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Contents

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

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

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

Enteric Viruses and Microbiological Indicators of Fecal Contamination ................................... 3

Previous Studies .................................................................................................................................. 4

Enteric Viruses in Ground-Water Supplies ............................................................................ 4

Factors that Affect the Presence of Enteric Viruses in Ground-Water Supplies ............ 6

Indicators of Fecal Contamination in the Subsurface .......................................................... 7

Environmental setting ......................................................................................................................... 7

Methods of Study .......................................................................................................................................... 9

Sampling procedures .......................................................................................................................... 9

Microbiological analyses ................................................................................................................. 10

Enteric Virus Analysis by RT-PCR .......................................................................................... 10

Enteric Virus Analysis by Cell Culture ................................................................................... 11

Indicator Bacteria ..................................................................................................................... 11

Coliphage ................................................................................................................................... 11

Ancillary Environmental and Water-Quality Information ............................................................ 11

Quality Assurance and Quality Control .......................................................................................... 12

Enteric Viruses and Microbiological Indicators of Fecal Contamination in Small Public

 Ground-Water Supplies .............................................................................................................. 12

Quality-Control Considerations for Enteric Viruses by RT-PCR ................................................. 14

Occurrence of Enteric Viruses and Microbiological Indicators of Fecal Contamination ...... 15

Microbiological Indicators of Fecal Contamination as Predictors of the Presence of

 Enteric Viruses ...................................................................................................................... 16

Environmental Factors and Chemical-Quality Constituents Related to the Presence of 

 Indicators and Enteric Viruses ................................................................................................... 17

Summary, Conclusions, and Suggestions for Future Study ................................................................. 23

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

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

Figures

 1. Map showing well location and surficial geology of the study area ................................... 8 

 2. Example of hybridization results from reverse transcriptase-polymerase chain

 reaction (RT-PCR) analysis for enteric viruses ............................................................. 13

 3. Graph showing chloride concentrations in relation to chloride to bromide (Cl:Br)

 ratios for small-public-supply wells sampled July 1999 through July 2001:

 Distribution of wells by map number, and at least one detection of total

 coliforms, enterococci, Escherichia coli, and (or) coliphage, and enteric

 viruses .................................................................................................................................. 21
iv

Tables

 1. Summary of literature on the occurrence of enteric viruses, bacterial indicators, 

 and coliphage in ground-water-supply wells ......................................................................... 5

 2. Results of enteric virus analysis by reverse transcriptase-polymerase chain 

 reaction (RT-PCR) of regular and replicate spike samples collected from small-

 public-supply wells in southeastern Michigan, July 1999 through July 2001 .................. 14

 3. Detections of enteric virus by reverse transcriptase-polymerase chain reaction 

 (RT-PCR), culturable viruses, and microbiological indicators of fecal 

 contamination from small-public-supply wells in southeastern Michigan, 

 July 1999 through July 2001 ..................................................................................................... 15

 4. Small-public-supply wells in southeastern Michigan with detections of 

 enteric viruses, July 1999 through July 2001, and detections of 

 microbiological indicators from the same well .................................................................... 16

 5. Predictive accuracy of microbiological indicators for virus occurrence in the 

 same small-public-supply well in southeastern Michigan, July 1999 through 

 July 2001 ...................................................................................................................................... 17

 6. Rankings of environmental and chemical-quality categorical variables by risk 

 of fecal contamination and relations to detections of microorganisms in small-

 public-supply wells in southeastern Michigan, July 1999 through July 2001 .................. 18

 7. Relations between detections of microorganisms and continuous environmental 

 and chemical-quality variables of small-public-supply wells in southeastern 

 Michigan, July 1999 through July 2001 .................................................................................. 20

 8. Characteristics of small-public-supply wells in southeastern Michigan 

 sampled from July 1999 through July 2001 ............................................................................ 29

 9. Oligonucleotide primers and probes used for virus detection in the reverse 

 transcriptase-polymerase chain reaction (RT-PCR) method ............................................. 35

 10. Quality-control samples for reverse transcriptase-polymerase chain reaction 

 (RT-PCR) analysis ...................................................................................................................... 36

 11. Field water-quality measurements and microbiological data for small-public-

 supply wells sampled July 1999 through July 2001 .............................................................. 37

 12. Chemical-quality data for small-public-supply wells in southeastern Michigan 

 sampled from July 1999 through July 2001 ............................................................................ 51
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YGTGCPCN[\GFHQTEQNKRJCIGD[VJG5#.OGVJQFCPFUCORNGUEnteric Viruses and Microbiological Indicators 13

Figure 2. Example of hybridization results from reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for enteric 

viruses.Columns 1 and 2 show results for enterovirus; 3 and 4 for reovirus; 5 and 6 for rotavirus; 7 and 8 for hepatitis A virus; 9 and 10 for 

Norwalk virus.) 

, ~'O 

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8 

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f € 

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H ' \\ 

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1 2 3 4 5 6 7 8 9 10 

Negative Matrix spike Negative Matrix spike Negative Matrix spike Negative Matrix spike Negative Matrix spike 

A process 29 process 29 process 29 process 29 process 29 control control control control control 

Sample 28 Matrix spike Sample 28 Matrix spike Sample 28 Matrix spike Sample 28 Matrix spike Sample 28 Matrix spike 

B 30 30 30 30 30 

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C 36 36 36 36 36 

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D 37 37 37 37 37 

Sample 36 peR negative Sample 36 peR negative Sample 36 peR negative Sample 36 peR negative Sample 36 peR negative 

E control control control control control 

Hybridization Hybridization Hybridization Hybridization Hybridization 

F Sample 37 negative Sample 37 negative Sample 37 negative Sample 37 negative Sample 37 negative 

control control control control control 

Seeded peR positive Seeded peR positive Seeded peR positive Seeded peR positive Seeded peR positive 

G process control process control process control process control process control control control control control control 

Matrix spike --- Matrix spike - Matrix spike 

H 28 28 28 --- Matrix spike 

28 - Matrix spike 28 ---
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Environmental Factors and Chemical-Quality Contituents 21

B

Maximum total coliform concentration 81

No detection of total coliforms

Cl:Br = 400

23

5

18

35

32

22

24

1

16

20

29

14

17

25

34

12

19

28

11 8

37

36

7

33 21

30

3

9

13

10

6

38

Sewage + halite

100

1,000

10,000

Background GW

GW + sewage

GW + halite

 GW+brine upper limit

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Sewage

10

1 10 100 1,000

100

1,000

10,000

Background GW

GW + sewage

Sewage + halite

CHLORIDE CONCENTRATION, IN MILLIGRAMS PER LITER

GW + halite

Sewage

10

 GW+brine upper limit

 GW+brine lower limit

CHLORIDE TO BROMIDE RATIO

18

5

17

1

250

4

1

81

1

CHLORIDE TO BROMIDE RATIO

1 Map identifier

Figure 3. Chloride concentrations in relation to chloride to bromide (Cl:Br) ratios for small-public- supply wells sampled 

July 1999 through July 2001: A, Distribution of wells by map number, and at least one detection of B, total coliforms, 

C, enterococci, Escherichia coli, and (or) coliphage, and D, enteric viruses. (Halite end-member from Knuth and others, 

1990; domestic sewage end-member from Davis and others, 1998, and Peavy, 1978; sewage has a Cl:Br ratio of 

30–600 and chloride concentration of 37–101 mg/L (Peavy, 1978, as cited in Thomas, 2000); brine end-members from 

Eberts and others, 1990; a Cl:Br ratio of 400 is the theoretical maximum for natural water (Thomas, 2000).)

A

Cl:Br = 400
22 Enteric Viruses in Ground Water from Small Public Water Supplies in Southeastern Michigan

1 10 100 1,000

Sewage + halite

100

1,000

10,000

Background GW

GW + sewage

GW + halite

 GW+brine upperlimit

 GW+brine lower limit

Sewage

10

Enteric virus detection

D

C

100

1,000

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Sewage + halite

GW + halite

10

 GW+brine upper limit

 GW+brine lower limit

1 2

6

88

1

1

9

Sewage

Enterococci concentration

1 Escherichia coli concentration

1

No detection of Escherichia coli, enterococci or coliphage

Coliphage detected

CHLORIDE CONCENTRATION, IN MILLIGRAMS PER LITER

CHLORIDE TO BROMIDE RATIO

Cl:Br = 400

Cl:Br = 400

CHLORIDE TO BROMIDE RATIO

Figure 3. Chloride concentrations in relation to chloride to bromide (Cl:Br) ratios for small-public- supply wells sampled 

July 1999 through July 2001: A, Distribution of wells by map number, and at least one detection of B, total coliforms, 

C, enterococci, Escherichia coli, and (or) coliphage, and D, enteric viruses. (Halite end-member from Knuth and others, 

1990; domestic sewage end-member from Davis and others, 1998, and Peavy, 1978; sewage has a Cl:Br ratio of 

30–600 and chloride concentration of 37–101 mg/L (Peavy, 1978, as cited in Thomas, 2000); brine end-members from 

Eberts and others, 1990; a Cl:Br ratio of 400 is the theoretical maximum for natural water (Thomas, 2000).) – Continued.
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Donna S. Francy, Rebecca N. Bushon, Julie Stopar, Emma J. luzano, and G. Shay Fout-Enteric Viruses in Ground Water From Small Public Water Supplies in 

Southeastern Michigan Scientific Investigations Report 2004-5219 

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