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Title:
The development of a human polyomavirus quantitative pcr assay to assess viral persistence, water quality, and human health risks
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English
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McQuaig, Shannon
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Real-time PCR
Human-associated
Indicator
Microbial source tracking
Environmental effects
Dissertations, Academic -- Biology -- Doctoral -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Microbial water quality is generally assessed using fecal indictor organisms; however host-specific microbial source tracking (MST) methodologies can be employed to differentiate sources of fecal pollution. The central goal of this research was to develop and validate a QPCR assay for the quantification of two human-specific polyomaviruses (HPyVs) in environmental water samples. These viruses are prevalent worldwide and produce lifelong, asymptomatic viruria in immunocompetent individuals. A Taqman quantitative PCR (QPCR) assay based on the conserved T-antigen of two HPyVs (JCV and BKV) was developed and optimized (Chapter 2). HPyVs were detected in a high proportion of human-associated waste samples (e.g. sewage) and were not detected in animal excrement samples (Chapter 2). The effects of ultraviolet radiation, temperature, and salinity on the persistence of HPyVs in water were reported in Chapter 3. Laboratory studies analyzing the effects of various UV doses, temperatures, and/or salinities demonstrated high doses of UV were required to significantly decrease the detection of HPyVs DNA and salinity stabilized pure cultures of HPyVs virus particles at high temperatures (25 and 35 degrees Celsius). Solar radiation as well as potential predation from microorganisms in sewage significantly reduced the persistence of HPyVs DNA in outdoor mesocosm studies (Chapter 3). An improved method to extract human polyomavirus (HPyVs) DNA from environmental water samples was developed, and the recoveries were larger and more consistent over a range of DNA concentrations as compared to the standard protocol (Chapter 4). In the California beaches study (Chapter 4), the presence of HPyVs by either QPCR or PCR had a high degree of matching results with the adenoviruses (83-91 percent), Methanobrevibacter smithii marker (82-92 percent) and moderate degree of matching results with the human-associated Bacteroidales spp. marker (57-80 percent) (Chapter 4). HPyVs were detected in the presence of various pathogens including: Giardia spp., Cryptosporidium spp., Vibrio spp., enteroviruses, and noroviruses (Chapter 5). The presence of HPyVs in relatively high concentrations of sewage and the specificity of HPyVs combined with the relatively conservative persistence of HPyVs when exposed to environmental conditions and the correlation of HPyVs with pathogens demonstrates that this assay is a useful MST method to detect human sewage.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2009.
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by Shannon McQuaig.
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Includes vita.

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The Development of a Human Polyomavirus Quantitative PCR Assay to Assess Viral Persistence, Water Quality, and Human Health Risks by Shannon M. McQuaig A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Integrated Biology College of Arts and Sciences University of South Florida Major Professor: Valerie J. Harwood, Ph.D. John Lisle, Ph.D. K.T. Scott, Ph.D. Degeng Wang, Ph.D. Date of Approval: November 6, 2009 Keywords: real-time PCR, human-associated, indicator, microbial source tracking, environmental effects Copyright 2009, Shannon M. McQuaig

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To the ones that have been there the entire way, without falter: My Mom, My Dad, and My Uncle Jimmy

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ACKNOWLEDGEMENTS I will first like to thank my adviso r Dr. Valerie Harwood for her guidance throughout the past four years. I also acknowledge my supervisory committee members, Dr. Wang, Dr. Lisle and Dr. Scott. I woul d like to acknowledge my fellow laboratory associates, Asja, Bina, Brian, Ch ris, Katrina, Miriam, and Zach. In addition, I would like to thank the entire Departme nt of Integrated Biology. I must thank Dr. Farrah and Dr. Troy Scott for their continued support and encouragement. I also must thank Dr. John Paul as well as his enti re laboratory; Lauren, Brian, Jenny, Beth, and Dave. Without their hospitality I would not have been able to complete my research in such a timely ma nner. I also acknowledge my friends Kindra Kelly, Rene Nortman, and Lyann Balert, as we ll as my pseudo-family Josephine and Ed Mattson and Michelle, Ryan and Morgan Golin ski who were always willing to lend an ear and provide much needed comic relief. I will forever be indebted to Robert (B obby) Ulrich for his patience, never ending encouragement, and the countless porch nights over the past three years. In times of doubt he provided relentless undue support and more importantly he made me laugh when I thought else nothing could. Lastl y, I thank my parents, Kathy and Gary McQuaig, and my uncle, James McQuaig (Uncle Jimmy). I owe them much appreciation and gratitude because collectiv ely they gave me a great childhood, great love, and the foundation needed to pursue this epic venture.

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TABLE OF CONTENTS LIST OF TABLES ............................................................................................................... vii LIST OF FIGURES ................................................................................................................x ABSTRACT ...................................................................................................................... .... xi BACKGROUND ....................................................................................................................1 Bacterial Waterborne Pathogens .................................................................................2 Campylobacter ................................................................................................2 Escherichia coli ...............................................................................................2 Leptospira species ...........................................................................................3 Plesiomonas shigelloides ................................................................................3 Salmonella species ..........................................................................................4 Shigella species ...............................................................................................5 Vibrio species..................................................................................................5 Viral Waterborne Pathogens .......................................................................................6 Adenovirus ......................................................................................................6 Astrovirus ........................................................................................................7 Enterovirus ......................................................................................................7 Hepatitis A virus .............................................................................................8 Hepatitis E virus ..............................................................................................9 Norovirus ........................................................................................................9 Rotavirus .........................................................................................................9 Protozoan Waterborne Pathogens .............................................................................10 Cryptosporidium parvum ..............................................................................10 Entamoeba histolytica ...................................................................................11 Giardia species ..............................................................................................11 Miscellaneous Waterborne Pathogen ........................................................................12 Schistosoma species ......................................................................................12 Surveillance of Illness Outbreaks Associated with Recreational Water Use ...........13 History of Traditional Water Qua lity Standards and Indicators ...............................15 Florida Water Quality Standards ..............................................................................17 Total Maximum Daily Loads (TMDLs) ...................................................................18 i

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Traditional Microbial Wa ter Quality Indicators .......................................................20 Overview of Microbial Source Tracking ..................................................................21 Phenotypic Library-De pendent Methods ..................................................................24 Antibiotic resistance profiles ........................................................................24 E. coli serotyping ..........................................................................................25 Phenotypic Library-Independent Method .................................................................26 Bifidobacteria ................................................................................................26 Genotypic Library-Dependent Methods ...................................................................27 Pulsed field gel electrophoresis (PFGE) .......................................................27 Repetitive element PCR (rep-PCR) ..............................................................28 Ribotyping.....................................................................................................28 Genotypic Library-Independent Methods .................................................................29 F-specific coliphage genotyping ...................................................................29 Enterococcal surface protein gene ................................................................30 Bacteroidales marker ....................................................................................31 Methanobrevibacter smithii ..........................................................................31 Human enteric viruses...................................................................................32 Study Rationale .........................................................................................................33 Polyomaviruses .........................................................................................................34 Human polyomaviruses ............................................................................................34 JC virus .........................................................................................................35 BK virus ........................................................................................................36 Objectives of Study ...................................................................................................38 QUANTIFICATION OF HUMAN POLYOM AVIRUSES, JCV AND BKV, BY TAQMAN QUANTITATIVE PCR AND COMPARISON TO OTHER WATER QUALITY INDICATORS IN WATER AND FECAL SAMPLES ................55 ABSTRACT ..............................................................................................................56 INTRODUCTION ....................................................................................................57 MATERIALS AND METHODS ..............................................................................59 Virus strains and viral DNA extraction ........................................................59 Primer and probe design ...............................................................................60 Quantitative PCR ..........................................................................................60 Sequencing of QPCR amplicon ....................................................................61 Standard curve and sensitivity of QPCR ......................................................62 Specificity of HPyVs QPCR .........................................................................62 ii

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Human target samples ...................................................................................64 Analysis of water quality indica tors in sewage over a 28-day period (sewage holding time experiment) ...........................................65 Environmental water samples .......................................................................66 Comparison of HPyVs QPCR to other methods ...........................................67 Enumeration of indicator bacteria .................................................................67 Detection of human-asso ciated Bacteroidales ..............................................67 Detection of the human-associated M. smithii .............................................68 Adenovirus nested PCR ................................................................................68 Statistical analysis .........................................................................................69 RESULTS .................................................................................................................70 Sensitivity of QPCR and calculation of standard curves ..............................70 Specificity of HPyVs QPCR and human-associated markers .......................70 HPyVs QPCR analysis of human waste samples .........................................71 Analysis of other water quality i ndicators in human waste samples ............71 Correlations among bacterial indica tors and HPyVs concentrations in human waste samples ..........................................................................72 Analysis of water quality indica tors in sewage over a 28-day period ......................................................................................................73 Environmental water samples .......................................................................74 Sequencing analysis of QPCR amplicons .....................................................75 DISCUSSION ...........................................................................................................84 ACKNOWLEDGEMENTS ......................................................................................88 THE EFFECTS OF TEMPERATURE, SALINITY, AND ULTRAVIOLET RADIATION ON THE PERSISTENCE OF HUMAN POLYOMAVIRUSES AND OTHER WATER QUALIT Y INDICATORS ......................................................89 ABSTRACT ..............................................................................................................90 INTRODUCTION ....................................................................................................91 MATERIALS AND METHODS ..............................................................................94 Virus strains and viral DNA extraction ........................................................94 Positive PCR controls ...................................................................................94 Human polyomavirus QPCR ........................................................................95 Human polyomavirus QPCR standard curve ................................................95 Effects of ultraviolet radiation x salinity on the detection of BK virus DNA by QPCR: laboratory expe riment with BK viruses ..............96 iii

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Effects of salinity x temperature on the persistence of BK viruses: laboratory experiment with BK viruses ..................................................97 Effects of salinity x natural sun light on the persistence of BK viruses: natural sunlight experiment with BK viruses ............................97 Effects of salinity x natural sunlig ht on the persistence of HPyVs, culturable bacteria, and other wa ter quality indicators: natural sunlight experiment with sewage ............................................................98 Enumeration of culturable indicator organisms ............................................99 Concentration of MST indicators ................................................................100 DNA extraction ...........................................................................................100 Detection of human-asso ciated Bacteroidales ............................................101 Detection of the human-associated M. smithii ...........................................102 Statistical analysis .......................................................................................103 RESULTS ...............................................................................................................104 Effects of ultraviolet radiation x salinity on the detection of BK virus DNA by QPCR: laboratory expe riment with BK viruses ............104 Effects of salinity x temperature on the persistence of BK viruses: laboratory experiment with BK viruses ................................................106 Physical parameters for all na tural sunlight experiments ...........................107 Effects of salinity x natural sun light on the persistence of BK viruses: natural sunlight expe riment with inoculated BK viruses ...................................................................................................107 Effects of salinity x natural sun light on the persistence of HPyVs persistence: natural sunlight experiment with sewage ..........................108 Effects of salinity x natural su nlight on the persistence of culturable fecal coliforms: natura l sunlight experiment with sewage ...................................................................................................109 Effects of salinity x natural sunlight on the persistence of E. coli: natural sunlight experiment with sewage ..............................................110 Effects of salinity x natural su nlight on the persistence of enterococci persistence: natura l sunlight experiment with sewage ...................................................................................................111 Effects of salinity x natural sun light on the persistence of humanassociated Bacteroidales marker persistence: natural sunlight experiment with sewage ........................................................................111 Effects of salinity x natural sun light on the persistence of M. smithii marker persistence: natu ral sunlight experiment with sewage ...................................................................................................112 iv

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Effects of salinity x natural sunlig ht on the persistence of HPyVs, culturable bacteria, and othe r water quality indicators: relationships among indicators and markers .........................................113 ACKNOWLEDGEMENTS ....................................................................................132 A TOOLBOX APPROACH TO A SSESS WATER QUALITY AT TWO CALIFORNIA BEACHES: A CASE STUDY AT DOHENY AND AVALON BEACHES ....................................................................................................................133 ABSTRACT ............................................................................................................134 INTRODUCTION ..................................................................................................135 MATERIALS AND METHODS ............................................................................139 Construction of recombinant plasmid for HPyVs control and QPCR standard curve ............................................................................139 Positive PCR controls .................................................................................139 Negative controls ........................................................................................140 Comparison of DNA ex traction protocols ..................................................140 Standard MO BIO PowerSoil Kit Protocol .................................................141 MO BIO/Qiagen Hybrid (MQH) Protocol ..............................................141 Human polyomavirus QPCR ......................................................................142 Human polyomavirus QPCR standard curve ..............................................143 Comparison of viral DNA recovery from water concentrates containing BK viruses ...........................................................................143 Comparison of viral DNA recovery from water concentrates containing raw sewage ..........................................................................144 Doheny Beach sites and sampling schedule ...............................................144 Avalon Beach sites and sampling schedule ................................................144 Sample collection ........................................................................................145 Enumeration of culturable indicator organisms ..........................................145 Concentration of MST indicators ................................................................145 Detection of human-asso ciated Bacteroidales ............................................146 Detection of the human-associated M. smithii ...........................................146 Adenovirus nested PCR ..............................................................................147 Detection of human polyomaviruses ..........................................................147 Quantification of human polyomaviruses in beach samples .......................148 Statistical analysis .......................................................................................148 RESULTS ...............................................................................................................149 Positive and negative controls ....................................................................149 v

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Comparison of DNA ex traction protocols ..................................................149 Comparison of viral DNA recovery from water concentrates containing BK viruses ...........................................................................150 Comparison of viral DNA recovery from water concentrates containing raw sewage ..........................................................................150 Bacterial water quality indicator concentrations at Doheny Beach sites .......................................................................................................151 Correlations of bacterial indica tors among Doheny Beach sites ................152 QPCR detection of HPyVs at Doheny Beach sites .....................................152 PCR detection of human-associat ed water quality indicators at Doheny Beach sites ...............................................................................153 Relationships among indicators a nd markers at Doheny Beach .................153 Bacterial water quality indicator concentrations at Avalon Beach sites .......................................................................................................155 Correlations of bacterial indica tors among Avalon Beach sites .................155 QPCR detection of HPyVs at Avalon Beach sites ......................................156 PCR detection of human-associat ed water quality indicators at Avalon Beach sites ................................................................................156 Relationships among indicators and markers at Avalon Beach ..................156 Comparison of Doheny and Aval on indicators and markers ......................157 DISCUSSION .........................................................................................................167 ACKNOWLEDGEMENTS ....................................................................................173 FINAL CONCLUSIONS AN D SUMMARY OF COLLAB ORATIVE STUDIES ..........174 ABOUT THE AUTHOR .......................................................................................... End Page vi

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LIST OF TABLES Table 1. Bacterial and prot ozoan associated disease outbreaks after exposure to recreational water bodies in th e United States from 1991-2006. .........................40 Table 2. Viral associated disease outb reaks after exposure to recreational water bodies in the United States and Mexico from 1991-2006. ...................................44 Table 3. Disease associated with un identified pathogens after exposure to recreational water bodies in th e United States from 1991-2006. .........................46 Table 4. The number of reported cases of Vibrio spp. associated disease after exposure to recreational water bodies in the United States from 20032006. .....................................................................................................................48 Table 5. Summary of contamination sources in recreational water outbreaks in the United States from 1971-2000. .......................................................................49 Table 6: United States Environm ental Protection Agency recommended regulatory standards for E. coli and enterococci in surface waters. .....................50 Table 7. Current Florida regulatory standa rds for indicator bacteria set by Florida Administrative Code 62-302.530 with modifications to meet EPAs BEACH Water Program requirements. ................................................................51 Table 8. An overview of the types of microbial source tracking methods. ........................52 Table 9. The observed geographical di stribution of JC virus genotypes. ...........................54 Table 10. Primers and probe sequences used in the McQuaig et al. 2009 study. ................77 Table 11. Results of QPCR assays for known viral numbers (100, 10, 1 or 0.1 particles) and detection limits determ ined for BK virus and JC virus. ................78 Table 12. Result of human-associated markers PCR assays on non-target and target samples. ......................................................................................................79 Table 13. Concentrations of human polyomaviruses (HPyVs), E. coli, enterococci and fecal coliforms in human waste samples. ......................................................80 Table 14. Correlation of HPyVs and indi cator bacteria in human sewage. .........................81 Table 15. Physical and microbial data for all environmental samples collected in McQuaig et al. 2009 study. ..................................................................................82 vii

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Table 16. The log decrease of BK viruse s with exposure to various ultraviolet radiation doses. ...................................................................................................115 Table 17. The decay rates of BK virus copy numbers over a seven day period for the effects of temperature x salinity. ..................................................................117 Table 18. The physical data recorded ove r three natural sunlight experiment trials. ...................................................................................................................118 Table 19. The decay rates of BK virus copy numbers, human polyomavirus copy numbers, culturable fecal coliforms, culturable E. coli and culturable enterococci over a seven day period for the natural sunlight experiment. .........124 Table 20. The persistence of human-associated Bacteroidales spp. and Methanobrevibacter smithii marker detection for the natural sunlight experiments. .......................................................................................................125 Table 21. The relationships between cultura ble bacterial indicator concentrations, HPyVs concentrations by QPCR an d other human-associated markers of fecal pollution (+ or by PCR) over a seven day period analyzed using binary logist ic regression. .........................................................................126 Table 22. Primers and probe sequences used in the California beaches study. .................158 Table 23. Comparison of the DNA extraction efficiency of the standard MO BIO protocol and the improved MO BI O/Qiagen (MQH) protocol using plasmids pipetted directly into bead tubes .........................................................159 Table 24. Comparison of the standard MO BIO protocol and the improved MO BIO/Qiagen (MQH) protocol viral DNA recovery from water samples (500 ml) inoculated with different volumes of BK virus dilutions. ...................160 Table 25. Comparison of the standard MO BIO protocol and the improved MO BIO/Qiagen (MQH) protocol viral DNA recovery from water samples inoculated with different volumes of raw sewage. .............................................161 Table 26. The co-occurrence of human-a ssociated markers in the Doheny and Avalon Beach samples. ......................................................................................162 Table 27. Positive and negative results for human polyomaviruses and pathogens in the 2007 Abdelzaher et al. (subm itted for publication) collaborative study at the Hobie Cat Beach (Miami FL) study with collaborators at the University of Miami. ....................................................................................182 viii

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Table 28. Results for indicator bacteria, human polyomaviruses, and pathogens in the 2007 Southern California Coastal Water Research Project (SCCWRP) collaborative study in Costa Mesa, California with collaborators including the laborator ies of Drs. Stewart, Fuhrman and Sobsey. ...............................................................................................................183 Table 29. Correlations of HPyVs with indicator bacteria and various pathogens in the 2007 Southern California Coastal Water Research Project (SCCWRP) collaborative study in Costa Mesa, California. ..............................185 Table 30. Reproduced from Report of Epidemiological Analyses: Doheny Beach, 2007 PRELIMINARY RESULTS ......................................................186 Table 31. Reproduced from Report of Epidemiological Analyses: Avalon Beach, 2007 PRELIMINARY RESULTS ......................................................187 Table 32. HPyVs concentrations in DNA from homogenized oyster samples. .................188 ix

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LIST OF FIGURES Figure 1. Concentration of indicator bacteria and human polyomaviruses (HPyVs) in sewage coupled with the PCR detection of humanassociated Bacteroidales M. smithii and adenovirus over a 28 day period. ...................................................................................................................83 Figure 2. Persistence of BK virus copy nu mbers in sterile dechlorinated tap water samples for the temperature x sali nity laboratory experiments. .........................116 Figure 3. Persistence of BK virus copy nu mbers in sterile dechlorinated tap water samples for the natural s unlight experiments. ....................................................119 Figure 4. Persistence of human polyoma virus (HPyVs) copy numbers in sewage inoculated water samples for the natural sunlight experiments. ........................120 Figure 5. Persistence of culturable fecal coliforms in sewage inoculated water samples for the natural s unlight experiments. ....................................................121 Figure 6. Persiste nce of culturable E. coli in water samples inoculated with sewage for the natural su nlight experiments. .....................................................122 Figure 7. Persistence of culturable enterococci in water samples inoculated with sewage for the natural su nlight experiments. .....................................................123 Figure 8. Sites of sample collecti on for the California beaches study. ..............................164 Figure 9. The average log10-transformed concentration of enterococci, fecal coliforms, total coliforms, and human polyomaviruses (HPyVs) at the (A) Doheny Beach sites and (B) Avalon Beach sites. ........................................165 Figure 10. Frequency of detection of human-associated MST markers and adenoviruses at the (A) Doheny B each sites and (B) Avalon Beach sites. ....................................................................................................................166 x

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The Development of a Quantitative PCR for th e Detection of Human Polyomaviruses in Water Shannon M. McQuaig ABSTRACT Microbial water quality is generally assessed using fecal indictor organisms; however host-specific microbi al source tracking (MST) meth odologies can be employed to differentiate sources of fecal pollution. The central goal of this research was to develop and validate a QPCR assay for th e quantification of two human-specific polyomaviruses (HPyVs) in environmental water samples. These viruses are prevalent worldwide and produce lifelong, asymptomatic vi ruria in immunocompetent individuals. A Taqman quantitative PCR (QPCR) assay based on the conserved T-antigen of two HPyVs (JCV and BKV) was developed a nd optimized (Chapter 2). HPyVs were detected in a high proportion of human-associ ated waste samples (e.g. sewage) and were not detected in animal excrement samples (Chapter 2). The effects of ultraviolet radiation, temperature, and salin ity on the persistence of HPyV s in water were reported in Chapter 3. Laboratory studies analyzing the effects of va rious UV doses, temperatures, and/or salinities demonstrated high doses of UV were required to significantly decrease the detection of HPyVs DNA and salinity st abilized pure cultures of HPyVs virus particles at high temperatures (25C and 35 C). Solar radiation as well as potential xi

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xii predation from microorganisms in sewage significantly reduced the persistence of HPyVs DNA in outdoor mesocosm studies (Chapter 3). An improved method to extract hum an polyomavirus (HPyVs) DNA from environmental water samples was developed, a nd the recoveries were larger and more consistent over a range of DN A concentrations as compared to the standard protocol (Chapter 4). In the California beaches study (Chapter 4), the presence of HPyVs by either QPCR or PCR had a high degree of matching results with the adenoviruses (8391%), Methanobrevibac ter smithii marker (82-92%) and modera te degree of matching results with the human-associated Bacteroidales spp. marker (57-80%) (Chapter 4). HPyVs were detected in the presence of various pathogens including: Giardia spp., Cryptosporidium spp., Vibrio spp., enteroviruses, and noroviruses (Chapter 5). The presence of HPyVs in relatively high con centrations of sewage and the specificity of HPyVs combined with the relatively conserva tive persistence of HPyVs when exposed to environmental conditions and the correlation of HPyVs with pathogens demonstrates that this assay is a useful MST method to detect human sewage.

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BACKGROUND Polluted water bodies can have devastati ng effects on ecosystems, as well as on businesses that are dependent on water systems (e.g. fishing industries and beach-related tourism). Therefore, maintaining healthy environmental water bodies is essential for aquatic conservation efforts, economy of coastal communities, and in some instances human health. Water systems can become th reatened when pollutants found in storm water runoff, agricultural runoff, or failing sewage handling systems enter the water. These sources of contamination can lead to an influx of excess nutri ents and subsequent eutrophic conditions in lakes, rivers, and coastal waters; which can have detrimental effects on aquatic plants and wildlife (1, 16, 102, 126, 137, 307). The introduction of anthropogenic contaminants into water sy stems may not only cau se eutrophication, but can also pose serious human health risks fo r shellfish consumers, swimmers, and other recreational water users (275). Various human pathogens can be shed in human or animal excrement and transmitted through contaminated water or sh ellfish. Microorganisms that enter the gastrointestinal tract and subs equently establish infection ar e termed enteric pathogens. In general, enteric pathogen infections ar e the result of poor sanitary conditions or consumption of contaminated food or water. Clinical symptoms associated with enteric pathogen infections can be intestinal (e .g. abdominal cramps, gastroenteritis) or extraintestinal symptoms (e.g. fever, head ache, jaundice). In addition to enteric pathogens, zoonotic pathogens can also be tr ansmitted through poor sanitary conditions 1

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or consumption of contaminated food or wate r. Human infections caused by waterborne pathogens can cause mild to serious illnesses, and in some cases may even be fatal. The following is a brief overview of the characte ristics of common water-borne pathogens. Bacterial Waterborne Pathogens Campylobacter The genus Campylobacter belongs to the family Campylobacteraceae and consists of motile, curved, gram-negative bacill i, with a polar flagellum (218). Currently, there are 17 recognized species of Campylobacter (253). Campylobacter spp. can infect poultry, livestock, domestic animals and humans. Campylobacter spp. (primarily C. jejuni and C. coli ) are the causative agents of campylobacterosis. The clinical symptoms of this disease are normally abdominal cr amps, fever, and gastroenteritis (218). Campylobacter infections are the most common cause of bacterial gastroenteritis in the United States. The Centers for Disease C ontrol (CDC) estimates that there are 2.4 million cases of campylobacteriosis every year (61, 206). Escherichia coli Escherichia coli belongs to the family Enterobacteriaceae This facultative anaerobic, gram-negative bacillus is a part of the intestinal flora of both humans and other warm-blooded animals (218), and some cold-blooded animals (84, 130). In general, E. coli are nonpathogenic; however some strains of E. coli have acquired genes for virulence factors such as toxins and/or adhesion molecules. These strains include enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC) (309, 314). These organisms can cause sympto ms ranging from mild diarrhea to kidney 2

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failure. The most notorious strain of E. coli is the verotoxin-producing EHEC strain 0157:H7. Ingestion of less than 100 bacil li can cause infection with severe bloody diarrhea and abdominal cramps (218). Imm unocompromised individuals infected with E. coli 0157:H7 often develop hemolytic uremic s yndrome and have a high mortality rate (351). The CDC estimates 70,000 cases of E. coli 0157:H7 per year in the Unites States (62). Leptospira species Leptospira spp. belong to the family Leptospiraceae (29). Leptospira spp. are motile, obligately aerobic spirochete s. There are 13 known species of Leptospira (29), with over 260 serotypes (3). Leptospira spp. are the etiological ag ents of leptospirosis and are the leading cause of zoonotic disease wo rldwide (353). After infecting an animal (e.g. dogs, rats, swine, cattle, and raccoons) Leptospira colonize the proximal renal tubules of the kidneys, and are excreted in urine. Human infection usually occurs through the exposure of skin abrasions to wa ter contaminated with urine of animal reservoirs (3). Humans are not carriers of Leptospira spp. and infection usually leads to a range of symptoms which may include fever, acute respiratory illne ss, liver and kidney failure, and sometimes death (29). The Wo rld Health Organization (WHO) estimates 500,000 cases of leptospirosis annually with a 10% mortality rate (353). Plesiomonas shigelloides Plesiomonas shigelloides are gram-negative bacilli curr ently placed in the family Vibrionaceae (160); however molecular studies have shown a closer re lationship to the family Enterobacteriaceae (192). Many strains of P. shigelloides cross react with Shigella spp. in serology-based tests (70). P. shigelloides can infect both humans and a 3

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wide variety of animals (e.g. cattle, goats, swine, cats, dogs, monkeys, vultures, snakes, and toads). This organism is ubiquitous and has been isolated from surface waters and soils (292). P. shigelloides infections in humans can cause gastroenteritis or septicemia (152). Little is currently know n about the pathogenesis of P. shigelloides infections, however these organisms have been isolated fr om the stool of infect ed individuals (160). Infection occurs afte r the ingestion of water or food containing the microorganism. While cases of P. shigelloides infections are not regularly re ported in the United States, two outbreaks affecting 5 individu als were reported in 2003 (93). Salmonella species Salmonella spp. are gram-negative bacilli in the family Enterobacteriaceae These organisms can colonize an array of hos ts including: humans, domestic animals, birds, reptiles, livestock, and rodents (218). Initial nomenclature was based on clinical manifestations, serology and putativ e host or geographic origin (e.g. Salmonella typhimurium mouse typhoid fever); however the lack of host specificity of these bacteria and the advent of reliable and highly discrimi natory molecular techniques has lead to classification of Salmonella as two species ( S. enterica and S. bongori ) with serotype designations (44). Salmonella infections are referred to as salmonellosis. After ingestion by humans, Salmonella pass through the gastrointestinal tract and invade the M cells of the small intestines which can cause chronic colonization, enteritis, or enteric fever (218). Although many Salmonella spp. infections are limited to the gastrointestinal tract, S. enterica serotype Typhi produces a generalized infection (typhoid fever) which can result in septicemia. The CDC receives reports of ~40,000 cases of salmonellosis per year in 4

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the Unites States (63); however it estimates that infection rates are up to 30 times greater due to underreporting of Salmonella infections. Shigella species Shigella spp. also belong to the Enterobacteriaceae family. The genus currently includes four recognized species; S. dysenteriae, S. flexneri S. boydii and S. sonnei. Humans and other primates are the only known host of Shigella spp. (176). These gramnegative organisms are the causative agent of shig ellosis. The onset of shigellosis can be caused by as few as 10-100 bacilli, and sympto ms range from mild abdominal discomfort and fever, to vomiting an d bloody diarrhea (176). S. sonnei and S. flexneri are the most common causes of shigellosis in the United States (223), while S. boydii is the least common with the majority of cas es occurring in India (225). S. dysenteriae produces Shiga toxin which can cause severe dysente ry (218). The CDC estimates that 14,000 cases of shigellosis occur in the United Stat es every year (151), however in developing countries the World Health Organization estim ates 163.2 million cases of shigellosis each year (168). Vibrio species Vibrio spp. are curved baci lli in the family Vibrionaceae These bacteria have the ability to grow naturally in estuarine a nd marine environments (193). Clinical manifestations of Vibrio spp. infections include gastro enteritis after ingestion or septicemia after wound exposure. Vibrio spp. pathogenic to humans include: V. parahaemolyticus V. cholerae and V. vulnificus (218) Most V. parahaemolyticus found in the environment are nonpathogenic; however strains with genes encoding a thermostable hemolysin can cause acute gastroenteritis when ingested (255) V. cholerae 5

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is the most well-known of the Vibrio species and is the causati ve agent of cholera, a pandemic diarrheic disease (333). In immunocompromised individuals, V. vulnificus can cause sepsis after ingestion (218). For both immunocompromised and immunocompetent individuals, V. vulnificus has the potential to cause seve re wound infections leading to amputation and sepsis (121). The CDC estimates approximately 6,000 cases of Vibrio spp. infections per year in the United States (204). Viral Waterborne Pathogens Adenovirus Pathogenic human adenoviruses belong to the genus Mastadenovirus and the family Adenoviridae (154). There are six specie s of human adenoviruses (Human adenovirus A, B, C, D, E, and F) divide d into 51 serotypes (51). The adenovirus icosahedral capsid is non-enveloped and is 80100 nm in diameter with spike-like fibers protruding from each of the 12 penton vertices (297). The capsid surrounds a 35 kbp double-stranded DNA genome (218). Huma n adenoviruses can cause a range of symptoms including respiratory illness, conjunc tivitis, and gastroenteritis. The clinical manifestation is dependent on serotype. Human adenovirus F serotypes 40 and 41 infection can cause gastroenteritis (218, 284). Serotypes 8, 19, or 37 infections can lead to conjunctivitis and serotypes 3, 4, or 7 inf ections can produce acute respiratory disease (218). Infection with serotypes 40 and 41 can al so lead to an asymptomatic infection of the gastrointestinal tract and excretion of vi ruses in the feces (viral shedding). Viral shedding can last months to years. Serotype s 40 and 41 are the most frequently detected adenoviruses in sewage (35, 154, 155, 172, 218); and have been detected in natural waters (69, 71, 101, 153, 154, 251). While serot ypes 40 and 41 are readily present in sewage, serotypes 3, 4, and 7 have been the causative agent of several outbreaks 6

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associated with recreational water use ( 18, 82, 201, 242, 315). In developed countries adenovirus 40 and 41 are responsible for an es timated 1-8% of acute viral diarrhea (39, 120, 349), and adenovirus is the fourth-leading cause of viral respiratory illness in children (113). Astrovirus Pathogenic human astroviruses belong to the genus Mamastrovirus and the family Astroviridae. Currently there are eight known serot ypes of human astroviruses (Human astrovirus 1-8) (52). The as trovirus icosahedral capsid is non-enveloped with a diameter of 28-33 nm (218). Negative stain electron microscopy reveals that the core of the astrovirus has the appearance of 5 or 6 point star (337). This co re surrounds a 6-8 kb single-stranded, positive-sense RNA genome ( 98). Symptoms of astrovirus infection include diarrhea, vomiting, and fever. Astroviruses account for 5-10% of viral gastroenteritis in ch ildren worldwide (212). Enterovirus The Enterovirus genus belongs to the family Picornaviridae (48). Enteroviruses, polioviruses, coxsackieviruses, and echovi ruses are species within the genus of Enterovirus. Enteroviruses have single-stranded, positive-sense, 7-8.5 kb RNA genomes surrounded by an non-enveloped icos ahedral capsid (218). The Enterovirus capsids are approximately 30 nm in diameter and have the ability to resist a pH as low as 3, enabling them to withstand the conditions of the gast rointestinal tract (218) These viruses are primarily transmitted by the fecal-oral route; however, infections with enteroviruses usually do not lead to intestinal symptoms. In contrast to traditional pathogenic enteric viruses, Enterovirus infections lead to extra-intestinal illness which may be established in 7

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organs such as the nervous sy stem, heart, and skin (218). Enterovirus infection may be asymptomatic, and symptoms range from gastro enteritis, hand foot and mouth disease, respiratory illness, and conjunctivitis to meningitis, myocarditis, poliomyelitis, and paralysis (232). These viruses can replicat e in the alimentary canal and be shed asymptomically for up to a month after infec tion (218). In the past poliovirus infection was relatively common; however, the advent of a safe and effective vaccine has lead to a great reduction of cases in the United States. The Office of Rare Diseases of the National Institutes of Health deems po liovirus infection a rare disease in the United States with the last natural inf ection (i.e. infection not caused by live vaccine) occurring in 1993 (220). Conversely, the CDC estimates 10-15 million non-polio Enterovirus infections every year in the United States (162). Hepatitis A virus Hepatitis A viruses belong to the genus Hepatovirus and the family Picornaviridae (49). Hepatitis A virus has a single-stranded, positive sense, 7.5 kb RNA genome surrounded by a 27 nm diameter, non-enveloped icosahedral capsid (218). Like other members of the Picornaviridae family, hepatitis A viruses are stable at a low pH and are not degraded by stomach acids (218). The hepatitis A virus is usually acquired by ingestion, then enters the bloodstream and establishes infection in the liver. Viruses produced in the liver are released in bile and therefore can be isolated from stool in high titers (218). Individuals infected with hepatitis A experience acute hepatitis with symptoms including fever, fatigue, nausea, abdominal pain, and ja undice. Worldwide, approximately 10 million people experience hepatitis due the hepatitis A virus (2). 8

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Hepatitis E virus The hepatitis E viruses belong to the genus Hepevirus and the family Hepeviridae (53). Like the hepatitis A virus, the hepati tis E virus has a single-stranded, positive sense RNA genome. The 7.2 kb genome is surrounded by a non-enveloped icosahedral capsid with a diameter of 27-34 nm (53). Hepatitis E causes symptoms comparable to hepatitis A; however, pregnant women are more susceptible to severe clinical manifestations. Infection in pregnant wome n can lead to fulminant hepatic failure and the mortality rate is approximately 20% in th ird trimester pregnancies (218). Hepatitis E viral infections are leading cau se of acute hepatitis in deve loping countries (177). In the United States, hepatitis E outbreaks are rare ly reported; nonetheless, studies have reported 19-23% of the United States population is seropositive for the hepatitis E virus (174) Norovirus Formerly known as Norwalk-like viruses the Norovirus genus is a member of the family Calicivirdae (47). Noroviruses have a 35-39 nm non-enveloped, icosahedral capsid surrounding a 7.3-7.7 kbp single -stranded RNA genomes (47). Noroviruses are clustered into 5 genogroups (GI-GV) (247). Genogroups GI, GII, and GIV are capable of producing infections in humans (247). Norovirus infection usually presents as vomiting, watery non-bloody diarrhea, and abdominal cr amps and nausea. Infections in young children and the elderly can lead to compli cations such as severe dehydration (218). Noroviruses are the leading cause of viral gastroen teritis in the United States (311). Rotavirus The Rotavirus genus belongs to the family Reoviridae (50). Rotaviruses have double-stranded RNA genomes comprised of 11 segments. These viruses have a non9

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enveloped, triple-layered prot ein capsid that is approximate ly 60 to 80 nm in diameter (119). Currently, there are seven groups in the Rotavirus genus (RV-A to RV-G); however, only RV-A, RV-B, and RV-C have been known to cause human infection (249). Rotaviruses are stable at pH 3.5 to 10, which allo ws the virus to survive the acidic environment of the stomach (218). Once inside the small intestines, Rotaviruses adsorb to the columnar cells and begin replication. At the height of disease as much as 1010 viral particles per gram of stool can be shed (218). Clinical manifestations of Rotavirus infections include diarrhea, fever, vomiting, and in some cases severe dehydration and death. Rotaviruses are the leading cause of viral gast roenteritis in children worldwide (119, 249). Protozoan Waterborne Pathogens Cryptosporidium parvum Cryptosporidium parvum is a eukaryotic parasite in the order of Eucoccidiorida (38). C. parvum can infect a range of hosts including humans, cattle, sheep, rodents, and birds (38). The life cycle of C. parvum is relatively complex with various morphological stages including oocyst, spor ozoite, trophozoite, and merozoite The 4-5 m oocysts of C. parvum are infectious. Once ingested, sporozoite s are formed and then attach to the epithelial cells of the ileum and colon (38). Several metamorphic events occur in which the sporozoites become trophozoites and then me rozoites. Merozoites are released, infect adjacent cells, transform into trophozoite s, and undergo schizogony to again produce merozoites (38). The second generation merozo ites again infect adjacent cells and then form gametocytes that produce oocysts (38). The oocysts are released and then excreted in the feces. C. parvum is the causative agent of cryptos poridiosis, whic h can result in asymptomatic infection or cause symptoms of severe diarrhea w ith vomiting, fever, and 10

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weight loss (95). Severe dehydration and death can occur in immunocompromised patients (38). The CDC estimates 300,000 cases of cryptosporidiosis annually in the United States (357). Entamoeba histolytica Entamoeba histolytica are eukaryotes in the order of Amoebida (38). E. histolytica infects humans and primates, and can transien tly infect dogs and cat s (281, 296). The life cycle of E. histolytica involves several stages, includi ng the production of infectious cysts, excystation, maturation into trophozoites, and subsequently the formation of more infectious cysts (38). The life cycle begins with ingestion of a mu ltinucleated cyst that has a diameter of 12 m. These cysts can withstand the acidic environment of the stomach, which allows them to pass through th e stomach to the ileum where excystation occurs (38). The excystation results in form ation of trophozoites with a diameter of 1560 m. These trophozoites can reproduce by binary fission and colonize the intestinal mucosa of the colon. After colonizati on, new infectious cysts are produced by encystation and then excreted in the feces (38) A majority of individuals infected with non-invasive strains of E. histolytica are asymptomatic (355). Invasive strains of E. histolytica can cause hepatic amoebasis, pulmona ry amoebasis, and amoebic dysentery (38). With an estimated 50 million sympto matic infections per year and 100,000 annual deaths worldwide, E. histolytica is the second leading cause of death due to parasitic infections (147, 293). Giardia species Giardia spp. are eukaryotes in the order of Diplomonadida (38). Currently, taxonomic revisions have been suggested regarding members of the genus Giardia (211). 11

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While a standardized nomenclature has not yet been adopted, the common names of Giardia that infect humans include: G. lamblia G. duodenalis G. intestinalis, and G. enterica (211, 310). The life cycle of Giardia spp. is similar to E. histolytica and includes the production of infectious cysts, excystation, maturation into trophozoites, and subsequently the formation of more infecti ous cysts (38). The life cycle begins with ingestion of a multinucleated cyst that has a diameter of 11 m. Af ter ingestion, the cysts pass through the stomach and enter the small intestines. The cysts form 14 m long trophozoites that attach to the intestinal walls (38). The trophozo ites reproduce by binary fission and eventually undergo encystation to form more cysts. Both trophozoites and cysts are excreted in the feces; however, only the cysts can survive outside the host. The adherence of the trophozoites on the intestinal wall can disr upt adsorption of nutrients and cause diarrhea, malnourishment, and dehydration (38). These symptoms are referred to as giardiasis. The CDC has reported that Giardia spp. infections are the most common parasitic disease in the United States (358). Miscellaneous Waterborne Pathogen Schistosoma species Schistosoma spp. are helminths belonging to th e class Trematoda (193). These organisms are bilaterally symmetrical wo rms, commonly referred to as blood flukes (193). These organisms are the etiological agents of schistosomiasis. Three Schistosoma spp. are medically important in humans: S. japonicum S. haematobium and S. mansoni (218). The life cycle of these Schistosoma spp. are similar, and involve several stages including the production of eggs, formation of miracidia, infection of an intermediate host (snail), formation of sporocysts, matura tion into cercariae, and infection of humans through the skin. The life cycle begins as eggs excreted in feces or urine enter 12

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environmental water bodies. Contact with water initiates hatching, which produces freeswimming larvae, called miracidia. The larvae must then infect a specific intermediate host, a snail. After infecti ng the snail, miracidia undergo a series of asexual reproduction stages resulting in the formation of sporocys ts (114). The maturation of the sporocysts results in the formation and re lease of cercaria. Cercaria have the ability to penetrate skin, and human infection occurs through exposure to water containing cercaria. Once the cercaria enters the skin s it undergoes several physical changes and becomes a wormlike schistosomule. The schistosomule migr ates through the circulatory system, enters the liver, and matures into schistosomes ( 191). The schistosomes then migrate to the species specific final infection site (bladder or intestines). Once the schistosome reaches the final site, egg production begins (191). The eggs migrate through the tissues and are released in the feces or urine, and the life cycle begins again. Humans can also be infected by avian and mammal schistosomes. These schistosomes cannot mature in the human body, however they can penetrate the sk in and cause local ir ritation leading to cercarial dermatitis (i.e. swimmers itch) (193). Swimmers itch occurs throughout the world and is more frequent in the summer months (65). Sc histosomiasis is relatively uncommon in the United States; however the CDC estimates an annual 200 million cases worldwide (64). Surveillance of Illness Outbreaks Associated with Recreational Water Use In 1971, a water-borne disease survei llance system was initiated by the CDC, United States Environmental Protection Agen cy (EPA), and the Council of State and Territorial Epidemiologists (184, 185, 213, 360) A water-borne disease outbreak is defined as more than one individual experi encing a similar illness after ingesting or 13

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exposure to a common water source. In 1978, th e surveillance system was broadened to include water-borne illness out breaks resulting from recreatio nal water use. Recreational waters are separated into two categories: tr eated or untreated wate rs. Treated waters include swimming pools, water parks, whirlpools and hot tubs. Untreated waters include lakes, rivers, ditches, ponds, and the ocean. For the purposes of this introduction, only untreated recreational waters will be discu ssed because the study focuses exclusively on untreated waters. From 1991-2006, a total of 77 outbreaks from nonVibrio bacterial and protozoan pathogens were reported after exposure to untreated recr eational waters ( Table 1 ). These outbreaks resulted in over 3,360 i ndividual cases of illness. A majority of the outbreaks were attributed to pathogenic E. coli (n=20) and Shigella spp. (n=20), followed by both Schistosoma spp. (n=10) and Cryptosporidium spp. (n=10). The remaining outbreaks were caused by Giardia spp. (n=8), Leptospira spp. (n=7), and P. shigelloides (n=2). In the same time period, 26 outbreaks from viral pathogens were reported ( Table 2 ). These outbreaks resulted in 1,889 indivi dual cases of illness. Norovirus was the leading virus genus associated with outbreaks (n=22). Echovirus (n=2), coxsackievirus A1 (n=1), and adenovirus 3 (n=1) also contribut ed to viral recreational wate r-borne outbreaks. Twentyseven outbreaks from unidentified etiol ogical agents were reported during 1991-2006 ( Table 3 ). A total of 252 cases of Vibrio spp. related illnesses after recreational water exposure were reported from 2003-2006 ( Table 4 ). In most cases the specific cause of water contamination was not identified; however, Craun et al. (78) compiled available information from 1971-2000 to determine main sources of water contamination when they could be determined ( Table 5 Table 5 ). 14

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These sources of contamination included bather overloading, fecal contamination of water by an infected person( s), children in diapers ente ring the water, malfunctioning sewage handling systems, fecal input by animals, and rainfall (78). Craun et al. (78) also noted a seasonal distribution of the 1971-2000, outbreaks with >70% of the outbreaks occurring during June, July, and August. Furthermore, Craun et al. (78) complied available water quality data from 38 of the 1971-2000 outbreaks in terms of fecal coliform or E. coli concentrations. Almost half (42.1 %) of the outbreaks occurred after exposure to water containing concen trations of fecal coliforms or E. coli 126 colony forming units (CFU)/100 ml, 18.4% occurred after exposure to water containing concentrations of fecal coliforms or E. coli ranging from 127 to 499 CFU/100 ml, and 39.5% occurred after exposure to water contai ning concentrations of fecal coliforms or E. coli 500 CFU/100 ml (78). The significance of these bacterial concen trations in terms of water quality is explained below. History of Traditional Water Quality Standards and Indicators The advancement of civilization brought la rge numbers of individuals into close proximity, which resulted in unsanitary condi tions and outbreaks of diseases due to consumption of water contaminated with ente ric pathogens from human excrement. Prior to the 1850s, little was known about water-bor ne diseases, and thus water quality was based on clarity, smell, and taste (254). During the late 1840s and early 1850s a large cholera outbreak occurred in London. Dr. J ohn Snow, a local physician, established a commonality among his patients; all had cons umed water from the Brood Street water pump. He surmised the water was the source of the ailment. Subsequent investigations uncovered a nearby sewer line was damaged and contaminating the well (254). 15

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In the United States, the first microbial standards for drinking water were initiated in 1914 by the United States Public Health Service (USPHS). Recognizing the extent and cost of monitoring water for pathogens the USPHS recommended total coliforms as bacterial indicators of fecal contamination ( 254). Despite regulations on drinking waters, surface waters were unregulated. Before the advent of wastewater treatment facilities surface waters commonly received direct untreated sewage discharge. Consequently, large numbers of water bodies were severely polluted. In 1948, the Federal Water Pollution Control Act (FWPCA) was issued by the EPA in an effort to grant the federal government authority over pollu tion control of environmental waters (318). Over the next 25 years the FWPCA underwent many amendments which gradually broadened the federal governments authority and increased restrictions on sewer and industrial drainage into receiving waters. In 1972, th e FWPCA evolved to prohibited the discharge of raw sewage into any environmental waters and stated discharged wastewater must, at minimum, undergo secondary treatment before being discharged in to receiving waters (318). In addition, FWPCA set forth specific criteria for biochemical oxygen demand (BOD), suspended solids (SS), and fecal colifor ms in secondary-treated effluent (327). In 1973, the Safe Drinking Water Act (SDWA) was passed, allowing the EPA to set and legally enforce standards for drinking wate r quality (e.g. zero dete ctable total coliform colony forming units (CFU) per 100 ml of water) (321). In 1976, the EPA released The Clean Water Act (CWA) (325). The CWA specified acceptable levels of biological a nd chemical contaminants in environmental waters, and gave the EPA authority to regulat e the discharge of va rious pollutants into these waters. Under section 303(d) of the CWA, each State was required to set 16

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regulatory standards for indigenous water bod ies and identify waters not meeting the criteria (termed impaired waters). The CWA stated that each State must set total maximum daily loads (TMDLs) of pollution that impaired waters could receive without exceeding the regulatory standards. In additi on, the report suggested the use of total and fecal coliform concentrations as i ndicators of fecal pollution (325). During the 1970s and1980s, several epidem iological studies were conducted to assess risk associated with human exposure to waters impacted by poi nt source (sewage) pollution, which also contained high concen trations of total and fecal coliforms, Enterococcus species, and E. coli (323). In 1986, the CWA was modified based on the correlations found in these epidemiological st udies (323). The resu lting report, entitled Ambient Water Quality Criteria for Bacteria (AWQCB), refined the existing criteria utilized for monitoring recreational water qua lity, and reported correl ations of bacterial densities and increased risk of swimming-associated gastroenteritis. Based on the findings, the EPA recommende d regulatory standards of E. coli or enterococci concentrations for fresh water and enterococci concentrations for marine water ( Table 6Table 6 ) (323). In 2000, the EPA re quired coastal states to c hoose water quality criteria that complied with Section 303(i) of the Cl ean Water Act (326). Despite EPA reports and recommendations standard indicator organi sms for microbial water quality were not mandated, and therefore water quality sta ndards are governed at a state level. Florida Water Quality Standards The state of Florida has approximately 11,000 miles of rivers and streams, 7,700 lakes, 4,000 square miles of estuaries, 8,000 m iles of coastline, and nearly 1,000 miles of beaches (295). In 2002, Florida develope d Florida Administrative Code 62-302 (FAC) 17

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which stated water classifications, criter ia, and anti-degrada tion policies (100). According to the FAC, all surface waters in th e state of Florida are classified according to their intended use ( Table 7 ). Class I designates water us ed to augment potable water supplies. Class II waters include all areas of water used to propagate and harvest shellfish. Class III waters c onsist of any waters used for recreation and the propagation or harvesting of fish. Any waters used for agricultural water supplies are categorized as Class IV. Class V waters comprise surface waters used for utilities, navigation or industrial purposes. Florida water quality criteria are depende nt on the intended use of the water with some criteria in place to protect aquatic lif e, and others are intended to protect human health. In 2002, Florida Ad ministrative Code 62-302.530 defined fecal coliforms as indicators of water quality for Class I, II, and III waters. The FAC code was subsequently modified after the EPA require d enterococci concentrations found in the AWQCB report be utilized to assess Florid as coastal recreation water quality by 2004 (326). Current Florida water regulatory standards for E. coli in freshwater and enterococci in marine waters and beaches are summarized in Table 7 Total Maximum Daily Loads (TMDLs) Routine water quality monitoring allows officials to determine temporal and spatial trends of microbial inputs and identify wate r bodies that do not meet designated classspecific water quality criteria. Microbial wa ter quality monitoring involves enumerating indicator bacteria as surroga te pathogens. The CWA requires the State to report water quality findings every two years. Waters containing high levels of indicators are considered substandard due to pathogen im pairment. Substandard waters are placed on 18

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the impaired waters (303(d)) list. Currently in Florida, 827 water bodies have been included on the 303(d) list with 18.2% of these waters path ogen impaired (316). Once on the list, Total Maximum Daily Loads (TMD Ls) must be developed for the water body by the responsible State agency. In Florida, the Florida Departme nt of Environmental Protection (FDEP) is the agency responsible for establishing TMDLs. The process of defining TMDLs is based on determining the source(s) and load allocations of the contaminant. After TMDLs have been established a plan of action (TMDL implementation) is then employed to reduce target contaminants. Bacterial indicator organisms are the sta ndard measure of microbial water quality ( Table 6 and Table 6 ); however these measurements do not give any insight as to the source of pollution or the actua l presence of pathogens. High b acterial concentrations can be attributed to many sources including agricu ltural runoff, storm water runoff, wildlife, pets, faulty septic tanks, and failing sewers (16, 45, 200). The ubiquitous presence of these bacteria in fecal matter from all sour ces confounds remedial efforts and limits the ability to implement TMDL goals. To address the issue of source identification, researchers have developed methods in whic h the biochemical or genetic features of certain microorganisms are used to indirectly determine the origin of fecal pollution; these methods are collectively termed micr obial source tracking (M ST). The following provides a description of traditional culture dependent water quality indicators, microbial source tracking methods, and the advantages and limitations of each indicator and method. 19

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Traditional Microbial Water Quality Indicators Culture-dependent enumeration of to tal coliforms, fecal coliforms, E. coli, and enterococci has traditionally been used to a ssess microbial water quality. Total coliforms consist of all aerobic and facultative anaer obic, gram-negative, nonspore-forming, rodshaped bacteria that ferment lactose with ga s formation within 48 hours of inoculation at 35C. Fecal coliforms, which include E. coli, are distinguished from total coliforms by growth at a higher temperature (44.5 0.2C) (73). Enterococci are a subgroup of fecal streptococci and are distinguished by their abi lity to grow in 6.5% sodium chloride, at 10C and 45C, and at a pH of 9.5 (272). Coliform bacteria and enterococci share a common feature; they predominantly inhabit th e intestines of warm-blooded animals. The exclusive association of these or ganisms with fecal contamination is questionable for several reasons. These reason s include the isolation of fecal bacteria from non-fecal sources and the detection of fecal bacteria in secondary habitats (e.g. lake sediments). Fecal coliforms, specifically E. coli have been isolated from paper mill effluents in areas presumed absent of hu man fecal impact (25, 106), and studies have reported the detection of fecal coliforms, E. coli, and enterococci in waters with no history of anthropogenic impact (56, 260, 354, 356). In addition, natural reservoirs of coliform bacteria have been found in the e nvironment (e.g. in soil and on vegetation) (43, 109), and studies have demonstrated these ba cteria have the abili ty to survive and potentially proliferate in tropical environments (15, 42, 86, 197). The reliability of assessing water quality in warmer areas (e.g. Florida, southern California, and Hawaii) with regulatory standa rds using traditional indicator organisms is confounded by the possibility of occurrence of native populations and survival and regrowth of indicators from fecal sources in tropical climates (68). The regulatory 20

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standards may ill represent wa ter quality and human health risks in warmer climates particularly since EPAs gui delines were based on studies performed in non-tropical areas of the United States including Bo ston, New York and New Orleans (280, 323). Although there have been some associations be tween high levels of indicator bacteria and disease outbreaks (68, 72, 302), there is lit tle or no prediction of specific sources of contamination, and questionable correlati on with human pathogens (97, 275, 283, 287, 305). Therefore, indicator organism con centrations alone do not provide an ideal assessment of water quality or prediction of human health risks. Overview of Microbial Source Tracking In addition to the inability of traditional indicators to predict human health risks, these organisms do not provide insight on the source of water quality impairment. The failure of these indicators to distinguish the contributions of hu man, domestic animal, wildlife, agricultural, and stormwater pollution is a major shortcoming. This shortcoming has given rise to the field of microbial source tracking (M ST), a collective term for methodologies employed to detect and differen tiate sources of fecal pollution in waters (i.e. using microorganisms as markers of source-associated contamination) (319). The concept of MST is based on the premise that diffe rences in the intestin al flora of different hosts occur because of the availability of distinct microbial habitats including temperature, food supply, and specific host digestive systems (319). Some methods exploit differences arising from natu ral selection including competition among microorganisms for space and nutrients, or exposure to antibiotics. The process of developing MST methods includes identifying a microorganism(s) with a characteristic and unique phenotypic an d/or genotypic referen ce feature. Ideally, 21

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the reference feature must be present in a ma jority of the target microorganisms found in excrement of the specific host group, and the re ference feature must solely discriminate the source-associated target organism(s) (319). Water is then tested for fecal pollution based on the association of fecal contamina tion with the presence or absence of the targeted reference feature. An ideal MS T assay utilizes a microorganism with the following features: (1) it correla tes with the presence of en teric pathogens, (2) is well characterized, (3) is shed by a majority of the indicative species, (4) has validated temporal and geographical consistency, (5) is stable in fresh and marine water environments, and (6) has a well-defined pers istence (273, 283). In addition, the method to detect the indicator should be standardized, reliable, reproducible, and cost effective (319). Methodologies used in MST can be categor ized into phenotypi c or genotypic, library-dependent or library-i ndependent methods (267, 275, 283) The characteristics of phenotypic library-dependent, phenotypic library-independent, genotypic librarydependent, and genotypic lib rary-in dependent MST me thods are summarized in Table 8 Phenotypic methods categorize microorganism s based on biochemical based tests and genotypic methods are based on molecular tech niques. In general, library-dependent methods rely on the compiling of characterist ics of bacterial isol ates (e.g. antibiotic resistance pattern or genotype) into geogr aphical and source-specific (e.g. animal vs. human) databases. In comparison, library-independent methods are based on a single common characteristic (e.g. presence of a specific gene or gene variant). The fundamental difference between library-depe ndent and library-independent methods is that library-dependent methods utilize compara tive traits of cultured isolates from fecal 22

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samples to determine sources of fecal input whereas library-inde pendent methods use a common genetic marker(s) to determine sour ces of fecal input. A major advantage of most library-independent methods is th e omission of isolating and culturing microorganisms, as well as the lack of re liance on expensive, maintenance-intensive databases (libraries). In the past, the most common MST methods were library-based. Libraries are compilations of fingerprints based on phenot ypic or genotypic tr aits from bacteria isolated from known hosts (e.g. humans, chic kens, cows, horses, etc.). Typically, representative libraries cont ain hundreds or thousands of known-source isolates. The fingerprints of unknown-source isolates are then compared to the library and statistical analysis is used to determine the similarity among the fingerprints and subsequently the origin of the unknown-source isolate. Various drawbacks of library-dependent methods have been identified and include: methods are labor intensive and time consuming, methods are not accurate over broad geographi cal areas, libraries must be continuously updated, and initial library construction is costly (267, 275, 299). In comparison, libraryindependent methods tend to be less labor inte nsive, more cost efficient, and applicable over broader areas. Library-independent methods target a species-specific phenotypic or genotypic marker. The presence or absence of this marker indicates the fecal input by associated species. In addition, some genotypic library-independent methods have expanded to quantifying the genetic marker us ing QPCR. Quantification of the marker may allow for the assessment of pollution inte nsity. The following is an overview of both relatively tenured and newl y developed MST methods. 23

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Phenotypic Library-Dependent Methods Antibiotic resistance profiles Commercially available penicillin was in troduced into the clinical field as an antibiotic in 1943. The effectiveness of penicillin led to incr eased interest in antibiotic discovery and synthesis. Since then many ot her antibiotics, both na tural and synthetic, have been introduced (e.g. cepha losporin, isoniazid, bacitracin, tetracycline, rifampin). Currently in the United States, an estima ted 11 million kilograms of antibiotics are prescribed for human use every year (340). In addition, farm animals, such as beef cattle, swine, and poultry, are frequently given antib iotics to promote rapid weight gain for quicker time to slaughter (261, 263). Conse quently, exposure of human and domestic animals to antibiotics has become frequent, and many antibiotic resistant strains of pathogenic and commensal enteric bacteria have emerged (224). This has led to intestinal flora that exhibit characteristic antibiotic resistance patterns in humans and domestic animals (275, 346). In contrast, ba cteria from wildlife species tend to be somewhat more susceptible to antibiotics, pa rticularly synthetic a nd semi-synthetic ones (275, 347). Escherichia coli and Enterococcus spp. are the primary organisms used in multiple antibiotic resistance analysis (MAR ) or antibiotic resistance an alysis (ARA). In the case of MAR, bacterial isolates from known sour ces are plated on agar containing various types of antibiotic disks at a single concen tration (245). The ARA pr otocol is slightly different from MAR, employing multiple antibiotics at various concentrations (346). Growth patterns are observed and a resistance pattern emerges that can be used in source differentiation (76, 77, 132, 161, 244, 346). Larg e libraries of isolates from known 24

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sources are constructed for a watershed. U nknown isolates are then compared to the known source profiles using st atistical analyses. Several studies have demonstrated succe ssful source identifi cation using ARA or MAR (132, 245, 346, 347). In 2003, Wiggins et al. (348) found large libraries (e.g. > 2,900 isolates) were able to classify unknown isolates more successfully as compared to smaller libraries (e.g. < 2,300) in six Virginia watersheds. Ho wever, Wiggins et al. (348) also reported larger libraries have low average rate of correct classification (ARCC). Studies report successful source identific ation using antibiotic resistance profiling based on ARCC values ranging from 62 to 84% (124, 245, 346, 347). However, Meays et al. (205) questioned the ability of the met hod to identify sources of fecal pollution from mixed samples (i.e. water containing isolates from various sources). In addition, Olivas et al. (235) reported signifi cant misclassifications in wate rsheds with low antibiotic resistance. The disadvantages of this met hod include costs associated with the initial construction of the known library, low AR CC, low reproducibility of results, high variability among statistical methods, and indeterminate results (133, 259, 265). E. coli serotyping The immune systems of both human s and animals respond to foreign carbohydrate and protein molecules by producin g antibodies (218). These antibodies circulate through the blood and can be isolated in serum. E. coli serotyping involves exposing various serums of either animal or human origin with the somatic antigens of E. coli isolates (79). The origin of the isolate can be determined based on reactions with specific animal or human antisera (243). Several studies have associated specific E. coli serotypes with either human or non-human sources (79, 115, 243). A serious limiting 25

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factor of E. coli serotyping is the requirement of a large antisera bank to achieve a complete and representative analysis (275). In addition, documented cross-reactivity of human isolates with isolates of nonhuman origin limits th e human-specificity of the method (28, 128, 237). Phenotypic Library-Independent Method Bifidobacteria Bifidobacteria spp. are commonly found in feces of warm-blooded animals and are among the dominant microbiota in the human intestines (250). These gram-positive bacteria are non-spore-forming obligate anaero bes (288). Sorbitol is a sugar substitute found in products made for human consump tion (e.g. diet drinks, sugar-free chewing gum, etc.) (188), and Bifidobacteria isolates with the abilit y to ferment sorbitol are hypothesized to be of human origin (55, 222, 258). Accordingly, researchers have suggested the presence of sorbitol-fermenting Bifidobacteria may indicate human fecal pollution (41, 196). Several researchers have reported consistent detection of sorbitolfermenting Bifidobacteria in human feces and sewage (150, 188). While sorbitolfermenting Bifidobacteria are commonly isolated in human feces, these organisms have also been found at relatively high concentratio ns in slaughterhouse wa stewater (41). To address this concern, researchers comp ared ratios of sorbitol-fermenting Bifidobacteria to total Bifidobacteria in raw sewage and slaughterhouse effl uents (41). Ratios greater than 0.2 were defined as human-derived wastes, and ratios less than 0.05 were defined as animal-derived slaughterhouse effluents ( 33, 41). To determine the source of the Bifidobacteria species, Lamendella et al. (179) designed Bifidobacteria species specific primers, and reported host specificity of B. thermophilum and B. boum However, Lamendella et al. (179) also reported the absence of Bifidobacteria in the presence of 26

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other fecal anaerobic bacteria in surface waters suggesting a low sensitivity. The utilization of this method is hindered by the lack of data on the persistence of Bifidobacteria in environmental water systems and the potential lack of sensitivity of the assay (56, 179). Genotypic Library-Dependent Methods Pulsed field gel electrophoresis (PFGE) Pulsed field gel electrophoresis (PFGE) is a molecular method used to create discriminatory DNA fingerprints from culture d isolates. Bacteria are isolated from various sources; DNA is extracted from the isol ates and then treated with restriction endonucleases (e.g. EcoRI, SmaI) to produce large fragments. The resulting high molecular weight restriction fragments are separated using pulsed -field electrophoresis (193). Large libraries of DNA fingerprints from known sources isolates are constructed for a specific watershed. DNA fingerprints from unknown source isolates are compared to the library using statistical analysis. Several studies have found associations be tween PFGE profiles and isolate source (219, 282, 299). In contrast, some researcher s have found no significant association of PFGE profiles and isolate source (219, 243). La ck of standardized protocols (e.g. choice of restriction enzyme), string ency levels, and statistical analysis leads to irreproducible results among laboratories (275). In addition, known source libraries are not applicable over large geographical areas (275). While PFG E has the ability to discriminate small genetic differences, the sensitivity may hinder broad discrimination of isolates necessary in MST (i.e. two isolates from one source ma y be classified as two distinct groupings based on minute genetic differences) (275). Moreover, the construc tion of the initial known source library is costly and time consuming. 27

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Repetitive element PCR (rep-PCR) Prokaryotic genomes contain interspersed repetitive extragenic palindromic (rep) elements (190). Repetitive element PCR (rep-PCR) utilizes one degenerate primer specific to the palindromic repetitive elem ents. In rep-PCR, DNA between adjacent extragenic palindromic repetitive elements is amplified. The PCR amplicons generated are then analyzed using gel el ectrophoresis, result ing in a DNA fingerprint. In a process similar to that used for PFGE, bacteria are isolated from various sources and the genomic DNA is extracted from the isolates. The DNA is then used as template in the rep-PCR. Isolates from known sources are used to construct large libraries of DNA fingerprints. DNA fingerprints from unknown sour ce isolates are compared to the library to identify the source of the bacterial isolate. The high discriminatory potential of the method warranted researchers to develop assays for E. coli and enterococci for source identification in water (89, 194). Rep-PCR of E. coli isolates has been used with some success (157, 167, 210, 299). In addition, rep-PCR of enterococci isolates has been utilized to identify sources of contamination in water (110). Limitations of this method include high costs of library construction, lim ited reproducibility of re sults, and confined geographical application of know n source library (275). Ribotyping Ribotyping is a relatively labor-intensi ve genotyping method that yields DNA fingerprints of ribosomal RNA. The me thod requires the cult ure/isolation of E. coli and restriction enzyme digestion of the genomic DNA. The resulting fragments are separated by gel electrophoresis. The sepa rated DNA is transferred to a nitrocellulose membrane and hybridized with a probes specific to a fragment of the rRNA gene. Analogous to PFGE and rep-PCR, a database of known sour ce isolates must be constructed for 28

PAGE 44

comparison and identification of unknown isol ates (274). Ribotyping has been employed in many studies with the average rate of correct classification of human and animal isolates ranging from 13-99% (58, 127, 219, 246, 265, 299). Initially this method was manually performed and required a significan t amount of time and labor, however the advent of automated systems (e.g. Riboprinter) has alleviated the extensive labor. This method has met with some success; however several glaring disadvantages such as high costs and questionable reproducibility make this method unfavorable. Genotypic Library-In dependent Methods F-specific coliphage genotyping The virus families, Inoviridae and Leviviridae, are composed of single-stranded DNA and RNA coliphages respectively. F-spec ific (F+) coliphage are viruses that primarily infect Escherichia possessing a plasmid coding for a sex pilus (193). In comparison to F+ DNA coliphages, the F+ RNA coliphages are found at high concentrations in sewage (144, 183). In addition, F+ RNA coliphages have been more fully characterized, and therefore have received more attention in regards to MST methods. The F+ RNA coliphages are divided into four genogroups: group I, group II, group III, and group IV (144). Group I colipha ges have been isolated from both human and animal fecal wastes. Groups II and III ar e primarily associated with human wastes, whereas group IV has been associated with fecal wastes of animal origin (269, 275). The presence of a certain group of coliphage is used to indicate the source of fecal contamination of water. F+ coliphages are plated on a bacterial hos t, incubated and the resulting plaques are transfe rred to a nylon membrane. The capsids are denatured, after which the phage nucleic acid is cross-linked on the membrane. Detection and 29

PAGE 45

differentiation of the F+ coliphages is accomplished using group-specific 32Por digoxigenin-labeled oligonucle otide probes (319). Several issues have arisen in regards to the use of F+ coliphages as indicators. Group II and III F+ RNA coliphages are isolated from only a small percentage of human fecal samples, however these are the predomin ant bacteriophage in sewage; this leads to the suspicion of proliferation when these ba cteriophage are introduc ed to sewage (111, 136, 273). Furthermore, Jiang et al. (155) found a low frequency of detection during summer months and a lack of correlation with the presence of hu man adenoviruses and enteroviruses. Studies have documented high bacteria titers in stormwater runoff (195, 200), which may increase coliphage concentrat ions and falsely indicator poor water quality. Survival characteristics of coliphage in the environment should be investigated before this method is used as a reliable indicator of fecal pollution. Enterococcal surface protein gene The enterococcal surface protein is a vi rulence factor associated with humansource, clinical isolates of Enterococcus faecium (231). In 2005, a method to detect the enterococcal surface protein ( esp ) gene in Ent. faecium of water samples was developed (272). Enterococci are isolated from wate r by membrane filtration. The membranes are placed on mEI agar and incubated at 41 C for 48 hours. The filters containing enterococci are lifted and placed in tryptic soy broth for 3 hours at 41 C. DNA extractions are performed on the resu lting culture and the presence of the esp gene is determined using PCR and gel electrophoresis In 2008, Ahmed et al. developed a real time (quantitative) assay to quantify the esp gene (10). Ent. faecium harboring the esp gene has been isolated from a high percentage of sewage samples and there have been 30

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several studies reporting successful use of the esp marker as an indicator of fecal pollution in water (12, 45, 202). Howeve r, many studies have detected the esp marker in non-human sources (54, 182, 352). The 48 hour incubation for the conventional PCR method and lack of host specificity severely hinder the reliability of this MST method. Bacteroidales marker The Bacteroidales are gram-negative, anaerobic non-spore forming bacilli. Members of this group are found at orders of magnitudes higher concentrations than coliforms in both human and animal feces (141 ). The use of this group to detect human fecal pollution was first suggested in 1998 ( 170). The method includes concentration, DNA extraction, and molecular detection. Bacteroidales spp. are concentrated from water using a 0.45 m pore-size filter. DNA is extracted from the f ilter and the presence of the human-associated Bacteroidales (HumBac) marker is detected using PCR or QPCR (27, 88, 163, 181, 234, 277). This approach has been widely used and these organisms have been found at high concentr ations of sewage (129); however, recent studies have reported a high frequency of false positives for non-human fecal samples making the discriminatory powers of this marker imperfect (57, 203). Methanobrevibacter smithii Methanobrevibacter is a major genus of methanogens found in intestines. Other than M. ruminantium M. gottschalkii, and M. thaueri most Methanobrevibacter spp. are thought to be host specific (186). M. smithii is the prominent methanogen in the human intestines and has been f ound at concentrations of 10710 organisms per gram of feces (40, 186). The use of this organism to detect human fecal pollution was first suggested in 2006 (330). Like the HumBac marker, this method includes concentration, DNA 31

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extraction, and molecular detection. M. smithii are concentrated from water using a 0.45 m pore-size filter. DNA is extracted fr om the filter and the presence of the M. smithii marker is detected using PCR. The PCR primers target the nifH gene (330), which was chosen based on sequence differences of va rious methanogens (233, 330). The use of these primers to identify sources of human-associated fecal pollution has been limited but successful in preliminary studies (330). Recently, Harwood et al. (129) reported M. smithii primer specificity tests to be 98% speci fic, with minimal cross-reactivity in several animal fecal samples (e.g. cow, dog, a nd seagull). Further analysis of marker persistence in the environment, and more app lications to water quality are necessary to validate this marker. Human enteric viruses Over 100 different enteric viruses exist. The most prevalent enteric viruses in human derived sewage include enteroviruses, adenoviruses, and ro taviruses. Direct monitoring of these viruses ha s been performed using integr ated cell culture methods and PCR or QPCR detection. Approximately 80% of the viral particles shed in feces are adenoviruses (146), and the pres ence of enteric adenovirus type 40 and 41 (Ad 40 and Ad 41) has been implemented as an indication of human fecal pollution (69, 153, 251). Ad 40 and Ad 41 have been found in surface wate rs contaminated with fecal pollution (59, 153, 154). In addition, enteroviruses have been utilized as indicators of human fecal pollution (142, 143, 271). Using human pathogenic enteric viruses as indicators of water quality can allow for direct human health risk assessments; however, only a small percentage of the population excretes these viruses, which can lead to inconsistent 32

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detection (101). In addition, st andard methods require large quantities of water (~100 L) to be filtered for analysis (73). Study Rationale Library-dependent methods have demonstr ated limitations including (1) the timeconsuming and labor-intensive nature of lib rary construction, (2 ) the limited geographic areas to which libraries can be applied, and (3) the cost of the methods and need for periodic updates of th e libraries (205, 275). In genera l, library-independent methods are less time consuming, potentially applicable ove r large areas, and in most cases are more cost effective than library-dependent methods (275, 319). In addition, large representative libraries have a high probability of containing cosmopolitan strains that inhabit the gastrointestinal tract of more than one type of host (319). The cosmopolitan nature of library-dependent methods, coupled with the questionable host specificity of established library-independe nt methods warrant the addi tion of a library-independent, human-specific marker that can be readily used in situations when more than one fecal contribution may be polluting a water system. Moreover, the advent of quantitative PCR (QPCR) has lead to the suggested modificat ion of MST methods toward those that are quantitative-based (10, 319). The benefits of incorporating QPCR and quantifying MST markers include an enhanced understanding of pollution intensity and an improved assessment of marker correlation with ot her water quality indicators, and more importantly, waterborne pat hogens. Also, compared to PCR, QPCR allows for a significant reduction of assay time. The focus of this research was the development of a QPCR assay for simultaneous detection of 2 hu man polyomaviruses as a rapid, sensitive, library-independent, human-specific indicator of water quality. 33

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Polyomaviruses Polyomavirus is the sole genus in the family Polyomaviridae (148) Polyomaviruses are host specific and can esta blish chronic infections in their natural hosts. These viruses are non-enveloped with an icosahedral capsid. The capsid is approximately 40-55 nm in diameter. The polyomaviruses have a circular, doublestranded DNA genome approximately 5,000 bp in length. The genome encodes for a large T antigen, small t antigen, and three vi ral proteins (VP1, VP2, and VP3) (264, 278). The International Committee on Taxonomy of Viruses curre ntly recognizes 16 polyomaviruses. The list includes BK virus (B KV), JC virus (JCV), KI virus (KIV), WU virus (WUV), Merkel cell virus (MCV), si mian vacuolating virus 40 (SV40), murine polyomavirus (PyV), hamster polyomavirus (HaPV), lymphotrophic papovavirus (LPV), budgerigar fledgling disease virus (BFDV), bovine polyomavirus (BPyV), kilham virus (KV), baboon polyomavirus-2 (PPV-2), rabbit kidney vacuolating virus (RKV), simian virus agent 12 (SA12), and ra t polyomavirus (RPV). In addition, an undefined polyomavirus has recently been detected in bat tissue samples (209). Human polyomaviruses Currently, there are five recognized hu man polyomaviruses (HPyVs); JCV (238), BKV (104), KIV (13), WUV (107), and MC V (46, 96, 105, 171). KIV ,WUV, and MCV are the most recently identified human polyomaviruses. KIV and WUV have been detected in respiratory samp les (31, 107). MCV has been associated with Merkel cell carcinoma and only been detected in tumor biopsy tissues (46, 96, 105, 171). WUV has been detected in human fecal samples; howev er, unlike JCV and BKV, these viruses have not been found in urine samples (30). The JCV and BKV human polyomaviruses (HPyVs) have similarly structured genomes that show 75% identity (80, 278). A 34

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symptomless primary infection typically occurs during childhood, followed by latent infections in the renal tissue, which can pe rsist indefinitely (9 1, 278). Asymptomatic viruria can occur occasionally or conti nuously in infected individuals (17, 140, 199, 252, 332). Disease generally occurs only wh en the hosts immune system becomes suppressed by conditions such as AIDS (278, 344). Serological studies have reported that 60-90% of the adult population harbor antibodies against human polyomaviruses (HPyVs) (139, 252, 279). The prevalence of th ese viruses in the human population is worldwide (37, 85, 248, 301). In general, HP yVs are associated with low morbidity, latency, symptomless reactivation, and life-l ong asymptomatic viruria. JC virus Disease caused by JCV is limited to indivi duals with comprised immune systems. The JC virus was first isolated in 1971 fr om a patient suffering from progressive multifocal leukoencephalopathy (PML) (240). JCV is now recognized as the pathogenic agent associated with PML in the immunocompromised (334). JCV isolated from the brain tissue of PML patients diffe rs from JCV isolated from urine and kidney samples. All non-PML JCV isolates contain a highly conserved regulatory region called the archetypal regulatory region ( 8, 67, 99, 198). In contrast, th e regulatory region of PMLassociated JCV contains a hypervariable regi on (6, 7, 20, 304). Studies have determined PML-associated JCV evolve within the patien t and are not circulating within the human population (278). The genome of all archetype JC viruses differs by only 1-2%, and these viruses do not differ antigenically (4, 278) Currently, there are 8 genoty pes and several subtypes of archetypal JCV known to be distributed with in the human population. Three methods 35

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have been used to type JC viruses. Thes e methods include comparison of sequence data based on: (1) a 215 bp region of the VP1 gene (7), (2) a 610 bp region of the VP1 and Tantigen genes (20, 122), and more recently (3) the entire genome (156). JC viruses are thought to have co-evolved with humans over the past 50,000 years, and consequently the different genotypes are associated with sp ecific geographical areas (164, 278). The genotypes and associated locations are summarized in Table 9 JCV excretion is not contingent on a suppressed immune system and JCV DNA is often found in the urine of healthy individuals (8, 37, 199, 252). The excretion of this virus in healthy individuals has allowed researchers to analyze urine fr om household family members over time. These studies report that second and third ge neration Japanese-Americans shed JCV with the Japan-associated genotype rather than JCV with the America-associated genotype (306). These findings, along with other longterm cohabitation studies in which JCV genotypes were household specific rather than geographically specific, suggest transmission occurs from parent to ch ild (36, 175, 306, 363). The genetic stability, worldwide distribution, and fre quent excretion in urine ma ke JCV a potentially ideal MST marker. BK virus The BK virus was also initi ally isolated in 1971. The virus was found in the urine of a male that had undergone a renal transplant (104). It is now documented that BKV reactivation can lead to inters titial nephritis (i.e. inflammation affecting the tubules of the kidney) and ureteral stenosis (i.e. narrowing of the ureter) in kidney transplant patients (139, 252). Disease caused by BKV is restrict ed to immunocompromis ed individuals. BKV is more readily excreted in imm unocompromised patients, including organ 36

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37 transplant patients, HIV patients and pre gnant women (165); however, excretion in immunocompetent individuals has been doc umented (30). Excretion in healthy individuals is less frequent than JCV though the seroprevelance of BKV is also worldwide. Moreover, BKV, unlike JCV, has been detected in feces of immunocompromised individuals (331, 350). Four BKV serotypes are known to exist in the human population. The serotypes include subtype I (BKV), subtype II (SB), s ubtype III (AS) and subtype IV (IV). The genetic difference among the 4 serotypes is <4% across the genome, with the primary source of variation in VP 1 gene sequence (80, 81). Based on genetic analysis, subtypes I and IV have been further divided into subgroups (308, 366). BKV infection is widespread in populations around the worl d. Recent evidence suggests that BKV subgroups, like JCV, are geographically associ ated (308, 366). In general subtype I is detected throughout the world, whereas subtype IV is prevalent in Asia and Europe, and subtype II and III are infrequently detected (278, 366). Subgroup I/b-2 is ubiquitous in Europeans and Americans of European de scent (366). Subgroup I/c is the most established BKV type in Asia (308, 366) Geographically-associated subgroups combined with early age seroprevalence imply familial transmission or congenital infection; however, the exact mode of transm ission has yet to be defined. While BKV is excreted less frequently than JCV, the genetic stability and worldwide distribution of both viruses make the simultaneous detection of both viruses a poten tially sensitive and reliable MST marker for human sewage contamination.

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Objectives of Study The purpose of this study was to develop a viral QPCR assay to detect human fecal pollution in environmental waters. Studies have reported that viruses, as compared to bacteria, have increased resistance to environmental stresses such as ultraviolet radiation and variable temperatures (136, 154, 256). Therefore, the utilization of a viral marker may more accurately mimic the fate a nd predict the presence of viral pathogens. Human polyomaviruses (JCV and BKV) were chosen as vira l indicators of human fecal pollution because they are unique to humans and exist in a high percentage of the human population (278). As stated previously, JCV a nd BKV are excreted in urine. An average adult produces approximately 1,200-1,500 ml of urine a day. The high volume of urine combined with the high prevalence of HPyVs ge nerates the possibility of potentially high titers of HPyVs in sewage. Efforts to estab lish water quality information using HPyVs as indicators may be confounded by excretion from recreational water users, thus the study design was modified to address this issue. The first part of this study was designed to confirm the presence of HPyVs in domestic sewage and septic tank wastes. In addition, investigations were made to determine whether or not the excretion of HPyVs underwent a seasonal occurrence. After preliminary studies, an improved method was developed to concentrate and detect HPyVs in water samples. The method devel oped to detect HPyVs in water samples was then applied to environmental samples susp ected of human fecal contamination. This study also compared results obtained from the detection of HPyVs in environmental water samples to results of the de tection of the human-associated Bacteroidales marker, 38

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39 M. smithii and/or adenovirus. Correlations amo ng the MST markers were assessed and compared to pathogen presence (i.e. adenovirus). In addition, mesocosm studies designed to examine marker persistence unde r various environmental conditions were performed. To address the potentially c onfounding excretion of HPyVs by individuals while swimming and assess the capacity of HP yVs to predict presen ce of pathogens; the latter part of this study included an epid emiological study comparing the presence and concentrations of HPyVs w ith human health risks.

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Table 1.Bacterial and protozoan associated disease outbreaks after exposure to recreati onal water bodies in the United States f rom 1991-2006. Year State Primary Etiologic Agenta Type of Illnessb Reported Cases Exposure Possible Source(s) of Contamination Reference 1991 Oregon E. coli O157:H7 AGI 80 Lake Unknown (213) 1991 Washington Giardia AGI 4 Lake Unknown (213) 1991 Washington Giardia AGI 4 Lake Unknown (213) 1991 Illnois Leptospira Lep 6 Pond Unknown (213) 1991 Pennsylvania S. sonnei AGI 203 Lake Unknown (213) 1991 Rhode Island S. sonnei AGI 23 Lake Unknown (213) 1991 Detroit Schistosome Skin 30 Ocean Unknown (213) 1991 Utah Schistosome Skin 5 Lake Unknown (213) 1992 New Jersey S. sonnei AGI 54 Lake Unknown (213) 1992 Virginia S. sonnei AGI 9 Lake Unknown (213) 1992 New Jersey Schistosome Skin 40 Lake Unknown (213) 1993 Washington Giardia AGI 6 River Unknown (169) 1993 New Jersey Giardia AGI 43 Lake Unknown (169) 1993 Maryland Giardia AGI 12 Lake Unknown (169) 1993 Ohio S. sonnei AGI 160 Lake Unknown (169) 1994 New Jersey C. parvum AGI 418 Lake Unknown (169) 1994 Pennsylvania C. parvum AGI 8 Lake Unknown (169) 1994 New York E. coli O157:H7 AGI 166 Lake Unknown (169) 1994 Minnesota E. coli O157:H7 AGI 5 Lake Unknown (169) 1994 New Jersey S. sonnei AGI 300 Reservoir Unknown (185) 1994 New Jersey S. sonnei AGI 242 Lake Unknown (169) 1994 Minnesota S. flexneri AGI 35 Lake Unknown (169) 40

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41 Year State Primary Etiologic Agenta Type of Illnessb Reported Cases Exposure Possible Source(s) of Contamination Reference 1995 Illnois E. coli O157:H7 AGI 12 Lake Unknown (185) 1995 Wisconsin E. coli O157:H7 AGI 8 Lake Unknown (185) 1995 Minnesota E. coli O157:H7 AGI 6 Lake Unknown (185) 1995 Minnesota E. coli O157:H7 AGI 2 Lake Unknown (185) 1995 Pennsylvania S. sonnei AGI 70 Lake Unknown (185) 1996 Indiana C. parvum AGI 3 Lake Unknown (185) 1996 Minnesota E. coli O157:H7 AGI 6 Lake Unknown (185) 1996 Colorado S. sonnei AGI 81 Lake Unknown (185) 1996 Colorado S. sonnei AGI 39 Lake Unknown (185) 1996 Oregon Schistosome Skin 71 Lake Unknown (185) 1996 Oregon Schistosome Skin 50 Lake Unknown (185) 1997 Tennessee Cryptosporidium AGI 28 Lake Unknown (360) 1997 Missouri E. coli O157:H7 AGI 8 Lake Unknown (24) 1997 Oregon Schistosome Skin 2 Lake Unknown (24) 1998 Illnois Leptospira Lep 375 Lake Unknown (24) 1999 Connecticut E. coli O121:H19 AGI 11 Lake Fecal contamination of water by infected person(s)c (184, 320) 1999 Washington E. coli O157:H7 AGI 36 Lake Fecal contributions by humans and ducks (184, 266) 1999 Washington E. coli O157:H7 AGI 36 Lake Fecal contamination of water by infected person(s)d (184, 320) 1999 Wisconsin E. coli O157:H7 AGI 5 Lake Unknown (184) 1999 Florida E. coli O157:H7 AGI 2 Ditch Unknown (184) 1999 Massachusetts Giardia AGI 18 Pond Unknown (184) 1999 Oregon Schistosome Skin 2 Lake Unknown (184) 2000 Minnesota C. parvum AGI 220 Lake Washing children in water while changing diapers (184, 320) 2000 Guam Leptospira Lep 21 Lake Unknown (184)

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42 Year State Primary Etiologic Agenta Type of Illnessb Reported Cases Exposure Possible Source(s) of Contamination Reference 2000 Minnesota S. sonnei AGI 25 Lake Fecal contamination of water by infected person(s) (184, 320) 2000 Minnesota S. sonnei AGI 15 Lake Unknown (184) 2000 California Schistosome Skin 6 Pond Unknown (184) 2000 California Schistosome Skin 4 Pond Unknown (184) 2001 Wyoming Cryptosporidium AGI 2 Hot Spring Unknown (359) 2001 Minnesota E. coli 026:NM AGI 4 Lake Unknown (359) 2001 South Carolina E. coli O157:H7 AGI 45 Lake Fecal contamination (320, 359) 2001 Minnesota E. coli O157:H7 AGI 20 Lake Geese (320, 359) 2002 Oregon Schistosome Skin 19 Lake Unknown (359) 2002 Wyoming Cryptosporidium AGI 3 Lake Unknown (360) 2002 Wyoming Giardia AGI 2 River Human or animal contamination (320, 360) 2002 New York S. sonnei AGI 20 Lake Unknown (360) 2003 Idaho Cryptosporidium AGI 4 Lake Bathers (93, 320) 2003 Ohio P. shigelloides AGI 3 Lake Unknown (93) 2003 Wyoming P. shigelloides AGI 2 Reservoir Unknown (93) 2003 Georgia S. sonnei AGI 13 Lake Fecal contamination of water by infected pe rson(s) (93, 320) 2003 Maryland S. sonnei AGI 65 Lake Dumping fecal wastes into watere (93, 320) 2004 Missouri Giardia AGI 9 Lake Unknown (93) 2004 Guam Leptospira Lep 3 River Unknown (93) 2004 Hawaii Leptospira Lep 2 Stream Unknown (360) 2004 Arkansas S. flexneri AGI 10 Lake Unknown (93) 2005 New York Cryptosporidium AGI 27 Lake Unknown (360) 2005 Minnesota E. coli O157:H7 AGI 4 Lake Unknown (360) 2005 Florida Leptospira Lep 43 Stream Unknown (360)

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43 Year State Primary Etiologic Agenta Type of Illnessb Reported Cases Exposure Possible Source(s) of Contamination Reference 2005 California Leptospira Lep 3 Stream Unknown (360) 2005 Pennsylvania S. sonnei AGI 15 Lake Unknown (360) 2005 Minnesota S. sonnei AGI 12 Lake Unknown (360) 2005 Massachusetts S. sonnei AGI 5 Lake Unknown (360) 2006 Massachusetts Cryptosporidium AGI 6 Pond Unknown (360) 2006 Tennessee E. coli O157:H7 AGI 3 Lake Unknown (360) 2006 Wisconsin E. coli O157:H7 AGI 3 Lake Unknown (360) a E. coli, Escherichia coli ; S. sonnei Shigella sonnei ; S. flexneri Shigella flexneri ; C. parvum Cryptosporidium parvum; P. shigelloides Plesiomonas shigelloides b AGI, acute gastroenteritis illness; Skin, clinical sy mptom(s) related to skin; Lep, leptospirosis c Toddler with severe diarrhea d no agricultural sources identified eDiapers

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Table 2. Viral associated disease outbreaks after exposure to recreational water bodies in the United States and Mexico from 19 912006. Year Location Primary Etiologic Agent Type of Illnessa Reported Cases Exposure Possible Source(s) of Contamination Reference 1991 North Carolina Adenovirus 3 ARI, Eye 595 Pond Unknown (213) 1994 Washington Norovirus AGI 18 Lake Unknown (169) 1994 Ohio Norovirus AGI 30 Lake Unknown (169) 1996 Idaho Norovirus AGI 55 Lake Unknown (185) 1998 Minnesota Norovirus AGI 15 Lake Unknown (359) 1998 Wisconsin Norovirus AGI 18 Lake Unknown (24) 1998 Ohio Norovirus AGI 30 Lake Unknown (24) 1999 Idaho Norovirus AGI 25 Hot springs Unknown (359) 1999 Idaho Norovirus AGI 25 Hot springs Unknown (184) 1999 New York Norovirus AGI 168 Lake Fecal contamination of water by infected person(s) (320, 359) 2001 Minnesota Norovirus AGI 40 Lake Unknown (359) 2002 Minnesota Norovirus AGI 11 Lake Unknown (359) 2002 Minnesota Norovirus AGI 11 Lake Unknown (359) 2002 Wisconsin Norovirus AGI 44 Lake Bathers and/or dumping fecal waste from boats (320, 359) 2002 Arizona Norovirus AGI 130 River Unknown (284) 2003 Arizona Norovirus AGI 22 River Unknown (284) 2004 Minnesota Norovirus AGI 9 Lake Unknown (93) 2004 Mexico Coxsackievirus A1 AGI 21 Ocean Unknown (26) 2004 Mexico Echovirus 30 AGI 21 Ocean Unknown (26) 2004 Oregon Norovirus AGI 39 Lake Unknown (93) 2004 Oregon Norovirus AGI 150 Lake Unknown (284) 2004 Siberia Echovirus AGI 294 Lake Unknown (284) 2005 Minnesota Norovirus AGI 8 Lake Unknown (360) 44

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45 Year Location Primary Etiologic Agent Type of Illnessa Reported Cases Exposure Possible Source(s) of Contamination Reference 2006 Minnesota Norovirus G1 AGI 10 Lake Unknown (360) 2006 Florida Norovirus AGI 50 Lake Unknown (360) 2006 Florida Norovirus G2 AGI 50 Lake Unknown (360) a ARI, acute respiratory illness; Eye, clinical symptom(s) re lated to the eye; AGI, acute gastroenteritis illness

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Table 3. Disease associated with unidentified pathogens after e xposure to recreational water bodies in the United States from 19912006. Year State Etiologic Agent Type of Illnessa Reported Cases Exposure Possible Source(s) of Contamination Reference 1992 Maryland Unidentified AGI 15 Creek Unknown (213) 1994 Maine Unidentified AGI 650 Lake Unknown (169) 1994 Washington Unidentified AGI 248 Lake Unknown (169) 1994 Washington Unidentified AGI 41 Lake Unknown (169) 1994 Florida Unidentified AGI 12 Lake Unknown (185) 1995 Pennsylvania Unidentified AGI 17 Lake Unknown (185) 1995 Minnesota Unidentified AGI 12 Lake Unknown (185) 1996 Oregon Unidentified AGI 32 Lake Unknown (185) 1996 Indiana Unidentified AGI 4 Lake Unknown (185) 1999 New York Unidentified AGI 140 Lake Unknown (360) 1999 Illnois Unidentified AGI 25 Lake Unknown (184) 2000 Maine Unidentified AGI 32 Lake Unknown (184) 2000 Florida Unidentified AGI 2 Lake Unknown (184) 2001 New Hampshire Unidentified AGI 42 Lake Unknown (359) 2002 Maine Unidentified AGI 33 Puddle Unknown (359) 2002 Florida Unidentified AGI 7 Lake Unknown (359) 2003 Florida Unidentified AGI 20 Lake Unknown (93) 2003 Florida Unidentified AGI 10 Lake Unknown (93) 2003 California Unidentified Skin 9 Lake Unknown (93) 2003 Ohio Unidentified Skin 6 Lake Unknown (93) 2004 Wisconsin Unidentified AGI 18 Lake Unknown (93) 2004 Georgia Unidentified Ear 9 Lake Unknown (93) 46

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47 Year State Etiologic Agent Type of Illnessa Reported Cases Exposure Possible Source(s) of Contamination Reference 2005 Florida Unidentified AR I 24 Ocean Unknown (360) 2005 New York Unidentified AGI 13 Lake Unknown (360) 2005 Maine Unidentified AGI 10 Lake Unknown (360) 2005 California Unidentified Skin 2 Lake Unknown (360) 2006 Ohio Unidentified Skin 2 Pond Unknown (360) aAGI, acute gastroenteritis illness; Skin, clinical symptom(s) related to the skin; Ear, clinical symptom(s) related to the ear

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Table 4. The number of reported cases of Vibrio spp. associated disease after exposure to recreational water bodies in the United States from 2003-2006. Years Location Etiologic Agent Reported Cases Exposure Reference 2003-2004 United States Vibrio alginolyticus 24 Recreational water (93) 2003-2004 United States Vibrio chlorae non O1, non O139 3 Recreational water (93) 2003-2004 United States Vibrio damsela 1 Recreational water (93) 2003-2004 United States Vibrio fluvialis 1 Recreational water (93) 2003-2004 United States Vibrio parahaemolyticus 12 Recreational water (93) 2003-2004 United States Vibrio vulnificus 20 Recreational water (93) 2003-2004 United States Vibrio species not identified 2 Recreational water (93) 2005-2006 United States V. alginolyticus 60 Recreational water (360) 2005-2006 United States V. parahaemolyticus 33 Recreational water (360) 2005-2006 United States V. vulnificus 67 Recreational water (360) 2005-2006 United States Vibrio, species not identified 29 Recreational water (360) 48

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Table 5. Summary of contamination s ources in recreational wa ter outbreaks in the United States from 1971-2000.* Source of Contamination Percentage of Outbreaks w ith Listed Contamination (%)a Bather overloading 34 Fecal contamination of water by infected person(s) 31 Children swimming/wading in diapers 25 Malfunctioning sewage systems 21 Animals 18 Rainfall 3 49 *Adopted from Craun et al. (78) aSome outbreaks had >1 contamin ation contributions, therefore the total percentage is >100

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Table 6: United States Environmental Protection Agency r ecommended regulatory standards for E. coli and enterococci in surface waters (323). CFU per 100 mla Indicator Guidelines Freshwater Marine Water E. coli Monthly Geomean b 126 N/Ac Single Day Maximum 235 N/A Enterococci Monthly Geomean 33 35 Single Day Maximum 61 104 aCFU, colony forming unit bMonthly Geomean, the geometric mean of at least 5 samples taken on different days over a 30 day period cN/A, Specific criterion is not defined 50

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Table 7. Current Florida regulat ory standards for indicator bacteria set by Fl orida Administrative C ode 62-302.530 with modific ations to meet EPAs BEACH Water Pr ogram requirements (100, 326). Class III Indicatora Guidelines Class I Class II Freshwaters Marine Waters Fecal Coliforms Monthly Geomeanb 200 14 200 N/Ac Exceptions 400 in 10% of the samples 43 in 10% of the samples 400 in 10% of the samples N/A Single Day Maximum 800 800 800 N/A Enterococcid Monthly Geomean N/A N/A 33 35 Single Day Maximum N/A N/A 61 104 51 aIndicator regulatory standards reported as colony forming units (CFU) per 100 ml bMonthly Geomean, the geometric mean of at least 5 samples taken on different days over a 30 day period cN/A, Specific criterion is not defined d Enterococci marine water quality criteria apply to all coastal public bathing beaches

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Table 8. An overview of the types of microbial source tracking methods. Description Type of Method Library construction Samples Examples References Phenotypic Library-dependent Target microorganism(s) are collected from know n fecal sources and typed based on defined biochemical analyses. The results of the analyses are compiled into a database and categorization by source is done statistically or by direct matching. Microorganism(s) isolated from water samples are analyzed using the defined biochemical analysis. The source of the microorganism(s) is determined based on significant similarities of phenotypic patterns compared to the results of the known-source database microorganisms. Antibiotic resistance patterns of E. coli or enterococci E. coli serotyping Harwood et al. (132) Parveen et al. (245) Wiggins et al. (347) Crichton et al. (79) Gonzalez et al. (115) Phenotypic Library-independent None. Water samples are analyzed for the direct presence of certain culturable organism(s) with a distinguishable source-specific biochemical reaction. Sorbital fermenting Bifidobacteria Bonjoch et al. (41) Mara et al. (196) Genotypic Library-dependent Target microorganism(s) are collected from know n fecal sources. DNA fingerprints from knownsource isolates are produced by molecular typing methods (e.g. endonucleases and/or PCR). DNA fingerprints are compiled into a database and categorization by source is done statistically or by direct matching. DNA fingerprints are produced from bacteria isolated from water. The source of the microorganism(s) is determined based on significant similarities of its DNA fingerprint compared to the results of the known-source DNA fingerprint database. Pulsed field gel electrophoresis Repetitive element PCR Ribotyping Myoda et al. (219) Simmons et al. (282) Stoeckel et al. (299) Dombek et al. (89) Malathum et al. (194) Carson et al. (58) Hartel et al. (127) Moore et al. (215) Myoda et al. (219) Parveen et al. (246) 52

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53 Description Type of Method Library construction Samples Examples References Genotypic Library-independent None. Molecular techniques are used to target a unique, species-specific microorganism(s) or a molecular marker(s) (e.g. gene) in a microorganism(s). The specific source is distinguished by the presence/absence (or quantity) of the unique target. F-specific coliphage genotyping Enterococcal surface protein Bacteroidales genotyping Methanobrevibacter smithii Human enteric viruses Cole et al. (74) Hsu et al. (144) Schaper et al. (269) Scott et al. (272) Kreader et al. (170) Bernhard et al. (27) Ufnar et al. (330) Chapron et al. (69) Jiang et al. (153) Pina et al. (251)

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Table 9. The observed geographical dist ribution of JC virus genotypes. Genotypesa Subtypesa Location References 1 Europe; United States b (5, 164, 278) 1A [A, EU] East Europe (5) 1B [A, EU] Southwest Europe (5) 2 Asia (5, 164, 278) 2A [B, MY] Americasc; Asia: China, Japan, South Korea (92, 278, 361) 2B [B1-c] Europe and Asia (92, 278) 2D [B1-b] Mongolia and India (122, 334) 2E [B3-b] Oceania: Austra lia, Mindanao, Guam (208) 3 Africa (5, 164, 278) 3A [B, AF2] Africa (134) 3B [B, AF2] Africa (134) 4 [A, EU] United States b ; Europe: Germany, Poland, Spain, Basque, Italy (5, 92, 164, 278) 5 Asia (134, 164) 6 C (AF1) Africa (5, 134, 164, 278) 7 Asia (5, 164, 278) 7A [B, SCf] Southeast Asia: China, Taiwan, Philippines, Nepal (122, 208, 268, 278) 7B [B, CY] China; Vietnam (122, 268) 7C1 [B1-a] China (122, 268) 7C2 [B2] China; Nepal (268, 278) 8 Papua New Guinea (164, 278) 8A Papua New Guinea (278) 8B Papua New Guinea (278) a Alternate nomenclature is presented in the brackets. b Genotype only found in individuals of European descent. c Genotype found in Native American Indians. 54

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CHAPTER 2: QUANTIFICATION OF HUMAN POLYOM AVIRUSES, JCV AND BKV, BY TAQMAN QUANTITATIVE PCR AND COMPARISON TO OTHER WATER QUALITY INDICATORS IN WATER AND FECAL SAMPLES Shannon M. McQuaig1, Troy M. Scott2, Jerzy O. Lukasik2, John H. Paul3, and Valerie J. Harwood1* 1Department of Biology, University of South Florida, Tampa, FL 33620 2Biological Consulting Services of No rth Florida, Gainesville, FL 32609 3College of Marine Science, University of South Florida, St. Petersburg, FL 33701 Published in: Applied and Environmental Microbiology, June 2009, Vol. 75 (11):3379-88 55

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Abstract Total maximum daily load (TMDL) sta ndards for water bodies that do not meet bacterial water quality standards are set by each State. The presence of human polyomaviruses (HPyVs) can be used as an indicator of human-associated sewage pollution in these waters. We have deve loped and optimized a Taqman quantitative PCR (QPCR) assay based on the conserve d T-antigen to quantify as well as simultaneously detect two HPyVs; JCV a nd BKV. The QPCR assay was able to consistently quantify > 10 gene copies per reaction and is linear over five orders of magnitude. HPyVs were consistently detect ed in human waste samples (57 of 64) and environmental waters with known human fecal contamination (5 of 5), and were not amplified in DNA extracted from 127 animal waste samples from fourteen species. HPyVs concentrations in sewage decreased 81.2% and 84.2% over 28 days incubation at 25C and 35C, respectively. HPyVs results were compared to Escherichia coli fecal coliform and enterococci concentrations and the presence of 3 other human-associated microbes: human-associated Bacteroidales, Meth anobrevibacter smithii and adenovirus. HPyVs were the most frequently detected of these in human and contaminated environmental samples, and were more human-specific than the Bacteroidales (HF183) or M. smithii HPyVs and M. smithii more closely mimicked the persistence of adenovirus in sewage than the other microbes. The use of this rapid and quantitative assay in water quality research could help regulatory agencies to identify sources of water pollution for improved remediation of cont aminated waters and ultimately protect humans from exposure to pathogens. 56

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Introduction Maintaining healthy coastal water systems is essential, as poor water quality can have detrimental effects on mangroves, seagrass beds, coral reefs, fishing and shellfish harvesting industries, and the health of recreational water users (1, 16, 102, 126, 137, 307). Since 1972, each State has been requi red to set total maximum daily loads (TMDLs) for pollutants in water bodies accordin g to section 303(d) of the Clean Water Act (325). The probability that microbial pathogens are present is estimated by enumerating indicator bacteria, which are shed in the feces of humans and most animals. The Environmental Protection Agency (EPA) recommends Escherichia coli and enterococci to assess the quality of fresh and saline water, respectively (323); however Florida currently employs fecal coliforms and enterococci as indicators of fecal pollution (323). When bacterial indicators exceed re gulatory levels, a plan of action (TMDL implementation) must be developed to re duce pathogens. TMDL plans for pathogen reduction are particularly problematic becau se they rely upon surrogate indicator bacteria, which yield little or no insight as to the source of pol lution. High indicator bacteria concentrations can be attributed to many sources including agricultural runoff, storm water runoff, wildlife, pets, faulty septic systems (onsite wastewater treatment and disposal systems), and failing central sewer infrastructure (16, 45, 200). To address the issue of source identification, methods have b een developed in which the biochemistry or genetics of certain microorganisms are used to indirectly identify probable source(s) of fecal pollution, which is termed microbial source tracking (MST) (319). MST methods based on detection of a source-associated ge ne (marker) by PCR have proliferated over the past ten years due to the additional in formation they can provide to watershed managers on fecal contamination sources (298 ). While marker detection by end-point 57

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(binary) PCR can give important insights on the source(s) of fecal contamination, quantitative measurements can provide info rmation about the relative magnitude of contamination from various sources. Mo reover, epidemiological studies on the correlation between recreational water use, microbial contamination and the risk of illness will greatly benefit from the ability to quantify MST markers, rather than simply assessing binary (+/-) detection. Although many bacterial targets have been proposed for MST of human sewage (27, 272, 330), fewer viral targets have been investigated (136, 155, 226). Polyomavirus is the sole genus in the family Polyomaviridae (148) These viruses have a 5-kbp doublestranded DNA genome surrounded by an 4050nm icosahedral capsid (264). The JCV and BKV human polyomaviruses (HPyVs) have similarly structured genomes that show ~75% identity (139). BKV and JCV gained much attention in the late 1970s as the etiological agents of kidney nephritis (i.e. BKV reactivation in the kidneys) and progressive multifocal leukoencephalopathy (i.e JCV reactivation in brain tissue) in the immunocompromised (104, 239). Serological st udies have shown that >70% of adults harbor antibodies against BKV or JCV ( 27, 30, 44). These viruses are known for producing lifelong, asymptomatic viruria in immunocompetent indivi duals (252). In 2000 it was first suggested that JCV would be a useful indicator of human sewage in water (37). The obligate host specificity a nd abundance of BKV and JCV in municipal sewage has lead to the successful use of these viruses to indi cate human fecal pollution in environmental water samples (45, 202). Due to the health implications of B KV and JCV, several methods have been developed to rapidly detect either BKV or JCV in clinical samples (17, 216, 241, 343). 58

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However, from a microbial source tracking stand point, it is advantage ous to target both BKV and JCV. BKV has been found in feces (3 31) and both viruses are excreted in the urine (17, 37, 252, 332, 367) either simultaneously or individually. The focus of this research was the modification of the previ ously developed nested PCR protocol for HPyVs detection (202) to a Taqman quantitative PCR (QP CR) assay to simultaneously detect and quantify both BKV and JCV. Furthermore, this study compared measurements obtained with the newly deve loped QPCR assay to those of other water quality indicators and MST markers. Thes e indicators included bacterial indicator concentrations (323), and PCR detection of human-associated markers currently used for MST. These included human-associated Bacteroidales (27), Methanobrevibacter smithii (330), and adenovirus (251). To assess the pot ential of HPyVs to mimic the fate of pathogens in water, persiste nce of all the water qualit y indicators was assessed and relationships between bacter ial indicator organisms and MST markers in both human waste samples as well as contaminated e nvironmental samples were examined. Materials and Methods Virus strains and viral DNA extraction BK virus (ATCC VR-837), JC virus (ATCC VR-1583) and adenovirus type 2 (ATCC VR-846) were obtained from the American Type Culture Collection (Manassas, VA). BK viruses were propagated in HEL299 cells (ATCC CCL-137). JC viruses were propagated in COS-7 cells (ATCC CRL-1651). Adenoviruses were propagated in MRC5 cells (ATCC CRL-171). Cell lines were gr own in Eagle minimum essential medium (Sigma, St. Louis, Mo.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Inc., Carlsbad, CA). Cell lines were maintained in Eagle minimum 59

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essential medium containing 2% FBS. Vira l DNA was extracted from cell culture using DNeasy Blood & Tissue Kit (Qiagen, Inc., Valenc ia, CA), and stored at -20C. Primer and probe design Primers P5 and P6 have been used successfully in conventional PCR reactions to simultaneously detect both JCV and BKV in clinical (19) and environmental samples (202). However, based on stipulations of Taqman PCR assay design, P5 was modified to increase the optimum annealing temperatur e. The primers target a region of the Tantigen in both JCV and BKC. JCV and BKV DNA sequences within the amplified partial T-antigen were obtained from GENBANK. Approximately 10 JCV and 10 BKV sequences were analyzed. The sequences were aligned using the ClustalW Program (European Bioinformatics Institute, UK), a nd probe sequences were chosen based on identical areas of the gene se quence. Probe sequences were also chosen based on melting temperature (Tm) stipulations (5-10C higher than Tm of primers) and base stipulations [(a) no long runs of the same base, (b) 2 G or C nucleotides in th e final 5 bases of the 3 end; and (c) A, C, or T on the 5 end]. Th e probe was labeled with a fluorescent reporter molecule, 6-carboxyfluorescein (FAM), at the 5 end and a minor groove binding, nonfluorescent quencher molecule (MGBNFQ) at the 3 end. Primers were purchased from Integrated DNA Technologies (http://www. idtdna.com, Coralville, IA), and probes were purchased from Applied Biosystems (Fos ter City, CA). Primers (SM2 and P6) and probe (KGJ3) sequences used in this study are presented in Table 10 Quantitative PCR The QPCR mixtures were prepared using 25 l TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems Foster City, CA), 0.5 M primer 60

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concentrations, 0.4 M labeled probe concentr ation, 5 l of template DNA (5-15 ng/l), and the volume was adjusted to 50 l using reagent grade water. Amplification was performed in the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). Optimal temperatures and primer and probe concentrations were determined by varying conditions over both temperat ure and concentration gradients (data not shown). The QPCR reaction conditions were as follows: DNA polymerase activation at 95C for 10 min, followed by 40 cycles of DNA melting at 95 C for 15 sec, then annealing at 55C for 15 sec, and extension at 60C for 60 sec. Sequencing of QPCR amplicon DNA was extracted from 0.1 ml of the BK virus and JC virus stock using DNeasy Blood & Tissue Kit (Qiagen, Inc., Valencia, CA ), and used as template in a real time PCR assay. The resulting 176-bp and 173-bp amp licons from the BK virus and JC virus reactions, respectively, were purified using QIAquick PCR Puri fication Kit (Qiagen, Inc.) and then cloned into pCR-TOPO vector (Invitrogen, Inc.) and transferred into E. coli One Shot chemically competent cells, a nd plated on LB agar containing 100 gml-1 ampicillin. Recombinant plasmids with a single copy of the partial T-antigen were purified using GenElute Five-Minute Plasmid Miniprep Kit (Sigma, St. Louis, MI) following manufacturers instructions. Plas mids containing the BKV (176 bp) or JCV (173 bp) insert sequence were sequenced at Macrogen U.S.A. (Rockville, MD). All sequences were subjected to BLAST search (www.ncbi.nlm.nih.gov/BLAST) for comparison to published sequences. Query a nd matching sequences were aligned using CLUSTAL W 61

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Standard curve and sensitivity of QPCR Purified recombinant plasmid DNA containing the BKV or JCV insert was quantified using a QubitTM fluorometer (Invitrogen, Inc., Carlsbad, CA). DNA quantification was performed in triplicate and averaged to determine the estimated total DNA concentration. The recombinant plasmids were serially diluted and the limit of detection for both inserts was determined base d on the lowest level of detection in the real time PCR reaction. Insert copy numbers were estimated by multiplying the average DNA concentration by Avogadros number then dividing by the product of the length and average weight of a base pa ir (362). To produce a sta ndard curve, the recombinant plasmid DNA was serially diluted in nucl ease-free reagent grade water to a final concentration ranging from 102 to 106 gene copiesl-1. The plasmid containing the BK insert was used to produce standard curves for both the sewage holding time experiments and analysis of HPyVs concentr ations of environmental waters. Five microliters of each dilution were used as template in the Taqm an real time standard curve PCR reactions. Each dilution was run in duplicat e. Applied Biosystems defau lt settings for the threshold cycle (Ct) were used for data analysis. Th e Ct values were plotted against copy number to generate the standard curve. Linear re gression was used to assess the relationship between Ct values and copy number. Diluted DNA was used in all subsequent reactions to create the standard curve for quantif ication of HPyVs in unknown samples. Specificity of HPyVs QPCR Fresh animal waste samples (e.g. dog feces and cow manure) were collected from local farms, wooded areas, and households. Individual fecal samples were collected from cats (n=5), chicken (n=1), cows (n=24) cranes (n=2), deer (n=3), dogs (n=55), 62

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ducks (n=4), fox (n=1), horses (n=8), raccoon (n=1), seagulls (n=6), sparrows (n=3), and feral pigs (n=2). Samples were collected in sterile propylene tubes, placed on ice and transported or shipped to the laboratory. All fecal samples were proc essed within 24 h of collection. Approximately 0.3g of animal waste was used for DNA extraction. DNA was extracted using MO BIO UltraClean Fecal DNA kit (Carlsbad, CA), per manufacturers instructions. Composite cow and pig fecal samples were collected from flushed manure holding tanks at the Universi ty of Floridas Dairy Research Unit and Swine Unit. DNA was extracted from 1 ml of the composite sample using QIAamp Blood DNA Midi Kit, with minor modifications. Briefly, 1 ml of the sample and 1 ml of phosphate buffered saline (PBS) were added to a 15 ml tube, then 200 l of Qiagen proteinase and 2.4 ml of Buffer AL were added to the tube containing the sample. The tube was then incubated at 70C for 15 min. DNA was then extracted from the lysate as per manufacturers suggestions. To assess the condition of samples and presen ce of PCR inhibitors, all animal fecal DNA was first subject to total Bacteroidales spp. PCR (27). Briefl y, the PCR reactions were prepared using 12.5 l GoTaq Green Master Mix (Promega Corporation, Madison, WI), 0.5 M primer concentrations ( Table 10 ), 2 l of template DNA (5-15 ng/l), and the volume was adjusted to 25 l using reagent grade water. The PCR reaction conditions were as follows: DNA polymerase activation at 95C for 3 min, followed by 30 cycles of DNA melting at 94C for 45 sec, then annealing at 55C for 45 sec, and extension at 72C for 60 sec, and a final extension at 72C for 5 min (Eppendorf Mastercycler Thermocycler, Eppendorf International, Hamburg, Germany). PCR products were separated by agarose gel elect rophoresis (2%). DNA was viewed using 63

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ethidium bromide under UV light. Amplicons were identified visually by comparison to a BenchTop 100bp DNA ladder (Promega Corpor ation) and a 676 bp positive control. DNA from fecal samples was not used for further analysis when the total Bacteroidales target was not amplified by PCR. Urine samples from both cats (composite of 3 cats) and dogs (n=9) were obtained from post-euthanized (within 1 hr) animals at animal shelters. The bladder was expressed manually and urine was caught directly in a sterile specimen cup. Samples were then placed on ice, shipped to the laboratory and stored at -20C until processed. All urine samples were processed within 7 days of co llection. DNA was extracted from 1 ml of the urine sample using QIAamp Blood DNA Midi Kit, as previously described. To test for inhibition, samples were spiked with 102 BKV particles and processed in tandem with samples. Extracted DNA was used as template in QPCR assays as described above. Human target samples A total of 90 human waste and human ur ine samples were collected. Of the human waste samples collected, 41 were ra w sewage (wastewater treatment plant (WWTP) influents or li ft stations), 9 were dechlorinate d tertiary treated WWTP effluent, 9 were from septic tank pump trucks (composite septic tank samples), and 5 were from individual septic tanks (2-4 individuals in the households). Raw sewage was collected from Brandon, FL (n=21); Costa Mesa, CA (n =3); Gainesville, FL (n=10); Oldsmar, FL (n=3); Orlando, FL (n=2); and Tampa, FL (n =2). Dechlorinated tertiary treated WWTP effluent was collected from Brandon, FL (n=9). Pump truck sample s were acquired from the greater Tampa, FL area (n=9). Septic tank samples were collected from Cocoa, FL (n=2) and the greater Tampa, FL area (n=3). All samples were collected in sterile 64

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propylene tubes or liter bottles, placed on ice, and transported or shipped to the laboratory. A total of 26 indivi dual urine samples were analyzed. Samples were collected from healthy volunteers ranging in age from 2-57 years old. Volunteers collected urine in sterile specimen cups, and then placed sa mples at 4C. All samples were processed within 24 h of collection. DNA was extracted from sewage, septic tank, pump truck and urine using the QIAamp Blood DNA Midi Kit with minor modifi cations, as previously described. Virus particles were concentrated from 500 ml of d echlorinated tertiary treated WWTP effluent. The pH of the water was adjusted to 3.5 us ing 2.0 N HCl and was filtered through a 0.45 m pore size, 47 mm diameter nitrocellulose filter. The filter was placed into a 2 ml microcentrifuge tube and placed at -20C. DNA was extracted from the filter within 24 h of filtration using MO BIO Powersoil Kit (Carlsbad, CA), per manufacturers instructions. Extracted DNA was used as template in QPCR assays as previously described. Six amplicons from human-associated samples were confirmed by sequencing and analysis by MEGA 4 (as previously described). One transformed E. coli colony from each cloning reaction was analyzed. Analysis of water quality indicators in se wage over a 28-day period (sewage holding time experiment) Three 100 ml aliquots of ra w sewage were placed in a dark area at room temperature (23.6.8C) or in an incubator ( 34.0.1C) to represent a range of ambient water temperatures in Florida during summer months. Room temperature mimics coastal water temperatures common to Florida coastlines, while the hot temperature more closely mimics temperatures of swallow waters (e.g. ditches, small streams, etc.) that can become extremely warm during mid-summer days One 1 ml aliquot was collected from 65

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each replicate on days 0, 7, 14, 21 and 28. DNA was extracted from the samples using the QIAamp DNA Blood Midi Kit and used as template in QPCR assays as previously described. Environmental water samples Five environmental water samples w ith high probability of human fecal contamination were collected from various loca tions in the state of Florida. DHR4a was collected from Hillsborough River (Tampa, FL ) near an overflowing lift station. Env1 and Env2 were collected from a beach (Tampa, FL) receiving contamination due to a broken sewer line. EJ3 was collected in Char lotte County, FL near re sidential areas with faulty septic tanks. SJH8 was collected from a tributary of St. Johns River (St. Augustine, FL) near a malfunctioning septic tank. Approximately 500 ml of water was placed into a sterile one liter polypropylene co ntainer and transported to the laboratory on ice. Salinity was measured using a hand refr actometer (Fisher Scien tific, Pittsburgh, PA). All environmental samples were processed within 6 hrs of collection. To promote electrostatic interactions betw een the viral capsid and nitroce llulose filter, the pH of the water was adjusted to 3.5 using 2.0 N HCl and was filtered through a 0.45 m pore size nitrocellulose filter (14, 189). The filter was pl aced into a 2 ml micr ocentrifuge tube and placed at -20C. DNA was extracted from the filter using MO BIO Powersoil Kit (Carlsbad, CA), per manufacturers instructi ons. HPyVs copy numbers were enumerated by the QPCR assay as described above. Four amplicons from contaminated environmental water samples were conf irmed by DNA sequencing and analysis by MEGA 4 (as previously described). 66

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Comparison of HPyVs QPCR to other methods HPyVs QPCR results were compared to c oncentrations of indicator bacteria and PCR detection of human-associated Bacteroidales, Methanobrevibacter smithii and adenovirus (see below for methods). Indicato r bacteria were enumerated in all human waste, sewage holding time and environmental water samples. PCR was used to detect human-associated Bacteroidales M. smithii, and adenovirus in human waste, sewage holding time and environmental water samples. In addition, PCR was used to examine the presence or absence of human-associated Bacteroidales and M. smithii in all animal samples. Enumeration of indicator bacteria Escherichia coli fecal coliforms, and enterococci were enumerated in human waste samples and environmental water samples. In addition, sewage samples held at room temperature or 35C were filtered weekly for the enumeration of all indicator bacteria. Fecal coliform concentrations were determined by membrane filtration using mFC agar (14), with incubation at 44 0.5C for 24 h. E. coli were enumerated by membrane filtration on modified mTEC agar, with incubation at 35C for 2 h to resuscitate any injured or stressed cells, followed by incubation at 44.5C for 22 h in a water bath (329). Enterococci were enumerated by membrane filtrati on on mEI agar, with incubation at 41 0.5C for 48 h (328). Detection of human-associated Bacteroidales Previously published primers specific fo r a partial region of 16S rRNA gene of human-associated Bacteroidales were used in a touchdown PCR (27) ( Table 10 ). PCR reactions were performed in a 25 l mixture containing 12.5 l GoTaq Green Master Mix (Promega Corporation), 0.5 M of each primer, and 2 l of template DNA. The 67

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touchdown PCR reaction conditions were as follows: DNA polymerase activation at 95C for 3 min, followed by 43 cycles of DNA melting at 94C for 45 sec, then annealing for 45 sec, and extension at 72C for 30 sec. Annealing temperatures ranged from 6555C. Cycles were performed twice at temp eratures 65-63C, once at temperatures 6256C, and 30 times at temperature 55C; fo llowed by a final elongati on at 72C for 5 min (Eppendorf Mastercycler Thermocycler). P CR products were separated by 2% agarose gel electrophoresis and viewed using ethidium bromide under UV light. Amplicons were identified visually by comparison to a BenchTop 100bp DNA ladder (Promega Corporation) and a 525 bp positive control. Detection of the human-associated M. smithii Previously published primers specific for the nifH gene of human-associated M. smithii were used in the touchdown PCR (330) (Table 10 ). PCR reactions were performed in a 25 l mixture containing 12.5 l GoTaq Green Master Mix (Promega Corporation), 0.5 M of each primer, and 2 l of template DNA. The touchdown PCR reaction conditions were the same as previous ly described. PCR products were separated by 2% agarose gel electrophoresis and viewed using ethidium bromide under UV light. Amplicons were identified visually by comparison to a BenchTop 100bp DNA ladder (Promega Corporation) and a 221 bp positive control. Adenovirus nested PCR Previously published primers specific fo r the hexon gene of human adenoviruses were used in the nested PCR (251) ( Table 10 ). Amplification was carried out in a 50 l reaction mixture containing 25 l GoTaq Green Master Mix (Promega Corporation), 0.08 M of each primer, and 5 l of template DNA. In both P CR reactions, the first round of 68

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denaturation was carried out for 4 min at 94 C followed by 30 cycles of denaturing at 94C for 90 sec, annealing at 55C for 90 sec, and extension at 72C for 120 sec, followed by a final elongation at 72C for 5 min. For the nested PCR, the 50 l reaction mixture contained: 25 l GoTaq Green Master Mix (Promega Corporation), 0.16 M of each primer, and 1 L of template from first roun d of PCR. PCR products were separated by 2% agarose gel electrophoresis and viewed using ethidium bromide under UV light. Amplicons were identified visu ally by comparison to a BenchTop 100bp DNA ladder (Promega Corporation) and a 143 bp positive control. Statistical analysis Summary statistics were computed for vari ables of interest using GraphPad InStat version 3.00 (GraphPad Software, San Di ego, CA). Differences among standard deviations of HPyVs and bacterial concentrations in either septic or sewage samples were determined using the Bartlett Statis tic. When the Bartlett Statistic P -value was <0.05, then the standard deviations among the sample s were considered significantly different and means were compared using nonparame tric repeated measures ANOVA with Dunns Multiple Comparison Test. When the Bartlett Statistic P-value was >0.05, then the standard deviations among the samples were considered not significantly different and means were compared using repeated m easures ANOVA with Tukey-Kramer Multiple Comparison Test. Means were considered significantly different when P <0.05. Linear relationships between HPyVs and IOs we re determined by calculating Pearson correlation coefficients. Differences were considered significant when P <0.05, and twosided tests were performed for all analysis. Comparisons of concentrations of water quality indicators over time at select temper atures were made using repeated measures 69

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ANOVA (InStat version 3.00). Differences of indicator c oncentrations among temperatures were compared using paired t test (InStat version 3.00). Observations of human associated markers were converted to binary data, and binary logistic regression models (SPSS version 12.0) were used to assess relationships be tween HPyVs or IOs concentrations and presence or absence of human-associated markers. Nagelkerkes R square, which can range from 0.0 to 1.0, denotes the effect size (the strength of the relationship); stronger associ ations have values closer to 1.0. Relationships were considered significant when the P value for the model chi square was 0.05. Results Sensitivity of QPCR and calculation of standard curves Standard curves constructed from analysis of BKV or JCV viral particles displayed a linear relationship fo r the QPCR assay, with an average Rsquared value 0.992 0.008. Under these conditions, the de veloped QPCR protocol was able to consistently quantify > 10 gene copies per reaction for both BK and JC viruses ( Table 11 ). For all standard curves generated during this study the average values for the variables in the linear regression were as follows: Y-inte rcept of 44.7 2.3 and slope of -3.61 0.22. Specificity of HPyVs QPCR and human-associated markers Total Bacteroidales, which is indicative of nons pecific fecal contamination, was detected by binary PCR in all animal fecal samples assayed ( Table 12 ). HPyVs were not amplified in any of the individual animal f ecal samples (n=115), composite animal fecal samples (n=2), or animal urine samples; and the assay did not amplify adenovirus DNA ( Table 12 ). In contrast, the human-associated Bacteroidales was detected by PCR in 13.0% (n=15) of the animal fecal samples (c ats and dogs). Moreover, human-associated 70

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M. smithii was detected in 0.9% (n=1) of the anim al fecal samples (individual cow fecal sample). The detailed results of the mark er specificity testing are presented in Table 12 HPyVs QPCR analysis of human waste samples HPyVs were detected in 23.1% (6 of 26) of human urine samples collected from healthy individuals (Table 12 ). HPyVs were detected in 17% (1 of 6) of samples collected from 0-20 age group, 25% (3 of 12) of the 21-40 age group a nd 25% (2 of 8) of the >40 age group. The concentration of HPyV s in positive urine samples ranged widely, from 6.61 x 102 to 1.20 x 107 copiesml-1, with a mean concentration of 2.71 4.72 x 106 copiesml-1. HPyVs were ubiquitous in untreated se wage and septage (septic tank pump truck or individual septic tank) samples, but were detected in only 22.2% of tertiary treated WWTP effluent samples (hereafter re ferred to as effluent samples) ( Table 12 ). HPyVs were found in relatively high concentr ations in raw sewage samples ( Table 13 ). They were the same order of magnitude in raw in fluent samples and septic tank samples (~104 copiesml-1); however, the mean concentrati on of HPyVs in raw influent was significantly higher than that in septage samples (P <0.05). As expected, HPyVs were present at a significantly lower concentration in WWTP effluent than in the influent or septage samples. Analysis of other water quality indicators in human waste samples The frequency of human-associated Bacteroidales, M. smithii and adenovirus detection by PCR is summarized in Table 12 Both human-associated Bacteroidales and M. smithii were detected in 100% of sewage influe nt samples and the majority of septage samples; however, neither marker was detect ed in any disinfected effluent sample. 71

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Adenoviruses were detected in 100% of sewage influent samples. Moreover, adenoviruses were detected in 77.8% and 11.1% of septic tank pump truck samples and disinfected effluent samples, respectively, but were not dete cted in any individual septic tank samples ( Table 12 ). The average concentrations of enterococci, E. coli and fecal coliforms are summarized in Table 13 All statistical comparisons within a sample type were performed by repeated measures ANOVA because severa l observations on one subject (i.e. sewage sample) were compared (217). In untreated sewage samples, fecal coliforms and E. coli tended to be more concentrated than en terococci; however, enterococci were more concentrated in the disinfected wastew ater. HPyVs gene copy concentration was comparable to that of the indicator bacteria for all sample types (within the same order of magnitude) ( Table 13 ). A comparison of indicator bacteria c oncentrations across the sewage types revealed that E. coli and enterococci concentrations we re significantly greater in raw sewage influent than in septage samples ( P< 0.05). Results for fecal coliforms were similar, except that their concentrations in raw influent were not significantly different from those in septic tank pump truck samp les. As expected, all indicator bacteria concentrations were significantly (and at least four orders of magnitude) lower in disinfected sewage than in influent or septage. Correlations among bacterial indicators an d HPyVs concentrations in human waste samples Correlations among bacterial concentrations and HPyVs gene copy concentrations were determined for sewage, disinfect ed wastewater and septage samples (Table 14 ). In sewage samples, despite concentrations with in the same order of magnitude, HPyVs gene 72

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copy concentrations were negatively correla ted with each bacterial indicator. As expected, all bacterial indicator concentrations in sewage were posit ively correlated with each other. In disinfected wastewater samples, HPyVs gene copy concentrations were negatively correlated with fecal coliforms, and while not significant a similar tendency was observed for the relationship between HPyVs and E. coli. Enterococci concentrations were also negatively correlate d with fecal coliform concentrations in WWTP effluent, and while HPyVs and enteroco cci concentrations were not significantly correlated there was a positive trend. A small number of individual septic tank samples were collected (n=5) which lead to non significant corr elations among HPyVs gene copy c oncentrations a nd all bacterial concentrations, as well as E. coli and both fecal coliforms and enterococci concentrations. However, enterococci were positively correlate d with fecal coliform concentrations. In septic tank pump truck samples, E. coli was significantly and positively correlated with both HPyVs gene copy concentrations and fecal coliforms. No other significant relationships where found with in the pump truck samples. Analysis of water quality indicato rs in sewage over a 28-day period To better understand the ne gative correlation between indicator bacteria and HPyVs in sewage the effects of time and te mperature was assessed. There was a larger percent decrease in samples exposed to the higher temperature (35 C) as compared to room temperature (25C) for all water quality indicators ( Figure 1 ). At the higher temperature, enterococci ha d the largest decrease with a change of -98.0% followed closely by both E. coli and fecal coliforms (98.0% and 96.3% decrease, respectively). 73

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HPyVs were slightly more resilient to highe r temperatures, with an 84.2% decrease. At room temperature, E. coli decreased 85.4%, fecal colifor ms decreased 84.8%, HPyVs decreased 81.2%, and enterococci decr eased 72.8%. Comparison of HPyVs concentrations at 25C vs. 35C showed no significant difference over the 28 day period ( P =0.4148). However, there was a significant difference between the temperatures for the concentrations of E. coli ( P =0.0068), enterococci ( P <0.0001) and fecal coliforms ( P =0.0296). Under both temperature conditi ons, HPyVs concentrations did not significantly decrease until Day 21 ( P <0.05), whereas significant decreases in indicator bacteria concentrations occurred at Day 7 ( P <0.01). Human-associated Bacteroidales, M. smithii, and adenovirus were detected by conventional PCR through Day 28 at room temperature (Figure 1 A). At the high temperature both M. smithii and adenovirus were detected through Day 28, however human-associated Bacteroidales was only detected until Day 14 ( Figure 1 B). Environmental water samples Salinities, bacterial and HPyVs concentr ations, and binary detection of humanassociated markers in environmental waters with a high probability of human sewage contamination are summarized in Table 15 Environmental samples were collected over a range of salinity from fresh to marine wate r. All indicator bacterial concentrations exceeded regulatory standards ( 294, 317). Human associated Bacteroidales and M. smithii were detected in 80% of the environm ental samples, while adenoviruses were detected in 60% of the samples. HPyVs were detected in all samples, at concentrations up to 1.4 x 106 copies ml-1. 74

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Binary logistic regression was used to assess the predictive relationship between HPyVs or bacterial indicators concentrati ons and the presence or absence of humanassociated indicators (Bacteroidales M. smithii and adenovirus). HPyVs, enterococci, and fecal coliform concentrations were indepe ndently strong predictors of the presence or absence of both human associated Bacteroidales and M. smithii (Nagelkerkes R2=1.000, P =0.025 for all relationships). In addition, the presence of adenovirus was strongly correlated with the concentrations of E. coli (Nagelkerkes R2=1.000, P =0.034), enterococci (Nagelkerkes R2=1.000, P =0.009), and fecal coliforms (Nagelkerkes R2=1.000, P =0.034). The presence of adenovirus and the concentrations of HPyVs were not correlated in environmental samples (Nagelkerkes R2=0.090, P =0.552). Since there must be at least one 1.0 or 0.0 when analyzi ng data using binary l ogistic regression the relationship between E. coli and both Bacteroidales and M. smithii could not be determined because both human associated markers were found in all 4 samples in which E. coli was enumerated. Sequencing analysis of QPCR amplicons Sequences of amplicons generated fro m cultured JCV and BKV (n=2), humanassociated waste (n=6), and environmental samples (n=4) were either 173-bp or 176-bp in length. The 176-bp and 173-bp amplicon sequences showed 99% identity to published BKV or JCV sequences, respectivel y. Specifically, the sequence derived from cultured BK viruses (GenBank No. FJ666992) sewage (GenBank No. FJ666993), human urine (GenBank No. FJ666994) and envir onmental water samples (GenBank No. FJ666995, FJ666997 and FJ666998) showed 99% identity to published BKV sequences. Sequences derived from cultured JC viru ses (GenBank No. FJ666999), sewage (GenBank 75

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76 No. FJ666991), individual septic tank (Gen Bank No. FJ667002), septic tank pump truck effluent (GenBank No. FJ667001), tertiary treated wastewater (GenBank No. FJ667000) and an environmental sample (GenBank No. FJ666996) showed 99% identity to published JCV sequences.

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Table 10. Primers and probe sequences used in the McQuaig et al. 2009 study. Assay Primers and Probe Sequence Product length (bp) Reference Total Bacteroidales PCR Bac32F 5-AACGCTAGCTACAGGCTT-3 676 (27) Bac708R 5-CAATCGGAGTTCTTCGTG-3 Human-associated Bacteroidales PCR HF183 5-ATCATGAGTTCACATGTCCG-3 525 (27) Bac708R 5-CAATCGGAGTTCTTCGTG-3 M. smithii PCR Mnif-342f 5-AACAGAAAACCCAGTGAAGAG-3 221 (330) Mnif-363r 5-ACGTAAAGGCACTGAAAAACC-3 Adenovirus PCR hexAA1885 5-GCCGCAGTGGTCTTACATGCACATC-3 301 (251) hexAA1913 5-CAGCACGCCGCGGATGTCAAAGT-3 Adenovirus nested PCR nehexAA1893 5-GCCACCGAGACGTACTTCAGCCTG-3 143 (251) nehexAA1905 5-TTGTACGAGTACGCGGTATCCTCGCGGTC-3 Human polyomavirus QPCR SM2 5-AGTCTTTAGGGTCTTCTACCTTT-3 173 (JCV) this study P6 5-GGTGCCAACCTATGGAACAG-3 176 (BKV) (19) KGJ3 5-(FAM)-TCATCACTGGCAAACAT-(MGBNFQ)-3 this study 77

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Table 11. Results of QPCR assays for known viral numbers (100, 10, 1 or 0.1 particles) and detection limits determined for BK virus and JC virus. Estimated Gene Copy Number Per Reaction 100 10 b 1 0.1 Replicate Measured Gene Copy Number Per Reaction BK virus A 76.2 31.8 0 0 B 143.6 21.6 0 0 C 82.1 13.5 0 0 Average S.D.a100.6.4 22.3.2 0 0 JC virus A 76.8 4.3 0 0 B 77.8 6.7 0 0 C 112.4 8.4 0 0 Average S.D.a89.0.3 6.5.0 0 0 a S.D., standard deviation bdetection limit of the assay 78

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Table 12. Result of human-associated markers P CR assays on non-target and target samples.a Sample Source n Total Bacteroidales PCRb HPyVsc QPCRb Humanassociated Bacteroidales PCRb M. smithii PCRb Adenovirus PCRb Non-target virus Adenovirus ATCC VR-846 1 ND 0 ND ND 1 Target viruses JC virus ATCC VR-1583 1 ND 1 ND ND ND BK virus ATCC VR-837 1 ND 1 ND ND ND Animal fecal samples Cat ( Felis catus) individuals 5 5 0 1 0 ND Chicken ( Gallus gallus ) individuals 1 1 0 0 0 ND Cow ( Bos taurus) individuals 24 24 0 0 1 ND Sandhill crane ( Grus canadensis ) individuals 2 2 0 0 0 ND Deer ( Odocoileus virginianus ) individuals 3 3 0 0 0 ND Dog ( Canis lupus familiaris ) individuals 55 55 0 14 0 ND Duck (Anas platyrhynchos ) individuals 4 4 0 0 0 ND Fox (Vulpes vulpes ) individuals 1 1 0 0 0 ND Horse ( Equus caballus ) individuals 8 8 0 0 0 ND Racoon ( Procyon lotor ) individuals 1 1 0 0 0 ND Seagull ( Larus atricilla ) individuals 6 6 0 0 0 ND Sparrow ( Ammodramus savannarum ) individuals 3 3 0 0 0 ND Wild Pig ( Sus scrofa ) individuals 2 2 0 0 0 ND Animal composite waste samples Cow ( Bos taurus) composite d 1 1 0 0 0 ND Pig (Sus scrofa domestica ) composite d 1 1 0 0 0 ND Animal urine samples Dog ( Canis lupus familiaris ) individuals 9 ND 0 ND ND ND Cat ( Felis catus) compositee1 ND 0 ND ND ND Target human samples Lift Station composite 2 ND 2 2 2 2 Sewage Influent composite 39 ND 39 39 39 39 Septic Tank Pump Truck composite f 9 ND 9 9 9 7 3 WWTP Effluentg effluent 9 ND 2 0 0 1 Septic Tanks compositeh 5 ND 5 3 4 0 Urine Samples individuals 26 ND 6 ND ND ND 79 aGrey boxes indicate amplification in non-target samples (false-positive results); bNumber of samples quantifiable by QPCR or am plified by conventional PCR; ND indicates sample not analyzed for respective assay; cHPyVs; human polyomaviruses; dFarm composite of >20 individuals; eComposite of 3 cats; fComposite of 2-3 septic tanks; gDechlorinated Tertiary Treated Wastewater Treatment Plant Effluent, hcomposite of 2-4 individuals

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Table 13. Concentrations of human polyomaviruses (HPyVs), E. coli, enterococci and fecal coliforms in human waste samples. Raw sewage influenta,b ( S.D.) Septic tank pump truck samplesa,b ( S.D.) Individual septic tanksa,b ( S.D.) 3 WWTP Effluenta,c,d ( S.D.) HPyVs (copiesml-1) 3.0 1.7 x 104 [A] 1.1 1.0 x 104 [D] 1.4 1.6 x 104 [G, H, I ] 1.2 2.5 x 10-1 [L, M] E. coli (CFUml-1) 4.6 1.8 x 104 [B] 3.1 1.9 x 103 [D, E] 9.7 9.1 x 103 [G, J, K] 2.0 1.4 x 10-2 [L, N] Enterococci (CFUml-1) 2.5 0.9 x 104 [A] 1.5 1.1 x 103 [E] 5.8 7.8 x 103 [H, J] 1.8 1.2 x 10-1 [O] Fecal coliforms (CFUml-1) 7.4 4.3 x 104 [C] 3.0 2.0 x 104 [F] 3.5 2.7 x 104 [I, K] 8.4 4.5 x 10-2 [M, N, O] a Values in the same column followed by the same letter (i.e. [A] through [O] ) are not statistically different at P < 0.05 b Repeated measures ANOVA was used to determine st atistical significance; S.D. = standard deviation c Tertiary treated wastewater treatment plant effluent dNonparametric repeated measures ANOVA was used to determine statistical significance 80

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Table 14. Correlation of HPyVs and indi cator bacteria in human sewage. Fecal coliformsaEnterococcia E. colia HPyVsa Sewage Pearson Correlation -0.6166 -0.3279 -0.5852 P value 0.0001 0.0363 0.0001 R-squared 0.3802 0.1075 0.3425 WWTP effluent Pearson Correlation -0.7131 0.2812 -0.2836 P value 0.0310 0.4636 0.4596 R-squared 0.5085 0.0791 0.0804 Septic Tanks Pearson Correlation 0.0376 -0.2139 -0.3946 P value 0.9522 0.7297 0.5110 R-squared 0.0014 0.0458 0.1557 Pump Trucks Pearson Correlation 0.4498 -0.6062 0.6855 P value 0.2245 0.0835 0.0415 R-squared 0.2023 0.3675 0.4699 E. coli Sewage Pearson Correlation 0.8718 0.3281 P value 0.0001 0.0362 R-squared 0.7600 0.1077 WWTP effluent Pearson Correlation 0.1035 0.2458 P value 0.7910 0.5238 R-squared 0.0107 0.0604 Septic Tanks Pearson Correlation 0.8683 0.8493 P value 0.0563 0.0686 R-squared 0.7539 0.7212 Pump Trucks Pearson Correlation 0.8145 -0.6077 P value 0.0075 0.0826 R-squared 0.6634 0.3693 Enterococci Sewage Pearson Correlation 0.4697 P value 0.0019 R-squared 0.2206 WWTP effluent Pearson Correlation -0.7167 P value 0.0298 R-squared 0.5137 Septic Tanks Pearson Correlation 0.9297 P value 0.0221 R-squared 0.8644 Pump Trucks Pearson Correlation -0.4691 P value 0.2027 R-squared 0.2201 a Statistics were calculated for relationships between organisms in sewage, dechlorinated tertiary treated wastewater treatment plant effluent, individual septic tanks and septic tank pump truck samples. Relationships were considered significant when P<0.05. Bolded values indicate significant relationships. 81

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Table 15. Physical and microbial data for all environmental samples collected in McQuaig et al. 2009 study. Site Salinity (ppt) Suspected source of contamination Enterococcia ( S.D.b) E. colia ( S.D.) Fecal coliformsa ( S.D.) HPyVs QPCR resulta ( S.D.) Adenovirus PCR resulta Humanassociated Bacteroidales PCR resulta M. smithii PCR resulta Dhr4A 3 faulty sewer line 3.5 3.2 x 104 3.0 2.1 x 104 7.8 3.5 x 104 1.4 1.1 x 106 + + + env1 33 faulty sewer line 1.6 0.7 x 105 1.1 0.4 x 105 1.9 1.1 x 105 3.5 0.3 x 102 + + + env2 32 faulty sewer line 5.8 0.3 x 104 4.5 0.4 x 104 6.1 2.4 x 104 2.2 0.1 x 102 + + + EJ3 25 faulty septic tank 1.3 1.0 x 104 1.0 0.2 x 104 1.3 0.3 x 104 1.2 2.0 x 104 + + SJH8 0 faulty septic tank 5.8 1.5 x 103 ND 3.9 0.1 x 103 9.2 0.2 x 101 a Bacteria concentrations reported as CFU100ml-1; HPyVs reported as gene copies number in 500ml sa mple; PCR results reported as + for a positive PCR result or - for a negative PCR result; ND, assay not done 82 b S.D., standard deviation

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(A) (B) Figure 1. Concentration of indicator bacteria and human polyomaviruses (HPyVs) in sewage coupled with the PCR detection of human-associated Bacteroidales, M. smithii and adenovirus over a 28 day period. (A) Samples held at room temperature (25C); (B) Samples incubated at high temperature (35C). E. coli Enterococci Fecal coliforms HP y Vs Adenovirus Human Bacteroidales M. smithii log10 (y+1) microorganisms per ml E. coli Enterococci Fecal coliforms HP y Vs Adenovirus Human Bacteroidales M. smithii log10 (y+1) microorganisms per ml 83

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Discussion This study introduces a new QPCR assa y for the quantification of human polyomaviruses BKV and JCV in environm ental water samples and summarizes relationships among bacterial and viral water quality indicators and pathogens (adenovirus) in various human waste samples. Initial attempts in our laboratory to develop a QPCR assay for HPyVs employed SYBR Green PCR chemistry and previously published primers (202), but it was found that nonsp ecific fluorescence occurred due to primer dimer formation (data not shown). The TaqMan QPCR assay coupled with a minor groove binding non-fl uorescent quencher (MGBNFQ) eliminates this problem and increases assa y specificity. The QPCR assay can detect as little as 10 JCV and BKV particles, which is comparab le to other published QPCR methods (that detect either JCV or BKV) in terms of dynamic range and precision (94, 241, 276). Sequencing analysis of randomly chosen clones from various human and environmental samples showed that 50% of the sequences aligned with BKV sequences and 50% aligned with JCV sequences. These re sults indicate widesp read distribution of both BKV and JCV in humans in the United St ates, supporting the desirability of an assay that detects both viruses. Furthermore, widespread geographic distribution provides some confidence that the assay can be useful worldwide. To date, JCV or BKV have been detected in raw sewage from Cair o, Egypt; Patras, Greece; Barcelona, Spain; Nancy, France; Pretoria, S outh Africa; Umea, Sweden; and Washington, D.C using conventional PCR (36, 37). The method describe d in this work has quantified HPyVs in samples from California and across Florida. 84

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The PCR results from previously pu blished studies estimate BKV and JCV concentrations in sewage ranging from 101 to 103 BKVml-1 and 102 to 104 JCVml-1 (37), which is within the range of the averag e concentration of HPyVs in sewage found in this study (3.0 x 104 copiesml-1). There are no previously published data documenting the concentrations of HPyVs in onsite wastew ater treatment and disposal (septic tank) systems and septic tank pump trucks. HPyVs we re consistently detected in these samples and the concentration was consistently high (~104 copiesml-1). These concentrations are also comparable to concentrati ons of culturable indicator bacter ia in sewage. In contrast, the detection of human-associated Bacteroidales and M. smithii in septic tank samples was inconsistent and detection of adenovirus in septic tank and pu mp truck samples was sparse. Furthermore, only HPyVs were de tected in an environmental sample contaminated by faulty septic tanks. These da ta suggest that the HPyVs assay is a useful marker for both sewage and septic system contamination. The obligate host specificity of viruses such as HPyVs is advantageous for specific identification of human sources. It is, however, possible that animals could ingest and excrete HPyVs without infection, leading to a transi ent association with feces. In addition, some have speculated that kidney infection and subseque nt viruria in these animals could occur despite the documented host specificity of HPyVs (278). Areas frequented by dogs (e.g. backyards or dog parks) may introduce such cosmopolitan organisms to nearby water sources during rain events, confounding attempts to identify contamination sources. In light of the recent studies reporting human-associated bacterial markers detected in dog fecal samples (11, 345) we were most concerned with crossreactivity in dog fecal and urine samples. A ll of these samples, as well as all animal85

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derived samples tested in this study were negative for HP yVs, further emphasizing the potential of these viruses as indicators for human-specific fecal contamination. The human-associated Bacteroidales assay was less specific, as the amplicon was detected in a cat fecal sample, as well as several dog fecal samples. The cross reactivity of the human-associated Bacteroidales has been previously documented (11, 163). The incomplete specificity of this marker may pose problems with water quality assessment in areas frequented by these animals (e.g. dog parks and animal friendly public beaches), and it may be ideal to augment water quality an alysis with a more human specific marker. Human-associated M. smithii was more human-specific than the Bacteroidales assay, as it was detected in only one cow fecal sample. The previously published M. smithii PCR protocol developed for MST (330) utilized a prefiltration step due to previous detection of M. smithii in ruminant ciliated protozoans (149). Prefiltration was not used in this studys protocol, and the M. smithii protocol may have increased specificity with an additional prefiltration step. However, the use of prefiltration tends to increase the assays limit of detection (unpublished da ta), leading to a probable tradeoff between sensitivity and specificity. While many MST methods focus on bacter ial markers, these indicators are not accurate predictor of some human pathogens, particularly viruses and protozoa (16, 90, 131). Doublestranded DNA viruses tend to be more resilient to environmental stresses such as temperature, ultraviolet light, and disinfectants compared to bacteria and therefore may more accurately mimic surviv al of viral and protozoan pathogens in environmental water systems (32, 207, 312, 313). Moreover, in tropical climates bacterial indicators have the potential to survive and perhaps even multiply in 86

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87 environmental waters (15, 180, 354) which may cause overestimation of public health risks. In this study, we found negative correlations between HPyVs and indicator bacteria in sewage and septic tank samples (e.g. Table 14 ), while all correlations among indicator bacteria were positive. The results of the sewage holding time experiment demonstrate that the kinetic s of viral DNA (adenoviruses an d HPyVs) decay were quite dissimilar from decay rates of culturable indicator bacteria. Furthermore, HPyVs mimicked the persistence of adenovirus at bo th warm and room temperature, while the bacterial indicators did not. The fate of HPyV s in sewage may be a better predictor of the fate and persistence of pathogenic enteric viru ses than indicator bacteria concentrations, since unlike the indicator bacteria the viru ses lack the potential for growth in the environment and are more similar in size and st ructure to each other than to bacteria. In addition, both adenovirus and HPyVs were detected in tertiary tr eated waste water, indicating the potential of these viruses to mimic the persistence of relatively disinfection-resistant microorganisms su ch as double-stranded DNA viruses. The success of this QPCR assay in quantifying low numbers of HPyVs in combination with HPyVs host specificity, a nd abundance in human-associated waste will allow for a rapid, quantitative, and cost-e ffective assessment of water quality. The benefits of incorporating the HPyVs Ta qman QPCR assay with the expanding MST toolbox include reduction of a ssay time, increased specific ity for human pollution, and quantification. These benefits will in turn allow a better understanding of relationships with other MST markers and waterborne pathog ens and ultimately a better perspective on the proportion of microbial contam ination from human sources.

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Acknowledgements We would like to thank Robert Ulrich fo r his time and insights. In addition we would like to thank Brittany Sears, Bina Nayak, Zach Staley, Katrina Gordon, Asja Korajkic, Phoebe Koch, Chris Staley, Br ian Badgley, Miriam Brownell, Sam Farrah, Kevin Grant, John Griffith, Kathy and Gary McQuaig, the Oldsmar Wastewater Reclamation Facility, Advanced Septic Tanks and Teds Tanks for help with sample collection. Funding for this study was provided in part by the Southeastern Branch American Society of Microbiology 2007 Henry Aldrich Research Award and Cooperative Institute for Coastal and Estuarine Environm ental Technology (CICEET) NA05NOS4191149 subaward 07-092. 88

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CHAPTER 3: THE EFFECTS OF TEMPERATURE SALINITY, AND ULTRAVIOLET RADIATION ON THE PERSISTENCE OF HUMAN POLYOMAVIRUSES AND OTHER WATER QUALITY INDICATORS Shannon McQuaig1 and Valerie J. Harwood1* 1Department of Biology, University of South Florida, Tampa, FL 33620 *Corresponding author. Mailing address: Depa rtment of Biology, SCA 110, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620. Phone: (813) 974-1524. Fax: (813) 974-3263. E-mail: vharwood@cas.usf.edu Running Title: HPyVs persis tence in the environment Prepared for submission to: Environmental Microbiology 89

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Abstract Water quality is commonly assessed using fecal indictor organisms (IOs) such as fecal coliforms, Escherichia coli and enterococci. Microbial source tracking (MST) methodologies can be employed to detect and differentiate specific sources of fecal pollution. Human polyomaviruses BKV and JCV (HPyVs) are specific indicators of human-sewage contamination; however, little is known about the persistence of these viruses in environmental waters. This study as sessed the effects of ultraviolet radiation, temperature, and salinity on the persistence of HPyVs in water, and compared it to that of cultured IOs, Methanobrevibacter smithii and the human-associated Bacteroidales marker. UV irradiation (800-1200 mWs/cm2) resulted in a 3 log reduction of HPyVs DNA detected by quantitative PCR (QPCR). Th e salinity condition of 10 ppt decreased the log decrease of BK virus gene copy numbers compared to 0 or 35 ppt. In laboratory microcosms HPyVs DNA detection at 4C and 25C over a salinity range of 0 ppt to 35 ppt was relatively constant over seven days ( k = -0.04 to -0.16). The persistence of HPyVs DNA was relatively stable at 35C a nd 35 ppt salinity; however it significantly decreased at lower salinities. In natural sunlight the detection of HPyVs DNA followed the general trends of IOs and the M. smithii marker when exposed to environmental conditions (e.g. solar radiation, predation, temperature), persisting no more than 5 days. The human-associated Bacteroidales marker was detected through day 7 in 1 of the 3 trials. This study fostered a better understanding of the influence of environmental factors on water quality indicators and furthe r demonstrated that HPyVs are a useful indicator of environm ental water quality. 90

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Introduction The quality of water systems (e.g. beach es, lakes, and rivers) is commonly monitored using fecal indictor organi sms (IOs) such as fecal coliforms, E. coli and enterococci (324). Despite the extensive utilization of IOs as water quality indicators, the ability of these organisms to exclusively signify sewage and/or fecal contamination is questionable for several reasons. These reas ons include the isolat ion of IOs from non fecal sources and the detection of IOs in s econdary habitats (e.g. environmental waters) (205, 275, 283). Fecal coliforms, specifically E. coli have been isolated from paper mill effluents in areas presumed absent of hu man fecal impact (25, 106), and studies have reported the detection of fecal coliforms, E. coli, and enterococci in waters with no history of anthropogenic impact (56, 260, 354, 356). Natural reservoirs of coliform bacteria have also been found in the envi ronment (e.g. in soil and on vegetation) (43, 109), and studies have demonstrated that thes e bacteria have the ability to survive and potentially proliferate in tropical envi ronments (15, 42, 86). Although correlations between increased gastroenteritis rates in re creational water users a nd IO concentrations have been noted in waters impacted by point sources of pollution (55, 336), IOs give little or no indication of specific sources of contamination, and questionable correlation with human pathogens under many circumstances (108, 116, 130, 270, 285, 287). In response to the limitations of traditi onal IOs, alternate methods ha ve been developed to more completely assess water quality. Microbial source tracking (MST) is the collective term for methodologies employed to detect and di fferentiate sources of fecal pollution in waters (i.e. using microorganisms as markers of source-associated contamination). In general, MST methods targeting a single sp ecific gene by PCR or QPCR (i.e. libraryindependent) are less time consuming, potentially applicable over large areas, and in most 91

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cases are more cost effective than methods requiring databases (i .e. library dependent methods) (275, 319). Recently, we have proposed the quantific ation of human polyomaviruses (HPyVs) as a human-specific, library-independent wate r quality indicator (203). HPyVs belong to the family Polyomaviridae (148) These viruses are non-enveloped with a 40-55 nm icosahedral capsid surrounding a circular, double-stranded 5-kbp DNA genome (264, 278). There are currently five recognized human-specific polyomaviruses; JCV (238), BKV (104), KIV (13), WUV (107), and MC V (46, 96, 105, 171). Both JCV and BKV have been detected in sewage (35, 37, 203) and both can be excreted in urine (278), therefore our HPyVs QPCR method targets both JCV and BKV, but not the other HPyVs. The JCV and BKV HPyVs have similarly stru ctured genomes that show 75% identity (80, 278), can establish latent in fections in the renal tissue and can persist indefinitely (91, 278), maintain worldwide prevalence ( 139, 252, 279), and in general are associated with life-long asymptomatic viruria. While the HPyVs PCR and QPCR methods have been shown to be highly sp ecific (129, 202, 203) and studies have reported the detection of HPyVs in low volumes of sewage (129, 202, 203), there is no published data regarding the fate of these viruses in environmen tal waters. HPyVs are exposed to many environmental stressors afte r entering a water body, incl uding ultraviolet radiation, fluctuating temperatures, and possibly salin ity changes. These conditions may have direct influences on the pers istence of HPyVs vi ral particles as well as viral DNA, and may affect the ability of th e assay target (i.e. HPyVs DN A) to accurately assess water quality. Studies have reported that viruses are more resilient to certain environmental stresses than IOs (22, 202, 312) [reviewed in (138, 154)] and subsequently may persist 92

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for long periods. Therefore, it is imperati ve to compare the eff ects of environmental factors on HPyVs persistence vs. that of IOs and wate rborne pathogens. Harwood et al. (129) reported the sensitivity and specificity of two other libraryindependent MST markers that were detected by PCR. These mark ers include targeting the nif gene of Methanobrevibacter smithii and the 16S rRNA gene of human-associated Bacteroidales PCR to detect human-specific fecal contamination. M. smithii is the prominent methanogen in the human intestines and have been found at concentrations of 10710 organisms per gram of feces (40, 186). In 2006, this organism was first suggested as an indicator of human fecal pollution (330). The use of this method to identify sources of human-associated fecal pollution has been limited but successful in preliminary studies (129, 330). The Bacteroidales group consists of gram-negative, anaerobic non-spore forming bacilli. Memb ers of this group are found at orders of magnitudes higher concentrations then coliforms in both human and animal feces (141). The identification of human-associated Bacteroidales pp and the use of this group to detect human fecal pollution was first suggested in 1998 (170), and since then numerous studies have utilized the marker as indicator of human-associated fecal pollution (27, 88, 163, 181, 234, 277). This marker has been widely used and is found in high concentrations of sewage (129). Recentl y, studies have been conducted assessing the environmental effects on the persistence of the human-associated Bacteroidales marker (21, 338, 339); however, conflicting reports ha ve emerged regarding the effects of sunlight. Bae et al. (21) and Walters et al. (338) reported th at sunlight ha d no significant effect on the persistence of Bacteroidales DNA; however Walters et al. (339) report a significant increase of decay rate following exposure to sunlight. 93

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The goal of this study was to assess the dire ct and interactive e ffects of ultraviolet radiation, temperature, and sa linity on the persistence of HPyVs in laboratory microcosm and mesocosm studies. Furthermore, the effects of sunlight, salinity, and time on the persistence of IOs and HPyV s and the detection of both M. smithii and human-associated Bacteroidales marker was assessed in outdoor mesocosm experiments. Materials and Methods Virus strains and viral DNA extraction BK viruses (ATCC VR-837) were obtain ed from the American Type Culture Collection (Manassas, VA). BK viruses we re propagated and harvested from HEL-299 cells. After cytopathic eff ects were observed, cell culture medium containing suspended viral particles was harvested fr om the cell culture flasks in 1 ml aliquots and placed at 20C (BK virus stock). For experiments requirin g the use of BK viruses, the virus stocks were defrosted and 1 ml was transferred into a 1.5 ml microcentrifuge tube. The viruses underwent centrifugation for 10,000 x g for 5 min to pellet any remaining mammalian cells. The supernatant was then transferred to a clean 1.5 ml microcentrifuge tube and again underwent centrifugation for 5 min at 10,000 x g The remaining supernatant was again transferred to a clean 1.5 ml microcen trifuge tube, and subsequently used in downstream applications. Positive PCR controls To construct clones for use as positive controls, a specific gene fragment for each MST target (human-associated 525-bp region of the 16S rRNA gene of Bacteroidales or 221-bp region of the nifH gene of M. smithii ) was amplified using the primers described below. The 525-bp and 221-bp amplicon were then cloned into a 94

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pCR-TOPO vector and E. coli One Shot chemically competent cells were transformed as described above. Recombinan t plasmids were propagated and purified as described above. Plasmids containing insert s were confirmed by sequencing at Macrogen USA (Rockville, MD). All sequences were subjected to BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) for comparison with published sequences. Human polyomavirus QPCR The HPyVs QPCR mixtures were prepar ed using 25 l TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems, Foster City, CA), 0.5 M primer concentrations (SM2: 5-AGT CTTTAGGGTCTTCTACCTTT-3 and P6: 5GGTGCCAACCTATGGAACAG-3), 0.4 M labeled probe (KGJ3: 5-(FAM)TCATCACTGGCAAACAT-(MGBNFQ)-3) concentr ation, 5 l of template DNA, and the volume was adjusted to 50 l using nuclease-free water (203). Amplification was performed using a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). The QPCR thermocycle profile was as follo ws: DNA polymerase activation at 95C for 10 min, followed by 40 cycles of DNA melting at 95C for 15 sec, then annealing at 55C for 20 sec, and extension at 60C for 60 sec. Human polyomavirus QPCR standard curve To produce a standard curve, the recomb inant plasmid DNA was serially diluted in nuclease-free water to final concentrations ranging from 102 to 106 gene copiesl-1. Five microliters of each dilution were used as template in the Taqman real time standard curve PCR reactions. Each dilution was run in duplicate. Applied Biosystems default settings for the threshold cycle (Ct) we re used for data analysis. The Ct values were plotted against copy number to generate the standard curve. Linear regression was 95

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used to assess the relationship between Ct values and copy number. The average R2 of the standard curve was 0.9899 0.0100. Effects of ultraviolet radiation x salinity on the detection of BK virus DNA by QPCR: laboratory experiment with BK viruses The salinity of sterile dechlorinated tap water was adjusted to 10 parts per thousand (ppt) or 35 ppt using Instant Ocean (Fisher Scientific, Pittsburgh, PA) and a hand refractometer (Fisher Scientific). Five hundred milliliters of dechlorinated water with defined salinities (0 ppt, 10 ppt, and 35 ppt ) were placed into 3 separate 1 L sterile beakers containing a magnetic stir bar. BK vi rus particles were adde d to a concentration of approximately 2 x 107ml-1. The beakers containing th e water and viral particle mixtures were placed on a stir plate and mixed for 10 min. Two-hundred microliter samples were taken from each of the 3 salinity beakers to assess in itial concentration. Seven milliliters of the suspended virus solution were pipetted into sterile 50 mm petri plate bottoms. The plates containing each sa linity and virus mixture were then placed into a Spectrolinker XL-1000 UV Crosslinker (Karackeler Scientific, Inc., Albany, NY) and exposed to UV doses of 100, 400, 800, and 1200 mWs/cm2. UV doses are a function of time and UV intensity. The Spectrolinker XL-1000 UV Crosslinker determined intensity and time of exposure to ensure appropriate dose. All UV dose exposures were performed in triplicate. After UV exposure, 200 l samples were taken from each plate and placed into a sterile 2.0 ml tube. A final 200 l sample was taken from the 1 L beakers to assess the effects of ambient laboratory condi tions on the viral suspension stock. Viral DNA was extracted from the t ube using the DNeasy Tissue Kit (Qiagen, Inc., Valencia, CA). BK virus copy numbers were determined using the HPyVs QPCR (as described above). 96

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Effects of salinity x temperature on th e persistence of BK viruses: laboratory experiment with BK viruses The salinity of sterile dechlorinated tap water was adjusted to 10 ppt or 35 ppt using Instant Ocean (Fisher Scientific, Pittsburgh, PA) and a hand refractometer (Fisher Scientific). Nine milliliters of dechlorinate d water with defined salinities were placed into 9 separate 15 ml centrifuge tubes. The defined salinities included 0 ppt, 10 ppt, and 35 ppt. BK virus particles were diluted to approximately 107ml-1 and 1 ml was added to each tube. Three tubes of all 3 salinities were placed in the dark at 4C, 25C, and 35C. Two-hundred microliter samples were take n daily from each tube over a seven day period. Viral DNA was extracted from the t ube using the DNeasy Tissue Kit (Qiagen, Inc., Valencia, CA). BK virus copy numbers were determined using the HPyVs QPCR (as described above). Effects of salinity x natural sunlight on the persistence of BK viruses: natural sunlight experiment with BK viruses The salinity of sterile dechlorinated tap water was adjusted to 10 ppt or 35 ppt using Instant Ocean (Fisher Scientific) and a hand refractometer (Fisher Scientific). Aliquots of 500 ml of dechlorinated water with defined salinities (0 ppt, 10 ppt, and 35 ppt) were placed into separate 1 L sterile beakers containing a magnetic stir bar. A total of 6 1 L beakers were used in this st udy, with a beaker used for each salinity (0 ppt, 10 ppt, and 35 ppt) under each light exposure (Light and Dark). Beakers were labeled with specific salinity and light expos ure (e.g. 0 ppt Dark). BK virus particles were added to the labeled beakers at a concentration of approximately 104 ml-1. The beakers containing the water and viral particle mixture were then placed on a stir plate and mixed for 10 min. Two-hundred microliter samples were taken from each of the beakers to assess initial concentration. To bl ock sunlight, opaque covers were placed on 97

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the dark beakers. Beakers under light exposure were covered with a transparent plastic film to prevent large volume loss from evaporation. The beakers were then placed outdoors at the University of South Florida Botanical Gardens (Tampa, FL). Beakers were placed under direct sun light in random order. Temperatures were measured each day using a thermometer, and UV intensities were measured using a Blak-Ray Model J225 (UVP, Upland, CA). High and low atmo spheric temperatures and UV indexes obtained from the National Oceanic and At mospheric Administration (NOAA) website ( http://www.noaa.gov/wx.html) were recorded each day. Th e persistence of BK viruses was assessed over a 7 day period. Two-hundred microliter samples were taken every day at 10:30 AM. Viral DNA was extracted from 200 l samples using the DNeasy Tissue Kit (Qiagen, Inc., Valencia, CA). BK vi rus copy numbers were determined using the HPyVs QPCR (as described above). The out side natural sunlight experiment was repeated in triplicate over three time periods: March 16-23, 2009; March 30-April 6, 2009; and April 16-23, 2009. Effects of salinity x natural sunlight on the persistence of HPyV s, culturable bacteria, and other water quality indicators: natu ral sunlight experiment with sewage The salinity of sterile dech lorinated tap water was adju sted to 10 ppt or 35 ppt using Instant Ocean (Fisher Scientific) and a hand refractometer (Fisher Scientific). Aliquots of 18.5 L of dechlorinated water with defined salinities (0 ppt, 10 ppt, and 35 ppt) were placed into separate high density po lypropylene 5 gallon buckets. A total of six buckets were used in this study; with a buc ket for each salinity (0 ppt, 10 ppt, and 35 ppt) under each light exposure (Light and Dark). Buckets were labeled with specific salinity and light exposur e (e.g. 0 ppt Dark). 98

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Two hours prior to the star t of the experiment, untreat ed sewage influent was collected from Falkenburg Advanced Wast ewater Treatment Plant (Brandon, FL), designed for 9 million gallons per day aver age daily flow. Two hundred milliliters of untreated sewage was added to each 5 gallon bucket. The water and sewage mixture was stirred manually with a sterile 50 ml pipette for 2 minutes. One liter of water was then collected to asse ss the presence of M. smithii and human-associated Bacteroidales spp. and initial concentratio ns of fecal coliforms, E. coli, enterococci, and HPyVs (as described below). To block sunlight, opaque covers were placed on the dark buckets. Buckets under light exposure were covered with a transpar ent plastic film to prevent large volume loss from evaporation. The buckets were then placed outdoors at the University of South Florida Botanical Gardens (Tampa, FL). Temperatures, UV indexes, UV intensities were recorded each day as described above. The persistence of indicator bacteria, M. smithii, human-associated Bacteroidales spp., and HPyVs was assessed each day over a 7 day period. One liter samples were taken every day at 10:30 AM. Indicator bacteria, HPyVs, M. smithii human-associated Bacteroidales spp. were enumerated or detected as described below. The outside natural light expe riment with sewage was run in parallel with the outside natural light e xperiment with BK viruses over three time periods: March 16-23, 2009; March 30-April 6, 2009; and April 16-23, 2009. Enumeration of culturable indicator organisms Culturable concentrations of all indicato r organisms were obtai ned using standard methods. Enterococci were enumerated by membrane filtration on mEI agar, with incubation at 41 0.5C for 24 h (328). Fecal co liform concentrations were determined by membrane filtration using mFC agar, with incubation at 44.5 0.5C for 24 h (14). 99

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Total coliform concentrations were enumerat ed by membrane filtration on mEndo agar, with incubation at 35 0.5C for 24 h (14). Concentration of MST indicators Bacteria and viruses from 500 ml sample s were concentrated simultaneously on a 0.45 m pore size nitrocellulose filter. Bacter ia were concentrated by physical capture on the filter. To concentrate the viruses, the pH of the water was adjusted to 3.5 using 2.0 N HCl prior to filtration (14, 202). The low pH did not affect the concentration and detection of the bacteria (129). After filtration, the filter was placed into a 2 ml microcentrifuge tube, and DNA was extracted using the MQH protocol, and was used as template for human-associated Bacteroidales PCR assay (described below), M. smithii PCR assay (described below), and HPyVs QPCR assay (described above). DNA extraction The DNA extraction protocol combined components of the MO BIO Powersoil Kit (MO BIO Laboratories, Carlsbad, CA) and the Qiagen DNeasy Tissue Kit (Qiagen, Inc., Valencia, CA). Sixty microliters of solution C1 (MO BIO kit) was added to the PowerBead tube (MO BIO kit) containing th e DNA sample. The tube was vortexed for 3 sec, then placed into FastPrep FP 120 (Qbiogene Inc., Carlsbad, CA), and run for 40 sec. The bead tube was spun at 10,000 x g for 30 sec. All the supernatant (~ 800 l) was transferred to a steril e 2.0 ml tube (provide d in the MO BIO kit) containing 250 l of solution C2 (MO BIO kit) and then vortexed fo r 5 sec. The tube was placed at 4C for 5 min., and was then centrifuged at 10,000 x g for 1 min. All the supernatant was transferred to a new sterile 2.0 ml tube (pr ovided in the MO BIO kit) containing 285l of solution C3 (MO BIO kit) and vortexed for 2 s ec. The tube was incubated again at 4C 100

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for 5 min, and spun at 10,000 x g for 1 min. A ll the supernatant was transferred to a 50 ml tube containing 1.6 ml solution C4 (MO BI O kit) and vortexed for 5 sec. The 50 ml tube was then spun at 2,000 x g for 1 min. Qiagen DNeasy Tissue spin columns (Qiagen, Inc.) were attached to a QIAvac 24 vac uum manifold (Qiagen, Inc.) by VacValves (Qiagen, Inc.) outfitted with VacConnectors (Qiagen, Inc.). The manifold was connected to a vacuum, and the vacuum was turned on and the individual samples were continuously loaded (~ 750 l at a time) into separate Qiagen DNeasy Tissue columns until all the sample had passed through the co lumn. The pressure did not exceed 25 in. Hg. The vacuum was then turned off and the pressure was relieved. The column was then washed by passing 750 l of solution C5 (MO BIO kit) through the column. The pressure was relieved and the wash step was repeated two additional times. Once the three washes were completed, the Qiagen column was detached and placed into a collection tube (Qiagen kit). The collection tube contai ning the column was spun at 14,000 x g for 3 min to completely remove so lution C5. After centr ifugation, the column was carefully removed from the collection t ube and placed into a new sterile 2.0 ml microcentrifuge tube. A 100 l aliquot of nuc lease-free water was a dded to the center of the column filter, and incubate d at room temperature for 5 min. The tube containing the column was then spun at 10,000 x g for 1 min. Eluted DNA was captured in the tube and the Qiagen column was removed and discarde d. DNA was stored at -20C until used as template. Detection of human-associated Bacteroidales Previously published primers specific for a region of the 16S rRNA gene of human-associated Bacteroidales (27) were used in a touchdown PCR (129). PCR 101

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reactions were performed in a 25 l mixture containing 12.5 l GoTaq Green Master Mix (Promega Corporation), 0.5 M concentrations of each primer (HF183F: 5ATCATGAGTTCACATGTCCG-3 and Bac708R: 5-CAATCGGAGTTCTTCGTG-3), and 2 l of template DNA. The touchdown P CR reaction conditions were as follows: DNA polymerase activation at 95C for 3 min, followed by 43 cycles of DNA melting at 94C for 45 sec, then annealing for 45 sec, a nd extension at 72C for 30 sec. Annealing temperatures ranged from 65-55C. Cycles were performed twice at temperatures 6563C (at a decrease rate of 1 degree), once at temperatures 62-56C (at a d ecrease rate of 1 degree), and 30 times at temperature 55 C; these steps were followed by a final elongation at 72C for 5 min (Eppendorf Mastercycler Ther mocycler) PCR products were separated by 2% agarose gel electrophor esis and viewed using ethidium bromide under UV light. Amplicons were identified visually by comparison to a BenchTop 100bp DNA ladder (Promega Corporation) and a plas mid containing the 525 bp positive control. Detection of the human-associated M. smithii Previously published primers specific for the nifH gene of human-associated M. smithii (330) were used in the touchdown PCR (129). PCR reactions were performed in a 25 l mixture containing 12.5 l GoTaq Green Master Mix (Promega Corporation), 0.5 M concentrations of each primer (Mnif-342f: 5-AACAGAAAACCCAGTGAAGAG3and Mnif-363r: 5-ACGTAAA GGCACTGAAAAACC-3), and 2 l of template DNA. The touchdown PCR reaction conditions were the same as previously described for the Bacteroidales. PCR products were separated and vi sualized as above, except a plasmid containing the 221 bp positive control was used. 102

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Statistical analysis Summary statistics were computed for vari ables of interest using GraphPad InStat version 3.00 (GraphPad Software, San Diego, CA). BK virus and HPyVs copy numbers were log10 (y+1) transformed. All ANOVA and t-te st analysis were performed using GraphPad InStat version 3.00. Univariate and multivariate ANOVA was performed using SPSS version 17.0 (SPSS Inc., Chicago, IL). All statistical analyses were considered significantly different at the alpha level of 0.05. Differences among the log decrease of BK copy numbers at the three salinities in the ultraviolet radiation x salinity: labor atory experiment were determined using ANOVA. Differences of the log deceases among UV doses at each salinity level and among salinities at each UV dose were de termined using ANOVA. The main and interactive effects of UV dose and salinity on the log decrease were determined using the univariate ANOVA. Cumulative (overall) decay rates were calculated for experiments involving persistence of organisms over seven days or until the analyte could no longer be detected, whichever came first. Decay rates were calculated by di viding the change of the log10 (y+1) transformed concentrations of th e microorganism over the 7-day period by 7 (217). Decay rates for microorganisms that were not detected prior to the end of the 7day period were determined by di viding the change of the log10 (y+1) transformed concentrations of the organism over the 7-day period by number of days the organism persisted (i.e. time to extinction). Differences among decay rates at the different test variables (e.g. salinity and te mperature) were compared using ANOVA. The main and interactive effects for each test variable in the "temperature x salinity: laboratory experiment" and "salinity x ultraviolet radiat ion: natural sunlight experiment with BK 103

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viruses" were determined using univariate ANOVA. For "salinity x ultraviolet radiation: natural sunlight experiment with sewage" th e main and interactive effects of the test variables were determined using multivar iate ANOVA. Multivariate ANOVA was used because values obtained for microorganisms sampled from the same container are not considered independent observations. Observ ations of human associated markers were converted to binary data, and binary logist ic regression models (SPSS version 17.0) were used to assess relationships between HPyVs or IOs concentrations and presence or absence of human-associated markers. Nagelkerkes R square, which can range from 0.0 to 1.0, denotes the effect size (the strength of the relationship); stronger associations have values closer to 1.0. All means and effects we re considered significan tly different at the alpha level of 0.05. Relationships we re considered significant when the P value for the model chi square was 0.05 and the confidence interval fo r the odds ratio did not overlap 1.0. Results Effects of ultraviolet radiation x salinity on the detection of BK virus DNA by QPCR: laboratory experiment with BK viruses In general, the log decrease of BK copy numbers increased as ultraviolet radiation (UV) doses increased ( Table 16 ). At 0 ppt the log decrea se of BK viruses at the 100 mWs/cm2 UV dose was significantly lower than 400, 800, and 1200 mWs/cm2 doses ( P <0.001). There were no significant differences among the 400, 800, and 1200 mWs/cm2 doses. The log decrease at the 1200 mWs/cm2 dose was significantly larger than the 800 mWs/cm2 dose ( P <0.05). At 10 ppt the log de crease of the 100 mWs/cm2 dose was significantly lower than all other doses ( P <0.01 for all). The log decrease at the 400 mWs/cm2 dose was significantly lower than the 800 and 1200 mWs/cm2 doses 104

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( P <0.01 and P <0.001, respectively). The l og decrease at the 1200 mWs/cm2 dose was significantly larger than the 800 mWs/cm2 dose ( P <0.05). The same relationships among UV doses were also observed at 35 ppt with the log decreas e of the 100 mWs/cm2 dose significantly lower than all other doses ( P <0.001 for all). The log decrease at the 400 mWs/cm2 dose was significantly lower than the 800 and 1200 mWs/cm2 doses ( P <0.001 for all). The log decrease at the 1200 mWs/cm2 dose was significantly larger than the 800 mWs/cm2 ( P <0.001). Among the 100 mWs/cm2 UV doses the log decreases between all the salinities were not significantly different (P =0.2464). Among the 400 mWs/cm2 UV doses the log decrease at 0 ppt was significantly la rger than both 10 ppt and 35 ppt ( P <0.001 for both); however, there was no significant differen ce between 10 ppt and 35 ppt. Among the various salinities at the 800 mWs/cm2 dose, the log decreases at 10 ppt was significantly lower than both the 0 ppt and 35 ppt. The log decreases of BK viruses at 0 ppt and 35 ppt were not significantly differe nt. As with the 800 mWs/cm2 dose, the log decreases at 10 ppt was significantly lower than bo th 0 ppt and 35 ppt at 1200 mWs/cm2 ( P <0.05 and P <0.01, respectively) and there wa s no significant difference betw een 0 ppt and 35 ppt. The log decreases at each level of UV treatment were compiled for each level of salinity for statistical analysis. Overall, decreases at 10 ppt were significantly lower than both 0 ppt and 35 ppt ( P =0.0013 and P <0.0001, respectively); however, there was no significant difference between 0 ppt and 35 ppt Salinity alone did not affect the log decrease of BK copy numbers (F2,24=3.704, P =0.090). The log decreases at each level of salinity were compiled for each level of UV dose for statistical analysis. UV doses had significant effects on the log decrease of BK copy numbers (F3,24=19.758, P =0.002). In 105

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addition, the interaction of UV dose and sali nity had significant effects on the log decrease of BK copy numbers (F6,24=11.550, P <0.005). Based on the univariate analysis results, an increasing UV dose over all salinitie s increases the log d ecrease of BK virus gene copy numbers. As compared to the salin ities of 0 ppt and 35 ppt, the effects of UV were diminished at the 10 ppt salinity condition. Effects of salinity x temperature on th e persistence of BK viruses: laboratory experiment with BK viruses There was a pronounced decrease of BK viru ses at 35C at the salinity level of 0 ppt and 10 ppt, but not 35 ppt. In general, BK virus gene copy numbers were relatively stable over the 7-day period among all saliniti es at 4C; however, fewer copy numbers were detected as the salinity decreased at 35C ( Figure 2 ). The decay rate of the BK virus gene copy number s are summarized in Table 17 At 0 ppt the BK virus gene copy number decay rate was not si gnificantly different between 4C and 25C. However, the average decay rate at 0 ppt for the 35C treatme nt was significantly larger than at either 4C or 25C ( P <0.001 for each). The same relationship was reported for the 10 ppt treatment, as the decay rates at 4C and 25 C were not significantly different, while the average decay rate at 35C was significan tly larger than either 4C or 25C ( P <0.001 for each). At 35 ppt there was no significan t difference among the decay rates at any temperature (P =0.3961), and copy numbers were very consistent over the seven-day experiment. Among the BK virus gene copy number decay rates determined for 4C and 25C, there were no significant differen ces at any level of salinity ( P =0.2077 and P =0.1545, respectively). At the higher temperature, 35C, the average decay rates among 0 ppt and 106

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10 ppt were not significantly different; howev er the average decay rate at 35 ppt was significantly smaller than 0 ppt and 10 ppt ( P <0.001 and P <0.0001, respectively). The decay rates of the BK virus gene copy numbers were compiled for each level of salinity and temperature. Temperature or salinity alone had no significant effects on the decay rates (F2,18=3.002, P =0.160 and F2,18=0.932, P =0.465). However, the interaction of temperature and salinity had significant effects on th e decay rates of BK copy numbers (F4,18=42.068, P <0.0005). Based on the univariate analysis results, an increase of temperature coupled with a decrease of salinity increases decay rates of BK virus gene copy numbers. Physical parameters for all natural sunlight experiments The physical parameters collected duri ng each trial of the natural sunlight experiment are summarized in Table 18 The average high and low outside temperatures over the 3 trials were 29.0 2.2C and 15.1 2.9C, respectively. The average UV index and UV intensity were 8.0 1.4 and 0.60 0.27 mW/cm2, respectively. The average water temperatures of the samples cont aining BK viruses exposed to light or dark conditions were 27.7 3.5C and 25.5 2.6C, respectively. The average water temperatures of the samples containing sewage exposed to light or dark conditions were 25.9 3.4C and 23.6 2.1C, respectively. Effects of salinity x natural sunlight on the persistence of BK viruses: natural sunlight experiment with inoculated BK viruses BK virus gene copy number concentrations at the three levels of salinity in the natural light experiment followed the same general, fairly linear decline trend as observed at 35C and lower salinity in the e ffects of temperature x salinity: laboratory experiment with BK viruses ( Figure 3 ). The decay rate of the BK virus gene copy 107

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numbers are summarized in Table 19 At 0 ppt the BK virus gene copy number decay rates were not significantly different among th e two levels of light exposure (light and dark) ( P =0.5258). At both 10 ppt and 35 ppt salin ity levels, the decay rates of the light exposures were significantly la rger than the dark exposures ( P =0.0006 and P =0.0012). Among the average decay rates determined for the light exposures, the decay rate of the 0 ppt treatment was significantly larger than either 10 ppt or 35 ppt ( P <0.05 and P <0.01, respectively), and there was no significant difference between the 10 ppt and 35 ppt treatments. Comparisons of the average decays rates for the dark exposures followed similar trends; the decay rate of the 0 ppt treatment was significantly larger than either 10 ppt or 35 ppt ( P <0.001 for both), and there was no significant difference between 10 ppt and 35 ppt. The decay rates of the BK virus gene copy numbers were compiled for each level of salinity and light exposure to examine intera ctions of the variable s. Salinity alone and light exposure alone had significan t effects on the decay rates (F2,12=149.542, P =0.007 and F1,12=57.775, P =0.017). However, the interaction of salinity and light exposure had no significant effect on the decay rates of BK copy numbers (F2,12=0.043, P =0.958). Based on the univariate analysis results, a decrease of salinity or light conditions increases decay rates of BK virus gene copy numbers. Effects of salinity x natural sunlight on the persistence of HPyVs: natural sunlight experiment with sewage HPyVs gene copy number concentrations in mesocosms inoculated with sewage persisted longer under dark conditions ( Figure 4 ) than when exposed to sunlight. The decay rate of the HPyVs gene copy numbers are summarized in Table 19 At all salinities, HPyVs gene copy number decay rate s were significantly greater for the light 108

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exposure treatment compared to the dark ( P =0.0008, P =0.0032, and P =0.0004, respectively). There were no significant di fferences among all the salinities for the average decay rates in the light exposures ( P =0.2704). In addition, among the average decay rates determined for the dark e xposures, there were also no significant differences among all the salinities (P =0.4235). The decay rates of the HPyVs gene copy numbers were compiled for each level of salinity and light exposure to determine interactions. Light exposure alone had significant effects on the decay rates of HPyVs (F1,12=15.628, P =0.002). However, the salinity alone and interaction of salinity and light exposure had no significant effect on the decay rates of HPyVs gene copy numbers (F2,12=1.314, P =0.305 and F2,12=1.331, P =0.301, respectively). Based on the multivariate analysis results, light conditions increases decay rates of HPyVs gene copy numbers. Effects of salinity x natural sunlight on th e persistence of culturable fecal coliforms: natural sunlight experiment with sewage In general, fecal coliform concentrations persisted longer under dark conditions than when they were exposed to sunlight ( Figure 5 ). The decay rates of the fecal coliform concentrations are summarized in Table 19 As compared to dark exposures, fecal coliform decay rates were significantly larger under the light exposure at all levels of salinity ( P <0.0001 for all). Among the average decay rates determined for the light exposures, there we re no significant differences among all the salinities ( P =0.2671). In addition, among the average de cay rates determined for the dark exposures at each level of salinity, there were also no significant differences among all the salinities ( P =0.0573). 109

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The decay rates of the fecal coliform con centrations were compiled for each level of salinity and light exposure. Light exposur e alone had significant effects on the decay rates of fecal coliforms (F1,12=45.399, P <0.0005). However, the salinity alone and interaction of salinity and light exposure had no significant effect on the decay rates of fecal coliforms (F2,12=2.774, P =0.102 and F2,12=1.548, P =0.252, respectively). Based on the multivariate analysis results, the dark conditions decreases decay rates of fecal coliform concentrations. Effects of salinity x natural sunlight on th e persistence of E. co li: natural sunlight experiment with sewage In general, culturable E. coli populations persisted longer under dark conditions than light ( Figure 6 ). The decay rates of the E. coli concentrations are summarized in Table 19 At 0 ppt, 10 ppt, and 35 ppt E. coli decay rates were sign ificantly larger among the light exposures ( P <0.0001 for all). Among the average decay rates determined for the light exposures, there we re no significant differences am ong all the salinities at the alpha level of 0.05; however the P value shows that a biological difference probably does exist (P =0.0524) with E. coli concentrations decreasing the fastest at 35 ppt. Among the average decay rates determined for the dark exposures, there were also no significant differences among all the salinities (P =0.8158). The decay rates of the E. coli concentrations were compiled for each level of salinity and light exposure. Sa linity alone, light exposure al one, and the interaction of salinity and light exposure had signif icant effects on the decay rates of E. coli (F2,12=6.834, P =0.010, F1,12=34.610, P <0.0005, and F2,12=6.967, P =0.010, respectively). Based on the multivariate analysis results low salinity, dark conditions, and the combination of both can decrease decay rates of E. coli concentrations. 110

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Effects of salinity x natural sunlight on the persistence of ente rococci persistence: natural sunlight experiment with sewage In general, enterococci concentra tions persisted longer under the dark conditions than under light ( Figure 7 ). The decay rates of the enterococci concentrations are summarized in Table 19 At 0 ppt, 10 ppt, and 35 ppt, enterococci decay rates were significantly larger among the light exposures ( P <0.0001, P =0.0009, and P <0.0001, respectively). Among the average decay rates determined for the l ight exposures, there were no significant differences among all th e salinities at the alpha level of 0.05; however, like the E. coli data, a biologically significan t effect was probably present ( P =0.0626) with the decay rate of 10 ppt and 35 ppt almost double the decay rate at 0 ppt. Among the average decay rates determined for the dark exposures, there were no significant differences among all the salinities ( P =0.2647). The decay rates of the enterococci concen trations were compiled for each level of salinity and light exposure. Light exposure alone had significant effects on the decay rates of enterococci (F1,12=23.628, P <0.0005). However, salinity alone and the interaction of salinity and light exposure had no significant effect on the decay rates of enterococci (F2,12=2.068, P =0.169 and F2,12=1.303, P =0.308, respectively). Based on the multivariate analysis results, dark conditi ons decrease decay rates of enterococci concentrations. Effects of salinity x natural sunlight on the persistence of human-associated Bacteroidales marker persistence: natura l sunlight experiment with sewage The human-associated Bacteroidales PCR results collected over the seven day period for all three trials is summarized in Table 20 The human-associated Bacteroidales marker was detected in 100% of the samples from all tria ls over all three 111

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salinities under the d ark exposure. Under light exposure the human-associated Bacteroidales marker was consistently detected at al l three salinities through Day 4. At 0 ppt and 10 ppt the marker was detected in 66.7% of the samples on Day 5 and 6, and only 33.3% of the samples on Day 7. At 35 ppt the marker was detected in 33.3% of the samples from Day 5 to Day 7. Binary detections of the human-associated Bacteroidales marker were compiled for each level of salinity and light exposure over the seven day period. Light exposure alone, time, and the interaction of light e xposure and time had significant effects on the detection of the marker (F1,96=25.000, P <0.0005, F7,96=6.206, P <0.0005, and F7,96=6.206, P <0.0005, respectively). However, salinity alone and interactions of salinity and light exposure, salinity and time, and salinity, ti me, and light exposure had no significant effect on the detection of the marker (F2,96=0.333, P =0.717 for salinity alone and the interaction of salinity and light; F14,96=0.206, P =0.999 for the interaction of salinity and time and the interaction of all three). Based on the multivariate analysis results, light conditions, increased time of exposure, and the coupling of both light and time decreases the detection of the human-associated Bacteroidales marker. Effects of salinity x natural sunlight on the persistence of M. smithii marker persistence: natural sunlight experiment with sewage The M. smithii PCR results collected over the seven day period for all three trials are summarized in Table 20 The M. smithii marker was detected in 100% of the samples from all trials over all thr ee salinities under the dark e xposure through Day 5. At 0 ppt and 10 ppt M. smithii was detected in 33.3% of the samples from Day 6 to Day 7. At 35 ppt the marker was detected in 66.7% of the samples from Day 6 to Day 7. Under light exposure the M. smithii marker was consistently detected at all three sa linities through 112

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Day 2. At 0 ppt and 35 ppt the marker was detected in 33.3% of the samples on Day 3, and 0% of the samples from Day 4 through 7. At 10 ppt the marker was detected in 66.7% of the samples on Day 3, 33.3% of the samples on Day 4, and 0% of the samples from Day 5 to Day 7. Binary detections of the M. smithii marker were compiled for each level of salinity and light exposure over the seven day period. Light exposure alone, time, and the interaction of light exposure and time had si gnificant effects on the detection of the marker (F1,96=96.000, P <0.0005, F7,96=28.514, P <0.0005, and F7,96=10.229, P <0.0005, respectively). However, salinity alone and in teractions of salinity and light exposure, salinity and time, and salinity, time, and light exposure had no si gnificant effect on the detection of the marker (F2,96=0.400, P =0.671; F2,96=1.200, P =0.306; F14,96=0.400, P =0.972; F14,96=0.286, P 0.994, respectively). Based on the mu ltivariate analysis results, light conditions, increased time of exposur e, and the coupling of both light and time decreases the detection of the M. smithii marker. Effects of salinity x natural sunlight on the persistence of HPyV s, culturable bacteria, and other water quality indicators: rela tionships among indicators and markers The decay rates of BK virus gene copy numbers were not significantly different from the decay rates of HPyVs gene copy num bers, fecal coliform concentrations, and E. coli concentrations ( P =0.437, P =0.969, and P =0.265, respectively). However, the decay rates of BK virus gene copy numbers were significantly smaller than the decay rates of enterococci concentrations ( P =0.045). The decay rates of HPyVs gene copy numbers were not significantly different from the decay rates of fecal coliform and E. coli concentrations ( P =0.128 and P =0.348, respectively). The decay rates of E. coli concentrations were not signi ficantly different from the decay rates of enterococci 113

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114 concentrations ( P =0.128 and P =0.348, respectively). However, the decay rates of enterococci concentrations were significantly larger than both HPyVs and fecal coliform decay rates ( P =0.008 and P =0.005, respectively). The predictive relationships among human-associated markers and indicator bacteria and HPyVs concentrations were de termined using binary logistic regression ( Table 21. ). In general, both human-associated Bacteroidales and M. smithii markers were moderately to strongly co rrelated with bacterial indica tors as well as HPyVs. Overall, the M. smithii marker had the highest correla tion with HPyVs concentrations, while the human-associated Bacteroidales marker had the highest correlation with fecal coliform concentrations.

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Table 16. The log decrease of BK viruse s with exposure to various ultraviolet radiation doses. Log Decrease a,b: UV Dose (mWs/cm2) 100 400 800 1200 Salinity 0 ppt 0.76 0.23 [A,B] 3.24 0.20 2.80 0.22 [E] 3.50 0.35 [F] 10 ppt 0.49 0.24 [A,C] 1.41 0.11 [D] 2.24 0.23 2.84 0.14 35 ppt 0.76 0.13 [B,C] 1.73 0.11 [D] 2.91 0.07 [E] 3.73 0.13 [F] a All assays were performed in triplicat e; log decrease reported as the average log10 (y+1)ml-1 standard deviation b Relationships among log decreases fo r each salinity at each UV dose was determined using ANOVA; values in the same column followed by the same letter (i.e. [A] through [F] ) are not statistically different at P < 0.05 115

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0 1 2 3 4 5 6 7 01234567log10 (y+1) BK copies per ml log10 (y+1) BKV copies per ml Days Figure 2. Persistence of BK virus copy numb ers in sterile dechlorinated tap water samples for the temperature x sali nity laboratory experiments. Each point is the mean of three replicat es S.D. (blue=4C; green=25C; red = 35C; solid line=0 ppt; dashed line=10 ppt; dotted line=35 ppt). 0 ppt at 4C 10 ppt at 4C 35 ppt at 4C 0 ppt at 25C 10 ppt at 25C 35 ppt at 25C 0 ppt at 35C 0 ppt at 4C 0 ppt at 25C 0 ppt at 35C 10 ppt at 35C 10 ppt at 4C 10 ppt at 25C 10 ppt at 35C 35 ppt at 35C 35 ppt at 4C 35 ppt at 25C 35 ppt at 35C 116

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Table 17. The decay rates of BK virus copy numbers over a seven day period for the effects of temperature x salinity. DECAY RATESa,b 4C 25C 35C Salinity 0 ppt -0.04 0.03* -0.16 0.01* -0.71 0.13 [A] 10 ppt -0.08 0.03* -0.08 0.04* -0.59 0.03 [A] 35 ppt -0.08 0.03* -0.11 0.06* -0.05 0.04 a All assays were performed in triplicate; decay rates are reported as average standard deviation b Relationships among decay rates for each salinity at each temperature were determined using ANOVA; Values in the same column followed by an asterisk (*) indicates all values in the column are not significantly different, values in the same column followed by the same letter (i.e [A]) are not statistically different at P < 0.05; letters were omitted from columns in which all data was not significantly different 117

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Table 18. The physical data recorded over th ree natural sunlight experiment trials. Days Conditions Analytesa 0 1 2 3 4 5 6 7 Outside Low temperature 15.0 2.0 16.0 3.5 15.7 2.9 17.3 4.0 15.0 1.7 14.0 2.6 15.3 4.9 12.3 1.5 Outside High temperature 29.3 1.5 29.0 1.7 30.3 1.2 30.0 2.6 28.3 0.6 27.7 1.2 29.3 3.5 28.3 4.5 Dark: BK viruses Water temperature 24.3 1.2 24.0 1.0 26.7 0.6 25.3 1.2 26.3 0.6 26.0 6.0 27.7 1.5 24.0 3.6 Dark: sewage Water temperature 20.0 0.0 23.0 0.0 25.7 1.2 24.3 0.6 24.7 0.6 24.0 2.6 24.3 1.2 22.5 3.5 Light: BK viruses Water temperature 24.3 1.2 26.0 0.0 30.0 2.6 28.0 1.0 29.0 1.7 28.7 8.6 30.0 1.0 25.3 2.5 Light: sewage Water temperature 20.0 0.0 25.0 1.0 28.7 1.5 27.7 0.6 27.3 2.1 26.0 4.6 28.7 2.1 24.0 2.6 Light UV index 8.3 1.2 7.7 1.5 8.3 0.6 8.3 0.6 7.3 0.6 9.3 0.6 8.7 1.5 6.3 2.5 Light UV intensity 0.78 0.160.39 0.190.71 0.140.62 0.13 0.52 0.220.76 0.450.71 0.280.31 0.23 118 a All data is reported as an average sta ndard deviation of the 3 data points collect ed for each trial; temperatures reported in units of C; UV intensity reported in units of mW/cm2

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0 1 2 3 4 5 6 7 01234567log10 BK virus copy numbers/100 ml log10 (y+1) BKV copies per 100 ml DaysFigure 3. Persistence of BK virus copy numb ers in sterile dechlorinated tap water samples for the natural s unlight experiments. Each point is the mean of three replicat es S.D. (yellow= light, exposed to sunlight; black=dark, not exposed to sunlight; solid line=0 ppt; dashed line=10 ppt; dotted line=35 ppt). Data was sampled approximately the same time each day for all three trials, data reported fo r each day is approximately 24 1.3 hr from the previous data point. 0 ppt in Dark 10 ppt in Dark 35 ppt in Dark 0 ppt in Light 0 ppt in Dark 0 ppt in Light 10 ppt in Light 35 ppt in Light 35 ppt in Dark 35 ppt in Light 10 ppt in Dark 10 ppt in Light 119

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0 1 2 3 4 5 01234567log10 HPyVs copy numbers/100 mlDays 0 log10 (y+1) HPyVs copies per 100 ml Figure 4. Persistence of hu man polyomavirus (HPyVs) copy numbers in sewage inoculated water samples for the na tural sunlight experiments. Each point is the mean of three replicat es S.D. (yellow= light, exposed to sunlight; black=dark, not exposed to sunlight; solid line=0 ppt; dashed line=10 ppt; dotted line=35 ppt). Data was sampled approximately the same time each day for all three trials, data reported fo r each day is approximately 24 1.3 hr from the previous data point. ppt in Dark 10 ppt in Dark 35 ppt in Dark 0 ppt in Light 10 ppt in Light 10 ppt in Dark 10 ppt in Light 35 ppt in Light 35 ppt in Dark 35 ppt in Light 0 ppt in Dark 0 ppt in Light 120

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0 1 2 3 4 5 01234567log10 (y+1) CFU/100 mlDays log10 (y+1) CFU per 100 ml Figure 5. Persistence of culturable fecal coliforms in sewage inoculated water samples for the natural s unlight experiments. Each point is the mean of three replicat es S.D. (yellow= light, exposed to sunlight; black=dark, not exposed to sunlight; solid line=0 ppt; dashed line=10 ppt; dotted line=35 ppt). Data was sampled approximately the same time each day for all three trials, data reported fo r each day is approximately 24 1.3 hr from the previous data point. 0 ppt in Dark 10 ppt in Dark 35 ppt in Dark 0 ppt in Light 0 ppt in Dark 0 ppt in Light 10 ppt in Light 10 ppt in Dark 10 ppt in Light 35 ppt in Light 35 ppt in Dark 35 ppt in Light 121

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0 1 2 3 4 5 01234567log10 (y+1) CFU/100 mlDays 0 log10 (y+1) CFU per 100 ml Figure 6. Persistence of culturable E. coli in water samples inoculated with sewage for the natural sunlight experiments. Each point is the mean of three replicat es S.D. (yellow= light, exposed to sunlight; black=dark, not exposed to sunlight; solid line=0 ppt; dashed line=10 ppt; dotted line=35 ppt). Data was sampled approximately the same time each day for all three trials, data reported fo r each day is approximately 24 1.3 hr from the previous data point. ppt in Dark 10 ppt in Dark 35 ppt in Dark 0 ppt in Light 0 ppt in Dark 0 ppt in Light 10 ppt in Light 10 ppt in Dark 10 ppt in Light 35 ppt in Light 35 ppt in Dark 35 ppt in Light 122

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-1 0 1 2 3 4 5 01234567log10 (y+1) CFU/100 mlDays log10 (y+1) CFU per 100 ml 0 ppt in Dark 10 ppt in Dark 35 ppt in DarkFigure 7. Persistence of culturable enterococci in water samples inoculated with sewage for the natural sunlight experiments. Each point is the mean of three replicat es S.D. (yellow= light, exposed to sunlight; black=dark, not exposed to sunlight; solid line=0 ppt; dashed line=10 ppt; dotted line=35 ppt). Data was sampled approximately the same time each day for all three trials, data reported for e ach day is approximately 24 1.3 hr from the previous data point. 0 ppt in Light 0 ppt in Dark 0 ppt in Light 10 ppt in Light 10 ppt in Dark 10 ppt in Light 35 ppt in Light 35 ppt in Dark 35 ppt in Light 123

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Table 19. The decay rates of BK virus copy numbers, human polyom avirus copy numbers, culturable fecal coliforms, culturable E. coli and culturable enterococci over a seven day period for the natural sunlight experiment. Analyte DECAY RATESa: Dark Light 0 ppt 10 ppt 35 ppt 0 pptb 10 ppt 35 ppt BK viruses -1.22 0.65 -0.42 0.24 [G,H,I,J] -0.24 0.04 [K,L] -1.73 1.11 -0.80 0.12 [P,Q] -0.57 0.25 [U] HPyVs -0.34 0.12 [A,B,C] -0.59 0.37 [G] -0.44 0.19 [M,N] -1.21 0.62 -1.30 0.49 [R,S] -2.21 1.17 [V,W,X] FCc -0.30 0.06 [A,D,E] -0.22 0.02 [H] -0.35 0.03 [K,M,O] -1.17 0.14 -1.09 0.11 [P,R,T] -1.81 0.80 [U,V] E. coli -0.38 0.06 [B,D,F] -0.40 0.08 [I] -0.38 0.08 [L,N,O] -1.62 0.60 -1.00 0.11 [Q,S,T] -3.43 1.30 [W,Y] Enterococci -0.48 0.02 [C,E,F] -0.69 0.23 [J] -0.67 0.21 -1.61 0.60 d -3.13 1.79 d -3.38 1.17 [X,Y] 124 a All enumeration (e.g. QPCR or membrane filtra tion techniques) assays for each trial were performed in triplicate; decay rates a re reported as average standard deviation; relationships among decay rates for analyte at each salinity were determined using AN OVA; values in the same column followed by the same letter (i .e. [A] through [Y] ) are not statistically different at P < 0.05 bColumns highlighted in grey indicat e there was no significance difference among the decay rates of the column cFC, fecal coliforms dBased on Figure 7 enterococci decay rates of 0 ppt and 10 ppt should be similar; however the high variability among the three trials lead to the dissimilar rates

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Table 20. The persistence of human-associated Bacteroidales spp. and Methanobrevibacter smithii marker detection for the natural sunlight experiments. No. of PCR positive s over 3 trials Days Assay Salinity Sun Exposure 0 1 2 3 4 5 6 7 Humanassociated Bacteroidales spp. PCRa 0 ppt Dark 3 3 3 3 3 3 3 3 Light 3 3 3 3 3 2 2 1 10 ppt Dark 3 3 3 3 3 3 3 3 Light 3 3 3 3 3 2 2 1 35 ppt Dark 3 3 3 3 3 3 3 3 Light 3 3 3 3 3 1 1 1 M. smithii PCRa 0 ppt Dark 3 3 3 3 3 3 1 1 Light 3 3 3 1 0 0 0 0 10 ppt Dark 3 3 3 3 3 3 1 1 Light 3 3 3 2 1 0 0 0 35 ppt Dark 3 3 3 3 3 3 2 2 Light 3 3 3 1 0 0 0 0 125 aThe data points reported for each day are compiled from each of th e three natural sunlight experi ment trials; +, positive PCR result; -, negative PCR result

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Table 21. The relationships between culturable bacterial indicator concentrations, HPyVs concentrations by QPCR and other human-asso ciated markers of fecal pollution (+ or by PCR) over a seven day period analyzed using binary logi stic regression. Dependent Variable Predictor Variablea Statistic Light Exposuresb Dark Exposuresb Combined Datab Humanassociated Bacteroidales marker FC Nagelkerkes R2 0.422 n/ac 0.553 P value <0.0005 <0.0005 odds ratio 5.54 x 1014 2.67 x 1013 E. coli Nagelkerkes R2 0.304 n/ac 0.477 P value <0.0005 <0.0005 odds ratio 3.80 x 1027 6.26 x 1024 Enterococci Nagelkerkes R2 0.213 n/ac 0.358 P value 0.001 <0.0005 odds ratio 1.38 x 1013 1.13 x 1023 HPyVs Nagelkerkes R2 0.318 n/ac 0.436 P value <0.0005 <0.0005 odds ratio 7.96 x 1017 1.11 x 1016 M. smithii marker FC Nagelkerkes R2 0.716 0.149 0.612 P value <0.0005 0.013 <0.0005 odds ratio 9.087 2.459 3.220 E. coli Nagelkerkes R2 0.696 0.363 0.620 P value <0.0005 <0.0005 <0.0005 odds ratio 2.05 x 103 5.182 4.325 Enterococci Nagelkerkes R2 0.569 0.387 0.593 P value <0.0005 <0.0005 <0.0005 odds ratio 8.94 x 1014 3.491 6.005 HPyVs Nagelkerkes R2 0.654 0.509 0.695 P value <0.0005 <0.0005 <0.0005 odds ratio 5.917 4.560 5.245 aFC, fecal coliforms; HPyVs, human polyomaviruses bAll significant Nagelkerkes R2 and corresponding P values are bolded and highlighted green; all significant odds ra tios are bolded and highlight ed yellow, odds ratio were considered significant at an alpha level of 0.05. cHuman-associated Bacteroidales were detected in 100% of the dark exposure samples, binary logistic regression requires two values among the dependent variables being processed. 126

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Discussion The objective of this study was to determine the effect s of environmental factors (e.g. ultraviolet radia tion, temperature, salinity) on the detection of human polyomavirus DNA and to compare it with general and speci fic indicators of mi crobial pollution. Numerous studies have been conducted on the survival of indicator bacteria (23, 103, 228, 286, 339), adenoviruses (112, 166, 313), enteroviruses (112, 187), and Bacteroidales spp. (21, 170, 339) under various conditions; however little is known about the persistence of HPyVs DNA. Initial concerns for utilizing a viral indicator excreted in urine included inaccurate estimations of fecal input in recreational waters because of the possible persistence of HPyVs DNA coupled with the high probability of swimmer/bather excretion. However, we found the detection of HPyVs DNA mimicked the general trends recorded for indicator bacteria and M. smithii when exposed to natural environmental conditions (e.g. solar radia tion, predation, temper ature). Both adenoviruses and HPyVs are double-stranded DNA viruses. Doublestranded DNA viruses tend to be more resistant to UV disinfection as compared to bacteria and RNA viruses (112, 154, 166, 286). Gerba et al. (112) reported a 4 log decrease of viable adenovirus type 2 with a UV dose of 160 mWs/cm2, while a UV dose 36 mWs/cm2 induced a 4 log decrease of viable echoviruses, coxsackieviruses, and polioviruses. Ko et al. (166) also reported a large log-reduction of viable adenovirus type 41 at 150 mWs/cm2 dose. Our laboratory found a larger UV dose (800-1200 mWs/cm2) was required for a 3 log reduction of the de tection of HPyVs DNA as compared to UV doses previously reported to reduce adenoviruses. To enumerate HPyVs, our study utilized a QPCR-based method that theoretical ly detects both viable and non-viable cells; 127

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conversely the previously mentioned studi es were based on viability assays (e.g. integrated cell culture PCR). UV exposur e induces thymine dimers throughout the genomic DNA. Viruses undergoing active re plication in a host ce ll have potential DNA replicative proofreading capabi lities; however the introductio n of a thymine dimer in an area of the DNA necessarily for initiation of replication may prevent the correction of damaged DNA and subsequently in activate the virus. In contrast, the effect of thymine dimers in DNA based assays (e.g. PCR and QP CR) is solely steric hindrance. The HPyVs QPCR targets approximately 180 bp of a 5,000 bp genome, within the targeted region there are approximately 60 adjacent th ymines (based on sequence analysis) and therefore the probability of a thymine dimer in the region is relatively small at low doses of UV. In addition, mid-range salinity seemed to decrease the effects of higher UV doses (at 800 and 1200 mWs/cm2 only) on the detection of HPyVs DNA. It has been reported that double stranded DNA complexes are more stable at higher NaCl concentrations and solutions containing 0.9 M Na+ are commonly used in DNA:DNA hybridizations (125). The stabilizing properties of NaCl may have contributed to the decreased UV effects at higher salinity. This study documented the decrease of HPyVs DNA detectability in dechlorinated tap water over a 7 day pe riod, which was most notable at higher temperatures and lower salinities. Interestin gly, temperature or salinity alone did not have a significant effect on the persistence of HPyVs; how ever, the interaction of both (higher temperature coupled with low salinity) caused a significant decrease of the concentrations of HPyVs DNA. The sta ble persistence of HPyVs DNA at higher temperatures and higher salinities (35C and 35 ppt) may be attributed to the evolution of 128

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these viruses with humans. These viruse s remain latent in the kidneys, undergo reactivation, and then are ex creted in the urine (278). The human body is approximately 37C and urine has a salinity of approximate ly 20 ppt (322); therefore HPyVs must be able to endure these conditions, po tentially for long time periods. As compared to the effects of temperatur e x salinity: laborato ry experiment, the concentrations of BK virus DNA followed the sa me general trend in the natural sunlight experiment under both dark and light condi tions, i.e. higher salinities resulted in lower decay rates. However, there was also an effect of light exposure which increased the rate of decay in samples exposed to s unlight. HPyVs enumerat ed in the parallel natural sunlight experiment with sewage inoculum did not follow the same trend in regards to salinity; in fact the opposite effect of salinity was observed and light exposure was the main contribution to th e decline of HPyVs concentrations. Interestingly, the same general trend was doc umented with all three indicator bacteria. Fujioka et al. (103) reported similar findings of the effects of sunlight on the survival of enteric bacteria introduced to seawater and fresh water environments. The study reported a higher rate of decay under l ight exposure in seawater as compared to freshwater. Since the 1981 study, many researchers have repo rted the decreased cu lturability of fecal bacteria after sunlight exposure (23, 66, 83, 228, 286). Barcina et al. (23) suggested that enterococci are more resilient to sunlight than E. coli ; however, Noble et al. (228) report the contrary. Our results indicated there was no significant difference among the decays rates of enterococci and E. coli under light conditions; however, higher salinities contributed to higher decay rates for E. coli while enterococci decay rates were mainly affected by light exposure. The decay rate of HPyVs, E. coli, and enterococci 129

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concentrations under all conditions were not significantly different, which also may suggest potential contribution of predation or presence of degradative enzymes from other organisms found in sewage (32). HPyVs could not be detected by QPCR afte r Day 5 when exposed to sunlight, and M. smithii could not be detected by PCR afte r Day 4. The human-associated Bacteroidales marker was detected by PCR thr oughout the 7 day study. The difference among the detection persistence may be attributable to the relatively high concentrations of human-associated Bacteroidales in sewage as compared to both HPyVs and M. smithii (129). Wetz et al. (341) inoc ulated enteroviruses into unfiltered seawater and found a 4 log reduction of infectious particles after 4.5 days (30C, no light exposure). The concentration of enteroviruses in un treated sewage is approximately 103 MPN100 L-1 (131). Compared to human-associated Bacteroidales decay rates, the rate of HPyVs and M. smithii decay at higher salinities may more closely mimic the persistence of infectious enterovirus in areas affected by direct sewage contamination. Walters et al. (339) examined the persistence of th e human-associated Bacteroidales marker in sewage inoculated seawat er and found the marker was detected through Day 8. In addition, the study reporte d that sunlight expos ure resulted in an increased decay rate of the marker. In contrast, Bae and Wuertz (21) found sunlight did not affect the decay rate of the marker in seawater microcosms and Walters and Field (338) also did not find a difference of decay rates under sunlight exposure in fresh water microcosms. While the binary PCR us ed to detect the human-associated Bacteroidales marker in this study does not allow calculation of a decay rate for direct comparison with the last two studies, the decreased detection of the marker in 2 of the 3 trials upon 130

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exposure to sunlight and the continuous de tection of the marker in dark conditions strongly suggests that light exposure limits th e persistence of the marker, which agrees with the findings of Walters et al. (339). Overall, mesocosm studies demonstrated the HPyVs marker persistence is similar to that of culturable fecal bacteria. In addition, the persis tence of HPyVs DNA is comparable to previously published data on the persistence of pa thogens. In addition, this study further emphasized the se nsitivity of the human-associated Bacteroidales marker for detection of sewage contaminati on, as the marker persisted longer than the other two human-associated markers. Previous studies have demonstrated the imperfect human specificity of both the human-associated Bacteroidales and M. smithii marker, and the 100% human specificity of the HPyVs ma rker (129, 203). Therefore, our suggestion is the incorporation of human-associated Bacteroidales M. smithii and HPyVs marker in a multi-indicator toolbox. The utilization of all three markers may allow for a more accurate assessment of water qua lity, as well as information regarding the recentness of the contamination. Understanding the infl uence of environmental factors on water quality indicators leads to more accurate in terpretation of results of the many emerging methods for measurement of sources of f ecal pollution in environmental waters and subsequently allows for a more accurate assessm ent of human health risks. However, as with all water quality analys es the careful consideration careful consideration of the location, climate, historical water quality data, and possible sour ces of contamination should be made before water qualit y indicators are selected. 131

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132 Acknowledgements We would like to thank Dr. John Paul for the use of the ABI 7500 real time machine. In addition, we would like to thank Jenny Delaney, Dave John, Lauren McDaniel, Robert Ulrich, Beth Young, and Brian Zielinski fo r technical and logistical support. Funding for this study was provided in part by EPA grant (MX-96478707-0).

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CHAPTER 4: A TOOLBOX APPROACH TO ASSESS WATER QUALITY AT TWO CALIFORNIA BEACHES: A CASE STUDY AT DOHENY AND AVALON BEACHES Shannon McQuaig1, John Griffith2, and Valerie J. Harwood1 1Department of Biology, University of South Florida, Tampa, FL 33620 2 Southern California Coastal Water Research Project Costa Mesa, CA 92626 Prepared for submission to: Applied and Environmental Microbiology 133

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Abstract An improved method to extract hum an polyomavirus (HPyVs) DNA from environmental water samples was develope d by coupling protocols from the MO BIO PowerSoil kit and DNeasy Tissue kit. The de veloped protocol was utilized in a water quality study at two marine beaches in California (USA) during May-September, 2008. Water quality was assessed using fecal indicat or bacteria concentra tions and four humanassociated, library-independe nt microbial source tracking (MST) methods (HPyVs PCR and QPCR, Methanobrevibacter smithii PCR, human-associated Bacteroidales PCR, and adenovirus nested PCR). One hundred thirty sa mples were collected from five sites at Doheny Beach and 120 samples were collect ed from four sites at Avalon Beach. Excedances of water quality standards were noted at Doheny and Avalon Beaches for enterococci (28.5% and 31.7%, respectively) and fecal coliforms (20% and 5.8%, respectively). Human-associated MST markers were detected by PCR more frequently at Avalon Beach (55% of all Avalon samples) as compared to Doheny Beach (33.8% of all Doheny samples). The human Bacteroidales marker was the most frequently detected marker at both beaches. Based on the compiled results we suggest that poor water quality at Doheny Beach is a combination of poor wa ter circulation, urban runoff from San Juan Creek, and bather overloading. In comparis on, we suggest that poor water quality at Avalon Beach is dominated to a greater extent by human sources, perhaps from overflowing low-flow sewage and stormwater diverters and wastes from boaters. We strongly recommend the use of a multitiered approach that meas ures overall fecal contamination and contamination sources when assessing recreational water quality. 134

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Introduction During the 1970s and1980s, several epidem iological studies were conducted to assess risks associated with human exposure to water containing fecal contamination and high concentrations of to tal and fecal coliforms, Enterococcus species, and E. coli (55, 323). The resulting report refined existing criteria utilized for monitoring recreational water quality, and reported correl ations of bacterial densiti es and swimming-associated gastroenteritis (323). Based on the findings, the U.S. Environmental Protection Agency (EPA) recommended regulatory standards of ente rococci concentrations for marine water (323). However, the reliability of regulatory standards based on bacterial indicators is uncertain due to the high densit ies of fecal indicator bacteria in non-point sources such as animal excrement and stormwater runoff (45, 178, 229, 230, 257), coupled with lack of knowledge about pathogens in such sources. The possibility also exists of survival and regrowth of indicator bacteria in tropical climates (68). To address limitations of traditional bacterial indicators, molecular assays for human-associated microbes (i.e. microbial source tracking methods ) have been developed to detect impacts from human sewage. This study employed established and em erging human-associated MST assays which included: human-associated Bacteroidales PCR (27), Methanobrevibacter smithii PCR (330), adenovirus nested PCR (251), human polyomavirus (HPyVs) PCR, and HPyVs QPCR (202, 203). Bacteroidales spp. are gram-negative, strictly anaerobic nonspore forming bacilli that out-number coliform s in both human and animal feces and have been isolated at concentrations of 109 to 1011 organismsg-1 in feces (141, 342), and 109 Bacteroidales100 ml-1 (75). Due to a high degree of sens itivity and its position as one of the first library-independent MST methods directed against human fecal sources, the 135

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human-associated Bacteroidales assay has been widely used (11, 27, 34, 87, 88, 117, 163, 181, 234, 277). M. smithii are the most prominent methanogen in the human intestines and have been found at concentrations of 10710 organismsg-1 in feces (40, 186). The use of M. smithii to identify human-associated fe cal pollution has been limited but successful in MST studies conducted to date (129, 330). Adenovirus type 40 and 41 are the etiological agents of viral gastroenteritis. The presence of enteric adenovirus type 40 and 41 has been utilized as an indicator of human fecal pollution in water (69, 153, 251), and allows for direct human health risk assessments. The HPyVs PCR and QPCR assay used in this study targeted JCV and B KV (202, 203), which are both widespread in sewage and are excreted in urine (8, 37, 199, 252). BKV has also been detected in feces (331, 350). Both viruses are genetically stable distributed worldwid e, and maintain high seropositive rates in the human population (5, 37, 85, 248, 301, 308, 366). The simultaneous use of both JCV and BKV as indi cators of human fecal pollution has been successful in several laboratory and field studies (35, 129, 202, 203). Research to define the most appropriat e microbial source tracking (MST) assay is on-going, and several researchers have sugge sted a toolbox or multitiered approach (226, 227, 267, 335). Toolbox studies incorporat e multiple indicators or markers to assess water quality (226, 335). Multitiered approaches usually assess water quality in tiers, using between one and several indicators or markers during each tier and generally progressing from less expensive to more e xpensive methods as possible sources are narrowed from many to a few (34, 227). To date several studies have been conducted in which non-point or point source contributions to water quality degr adation have been determined utilizing a microbial so urce tracking toolbox (34, 132, 145, 202, 227). 136

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However, to our knowledge, studies using the toolbox approach to as sess water quality of both non-point and point source contaminated waters within the same study period have not been performed. This study incorporated data from two California beaches impacted by non-point source (Doheny State Beach, Dana Point, CA) or point source pollution (Avalon Beach, Catalina Island, CA). Indicator bacteria concentrations in th e waters of Doheny State Beach routinely exceed regulatory standards for microbial water quality (221). The poor water quality has been attributed to several factors incl uding limited water circulation caused by a jetty located at the north-west end of the beach, high concentrations of seagulls releasing fecal matter into the water and sediments, and urba n runoff from the San Juan Creek watershed (221, 236). San Juan Creek is a 29-mile stream that runs through Orange County, California. The watershed encompasses approximately 125 square miles (236). The stream discharges into a fresh-water lagoon located at the north end of Doheny Beach. Occasionally, under high tide or wet conditi ons, the lagoon breaches the beach and the creek outflows into the P acific Ocean (214). Water quality monitoring around the outflow area has reported consistently higher co ncentrations of fecal indicator bacteria in the creek as compared to the ocean (221). In a 2003, antibiotic resistance analysis and ribotyping of E. coli and enterococci in the area suggested that the high concentrations of indicator bacteria were due to intervening storm drains, direct fecal contamination by avian sources (e.g. seagulls), and survival a nd proliferation of adaptive fecal bacteria (236). A wastewater treatment plant is locate d near the San Juan Creek less than a mile upstream of Doheny Beach; however the effluent is discharged into the San Juan Creek Ocean Outfall and not the creek (60). The O cean Outfall is located approximately 3.2 km 137

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offshore in a southwest direction from Dohe ny Beach at San Juan Creek and has a flow rate of 19.1 mgd (289, 290). The waters of Avalon Beach also routin ely exceed regulatory standards (221). However, in contrast to Doheny Beach, Avalon Beach waters circulate relatively freely (158). Sewer lines run within 20 m parallel to the beach and stormwater runoff is channeled into the sewer lines using low-flow diverters (34, 118). When the low-flow diverters reach maximum capacit y, runoff enters small drains that discharge into the ocean through the sand (118). By way of th ese drains, untreated runoff and sewage can enter the ocean and degrade water quality. Large numbers of s ea gulls and pigeons accumulate around restaurants near the beach an d may also contribute to fecal bacteria inputs (34). Boehm et al. ( 34) reported the presence of human-associated markers and human pathogens at several sites, emphasi zing the impact of human fecal input. Descanso Beach, which is north of Avalon Beach, was used a control site due to historically low levels of f ecal bacteria (221). The goals of this study were to assess the presence and absence of several MST markers, enumerate bacterial indicators, dete rmine any correlations among indicators and markers, and examine the differences in o ccurrence of indicators and markers between the non-point and point source locations. The amalgamation of bacterial, viral, and methanogen-based MST applications may allo w for a more complete perspective of microbial levels and a better interpretation of water quality and hum an health risks at beaches with various contamination sources. 138

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Materials and Methods Construction of recombinant plasmid fo r HPyVs control and QPCR standard curve BK virus (ATCC VR-837) was obtained from the American Type Culture Collection (Manassas, VA), and propagat ed in HEL-299 cells (ATCC CCL-137). The cell line was grown in Eagle minimum esse ntial medium (Sigma, St. Louis, Mo.) supplemented with 10% heat-inactivated fe tal bovine serum (FBS) (Invitrogen, Inc., Carlsbad, CA). Cell lines were maintained in Eagle minimum essential medium containing 2% FBS. DNA was extracted from 0.1 ml of the BK virus culture using DNeasy Blood & Tissue Kit (Qiagen, Inc., Valenc ia, CA), and used as template in the HPyVs PCR assay (202). The resulting 176-bp amplicon was purified using QIAquick PCR Purification Kit (Qiagen, Inc.) and th en cloned into pCR-TOPO vector (Invitrogen, Inc.). The vector was then transferred into E. coli One Shot chemically competent cells, and plated on LB agar containing 100 gml-1 ampicillin. Recombinant plasmids with a single copy of the amplicon were purified using Ge nElute Five-Minute Plasmid Miniprep Kit (Sigma, St. Louis, MI ) following manufacturers instructions. Purified recombinant plasmi d DNA containing the insert was quantified using a QubitTM fluorometer (Invitrogen, Inc., Carlsbad, CA). DNA quantification was performed in triplicate and averaged to de termine the estimated total DNA concentration. Insert copy numbers were estimated by multiplying the average DNA concentration by Avogadros number then dividing by the pr oduct of the entire plasmid a nd average weight of a base pair (362). Positive PCR controls To construct clones for use as positive controls, a specific gene fragment for each MST target (human-associated 525-bp region of the 16S rRNA gene of 139

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Bacteroidales or 221-bp region of the mnif gene of M. smithii) was amplified using the primers described below. The 525-bp and 221-bp amplicon were then cloned into a pCR-TOPO vector and E. coli One Shot chemically competent cells were transformed as described above. Recombinan t plasmids were propagated and purified as described above. Plasmids containing insert s were confirmed by sequencing at Macrogen USA (Rockville, MD). All sequences were subjected to BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) for comparison with published sequences. Negative controls The freedom of each DNA extraction from contamination was tested using sterile, DNA-free water. Sterile water was placed into the DNA extraction tube and processed in parallel with all samples. In addition, c ontamination of water sa mples during collection or filtration was ruled out using method blanks. Method blanks were processed in parallel with all water samples. DNA fr om the extraction blank and method blank was used as template in each respectiv e PCR or QPCR reaction. Comparison of DNA extraction protocols Plasmids containing the BK virus insert were serially diluted in nuclease-free, reagent grade water to achieve 7.7 x 1037.7 x 105 target copy numbers in 100 l. One hundred microliters of each dilution was then inoculated into MO BIO Powersoil Kit bead tubes (MO BIO Laborator ies, Carlsbad, CA). All analyses were conducted in triplicate. The efficacy of the standard MO BIO Powersoil kit protocol was compared to a modified protocol (MO BIO/Qiag en Hybrid protocol; see below). 140

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Standard MO BIO PowerSoil Kit Protocol The 100 l aliquot of plasmid DNA wa s processed as per manufacturers instructions with some minor modifications. After the addi tion of 60 l of solution C1, the bead tube was placed into FastPrep FP 120 (Qbiogene, Inc., Carlsbad, CA), and run for 40 sec. The sample was then processed according to manufacturers instructions. MO BIO/Qiagen Hybrid (MQH) Protocol The modified protocol utilized both MO BIO Powersoil Kit and Qiagen DNeasy Tissue Kit (Qiagen, Inc., Valencia, CA) components. Sixty microliters of solution C1 (MO BIO kit) was added to the PowerBead tube (MO BIO kit) containing the DNA sample. The tube was vortexed for 3 sec, then placed into FastPrep FP 120, and run for 40 sec. The bead tube was then centrifuged at 10,000 rpm for 30 sec. All the supernatant was transferred to a sterile 2.0 ml tube (provi ded in the MO BIO kit) containing 250 l of solution C2 (MO BIO kit) and then vortexed fo r 5 sec. The tube was placed at 4C for 5 min., and was then centrifuged at 10,000 rp m for 1 min. All the supernatant was transferred to a new sterile 2.0 ml tube (pr ovided in the MO BIO kit) containing 285l of solution C3 (MO BIO kit) and vortexed for 2 s ec. The tube was then incubated again at 4C for 5 min, and centrifuged at 10,000 rpm for 1 min. All the supernatant was transferred to a 50 ml tube containing 1.6 ml solution C4 (MO BIO kit) and vortexed for 5 sec. The 50 ml tube was then centri fuged at 2,000 rpm for 1 min. Qiagen DNeasy Tissue spin columns (Qiagen, Inc.) were at tached to a QIAvac 24 vacuum manifold (Qiagen, Inc.) by VacValves (Qiagen, Inc.) out fitted with VacConnectors (Qiagen, Inc.). The vacuum manifold was turned on and the individual samples were continuously loaded (~ 750 l at a time) into separa te Qiagen DNeasy Tissue columns until all the sample had passed through the column. The pressure did not exceed 25 in. Hg. The 141

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vacuum was then turned off and the pressure was relieved. The column was then washed by passing 750 l of solution C5 (MO BIO k it) through the column. The pressure was relieved and the wash step was repeated 2 additional times. Once the 3 washes were completed, the Qiagen column was detached and placed into a collection tube (Qiagen kit). The collection tube containing the column was centrifuged at 14,000 rpm for 3 min to completely remove solution C5. Afte r centrifugation, the co lumn was carefully removed from the collection tube and placed into a new sterile 2.0 ml microcentrifuge tube. A 100 l aliquot of nuclease-free, r eagent grade sterile wa ter was added to the center of the column filter, and incubated at room temperature for 5 min. The tube containing the column was then centrif uged at 10,000 rpm for 1 min. Eluted DNA was captured in the tube and the Qiagen co lumn was removed and discarded. DNA was stored at -20C until used as template in the HPyVs QPCR. Human polyomavirus QPCR The HPyVs QPCR mixtures were prepar ed using 25 l TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems, Foster City, CA), 0.5 M concentrations of each primer ( Table 22 ), 0.4 M labeled probe concentration, 5 l of template DNA (1-25 ng), and the volume was adjusted to 50 l using reagent grade water. Amplification was performed in the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). The QPCR reaction conditions were as follows: DNA polymerase activation at 95C for 10 min, fo llowed by 40 cycles of DNA melting at 95C for 15 sec, then annealing at 55C for 20 sec, and extension at 60C for 60 sec. 142

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Human polyomavirus QPCR standard curve To produce a standard curve, the recomb inant plasmid DNA was serially diluted in nuclease-free reagent grade water to a final concentration ranging from 102 to 106 gene copiesl-1. Five microliters of each dilution were used as template in the Taqman real time standard curve PCR reactions. Each dilu tion was run in duplicate. A standard curve was run with every QPCR assay. Applied Bios ystems default settings for the threshold cycle (Ct) were used for data analysis. Th e Ct values were plotted against copy number to generate the standard curve. Linear re gression was used to assess the relationship between Ct values and copy number. Comparison of viral DNA recovery from wa ter concentrates containing BK viruses BK viruses were propagated and harveste d from HEL-299 cells. After cytopathic effects were observed, cell culture medium containing suspended viral particles was harvested from the cell culture flasks and fr ozen in 1 ml aliquots (BK virus stock). Different BK virus stock solutions were d iluted and used throughout the experiments. BK virus stock solutions contained 1.3-5.9 x 1011 HPyVs copy numbersml-1. Before dilution, a 2 ml aliquot of BK virus stock was centrifuged at 10,000 rpm for 5 min. Approximately 1.5 ml of the supernatant was transferred into a st erile microcentrifuge tube and again centrifuged at 10,000 rpm for 5m in. From this sample, 1 ml of the supernatant was serially diluted 10 times. One mill iliter of the 10-5, 10-6, 10-7, 10-8, 10-9 and 10-10 dilutions were inoculated into 500 ml of sterile dechlorinated tap water. The viruses were concentrated using 0.45 m pore size nitrocellulose filters. To promote electrostatic interactions betw een the viral capsid and nitroce llulose filter, the pH of the water was adjusted to 3.5 using 2.0 N HCl pr ior to filtration (14, 202). The filter was placed into a 2 ml microcentr ifuge tube and placed at 20C. DNA was extracted from 143

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the filter using either the standard MO BIO Powersoil Kit protocol or the modified MQH protocol. HPyVs copy numbers were enumerated by the QPCR assay as described above. Comparison of viral DNA recovery from wa ter concentrates containing raw sewage Untreated sewage influent was collecte d from Falkenburg Advanced Wastewater Treatment Plant (Brandon, FL), designed for 9 millions gallons per day average daily flow. Various aliquots (0.01-1.00 ml) of sewage were inoculated into 500 ml of sterile dechlorinated tap water. The viruses we re concentrated from the water, DNA was extracted from the filters, and HPyVs were enumerated; all as described above. Doheny Beach sites and sampling schedule Water was collected every weekend at five sites along Doheny Beach over a four month period (May-September 2008). Site s were designated A, B, C, D and E ( Figure 8A). Sites A, B, D and E samples were colle cted along the beach. Site A samples were collected adjacent to the jetty. Site B samples were collected at the north beach between Site A and D. Site C samples were collected in the San Juan Creek Lagoon approximately 50 m prior to its discharge acro ss the beach into the ocean. Site D samples were collected on the north end of the south beach adjacent to lagoon. Site E samples were collected at the south end of the south beach. Avalon Beach sites and sampling schedule Water was collected every Thursday through Sunday at 4 sites along Catalina Island over a 2 month period (June-August 2008). Sites were designated A, B, C, and D ( Figure 8 B). Samples from sites A, B, and C we re collected along Avalon Beach. Site A samples were collected at the south beach in the southernmost corner of the beach. Site 144

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B samples were collected on the south side of the pier (located between sites B and C). Site C samples were collected on the north side of the pier. Site D samples were collected north of Avalon Bay near Descanso Beach Club. Sample collection Sampling was conducted from 8:00-9:00 AM. All samples were taken at 0.5 meter depth. Approximately one liter of water was placed into a sterile one liter polypropylene container and transported to th e laboratory on ice. Water samples were processed within 3 hours of co llection. All samples were analyzed for enterococci, total coliforms, fecal coliforms, HPyVs (quantif ied by QPCR and presence/absence detection by PCR), human-associated Bacteroidales M. smithii and adenovirus (as described below). Enumeration of culturable indicator organisms Culturable concentrations of all indicato r organisms were obtai ned using standard methods. Enterococci were enumerated by membrane filtration on mEI agar, with incubation at 41 0.5C for 24 h (328). Fecal co liform concentrations were determined by membrane filtration using mFC agar, with incubation at 44.5 0.5C for 24 h (14). Total coliform concentrations were enumerat ed by membrane filtration on mEndo agar, with incubation at 35 0.5C for 24 h (14). Concentration of MST indicators Bacteria, methanogens, and viruses fro m 500 ml samples were concentrated simultaneously on a 0.45 m pore size nitrocel lulose filter. Bacteria and methanogens were concentrated by physical capture on the filt er. To concentrate the viruses, the pH of the water was adjusted to 3.5 using 2.0 N HC l prior to filtration (14, 129, 202). The low 145

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pH did not affect the concentr ation and detection of the bacteria and methanogens (129). After filtration, the filter wa s placed into a 2 ml microcentrifuge tube. The tube containing the filter were placed on dry ice and shipped from California to the Harwood lab (Tampa, Florida). DNA was extracted fr om the filter using the MQH protocol, and was used as template for human-associated Bacteroidales PCR, M. smithii PCR, adenovirus nested PCR, HPyVs PCR and HPyVs QPCR assays. Detection of human-associated Bacteroidales Previously published primers specific for a region of the 16S rRNA gene of human-associated Bacteroidales (27) were used in a touchdown PCR (129) ( Table 22 ). PCR reactions were performed in a 25 l mixture containing 12.5 l GoTaq Green Master Mix (Promega Corporation), 0.5 M concentrations of each primer, and 2 l of template DNA. The touchdown PCR reac tion conditions were as follows: DNA polymerase activation at 95C for 3 min, fo llowed by 43 cycles of DNA melting at 94C for 45 sec, then annealing for 45 sec, and extension at 72C for 30 sec. Annealing temperatures ranged from 65-55C. Cycles were performed twice at temperatures 6563C, once at temperatures 62-56C, and 30 times at temperature 55C; followed by a final elongation at 72C for 5 min (Eppe ndorf Mastercycler Thermocycler). PCR products were separated and vi sualized as above, except a plasmid containing the 525 bp positive control was used. Detection of the human-associated M. smithii Previously published primers specific for the nifH gene of human-associated M. smithii (330) were used in the touchdown PCR (129) ( Table 22 ). PCR reactions were performed in a 25 l mixture containing 12.5 l GoTaq Green Master Mix (Promega 146

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Corporation), 0.5 M concentrations of each primer, and 2 l of template DNA. The touchdown PCR reaction conditions were the same as previously described. PCR products were separated and vi sualized as above, except a plasmid containing the 221 bp positive control was used. Adenovirus nested PCR Previously published primers specific fo r the hexon gene of human adenoviruses were used in the nested PCR (251) ( Table 22 ). Initial amplification was carried out in a 50 l reaction mixture containing 25 l GoTaq Green Master Mix (Promega Corporation), 0.8 M concentrations of each primer (hexAA 1885 and hexAA 9113), and 5 l of template DNA. In both PCR reactions, the first round of denaturation was carried out for 4 min at 94C followed by 30 cycles of denaturing at 94C for 90 sec, annealing at 55C for 90 sec, and extension at 72C fo r 120 sec, followed by a final elongation at 72C for 5 min. For the nested PCR, the 50 l reaction mixture contained: 25 l GoTaq Green Master Mix (Promega Corporation), 0.4 M concentrations of each primer (nehexAA 1893 and nehexAA 1905), and 1 L of template from the first round of PCR. The PCR products were visualized as above except a plasmid containing the 143 bp positive control was used. Viral DNA was ex tracted from cell culture using DNeasy Blood & Tissue Kit (Qiagen, Inc., Valencia, CA), and stored at -20C. Detection of human polyomaviruses Primers specific for a partial region of th e HPyVs T-antigen gene were used in a PCR (203) ( Table 22 ). PCR reactions were performed in a 50 l mixture containing 25 l GoTaq Green Master Mix (Promega Corporation), 0.4 M of each primer, and 5 l of template DNA. The PCR reaction conditions were as follows: DNA polymerase 147

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activation at 95C for 2 min, followed by 45 cy cles of denaturing at 94C for 20 sec, annealing at 55C for 20 sec, and extensi on at 72C for 20 sec, followed by a final elongation at 72C for 2 min. PCR products we re separated and visualized as above, except a plasmid containing the 173 bp positive control was used. Quantification of human polyomaviruses in beach samples QPCR reactions were carried out in a 25 l volume. The HPyVs QPCR mixtures were prepared using 12.5 l TaqMan Un iversal PCR Master Mix, No AmpErase UNG (Applied Biosystems, Foster City, CA), 0.5 M primer concentrations, 0.4 M labeled probe concentration, 2 l of template DNA, and the volume was adjusted to 25 l using reagent grade, nuclease-free steril e water. HPyVs QPCR amplification and quantifications were performed as described above. Standard curve reactions were run in duplicate for each QPCR run performed, and the average R2 was 0.9879 0.0103. Statistical analysis Summary statistics were computed for variables of interest using GraphPad InStat version 3.00 (GraphPad Software, San Diego, CA ). Bacterial concen trations and HPyVs copy numbers were log10 transformed and differences among concentrations and copy numbers were determined using paired or unpaired t-tests. Means were considered significantly different at the alpha level of 0.05. Relationships between indicators and markers were determined by calculating Pear son correlation coefficients. Differences were considered significant when P <0.05, and two-sided tests were performed for all analyses. Observations of human associated markers were converted to binary data, and binary logistic regression m odels (SPSS version 17.0) were used to assess relationships between HPyVs or IOs concentrations and presence or absence of human-associated 148

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markers. Nagelkerkes R square, which can range from 0.0 to 1.0, denotes the effect size (the strength of the relati onship); stronger associations have values closer to 1.0. Relationships were considered significant when the P value for the model chi square was 0.05 and the confidence interval for the odds ratio did not overlap 1.0. The odds ratio is the measure of the effect size and an estimati on of the probability of the same response of the two variables. Odds ratios were determin ed for all binary logist ic regression analysis and significant odds ratio (<0.05 with a confiden ce interval that did not include 1.0) were reported. Contingency tables were used to assess significant co rrelations among binary marker data. Relationships were considered significant when P <0.05. Results Positive and negative controls All constructed plasmids used as PCR or QPCR positive controls contained the correct sequence. All DNA extraction blanks and method blanks were negative in all assays. Comparison of DNA extraction protocols The MO BIO/Qiagen hybrid (MQH) protocol outperformed the standard MO BIO protocol ( Table 23 ). The percent recovery of the M QH protocol samples increased as the initial target concentr ation decreased, ranging from 82.3% at 7.7 x 105 expected gene copies to 99.6% at 7.7 x 103 expected gene copies. In contrast, the percent recovery of the standard MO BIO protocol samples decr eased as the initial target concentration decreased, ranging from 65.8% at 7.7 x 105 expected gene copies to 50.6% at 7.7 x 103 expected gene copies. In addition, the av erage percent recoveries achieved with the MQH protocol were signifi cantly greater than the sta ndard MO BIO protocol ( P =0.0126). 149

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Comparison of viral DNA recovery from wa ter concentrates containing BK viruses Initially, a broad range of suspended BK viral particles (dilutions from 10-5 to 10-10, 1.5 x 101 to 6.2 x 106 particles) were inoculated in to 500 ml volumes of sterile dechlorinated tap water samples, filtered, and quantified. The measured copy numbers and percent recoveries for each dilution are summarized in Table 24 Based on the high concentration of viral particles in the 10-5 and 10-6 dilutions, these samples were omitted from subsequent analysis. Different BK virus stocks were used throughout the experiments resulting in varying multipliers; however the magnitude of the virus stock was consistent throughout the study (see Table 24 ). BK viruses were consistently detected at the 10-7 dilution (corresponding to 2.8-2.9 x 104 viral particles) using both the standard MO BIO and MQH prot ocol. Moreover, BK viruses were not detected at the 1010 dilution (1.5 x 101 viral particles) for both protocols. However, the number of BK viruses detected was consistently larger at every dilution using the MQH protocol ( Table 24). The MQH protocol was able to enumer ate BK viruses from 60% and 40% of water samples containing 10-8 and 10-9 virus dilutions, respectively; as compared to the 50% and 0% frequency achieved using the standard MO BIO protocol. Furthermore, the samples processed via the MQH protocol had a str onger correlation of virus copy numbers vs. virus inoculum (r=0.9894, R2=0.9788, P <0.05) when compared to the MO BIO protocol (r=0.9012, R2=0.8121, P <0.05). Comparison of viral DNA recovery from wate r concentrates containing raw sewage Initially, a broad range of volumes of unt reated sewage influent (0.01-1.0 ml) was inoculated into 500 ml sterile dechlorinate d tap water samples, filtered and quantified ( Table 25 ). Based on the high concentration of HPyVs in the 0.25-1 ml volumes, these samples were omitted from subsequent analyses. HPyVs were consistently detected in 150

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water samples inoculated with 0.1 ml of se wage for both protocols. Using the MQH protocol, HPyVs were detected in 100% of samples inoculated with 0.05 ml sewage (10-4 dilution), and 66.7% of those inoculated with 0.01ml. Conversely, using the standard MO BIO protocol, HPyVs were detected in only 66.7% and 33.3% of the 0.05 and 0.01 ml concentrates, respectively. The HP yVs gene copy number estimated by QPCR was consistently and significantly larger at every volume of sewage using the MQH protocol ( P =0.0198). Furthermore, the samples processe d via the MQH protoc ol had a stronger correlation of virus copy number s vs. initial inoculum (r=0.8690, R2=0.7551, P <0.05) when compared to the MO BIO protocol (r=0.7888, R2=0.6222, P <0.05). Bacterial water quality indicator con centrations at Doheny Beach sites One hundred thirty samples were collected from Doheny Beach over the study period, with 26 samples collected from each of the sites. The average log10-transformed concentrations of all bacterial indicator organisms (IOs) at each Doheny site are summarized in Figure 9 A. The average concentrati on of each IO at site C was significantly greater than all other Doheny sites ( P <0.001). Enterococci at site C exceeded regulatory standards for one-time sampling (104 CFU/100ml) in 88.5% of the samples (221). Fecal coliform concentrations at site C exceeded regulatory standards (400 CFU/100 ml) in 76.9% of the samples (221). While total coliform regulatory standards are no longer in practice, at site C total coliform concentr ations exceeded the retired standard of 2300 CFU/100 ml in 76.9% of the samples. The average concentrations of both enterococci and fecal co liforms at site D were significantly larger than sites A, B, and E (P <0.05). Site D exceeded enterococci and fecal coliform regulatory standards in 30.8% and 15.4% of the samples, respectively. The average 151

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concentrations of enterococci and fecal coliforms among sites A, B, and E were not significantly different. The individual enteroco cci concentrations at sites A, B, and E exceeded regulatory standards in only 11.5%, 7.7%, and 3.8% of the samples, respectively. Fecal coliform concentrati ons at sites B and E exceeded regulatory standards in only 3.8% of the samples; while no exceedances were detected at site A. Aside from site C, total coliform concen trations exceeded 2300 CFU/100 ml only one time at site A. Correlations of bacterial indica tors among Doheny Beach sites The concentrations of enterococci at site A were positively correlated with site C (r=0.4113, R2=0.1692, P =0.0368) and site E (r=0.6390, R2=0.4084, P =0.0004). In addition, enterococci concentrations at site E were also positively correlated with site B (r=0.4557, R2=0.2076, P =0.0193). Moreover site C was pos itively correlated with site D (r=0.5837, R2=0.3407, P =0.0017). The concentrations of fecal coliforms at site D were positively correlated with site B (r=0.4717, R2=0.2225, P =0.0150), site C (r=0.4181, R2=0.1748, P =0.0335), and site E (r=0.4122, R2=0.1699, P =0.0364). The concentrations of total coliforms were positively co rrelated between all sites (r=0.4949-0.6581, R2=0.2449-0.4330, P <0.0003-0.0102) except sites C and E ( P =0.1237). In addition, enterococci, fecal coliforms, and total coli forms were positively correlated among each other at each site (r=0.4753 to 0.8446, R2=0.2489-0.7310 P =<0.0001-0.0143). QPCR detection of HPyVs at Doheny Beach sites HPyVs were rarely detected by QPCR at any Doheny site ( Figure 10. A). HPyVs were not detected at sites C and D, were de tected once at site A and E, and detected 3 times at site B. Quantities of HPyVs dete cted ranged from 125-2,884 copy numbers100 152

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ml-1. There were no significant differences in the concentrations of HPyVs copy numbers detected among the sites. In addition, ther e were no significant co rrelations of HPyVs copy numbers between sites where HPyVs were detected. PCR detection of human-associated water quality indicators at Doheny Beach sites The frequency of human marker detection at each site is summarized in Figure 10.A. HPyVs were detected by PCR in 4 samples at site A, 3 samples at site B, 1 sample at sites D and E, and were not detect ed at site C. Human-associated Bacteroidales were detected in 9 samples at site C, 8 samples at site D, 4 samples at site B, and 2 samples at both sites A and E. The M. smithii marker was detected in Doheny Beach only once at site C. Adenovirus was detected in 3 samples at both sites B and D, 2 samples at site A, 1 sample at site C, and was not detected at site E. The frequency of human-associated Bacteroidales detection was significan tly more frequent than M. smithii detection ( P <0.01). All other detection frequencie s were not significantly different. Relationships among indicators and markers at Doheny Beach At site A, PCR detection of HPyVs and th e concentrations of fecal coliforms were strongly correlated (Nagelkerkes R2=0.567, P <0.001, odds ratio=246.100, P =0.039). At site B, the concentrations of HPyVs were moderately correl ated with the presence of adenovirus (Nagelkerkes R2=0.413, P <0.013, odds ratio=3.653, P =0.025). In addition, the occurrence of HPyVs (by PCR detecti on) was significantly correlated with the occurrence of adenovirus (Likelihood ratio 15.333, R2=0.3884, P =0.0269). Several correlations were documented at site C. These correlations were positive and included enterococci concentrations and the occurrence of the human-associated Bacteroidales marker (Nagelkerkes R2=0.204, P =0.041), M. smithii (Nagelkerkes R2=1.000, P =0.004), 153

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and adenovirus (Nagelkerkes R2=1.000, P =0.004). The human-associated Bacteroidales marker was also significantly correlated with fecal coliform concentrations (Nagelkerkes R2=0.191, P =0.049). No significant relationships were observed at site D or E. Bacterial indicator concentr ations and marker detection at each Doheny site were compiled for all sites combined. Enterococci concentrations were strongly correlated with fecal coliforms (r=0.8620, R2=0.7430, P <0.005), total coliforms (r=0.8480, R2=0.7180 P <0.0005), and M. smithii (Nagelkerkes R2=1.000, P <0.001). Fecal coliforms were also strongly corr elated with total coliforms (r=0.8780, R2=0.771, P <0.0005). Total coliforms were moderately correlated with M. smithii (Nagelkerkes R2=0.466, P =0.021). The human-associated Bacteroidales marker was weakly correlated with enterococci (Nagelkerkes R2=0.192, P =0.004), fecal coliforms (Nagelkerkes R2=0.190, P <0.001, odds ratio=2.383, P <0.0005), and total coliforms (Nagelkerkes R2=0.137, P =0.001, odds ratio=1.858, P =0.001). HPyVs detected by PCR and HPyVs copy numbers were significantly correlated (Nagelkerkes R2=0.521, P <0.001). The presence of adenovirus was correlated with both HPyVs concentrations (Nagelkerkes R2=0.087, P <0.033) and detection of human-associated Bacteroidales (Likelihood ratio=2.307, R2=0.1078, P =0.0016). Overall, human-associated Bacteroidales were the most frequently detected marker of human sewage pollution at all the Doheny sites (n=25). The M. smithii marker was the least frequently detected marker (n =1). Adenovirus and HP yVs were detected by PCR were detected in 9 samples. HPyVs were detected by QPCR in 5 samples. The cooccurrence of human markers is summarized in Table 26 . 154

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Bacterial water quality indicator con centrations at Avalon Beach sites One hundred twenty samples were collect ed from Avalon Beach over the entire study period, with 30 samples collected fr om each of the sites. The average concentrations of all bacterial indicator organisms (IOs) at each Avalon site are summarized in Figure 9 B. Enterococci, fecal coliform, and total coliform concentrations at site C were significantly gr eater than at site A and D ( P <0.01). Enterococci at site C exceeded regulatory standards in 60.0% of the sa mples. Fecal coliform concentrations at site C exceeded regulatory standards in 10.0% of the samples, and total coliform concentrations exceeded 2300 CFU100 ml-1 in 20.0% of the samples. Enterococci and total coliform concentrations at sites A a nd B were not significantly different; however fecal coliform concentrations at site B were significantly larger than site A (P <0.05). Enterococci and fecal coliforms at site B exceeded regulatory standards in 46.7% and 13.3% of samples, respectively. Tota l coliforms exceeded 2300 CFU100 ml-1 in 6.7% of the samples. At site A, fecal and total coliform exceedances were not observed; while enterococci exceedances were only observed in 20.0% of the samples. The average concentrations of all IOs at site D were significantly lower than all other Avalon sites ( P <0.001). Exceedances were not noted for any bacterial indicators at site D. Correlations of bacterial indica tors among Avalon Beach sites The concentrations of enterococci at site D were negatively correlated with site A (r= -0.4813, R2=0.2316, P =0.0071). No significant correlations were found for fecal coliforms among the Avalon sites. Total coliform concentrations were positively correlated at sites B and C (r=0.4171, R2=0.1739, P =0.0219). In addition, enterococci, fecal coliforms, and total coliforms were pos itively correlated among each other at sites 155

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A, B, and C (r=0.5431 to 0.9239, R2=0.3404-0.5315, P <0.0019). At site D, only fecal and total coliforms were si gnificantly correlated (r=0.6211, R2=0.3858, P =0.0002). QPCR detection of HPyVs at Avalon Beach sites HPyVs were detected by QPCR at every Avalon site (Figure 10. B). HPyVs were detected 7 times at site C, 6 times at site B, and 4 times at both sites A and D. Quantities of HPyVs detected ranged from 50-35,481 copy numbers100 ml-1. There were no significant differences in the concentrations of HPyVs copy numbers detected among the sites. In addition, there were no significant correlations of HPyVs copy numbers among the sites. PCR detection of human-associated water quality indicators at Avalon Beach sites The frequency of human marker detec tion at each site is summarized in Figure 10.B. HPyVs were detected by PCR in 7 samples at site B, 6 samples at site C, 5 samples at site D, and 4 samples at site A. Human-associated Bacteroidales were detected in 13 samples at site A, 12 samples at site B, 11 samp les at site C, and 6 samples at site D. The M. smithii marker was detected once at sites B and D, and was not detected at either site A or D. Adenovirus was not detected at any Avalon Beach site. The frequency of human-associated Bacteroidales detection was significantly mo re frequent than HPyVs, M. smithii and adenovirus detection ( P <0.01). The frequency of HPyVs detection was significantly more frequent than M. smithii and adenovirus detection ( P <0.01). Relationships among indicators and markers at Avalon Beach Unlike the Doheny Beach sites, there we re no significant relationships between indicators or human-associated markers at any of the sites at Av alon Beach. Bacterial indicator concentrations and marker dete ction at all Doheny sites were compiled. 156

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157 Enterococci concentrations were strongly correlated w ith fecal coliforms (r=0.7282, R2=0.5303, P <0.0001) and total coliforms (r=0.6277, R2=0.3940, P <0.0001). Fecal coliforms were also strongly corr elated with total coliforms (r=0.8926, R2=0.7967, P <0.0001). The presence of the human-associated Bacteroidales marker was weakly correlated with fecal coliforms (Nagelkerkes R2=0.0.074, P =0.010, odds ratio=1.827, P =0.014) and total coliforms (Nagelkerkes R2=0.061, P =0.020, odds ratio=1.774, P =0.027). HPyVs detected by PCR and HP yVs copy numbers were significantly correlated (Nagelkerkes R2=0.361, P <0.001, odds ratio=2.777, P <0.0005). Overall, human-associated Bacteroidales were the most frequently detected at all the Avalon sites (n=42). HPyVs were detected by PCR were detected in 22 samples, M. smithii was detected in 2 samples, and adenoviruses were not detected at any site. The co-occurrence of human markers and pat hogens is summarized in Table 26 Comparison of Doheny and Avalon indicators and markers The concentrations of enterococci, fecal coliforms, and total coliforms were not significantly different between Doheny a nd Avalon Beaches. In contrast, the concentrations of HPyVs copy numbers was significantly larger at Avalon Beach ( P =0.0006). In addition, the frequency of PC R detection of HPyVs was significantly higher at Avalon Beach sites ( P =0.0181). The frequency of human-associated Bacteroidales and M. smithii detection was not significantly different between Avalon and Doheny Beaches. Adenovirus was not detected at any Avalon Beach site and therefore statistical analysis c ould not be performed; however it was detected in 9 out of 130 samples at Doheny Beach, indicating a higher frequency of occurrence than at Avalon Beach.

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Table 22. Primers and probe sequences us ed in the California beaches study. Assay Primers and Probe Sequence Reference Human Bacteroidales PCR HF183 5-ATC ATG AGT TCA CAT GTC CG-3 (27) Bac708R 5-CAA TCG GAG TTC TTC GTG-3 M. smithii PCR Mnif-342f 5-AAC AGA AA A CCC AGT GAA GAG-3 (330) Mnif-363r 5-ACG TAA AG G CAC TGA AAA ACC-3 Adenovirus nested PCR hexAA1885 5-GCC GCA GTG GTC TTA CAT GCA CAT C-3 (251) hexAA1913 5-CAG CAC GCC GCG GAT GTC AAA GT-3 nehexAA1893 5-GCC ACC GAG ACG TAC TTC AGC CTG-3 nehexAA1905 5-TTG TAC GAG TA C GCG GTA TCC TCG CGG TC-3 Human polyomavirus PCR/QPCR* SM2 5-AGT CTT TAG GGT C TT CTA CCT TT-3 (202, 203) P6 5-GGT GCC AAC CTA TGG AAC AG-3 KGJ3 5-(FAM)-TCA TCA CTG GCA AAC AT-(MGBNFQ)-3 158 *The SM2 and P6 primers were us ed in both the PCR and QPCR assay.

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Table 23. Comparison of the DNA extraction efficiency of the sta ndard MO BIO protocol and the improved MO BIO/Qiagen (MQH) protocol using plasmids pipetted directly into bead tubes. DNA Extraction Protocol Estimated HPyVs Copy Numbers Added to Bead TubeaAverage Percent Recovery 7.7 x 1037.7 x 1047.7 x 105 Measured HPyVs Copy Numbersb MO BIO Measured 3.1 2.0 x 103 3.5 1.9 x 104 4.3 2.2 x 105 Percent Recovery 50.6% 50.6% 65.8% 55.7% MO BIO/Qiagen Measured 7.6 1.3 x 1037.4 1.2 x 1046.0 1.5 x 105 Percent Recovery 99.6% 98.3% 82.3% 93.4% 159 aResults reported as extrapolated human pol yomavirus (HPyVs) copy numbers in the initia l 100 l sample standard deviation. bAll dilutions were performed in triplicate.

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Table 24. Comparison of the standard MO BIO protocol and the improved MO BIO/Qi agen (MQH) protocol viral DNA recovery from water samples (500 ml) inoculated with different volumes of BK virus dilutions. DNA Extraction Protocol Log (y+1) HPyVs Copy Number in Samplea Suspended Virus Dilution 10-510-6 10-7 10-8 10-9 10-10 MO BIO Initial 6.8 0.0 5.8 0.0 4.4 0.2 3.3 0.1 2.2 0.1 1.2 0.1 Measured 3.9 (n=1) 5.6 (n=1) 3.2 1.1 (n=5) 1.3 1.5 (2)c(n=4) 0.0 0.0 (4)c(n=4) 0.0 0.0 (3)c(n=3) Percent Recovery 57.8% 96.5% 72.2% 38.1% 0% 0% MO BIO/Qiagen Initial 6.8 0.0 5.8 0.0 4.5 0.2 3.3 0.1 2.3 0.1 1.2 0.1 Measured 6.7 (n=1) 5.7 (n=1) 4.2 0.4 (n=6) 1.9 1.7 (2)c(n=5) 1.2 1.6 (3)c(n=5) 0.0 0.0 (3)c(n=3) Percent Recovery 97.9% 99.2% 94.0% 56.8% 51.0% 0% 160 a Results reported as estimated human polyomav irus (HPyVs) copy numbers (transformed to log (y+1)) in the entire sample standard deviation. bRelationships between increasing virus inoculum and estimated HPyVs copy numbers were determ ined. All correlations were significant ( P <0.05). cSingle number in parentheses indicates num ber of samples that did not amplify.

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Table 25. Comparison of the standard MO BIO protocol and the improved MO BIO/Qi agen (MQH) protocol viral DNA recovery from water samples inoculated with different volumes of raw sewage. DNA Extraction Protocol HPyVs Copy Number in SampleaCorrelationb Sewage Volume (ml) 1.00 (n=1) 0.75 (n=1) 0.50 (n=1) 0.25 (n=1) 0.10 (n=4) 0.05 (n=3) 0.01 (n=3) MO BIO 4.5 4.0 4.3 3.6 3.3 0.4 1.8 1.6 (1)c 0.7 1.3 (2)c r=0.7888 R2=0.6222 MO BIO/Qiagen 4.7 4.6 4.4 3.9 3.4 0.1 2.6 0.2 1.8 1.6 (1)cr=0.8690 R2=0.7551 161 aResults reported as extrapolated human polyo mavirus (HPyVs) copy numbers (transformed to log (y+1) ) in the entire sample standard deviation. bCorrelation between HPyVs gene copy numbers detected vs. inoculum in 500 ml water; all correlations were st atistically signific ant ( P <0.05). cNumber in parentheses indicates number of samples that did not amplify.

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Table 26. The co-occurrence of human-associated ma rkers in the Doheny and Avalon Beach samples. Beach Marker A Marker B Percent of: Matching Results Co-occurrence Co-absence Absence of A in Presence of B Presence of A in Absence of B Doheny HPyVs # M. smithii 95.4 0 95.4 0.8 3.8 HPyVs # HPyVs 93.9 2.3 91.6 4.6 1.5 M. smithii Adenovirus 93.8 0.7 93.1 6.2 0 HPyVs M. smithii 92.3 0 92.3 0.8 6.9 HPyVs # Adenovirus 90.8 0.8 90.0 6.2 3.0 HPyVs Adenovirus 89.2 1.5 87.7 5.4 5.4 HBac Adenovirus 83.1 4.6 78.5 2.3 14.6 HBac M. smithii 81.5 0.8 80.7 0 18.5 HPyVs HBac 80.0 3.1 76.9 16.2 3.8 HPyVs # HBac 78.5 0.8 77.7 18.5 3.0 162

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163 Beach Marker A Marker B Percent of: Matching Results Co-occurrence Co-absence Absence of A in Presence of B Presence of A in Absence of B Avalon M. smithii Adenovirus 98.3 0 98.3 0 1.7 HPyVs # HPyVs 87.5 11.7 75.8 6.7 5.8 HPyVs # Adenovirus 82.5 0 82.5 0 17.5 HPyVs Adenovirus 81.7 0 81.7 0 18.3 HPyVs M. smithii 81.7 0.8 80.9 0.8 17.5 HPyVs # M. smithii 80.8 0 80.8 1.7 17.5 HBac Adenovirus 65.0 0 65.0 0 35.0 HBac M. smithii 65.0 0.8 64.2 0.8 34.2 HPyVs HBac 56.7 5.0 51.7 30.0 13.3 HPyVs # HBac 55.8 4.2 51.7 30.8 13.4 aHPyVs, human polyomaviruses detected by PCR; HPyVs #, human pol yomaviruses detected by QPCR; HBac, human-associated Bacteroidales marker

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164 Figure 8. Sites of sample collection for the California beaches study. (A) Doheny State Beach and San Juan Creek Lagoon; (B) Avalon Beach at Catalina Island. Images courtesy of the U.S. Ge ological Survey; http://terraserver-usa.com 164

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(A) (B) Figure 9. The average log10-transformed concentration of enterococci, fecal coliforms, total coliforms, and human polyomaviruses (H PyVs) at the (A) Doheny Beach sites and (B) Avalon Beach sites. Error bars represent standard deviation. Indicator b acteria are reported as log10 colony forming units (CFU) 100 ml-1. HPyVs are reported as log10 copy numbers 100 ml-1. 165

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(A) (B) Figure 10. Frequency of detection of human-associated MST markers and adenoviruses at the (A) Doheny Beach sites and (B) Avalon Beach sites. 166

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Discussion This study is among the first to compare the magnitude and frequency of observation of culturable indicator bacteria (IOs), multiple MST markers and a viral pathogen in recreational waters. Among the most important findings were the correlations of human-associated MST markers with each other and with adenovirus detection. This study also introduces an improved DNA extraction prot ocol that is less prone to contamination and decreases pro cessing time, allowing for greater sample throughput in less time. The MO BIO PowerSoil kit is frequen tly used for DNA extraction in water quality laboratories due to its ability to remove humic acids and other PCR inhibitors from DNA suspensions. However, all the supern atant from each step is not utilized in the standard MO BIO protocol, wh ich can lead to inconsistent results and inefficient recoveries (e.g. Table 24 and Table 25 ). Recent research has reported improved recoveries using a modified pr otocol in which the total volume of supernatant is used (300). Using the protocol suggested by Sto eckel et al. (2009) significantly increases precision and recovery efficiency, however th e time required to extract DNA is also significantly increased (data not shown). The substitution of the MO BIO spin filter column with the Qiagen DNeasy Tissue kit spin column allows the use of the QIAvac 24 vacuum manifold. The use of the manifo ld eliminates the re peated loading and centrifuging of the MO BIO spin filter, and subsequently reduces labor, time, and the potential for contamination. Moreover, by using the hybrid MO BIO/Qiagen DNA extraction protocol, BK viruses were quantif iable in a higher percentage of dilute samples. Based on previously published concentrations of HPyVs in sewage (~3 x 104 167

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copy numbersml-1) the incorporation of the MO BIO/Qiagen extraction protocol allowed the detection of as few as 400 virus particles in 500 ml of wa ter (203). The integration of the suggested MO BIO/Qiagen prot ocol allows for consistent and efficient recoveries. The Doheny State Beach sites were dissi milar in terms of IO, MST marker and pathogen variables. The San Juan Creek lagoon (s ite C) just upstream of the discharge to the ocean (site D) consistently had the highest concentrations of indicator bacteria, which were highly correlated with the nearest beach site (D) observations. Despite the high concentrations of IOs at sites C and D, the adenovirus and HPyVs markers were detected at a higher frequency at the northern b each sites (A and B), and the presence and concentration of the HPyVs marker was strongly correlated with adenovir uses at site B. In addition, the presence of human-associated Bacteroidales, HPyVs marker, and adenovirus co-occurred in 2 of the 26 samples collected at site B, but not at any other Doheny site. The nearshore water sites at north D oheny Beach are shallow and commonly used by children. Craun et al. (78) compiled s ources of recreational water contamination leading to water-borne illne ss outbreaks and reported that 25% of the outbreaks were attributed to children in diapers and 34% were due to bather overcrowding (78). Moreover, the San Juan Creek Ocean Outfall located off the beach may be contributing to the poor water quality. While Jones and Terr ill (159) report the discharge plume from the outfall does not directly impact Doheny Beach, it has been suggested that sewage plumes are complex with dispersal dynamics direc tly related to rapidly changing factors including stratification, currents, winds, wave s, seasonal and climatic conditions (135). The high frequency of adenovirus and HP yVs detection at the north beach sites 168

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may be caused by a combination of bather sh edding/excretion and the contaminants from the Ocean Outfall coupled with poor water circul ation at the jetty, rath er than direct input from San Juan Creek. Furthermore, bacteria l indicators at the southern Doheny site (E) were positively correlated with northern beach sites A and B, suggesting the possibility of a similar source of contamination. Similar frequencies of HPyVs and human-associated Bacteroidales detection at the southern (E) and northern sites (A and B) are further evidence these sites may be influenced in some measure by common source(s) of contamination. We suspect that the San Juan Creek outflow is degrading water at the closest site; however the poor water quality at the other si tes may be due to bather overcrowding, poor water circula tion, and the Ocean Outfall. At Avalon Beach, fecal coliform concen trations did not show any geographic relationship (correlation of values between proximal sites). The same observation was true of enterococci. The lack of correlat ion between sites suggests a separate or disproportionate fecal input(s) that affects each site individually. At least two of the three human markers were frequently detected at th e Avalon Beach sites (sites A, B, and C). The frequency of human marker detection at these sites strongly suggests contamination from a human source(s). While adenoviruses we re not detected at any Avalon Beach site, the small volume of sample analyzed may have limited the detection. Jiang et al. (153) suggest concentrating adenoviru ses from 20 L of water for a representative analysis of indirect anthropogenic input (e.g. leaking sewers). However, the absence of the adenovirus marker and the infrequent detection of the M. smithii marker may indicate human fecal pollution from the discharge of wa stes by boats into the bay, because both 169

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adenoviruses and M. smithii are excreted by a minority of the population and are less likely to be detected in n on-community wastes (203). The Avalon control site (site D), chosen fo r its historically low levels of fecal bacteria concentrations, maintained low bact erial levels and did not exceed regulatory standards for fecal coliforms or enterococci in this study. However, human-associated Bacteroidales, M. smithii and HPyVs were detected at th is site. Moreover, all three markers were detected in one of the samp les simultaneously. Th e detection of the human-associated markers coupled with the low levels of cu lturable bacterial indicators indicates the inability of IOs to detect human fecal contamin ation in certain conditions. The low levels of IOs and frequent humanassociated marker detection may indicate minimal seagull and pigeon fecal contributi ons and a strong huma n fecal input(s). However, recent sewage contamination would re sult in high levels of IO concentrations. The lack of elevated IO concentrations ma y be cause by the genera l inability of fecal bacteria to persist in conditions of high salin ity and exposure to sola r radiation (McQuaig et al. unpublished data). Overall, the human-associated Bacteroidales marker was the most frequently detected marker at both Doheny and Avalon site s. This marker also showed the highest co-occurrence with the adenoviru s marker detected at the Doheny sites; however since the human-associated Bacteroidales marker was detected at the highest frequency the coabsence with adenovirus was the lowest among all the markers ( Table 26 ). Recent studies have documented the detection of the human-associated Bacteroidales marker in a small percentage of animal fecal samples, indicating incomplete specificity of this marker (129, 203). Doheny State Beach is dog-friendly and also maintains a large 170

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seagull population near the San Juan Creek (per sonal communication with John Griffith). However, the levels of human-associated Bacteroidales spp. in raw sewage are several orders of magnitude higher than the other human-associated markers giving the marker a potentially higher sensitivity (129). The presence of the human-associated Bacteroidales marker should therefore be inte rpreted carefully, preferably in a quantitative format (75, 163, 181), and in conjunction with ot her human-associated markers. Recent studies have also reporte d the human specificity of the M. smithii assay is <100% (129, 203). Despite this caveat the M. smithii marker has been detected at relatively high concentrations in sewage, fo r instance Harwood et al (129) reported the detection of M. smithii in 10-3 and 10-4 dilutions of sewage (129) Throughout this study the M. smithii marker was infrequently detected sugge sting this marker may be relatively conservative. Compared to the human-associated Bacteroidales marker which has been detected in sewage diluted as low as 10-6 (129), the M. smithii marker is relatively insensitive. However, the M. smithii and adenovirus marker had the highest percent of matching results at both beaches with a major ity of the matching resu lts composed of coabsents. While the M. smithii marker showed the highest matching results, humanassociated Bacteroidales had the lowest percentage of absence in the presence of adenoviruses. This may indi cate the human-associated Bacteroidales marker is a more liberal indicator of human health risks than the M. smithii marker. The presence of HPyVs detected by PCR and the quantity of HPyVs by QPCR were highly correlated at both Doheny and Avalon Beaches, which is not surprising considering both assays utilize the same prim ers. However, the marker presence or absence with both assays was not 100%. We theorize the discrepancies observed could 171

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be attributed to the different template volumes used in each assay (2 l was used in the QPCR assay and 5 l was used in the PCR assa y). The interpretation of HPyVs data for recreational water quality assessment may be infl uenced by the fact that these viruses are excreted in urine; however the high percen t of analogous results with adenovirus at Doheny Beach (90.2%) suggests th at HPyVs have a strong corr elation with human health risks. The presence of adenoviruses is a dir ect assessment of human health risks; however only a small percentage of the popul ation excretes these viruses (154), which can lead to inconsistent dete ction in sewage-impacted wate rs. The lack of adenovirus detection at the Avalon Beach sites when the three human MST markers were present demonstrates that the adenoviruses should be used in conjunction with other pathogen assays for detection of sewage contamination. The potential of HPyVs to be excreted in urine of swimmers and the incomplete specificity of both the human-associated Bacteroidales and M. smithii marker can mean ambiguous results when only one marker is de tected. However, the predictive power of each marker is increased when more than one marker is detected at the same site. For all markers utilized in this study, an epidemiol ogical study assessing the human health risks associated with the presence or absence of the marker would more correctly define the usefulness of each assay. In addition, determ ining the concentrations of each marker by QPCR in future studies may provide a bett er understanding of relationships of one marker with other MST markers and waterborne pathogens and ultimately provide a better perspective on the propor tion of microbial contamination from human sources. 172

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173 This study has provided insight on the us efulness of traditi onal indicators and several human-associated microbi al source tracking methods to establish water quality at non-point and point source beaches. Determin ing water quality is a complex assessment of various indicators, and careful considerati on of the location, clim ate, historical water quality data, and possible sources of contamin ation should be made before water quality indicators are selected. In addition, we strongly recomme nd the use of a multi-indicator "toolbox" approach when assessing water quality. Acknowledgements We would like to thank all the Southern California Coastal Research Project (SCCWRP) personnel involved with the sample collection. We would also like to thank Dr. John Paul for the use of the ABI 7500 real ti me machine. In addi tion, we would like to thank Jenny Delaney, Dave John, Lauren McDaniel, Robert Ulrich, Beth Young, and Brian Zielinski for technical and logistical support. Funding for this study was provided in part by EPA grant (MX-96478707-0).

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CHAPTER 5: FINAL CONCLUSIONS AND SUMM ARY OF COLLABORATIVE STUDIES The central goal of this research was to develop and validate a QPCR assay for the quantification of human polyomaviruses (HPyVs) that quantifies both BKV and JCV in environmental water samples. The HPyV s QPCR assay is intended to serve as a human-specific, library-indepe ndent indicator of water quality, specifically for human sewage. Both the developed HPyVs QPCR assay and use of HPyVs as an indicator of water quality were scrutinized for accuracy and dependability. The validation of the marker included many assessments such as: se nsitivity testing (Chapter 2), specificity testing (Chapter 2), persisten ce analysis (Chapter 3), recovery testing (Chapter 4), and correlation with traditional water quality i ndicators, other human-associated microbial source tracking (MST) markers, pathogens, and human health risks (C hapter 4). The HPyVs QPCR was relatively sensitive in terms of its abi lity to detect low gene copy numbers, as the optimized primers and probe concentrations reliably detected as few as ten gene copy numbers. The utilization of the minor groove binding nonfluorescent quencher (MGBNFQ) allows for added stability (and consequently specificity) of the probe since the MGBNFQ binds the DNA and mismatched bases disrupt the binding of the probe. In additi on, the lack of fluorescence from the quencher decreases the background fluorescence of the as say. Therefore, the utilization of the MGBNFQ can both increase the specificity of the assay, as well as increase the 174

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sensitivity of relative fluorescence de tection because of decreased background fluorescence. The specificity of the QPCR a ssay was tested in conjunction with the specificity of the HPyVs marker by testing the urine and feces of both non-target and target species. HPyVs were detected and quantified in 100% of communal human waste samples (e.g. wastewater treatment plant in fluent, septic tank pump trucks) and a moderate proportion of individua l human-derived samples (e.g. urine). This coupled with the absence of HPyVs in all animal derived samples sugge sts this marker has a high degree of human specificity and is preval ent in the human population of the United States. The geographical boundaries of HPyVs di stribution were addressed through literature analysis, direct research, and work with collaborators. Bofill-Mas et al. (37) reported both JCV and BKV in raw sewage fr om Egypt, Spain, France, South Africa, and Sweden. In addition, JCV and BKV have been detected in urine of individuals from Europe, Asia, America and the Mediterranean (17, 123, 165, 173, 268, 303, 308, 363367). Analysis performed in this study f ound HPyVs present in sewage collected from California and across Florida (Jacksonville, Ga inesville, Miami, Tampa, and Oldsmar). The primers used in this study were also able to detect HPyVs in sewage from Mississippi (129) and New Zealand (9). The widespread geographic distribution of these viruses provides confidence that the assay may be applicable worldwide. The sensitivity, specificity, and widespread geographical app lication of this marker are ideal characteristics of a suc cessful MST marker; how ever, understanding the persistence and survival, or detectability of the marker in various water systems is pertinent to assess its reliabi lity to indicate environmental wa ter quality and human health 175

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risks in real world situations (e.g. beaches, rivers, lakes). It is imperative for water quality indicators to mimic the survival and persistence of pathogens. Markers that persist longer than pathogens in the environment may lead to false-positives (assumed health risks in the absence of pathogens). In contrast, mark ers that do not persist in the environment as long as pathogens may lead to false-negatives (assumed safe conditions in the presence of pathogens). Therefore, understanding the effects of environmental factors on the persistence of markers allows comparisons of the marker to pathogens, and consequently the efficacy of the marker to pr edict human health risks. As compared to bacteria and RNA viruses, double-stranded DNA viruses tend to be more resilient under conditions of high temperatures and ultr aviolet radiation (UV) (112, 154, 166, 207, 286). Initial studies in which raw sewage was he ld in the dark at 25C and 35C yielded unpromising results with HPyVs detected thr oughout the study (28 days). In addition, Bofill-Mas et al. (35) reported detection of JCV in sewage held at 20C for >120 days. Therefore, it was imperative to establis h the inactivation ra tes of HPyVs under environmental conditions. Preliminary studies analyzing the effects of various UV doses, temperatures, and/or salinities demonstrat ed high doses of UV were required to significantly decrease the de tection of HPyVs DNA. Moreover, salinity proved to stabilize pure cultures of HPyVs virus par ticles at high temperatures (25C and 35C). The longer persistence at higher salinities in laborator y settings initially made this marker seem unfavorable as a water quality indicator at beaches (i.e. environmental waters with high salinity). However, the introduction of so lar radiation as well as potential predation from microorganisms in sewage significantl y reduced the persistence of HPyVs DNA in outdoor mesocosm studies. Similar effects we re observed on the pers istence culturable 176

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indicator bacteria. The sim ilar persistence of HPyVs DNA compared to other indicator of fecal pollution in more environmentally realistic settings provides confidence that these viruses will indicate recent and relevant human-associated contamination of environmental waters. The use of HPyVs and other human-associat ed markers as indicators of water quality was complicated by inconsistent effi ciency of DNA extraction, which was caused in part by the methods utilized in our laborat ory. To address these issues a modified protocol was designed and tested. The recoveri es were larger and more consistent over a range of DNA concentrations as compared to the standard protoc ol. In addition, the modified protocol required less time to comple te and was able to detect fewer viruses in a 500 ml volume. The additional sensitivit y, reliability, and time efficiency of incorporating the modified protocol allow th e HPyVs method to be highly useful addition to the human-associated microbial source track ing toolbox. In addition, the utilization of the modified protocol may allow for more efficient DNA extractions for other library independent assays (e.g. M. smithii and human-associated Bacteroidales spp. assay). Throughout this study, comparisons of HPyV s concentrations versus traditional water quality indicator concen trations (fecal coliforms, E. coli, and enterococci) or the presence/absence of other MST markers were made. Interestingly, th e concentrations of HPyVs were the same magnitude as all th ree indicator organisms in raw sewage. However, in some environmental situations (e.g. Avalon and Doheny Beaches study, Chapter 4) indicator organism concentrations tended to be significantly larger than HPyVs concentrations. In thes e situations more than one fecal input was suspected at each location. In the same study, the presence of HPyVs by either QPCR or PCR had a 177

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high degree of matching results with the M. smithii marker (82-92%) and moderate degree of matching results w ith the human-associated Bacteroidales spp. marker (5780%). The detection of HPyVs mimicked the detection of the M. smithii marker in the initial sewage hold time experiment (Chapter 2), and mimicked the persistence of the M. smithii marker in the outside mesocosm study (Cha pter 3). Harwood et al. detected the M. smithii marker and HPyVs at the same level of diluted sewage. The results of that study combined with the similar results of HPyVs and M. smithii detection in experiments involving sewage in this study indicates these human-associated markers may be found at similar con centrations in sewage (129). While correlations among human-associated markers are noteworthy, correlations with pathogens and human health risks are ul timately the most important characteristic of a useful marker of water quality. In the California beaches study (Chapter 4), the presence of HPyVs by either QPCR or PCR ha d a high degree of matching results with the adenoviruses (83-91%), and the detection of HPyVs mimicked the detection of adenoviruses in the initial sewage hold tim e experiment (Chapter 2). Aside from adenoviruses, HPyVs were also detected in the presence of Giardia spp., Cryptosporidium spp., and pathogenic Vibrio spp. in Hobie Cat Beach (Miami, FL) samples (Abdelzaher et al. submitted for pub lication) during a collaborative study with the University of Miami. In this study, beach water and sediment samples were collected at high and low tide tw ice a day on two dates, July 18th and August 8th, 2007. All samples collected on July 18th were below detection limits for HPyVs and pathogens. Water samples collected at high tide (morning) of August 8th were positive for HPyVs, Vibrio spp., and Giardia spp. Sediment samples collected at the same time 178

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were also positive for HPyVs, Vibrio spp., and Cryptosporidium spp. Later the same day, under low tide conditions, HPyVs were detected in both water and sediment samples. In addition, Vibrio spp. and Cryptosporidium spp. were also detected in sediment samples. All results from the University of Miami collaborative study are summarized in Table 27 Our laboratory also collaborated with Southern California Coastal Water Research Project (SCCWRP) in a blinded study to assess the efficacy of various water quality indicators and human pathogens to detect human sewage and to differentiate it from other sources of fecal contamination. Results from our laboratory as well as several other laboratorie s are summarized in Table 28 HPyVs detection by PCR was strongly correlated with the presence of noroviruses; however using QPCR to quantify HPyVs lead to an increase of significant relationships ( Table 29 ). HPyVs concentrations determined by QPCR were co rrelated with enteroviruses, noroviruses, and adenoviruses ( Table 29 ). The strong and frequent correlations of HPyVs and pathogens in various studies suggest that HPyVs may accurately predict human health risks. To address correlations of HPyVs a nd human health risks our laboratory participated in two epidemiological studies in collaboration with SCCWRP. The studies took place in 2007 and 2008 at Doheny and Avalon Beaches (California). HPyVs were detected using PCR in the 2007 study and the developed HPyVs QPCR assay was utilized in the 2008 study to enumerate HPyV s. Results from the 2007 study indicate there was no significant co rrelation among HPyVs detecti on and human health risks (gastrointestinal illness) because the confid ence interval of the odds ratio included 1, 179

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( Table 30 and Table 31. ). After completion of the 2007 study, SCCWRP collaborators were dissatisfied with the num ber of participants interviewed (n=3,229 for Doheny Beach and n=2,548 for Avalon Beach) and consequent lack of correlations of human health risks with any indicator. Therefore the goal of the 2008 epidemiological study was to interview more participants and combine the results of the two year study. Due to the high volume of data collected in the 2008 study, to date a finalized report has not been released by SCCWRP for the 2008 study; however concentrati ons of HPyVs have shown a significant correlation with human health risks in preliminary statistical analyses (personal communications with John Griffin). Recently, Roslev et al. (262) found humanassociated enterococci in mussels and suggested mussels as additional matrix to target MST markers to assess water quality. Our laboratory analyzed DNA samples from homogenized oyster samples provided by Dr. Joe Lepos laboratory (Uni versity of West Florida). Oysters were homogenized and DNA was extracted from 100 l of the homogena te. HPyVs were quantified in 3 of 6 samples ( Table 32. Table 32.) at relatively high concentrations (103 to 109 HPyVs gene copy numbers per sample). While the use of filter-feeding organisms as a matrix for MST analysis is relatively nove l, the use of mussels, oysters and other filtrating bivalve mollusks may better represent water quality ove r extended periods as compared to grab samples of water. Further studies are needed to develop the use of filter-feeding organisms as a matrix for MST; however HPyV s may be utilized as a target due to the preliminary success of HPyV enumeration in oys ters and the potential of the marker to correlate with viral pathogens. 180

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181 The potential correlation of HPyVs concen trations with human health risks and the increase of corre lation among HPyVs and pathogens using concentrations versus binary data further emphasize the importanc e of incorporating quantification into MST methods. The presence of HPyV s in relatively high concentrations of sewage, the high frequency of HPyVs in human-derived wastes and the specificity of HPyVs combined with the relatively conservative persisten ce of HPyVs when exposed to environmental conditions and the correlation of HPyVs with pathogens and human health risks demonstrates that this assay is a useful MST method to detect human sewage. Studies conducted throughout this research have de monstrated that the use of a toolbox approach utilizing several MST methods ma y more accurately reflect overall fecal contamination and contamination sources in environmental waters and the predictive power of any human-associated marker is increased when more than one marker is detected within the same sample. With th e suggestion of a toolbox approach, we also suggest the incorporation of HPyVs QPCR as a standard toolbox method because the marker has had published success and is a powerful humanassociated MST tool.

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Table 27. Positive and negative results for human polyomaviruses and pathogens in the 2007 Abdelzaher et al. (submitted for publicatio n) collaborative study at the Hobie Cat Beach (Miami, FL) study with collaborators at the University of Miami. July 18th, 2007 August 8th, 2007 Sample Type Analytesa Low Tide Morning High Tide Afternoon High Tide Morning Low Tide Afternoon Water HPyVs -b+ +c Vibrio spp. + Giardia spp.(PCR) + Giardia spp. (microscopy) + Cryptosporidium spp. (PCR) Sand HPyVs + + Vibrio spp. + + Giardia spp. (PCR) Giardia spp. (microscopy) Cryptosporidium spp. (PCR) + + aHPyVs, human polyomavirus b - = MST marker/pathogen level was be low detection limit (HPyVs PCR limit of detection, <40 viruses) c + = MST marker/pathogen detected 182

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Table 28. Results for indicator bacteria, human polyomaviruses, and pathogens in th e 2007 Southern California Coastal Water Research Project (SCCWRP) collaborative study in Costa Mesa, California with collabora tors including the la boratories of Drs. Stewart, Fuhrman and Sobsey. Laboratory of Analysis: SCCWRP STEWART HARWOOD FUHRMAN SOBSEY Indicator Bacteria Enterovirus No rovirus HPyVs Hepatitis A Adenovirus Matrix Sewage (ml) or Guano E. colia Entb Volume filtered (L) PCR Volume filtered (L) PCR Volume filtered (L) PCR QPCR Volume filtered (L) PCR Volume filtered (L) PCR PBSc none <1 <1 15 15 0.6 0 N/Dd N/D 20 PBS none <1 <1 15 15 0.6 0 N/D N/D N/D N/D Imperial Beach none <2 <2 15 15 0.6 0 0.5 0 20 Imperial Beach none <2 <2 15 15 0.6 0 0.5 I N/D N/D Offshore seawater none <2 <2 15 15 0.6 0 0.5 0 20 Offshore seawater none <2 <2 15 15 0.6 0 0.5 0 N/D N/D Surfrider Beach none 6 8 15 15 0.6 0 N/D N/D 20 Surfrider Beach none 4 14 15 15 0.6 0 N/D N/D N/D N/D Doheny Beach none 56 18 15 15 I 0.6 0 N/D N/D N/D N/D Doheny Beach none 28 48 15 15 I 0.6 0 N/D N/D 20 Malibu Creek none 900 100 15 15 I 0.6 0 N/D N/D 20 Malibu Creek none 700 100 15 15 I 0.6 0 N/D N/D N/D N/D Ballona Creek none 190 630 15 15 0.6 + 35 N/D N/D 20 Ballona Creek none 60 720 15 15 0.6 0 N/D N/D N/D N/D Seawater 0.01 ml 26 22 15 15 + 0.6 0 0.5 0 N/D N/D Seawater 0.01 ml 24 100 15 15 0.6 0 0.5 0 20 Seawater 0.02 ml 58 46 15 15 0.6 + 0 0.5 0 N/D N/D Seawater 0.02 ml 32 68 15 15 + 0.6 + 0 0.5 0 20 Seawater 0.05 ml 280 120 15 15 + 0.6 + 39 0.5 0 20 Seawater 0.05 ml 380 50 15 15 + 0.6 + 67 0.5 0 N/D N/D Seawater 0.10 ml 860 1.1x103 15 15 + 0.6 + 34 0.5 0 N/D N/D Seawater 0.10 ml N/D N/D 15 15 + 0.6 + 54 0.5 0 20 + Seawater 1.00 ml 5.0x103 5.0x103 15 + 15 + 0.6 + 145 0.5 0 20 + Seawater 1.00 ml 8.0x103 6.0x103 15 + 15 + 0.6 + 641 0.5 0 N/D N/D Seawater guano N/D N/D 15 15 0.6 0 N/D N/D 20 Seawater guano N/D N/D 15 15 0.6 0 N/D N/D N/D N/D 183

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184 Laboratory of Analysis: SCCWRP STEWART HARWOOD FUHRMAN SOBSEY Indicator Bacteria Enterovirus No rovirus HPyVs Hepatitis A Adenovirus Matrix Sewage (ml) or Guano E. colia Entb Volume filtered (L) PCR Volume filtered (L) PCR Volume filtered (L) PCR QPCR Volume filtered (L) PCR Volume filtered (L) PCR Doheny Beach 0.05 ml 510 300 15 + 15 0.6 74 N/D N/D N/D N/D Doheny Beach 0.05 ml 650 340 15 15 I 0.6 45 N/D N/D 20 + Doheny Pond 0.10 ml N/D N/D 15 15 I 0.6 + 0 N/D N/D 20 Doheny Pond 0.10 ml N/D N/D 15 15 I 0.6 + 0 N/D N/D N/D N/D Doheny Pond none 1.4x104 1.2 x104 15 15 0.6 0 N/D N/D N/D N/D Doheny Pond none 1.1x104 13,800 15 15 I 0.6 0 N/D N/D 20 Doheny Beach guano N/D N/D 15 15 0.6 0 0.5 0 20 Doheny Beach guano N/D N/D 15 15 0.6 0 0.5 0 N/D N/D Tijuana River none 1.9x106 3.4x106 15 + 15 + 0.6 0 20 0 N/D N/D Tijuana River none 4.2x106 3.8x106 15 + 15 + 0.6 0 20 12 20 + aEC, E. coli bEnt, enterococci cPBS, phosphate buffer saline solution dN/D, not done

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Table 29. Correlations of HPyVs with indicato r bacteria and various pathogens in the 2007 Southern California Coastal Water Resear ch Project (SCCWRP) collaborative study in Costa Mesa, California. HPyVsa Binary detection by QPCRb Concentrations by QPCRb Analytes Nagelkerkes R -squared Odds Ratio Nagelkerkes R -squared Odds Ratio E. coli N/Ac N/A Enterococci N/A N/A Enterovirus 0.264 3.478 Norovirus 0.426 18.677 0.344 3.988 Hepatitis A Adenovirus 0.452 6.242 aHPyVs, human polyomaviruses bBinary logistic regression was used to asse ss relationships among binary (+/-) detection of human polyomaviruses and pathog ens; all significant Nagelkerkes R2 and corresponding odds ratios are bolded; Nagelkerkes R2 and odds ratio were considered significant at an alpha le vel of 0.05; - indicates no significant relationship cN/A, binary logistic regressi on could not be used to assess the relationship because both variables were continuous (non-binary) 185

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Table 30. Reproduced from Report of Epidemiological An alyses: Doheny Beach, 2007 PRELIMINARY RESULTS (291). 186

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Table 31. Reproduced from Report of Epidemiological An alyses: Avalon Beach, 2007 PRELIMINARY RESULTS (291). 187

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Table 32. HPyVs concentrations in DNA from homogenized oyster samples. Sample QPCR resulta Average HPyVs gene copy numbers in sample A B Oyster 1 undet. undet. <200 Oyster 2 undet. undet. <200 Oyster 3 undet. undet. <200 Oyster 4 1.8 x 1086.1 x 107 4.8 x 109 Oyster 5 2.3 x 108 9.4 x 107 6.5 x 109Oyster 6 8.8 x 101 9.2 x 101 3.6 x 103 a HPyVs gene copy numbers in 50 l reaction with 5 l reaction 188

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ABOUT THE AUTHOR In 2000, Shannon McQuaig began her college ca reer at the University of Florida, Gainesville, FL. In 2003, she earned a Bachel or of Science degree in Microbiology and Cell Science, and went on to earn a Master of Science degr ee from the Department of Microbiology and Cell Science in 2005. That same year she began as a Ph.D. student in the Department of Biology, at the Univ ersity of South Florida (USF). At USF, she taught for the Microbiol ogy and Physiology Laboratories. She was also a research assistant on grants invol ving water quality monitoring and microbial source tracking. She presented her research at several Southeastern branch (SEB) and General meetings of the American Society for Microbiology (ASM). She was awarded a student research grant from the SEB ASM. In addition, she has published several journal articles on her research and has earned the College of Arts and Sciences Publication Award.


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The development of a human polyomavirus quantitative pcr assay to assess viral persistence, water quality, and human health risks
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ABSTRACT: Microbial water quality is generally assessed using fecal indictor organisms; however host-specific microbial source tracking (MST) methodologies can be employed to differentiate sources of fecal pollution. The central goal of this research was to develop and validate a QPCR assay for the quantification of two human-specific polyomaviruses (HPyVs) in environmental water samples. These viruses are prevalent worldwide and produce lifelong, asymptomatic viruria in immunocompetent individuals. A Taqman quantitative PCR (QPCR) assay based on the conserved T-antigen of two HPyVs (JCV and BKV) was developed and optimized (Chapter 2). HPyVs were detected in a high proportion of human-associated waste samples (e.g. sewage) and were not detected in animal excrement samples (Chapter 2). The effects of ultraviolet radiation, temperature, and salinity on the persistence of HPyVs in water were reported in Chapter 3. Laboratory studies analyzing the effects of various UV doses, temperatures, and/or salinities demonstrated high doses of UV were required to significantly decrease the detection of HPyVs DNA and salinity stabilized pure cultures of HPyVs virus particles at high temperatures (25 and 35 degrees Celsius). Solar radiation as well as potential predation from microorganisms in sewage significantly reduced the persistence of HPyVs DNA in outdoor mesocosm studies (Chapter 3). An improved method to extract human polyomavirus (HPyVs) DNA from environmental water samples was developed, and the recoveries were larger and more consistent over a range of DNA concentrations as compared to the standard protocol (Chapter 4). In the California beaches study (Chapter 4), the presence of HPyVs by either QPCR or PCR had a high degree of matching results with the adenoviruses (83-91 percent), Methanobrevibacter smithii marker (82-92 percent) and moderate degree of matching results with the human-associated Bacteroidales spp. marker (57-80 percent) (Chapter 4). HPyVs were detected in the presence of various pathogens including: Giardia spp., Cryptosporidium spp., Vibrio spp., enteroviruses, and noroviruses (Chapter 5). The presence of HPyVs in relatively high concentrations of sewage and the specificity of HPyVs combined with the relatively conservative persistence of HPyVs when exposed to environmental conditions and the correlation of HPyVs with pathogens demonstrates that this assay is a useful MST method to detect human sewage.
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