81°06'21.4"

Santee NWR, South Carolina

SANWR-01 333314080262801 05/26/2006 Banding Pond 33°33'13.9" 80°26'27.9"

SANWR-02 333315080262601 01/17/2007 do. 33°33'14.8" 80°26'25.8"

Pee Dee NWR, North Carolina

PDNWR-01 350446080024501 05/26/2006 Arrowhead Lake 35°04'45.8" 80°02'44.6"

PDNWR-02 350540080010601 01/17/2007 Andrews Pond 35°05'40.1" 80°01 06.2"

Mason Neck NWR, Virginia

MNNWR-01 383728077120601 05/24/2006 Little Marsh Creek 38°37'27.7" 77°12'05.7"

Occoquan Bay NWR, Virginia

ONWR-01 383838077135701 05/24/2006 unnamed tidal creek 38°38'38.4" 77°13'56.8"

Blackwater NWR, Maryland

BWNWR-01 382627076071801 05/24/2006 Pool 3b 38°26'27.2" 76°07'18.2"

BWNWR-02 382635076072401 do. Pool 3a 38°26'35.3" 76°07'23.5"

BWNWR-03 382642076080201 do. Pool 5b 38°26'41.8" 76°08'01.5"

Chincoteague NWR, Virginia

CNWR-01 375432075201501 05/25/2006 Snow Goose Pool 37°54'32.0" 75°20'15.3"

CNWR-02 375406075210201 do. Swans Cove Pool 37°54'06.1" 75°21'02.5"

Purpose and Scope

This report presents results of a reconnaissance study 

by the USGS Georgia Water Science Center, Atlanta, Georgia, and USGS National Wildlife Health Center, Madison, 

Wisconsin, to analyze water and bed-sediment samples for the 

presence of AIV from selected water bodies along the Atlantic 

Flyway during February and May 2006 and January 2007. 

The selected water bodies were in National Wildlife Refuges 

(NWRs) in Georgia, South Carolina, North Carolina, Virginia, 

and Maryland (table 1; fig. 3). 
4 Occurrence of Viable Avian Influenza Viruses in Water and Bed Sediments from Selected Water Bodies

Sample Collection and Analysis

Feces shed from waterfowl settles in a water body and 

mixes with other organic material, such as detritus and aquatic 

vegetation, to form an organic layer that may be an important 

substrate for AIV to persist, protected from conditions that 

cause it to deteriorate. The methods used during this reconnaissance are preliminary in nature but constitute the first-ever 

successful detection of AIV from the benthic regions in bodies 

of water. The objective was to develop a method to collect 

samples from bed sediments and analyze the mixture for 

detection of AIV in a water body. 

Sampling Method 

For this reconnaissance, the term “sampling site” generally 

refers to an area within a water body where large populations of 

waterfowl were observed during regular feeding and migratory 

patterns, by a U.S. Fish and Wildlife Service biologist at each 

NWR (table 2). The term “water body” refers to a pond or 

managed impoundment at a selected NWR. A single water 

body was divided into multiple sampling sites if migratory 

waterfowl were observed to congregate in different areas of the 

water body. At each sampling site, three to five subsamples of 

bed sediments were collected and composited for processing. 

All sampling, collection, and processing equipment 

were cleaned and sterilized using a Barnstead/Thermolyne, 

Corp., Sterilemax tabletop steam sterilizer on liquid cycle at 

121 degrees Celsius (°C) for 15 minutes prior to field activities 

to control cross contamination of equipment. To avoid cross 

contamination in the field, dedicated cleaned sample bottles 

and tubing were assigned to each sampling site. Plastic bins 

and coolers used to transport sampling equipment were 

washed with soapy water, rinsed with tap water, and lined 

prior to being filled with equipment. 

N

Savannah National

Wildlife Refuge

(SNWR)

Occoquan Bay

National

Wildlife Refuge

(ONWR)

Chincoteague

National

Wildlife Refuge

(CNWR)

Santee National

Wildlife Refuge

(SANWR)

EXPLANATION

Pee Dee National

Wildlife Refuge

(PDNWR)

Mason Neck

National

Wildlife Refuge

(MNNWR)

Blackwater

National

Wildlife Refuge

(BWNWR)

Base from U.S. Geological Survey

1:2,000,000-scale digital data

0 4020 60 MILES

0 40 80 120 KILOMETERS

Water and bed-sediment

 sample location

Approximate location of

 Atlantic Flyway

Avian influenza virus 

 detected in bed sediments

Figure 3. Water and bed-sediment samples collected from 

selected water bodies from National Wildlife Refuges along 

the Atlantic Flyway for analysis of avian influenza viruses, 

February and May 2006 and January 2007. 

NC

SC

VA

WV

GA

MD

Figure 3. Water and bed-sediment samples collected from 

selected water bodies from National Wildlife Refuges along 

the Atlantic Flyway for analysis of avian influenza viruses, 

February and May 2006 and January 2007.
Sample Collection and Analysis 5

Water and bed-sediment samples in February and 

May 2006 were collected using a 1-liter plastic bottle attached 

with duct tape to a galvanized steel rod. The bottle was dredged 

along the bottom of the water body collecting as much sediment 

and organic matter as possible along with additional water that 

was removed after the samples were allowed to settle. Samples 

in January 2007 were collected similarly; however, the 1-liter 

plastic bottle was equipped with a gate valve that could be 

opened and closed remotely when near the bottom of the water 

body to reduce possible contamination from surface water. At 

each site, enough bed-sediment material was composited to 

fill a 3-liter Teflon bottle. After returning to the laboratory and 

allowing the bed material to settle to the bottom of the Teflon 

bottle, the remaining water was removed using a peristaltic 

pump. Once all the excess water was removed, bed-sediment 

material was collected in an autoclaved 250-milliliter plastic 

bottle and analyzed for the presence of AIV. For each sample, 

an aliquot of the decanted water was also analyzed for AIV. 

Field measurements of pH, specific conductance, temperature, and dissolved oxygen were made at each sampling site 

(table 3). Laboratory molecular and virus culture techniques 

were performed at the USGS National Wildlife Health Center 

in Madison, Wisconsin. 

Laboratory Analysis

Prior to analysis, the samples were centrifuged at 

800 times gravitational acceleration (9.81 meters per second 

squared) for 15 minutes to produce a supernatant that was 

analyzed for AIV. Molecular detection of AIV and the determination of the presence of H5 and H7 subtypes were performed 

according to methods described in the U.S. Interagency 

Strategic Plan (2006). Analysis for AIV included recovering 

possible viral ribonucleic acid (RNA) from 50-microliter 

aliquots of the whole or filtered water or supernatant using 

the Ambion® MagMaxTM viral RNA isolation kit. A 5-microliter sample of recovered RNA was analyzed using an AIV 

universal matrix-gene real-time reverse transcription polymerase-chain reaction (RT-PCR) test. The RT-PCR tests were 

done using a Stratagene Corporation MX3005PTM real-time 

PCR system with reference to samples with known AIV RNA 

concentrations (Spackman and others, 2002). All matrix-gene 

RT-PCR-positive samples were further subtyped using H5 and 

H7 RT-PCR tests, as these subtypes are of particular human 

and veterinary concern. 

Analysis for AIV also was done by viral isolation. The 

supernatant was filtered through a 0.2-micrometer filter. 

A small volume (0.2 milliliter) of the filtered sample then 

was inoculated into three 8-day-old specific pathogen-free 

embryonated chicken eggs. Following a 3-day incubation 

period at 7 °C, a sample of allantoic fluid was collected from 

the eggs and analyzed for the presence of AIV using a hemagglutination assay with rooster and turkey red-blood cells. 

The presence of AIV was confirmed by recovering RNA from 

the allantoic fluid using the Ambion®MagMaxTM viral RNA 

isolation kit and subjecting it to an AIV universal-virus matrixgene RT-PCR test. Hemagglutination assay-negative samples 

were retested by inoculation into embryonated eggs a second 

time before the sample was considered negative.

Hemagglutination assay and matrix-gene RT-PCRpositive samples were subtyped at the U.S. Department of 

Agriculture National Veterinary Services Laboratory in Ames, 

Iowa, against a panel of reference antisera. Antisera antibodies 

were developed using reference strains of each H and N subtype of AIV and were used to identify specific viral subtypes 

present in samples. 
6 Occurrence of Viable Avian Influenza Viruses in Water and Bed Sediments from Selected Water Bodies Table 2. Sampling-site conditions for water and bed-sediment samples collected from National Wildlife Refuges along the Atlantic Flyway for analysis of avian influenza viruses, February and May 2006 and January 2007. [NWR, National Wildlife Refuge; do., ditto; bold text indicates positive results for avian influenza type A viruses by viral isolation tests] Sample number Sample date Description Sample site condition Estimated water-body depth (meters) Migratory bird species common to this site (scientific name) Estimated total population Migratory bird activity Savannah NWR, Georgia and South Carolina SNWR-01 02/28/2006 Freshwater pond Full pool1 1 Ring-necked duck (Aythya collaris)

1 1About Departed 10 days prior to sample 

200 collection after over-wintering1

SNWR-02 do. Freshwater pond, do. 1 do. do. do.

very loose fine sediments

SNWR-03 do. Managed Full pool, 1 do. 15,000 to do.

freshwater rice-stalk mat 10,000

impoundment covering bottom1

SNWR-04 do. do. do. 1 do. do. do.

SNWR-05 do. do. do. 1 do. do. do.

SNWR-06 01/16/2007 do. Less than 0.5 do. 1About Present at time of sampling1

full pool1 8,000

SNWR-07 do. do. do. 0.1 do. do. do.

SNWR-08 do. do. do. 0.1 do. do. do.

Santee NWR, South Carolina

SANWR-01 05/26/2006 Managed Full pool2 1 Ring-necked duck (Aythya collaris), 22,000 to Departed about 6 weeks prior 

freshwater green-winged teal (Anas carolinensis)

2 6,000 to sample collection after 

impoundment over-wintering2

SANWR-02 01/17/2007 do. do. 1 Ring-necked duck (Aythya collaris)2 2About Present at time of sampling2

6,000

Pee Dee NWR, North Carolina

PDNWR-01 05/26/2006 Freshwater Full pool3 1 Wood duck (Aix sponsa), mallard (Anas 35,000 to Departed about 6 weeks prior 

pond platyrhynchos), green-winged teal (Anas 10,000 to sample collection after 

carolinensis), Canada goose (Branta over-wintering3

canadensis), northern pintail (Anas acuta)

3

PDNWR-02 01/17/2007 do. do. 1 Ring-necked duck (Aythya collaris), do. Present at time of sampling3

green-winged teal (Anas carolinensis), Canada goose (Branta canadensis)3

Mason Neck NWR, Virginia

MNNWR-01 05/24/2006 Managed Full pool4 2 American black duck (Anas rubripes), wood 4500 to Departed about 6 weeks prior 

freshwater duck (Aix sponsa), hooded merganser 1,000 to sample collection after 

impoundment (Lophodytes cucullatus)

4 over-wintering4
Sample Collection and Analysis

7

Table 2. Sampling-site conditions for water and bed-sediment samples collected from National Wildlife Refuges along the Atlantic Flyway for analysis of avian influenza viruses, February and May 2006 and January 2007.—Continued[NWR, National Wildlife Refuge; do., ditto; bold text indicates positive results for avian influenza type A viruses by viral isolation tests]

Sample 

number

Sample 

date Description Sample site condition

Estimated 

water-body

depth

(meters)

Migratory bird species common 

to this site (scientific name)

Estimated

total

 population

Migratory bird activity

Occoquan Bay NWR, Virginia

ONWR-01 05/24/2006 Main channel of 

marsh stream4

Low tide4 1 American black duck (Anas rubripes), wood 

duck (Aix sponsa), hooded merganser (Lophodytes cucullatus)6

4500 to 

1,000

Departed about 6 weeks prior 

to sample collection after over-wintering4

Blackwater NWR, Maryland

BWNWR-01 05/24/2006 Managed

freshwater

impoundment5

Dry except slough 

bordering impoundment, sampled slough5

1 American black duck (Anas rubripes), 

wood duck (Aix sponsa), mallard (Anas platyrhynchos), green-winged teal (Anas carolinensis), Canada goose (Branta canadensis), northern pintail (Anas acuta)5

5 5,000 to 

10,000

Departed about 6 weeks prior 

to sample collection after over-wintering5

BWNWR-02 do. do. Dry except 

isolated pools5

0.1 do. do. do.

BWNWR-03 do. do. do. 0.1 do. do. do.

Chincoteague NWR, Virginia

CNWR-01 05/25/2006 Managed

freshwater

impoundment6

Less than

full pool6

1 American black duck (Anas rubripes), 

wood duck (Aix sponsa), mallard (Anas platyrhynchos), green-winged teal (Anas carolinensis), Canada goose (Branta canadensis), northern pintail (Anas acuta)6

65,000 to 

10,000

Departed about 6 weeks prior 

to sample collection after over-wintering6

CNWR-02 do. do. do. 1 do. do. do.

1Russell Webb, Biologist, Savannah National Wildlife Refuge, U.S. Fish and Wildlife Service, oral commun., February 28, 2006, and January 16, 20072Lawrence A. Woodward, Biologist, Santee National Wildlife Refuge, U.S. Fish and Wildlife Service, oral commun., May 26, 2006, and January 17, 20073Matthew Garner, Biologist, Pee Dee National Wildlife Refuge, U.S. Fish and Wildlife Service, oral commun., May 26, 2006 , and January 17, 20074Marty McClevey, Biologist, Mason Neck and Occoquan Bay National Wildlife Refuges, U.S. Fish and Wildlife Service, oral commun., May 24, 20065Dixie L. Birch, Biologist, Blackwater National Wildlife Refuge, U.S. Fish and Wildlife Service, oral commun., May 16, 20066William Hogland, Biologist, Chincoteague National Wildlife Refuge, U.S. Fish and Wildlife Service, oral commun., May 25, 2006
8 Occurrence of Viable Avian Influenza Viruses in Water and Bed Sediments from Selected Water Bodies

Table 3. Field measurements of water-quality properties and results of matrix RT-PCR and viral isolation tests associated with water 

and bed-sediment samples collected from National Wildlife Refuges along the Atlantic Flyway for analysis of avian influenza viruses, 

February and May 2006 and January 2007.

[mg/L, milligrams per liter; μS/cm, microsiemens per centimeter; °C, degree Celsius; RT-PCR, reverse transcriptase-polymerase chain reaction; NWR, 

National Wildlife Refuge; do., ditto; NA, not available; bold text indicates positive results for avian influenza type A viruses by viral isolation tests]

Sample number 

(see table 1 

for location)

Date

Number

of

measurements

Median

dissolved 

oxygen

(percent

saturation)

Median

dissolved

oxygen 

(mg/L) 

Median 

pH 

Median 

specific

conductance 

(μS/cm)

Median 

temperature 

(°C) 

Matrix 

RT-PCR

test

Viral

isolation

through 

hemagglutination

Detected

avian

influenza

viral 

isolate

Savannah NWR, Georgia and South Carolina

SNWR-01 02/28/2006 5 43.9 4.6 5.2 34 13.6 Negative Negative

SNWR-02 do. 5 37.9 3.9 5.1 33 14.2 do. do.

SNWR-03 do. 5 51.7 5.5 6.1 827 12.9 do. do.

SNWR 04 do. 5 69.1 7.3 6.3 910 12.7 do. Positive H3N1

SNWR-05 do. 5 85.5 8.6 6.4 1,490 12.6 do. Negative

SNWR-06 01/16/2007 5 NA 0.2 6.4 281 16.5 do. do.

SNWR-07 do. 3 NA 2.3 6.3 245 18.9 do. do.

SNWR-08 do. 5 NA 5.8 6.5 336 19.0 do. do.

Santee NWR, South Carolina

SANWR-01 05/26/2006 1 73.5 5.6 3.8 112 29.1 Negative Negative

SANWR-02 01/17/2007 5 NA 7.2 8.0 108 12.5 do. Positive H3N8

Pee Dee NWR, North Carolina

PDNWR-01 05/26/2006 1 25.1 2.1 2.2 55 24.6 Negative Negative

PDNWR-02 01/17/2007 5 NA 7.0 7.1 82 11.8 Positive Positive H3N8

Mason Neck NWR, Virginia

MNNWR-01 05/24/2006 2 NA 7.1 6.7 102 18.7 Negative Negative

Occoquan Bay NWR, Virginia

ONWR-01 05/24/2006 1 57.0 5.6 7.2 149 16.5 Negative Negative

Blackwater NWR, Maryland

BWNWR-01 05/24/2006 1 40.3 3.3 6.0 625 25.4 Negative Negative

BWNWR-02 do. 1 51.5 4.6 7.4 333 20.9 do. do.

BWNWR-03 do. 1 97.6 8.1 7.0 1,708 24.3 do. do.

Chincoteague NWR, Virginia

CNWR-01 05/25/2006 1 97.6 8.1 9.7 2,930 19.3 Negative Negative

CNWR-02 do. 1 135.2 10.9 10.0 3,573 19.5 do. do.
Occurrence of Viable Avian Influenza Viruses 9

Occurrence of Viable Avian 

Influenza Viruses

Water and bed-sediment samples, evaluated for the 

presence of AIV, were collected during February and 

May 2006 and January 2007 from seven NWRs (table 1) 

along the Atlantic Flyway (fig. 3). None of the water samples 

collected tested positive for the presence of AIV. Three of the 

19 bed-sediment samples collected tested positive for AIV; 

water-chemistry data indicate that these positive samples 

were collected in water with temperatures ranging from 

11.8 to 12.7 °C, specific conductance ranging from 82 to 

910 microsiemens per centimeter (μS/cm), and pH ranging 

from 6.3 to 8.0 (table 3). Viral isolation and RT-PCR tests 

detected AIV H3N1 in a sample from the Savannah NWR in 

February 2006 and H3N8 in samples from both the Santee and 

Pee Dee NWRs in January 2007 (fig. 3; table 3). AIV was not 

detected in samples collected during May 2006. 

Savannah National Wildlife Refuge

Eight samples were collected during February 2006 

and January 2007 from the Savannah NWR, near Savannah, 

Georgia (table 1). Two samples, SNWR-01 and SNWR-02, 

were collected from Kingfisher Pond, during February 2006 

(table 2). Kingfisher Pond is a man-made freshwater pond with 

a loose-sediment bottom. NWR personnel estimated about 

200 ring-necked ducks had used the pond for over-wintering 

and departed about 10 days prior to sample collection. AIV 

was not detected in samples from Kingfisher Pond (table 3). 

Three samples, SNWR-03, SNWR-04, and SNWR-05, 

were collected from Pond number (#) 14 during February 2006 

(table 2). Pond #14 is a managed impoundment maintained 

by Savannah NWR personnel as a waterfowl habitat (Russell 

Webb, Biologist, Savannah National Wildlife Refuge, U.S. 

Fish and Wildlife Service, oral commun., February 28, 2006). 

NWR personnel estimated about 5,000 to 10,000 ring-necked 

ducks had used Pond #14 for 1.5 months and departed about 

10 days prior to sample collection. AIV H3N1 was isolated 

and detected in sample SNWR-04; which was collected when 

median water temperature was 12.7 °C, median specific 

conductance was 910 μS/cm, median pH was 6.3, and median 

dissolved oxygen was 7.3 milligrams per liter (mg/L; table 3). 

Two samples, SNWR-06 and SNWR-08, were collected 

from Pond #14 during January 2007 (table 1). NWR personnel 

estimated about 8,000 ring-necked ducks were using Pond #14 

during sample collection (table 2). AIV was not detected in 

samples SNWR-06 and SNWR-08 (table 3). 

One sample, SNWR-07, was collected from Pond #10 

during January 2007. Pond #10 is a managed impoundment 

maintained by Savannah NWR personnel as a waterfowl

habitat (Russell Webb, Biologist, Savannah National 

Wildlife Refuge, U.S. Fish and Wildlife Service, oral 

commun., January 16, 2007). NWR personnel estimated 

about 8,000 ring-necked ducks were using Pond #10 

during sample collection; however, AIV was not detected 

in sample SNWR-07 (table 3).

Santee National Wildlife Refuge

Two samples were collected from the Santee NWR, 

near Santee, South Carolina, one each in May 2006 and 

January 2007 (table 1). Both samples, SANWR-01 and 

SANWR-02, were collected from the Banding Pond, a 

man-made freshwater pond with a loose sediment bottom, maintained by Santee NWR personnel as a waterfowl 

habitat (Lawrence Woodward, Biologist, Santee National 

Wildlife Refuge, U.S. Fish and Wildlife Service, oral commun., May 26, 2006). Sample SANWR-01 was collected 

during May 2006. NWR personnel estimated about 2,000 to 

6,000 ring-necked ducks and green-winged teals had used the 

pond for over-wintering and departed about 6 weeks prior to 

sample collection (table 2). AIV was not detected in sample 

SANWR-01 (table 3).

Sample SANWR-02 was collected during January 2007. 

At the time of sample collection, NWR personnel estimated 

about 6,000 ring-necked ducks and green-winged teals were 

using the pond for over-wintering. AIV H3N8 was detected in 

sample SANWR-02, which was collected when median water 

temperature was 12.5°C, median specific conductance was 

108 μS/cm, median pH was 8.0, and median dissolved oxygen 

was 7.2 mg/L (table 3). 

Pee Dee National Wildlife Refuge

Two sediment samples were collected from the Pee Dee 

NWR, near Wadesboro, North Carolina, one each during 

May 2006 and January 2007 (table 2). Sample PDNWR-01 

was collected from Arrowhead Lake, a man-made freshwater

lake maintained by Pee Dee NWR personnel as a waterfowl habitat (Matthew Garner, Biologist, Pee Dee National 

Wildlife Refuge, U.S. Fish and Wildlife Service, oral commun., May 26, 2006). Sample PDNWR-01 was collected 

during May 2006. NWR personnel estimated about 5,000 to 

10,000 wood ducks, Canada geese, mallard ducks, northern 

pintail ducks, and green-winged teals had used the pond for 

over-wintering and departed about 6 weeks prior to sample collection. AIV was not detected in sample PDNWR-01 (table 3).

Sample PDNWR-02 was collected during January 2007 

from Andrews Pond, a man-made freshwater pond maintained 

by Pee Dee NWR personnel as a waterfowl habitat (Matthew 

Garner, Biologist, Pee Dee National Wildlife Refuge, U.S. 

Fish and Wildlife Service, oral commun., January 17, 2007). 

Sample PDNWR-02 was collected during January 2007. At 

the time of sample collection, NWR personnel estimated about 

5,000 to 10,000 wood ducks, Canada geese, and green-winged 

teals were using the pond for over-wintering. AIV H3N8 

was detected in sample PDNWR-02; which was collected 

when median water temperature was 11.8 °C, median specific 

conductance was 82 (μS/cm), median pH was 7.1, and median 

dissolved oxygen was 7.0 mg/L (table 3). 
10 Occurrence of Viable Avian Influenza Viruses in Water and Bed Sediments from Selected Water Bodies

Mason Neck and Occoquan Bay 

National Wildlife Refuges

Mason Neck and Occoquan Bay National Wildlife 

Refuges near Woodbridge, Virginia, are jointly maintained by 

Mason Neck NWR personnel (Marty McClevey, Biologist, 

Mason Neck National Wildlife Refuge, U.S. Fish and Wildlife 

Service, oral commun., May 25, 2006). One sediment sample 

was collected during May 2006 from the Mason Neck NWR 

(table 2). Sample MNNWR-01 was collected from Little 

Marsh Creek, a managed freshwater impoundment maintained 

by Mason Neck NWR personnel as a waterfowl habitat (Marty 

McClevey, Biologist, Mason Neck National Wildlife Refuge, 

U.S. Fish and Wildlife Service, oral commun., May 24, 2006). 

NWR personnel estimated about 500 to 1,000 wood ducks, 

American black ducks, and hooded mergansers had used the 

impoundment for over-wintering and departed about 6 weeks 

prior to sample collection. AIV was not detected in sample 

MNNWR-01 (table 3).

One sediment sample was collected during May 2006 

from the Occoquan Bay NWR (table 2). Sample ONWR-01 

was collected from an unnamed tidal creek maintained by 

Mason Neck NWR personnel as a waterfowl habitat (Marty 

McClevey, Biologist, Mason Neck National Wildlife Refuge, 

U.S. Fish and Wildlife Service, oral commun., May 24, 2006). 

NWR personnel estimated about 500 to 1,000 wood ducks, 

American black ducks, and hooded mergansers had used the 

creek for over-wintering and departed about 6 weeks prior to 

sample collection. AIV was not detected in sample ONWR-01 

(table 3).

Blackwater National Wildlife Refuge

Three sediment samples were collected during May 

2006 from the Blackwater National Wildlife Refuge, near 

Cambridge, Maryland (table 2). Sample BWNWR-01 was 

collected from Pool 3b, a managed freshwater impoundment 

maintained by Blackwater NWR personnel as a waterfowl 

habitat (Dixie Birch, Biologist, Blackwater National Wildlife Refuge, U.S. Fish and Wildlife Service, oral commun., 

May 16, 2006). NWR personnel estimated about 5,000 to 

10,000 wood ducks, American black ducks, Canada geese, 

mallard ducks, northern pintail ducks, and green-winged teals 

had used the impoundment for over-wintering and departed 

about 6 weeks prior to sample collection. AIV was not 

detected in sample BWNWR-01 (table 3).

Sample, BWNWR-02 was collected from Pool 3a, a 

managed freshwater impoundment maintained by Blackwater 

NWR personnel as a waterfowl habitat (Dixie Birch, Biologist, 

Blackwater National Wildlife Refuge, U.S. Fish and Wildlife Service, oral commun., May 16, 2006). NWR personnel 

estimated about 5,000 to 10,000 wood ducks, American black 

ducks, Canada geese, mallard ducks, northern pintail ducks 

and green-winged teals had used the pond for over-wintering 

and departed about 6 weeks prior to sample collection. AIV 

was not detected in sample BWNWR-02 (table 3).

Sample BWNWR-03 was collected from Pool 5b, a 

managed freshwater impoundment maintained by Blackwater 

NWR personnel as a waterfowl habitat (Dixie Birch, Biologist, 

Blackwater National Wildlife Refuge, U.S. Fish and Wildlife Service, oral commun., May 16, 2006). NWR personnel 

estimated about 5,000 to 10,000 wood ducks, American black 

ducks, Canada geese, mallard ducks, northern pintail ducks, 

and green-winged teals had used the impoundment for overwintering and departed about 6 weeks prior to sample collection. AIV was not detected in sample BWNWR-03 (table 3).

Chincoteague National Wildlife Refuge

Two sediment samples were collected during May 2006 

from the Chincoteague National Wildlife Refuge, near Chincoteague, Virginia (table 2). Sample CNWR-01 was collected 

from Snow Goose Pool, a managed freshwater impoundment 

maintained by Chincoteague NWR personnel as a waterfowl 

habitat (William Hogland, Biologist, Chincoteague National 

Wildlife Refuge, U.S. Fish and Wildlife Service, oral commun., May 25, 2006). NWR personnel estimated about 

5,000 to 10,000 wood ducks, American black ducks, Canada 

geese, mallard ducks, northern pintail ducks, and greenwinged teals had used the impoundment for over-wintering 

and departed about 6 weeks prior to sample collection. AIV 

was not detected in sample CNWR-01 (table 3).

Sample, CNWR-02 was collected from Swans Cove 

Pool, a managed freshwater impoundment maintained 

by Chincoteague NWR personnel as a waterfowl habitat 

(William Hogland, Biologist, Chincoteague National Wildlife Refuge, U.S. Fish and Wildlife Service, oral commun., 

May 25, 2006. NWR personnel estimated about 5,000 to 

10,000 wood ducks, American black ducks, Canada geese, 

mallard ducks, northern pintail ducks, and green-winged teals 

had used the impoundment for over-wintering and departed 

about 6 weeks prior to sample collection. AIV was not 

detected in sample CNWR-02 (table 3).
References 11

Summary 

During February and May 2006, and January 2007, a 

total of 19 water and bed-sediment samples were collected 

from selected water bodies from U.S. Fish and Wildlife 

Service National Wildlife Refuges along the Atlantic Flyway 

in Georgia, South Carolina, North Carolina, Virginia, and 

Maryland. The samples were analyzed for the presence of 

viable avian influenza viruses (AIV), which was detected in 

bed-sediment samples collected from the Savannah National 

Wildlife Refuge (NWR) in Georgia during February 2006 

and from the Santee NWR in South Carolina and the Pee 

Dee NWR in North Carolina during January 2007. AIV was 

not detected in any of the water samples collected for the 

study or in any of the bed-sediment samples collected during 

May 2006. 

These results indicate that the sampling and analytical 

methods used were successful in detecting AIV in the 

environment at three NWRs. The reconnaissance method used 

in this study combined the water column and bed-sediment 

materials into a single composited sample and thus could not 

differentiate the source of the AIV between the two media. 

However, water samples collected concurrently at each of the 

sampling sites were uniformly negative for AIV, suggesting 

that AIV resides in the solid-phase materials. Samplingmethod refinements could be implemented that would allow 

the determination of the relative association of AIV within the 

water column and sediment. Additional data collection would 

provide a better understanding of the occurrence of AIV in 

relation to water-quality conditions, climate conditions, and 

the presence of waterfowl. 

Acknowledgments

The authors would like to acknowledge the help and 

assistance of U.S. Fish and Wildlife Service biologists at the 

NWRs sampled during this study, including Russell Webb 

at the Savannah NWR, Lawrence A. Woodward at the Santee NWR, Matthew Garner at the Pee Dee NWR, Marty 

McClevey at the Mason Neck and Occoquan Bay NWRs, 

Dixie Birch at the Blackwater NWR, and William Hogland 

at the Chincoteague NWR. This study would not have been 

possible without their extensive knowledge of the migratory 

waterfowl in their respective NWRs and their willingness to 

assist the authors.

References 

Halvorson, D.A., Karunakaran, D., Hinshaw, V.S., and 

Newman, J., 1983, Epizootiology of avian influenza—

Simultaneous monitoring of sentinel ducks and turkeys 

in Minnesota: Avian Diseases, v. 27, p. 77–85. 

Hinshaw, V.S., Webster, R.G., and Turner, B., 1979, Waterborne transmission of avian influenza A viruses: Intervirology, v. 11, p. 66–68.

Ip, H.S., and Slota, Paul, 2005, The avian influenza H5N1 

threat, current facts and concerns about highly pathogenic 

avian influenza H5N1: U.S. Geological Survey, Fact 

Sheet 2005–3146, 2 p.

Ito, T., Okazaki, K., Kawaoka, Y., Takada, A., Webster, R.G., 

Kida, H., 1995, Perpetuatuion of influenza viruses in 

Alaskan waterfowl reservoirs: Archives of Virology, v. 140, 

no. 7, p. 1163–1172.

Karasin, A.I., Brown, I.H., Carmen, S., and Olsen, C.W., 2000, 

Isolation and characterization of H4N6 avian influenza 

viruses from pigs with pneumonia in Canada: Journal of 

Virology, Oct. 2000, p. 9322–9327.

Kelleher, C.J., Halvorson, D.A., Newman, J.A., and Senne, 

D.A., 1985, Isolation of avian paramyxoviruses from 

sentinel ducks and turkeys in Minnesota: Avian Diseases, 

v. 29, p. 400–407.

Markwell, D.D., and Shortridge, K.F., 1982, Possible waterborne transmission and maintenance of influenza viruses 

in domestic ducks: American Society for Microbiology, 

Applied and Environmental. Microbiology, January, v. 1, 

p. 110–116.

Sivanandan, V., Halvorson, D.A., Laudert, E., Senne, D.A., 

and Kumar, M.C., 1991, Isolation of H13N2 influenza A 

virus from turkeys and surface water: Avian Diseases, v. 35, 

p. 974–977.

Spackman, E., Senne, D.A., Myers, T.J., Bulaga, L.L., 

Garber, L.P., Perdue, M.I., Lohman, K., Daum, L.T., and 

Suarez, D.L., 2002, Development of a real-time reverse 

transcriptase PCR assay for influenza virus and the avian 

H5 and H7 hemagglutinin subtypes: Journal of Clinical 

Microvirology, v. 40, p. 3256–3260.
12 Occurrence of Viable Avian Influenza Viruses in Water and Bed Sediments from Selected Water Bodies

Stallknecht, D.E., Shane, S.M., Kearney, M.T., and Zwank, 

P.J., 1990, Persistence of avian influenza viruses in water: 

Avian Diseases, v. 34, p. 406–411.

U.S. Department of Health and Human Services, 2008, Pandemic flu, general information; accessed March 5, 2008, at 

http://www.pandemicflu.gov/general/.

U.S. Interagency Strategic Plan, 2006, An early detection 

system for highly pathogenic H5N1 avian influenza in 

wild migratory birds; accessed November 29, 2007, at 

http://www.nwhc.usgs.gov/publications/other/Final_

Wild_Bird_Strategic_Plan_0322.pdf.

Webster, R.G., Yakhno, M., Hinshaw, V.S., Bean, W.J., Jr., 

and Mutri, K.G., 1978, Intestinal influenza, replication and 

characterization of influenza viruses in ducks: Virology, 

v. 84, p. 268–278.

 World Health Organization, 2003, Influenza, Report of the 

Secretariat: March 13, 2003, A556/23, Fifty-sixth World 

Health Assembly.

 World Health Organization, 2006, Review of the latest available evidence on risks to human health through potential 

transmission of avian influenza (H5N1) through water and 

sewage: Geneva, Water, Sanitation and Health Protection of 

the Human Environment, March 24, 2006, p. 15. 

Zhang, G., Shoham, D., Gilichinsky, D., Davydov, S., 

Castello, J.D., and Rogers, S.O., 2006, Evidence of avian 

influenza A virus in Siberian lake ice: Journal of Virology, 

v. 8, no. 24, p. 12229–12235. 

Manuscript approved for publication, July 31, 2009

Graphics by Bonnie J. Turcott

Layout by Caryl J. Wipperfurth

For more information concerning the research in this report, contact:

USGS Georgia Water Science Center

3039 Amwiler Road

Atlanta, Georgia 30360

telephone: 770-903-9100

http://ga.water.usgs.gov