Proceedings
The U.S. EPA's
Research on Microorganisms
in Drinking Water Workshop
August 5-7, 2003
Cincinnati, Ohio

                           I



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                Research on Microorganisms in Drinking Water Progress Review Workshop
                                    Table of Contents
Introduction
Overview of the U.S. EPA's Drinking Water Research Program
    Fred S. Hauchman

Regional Concerns for Microorganisms in Drinking Water (See Appendix 1)
    Bruce Macler

Topic Area 1: Research Supporting Office of Water's Ground Water/Source Water Regulatory
Activities

Presentation Abstracts

SDWA Requirements & Microbial Research Needs (Surface Water, Ground Water, & Distribution Systems)
(See Appendix 1)
    Stig Regli

Microbial Dose-Response Modeling:  A Predictive Bayesian Approach
    James D. Englehardt, JeffSwartout

The Use of Randomized Trials of In-Home Drinking Water Treatment To Study Endemic Water Borne
Disease
    Timothy J. Wade, Rebecca Calderon, John M. Colford, Jr.

Screening Models To Predict Probability of Contamination by Pathogenic Viruses to Drinking Water Aquifers
    Bart Faulkner

Integrated Approach for the Control of Cryptosporidium parvum Oocysts and Disinfection By-Products
in Drinking Water Treated With Ozone and Chloramines
    Jason L. Rennecker, Amy (Driedger) Samuelson, Benito Corona-Vasquez, JaehongKim, Hongxia Lei,
    Roger A. Minear, Benito J. Marinas

Prevalence  and Distribution of Genotypes of Cryptosporidium parvum in United States Feedlot Cattle
    Robert Atwill

Poster Abstracts

Microbial Drinking Water Contaminants: Endemic and Epidemic Waterborne Gastrointestinal Disease
Risks in the United States
    Rebecca L. Calderon, Gunther Craun

Evaluating Microbial Indicators  and Health Risks Associated With Bank Filtration
    Floyd J. Frost

A Prospective Epidemiological Study of Gastrointestinal Health Effects Associated With Consumption
of Conventionally Treated Groundwater
    Christine Moe, Stuart Hooper, Deborah Moll, Debi Huffman, Ricardo Izurieta,
    Renea Doughton-Johnson, Tatiana Ochoa, Jim Uber, Dominic Boccelli, Joan Rose,
    Pierre Payment
           The Office of Research and Development's National Center for Environmental Research

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                Research on Microorganisms in Drinking Water Progress Review Workshop
Using Neural Networks To Create New Indices and Classification Schemes
    Gail Brian, Srini Lingireddy

Topic Area 2: Research Supporting Office of Water's Contaminant Candidate List (CCL)

Presentation Abstracts

The Contaminant Candidate List: Determining the Need for Future Drinking Water Standards (See
Appendix 1)
    Tom Carpenter

The Roles of Pathogen Risk Assessment in the Contaminant Candidate List Process
    Glenn Rice, Michael Wright, Brenda Boutin, Jeff Swartout, Michael Broder, Patricia Murphy, Jon Reid,
    Lynn Papa

Overview:  CCL Pathogens Research at NRMRL
    Donald J. Reasoner

Topic Area 2.1:  CCL Protozoa

Presentation Abstracts

Detection ofCyclospora cayetanensis and Microsporidial Species Using Quantitative Fluorogenic 5' Nuclease
PCR Assays
    Frank W. Schaefer, III, JeffD. Hester, Manju Varma, Michael W. Ware, Barley D.A. Lindquist

Development of Detection and Viability Methods for Waterborne Microsporidia Species Known To Infect
Humans
    Rebecca Hoffman, Marilyn Marshall, Mark Borchardt

Development and Evaluation of Procedures for Detection of Infectious Microsporidia in Source Waters
    Paul A. Roche lie

Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/
Infectivity of Three Protozoa From Large Volume of Water
    Saul Tzipori,  Udi Zuckermann

Topic Area 2.2:  CCL Viruses

Presentation Abstracts

Norwalk Virus Dose Response and Host Susceptibility
    Christine Moe, Lisa Lindesmith, Ralph Baric, Jacques LePendu, Peter Tennis

Development of a Rapid, Quantitative Method for the Detection of Infective Coxsackie and Echo Viruses
in Drinking Water
    Marylynn V. Yates, W. Chen, A. Mulchandani

Poster Abstracts

Dose-Response Assessments for NLV and Coxsackievirus in Drinking Water
    Brenda Boutin, Jeff Swartout
           The Office of Research and Development's National Center for Environmental Research

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                Research on Microorganisms in Drinking Water Progress Review Workshop
Methods Used To Analyze a Norovirus Outbreak
    Jennifer L. Cashdollar, Sandhya U. Parshionikar, Christina M. Newport, Sandra Willian-True,
    Daniel R. Dahling, G. Shay Font, Outbreak Investigation Team

Development of a Molecular Method To Identify Astrovirus in Water
    Ann C. Grimm, Jennifer L. Cashdollar, Frederick P.  Williams, G. Shay Font

Effectiveness of UV Irradiation for Pathogen Inactivation in Surface Waters
    Karl Linden, Mark Sobsey, Gwy-Am Shin

Topic Area 2.3: CCL Bacteria

Presentation Abstracts

Disinfection of Helicobacterpylori wdAeromonas Species
    Laura Boczek, Samuel L. Hayes, Clifford H. Johnson, Donald J. Reasoner, Eugene W. Rice,
    Sashi Sabaratnam

Genomic and Physiological Diversity ofMycobacterium avium Complex
    Gerard Cangelosi

Mycobacterium avium Complex (MAC) in Drinking Water: Detection, Distribution, and Routes
of Exposure
    Phanida Prommasith, Timothy E. Ford

Poster Abstracts

Sensitivity of Three Encephalitozoon Species to Chlorine and Chloramine Treatment as Detected by
an In Vitro Microwell Plate Assay
    Cliff H. Johnson, Marilyn M. Marshall, Jackie Moffet, Charles R. Sterling, Laura A. DeMaria,
    Gene  W. Rice

Inactivation ofAeromonas by Chlorine and Monochloramine
    Laura A. Boczek, Cliff H. Johnson, Eugene W. Rice

Mycobacterium paratuberculosis and Nontuberculous Mycobacteria in Potable Water
    Stacy Ffatter, Terry C. Covert

Detection of Helicobacter pylori Using a Highly Variable Locus Upstream of the 16S Ribosomal
RNA Gene
    M. Shahamat, M.R. Alavi, J.E.M. Watts, K.R. Sowers, D. Maeder, F. Robb

Using Real-Time PCR To Detect Toxigenic Strains of Microcystis aeruginosa
    Carrie Moulton

Role of Adaptive Response in the Kinetics of Mycobacterium avium Inactivation With Monochloramine
    Ning Tong, Lutgarde Raskin, Benito J. Marinas
           The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


Topic Area 3: Distribution Systems and Biofilms

Presentation Abstracts

The Effect of Chlorine, Chloramine, and Mixed Oxidants on Biofilms in a Simulated Water Distribution
System
    Mark C. Meckes, Roy C. Naught, David W. Cmehil, Leslie Wilsong, Janet C. Blannon,
    Mano Sivaganesan

Molecular Characterization of Drinking Water Microbial Communities
    Jorge Santo Domingo, Mark C. Meckes, Catherine Kelty, Margaret Williams, Joyce M. Simpson,
    Donald J. Reasoner

Poster Abstracts

Phylogenetic Analysis of Prokaryotic and Eukaryotic Microorganisms in a Drinking Water Distribution
System Simulator
    Margaret M. Williams, Mark C. Meckes, Cathy A. Kelty, HildredS. Rochon, Jorge W. Santo Domingo

Identification and Characterization ofAeromonas Isolates From Drinking Water Distribution Systems
    Jennifer Birkenhauer, M. Rodgers

Pathogenicity of Biofilm Bacteria
    Dennis Lye

Topic Area 4: Cross-Cutting Research and Emerging Topics

Presentation Abstracts

The Application of Mass Spectrometry to the Study of Microorganisms
    Jody A. Shoemaker, Susan T. Glassmeyer

Cyanobacteria and Their Toxins
    Elizabeth D. Hilborn

Transport of Chemical and Microbial Contaminants From Known Wastewater Discharges: Potential
Chemical Indicators of Human Fecal Contamination
    Susan T. Glassmeyer, Imma  Ferrer, Edward T. Furlong, Jeffrey D. Cahill, Steven D. Zaugg,
    Stephen L. Werner, Michael T. Meyer, Dana W. Kolpin, David D. Kryak

High Throughput DNA-Based Tools To Study Water Microbial Communities
    Jorge Santo Domingo, Joyce Simpson, Margaret Williams, Catherine Kelty

Detection of Emerging Microbial Contaminants in Source and Finished Drinking Water
Using DNA Microarrays
    Timothy M. Straub, Paul A. Rochelle, Ricardo DeLeon, DarrellP. Chandler

Mammalian Cell Response to Pathogens
    Samuel L. Hayes
           The Office of Research and Development's National Center for Environmental Research

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                Research on Microorganisms in Drinking Water Progress Review Workshop
Poster Abstracts

Effectiveness of UV Irradiation for Pathogen Inactivation in Surface Waters
    Karl Linden, Mark Sobsey, Gwy-Am Shin

Survey of U.S. Public Health Laboratories:  Microbial Pathogens on the Candidate Contaminant List
    Elizabeth D. Hilborn, Michael O. Royster, Doug J. Drabkowski

Comparative Diversity of Fecal Bacteria in Agriculturally Significant Animals To Identify Alternative
Targets for Microbial Source Tracking
    Joyce M. Simpson, Samuel P. Myoda, Donald J. Reasoner, Jorge W. Santo Domingo

Developing Dynamic Infection Transmission Models for Microbial Risk Assessment (MRA) Applications
    Patricia Murphy, Brenda Boutin, JeffSwartout, Glenn Rice, Jon Reid, Michael Broder

Virulence Factors ofAeromonas: A Molecular Genetic Characterization
    Keya Sen, Mark Rodgers

Effects of pH and Temperature on the Kinetics ofAeromonas hydrophila Inactivation With Combined
Chlorine
    Kwanrawee Sirikanchana, Benito J. Marinas

Agenda
Poster Titles and Sessions List
Participants List (including Remote Participants)

Appendix 1: Presentations of Regional Research Needs and Office of Water Regulatory Activities and
Research Needs

Regional Concerns for Microorganisms in Drinking Water
    Bruce Macler

SDWA Requirements & Microbial Research Needs (Surface Water, Ground Water, & Distribution Systems)
    Stig Regli

The Contaminant Candidate List: Determining the Need for Future Drinking Water Standards
    Tom Carpenter

Appendix 2: Additional NCER STAR Drinking Water Grant Microbial Research

Experimental Infection of Healthy Adults with a Cryptosporidium Genotype 1 Isolate (TU502)
    Cynthia Chappell, P. Okhuysen, R. Langer, D. Akiyoshi, S. Tzipori

Experimental Challenge of Healthy Adult Volunteers With Cryptosporidium muris Oocysts
    Cynthia Chappell, P. Okhuysen, R. Langer, S. Tzipori

Appendix 3: STAR Grant Presentation Abstracts and Agenda From the USEPA/USGS Meeting on
Cryptosporidium Removal by Bank Filtration, September 9-10, 2003

Abstract Not Provided
    William Blanford
           The Office of Research and Development's National Center for Environmental Research

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                Research on Microorganisms in Drinking Water Progress Review Workshop
Study of Particle and Pathogen Removal During Bank Filtration of River Waters
    Edward J. Bouwer, Charles R. O'Melia, W. Joshua Weiss, Kellogg J. Schwab, Binh T. Le,
    Ramon Aboytes

Evaluating Microbial Indicators and Health Risks Associated With Bank Filtration
    Floyd J. Frost

Application of a Multipath Microsphere Tracer Test To Understanding Transport of Bacteria and Protozoa
at a Bank Filtration Site
    Rick Langford, Dirk Schulze-Makuch, Suresh Pillai

Pathogenic Microbe Removal During Riverbank Filtration
    Joseph N. Ryan, Yumiko Abe, Rula Abu-Dalo, Menachem Elimelech, GarrettMiller,
    Zachary Kuznar, Ronald W. Harvey, David W. Metge

Event Description
Agenda
           The Office of Research and Development's National Center for Environmental Research

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                Research on Microorganisms in Drinking Water Progress Review Workshop
                                         Introduction

    One of the high-priority research areas identified by the U.S. Environmental Protection Agency's (EPA)
Office of Research and Development (ORD) is drinking water. Although the United States has one of the saf-
est water supplies in the world, drinking water quality varies from place to place, depending on the condition
of the source water and the treatment it receives. In 1996, Congress amended the Safe Drinking Water Act to
emphasize sound science and risk-based standard setting.

    Threats  to drinking water safety come from the occurrence of chemical contaminants or pathogens in
drinking water, and research is needed in a variety of areas to improve the ability to assess and thereby reduce
the public health risks from America's public water systems. The continued occurrence of waterborne disease
outbreaks demonstrates that the safety of drinking water might be threatened by pathogenic microorganisms if
treatment is inadequate or if the quality of water in the distribution system is compromised.

    ORD's research on  microorganisms that may impact human health through drinking water is conducted
across its three  national  laboratories  (National Health  and Environmental Effects  Research  Laboratory,
[NHEERL]; National Exposure Research  Laboratory [NERL]; and the National Risk Management Research
Laboratory [NRMRL])  and through two national centers  (National Center for Environmental  Assessment
[NCEA] and National Center for Environmental Research [NCER]). In addition, EPA's drinking water re-
search program may indirectly benefit from microbial research being conducted through its National Home-
land Security Research Center, although no individual research efforts from this center were presented at the
workshop. NCER is responsible for implementing the Science To Achieve Results (STAR) competitive grants
program; the remaining national laboratories and centers manage intramural research programs.

    This workshop was organized by representatives from  ORD's laboratories and centers to bring together
ORD's intramural and extramural scientists who are researching microorganisms in drinking water, staff from
the Office of Water (OW), and regional  office representatives. The meeting was open to the public. The work-
shop provided a forum for the scientists  to present their research, for OW to identify the research needs associ-
ated with their upcoming regulatory agenda, and for all participants to discuss applications of the research.

    The EPA uses meetings like  this one to discuss research progress on topics of major scientific interest to
the Agency.  The research reported is of critical importance to EPA, as it has the potential to strengthen the sci-
entific basis  for both assessing the risk from exposure to pathogenic microorganisms and developing appropri-
ate risk-management practices to mitigate their effects.

    The meeting had both platform and poster sessions. Presentations were provided by ORD intramural scien-
tists, STAR grantees, and representatives from a regional office and OW. The abstracts in this report are organ-
ized by  research topic area into platform presentations and poster presentations in the order of the Agenda or
poster listing. For the  one regional office  presentation and the two presentations by OW in which regulatory
agendas or research needs were identified, rather than an abstract, the full presentations are provided in Ap-
pendix 1, Presentations of Regional Research Needs and Office  of Water Regulatory Activities and Research
Needs. In addition, one STAR grantee  was unable to  attend the meeting; the abstracts of her two research
grants are provided in Appendix 2, Additional NCER STAR Drinking Water Grant Microbial Research.

    Finally,  for this meeting EPA arranged for Web-broadcast of the presentations,  enabling remote partici-
pants to hear and view the plenary presentations and discussions. Hence, you will see  a remote participants list
included in this proceedings document.

    Rather than participate in this meeting, STAR grantees doing riverbank filtration research were invited to
present  their research at a separate meeting arranged by EPA/ORD/NCER and EPA/OW jointly with the U.S
Geological  Survey (USGS), The USEPA/USGS Meeting on Cryptosporidium Removal by Bank Filtration.
This meeting was held on September 9-10, 2003, at the USGS facility in Reston, VA. The meeting consisted of
a series  of plenary presentations provided by researchers from OW, NCER's STAR program, USGS, U.S. De-
           The Office of Research and Development's National Center for Environmental Research

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                Research on Microorganisms in Drinking Water Progress Review Workshop
partment of Agriculture, universities, and states. This meeting also was open to the public. No formal proceed-
ings document was prepared; however, the event description, agenda, and abstracts of the STAR research pre-
sented are included in Appendix 3, STAR Grant Presentation Abstracts and Agenda From the USEPA/USGS
Meeting on Cryptosporidium Removal by Bank Filtration, September 9-10, 2003. In addition, pdf versions of
the full presentations can be found at: http://es.epa.gov/ncer/publications/meetings/drinking_sept9_03.html.

    For more information on ORD's drinking water research program, please contact the Acting National Pro-
gram Director for Drinking Water, Gregory Sayles, at 513-569-7607 (sayles.gregory@epa.gov). For more in-
formation  about EPA's STAR drinking water research grants program, you may contact Cynthia Nolt-Helms
at 202-343-9693 (nolt-helms.cynthia@epa.gov) or Angela Page at 202-343-9826 (page.angelad@epa.gov). For
more information on EPA's ORD, please visit our homepage: http://www.epa.gov/ord.
           The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


      Overview of the U.S.  EPA's Drinking Water Research  Program

                                         Fred S. Hauchman
      National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC

                                      Presentation Abstract

    Drinking water is one of the highest priority research programs of the U.S. Environmental  Protection
Agency's (EPA) Office of Research and Development (ORD). To address the wide range of issues  relating to
waterborne contaminants, the U.S. EPA has established an integrated, multidisciplinary drinking water re-
search program that is closely linked to the Office of Water's (OW) regulatory activities and  timelines. Drink-
ing water research is conducted or supported by ORD's six national laboratories and centers: the National
Exposure Research Laboratory, the National Health  and Environmental Effects Research Laboratory, the Na-
tional Risk Management Research Laboratory,  the National Center for Environmental Assessment, the Na-
tionational Center for Environmental Research, and the National  Homeland Security Research Center. The
research program supports OW decisionmaking and the implementation of EPA rules at the state and local
level through the development of new scientific data, innovative methods, and cost-effective technologies for
improving the assessment and control of drinking water risks.

    ORD's drinking water research program activities and plans for fiscal years 2003-2010 are described in a
new Drinking Water Research Multi-Year Plan (MYP). As described in the MYP, the Safe Drinking Water
Act (SDWA) provisions with the most significant implications for research on waterborne pathogens include
the Microbial/Disinfection Byproduct (M/DBP)  set of rules, the Contaminant Candidate List (CCL)  of unregu-
lated contaminants, distribution systems, and source water protection.  Research to address  key uncertainties
associated with the Source Water and Ground Water rules (part of the M/DBP cluster of rules) includes efforts
to: (1) improve methods  to detect Cryptosporidium in water matrices; (2) assess risks associated with exposure
to protozoa and viruses;  and (3) remove Cryptosporidium, particularly for small systems. Research on unregu-
lated pathogens is primarily focused on developing new or improved analytical detection methods, and on de-
termining the ability of conventional and advanced treatment to remove or inactivate microorganisms. Studies
also are being conducted on innovative molecular approaches to characterize and prioritize pathogens for pos-
sible listing on future CCLs. Distribution system studies include research on opportunistic pathogens in bio-
films, and on the relationship between Mycobacterium disease and isolates of this microorganism in the distri-
bution system. Finally, research on source water assessment methods, tools, and  best management practices is
being conducted to support both SDWA and Clean Water Act provisions that relate to the protection of drink-
ing water sources. This presentation will provide an overview of current and planned research activities in each
of these areas.
           The Office of Research and Development's National Center for Environmental Research

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    Research on Microorganisms in Drinking Water Progress Review Workshop


          Regional Concerns for Microorganisms
                       in Drinking Water

                            Bruce Macler
        U.S. Environmental Protection Agency, Region 9, San Francisco


              The full presentation can be found in Appendix 1.
The Office of Research and Development's National Center for Environmental Research

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Topic Area 1: Research Supporting Office
  of Water's Ground Water/Source Water
          Regulatory Activities

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


    SDWA Requirements & Microbial Research Needs
 (Surface Water, Ground Water, & Distribution Systems)

                            Stig Regli
  U.S. Environmental Protection Agency, Office of Water/Office of Ground Water
                         and Drinking Water


              The full presentation can be found in Appendix 1.
 The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


               Microbial Dose-Response Modeling:  A Predictive
                                    Bayesian Approach

                               James D. Englehardt1 and JeffSwartouf
      University of Miami, Miami, FL; Office of Research and Development, National Center for Environ-
               mental Assessment, U.S. Environmental Protection Agency, Cincinnati, OH

                                         Presentation Abstract

    Absolute dose-response assessments have not been possible for low doses of chemicals and microbes, due
to the infeasibility of direct testing of low-dose response. The problem may apply to high doses in microbial
risk assessment as  well, because health effects of microbes may not generally extrapolate from animals to hu-
mans as readily as for chemicals. The objective of this project is to develop a robust dose-response model for
human population  response across all doses following exposure to a pathogen. The goal is to  derive an ap-
proach that is physically and biologically plausible, and that accounts for variability in human susceptibility,
variability in microbial (intra-species) strain virulence, variability in human-pathogen interaction, and variabil-
ity in the form and quantity of available information.

    A predictive Bayesian approach has been selected as the most efficient means of integrating all the desired
factors into a framework that is independent of any arbitrarily  chosen confidence limit. Recently, a predictive
Bayesian method for absolute dose-response assessment from limited information has been proposed. Informa-
tion types can be diverse, such as epidemiologic results, genetic prevalence data, cell culture data, and medical
judgment, as well  as conventional dose-response data. All available information is integrated rigorously, and
the function narrows in response to information content. Response is measured in terms of believed risk, which
is slightly higher than the expected frequency of health response as would be estimated by traditional frequen-
tist methods, even with an allowance for uncertainty.

    Predictive Bayesian models based on both the infection and illness endpoints are demonstrated in this pro-
ject. The  exact form (based on  the  confluent hypergeometric function) of the beta-Poisson dose-response
model is used for modeling the infection endpoint. The model for the illness endpoint  is derived from a self-
organized critical pattern of pathogenic illness severities and is  demonstrated numerically. Results indicate that
self-organizing characteristics of pathogenesis result in a third parameter of the dose-response function for mi-
crobes corresponding to the assumed definition of illness (minimum severity). Information-limited predictive
Bayesian  dose-response assessments, obtained for Cryptosporidium parvum infection and illness endpoints,
are compared and contrasted. The dose corresponding to 10"  cases of waterborne cryptosporidiosis per capita-
year is estimated to be 0.002 oocysts per exposure. Figure 1 shows the predictive  dose-response function for
Cryptosporidiosis based on the human response data for three C. parvum isolates, which also are shown in the
figure.

    The findings are significant in that they incorporate varied information, rigorously accounting for limited
data in a manner that allows for straightforward updating with new information, without dependence on preset
confidence intervals. Follow-on activities include an analysis and integration of data for a new C. parvum iso-
late and application of the model to other pathogens. Investigation of more flexible, but still biologically rele-
vant models for better fitting of the entire dose-response curve is being pursued.
           The Office of Research and Development's National Center for Environmental Research

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                 Research on Microorganisms in Drinking Water Progress Review Workshop
  1

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                Figure 1.  Predictive dose-response function for C. parvum: illness endpoint.
            The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


               Microbial Dose-Response Modeling:  A Predictive
                                    Bayesian Approach

                               James D. Englehardt1 and JeffSwartouf
      University of Miami, Miami, FL; Office of Research and Development, National Center for Environ-
               mental Assessment, U.S. Environmental Protection Agency, Cincinnati, OH

                                        Presentation Abstract

    Absolute dose-response assessments have not been possible for low doses of chemicals and microbes, due
to the infeasibility of direct testing of low-dose response. The problem may apply to high doses in microbial
risk assessment as  well, because health effects of microbes may not generally extrapolate from animals to hu-
mans as readily as for chemicals. The objective of this project is to develop a robust dose-response model for
human population  response across all doses  following exposure to a pathogen. The goal is to derive an ap-
proach that is physically and biologically plausible, and that accounts for variability in human susceptibility,
variability in microbial (intra-species) strain virulence, variability in human-pathogen interaction, and variabil-
ity in the form and quantity of available information.

    A predictive Bayesian approach has been selected as the most efficient means of integrating all the desired
factors into a framework that is independent of any arbitrarily  chosen confidence limit. Recently, a predictive
Bayesian method for absolute dose-response assessment from limited information has been proposed. Informa-
tion types can be diverse, such as epidemiologic results, genetic prevalence data, cell culture data, and medical
judgment, as well  as conventional dose-response data. All available information is integrated rigorously, and
the function narrows in response to information content. Response is measured in terms of believed risk, which
is slightly higher than the expected frequency of health response as would be estimated by traditional frequen-
tist methods, even with an allowance for uncertainty.

    Predictive Bayesian models based on both the infection and illness endpoints are demonstrated in this pro-
ject. The  exact form (based on  the confluent hypergeometric function) of the  beta-Poisson dose-response
model is used for modeling the infection endpoint. The model for the illness endpoint  is derived from a self-
organized critical pattern of pathogenic illness severities and is  demonstrated numerically. Results indicate that
self-organizing characteristics of pathogenesis result in a third parameter of the dose-response function for mi-
crobes corresponding to the assumed definition of illness (minimum severity). Information-limited predictive
Bayesian  dose-response assessments, obtained for Cryptosporidium parvum infection and illness endpoints,
are compared and contrasted. The dose corresponding to 10"  cases of waterborne cryptosporidiosis per capita-
year is estimated to be 0.002 oocysts per exposure. Figure 1 shows the predictive dose-response function for
Cryptosporidiosis based on the human response data for three C. parvum isolates, which also are shown in the
figure.

    The findings are significant in that they incorporate varied information, rigorously accounting for limited
data in a manner that allows for straightforward updating with new information, without dependence on preset
confidence intervals. Follow-on activities include an analysis and integration of data for a new  C. parvum iso-
late and application of the model to  other pathogens. Investigation of more flexible, but still biologically rele-
vant models for better fitting of the entire dose-response curve is being pursued.
           The Office of Research and Development's National Center for Environmental Research

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                 Research on Microorganisms in Drinking Water Progress Review Workshop
      1


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                Figure 1. Predictive dose-response function for C. parvum: illness endpoint.
            The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


  The Use of Randomized Trials of In-Home Drinking Water Treatment
                      To Study Endemic Water Borne Disease

                    Timothy J. Wade1, Rebecca Colder on1, and John M. Coif or d, Jr.2
   1 Human Studies Division,  U.S. Environmental Protection Agency, Chapel Hill, NC; 2 School of Public
                        Health,  University of California—Berkeley, Berkeley, CA

                                      Presentation Abstract

    Randomized trials of water treatment have demonstrated the ability of simple water treatments to signifi-
cantly reduce the incidence of gastrointestinal illnesses in developing countries, where  drinking water is of
poor  quality. Whether or not additional  treatment  at the tap reduces enteric illness in areas where water is
treated to a higher degree has not been fully resolved. Randomized trials of in-home water treatment have been
conducted to determine how much enteric illness, if any, is transmitted through a public water system and to
determine whether or not current water treatment practices adequately protect public health.

    The goal of this project is to review randomized trials of in-home water treatment conducted in developed
countries, discuss key study design characteristics and sources of bias, and present preliminary data from sev-
eral ongoing trials. The advantages and limitations of this type of study design also will be presented. The ba-
sic study design is as follows: households are  randomly assigned to  receive a device that provides additional
treatment of pathogenic microorganisms  in the household. The control group receives either no device, or re-
ceives an identical looking placebo device. Household members record occurrences of gastrointestinal symp-
toms. Any statistically significant difference between the rate of illness in the control group and the rate of ill-
ness in the active group is considered to be the amount of excess illness attribut- able to drinking regularly
treated tap water. Clinical specimens also may be  collected and tested for common pathogenic microorgan-
isms.

    Features to consider when designing or interpreting the results include: source water quality, distribution
system water quality, study location, study population, sample size,  device design, use of a placebo, recruit-
ment methods, blinding of participants and investigators, randomization procedures, tracking water consump-
tion, collection of clinical specimens, collection of water samples, statistical analyses, and outcome measure-
ment and definition.

    Four trials have been completed and published, two in Canada, one in Australia,  and one small pilot study
in the United States (Walnut Creek, California). The two trials in Canada identified a significant excess risk of
illness in those receiving regular tap water. A  blinded study in Australia, however, found no such increase in
illness and, unlike the Canadian studies, included a placebo device and a blinded  control group. Several major
trials are ongoing or nearly completed in the United States. These include the full-scale followup to the pilot
study being conducted in Davenport Iowa. Results from a second trial in an HIV  infected population also will
be published soon, and a large multiyear  study in an elderly cohort is nearing completion. An additional study
is being conducted in a community with a groundwater drinking water supply.

    Results of these ongoing trials may provide a more complete picture of the adequacy of current microbial
drinking water regulations. It is unlikely that trial data alone can provide a complete picture of the risks associ-
ated with tap water consumption. The applicability of trials in restricted geographic locations to the entire U.S.
population is questionable. Moreover, the interpretation of null results must be fully considered, and alterna-
tive hypotheses must be developed to explain such findings.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


         Screening Models To  Predict Probability of Contamination
               by Pathogenic Viruses to Drinking  Water Aquifers

                                           Bart Faulkner
           Office of Research and Development, National Risk Management Research Laboratory,
                             U.S. Environmental Protection Agency, Ada, OK

                                      Presentation Abstract

    The purpose of this research is to develop simple, screening level models to predict the overall ability of
ground-water systems to attenuate viable viruses introduced to them. Compartmental modeling approaches are
used because of their simplicity. Stakeholders and decisionmakers often seek cursory screening level models to
identify at-risk drinking water supplies (see Figure  1). When applied under appropriate circumstances, com-
partmental models can  allow prioritization of groundwater systems for further investigation, especially if such
models employ probabilistic methods in their development that  can capture much of the uncertainty in input
parameters. The goals of this research are to develop such screening models and to perform sensitivity analyses
of the models, and to develop prior distributions that correspond to actual uncertainties in the domain of the
inputs.

    The approach used is to identify components of groundwater systems that can be considered to behave as
compartments,  contributing to a final catchment scale subsurface flow model. After these flow models are
developed, appropriate attenuation functions are derived and  applied.  In  most cases, these are based on
physical, deterministic governing differential equations. By invoking certain assumptions such as steady state,
or well-mixed reactors, the  equations can be solved to yield algebraic expressions. Uncertainty is captured in
final model estimates of attenuation by (1)  error propagation methods, or (2) Monte Carlo methods. Bayesian
approaches will be used to  specify prior  distributions for parameters to allow for sparse data and robustness,
and to quantify uncertainty.

    At present, a complete probabilistic modeling approach has been developed for homogeneous unsaturated
soils. Assuming gravity flow, a suite of prior distributions has been developed for each of the 12 U.S. Depart-
ment of Agriculture soil categories. Probabilistic outputs show that for one-half meter thick soils, most exhibit
probabilities of failure to attenuate most viruses to the 99.99 percent reduction demanded in many regulatory
compliance settings. Sensitivity analyses show that mean logio of saturated hydraulic conductivity and the wa-
ter-to-air mass transfer affected virus fate and transport about three times more than any other parameter, in-
cluding inactivation rate of percolating viruses. A user-friendly computer model with a graphical user interface
has been developed and released.

    The sensitivity analyses suggest extreme infiltration  events may  play a predominant role in leaching of
viruses in soils, as such events could impact hydraulic conductivity. The water-air interface also plays a large
role. The current research issue of accurate  estimation of the air-water interfacial area is an important one, not
only for modeling transport of contaminants subject to hydrophobicity effects,  but also for unsaturated-zone
virus transport modeling.

    Developing additional components of an overall catchment-scale groundwater model, including residence
time in the saturated zone, remains as an important next step. Challenges include developing appropriate as-
sumptions to make residence time prediction for spatially explicit virus loadings tractable, and yet still reason-
able for useful prediction.
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                Research on Microorganisms in Drinking Water Progress Review Workshop
                 Public Water
                 Supply Well
                                                       ^""/J.• IV'' :%• Proposed hydrogeologic barrier;
Figure 1.  A user-friendly Monte Carlo-based model (Virulo) has been developed by the U.S. EPA. It can be
          used to predict the probability of failure of a proposed hydrogeologic barrier to attenuate viruses to
          a given level.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


     Integrated Approach for the Control of Cryptosporidium parvum
    Oocysts and Disinfection By-Products in Drinking Water Treated
                            With Ozone and Chloramines

         Jason L. Rennecker, Amy (Driedger) Samuelson, Benito Corona-Vasquez, Jaehong Kim,
                        Hongxia Lei, Roger A. Minear, and Benito J. Marinas
    Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign,
                                           Vrbana, IL

                                     Presentation Abstract

    The overall goal of this project was to develop process design recommendations for the simultaneous con-
trol of Cryptosporidium parvum oocysts and disinfection by-products (DBFs) during ozone/chloramines se-
quential disinfection  of natural waters. Because the main objective of the study was to develop an integral con-
trol strategy, the scope of work included investigating a limited number of DBFs (bromate, formaldehyde, and
cyanogen halides) associated with the ozone/chloramines sequential disinfection process. This presentation
will emphasize experimental findings on the inactivation of C. parvum oocyst with  sequential ozone/chlor-
amines (as well as ozone/free chlorine) schemes. The DBF work  of the study will be presented at a future
NCER STAR meeting focusing on DBF aspects.

    C. parvum oocysts have emerged as the microbial water contaminant with greatest resistance to chemical
disinfectants. There is particular concern because both the free and combined forms of chlorine, most com-
monly used  as primary  inactivation  agents, are considered practically ineffective in controlling  C. parvum
oocysts under typical drinking water conditions. In contrast, ozone and chlorine dioxide are both considered
viable chemical disinfectants, but there is concern about potentially high disinfection requirements. A promis-
ing alternative for more efficient control of C. parvum oocysts is the sequential application of certain combina-
tions of disinfectants. The efficiency of C. parvum oocyst inactivation by combined as well as free chlorine can
be increased significantly after limited exposure to ozone.

    There are various factors that could affect the overall efficiency of sequential ozone/monochloramine as
well as ozone/free chlorine disinfection  processes. These include pH, temperature, ozone pretreatment level,
and oocyst resistance variability.  Experimental results showing the role of these various  parameters will be
presented.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


 Prevalence and Distribution of Genotypes of Cryptosporidium parvum
                            in  United  States Feed lot Cattle

                                           Robert Atwill
                              University of California at Davis, Davis, CA

                                      Presentation Abstract

    The overall goal for this research project is to characterize the ability of feedlot cattle in the United States
to load the environment with the protozoal parasite, Cryptosporidium parvum. Specific objectives are to estab-
lish the prevalence, intensity, and distribution of genotypes of C. parvum in populations of feedlot cattle and to
identify risk factors that are associated with feedlot cattle shedding C. parvum.

    Fecal samples were collected from 5,274 cattle, whereby approximately 240 cattle from 22 different feed-
lots located  in California, Washington, Texas, Oklahoma, Colorado, South  Dakota, and Nebraska were sam-
pled. Within each feedlot, fecal samples were typically collected from sets of cattle who had just arrived, had
been at the feedlot 4 to 8 weeks, and from cattle several weeks prior to slaughter to generate a comprehensive
survey of fecal shedding of C. parvum.

    Out of 5,274 fecal samples, only 9 (0.2%) had detectable levels of oocysts as measured by our standard
diagnostic test, direct immunofluorescent microscopy (see Table 1). This assay can reliably detect oocyst con-
centrations down to  about 600 oocysts per gram of feces. To determine if a percentage of cattle were shedding
small numbers of oocysts, 10 negative cattle were retested from each feedlot using immunomagnetic separa-
tion of oocysts followed by direct immunofluorescence (IMS-DFA). Our IMS-DFA method can detect as few
as 1 oocyst per gram of bovine fecal material, arguably the most sensitive method published to date for detect-
ing C. parvum in bovine  feces.  Using  this highly sensitive method, 2 out of 220 (0.9%) fecal samples con-
tained low levels of oocysts, indicating that false negatives were relatively uncommon in our data. This dataset
is being statistically modeled to estimate the environmental loading rate using methods developed in our labo-
ratory.1 Using a nested polymerase chain reaction technique associated with restriction fragment length poly-
morphism developed by Xiao et al. (1999) of the Centers for Disease Control and Prevention that targets the
18S rRNA gene, the genotype for these isolates was the bovine genotype A.

    Although we are still in the process of calculating the  environmental loading rate of C. parvum bovine
genotype A  from feedlot cattle, these preliminary data suggest that feedlot cattle are not heavily infected with
C. parvum in middle and western United States. This lack of substantial shedding of C. parvum among feedlot
cattle is a positive finding, given the fact that feedlots are located throughout the United States, feedlots can
produce large amounts of fecal  material on a per acre basis, and given the infectious potential of bovine  C.
parvum for susceptible humans.
Reference

1. Atwill, E.R., et al. 2003. Improved quantitative estimates of low environmental loading and sporadic peri-
   parturient shedding of Cryptosporidium parvum in adult beef cattle. Applied Environmental Microbiology
   69:4604-4610.
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                Research on Microorganisms in Drinking Water Progress Review Workshop
  Table 1. Prevalence of Fecal Shedding of Cryptosporidium parvum in Feedlot Cattle in the United States.
California
Washington
Texas
Oklahoma
Colorado
South Dakota
Nebraska
TOTAL
4
1
3
3
4
4
3
22
0/960
2/240 (0.8%)
0/711
0/722
6/957 (0.6%)
1/964(0.1%)
0/720
9/5,274 (0.2%)
0/40
0/10
0/30
1/30
0/40
1/40
0/30
2/220 (0.9%)
1  Diagnostic method was direct immunofluorescent assay.
2  Diagnostic method was immunomagnetic separation of oocysts coupled with a direct immunofluorescent
  assay, capable of detecting one oocyst per gram of feces.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


     Microbial Drinking Water Contaminants:  Endemic and  Epidemic
     Waterborne Gastrointestinal  Disease Risks in the United  States

                              Rebecca L. Calderon1 and Gunther Craun2
                  U.S. Environmental Protection Agency, Research Triangle Park, NC;
                              Gunther F. Craun Associates, Staunton, VA

                                         Poster Abstract

    In the United States, drinking water treatment is rightfully considered a major public health achievement
of the 20th century. However, a residual number of waterborne outbreaks continue to be reported when water
treatment systems fail or are poorly operated, and distribution  systems or sources become contaminated. In
some reported outbreaks, current drinking water standards had not been exceeded. Recent epidemiologic stud-
ies also have suggested that cases of mild, unreported gastroenteritis may be associated with  consumption of
tap water from systems where no outbreak was reported. The U.S. EPA has collaborated with the Centers for
Disease Control and Prevention for more than 30 years in the surveillance and investigation of waterborne dis-
ease outbreaks. The periodic analysis of waterborne outbreak statistics from 1971 to 2000 has assisted the U.S.
EPA in developing a research program and promulgating regulations for safe drinking water (e.g., the Surface
Water Treatment Rule, Ground  Water Treatment  Rule, Total Coliform Rule). The nature and magnitude of
endemic waterborne disease, however, is not well characterized in the United States. Recent studies conducted
in Canada suggest that in some  communities that meet all current drinking water regulations, drinking water
could be a significant cause of gastrointestinal (GI) disease. Because persons with GI  illnesses rarely visit a
physician for treatment, the causes of these illnesses in developed countries are difficult to study by traditional
observational epidemiologic designs.

    EPA's Surface Water Treatment Rule (SWTR) of 1989 requires all communities  that use surface water as
a source of their drinking water to filter their water, unless special criteria are met. The promulgation of this
rule provided an opportunity to use a natural experiment and a quasi-experimental  epidemiologic design to
evaluate endemic waterborne illness risks. After surveying water utilities affected by the SWTR, 21  utilities
were found that would be good candidates for an epidemiologic study of waterborne disease. A pilot epidemi-
ologic study was conducted in one community to evaluate the endemic waterborne  gastrointestinal illnesses
risks and obtain information  for use in designing  additional studies. This community intervention study col-
lected information about daily GI symptoms from families before and after filtration was added to the drinking
water treatment process  Information was obtained during July through December in 1996 and 1997. As a re-
quirement for participation, families had to include one  or more children in the study. Analyses of the pilot
study community indicate a significant reduction occurred in the rate of credible-gastrointestinal (CGI) illness
after filtration of drinking water. The relative risk of CGI before filtration was almost double  that after filtra-
tion (RR=1.8, 95% CI=1.5-2.1). The attributable risk of CGI associated with unfiltered  drinking water is esti-
mated to be 34 percent. We concluded that this is an efficient study  design and have moved forward with two
other community intervention studies. The second and third communities will include an assessment of CGI in
families  in a  control community to  control for community variability in the incidence  of GI illness and help
interpret the observed waterborne contribution to illness risks. The second study in Washington State has fin-
ished data collection, and the  third study in southwest Texas is in the middle of data collection.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


                Evaluating Microbial Indicators and Health Risks
                            Associated  With Bank Filtration

                                           Floyd J. Frost
                            Lovelace Clinic Foundation, Albuquerque, NM

                                          Poster Abstract

    The purpose of the proposed project is to compare serological responses to Cryptosporidium antigens in
users of bank-filtered water (one community with only bank filtration and disinfection and one community
with bank filtration, conventional filtration, and  disinfection) with the responses of similar people residing in
an area that uses disinfected but unfiltered high-quality groundwater. The hypothesis is that, if bank filtration
completely removes  Cryptosporidium oocysts, the serological responses of the three populations should be
similar. The specific goals of the study are to: (1) identify approaches to collecting sera from similar popula-
tions in different geographic locations so that rates of serological responses can be compared; (2) pilot test the
approach in three different geographical locations by collecting sera from cities that use bank filtration and
nearby cities that use high-quality groundwater for a drinking water source; (3) analyze the sera for serological
responses to Cryptosporidium and Giardia antigens and compare the frequency and intensity of responses be-
tween the bank filtration cities and the groundwater cities; and (4) compare serological responses in the same
cities at times when bank filtration efficacy is likely to be optimal and when it is likely to be least effective.

    Sera from 50 people from each of three communities (users of bank filtered and chlorinated, bank filtered
plus direct filtration plus ozonation, and chlorinated groundwater) will be collected at baseline and at five fol-
lowup blood draws. A questionnaire on risk factors will be collected at each blood draw. Sera will be tested for
the presence of antibody responses to two Cryptosporidium antigens (15/17-kDa and 27-kDa) and for serologi-
cal changes (seroconversion). The baseline level  of serological responses as well as the rates of seroconversion
will be compared for each population (50 baseline and 250 periods for estimating rates of seroconversion).
Comparisons will adjust for collected risk factor  data from each individual. For purposes of extrapolating these
results to other locations, a  series of source  and finished water quality indicators will be measured for each
water source.

    No results are available  at this time. Analysis of sera will take place once all sera are collected. Then, all
sera from a subject will be run on the same Western blot to reduce variations between blots. Blood draws will
continue every 4 months. Data entry protocols will be developed and implemented for data entry of the ques-
tionnaires. Sample analysis will commence once all of the samples are collected, because the analysis of each
subject's samples will be on  the same Western blot. The distribution systems analysis also will commence.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


     A Prospective Epidemiological Study  of Gastrointestinal  Health
          Effects Associated With Consumption  of Conventionally
                                  Treated Groundwater

           Christine Moe1, Stuart Hooper1, Deborah Moll2, Debi Huffman3, Ricardo Izurieta2'3,
              Renea Doughton-Johnson ,  Tatiana Ochoa , Jim Uber , Dominic Boccelli' ,
                                 Joan Rose , and Pierre Payment
          Collins School of Public Health, Emory University, Atlanta,  GA; 2 Centers for Disease
          Control and Prevention, Atlanta, GA;3University of South Florida, St. Petersburg, FL;
           4University of Cincinnati, Cincinnati, OH; 5Michigan State University, Lansing,  MI;
                               6Universite du Quebec, Quebec, Canada

                                        Poster Abstract

    The overall goal of this study is to estimate the risks of endemic  gastrointestinal (GI) illness associated
with the consumption of conventionally treated groundwater in the United States and determine the relative
contributions of source water quality, treatment efficacy,  and  distribution system vulnerability to endemic
waterborne disease.

    In the United States, nearly 89 million people depend on community groundwater systems for drinking
water. Epidemiology studies in communities using surface water sources have suggested that 10-40 percent of
GI illness may be associated with drinking water. Recent national groundwater surveys have found significant
occurrence of microbial contamination in groundwater sources, and there is no information about the endemic
illness that may be associated with consumption of treated groundwater. Also, there is uncertainty about the
relative magnitudes  of risks from problems  with distribution systems  and risks from treatment deficiencies.
The specific aims of this study are to: (1) compare GI illness rates in individuals drinking highly purified bot-
tled water to GI illness rates in individuals drinking conventionally treated groundwater bottled at the treatment
plant to determine the risk of GI illness associated with source water quality and treatment; and (2) compare GI
illness rates in individuals drinking bottled treatment plant water to GI illness rates in individuals drinking tap
water from selected areas of the distribution system to determine the risk of GI illness associated with distribu-
tion system vulnerability.

    This study is a  12-month, double-blinded, randomized intervention trial of 900 households in a large
metropolitan area in the southeastern United States with a community groundwater system that uses con-
ventional treatment, meets current water quality standards, and has a well-characterized distribution sys-
tem with  areas of vulnerability. Study households will  be  randomly divided into three  groups of 300
households: Group  1 households will drink bottled water that has been treated with ozonation and reverse
osmosis (O3-RO bottled water), Group 2 households will drink bottled water collected at the water treat-
ment  plant after treatment (WTP bottled water), and Group 3 households will drink tap water from their
home. Groups 1 and 2 will be blinded to their group assignment. One-half of the households within each
group will be  recruited from vulnerable areas  in the distribution system to examine the health risks from
distribution system  intrusion. A summary of the experimental design is shown in Figure 1. Study partici-
pants will  report GI illness symptoms and selected risk factors in  a  weekly health diary and  biweekly
telephone interview. Samples will be routinely collected from raw source water, treated water, distribu-
tion system water, and bottled water and analyzed for microbial indicators of fecal contamination and in-
trusion. Data analyses will compare GI illness rates and water quality among the three study groups and
among study households in different parts of the distribution system.

    Work to date has  involved developing  survey materials and obtaining  Institutional  Review Board ap-
proval, coordinating activities with the local utility, obtaining and analyzing historic and current water quality
data, and investigating areas of the distribution system that may be more vulnerable to intrusion. The vulner-
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                Research on Microorganisms in Drinking Water Progress Review Workshop
ability assessment has been performed using hydraulic  simulations of the distribution system, water quality
data, operations data (main breaks and repairs, customer complaints), and expert opinion provided by plant and
distribution system personnel.

    This is the first study to measure the risk of GI illness associated with the consumption of conventionally
treated groundwater and to distinguish between the risk from source water and treatment vs. the risk from the
distribution system. The results of this study will provide valuable information on the magnitude of endemic
GI illness associated with drinking water in the United States. The next steps in this study will be to recruit and
enroll households and begin the intervention and data collection.
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            Research on Microorganisms in Drinking Water Progress Review Workshop
                               Water Treatment Plant
Less-vulnerable distribution system
Vulnerable distribution system
                              300 Pure Bottled HH(A+B)
                                        vs.
                              300 WTP Bottled HH(A+B)
                               Water Treatment P ant
                                                         Vulnerable distribution system
                                                             150 WTP Bottled HHs
                                                              150 Tap Water HHs
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


                  Using  Neural Networks To Create New Indices
                              and Classification  Schemes

                                    Gail Brian and Srini Lingireddy
                Department of Civil Engineer ing, University of Kentucky, Lexington, KY

                                          Poster Abstract

    The hypothesis for this project is that shifts in indicator and indigenous bacterial populations can be relia-
bly and mathematically related by neural network models to  the presence, concentration, age, and source of
microbial pathogens in river water. To test this hypothesis in the Kentucky River, tools must be developed that
provide early warning of potentially "risky" conditions in source waters for Water Treatment Plants.  Specifi-
cally, the objectives are to: (1) collect and analyze surface water samples for a host of surrogate indices and
probable human pathogens over a multiyear period; (2) apply traditional modeling techniques and statistical
analysis to the database to find relationships between indices and pathogen presence; (3) define the correlation
between atypical coliform colonies and presence/ concentration of other surrogate  indicators and pathogens;
(4) confirm  the relationship between the ratio of atypical coliform colonies to total coliforms (AC/TC) with
time; and (5) apply neural network modeling (ANN) for identification of indices and combinations of indices
related to pathogen presence, concentration, and probable source as well as to gain  insight into guidelines for
the application of ANNs for surface water quality modeling.

    A multiparameter database will be created locally, while other databases are accumulated internationally.
ANNs will be applied to predict the presence of pathogens (enteric viruses, protozoa) from other water quality
parameters (i.e., turbidity, pH, alkalinity, indicator bacteria,  indicator bacteriophage, fecal  sterols, bacterial
ratios).  ANN modeling has been applied to a dataset provided by multiple investigators from Europe for the
prediction of enteric viruses from shellfish samples.  It has been shown that the ANN approach is superior to
that of logistic regression. Also, it has been demonstrated how ANNs can be used to determine which of the
variables are of  significance. A case  study related to the research objectives of this grant has shown that the
AC/TC ratio can find sources of human pollution in a creek where other proposed indicators cannot. Along the
waterway studied, levels of fecal coliforms and enterococci were statistically indeterminate. However, the
AC/TC ratio dropped whenever the waterway passed through  inadequately sewered towns with statistical sig-
nificance.

    Initial sampling of the Kentucky River has  shown a trend between drops in the AC/TC ratio and the pres-
ence of fecal sterols, FRNA phage, protozoa, and enteric viruses. However, many more samples are required
before this noted trend can be statistically verified. ANN modeling has  again shown itself to be superior to
other regression-based modeling methods for the precise prediction of pathogen presence. This provides the
basis for new monitoring and control models.  If the AC/TC ratio is upheld as a valid means of assessing the
presence of fresh fecal contamination, these new indices could be applied for initial watershed sampling to find
"hot spots" for applying remediation.  The ratio might provide the basis for new types of TMDL models that
consider the age  and source of fecal material as well as load.

    The next steps are to continue sampling on a weekly to biweekly basis and analyzing the in-house data-
bases accumulated, and to develop an LC/MS based methodology for fecal sterols.
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Topic Area 2: Research Supporting Office of
 Water's Contaminant Candidate List (CCL)

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


 The Contaminant Candidate  List:  Determining the Need for Future
                         Drinking Water Standards

                                  Tom Carpenter
         U.S. Environmental Protection Agency, Office of Water/Office of Ground Water
                                 and Drinking Water


                     The full presentation can be found in Appendix 1.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


       The Roles of  Pathogen  Risk Assessment in the Contaminant
                                 Candidate  List Process

      Glenn Rice, Michael Wright, Brenda Boutin, Jeff Swartout, Michael Broder, Patricia Murphy,
                                     Jon Reid, and Lynn Papa
  National Center for Environmental Assessment, U.S. Environmental Protection Agency, Cincinnati, OH

                                     Presentation Abstract

    Under the 1996 Amendments to the Safe Drinking Water Act, the U.S. EPA is required to make regular
determinations of whether or not to regulate contaminants on the Contaminant Candidate List (CCL). The pro-
vision of safe public drinking water includes ongoing monitoring for contaminants with the potential to cause
adverse health effects in humans through tap water exposures. The CCL includes 10 bacterial and viral patho-
gens. The U.S. EPA's Office of Research and Development (ORD) and Office of Water (OW) have published
a draft research strategy to identify and prioritize research for contaminants listed on the CCL. The CCL re-
search strategy highlights the need to conduct risk assessments at two different time points. The initial assess-
ment (see Figure 1) identifies and integrates all of the available information needed to estimate the risk posed
by a contaminant, and identifies and prioritizes the information gaps so that the most appropriate research can
be conducted. After the data identified in the initial assessment are collected, a second risk assessment is con-
ducted to  provide a more accurate estimate  of the risk posed by the contaminant than was determined by  the
initial assessment. The OW can use these assessments to decide whether or not a contaminant should be regu-
lated. EPA's National Center for Environmental Assessment (NCEA) is developing  pathogen assessments to
address this  initial assessment phase. A recently completed NCEA assessment of exposure to Mycobacterium
avium Complex (MAC) via tap water ingestion will be presented as an example of this process. After evaluat-
ing the available information, the infection risk from MAC-contaminated tap water appears to be limited to a
few populations including people with AIDS, transplant recipients and others patients receiving immunosup-
pressive therapies, individuals with compromised pulmonary systems, and children. CD4+ cell counts are a
strong predictor of MAC infection risk in the AIDS population; therefore, the MAC assessment targeted  the
fraction of the U.S. AIDS population having less than 100 CD4+  cells/mm3, the severely immunocompromised
fraction of the AIDS population. Members of this population would be expected to experience the most severe
responses to MAC infections. Two exposure assessments were developed to address MAC ingestion exposures
in this population (see Figures 2 and 3). Epidemiologic research designed to examine the association between
MAC infections in  the severely immunocompromised fraction of the AIDS population  and water-related
activities  is  needed. If water-related activities are risk factors  for MAC infection, then research assessing
pathogen exposure-response relationships in the AIDS population and in other susceptible populations also is
needed.
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      Research on Microorganisms in Drinking Water Progress Review Workshop
                                                               Are data available on
                                                                 treatability by
                                                                generally available
                                                                 technology?
                                                                                  LEGEND:

                                                                                  Activities

                                                                                  Scientific
                                                                                  Questions
                  Figure 1.   CCL Phase I decision making process.
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               Research on Microorganisms in Drinking Water Progress Review Workshop
CDC, 2002b
US AIDS Population
(mean =3 34,000)
Fraction of U.S. AIDS Population with
< 100 CD4+ cells/mm3 blood (20%)
                              Aragon et al., 2003; Kim et al., 1998
                              No Tap Water Consumptiorr^sfot At Risk (21-43%)
                              Always Consume Tap Water    1
                                 (12%)
                              Some Tap Water Consumption  J
                                 (remaining fraction, 45-67%)
                                                                                     Eisenberg et al., 2002
                                                                                 Further Treat>Not At Risk
                                                                                 Tap Water (50%)
                                                   At Risk Population
                                         (mean = 400 individuals with AIDS having
                                     CD4+ < 100/mm3 ingest detectable levels of MAC/day)
    Figure 2. Model 1: Severely immunocompromised AIDS population exposed to MAC via tap water
              ingestion.
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         Research on Microorganisms in Drinking Water Progress Review Workshop
CDC. 2002b
US AIDS Population
(mean = 334,000)
Fraction of AIDS population with
< 100 CD4+ cells/mm3 blood (20%)
                            Davis et al..
                            Percentage of Tap Water
                            fo/.
                            Consumed at Home
                                   100
                                   76-99
                                   51-75
                                   26-50
                                   1-25
                                   None
                Fraction of HIV Population
                       18
                       28
                       16
                       26
                                              At-Risk Population
                                    (mean = 1500 individuals with AIDS having
                               CD4+ < 100/mm3 ingest detectable levels of MAC/day)
Figure 3. Model 2:  Severely immunocompromised AIDS population exposed to MAC via tap water
        ingestion.
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                Overview:   CCL Pathogens  Research  at NRMRL

                                        Donald J. Reasoner
         Microbial Contaminants Control Branch,  Water Supply and Water Resources Division,
        National Risk Management Research Laboratory,  U.S. Environmental Protection Agency,
                                          Cincinnati, OH

                                     Presentation Abstract

    The Microbial Contaminants Control Branch (MCCB), Water Supply and Water Resources Division, Na-
tional Risk Management Research Laboratory (NRMRL), conducts research on microbiological problems as-
sociated with source water quality, treatment processes, distribution, and storage of drinking water. MCCB's
work on Contaminant Candidate List (CCL) microorganisms was formally initiated in 1998 coincident with
the publication of the CCL. The CCL included 10 microorganisms: Acanthamoeba (Guidance), Adenoviruses,
Aeromonas hydrophila, Caliciviruses, Coxsackieviruses, Cyanobacteria (blue-green algae), Echoviruses, Heli-
cobacterpylori, Microsporidia, andMycobacterium avium intracellulare. MCCB has completed planned disin-
fection studies on A. hydrophila and will complete similar studies with H. pylori next year. Disinfection stud-
ies with Adenovirus were poised  to begin shortly after September 11, 2001. Those studies were set aside to
conduct disinfection studies on surrogates for bioterrorism agents. When disinfection studies on Adenovirus
begin, they will be done as a collaborative effort between NRMRL and the National Environmental Research
Laboratory.  In collaboration with the University of Arizona, limited studies on the disinfection of micro-
sporidia have been completed using chlorine and chloramine. As a result of the events of September 11, disin-
fection studies on Coxsackievirus, Echovirus, and M. avium intracellulare will be delayed for 2 to 3 years.
Work on inactivation of cyanobacteria toxins by drinking water disinfection treatment is ongoing with the
University of Wisconsin and the Wisconsin State Health Department.
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Topic Area 2.1: CCL Protozoa

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


    Detection  of Cyclospora cayetanensis and Microsporidial  Species
          Using Quantitative Fluorogenic 5' Nuclease PCR Assays

    Frank W. Schaefer, III, JeffD. Hester, Manju Varma, Michael W. Ware, and Harley D.A. Lindquist
                       National Exposure Research Laboratory, Cincinnati, OH

                                      Presentation Abstract

    Both Cyclospora cayetanensis, a coccidian parasite, and Encephalitozoon spp., a microsporidian parasite,
have a fecal-oral life cycle. These parasites can be transmitted as contaminants of either food or water. Tradi-
tional microscopic  methods for detecting and identifying these organisms in water are tedious, time consum-
ing, and not always accurate. In the case of microsporidians, detection and identification of spores (see Figures
1 and 2) is usually done by transmission electron microscopy, which, although accurate,  is not  feasible for
rapid analysis of water samples. To address these shortcomings, we have developed molecular assays for these
organisms using 5'  nuclease PCR that incorporate both a primer set and a dual labeled fluorogenic probe. For
C. cayetanensis, both a species-specific primer set and fluorescent labeled probe were designed based on the
uniqueness of the 18S ribosomal gene sequence of this parasite. For Encephalitozoon spp. (E. hellem, E. intes-
tinalis, and E. cuniculi), species-specific primer sets and a genus-specific fluorogenic probe  designed to anneal
within the Encepahalitozoon 16S rRNA gene were used. Oocysts and spores were counted accurately on a
fluorescence activated cell sorter to determine the sensitivity of these assays. Results were that as  few as 1  C.
cayetanensis oocyst and 1  Encephalitozoon spp. spore could be detected per 5 uL reaction volume. Utilizing
standard curves, the quantity of the parasites detected can be estimated with these assays. Specificity of these
molecular assays were tested against DNA isolated from numerous other related and unrelated protozoa, fresh
water algae, and bacteria. In no  case were any cross-reactions detected. These assays, although sensitive and
specific, will not determine the viability or infectivity of the detected parasite. In addition, these are assays and
are not complete methods suitable for routine use with water samples. A complete method for analysis of water
samples will require insertion of both suitable water concentration steps as well as parasite purification steps
before the 5' nuclease PCR assay is utilized.
    Figure 1.  Transmission electron  micrograph of      Figure 2. Scanning electron micrograph
              an Encephalitozoon spp. spore with its               of an Encephalitozoon spp.
              polar tube extended.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


     Development of Detection and Viability Methods for Waterborne
                Microsporidia Species Known To Infect  Humans

                     Rebecca Hoffman1, Marilyn Marshalf, and Mark Borchard/
      WI State Laboratory of Hygiene, University of Wisconsin—Madison, Madison WI; Department
         of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ; Marshfield
                           Medical Research Foundation, Marshfield, WI

                                     Presentation Abstract

    Microsporidia are obligate intracellular parasites capable of initiating disease in a host of vertebrate and
invertebrate species following ingestion of a small, environmentally resilient spore. Waterborne transmission
of this organism is suspected. However, methods to detect Microsporidia in the aquatic environment are de-
velopmental at best. The objective of this project is to develop a robust strategy for detection of waterborne
Microsporidia using seeded model and natural waters, and ultimately perform method validation in unseeded
natural waters.

    Flow cytometry with cell sorting (FCCS) was used to generate precisely enumerated Encephalitozoon in-
testinalis seeding standards. Standards were spiked into 10 L of filtered tap water at a concentration of either
10 or 100 spores/L and concentrated using a modified continuous flow centrifuge (CFC). Retentate and rinse
volumes were further concentrated by centrifugation, dried on well slides, and examined microscopically for
the presence of spores. Recoveries ranged from 39 to 76 percent (n=24).

    Sample purification studies included the evaluation of several direct and indirect immunomagnetic separa-
tion (IMS) products. In reagent water samples, indirect methods using beads directed against rabbit IgG were
shown most promising with recoveries of spores ranging from 73 to 95 percent. Flow cytometry with cell sort-
ing also was evaluated for the ability to isolate E. intestinalis spores from water concentrates. Approximately
80 percent of spores seeded into reagent water were recovered using FCCS with subsequent microscopic detec-
tion. Further experiments combined CFC concentration of seeded filtered tap water samples with isolation by
FCCS and microscopic detection. Spore recoveries ranging from 31 to 77 percent (n=23) were achieved using
this approach.

    Molecular methods including polymerase chain reaction  (PCR) and reverse transcription-PCR (RT-PCR)
following cell culture were assessed for detection of Microsporidia and viability determination. Several primer
sets, including those directed against beta-tubulin, 16S rRNA, and hsp70, were  shown capable of detecting
Microsporidia; however, their usefulness as viability indicators differed. More recently, real-time PCR using
primers directed against 16S rRNA has been shown promising for detecting low levels of spores seeded into
reagent and source water.  Future studies will evaluate recoveries of spores seeded into natural water samples,
concentrated using the CFC, and detected using real-time PCR.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


 Development and Evaluation of Procedures for Detection of Infectious
                            Microsporidia in  Source Waters

                                          Paul A. Rochelle
                   Metropolitan Water District of Southern California, La Verne, CA

                                       Presentation Abstract

 The Microsporidia group of protozoa, particularly Enterocytozoon bieneusi and Encephalitozoon spp., are re-
 sponsible for a substantial human disease burden. Many animals can carry Microsporidia, so it is possible that
 source waters may be contaminated and consequently serve as  a route of transmission to humans. However,
 there are no routine methods for detection of Microsporidia in water and very little is known about their occur-
 rence. There is a critical need to determine the role that drinking water plays in the epidemiology of this group
 of parasites. The overall objectives of this study are to develop methods to recover Microsporidia from water,
 determine the viability and infectivity of detected spores, and use the methods to assess the occurrence of Mi-
 crosporidia in untreated source waters.

 The project involved:  (1) evaluation of filtration methods for recovery of Microsporidia spores from environ-
 mental water samples; (2) development and assessment of an immunomagnetic separation (IMS) procedure for
 purification and further concentration of spores; (3) evaluation of molecular and microscopic methods for de-
 tecting spores; and (4) development of an infectivity assay combining in vitro cell culture with a molecular
 detection assay.

 Recovery efficiencies for capsule filters ranged from an average of 13.2 percent for 0.8-1 um porosity filters
 to 38 percent for 0.55 um porosity filters. Initial trials with centrifugal filters, consisting of a modified nylon
 membrane  in a centrifuge tube, resulted in recovery efficiencies of 52 percent, 22 percent, and 36 percent with
 porosities of 0.2 jam, 0.3 jam, and 0.45 jam, respectively. A prototype ultrafiltration apparatus achieved up to
 40 percent recovery using 0.05 um hollow fiber filters. Most antibody preparations demonstrated considerable
 background staining, particularly with environmental samples. In addition, a significant number of spores did
 not stain. Non-antibody based stains, such as a modified trichrome method and Calcofluor, were found to be
 effective only in nonenvironmental samples. A quantitative molecular detection  assay was developed for
 E. intestinalis with a sensitivity of 10 spores. A  cell culture-based infectivity assay  also was developed for E.
 intestinalis spores. Infection in RK13 cells was rapid  and  led  to effective spore  propagation. A  confluent
 monolayer of RK13 cells in a 75-cm flask produced more than 1 x 10 spores within 1 week of inoculation
 with a low dose of E. intestinalis spores. The 50 percent infectious dose for this assay was 36 spores (see Fig-
 ure 1). The infectivity assay was used to measure the efficacy of ultraviolet  (UV) disinfection; at least 90 per-
 cent inactivation of E. intestinalis spores was obtained with aUV dosage of 3.3 mJ/cm2.

 This research project has made considerable progress in the development and evaluation of methods for detect-
 ing environmental Microsporidia spores and measuring their infectivity. These techniques (once optimized)
 can be used to assess the extent of Microsporidia contamination in water, which will allow the water industry
 and public health officials to determine whether water represents a significant route of transmission for these
 parasites.

 An efficient recovery procedure will need effective antibodies  with high avidity and specificity that can be
 used for both IMS purification and immunofluorescent detection of spores. Consequently, a wider array of an-
 tibodies  should be evaluated. In addition, a wider range of polymerase chain reaction primers needs to be
 screened for improved specificity. The cell culture-based infectivity assay can be used to assess spore survival
 in the environment and the efficacy of disinfectants such as ultraviolet light, ozone, and chlorine dioxide for
 inactivation of Microsporidia spores.
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       Research on Microorganisms in Drinking Water Progress Review Workshop
    2   •
o
to
c
o
Q.
D)

O
   -1
   -2   •
      0.5
               1               1.5               2


                      Spore dose (Log10)



Figure 1. Dose response curve for E. intestinalis in RK-13 cell culture.
2.5
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


     Development and Evaluation of Methods for the Concentration,
          Separation, Detection, and Viability/lnfectivity  of Three
                     Protozoa From  Large Volume of Water

                               Saul Tzipori and Vdi Zuckermann
   Division of Infectious Diseases, Tufts University School of Veterinary Medicine, North Grafton, MA

                                   Presentation Abstract

    The objective of this project was to evaluate and optimize a modified continuous flow centrifugation
(CFC) method for recovery of Cryptosporidium giardia and microsporidia from turbid and large volumes
of water. The CFC method allows for concentration of oocysts, cysts, and spores from large volumes of
water, and for continuous monitoring of their presence in water,  as opposed to one-time sampling of exist-
ing methods. This method  is efficient, portable, rapid, and easy  to operate. The third phase of this project
included further optimization of the CFC method for recovery of microsporidia from 10 to 50 L of water,
simultaneous recovery of all three pathogens from volumes of 10, 50, and 1,000 L of water, and viability
testing after recovery to ascertain that the CFC method does not  lead to parasite inactivation.

    A large number of spiked experiments were conducted over the period of support, and the table below
illustrates the rate of recovery of the three protozoa from 50 L of simultaneously spiked tap water.

          Table 1.  Percent recoveries by CFC when 50 L of tap water are spiked with three
                  parasites (Giardia, C. parvum, E.  intestinalis).
1
2
o
3
4
Average%±S.D
1,500
1,500
500
500

75
71
84
37.4
57.5±38.7
2,500
2,500
2,500
2,500

37.6
82.6
58.2
10.6
44.6±35
500
500
500
500

78.4
82.4
54
N.A.
53.6±46.5
    Recoveries for C. parvum,  Giardia lamblia, and E. intestinalis spiked various volumes of water and
turbidity were better than expected. The recoveries of all three pathogens were particularly impressive
after numerous repeated spiking experiments. The reproducibility and the consistency of this system also
were very impressive. In spiked experiments performed in parallel, the CFC outperformed other currently
used filtration methods in terms of efficiency of recovery, speed, and simplicity. The CFC method is cur-
rently undergoing validation by the U.S. EPA Office of Water in several water utilities. The results will
be presented at the meeting.
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Topic Area 2.2: CCL Viruses

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


            Norwalk Virus Dose Response  and Host Susceptibility

          Christine Moe1, Lisa Lindesmith2, Ralph Baric3, Jacques LePendu3, and Peter Teunis4
 Rollins School of Public Health, Emory University, Atlanta, GA; Department of Epidemiology, University
  of North Carolina, Chapel Hill, NC; Institute of Biology, Nantes, France; National Institute for Public
                          Health and Environment, Bilthoven, The Netherlands

                                       Presentation Abstract

    Human caliciviruses are a leading cause of epidemic and endemic acute gastroenteritis and are responsible for
numerous waterborne outbreaks. The overall objective of this research project is to develop our understanding of
the risks associated with exposure to waterborne human caliciviruses as a function of dose and host  susceptibility.
The dose-response was examined for two important human caliciviruses  (HuCVs), a prototype Genogroup I virus
(Norwalk virus (NV)) and a prototype Genogroup II viruses (Snow Mountain Agent (SMA)) to:  (1) identify the
dose range of NV and SMA (IDi0, ID50, and ID90) in human volunteers; (2) examine the immune response (serum
and secretory antibodies) and determine the characteristics of volunteers that are susceptible to infection; and (3)
evaluate the fit of several mathematical models of dose-infectivity to our data. This presentation focuses on the
dose-response results of the NV study.

    A double-blinded human challenge study was conducted to determine the dose-infectivity relationship for NV.
Subjects were given various doses of a suspension of NV, monitored in a  clinical  setting for gastrointestinal
symptoms for 5 days and returned for Day 8, 14, and 21 followup visits.  Stool specimens were assayed for NV by
reverse  transcription-polymerase chain reaction (RT-PCR). NV serum and salivary antibodies were measured by
enzyme immunoassay. Saliva samples were tested for secretor status presence of H type-1 antigen and FUT2 gene
as a marker of susceptibility. Infection was defined as excretion of NV or seroconversion.

    A total of 75 subjects were challenged with NV, and 22 became infected. Twenty-two of the 75  subjects were
secretor negative, and none became infected. NV doses ranged from 1 x 10"1 to 1 x 107 PCR  detectable units
(PDU).  Approximately 68 percent of the infected subjects had gastrointestinal symptoms. Most subjects shed the
virus for at least 8 days postchallenge, and several continued to shed the virus for 18-23 days post-challenge. Sub-
jects with an early (< 5 days post-challenge) anti-NV salivary IgA response appeared to have protective immunity
to NV infection as compared to subjects with a late anti-NV salivary IgA response.

    The infectivity response appeared not to be consistent with a simple dose-response model, indicating
considerable heterogeneity in  susceptibility  to  infection among  subjects.  Susceptibility to infection with
Norwalk virus appeared to  depend on a genetic marker (FUT2) for a probable virus receptor (H type-1 antigen)
on host epithelial cells in the gastrointestinal tract. The 22 subjects who did not have this gene appeared to be
completely resistant to infection, regardless of virus dose. When the analysis was restricted to the susceptible
fraction of  the challenged volunteers (n=53), a  dose-response effect  was  seen,  although  considerable
heterogeneity still remained. At low doses there was a considerable probability  of infection, but it takes very
high doses to reach an infection probability near 1.

    There seemed to be no indication of a dose response  for illness among infected subjects. When  pre-
challenge anti-NV serum IgG was used as an indicator of prior NV infection (assuming higher levels indicated
more recent infection or infection with a virus more closely related to NV), there  appeared to be weak evidence
of a protective effect against infection among those with higher levels of anti-NV IgG.

    When illness conditional on infection was considered, higher baseline IgG  seemed to be associated with
slightly increased risk. This suggests that a subject with a high baseline anti-NV  IgG level needs  a higher dose
to become infected, but then has a slightly elevated risk of becoming ill.
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                 Research on Microorganisms in Drinking Water Progress Review Workshop
    NV is highly infectious.  The low infectious dose, mild illness or asymptomatic infections, and prolonged
shedding facilitate waterborne and secondary transmission of this virus. A genetic marker of host susceptibility
was identified that suggests that 80 percent of the general population is susceptible to NV infection. However,
some persons exhibit an early, protective, salivary IgA response. Quantitative models that describe the interaction
between virus inoculation and  growth and observable immune variables may improve understanding of the
infection  process and consequently improve risk predictions. The results of these  studies  are  valuable for
estimating the risk of HuCV infection and gastroenteritis associated with exposure to contaminated water and to
establish safe exposure limits for HuCVs in water to reduce waterborne disease.

    The next steps of the project include completion of the dose-response analyses for the SMA challenge study
and completion of the examination of the role of T-cell mediated immunity in NV and SMA challenge and infec-
tion.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


      Development of a Rapid, Quantitative Method for the Detection
         of Infective Coxsackie and Echo Viruses in Drinking Water

                          Marylynn V. Yates, W. Chen, and A. Mulchandani
Department of Environmental Sciences and College of Engineering, University of California, Riverside, CA

                                      Presentation Abstract

    The objective of this research was to improve on the current analytical  methods for quantitative detection
of infective nonpolio enteroviruses (NPEV) in drinking water. The specific objectives of this research were to:
(1) develop a molecular-beacon-based (MB) RT-PCR method to detect NPEV; (2) establish a potential correla-
tion between IMS-MB-RT-PCR detection and cell culture detection for infective viruses; (3) using the molecu-
lar beacon, develop and evaluate real-time monitoring of virus replication in cell culture; and (4) evaluate the
above methods to quantify the presence of infective NPEV in concentrated drinking water samples. A set of
primers was designed to amplify a 155-base pair section of RNA of Echovirus 11 (Echo 11), the virus chosen
to represent the enterovirus group. An antisense-MB also was designed to specifically recognize a 25-base pair
sequence within the 5 noncoding region of Echo 11. Viral RNA was reverse-transcribed and subsequently am-
plified by polymerase chain reaction (PCR). Using the MB-based reverse transcription-PCR (RT-PCR) assay,
detection and quantification of the virus was achieved. A detection limit of 0.1 plaque-forming units  (PFU)
was  obtained for Echo 11.  Specificity testing  positively identified other members  of the enterovirus group
(Coxsackieviruses Bl, B3, and B6; Echoviruses 11, 17, and 19; Poliovirus 1); nonenteroviruses (Parechovirus
1; Adenoviruses  2  and 15;  Rotavitus WA; hepatitis A virus; MS2 and  phiX174 bacteriophages; E. coli
0157:H7 and Salmonella typhimurium) were not detected.

    After development of the MB-based assay,  IMS was added to the process to minimize the potential for the
amplification of noninfective viruses. Using the IMS-MB-RT-PCR assay, the detection limit for Echovirus 11
was  3 pfu (see Figure 1).  The method was then tested using surface and groundwater concentrates that were
spiked with echoviruses. The method was able to detect the viruses at a concentration of 3 pfu in the surface
water and less than 1 pfu in groundwater. In parallel, the spontaneous hybridization between molecular bea-
cons  and their target sequences was exploited  as a means for real-time detection  of virus replication  in situ.
Newly synthesized viral RNA was used as an indicator for viral infection. A molecular  beacon targeting a spe-
cific region of the enterovirus RNA was used for the initial demonstration.  Buffalo green monkey kidney cell
cultures with or without infection with Echo 11 were collected at various time points, fixed, and permeablized.
After introduction of molecular beacons, whole fluorescence was monitored with  a fluorescence microscopy.
The results demonstrated that only cells infected with Echo 11 were brightly fluorescent, and noninfected cells
were not fluorescent. These results could be achieved within  a few hours post-infection. This result suggests
the possibility to provide real-time and quantitative determination of infective viruses without the need to per-
form a conventional plaque assay, which takes days to complete.
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      Research on Microorganisms in Drinking Water Progress Review Workshop

         Detection  of echovirus 11 in spiked
        surface water sample using IMS-MB-
                         RT-PCR
           150
                          Cycles
Figure 1. Detection of echovirus 11 in spiked surface water sample using IMS-MB-RT-PCR.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


         Dose-Response Assessments for NLV and  Coxsackievirus
                                      in Drinking Water

                                   Brenda Boutin and JeffSwartout
  National Center for Environmental Assessment, U.S. Environmental Protection Agency, Cincinnati, OH

                                          Poster Abstract

    This poster presents the choices made for surrogate pathogen dose-response selection and the outcome of
those choices. The objective of both the calicivirus and coxsackievirus assessments is to determine an estimate
of the dose-response based on data available for selected surrogate pathogens.

    Dose-response data are limited or nonexistent for coxsackievirus and calicivirus. Drinking water exposure
is the only route presented in this poster. Potential surrogate pathogens that had available dose-response data
were considered for the coxsackievirus and calicivirus risk assessments. There is no formalized process or cri-
teria for selection of representative surrogates for Contaminant Candidate List pathogens. For the most part,
family/genus, relative infectivity, and similarity of disease endpoints may be the most data available, but these
data may not substantiate the selection of a surrogate. The National Center for Environmental Assessment
(NCEA) is now developing a white paper to recommend criteria and a selection process for surrogates.

    Coxsackievirus and echovirus-12 are both single-stranded, non-enveloped RNA viruses in the Picornaviri-
dae family, Enterovirus. The probability of infection by coxsackievirus was based on a dose-response relation-
ship developed from echovirus-12 enterovirus because no data associated with the ingestion of coxsackievirus
were available from the published literature. Both viruses replicate in the gastrointestinal tract;  infection is
mostly asymptomatic. However, disease endpoints for both viruses can range from mild unspecified febrile
illness to fatal central nervous system complications.1

    The dose-response model for calicivirus was developed based on data for rotavirus because dose-response
data are not available for Norwalk Like Virus (NLV) or calicivirus. Calicivirus belongs to  the Caliciviridae
family, of which human calicivirus/NLV are members. Rotavirus is an unrelated (family Reoviridae) but may
have  similar  infectivity, and serves as a conservative surrogate for the infectious calicivirus (attack rates in
drinking water outbreaks range from 31 to 87%).  The use of the rotavirus dose-response data may represent a
conservative  assessment for NLV infectivity in humans. All members of the population are at risk of infections
from exposure to either calicivirus or coxsackievirus.

    A number of models are used in the literature to describe microbial  dose-response data. This assessment
considers only the physically and biologically relevant dose-response models; these include the exponential
and beta-Poisson models. The Pareto II also is considered,  as it closely approximates the beta-Poisson over a
wide range of parameter values and is much more analytically tractable than the beta-Poisson.  The parameter
values of these models fitted to the rotavirus data are presented in Table 1.

    In addition, the results of fitting these models to the echovirus-12 human-infectivity data, used as a surro-
gate for coxsackievirus, are shown in Table 1. The table shows the fitted model parameters and selected infec-
tious dose estimates. Uncertainty in low-dose response was estimated by bootstrapping the data set based on
the initial beta-Poisson fit. The results of the bootstrap simulation indicate about a five-fold span in the 95 per-
cent confidence interval on risk of infection at low doses (< 0.01 pfu). The upper 95 percent bound on low-
dose risk for  echovirus-12 is still 260 times less than the maximum possible risk (where exposure to 1 pfu al-
ways results in infection). In contrast, a similar bootstrap simulation performed on the rotavirus data, used as a
surrogate for  NLV, yields an upper 95 percent confidence bound on low-dose risk that is only 20 percent lower
than maximum possible risk.
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                Research on Microorganisms in Drinking Water Progress Review Workshop
    The next steps include recommendations for research on exposure and dose response for calicivirus, cox-
sackievirus, and NLV. NCEA is working to develop surrogate selection criteria and a selection process, as well
as secondary transmission models for person-to-person transmission of infectious virus such as calicivirus.

References

1.  Embrey M. Coxsackievirus in drinking water (Literature  Summary). Final Report. The George Washing-
    ton University School of Public Health and Health Services, Department of Environmental and Occupa-
    tional Health, 1999.

2.  Embrey M., et al.  Caliciviridae in  drinking water. In: Handbook of CCL Microbes in Drinking Water.
    American Water Works Association, 2002.

        Table 1.  Maximum likelihood parameter estimates, predicted IDS and goodness-of-fit statistics for
                 each model (Infectivity data, Echovirus 12).
  Exponential
  r   = 0.000583
 ID50  =1187
  IDoi = 17.2
IDoooi =0.17
   D=22.3 (df=3)
    (p = 0.0002)
(significant lack of fit)
  Pareto II
  a   = 1.06
  P   = 994
  ID50 =918
  IDoi = 9.5
 IDoooi = 0.094
   D=3.21 (df=2)
     (p = 0.20)
  Beta-Poisson
  a   = 1.06
  P   = 994
  ID50 =918
  IDoi = 9.5
IDoooi =0.094
   D=3.21 (df=2)
     (p = 0.20)
3 Subscript indicates cumulative response percentile at which the ID is calculated (based on MLE parameters).
b Plaque-forming units.
°D = deviance (-2 x maximum log-likelihood); df = degrees of freedom for X2 statistic; p = significance of fit,
 where p < 0.05 indicates lack of fit.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


                Methods  Used To Analyze a Norovirus Outbreak

      Jennifer L. Cashdollar, Sandhya U. Parshionikar, Christina M. Newport, Sandra Willian-True,
                 Daniel R. Dahling, G. Shay Fout, and the Outbreak Investigation Team
                        U.S. Environmental Protection Agency, Cincinnati, OH

                                         Poster Abstract

    The goals and objectives of this project are to:  (1) isolate and identify the viral agents in well water sam-
ples associated with two outbreaks of acute gastroenteritis reported to the Wyoming Department of Health in
February 2001 and October 2001; (2) isolate and identify the viral agents in patient stool samples; and (3) de-
termine the link between water consumption and illness.

    The project had a three-way approach: (1) an epidemiological investigation was performed to identify any
common routes of exposure among those afflicted with gastroenteritis; (2) an environmental survey was done
of the premises involved in each outbreak to determine possible  sources of contamination; and (3) laboratory
analysis was performed on well water samples for coliform and viral detection using reverse transcription po-
lymerase chain reaction (RT-PCR) and DNA sequencing. Stool samples also were analyzed for the presence of
noroviruses.

    Epidemiological studies revealed a close association between water consumption  and illness. Environ-
mental surveys in both outbreaks determined that the water supply was vulnerable to fecal contamination. Well
water samples in both cases were positive for coliforms,  and RT-PCR and DNA sequencing revealed norovi-
ruses as the causative agents of acute gastroenteritis.

    This investigation demonstrates that the U.S.  EPA's viral concentration and molecular methods, in con-
junction with epidemiological and environmental  analysis, are very useful in outbreak  studies.  The methods
used in this study can be performed in most laboratories with trained personnel and appropriate equipment,
which would allow for routine monitoring of enteric viruses in drinking water, thus preventing any future out-
breaks from occurring.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


                 Development of a Molecular Method To Identify
                                    Astrovirus in Water

             Ann C. Grimm, Jennifer L. Cashdollar, Frederick P.  Williams, and G. Shay Fout
 Microbiological and Chemical Exposure Assessment Research Division, Biohazard Assessment Research
 Branch, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH

                                         Poster Abstract

    Astrovirus is a common cause of gastroenteritis that has been determined to be responsible for several out-
breaks. Because astrovirus can be waterborne, there is interest in testing  environmental water  for astrovirus.
We have developed a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay (see Figure  1)
that is designed to detect all known astrovirus strains.

    The assay was based on  a primer  set that contained multiple upper and  lower primers as well as multiple
probes. This would allow for amplification of all of the  known strains of astrovirus using a single reaction.
When tested, this  assay was able to detect strains from all eight serotypes. In addition, an internal control was
developed, so that it will be possible  to determine if the sample being tested contains PCR inhibitors. Most
probable number analysis determined that when amplified with the developed assay, a single DNA molecule of
the internal control could be detected if inhibitors were  not present. The assay was successfully adapted to
real-time PCR, and this method was used for integrated cell culture/RT-PCR detection of infectious virus. The
methods were successfully used to detect astrovirus present in clinical samples and spiked water samples.

    A simple, sensitive method for detecting all known astrovirus strains has been developed that can be used
to detect this virus in water. This assay will be field tested by analyzing environmental water samples.
                                  AstUl-U4       AstPl-P4  AstLl-L2
                                        RT-PCR product 0.2 kb
                                     Figure 1.  Assay design.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


          Effectiveness of UV Irradiation for Pathogen Inactivation
                                      in Surface Waters

                            Karl Linden1, Mark Sobsey2, and Gwy-Am Shin2
     1 Civil and Environmental Engineering, Duke University, Durham, NC; 2Environmental Sciences and
                Engineering, University of North Carolina-Chapel Hill, Chapel Hill, NC

                                          Poster Abstract

    Ultraviolet (UV) irradiation is now recognized as an  effective and cost-competitive measure to  achieve
significant level of inactivation of Cryptosporidium while not producing appreciable level of harmful disinfec-
tion by-products (DBFs) at practical doses. However, the effectiveness of UV technology against new and
emerging pathogens and uncertainties in application of UV disinfection for unfiltered surface waters still needs
to be assessed before widespread use of this technology as a primary disinfectant in drinking water treatment
processes. The primary objectives of this research project are to evaluate the susceptibility (or resistance) and
repair potential of select Contaminant Candidate List (CCL) pathogens and indicator microorganisms to or
after UV disinfection from low- and medium-pressure (LP and MP) UV sources, and to investigate the extent
of microbial  association with particles in unfiltered systems  and  the effects of this  particle association and
other water quality parameters on UV disinfection potential.

    Preliminary results indicate that  both LP and MP UV irradiation are very effective against most of the in-
dicator and emerging microorganisms tested. However, some of the indicator microorganisms like coliphage
MS2, bacteriophage PRD-1, and Bacillus subtilis endospores as well as CCL and emerging pathogens like My-
cobacterium  terriae (a substitute  for Mycobacterium avium complex), adenovirus type 2, and  Toxoplasma
gondii oocysts showed relatively high resistance against both UV irradiation. Although the effectiveness of LP
and MP UV appeared to be similar against most of the microorganisms tested, there was some remarkable dif-
ference between these two UV technologies in terms of their effectiveness against adenovirus 2. To determine
the level of particle association and  its effect on UV disinfection, raw surface water samples have been col-
lected from various utilities across the United States, and the waters have been examined for particle associated
coliform and aerobic endospores  using physical particle disruption techniques such as  homogenization and
blending. However, the levels of the indigenous microbes in the raw waters were typically low (< 1,000/100
mL), so  that it was not feasible to assess the degree of particle association in these waters based on those
physical methods.

    Currently, the use of microscopic techniques (nucleic acid staining/probes  along with confocal  micros-
copy) are being investigated to determine the level of particle association in those raw waters. Regarding the
development of new assay systems for some of the CCL microorganisms, we have been successful in develop-
ing a new assay system (Long-template [LT] RT-PCR) for Norwalk virus and a new molecular biology assay
(RT-PCR) for adenovirus 40 or 41,  which are being and  will be  used in the current and future  inactivation
study on these viruses by LP and  MP UV. In addition, a method has been established to perform wavelength
specific studies using a polychromatic UV light source (MP UV lamps) with a set of UV bandpass filters, and
this setup will be utilized to develop  wavelength effectiveness information for select CCL microorganisms like
adenovirus 2, M.  terriae, and T.  gondii  oocysts.  Finally,  protocols have been established to examine repair
phenomenon following UV disinfection in various conditions in real water treatment situations, and these pro-
tocols will be implemented to evaluate the presence and extent of repair after UV irradiation in the select CCL
and emerging microorganisms in the  later phases of this research.
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Topic Area 2.3:  CCL Bacteria

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


        Disinfection of Helicobacter pylori and Aeromonas Species

       Laura Boczek, Samuel L. Hayes, Clifford H. Johnson, Donald J. Reasoner, Eugene W. Rice,
                                      and Sashi Sabaratnam
         Microbial Contaminants Control Branch, Water Supply and Water Resources Division,
        National Risk Management Research Laboratory, U.S. Environmental Protection Agency,
                                         Cincinnati, OH

                                     Presentation Abstract

Helicobacter pylori and Aeromonas hydrophila are contaminants listed on the U.S. EPA's 1998 Contaminant
Candidate List (CCL). The sensitivity of H. pylori to chlorine and of Aeromonas spp. to inactivation by free
chlorine, chloramines, and ultraviolet (UV) was examined. Selective and nonselective monitoring media were
evaluated to assess recovery of chlorine or UV-stressed Aeromonas spp. Results of experiments using free
chlorine showed that the H. pylori and Aeromonas spp. were readily inactivated under all conditions studied.
H. pylori showed more than 3.5 orders of magnitude inactivation by 0.5 mg/L chlorine in 80 seconds at 5 °C.
The Aeromonas spp. were inactivated by more than 5 orders of magnitude within a 1-minute exposure to free
chlorine at pH 7 or 8, and at 5 °C or 25 °C. Reductions of the Aeromonas spp.  with 2.0 mg/L of monochlor-
amine reached approximately 2 orders of magnitude at pH 8.0 for 4 minutes and greater than 5 orders of mag-
nitude inactivation after 8  minutes of exposure.  Aeromonas spp. were found to be sensitive to UV irradiation,
with fluences of less than  7 mj/cm2, giving between 5-7 logio reductions. For free chlorine, there was no ob-
servable difference in recovery  of chlorine-stressed Aeromonas spp. organisms  between selective  and nonse-
lective media. However, with UV disinfection, some Aeromonas spp. counts on nonselective media were sig-
nificantly higher than those obtained on selective agar. These findings suggest that selective agars may under-
estimate the number of viable Aeromonas recovered after exposure to UV irradiation.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


                        Genomic and Physiological Diversity
                          of Mycobacterium avium Complex

                                          Gerard Cangelosi
                          Seattle Biomedical Research Institute, Seattle, WA

                                      Presentation Abstract


    The Mycobacterium avium complex (MAC) is an environmental pathogen of susceptible humans, espe-
cially AIDS patients, children, and the elderly. MAC infections are debilitating and very difficult to treat due
to intrinsic multi-drug resistance. Infections originating from potable water have been documented; however,
the occurrence of MAC in water does not always correlate with high rates of MAC disease. The ability to pre-
dict consequences of human exposure to MAC remains an elusive goal. One factor that may contribute to this
problem is the genetic and phenotypic heterogeneity of MAC isolates. Strains and colony types are thought to
vary with regard to infectivity, susceptibility to antibiotics, and ability to survive in various environments. The
goal of this research project is to understand the  genomic and physiological bases for these phenotypes. Such
an understanding could lead to refined methods for detecting environmental MAC populations that are likely
to cause disease in humans.

    Microarrays and restriction fragment length polymorphism are being used to  quantify the genomic diver-
sity of MAC isolates from clinical and environmental  sources. In addition, transposon mutagenesis and disease
models are being used to characterize the highly mutable properties of colony type, intrinsic drug resistance,
and virulence of MAC. By  combining these two lines of investigation, we hope to identify genomic markers
that can be used to identify virulent strains of MAC in the environment.

    Comparative genomic hybridization to  a MAC genomic microarray has revealed extensive strain-to-strain
diversity within MAC. There also is extensive heterogeneity within individual isolates, as evident from RFLP
analysis and the appearance of multiple colony types on laboratory growth media.  A novel colony type switch,
termed red-white, has been identified that affects virulence, drug susceptibility, and other phenotypes. To char-
acterize the genetic basis for these phenotypes,  a transposon mutagenesis system has  been developed with
which we have begun to identify MAC genes required for pathogenicity and intrinsic  multi-drug resistance
(see Figure 1).

    Microarray and mutational analysis will be expanded. Moreover, phenotypic and genomic diversity will be
examined among environmental isolates. Phenotypic  diversity will be measured by using intracellular growth
assays, and genomic diversity by microarray analysis. Through this investigation, we hope to:  (1) test the hy-
pothesis that some  environmental strains of  MAC are more virulent than others, and  (2)  identify genomic
markers that may be used to identify virulent strains.
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              Research on Microorganisms in Drinking Water Progress Review Workshop
                     RW-E
                  RW-F
                   WR2.55
                      RW1,
                      RW2
                                                          RW-J
                                 WR2.58
                                      M. avium 104
                                         5.48 mB
RRgi,
RRg2,
RRg6,
RRg-B,
RRg-D,
RRg-G,
WRg1,
WRg2
                                                        RRg4
Figure 1.  Map ofMycobacterium avium strain 104 genome, with positions of transposon insertions that affect
         colony morphotype and/or multi-drug resistance.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


  Mycobacterium avium Complex (MAC) in Drinking Water:  Detection,
                        Distribution,  and  Routes of Exposure

                             Phanida Prommasith1 and Timothy E. Ford2
          Harvard School of Public Health, Boston, MA; 2Montana State University, Bozeman, MT

                                        Presentation Abstract

    The objectives of this project are to:   (1) develop techniques and methods for detecting MAC in both
drinking water and biofilm samples; (2) investigate the presence and distribution of MAC in water distribution
systems in four Massachusetts towns; and (3) examine possible routes of exposure for residential end-users. A
method was developed to detect MAC from drinking water, standard filtering techniques have been modified,
and percent recovery was compared with DAPI direct counts. The most efficient protocol was compared with
selective culture on paraffin slides. Both environmental water samples and MAC-spiked autoclaved water were
used in these tests. Environmental biofilm samples were collected from residential sites and from hot water by-
pass systems.

    Two fluorescent oligonucleotide  probes  have  now been  designed  and  tested.  The probes target the
16sRNA genes of Mycobacterium avium  and M.  intracellulare. PCR-restriction enzyme pattern  analysis
(PRA) of the HSP65 gene also has been used to identify MAC. Identification of species has been confirmed by
comparison with the NCBI-GenBank database. To investigate the presence and distribution of MAC, potable
water samples have been systematically examined from four communities with different water supplies and
distribution characteristics. All samples have been analyzed for pH, temperature, free and total chlorine, alka-
linity,  ammonia-N, nitrite-N, nitrate-N, total iron, and assimilable organic carbon, MAC, and heterotrophic
plate counts.

    In addition, to investigate the possible routes of exposure, water samples have been collected from kitch-
ens and showers in the end-users' homes (cold and  hot water)  and  analyzed for the presence of MAC. Cur-
rently, biofilms are being grown in end-user toilet cisterns. Any residential sites found to have MAC in water
samples have been subject to be more frequent sampling.

    Small doses of antibiotics (nalidixic acid, ethambutol, ofloxacin) and anti-fungus (cyclohexamide) added
to 7H10 media were found to improve the detection of MAC. It is still inconclusive as to whether the paraffin
slide culture is a more sensitive technique. The FISH probe for the M. avium showed a positive hybridization
signal less than 35 percent FA stringency hybridization conditions on isolated cells. The probe forM intracel-
lulare needs to be redesigned and retested to ensure successful hybridization.

    Approximately 20 percent (n=861) of water samples collected to date were positive for Mycobacteria,
with 8.36 percent positive for MAC. The  household results ranged from 0 percent to more than 40 percent
positive for MAC; the distribution system results ranged  from 0 percent to  almost 30 percent positive for
MAC.  Of the MAC positive plates, 11 percent were found to haveM avium (20-450 cfu/L), and 3.5 percent
wereM intracellulare (4-12 cfu/L). No significant differences were detected in the presence of MAC between
kitchen faucets and showerheads,  although numbers of MAC were slightly higher in showerhead samples. Ap-
proximately 100 percent ofM. intracellulare were found in cold water (8-17 °C), whereas mostM avium (66
percent) were found in hot water (35-46 °C).

    The next steps will include: (1) concluding the investigation of the use of paraffin slide cultures; (2) final-
izing FISH protocols to directly hybridize to biofilm samples; (3) improving M. intracellulare probe and pro-
tocols; (4) evaluating biofilms grown in toilet tanks for MAC; and (5) examining relations of MAC coloniza-
tion to water quality parameters.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


         Sensitivity of Three Encephalitozoon Species to Chlorine
            and Chloramine Treatment as Detected by an In Vitro
                                  Microwell Plate Assay

    Cliff H. Johnson1, Marilyn M. Marshall2, Jackie Moffet2, Charles R. Sterling2, Laura A. DeMaria1,
                                        and Gene W. Rice1
       1U.S. Environmental Protection Agency, Cincinnati, OH; 2 University of Arizona, Tucson, AZ

                                        Poster Abstract

    Microsporidia are obligate intracellular parasites that form environmentally resistant, infectious spores.
These parasites are ubiquitous in the environment, infecting members of almost every class of vertebrates and
invertebrates. At least 14 microsporidian species are known to infect humans. Of primary concern are the mi-
crosporidian species that infect the human gastrointestinal tract, Entercytozoon bieneusi and Encephalitozoon
intestinalis. Spores are typically able to survive and maintain their infectivity for weeks. A modified in vitro
cell culture assay was performed on three species of Encephalitozoon (E. intestinalis, E. helium, and E.  cuni-
culi) after exposure to chlorine and chloramine at a concentration of 2 mg/L at 25EC, pH 7, and pH 8, respec-
tively.  Spores were harvested from RK-13 cell monolayers and Percoll purified assayed using an in vitro mi-
crowell plate viability procedure. Ten-fold dilutions of chlorine and chloramine treated spores were inoculated
onto RK-13 cell monolayers grown on 15mm sterile Thermanox coverslips in 24 well plates.  Five coverslips
were inoculated for  each spore dilution. After incubation, the coverslips were fixed with methanol, stained
with Giemsa stain, and examined with a light microscope. The percentage of infectivity was calculated by di-
viding  the number of positive (infected) wells by the total number of wells inoculated. Most probable number
determinations also were determined for each assay. For each treatment  and time period, two replicates of 5
coverslips were performed. Varying log reductions were observed for the three Encephalitozoon species.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


       Inactivation of Aeromonas by Chlorine and Monochloramine

                       Laura A. Boczek, Cliff H. Johnson, and Eugene W. Rice
                        U.S. Environmental Protection Agency, Cincinnati, OH

                                         Poster Abstract

    The Bacterial genus Aeromonas is currently listed on the U.S. EPA's Candidate Contaminant List (CCL).
Resistance to chemical disinfection is an essential aspect regarding all microbial groups listed on the CCL.
This study was designed to determine the inactivation kinetics of Aeromonas spp. for free available chlorine
and monochloramine. Three species, Aeromonas caviae, A. hydrophila, A. veronii, which are known to be as-
sociated with human infections, were studied in pure culture under oxidant demand-free conditions. Free chlo-
rine  experiments were conducted at pH 7 and 8, at 5  °C and 22 °C. Experiments using preformed mono-
chloramine also were conducted at both temperatures and at pH 8. Three media were evaluated for their ability
to recover chlorine-stressed organisms [nutrient agar (Difco), ampicillin dextrose agar (Biolife), and Ryan agar
(oxoid)]. Experiments using free chlorine indicated that the Aeromonas spp. were  readily inactivated under all
conditions studied.  The organisms were inactivated by more than five orders of magnitude within a 1-minute
exposure to free chlorine at both temperatures. Inactivation kinetics were similar for other bacterial organisms,
with greater inactivation occurring at lower pH values and at higher temperatures. For free chlorine, there was
no observable  difference in recovery of chlorine-stressed organisms on the three bacteriological media. Two
wells containing Aeromonas spp.  (approximately 103 CFU/mL) have  been located in a recent survey. Water
from these wells also was used in disinfection experiments.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


  Mycobacterium paratuberculosis and Nontuberculous Mycobacteria
                                      in Potable Water

                                  Stacy Pfotter and Terry C. Covert
            National Exposure Research Laboratory, U.S. Environmental Protection Agency,
                                          Cincinnati, OH

                                         Poster Abstract

    Nontuberculous mycobacteria (NTM) include Mycobacterium species that are not members of the Myco-
bacterium tuberculosis  Complex. Members of the NTM group are important causes of disease in birds and
mammals. Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium  paratuberculosis  are
NTM and members of the Mycobacterium avium Complex (MAC). These organisms are found in a variety of
environments, including soil and water, and are included on the Contaminant Candidate List (CCL). Earlier
exploratory occurrence studies suggest that NTM have widespread occurrence in potable water throughout the
United States. M. paratuberculosis  is the  causative agent for  Johne's disease in  cattle. In addition to well-
documented evidence ofM paratuberculosis as the causative agent of Johne's disease in cattle, there has been
evidence linking M. paratuberculosis with Crohn's disease, a  chronic inflammatory disease of the intestinal
tract in humans. Transmission of M paratuberculosis via water contaminated with cattle feces may be one
route of infection.

    Current NTM research focuses on three areas:  (1) development of an improved cultural method for isola-
tion of NTM in drinking water, (2) development of a rapid polymerase chain reaction (PCR) multiplex method
for detection of MAC organisms in  drinking water, and  (3) development of a molecular method for detection
of M. paratuberculosis in water.

Improved Cultural Method

    Current methods for isolating NTM from environmental  samples  require harsh decontamination tech-
niques to reduce the levels of background organisms often leading to loss of 50-70 percent of the target NTM.
The goal of this research is to develop improved selective method(s) that do not use classical decontamination
procedures. The use of antibiotics, dyes, detergents, and other growth inhibitors are being examined for their
ability to reduce background organisms and permit growth of NTM. A membrane filter  method approach has
been selected.  Screening studies with spiked drinking water samples comparing candidate methods to classical
decontamination techniques have been initiated. Candidate methods that permit better recovery of NTM and
better reduction of background organisms will be tested with additional recovery studies and analyses of drink-
ing water samples.

    Various antibiotics,  dyes, and detergents have been examined using a membrane filter cultural method
approach. Thus far, an  oxidizer  has shown promise for better recovery (80 percent) and reduction of back-
ground organisms than  the standard accepted cultural method. An improved cultural method would lead to
better estimates of the occurrence of NTM, better estimates of the numbers of NTM in  positive samples, and
the possibility of recovering NTM unusually sensitive to decontaminating agents. Future research will  entail
additional NTM recovery studies, followed by comparison studies with the standard cultural approach and the
improved method with distribution samples.

PCR Multiplex Method

    Current methods for detection of MAC organisms in drinking water typically  take from 3 to 8 weeks for
completion of analyses, with additional time for identification of the organisms. The goal of this research is to
develop a rapid PCR multiplex method for detection of M. avium andM intracellulare. Drinking  water sam-
ples (500 mL) are membrane filtered, and the filters are placed in modified 7H9 broth for 7-day enrichment.
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                Research on Microorganisms in Drinking Water Progress Review Workshop
After enrichment, the cells are centrifuged and lysed to harvest the genomic DNA. The DNA is amplified
(PCR) using primers specific for M. avium and M. intracellulare and all Mycobacteria. The PCR product is
visualized by gel electrophoresis. Sixty  samples  (reservoir and  drinking water) have  been analyzed by the
standard culture method and the multiplex PCR method. Nine samples were positive by both methods, seven
were positive only by multiplex PCR, and three were positive only by the cultural method. The use of multi-
plex PCR significantly decreases the time for analyses for these organisms, and is able  to detect MAC organ-
isms not detected by the culture method. The next steps include completion of detection  limit studies and addi-
tional comparison studies with the standard culture technique using drinking water samples.

Method for Detection of M paratuberculosis

    A new project in our laboratory involves the development  of a molecular detection and quantification
method  for M. paratuberculosis (MAP) in water. The method will be an important step in determining the sig-
nificance of exposure to MAP in contaminated water, and may help to establish the link  between contaminated
water and Crohn's disease. Current methods of detection, which include culture-based methods, are inade-
quate. A 16- to 20-week incubation time is required to grow the organism, during which other microorganisms
overgrow  the medium. Harsh decontamination  procedures used  to reduce background organisms also kill a
portion of MAP. This study proposes to develop a rapid molecular method to detect and quantify MAP in envi-
ronmental samples by targeting a genetic molecule specific to MAP. One potential target is the MAP-specific
insertion sequence IS900. The element is found only in MAP, and is present in 14 to 18 copies  per cell. Other
possible targets include  seven recently discovered MAP-specific gene segments. A quantitative PCR-based
method  would significantly reduce detection times from approximately 16 weeks to a few hours.
Figure 1.  Electrophoretic separation of multiplex PCR products obtained from drinking water isolates. Lanes
          contain the following: lanes 1, 2, 3, 4, 5, 7, 8, 9, and 10, M avium isolates; lane 11,M avium posi-
          tive control; lane 12, M. intracellulare positive control,  and lane 6,  cpX174 RF DNA/ Hae III mo-
          lecular weight marker (Gibco-BRL) containing fragments of 1353,  1078, 872, 603, 310, 271, 234,
          194, 118, and 72 base pairs.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


      Detection of Helicobacter pylori Using  a Highly Variable Locus
                    Upstream of the 16S Ribosomal RNA Gene

             M. Shahamat, M.R. Alavi, J.E.M. Watts, K.R. Sowers, D. Maeder, and F. Robb
     Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD

                                         Poster Abstract

    Helicobacter pylori  is a highly successful infectious agent, and it is the principal cause of gastritis and
peptic ulcer disease.  Strong associations also have been found with gastric adenocarcinoma and lymphoma.
Although regional rates of infection vary, H. pylori is still a major public health concern in many parts of the
world. This highly infectious bacterium is able to use multiple routes of transmission to infect a susceptible
host. It is thought to be transmitted by the fecal-oral route but also may be transmitted directly through saliva
or from the environment; however, the precise mode of transmission is not well understood. H. pylori has been
demonstrated to change morphologically (see Figure 1) and enters nonculturable stages of survival in the envi-
ronment  and still can become infective. Currently, it is difficult to elucidate the environmental route of H. py-
lori, as there is  no reliable  method for detecting  and differentiating H.  pylori strains from environmental
sources.

    To overcome culturability limitations, a molecular approach was utilized consisting of designing primers
complementary to  intergenic spicer region (ISR) between the 16S  and 23S to detect H. pylori from water
sources.  In the H. pylori genome, the 16S and 23S rRNA genes are not contiguous (see Figure 2). This study
found a highly conserved region followed by an intergenic spacer variable region upstream of the 16S rRNA
gene. Primers were designed for this conserved region  and to the 5'-end of the 16S rRNA gene to amplify the
variable region in between. Each  strain of H. pylori tested gave a positive amplification by polymerase chain
reaction  (PCR), using the primers mentioned above to amplify the variable region. A number of other species
tested, such as Campylobacter, Escherichia coli, Salmonella, Shigella, Vibrio, and Bacillus species, resulted in
no amplification. Tests performed using laboratory constructed mixtures of different bacterial species gave
only positive results if H. pylori was present. The sensitivity and specificity of this PCR method for direct de-
tection of H. pylori in environmental samples has been determined and found to be optimal for water samples.

    H. pylori strain differentation is possible using this molecular technique, and future studies will involve
terminal  restriction fragment length polymorphism analysis to detect and differentiate strains of H. pylori. This
analysis will provide a rapid and reliable detection tool for H. pylori strains in both clinical and environmental
samples, deepening our understanding of the source of infection.
                   Figure 1. Helicobacter pylori Strain RSB6 in River water for 140 days.
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          Research on Microorganisms in Drinking Water Progress Review Workshop
Typical organization of the ribosomal genes in microorganisms
                             Conserved Region
                                    23S
                                       tS
                   Inter Spaced Region (ISR; var iatole region)
Relative locations of the ribosomai RNA genes in H. pylori genome.
 Genomic map of H. pyiori strain 26695
                                                            23S-5S-1
   16S-1
               16S-2
                                                      23S-5S-2
            Figure 2. Relative location of ribosomal RNA in H. pylori and other bacteria.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


               Using  Real-Time  PCR To Detect Toxigenic Strains
                               of Microcystis aeruginosa

                                          Carrie Moulton
                Office of Water, U.S. Environmental Protection Agency, Cincinnati, OH

                                         Poster Abstract

    Both toxigenic and nontoxigenic strains of Microcystis exist in nature with a patchy distribution. Only
about 50 percent of all Microcystis blooms tested for heptotoxicity are positive by the mouse bioassay. The
goal of this project is to develop the capability to discern between toxic and nontoxic blooms of Microcystis.
Polynucleotide  sequences were used within the 16S ribosomal RNA gene and the mcyA gene in the micro-
cystin synthetase operon to develop primer/probe sets for a multiplex 5'-nuclease polymerase chain reaction
(PCR) assay. Presumably the primer/probe set developed for the mcyA gene will give positive results only with
strains that produce toxins.

    Nineteen polynucleotide sequences for the mcyA  gene and four sequences for the 16S gene were entered
into the Primer Express® software. Primer/probe sets were selected using two criteria:  (1) the lowest probabil-
ity of meeting the designated parameters  by  random chance; and (2) the highest number of M. aeruginosa
strains that contained the selected sequences as found in GenBank for each primer and probe. Eleven different
strains of M. aeruginosa, six of which were previously documented to be toxigenic, were obtained from the
University of Texas Culture Collection and established in BG-11 media. These strains were originally isolated
from samples collected in Canada, Australia,  South Africa, and the United States. PCR products for the 16S
gene found in the presence or absence of NMT products served as a positive control to show that PCR inhibi-
tors were not present and that analytical procedures were not compromised.

    As predicted, only the strains determined to be toxigenic by high performance liquid chromatography and
enzyme-linked immunosorbent assay were positive for detection of the NMT region of the mcyA gene (see
Table 1). In  addition, the NMT region of the mcyA gene has been detected in 18 toxigenic cultures and 2 non-
toxigenic cultures, but not in 17 other nontoxigenic cultures by other investigators. Using real-time PCR to
detect toxigenic strains of M aeruginosa was successful  and indicates the potential for a highly sensitive and
precise assay. Further testing to assess the correlation between the presence of this genetic region and toxi-
genicity of environmental samples is needed.

                                       Table 1. Multiplex PCR.
B2662
LB 2664
B2666
B2667
B2669
B2670
LB 2386
B2661
B2671
B2672
B2676
Negative control
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No

18.43
18.33
23.21
18.27
19.34
17.44
18.15
18.64
17.33
21.91
18.11
Negative
18.88
19.17
23.78
18.4
19.93
17.92
Negative
Negative
Negative
Negative
Negative
Negative
*Cycle threshold, Ct, is the first thermocycle in which there is a
significant increase in fluorochrome emission from the probe.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


   Role of Adaptive  Response in the Kinetics of Mycobacterium avium
                         Inactivation With Monochloramine

                         Ning Tong, Lutgarde Raskin, and Benito J. Marinas
                 Department of Civil and Environmental Engineering,  University of Illinois
                                   at Vrbana-Champaign, Vrbana, IL

                                         Poster Abstract

    Mycobacterium avium is a waterborne opportunistic pathogen commonly detected in drinking water. The
persistence of M. avium in drinking water distribution systems has been associated to its presence in biofilms,
inside which these microorganisms appear to gain protection against disinfectant attack. It is also suspected
that M.  avium detected in tap samples, also containing a measurable level of chlorine residual, survives em-
bedded  inside suspended particles detached from  biofilms. Because chlorine reacts  with biofilm or particle
organic matter, the embedded cells get exposed to lower disinfectant concentrations. Furthermore, exposure to
low concentrations of the disinfectant hydrogen peroxide has been shown to trigger an  adaptive response, re-
ferred to as the SOS response, with both A. hydrophila and Escherichia coli. The SOS response triggers the
synthesis of a number of proteins, some of which  can repair DNA damage induced by the disinfectants. The
main objective of this study is to characterize the effects of disinfectant concentration, temperature, and pH on
the inactivation kinetics of M avium (ATCC 15769) with combined chlorine. The occurrence and impact of a
biochemical adaptive response during  the inactivation of M avium with this disinfectant also has been as-
sessed.  Experiments were performed in batch reactors with the temperature controlled  at target values in the
range of 1-30 °C. The solution pH is maintained constant at target values in the range of 6-10 with phosphate
and borate buffers.  Chlorine concentrations range from 0.01  to 10 mg/L as  C12.

    Resulting inactivation curves were characterized by pseudo-first order kinetics without the occurrence of a
lag phase. A single curve was obtained  for relatively high concentrations (e.g., 5.0 mg/L and 10 mg/L at 20 °C
and pH 8) when plotting the natural logarithm of survival ratio versus CT. This finding confirmed the validity
of the CT concept (a fixed value of the product of the concentration and  contact time resulted in a fixed degree
of inactivation at a given temperature and pH, independently of the disinfectant concentration used) at rela-
tively high concentrations. In contrast,  the inactivation kinetics M. avium at low monochloramine concentra-
tion (e.g., <1 mg/L as C12 at 20 °C and pH 8) was only approximately 25 percent of that observed at high con-
centration at the same temperature and pH. The greater  resistance to inactivation observed at the lower
monochloramine concentration was consistent with the occurrence of an adaptive response similar to that re-
ported for other bacteria. However, additional work at the molecular level is necessary to assess if the adaptive
response mechanism is the SOS response observed with other bacteria.
           The Office of Research and Development's National Center for Environmental Research

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Topic Area 3: Distribution Systems
          and Biofilms

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


  The Effect of Chlorine,  Chloramine, and Mixed Oxidants  on Biofilms
                    in a Simulated Water Distribution System

           Mark C. Meckes, Roy C. Haught, David W. Cmehil, Leslie Wilsong, Janet C. Blannon,
                                       andMano Sivaganesan
          National Risk Management Research Laboratory, U.S. Environmental Protection Agency,
                                           Cincinnati, OH

                                       Presentation Abstract

    Throughout the world there are millions of miles of water distribution pipe lines that provide potable water
for use by individuals and industry. Some of these water distribution systems have been  in service well over
100 years. Treated water moving through a distribution system comes into contact with a  wide range of mate-
rials under a variety of conditions, which can affect water quality. Suspended solids in finished water can settle
out under low flow conditions and can be resuspended as flows increase. Disinfectants  and water additives
react with organic and inorganic materials within the distribution system, producing by-product compounds
that may be undesirable in the water supply. Oxidant resistant microorganisms can colonize; pipe surfaces,
cracks, and crevices produce a complex microenvironment known as "biofilm." These biofilms can be highly
resistant to many disinfection methods and techniques. This resistance to disinfection can extend to the entire
colony of microbes, which can include microbial indicators of contamination such as coliform bacteria.

    The extent of biofilm growth that can occur under conditions of limited nutrients and in the presence of
residual oxidizing agents was evaluated using U.S. EPA's water distribution system simulator (DSS). This
presentation describes the features of the DSS and how it was used to compare the effectiveness of three disin-
fecting agents on system biofilms. Results from this work suggest that chloramine and MIOX® were more
effective than free chlorine in reducing culturable drinking water biofilms within the DSS.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


                    Molecular Characterization of Drinking Water
                                  Microbial  Communities

     Jorge Santo Domingo, Mark C. Meckes, Catherine Kelty, Margaret Williams, Joyce M. Simpson,
                                     and Donald J. Reasoner
      National Risk Management Research Laboratory, Water Supply and Water Resources Division,
     Microbial Contaminant Control Branch, U.S. Environmental Protection Agency, Cincinnati, OH

                                     Presentation Abstract

    The objective of this study was to monitor the impact of chlorination and chloramination treatments on
heterotrophic bacteria (HB) and ammonia-oxidizing  bacteria (AOB) inhabiting a water distribution system
simulator (see Figure 1). HB densities decreased while AOB densities increased when chloramine was added.
AOB densities decreased below detection limits  after the disinfection treatment was switched back to chlorina-
tion. The presence of AOB was confirmed using a group-specific 16S rDNA-PCR method. 16S rDNA  se-
quence analysis showed that most bacterial isolates from feed water, discharge water, and biofilm samples
were a-Proteo-bacteria or (3-Proteobacteria. The latter bacterial groups also were numerically dominant among
the sequences recovered from water and biofilm 16S rDNA clone libraries. The relative frequency of each cul-
turable bacterial group was different for each sample examined. Denaturing gradient gel electrophoresis analy-
sis of total community 16S  rDNA genes showed notable differences between the microbial community struc-
ture of biofilm samples and feed water.  The  results of this study suggest that disinfection treatments could
influence the type of bacterial community inhabiting water distribution systems.
                     Phylogenetic analysis of natural microbial communities
                                 Total DMA from environmental sample
  PCR amplification   ^T

    Amplified rRNA_pr rDNA

Cloning

PCR clone library
                     Sequencing
                                                          L^   Direct cloning

                                                           Random clone library
                                                                         Screening

                                                            rDNA containing clones
                                                                        Sequencing
                               Phylogenetic identification of natural clones

                                   Clones specific primers and probes
                  Hybridization with
                  fluorescent probes
                            In situ detection
                                           PCR Amplification
                                      ^
                             Detection of cloned rDNA sequences
                             in environmental samples
                   Figure 1.  Phylogenetic analysis of natural microbial communities.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


 Phylogenetic Analysis of Prokaryotic and Eukaryotic  Microorganisms
               in a Drinking Water Distribution System Simulator

             Margaret M. Williams1, Mark C. Meckes2, Cathy A Kelty2, Hildred S. Rochon3,
                                   and Jorge W. Santo Domingo
   National Research Council, Cincinnati, OH; U.S. Environmental Protection Agency, Cincinnati, OH;
          3School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA

                                         Poster Abstract

    Within potable water distribution systems, opportunistic pathogens such as Legionella species infect pro-
tozoa, gaining protection from disinfectant residuals. Analyzing the prokaryotic and eukaryotic populations in
distribution system water provides a basis for understanding the interactions between these microorganisms.
Samples were obtained from the feed and discharge water of a ductile iron distribution system simulator that
receives drinking water containing a 0.5  ppm monochloramine  (NH2C1) residual.  Bacteria were isolated on
R2A agar, then identified by polymerase chain reaction amplification of the 16S rRNA gene (rDNA), followed
by sequence analysis. To determine a broader range of microbial populations present in the systems, clone li-
braries of 16S rDNA and 18S rDNA were made from sample water. The majority of the isolates  were closely
related to bacteria belonging to the alpha  proteobacteria, including Sphingomonas, Brevundimonas,  and Cau-
lobacter species. Sequence analysis of the clones obtained from the pipe loop discharge water showed a mix-
ture of alpha and beta proteobacteria, as well as the presence ofNitrospira sp., which are nitrite oxidizers. Us-
ing genus-specific 16S rDNA primers, two Legionella-like species were identified: Tatlockia micdadei and
Legionella-Like  Amoebal Pathogen  1 (LLAP1).  A wide range of eukaryotic microorganisms,  including
dinoflagellates such as Gymnodinium and Peridinium spp., have been identified from clones  obtained using
universal 18S rDNA primers (see Figure  1). Determination of the predominant protozoan species within the
distribution water will allow the identification of possible hosts for Legionella species and other opportunistic
pathogens. Prokaryotic and eukaryotic community analysis is a first step in elucidating the relative activity and
survivability of each group of organisms.
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               Research on Microorganisms in Drinking Water Progress Review Workshop
Figure 1.  Phylogenetic tree demonstrating the relationships among dinoflagellate sequences obtained from
          the feed (L3F samples) and discharge (L3D samples) of the distribution system simulator (DSS).
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


         Identification and  Characterization of Aeromonas Isolates
                    From  Drinking Water Distribution Systems

                              Jennifer Birkenhauer1 andM. Rodgers2
         1Oak Ridge Institute for Science and Education,  U.S. Environmental Protection Agency,
                Cincinnati, OH; 2U.S. Environmental Protection Agency, Cincinnati, OH

                                        Poster Abstract

    Members of the bacterial genus Aeromonas are commonly isolated from both fresh and salt waters world-
wide, and some are believed to cause infections in humans, including gastroenteritis and wound infections.
Currently, aeromonads are on the U.S. EPA's Contaminant Candidate List, and are suspected of contaminating
drinking water distribution systems. Identification of aeromonads  to the species level is difficult as new spe-
cies, taxa, and biogroups continue to be proposed. In this  study,  both metabolic and genomic fingerprinting
identification methods were employed to obtain an understanding  of the occurrence and types of aeromonads
in drinking water distribution systems in the United States.

    Water samples were analyzed from 18 drinking water distribution systems across the United States, eight
of which were found to contain aeromonads. All colonies were isolated from  ADA-V medium and were con-
firmed to be aeromonads as recommended in EPA Method 1605.  Confirmed  isolates, 212 in total, were then
subjected to both a Restriction Fragment Length Polymorphism (RFLP) analysis (Borrell, et al, 1997) and to a
carbon source utilization assay employing the BIOLOG microbial identification system.

    The BIOLOG microbial identification system offers a straightforward approach to identifying environ-
mental microbes. However, we found that only after compiling our own database were we able to gain confi-
dence in the system's ability to correctly identify each isolate. The RFLP analysis, while requiring much more
time and technical skill, was able to give a more consistent identification of each isolate, with the exception to
certain biotypes.

    Based on both the metabolic and genomic fingerprinting of these organisms, we were able to identify sev-
eral different biotypes, including A. hydrophila, A. bestiamm, and A. salmoncida from drinking water distribu-
tion systems.  Because some  of the species that were isolated have been implicated in human disease, the re-
sults from this study indicate  that  a more comprehensive  survey of drinking water utilities is warranted to
determine if aeromonads in drinking water pose a threat to public health.
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                Research on Microorganisms in Drinking Water Progress Review Workshop
                           Pathogenicity of Biofilm  Bacteria

                                            Dennis Lye
     National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH

                                          Poster Abstract

    There is a paucity of information concerning any link between the microorganisms commonly found in
biofilms of drinking water systems (see Table 1) and their impacts on human health. For bacteria, culture-
based techniques detect only a limited number of the total microorganisms associated with biofilms. The pos-
sibility of unknown opportunistic pathogens occurring in potable  water and biofilms within drinking water
systems still exists, but it is  unlikely that pathogenic microorganisms will be found using individual in vivo
culture-based techniques or by screening large numbers of isolates  using the currently available in vitro viru-
lence tests. A combination of molecular-based techniques and animal-exposure studies will provide the infor-
mation necessary  to  fully characterize the pathogenicity of microorganisms  commonly associated with
biofilms.
                 Table 1.  Opportunistic microbial pathogens that could be encountered in
                          biofilms of drinking water systems.
                 BACTERIA:

                 Helicobacter pylori
                 Escherichia coli
                 Mycobacterium avium complex
                 Legionella spp.
                 Pseudomonas aeruginosa
peptic ulcers
gastroenteritis
chronic diarrhea, lung disease
Legionnaires disease
burn infections
                 VIRUSES:

                 Polio
                 Coxsackie
                 Norwalk
                 Hepatitis A
poliomyelitis
upper respiratory
gastroenteritis
infectious hepatitis
                 PROTOZOA:

                 Cryptosporidium
                 Giardia
                 Entamoeba
                 Acanthamoeba
gastroenteritis
gastroenteritis
amoebic dysentery
eve infection
                 FUNGI:

                 Aspergillus
pulmonary disease
           The Office of Research and Development's National Center for Environmental Research

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Topic Area 4:  Cross-Cutting Research
       and Emerging Topics

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


              The Application of Mass Spectrometry to the Study
                                     of Microorganisms

                             Jody A. Shoemaker and Susan T. Glassmeyer
     National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH

                                       Presentation Abstract

    The purpose of this research project is to use state-of-the-art mass spectrometric techniques  such as
electrospray ionization  (ESI) and matrix assisted laser desorption  ionization  (MALDI) mass spectrometry
(MS), to provide "protein mass fingerprinting" and protein sequencing information for microorgansims listed
on the  1998  Contaminant  Candidate List (CCL) that  cause  waterborne disease.  The responsibility of
characterizing and investigating  microorganisms  has traditionally fallen to  microbiologists,  but recent
advances in mass spectrometry have allowed analytical chemists also to enter the realm of microorganisms.

    Protein mass fingerprinting libraries will be developed and evaluated to determine whether MS techniques
can identify protein fingerprints related to the infectivity/viability of selected microorganisms and whether they
can differentiate between species and strains of selected microorganisms. Sequence information for proteins
that are found to be specific or unique to species/strain and infectivity/viability also can be obtained with these
MS techniques.

    This global proteomic project has a number of subtasks for which preliminary results have been obtained
on microorganisms such as  coxsackievirus, Cryptosporidium parvum,  and enterococci. Through the use of
mass  spectrometry, a potential viral biomarker of coxsackievirus has been identified that may indicate whether
the virus is infectious. A unique mass spectral peak was observed in an infectious coxsackievirus, but was not
observed in a noninfectious coxsackievirus. This unique peak may be responsible for viral infectivity, thus, be
a potential biomarker.

    In addition to viruses, initial experiments were performed to determine the ability of MALDI to analyze C.
parvum both in  an intact form, as well as oocysts that have been rendered nonviable. MALDI analysis was
performed on several different harvests of the intact oocysts, as well as the separated cell walls and sporozoites
that make up the oocysts. The  analysis of the oocysts walls was inconclusive due to  lack of discernable mass
spectral peaks, but MALDI analysis of the sporozoites yielded reproducible mass spectra.

    Whole enterococci cell protein profiles were evaluated using MALDI as a tool to identify seven different
enterococci species. Many mass spectral peaks were shared among the different enterococci species; however,
each  species showed unique peaks, primarily in the 6,000 to 7,000 m/z region. When  environmental isolates
were  tested, the  signature peaks were observed in many of the different isolates, suggesting that these peaks
could be used for  species identification. Sequence analysis of the environmental isolates by  16S rDNA con-
firmed the identity of the strains tested, and matched the MALDI identity prediction in  75 percent of the sam-
ples.  The results from this study indicate that the  analysis of whole enterococci cells by MALDI generate
unique protein profiles, which  can be used for the rapid identification of fecal enterococci environmental  iso-
lates.

    Although mass spectrometry currently is not sensitive  enough to detect  single  cells in drinking water, the
basic  proteomic  information obtained with these mass spectrometric techniques can be used to develop more
sensitive and precise microbiological techniques that focus  on these unique proteins in drinking water samples.
These conventional microbiological methods can then be used to gather the occurrence data that will be used to
create better U.S. EPA regulations for protecting humans from microbiological contaminants in U.S. drinking
water supplies.
           The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


                           Cyanobacteria and Their Toxins

                                        Elizabeth D. Hilborn
  Office of Research and Development, National Health and Environmental Effects Research Laboratory,
                  U.S. Environmental Protection Agency, Research Triangle Park, NC

                                      Presentation Abstract

    Cyanobacteria and their toxins are listed as microbial contaminants on the Candidate Contaminant List.
Increasingly, toxic cyanobacterial blooms are being reported in surface fresh water bodies worldwide. It is be-
lieved that both increased occurrence associated with eutrophication  and climatic changes, and increased de-
tection due to improvements in scientific knowledge and methods to detect blooms are contributing  to this
trend. Recent studies suggest that consumers of drinking water derived from surface  sources in the United
States may be exposed episodically to low concentrations of these toxins. However, there is little  documented
information  about potential human health effects associated with exposure to these contaminants at ambient
concentrations. Characteristics of Cyanobacteria and their toxins, and recent U.S. EPA activities will be dis-
cussed.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


    Transport of Chemical and Microbial Contaminants  From  Known
           Wastewater Discharges:   Potential Chemical Indicators
                           of Human Fecal Contamination

     Susan T. Glassmeyer1, Imma Ferrer2, Edward T. Furlong2, Jeffrey D. Cahilf, Steven D. Zaugg2,
             Stephen L. Werner2, Michael T. Meyer3, Dana W. Kolpin4, and David D. Kryak5
      3U.S. Environmental Protection Agency, Cincinnati, OH; 2U.S. Geological Survey, Denver, CO;
               3U.S. Geological Survey, Ocala, FL; 4U.S.  Geological Survey, Iowa City, IA;
                  U.S. Environmental Protection Agency, Research Triangle Park, NC

                                     Presentation Abstract

    The quality of drinking and recreational water is currently ascertained using  indicator bacteria such as Es-
cherichia coli and fecal enterococci. However, the tests to analyze for these bacteria  require 24 to 48 hours to
complete,  and do not discriminate between human and animal  fecal material sources. One solution to these
problems is to use  chemicals that are commonly found in human wastewater as supplementary tracer com-
pounds. The chemicals have the advantage of requiring shorter analysis times (3-4 hours), and a suite of human
specific markers can be selected  that are unique to human  wastewater. For this project, compounds includes
those that are produced and excreted by humans (e.g.,  coprostanol), that are consumed and pass easily through
humans (e.g., pharmaceuticals and caffeine), and that are associated with humans and deposited into the com-
bined graywater/blackwater household septic waste stream  (e.g., surfactants). At 10  wastewater treatment fa-
cilities, a treated effluent sample, as well as surface water samples from upstream and at two successive points
downstream from the facility were collected. This longitudinal sampling scheme was used to determine the
persistence of the target compounds in streams. Compounds that are quickly removed or degraded may not be
persistent enough to serve  as tracers; those that are too recalcitrant would similarly not be suitable as they
would be present after the pathogens have been eliminated.

    To estimate the environmental persistence of pathogens, the water samples were analyzed for E. coli and
fecal enterococci in addition to the suite of chemicals being measured. For chemical analysis, the water sam-
ples were extracted using either solid phase extraction (for the pharmaceuticals) or liquid-liquid extraction (for
the other wastewater contaminants) and were analyzed using  either high-performance  liquid chromatogra-
phy/mass  spectrometry (HPLC/MS; pharmaceuticals) or gas chromatography/mass spectrometry (GC/MS;
other wastewater contaminants).  The concentration of microbial indicators was determined using modified
mTEC (E. coli) or  mEI (enterococci) media. Of the 114 chemical analytes investigated in this project, more
than 80 were found in at least one sample. Although most concentrations were in the range of 0.1 to 1.0 |_ig/L,
in some of the more highly contaminated samples, concentrations were in the range of 5-20 |J.g/L. The concen-
trations of the majority of the chemical compounds present in  the samples  generally  followed the expected
trend: they were either nonexistent or at only trace levels in the  upstream samples, had their maximum values
in the wastewater effluent samples, and then declined in the two downstream samples. However, at most loca-
tions, there were indicator bacteria in the upstream samples, illustrating some of the difficulty in using bacteria
to monitor water quality.

    This work indicates that these human wastewater constituents do have utility  as tracers of human wastewa-
ter discharge. However, until the  behavior of these chemical analytes is evaluated in  a rigorous epidemiologi-
cal study, their true potential as chemical indicators of human fecal contamination will not be determined. To
begin this assessment, samples are currently being  analyzed as part of the National Epidemiological and Envi-
ronmental Assessment of Recreational Water Study, which should determine if there is a correlation between
concentration of any of the chemicals and incidence of illness.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


              High Throughput DMA-Based  Tools To Study Water
                                 Microbial Communities

             Jorge Santo Domingo, Joyce Simpson, Margaret Williams, and Catherine Kelty
      National Risk Management Research Laboratory, Water Supply and Water Resources Division,
     Microbial Contaminant Control Branch, U.S. Environmental Protection Agency, Cincinnati, OH

                                      Presentation Abstract

    The demands for water safe for human consumption and recreational activities have increased rapidly in
recent years due to human exponential growth. The impact of this  growth has affected the performance of
wastewater treatment facilities and changed the biological and chemical stability of watersheds throughout the
nation. One of the most significant challenges facing the Nation is to meet the standards established by regula-
tory agencies, recognizing the shortcomings characteristic of current technologies used to monitor microbi-
ological water quality. One relevant shortcoming is the strict dependence on culturing techniques to determine
the presence and estimate densities of indicator bacteria and microbial pathogens. Culture-based methods tend
to underestimate the densities and diversity of microorganisms because they can only recover a  small number
of organisms. Frequently, accurate identification of microorganisms can take a day or two to several weeks.
Nucleic acid-based approaches can  circumvent many of the  shortcomings of the culture-based  methods.  For
instance, the possibility of rapidly and simultaneously monitoring for the presence of hundreds of microorgan-
isms and genes relevant to public health is now becoming a reality in light of the recent advances in microarray
technology. This presentation will review recent technological developments in nucleic acid research that  can
be used to assess the microbiological quality of water systems (see Figure 1). Examples will be provided to
illustrate the application of molecular tools to: (1) evaluate microbial changes in water quality of fecally  im-
pacted watersheds; and (2) study microbial diversity during treatment, distribution, and storage of drinking
water. The importance of constructing comprehensive molecular databases for water distribution systems  and
watersheds also will be discussed. These databases are necessary to optimize the various methods that will be
used in the years to come by  environmental microbiologists. In summary, we believe that the use of rapid  and
high-throughput methods will result in the development of risk management measures that are based on a
framework of sound science.
                          Figure 1. Microarray for water quality assessment.
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


          Detection of Emerging Microbial Contaminants in Source
             and Finished Drinking Water Using DMA Microarrays

            Timothy M. Straub1, Paul A. Rochelle2, Ricardo DeLeon2, and Darrell P. Chandler3
        Battelle Memorial Institute, Pacific Northwest, Richland, WA;  Metropolitan Water District
          of Southern California, Los Angeles, CA; Argonne National Laboratory, Argonne, IL

                                      Presentation Abstract

    DNA microarrays represent a potentially significant technology and analytical technique for the simulta-
neous detection of multiple pathogens in a single water sample, with the ability to incorporate live/dead dis-
crimination  using messenger RNA expression. The objectives of this project are to  develop  and use DNA
arrays for detecting pathogens in natural, turbid, and processed water supplies. Cryptosporidium parvum, Es-
cherichia coli 0157:H7, and Helicobacterpylori serve as model organisms.

    The genetic sequence differences for certain genes for strains of C. parvum and closely related nonpatho-
genic species are often single nucleotide substitutions  at various  locations within a gene. An array was de-
signed and tested to investigate these single nucleotide substitutions within the hsp70 (70 kilodalton heat shock
protein) gene for two  species of Cryptosporidium. A similar array was constructed to investigate both single
nucleotide substitution and the presence or absence of a virulence gene for H. pylori. For E.  coli 0157:H7,
certain strains lack either or both shiga-like toxin genes. An array was constructed for this pathogen and tested
with various strains of E. coli 0157:H7. For all arrays, polymerase chain reaction (PCR) using fluorescently
labeled primers for each organism was performed and then hybridized to the array. The resulting hybridization
patterns on the array were analyzed to determine if differences between species and strains could be elucidated.

    For C. parvum, differences between two species (C.  hominis and C. parvum) were easily differentiated by
both their pattern of hybridization to the array, and statistical discrimination of the data for single nucleotide
substitutions. Likewise, single nucleotide substitution also was possible  for H. pylori. For this organism, the
difference between strains also was readily apparent by the absence of a virulence factor gene.  For  E. coli
0157:H7, discrimination between strains that  lacked either or both shiga-like toxin genes was confirmed.
Specificity testing with nontarget organisms revealed extremely low false positive rates. Sensitivity of the ar-
rays was dependent, in part,  by the PCR process that was used to generate the fluorescent probes to hybridize
to the array, but it was in the general range of 10 to 100 cells.

    Microarray analysis of waterborne pathogens allows excellent discrimination between strains  and closely
related  species of organisms. In the context of these findings, the arrays can serve to potentially fingerprint
isolates in documented waterborne disease  outbreaks. Also, the presence or absence of virulence factor genes
in certain isolates may render these organisms more or less pathogenic. In this case, microarray analysis may
aid epidemiologists to link mild cases of gastroenteritis due to consumption of contaminated drinking water to
less virulent forms of known waterborne pathogens.

    The true ability of DNA microarrays for detection of any waterborne pathogen using just one assay
method has yet to be realized. This is due,  in part, to generation of labeled probes using PCR.  To realize this
goal, many different PCR primers, to cover known and emerging pathogens, must work within  the same reac-
tion vessel.  This is an extremely difficult task and will require next generation reagents and  bioinformatics
software to design PCR primers that will work in a reaction of this type. Currently, this avenue  is being inves-
tigated  as well as an alternate, where messenger RNA is hybridized directly to the arrays. Using the multi-
plexed PCR approach, a 6-plex PCR has been performed successfully. With the RNA approach  (see Figure 1),
the presence of 12 different gene sequences representing 8 unique genes for an E.  coli 0157:H7 isolate that
lacked its shiga-like toxin 2 gene were detected simultaneously.  The principle advantages of the RNA ap-
proach are the ease with which multiplexing can be achieved and the potential for live/dead discrimination.
           The Office of Research and Development's National Center for Environmental Research

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     Research on Microorganisms in Drinking Water Progress Review Workshop
                              uidA1 OraGreen

                      sit1              lamB
                                       '   .
                     eaeA    stxl    iacZ
                             ,;   ^
                      1IIC     hlyA    uidA3
                    fe> •**' W ':<-:  .     '•''''•  •"'  '••"•
                      rfbE    eaeA   uidA2    Cv3
        Figure 1. Detection of virulence and marker genes for E. coli 0157:H7
                 ATCC 43890 using the direct mRNA hybridization approach.
                 This strain lacks the shiga-like toxin 2  gene, and this is
                 confirmed in this image.
The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


                      Mammalian  Cell Response to Pathogens

                                          Samuel L. Hayes
                        U.S. Environmental Protection Agency, Cincinnati, OH

                                       Presentation Abstract

    The goal of microbiological water testing is to determine whether or not the consumption of and/or expo-
sure to a water sample will lead to health  problems in an exposed population. Water utilities currently rely on
the detection of indicator bacteria as a measure of the  potential presence of pathogenic microorganisms. Al-
though it is possible to directly analyze for the presence of specific pathogens, it is difficult to  design assays
that detect the full range of potential human pathogens and assess the virulence of each pathogen detected. A
better approach might involve measuring the interaction between waterborne microorganisms (i.e., potential
pathogens) and human cells as a measure of exposure assessment.

    Microbial pathogens have been shown elicit host responses resulting in shifts in cell metabolism and pro-
tein synthesis. These shifts are reflected in the messenger RNAs (mRNAs) produced by the responding cell or
tissue. Therefore, it should be possible to determine whether a disease response has occurred by monitoring the
expression of mRNA  molecules. DNA microarrays are one way to monitor changes in mRNAs. The specific
goal of this project is to look at mammalian cellular response to pathogens, specifically assessing gene expres-
sion. The focus will be on pathogens that are known (or suspected) to cause disease from exposure to contami-
nated water. These pathogens typically cause gastrointestinal illness. Thus, experimentation will focus on dis-
ease mechanisms associated with intestinal infections. The experimental design is as follows:

Stage 1:
    •   Establish appropriate animal model for intestinal infection
    •   Establish corresponding animal intestinal cell line
    •   Grow pathogen and prepare inoculum at the desired density
    •   Infect animal  model and cell lines
    •   Analyze mRNAs using species-specific cDNA microarrays
    •   Look for similar mRNA expressions between the animal model and tissue cell line.

Assuming similar gene expression patterns are found between the tissue culture and animal models, move to
Stage 2  testing.

Stage 2:
    •   Establish and infect human intestinal cell line
    •   Analyze mRNAs from human tissue cultures, look for similar patterns as seen in the animal model us-
        ing human cDNA microarray.

    The literature suggests that pathogens elicit characteristic  sets of mRNAs in host tissues. These mRNAs
could be used to define the pathogen present. Alternatively, a common set of mRNAs may be produced in re-
sponse to many different pathogens. In either case, the identification of specific mRNAs in host tissue will in-
dicate significant risk. By establishing a relationship between animal models and tissue culture, the case will
be made that human tissue culture  can serve as a  model for human infection in terms of mRNA expression.
This addresses effects of exposure and identifies disease mechanisms from exposure. An important secondary
goal of the project is to eliminate using animal testing for pathogen exposure research.
           The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


          Effectiveness of UV Irradiation for Pathogen Inactivation
                                      in Surface Waters

                            Karl Linden1, Mark Sobsey2, and Gwy-Am Shin2
     1 Civil and Environmental Engineering, Duke University, Durham, NC; 2Environmental Sciences and
                Engineering, University of North Carolina-Chapel Hill, Chapel Hill, NC

                                          Poster Abstract

    Ultraviolet (UV) irradiation is now recognized as  an  effective and cost-competitive measure to achieve
significant level of inactivation of Cryptosporidium while not producing appreciable level of harmful disinfec-
tion by-products (DBFs) at practical doses. However,  the effectiveness of UV technology against new and
emerging pathogens and uncertainties in application of UV disinfection for unfiltered surface waters still needs
to be assessed before widespread use of this technology as a primary disinfectant in drinking water treatment
processes. The primary objectives of this research project are to evaluate the susceptibility (or resistance) and
repair potential of select Contaminant Candidate List  (CCL) pathogens and indicator microorganisms  to or
after UV disinfection from low- and medium-pressure (LP and MP) UV sources, and to investigate the extent
of microbial  association with particles in unfiltered systems  and the effects of this  particle association and
other water quality parameters on UV disinfection potential.

    Preliminary results indicate that  both LP and MP UV irradiation are very effective against most of the in-
dicator and emerging microorganisms tested. However, some of the indicator microorganisms like coliphage
MS2, bacteriophage PRD-1, and Bacillus subtilis endospores as well as CCL and emerging pathogens like My-
cobacterium  terriae (a substitute  for Mycobacterium avium complex), adenovirus type 2, and  Toxoplasma
gondii oocysts showed relatively high resistance against both UV irradiation. Although the effectiveness of LP
and MP UV appeared to be similar against most of the microorganisms tested, there was some remarkable dif-
ference between these two UV technologies in terms of their effectiveness against adenovirus 2. To determine
the level of particle association and  its effect on UV disinfection, raw surface water samples have been col-
lected from various utilities across the United States, and the waters have been examined for particle associated
coliform and aerobic endospores  using physical particle disruption techniques such as  homogenization and
blending. However, the levels of the indigenous microbes in the raw waters were typically low (< 1,000/100
mL), so  that it was not feasible to assess the degree of particle association in these waters based on those
physical methods.

    Currently, the use of microscopic techniques (nucleic acid staining/probes  along with confocal micros-
copy) are being investigated to determine the level of particle association in those raw waters. Regarding the
development of new assay systems for some of the CCL microorganisms, we have been successful in develop-
ing a new assay system (Long-template [LT] RT-PCR)  for Norwalk virus and a new molecular biology assay
(RT-PCR) for adenovirus 40 or 41,  which are being and  will be used in the current and future  inactivation
study on these viruses by LP and  MP UV. In addition,  a method has been established to perform wavelength
specific studies using a polychromatic UV light source  (MP UV lamps) with a set of UV bandpass filters, and
this setup will be utilized to develop  wavelength effectiveness information for select CCL microorganisms like
adenovirus 2, M.  terriae, and T. gondii  oocysts.  Finally,  protocols have been established to examine repair
phenomenon following UV disinfection in various conditions in real water treatment situations, and these pro-
tocols will be implemented to evaluate the presence and extent of repair after UV irradiation in the select CCL
and emerging microorganisms in the  later phases of this research.
           The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


     Survey of U.S. Public Health  Laboratories:  Microbial Pathogens
                         on the Candidate Contaminant List

                 Elizabeth D. Hilborn1, Michael O. Royster2,  and Doug J. Drabkowski3
            Office of Research and Development, National Health and Environmental Effects
            Research Laboratory, U.S. Environmental Protection Agency, Research Triangle
            Park, NC; 2 Crater Health District, Petersburg, VA; 3Association of Public Health
                                   Laboratories, Washington, DC

                                         Poster Abstract

    During March 1998, the U.S.  EPA published the Candidate Contaminant List (CCL) of drinking water
contaminants; these chemical contaminants and microbial pathogens will be  evaluated by the U.S. EPA for
potential regulation. Microbial pathogens contained in  the list include: Aeromonas hydrophila, adenovirus,
calicivirus, coxsackie virus, echo virus, Helicobacter pylori, Microsporidia spp., and Mycobacterium  avium-
intracellulare complex (MAIC). Because few of these pathogens are reportable and detection methods  among
laboratories may vary, an estimate of the public health burden of illness is needed to prioritize pathogens for
regulatory action. State public health laboratories (SPHL) serve as reference laboratories in many states. If
SPHL are likely to receive requests to detect CCL pathogens in human clinical specimens, they may serve as
future active surveillance sites to help establish population-based estimates of illness with CCL pathogens.

    During early 2000, a survey of SPHLs was performed by the Association of Public Health Laboratories.
The survey goal was to ascertain the number of clinical  specimens submitted, the number of specimens in
which evidence of infection with a CCL pathogen was verified, and analytic methods used to detect evidence
of infection. Each state laboratory director was asked to report fiscal year 1999 (FY 99)  data via a self-com-
pleted questionnaire.

    Forty-seven of 50 (94 percent)  SPHL representatives completed and returned questionnaires. During FY
99, the  number of clinical specimen submissions, percent positivity, and analytic methods varied by CCL
pathogen. Number of submissions ranged from 1,009 for analysis  of calicivirus, to  199,641 for analysis of
MAIC.  Percent positivity ranged from less than 1 percent of specimens examined for evidence of A.  hydro-
phila, coxsackie  virus, and Microsporidia infection, to 40 percent of specimens examined for evidence of
calicivirus infection. Analytic methods used by SPHLs included: culture, immunologic and molecular  assays,
and direct visualization of pathogens. SPHLs solely reported using polymerase chain reaction (PCR) to detect
calicivirus in clinical specimens; this technique resulted in the highest percent detection  (40 percent as com-
pared to < 5 percent for all other pathogens).

    This survey provided information about which CCL pathogens are currently detected at SPHL and analytic
methods used during 1999. SPHL may be useful in active surveillance systems for nontuberculous Mycobacte-
rium  spp., adenovirus, and enteroviral (coxsackie virus, echo virus)  infections.  SPHL are least likely to be
good locations for surveillance ofH. pylori, Microsporidia spp., and calicivirus. The use of PCR to detect evi-
dence of calicivirus in clinical specimens resulted in the highest percent detection of calicivirus among all CCL
pathogens. However, PCR may be underutilized in SPHLs. Increased use of molecular techniques may in-
crease the diagnostic efficiency of CCL pathogens within SPHLs.
           The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


  Comparative Diversity of Fecal Bacteria in Agriculturally Significant
 Animals To  Identify Alternative Targets for Microbial Source Tracking

       Joyce M. Simpson1, Samuel P. Myoda2, Donald J. Reasoner1, and Jorge W. Santo Domingo1
      1 Water Supply and Water Resources Division, National Risk Management Research Laboratory,
      U.S. Environmental Protection Agency, Cincinnati, OH; 2Division of Water Resources, Delaware
             State Department of Natural Resources and Environmental Control, Dover, DE

                                        Poster Abstract

    Animals of agricultural significance contribute a large percentage of fecal pollution to waterways via run-
off contamination. The premise of microbial source tracking is to utilize fecal bacteria to identify target popu-
lations that are directly correlated to specific animal feces, thus permitting identification of contamination
sources and implementation of remediation practices.

    To identify alternative targets for source tracking studies, comparison of fecal bacterial  populations was
performed using  Denaturing Gradient Gel Electrophoresis (DGGE) targeting the V3 region of the 16S rDNA
gene. Fecal populations from individual horses, cattle, swine, sheep, and goats were compared (see Figure 1).

    The greatest diversity was found in the ruminant animal  species. Within the ruminants, between 40 and 51
percent of the bands within the fecal patterns were dominant populations (i.e., occurred in greater than 50 per-
cent of animals tested) and 7 percent were highly dominant (occurred  in greater than 80 percent of animals
tested). Within the non-ruminants, only 14 to 18 percent of the bands were  dominant, and 4 percent were
highly dominant. Eleven bands were common  to all fecal samples, and eight bands were present in ruminants
only. Another eight bands were predominantly found in ruminants, and three bands were predominant in non-
ruminants. No bands specific to non-ruminants  were found in any of the animals tested.

    A comparison using Dice's similarity coefficient and Ward's dendrogram algorithm indicated that fecal
patterns tended to cluster according to digestive physiology (i.e., ruminants clustered with ruminants) rather
than by species.  Non-ruminant species tended to cluster more closely within  species than to each other  and
were not as intermixed as ruminant results.

    Phylogenetic examination of the common  and divergent banding populations should provide information
to determine if there are suitable alternative organisms that may be used to track fecal pollution. Elucidation of
novel organisms  related to fecal contamination would potentially increase the ability to identify sources more
accurately, thereby allowing the appropriate remediation response to be expeditiously selected.
           The Office of Research and Development's National Center for Environmental Research

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           Research on Microorganisms in Drinking Water Progress Review Workshop
  COW    ]  I   GOAT   ]     SHEEP
HORSE  j  [     PIG    ]
             1


                                                                   H
            l-r
Figure 1.  DGGE gel representing typical fecal banding patterns observed for different animal species.
      The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


    Developing Dynamic Infection Transmission  Models for Microbial
                       Risk Assessment (MRA) Applications

        Patricia Murphy, Brenda Boutin, Jeff Swartout, Glenn Rice, Jon Reid, and Michael Broder
            Office of Research and Development, National Center for Environmental Assessment,
                            U.S. Environmental Protection Agency, Edison, NJ

                                         Poster Abstract

    Drinking water MRAs generally are conducted with methods and assumptions that are analogous to exist-
ing chemical risk  assessment methodologies. These assessments  usually consider only a primary environ-
mental transmission route, such as drinking water, where a human host is  exposed and infected solely through
this route. There are, however, several issues unique to infectious diseases that the traditional chemical frame-
work does not address, including:  (1) the potential for secondary transmission (ST), where the infectious agent
is passed directly or indirectly from an infectious human to other susceptible humans; (2) acquired immunity to
the infectious agent, where previous exposures render the human host either completely or partially resistant,
and the duration of that immunity; and (3)  the environmental population dynamics of pathogens, which  are
living organisms.

    To explore the influence of these additional, interdependent factors on traditional MRA approaches,  the
National Center for Environmental Assessment initiated collaborative research with investigators at the Uni-
versity of California-Berkeley (UCB) and the University of Michigan (UM) to develop and apply infection
transmission models for waterborne pathogen exposures. The academic research teams extended existing  ap-
proaches for compartmental modeling of dynamic infection transmission systems to include infection routes
appropriate for enteric waterborne pathogens, that is, exogenous sources, human-human contact, and human-
water-human pathogen  circulation. Using data from the scientific literature, computer simulation approaches
were used to study how model output changed in relation to  alternative assumptions for the studied exposure
scenarios, alternative values for the input variables, and alternative analytical forms of the model, that is,  de-
terministic or stochastic.

    This project has demonstrated that existing infection transmission models can be extended and modified to
integrate diverse and complex information on host, agent, and environmental characteristics that affect patho-
gen exposure and risk. Results show that ignoring or mis-specifying ST effects in the context of MRAs leads
to mis-characterization  of individual- and population-level risks and mis-estimation of the health benefits at-
tributable to different drinking water treatment interventions.

    Existing MRA methodologies for estimating health risks from waterborne exposure  to pathogens with a
significant potential for human-human transmission require conceptual and analytical modifications to accu-
rately capture host-agent-environment interdependencies that determine human exposure and risk. The final
report from this research is under development. It will provide the focus for an upcoming expert workshop that
will develop specific recommendations on when and how ST effects should be modeled and incorporated into
assessments and will form part of the basis for a 2008 EPA guidance document on MRA tools.
           The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


           Virulence  Factors of Aeromonas: A Molecular Genetic
                                      Characterization

                                    Key a Sen and Mark Rodgers
           Office of Ground Water and Drinking Water, U.S. Environmental Protection Agency,
                                          Cincinnati, OH

                                          Poster Abstract

    Surveys of finished drinking water from eight small systems (2001)  and eight large systems (2000) con-
ducted by the U.S. EPA, revealed that 7 out of 16 water utilities encompassing several states (NY, KY, IA,
OH) were  contaminated with Aeromonas species. Altogether 205 organisms were isolated by EPA Method
1605. The  goal of this project was to determine whether the Aeromonas  species isolated from these drinking
water utilities had the potential to be pathogenic.

    A molecular genetic  approach was chosen. Published literature was searched to identify the genes that
played a role in the pathogenesis of the organism. Only those genes were selected that definitively played a
role in the  virulence of the species when tested in animal models or cell cultures. The isolates were tested for
the virulence factors elastase, lipase, flagella A and B genes, the cytotoxic enterotoxin, Act (achytoen), and the
cytotonic enterotoxins, Alt and Ast. Oligonucleotide primers were developed against these genes, and when
used in the polymerase chain reaction (PCR), a portion of the gene was  amplified in the positive control. A
positive  control was an Aeromonas species known to  have the virulence gene being tested. PCR was per-
formed in three duplex assays with the 205 isolates using the  following primer sets together: elastase (ahyB)
and lipase (pla); fla and alt; act and ast.

    Preliminary findings showed that pla was present in 86 percent, act in 69 percent, fla in 55 percent, ahyB
in 40 percent, and alt and ast in 45 percent and 35 percent of the isolates, respectively. Only one isolate had all
six virulence genes. Multiple  species were isolated from most of the utilities. Different  combinations of viru-
lence factors also were observed, sometimes even in different strains of the same species. However, a domi-
nant strain having the same set of virulence factors was usually isolated from different rounds of sampling
from a single tap.

    These  results  suggest the  importance of examining  as many Aeromonas isolates  as possible from a water
sample, as  within the same species the occurrence of certain virulence factors may vary. The results also sug-
gest that the Aeromonas strains isolated from water utilities have the potential to be pathogenic. However, ad-
ditional virulence factors, which have not yet been identified or characterized, may be needed to cause actual
disease.

    Isolates having different combinations of virulence factors will be tested in animal models to determine
whether there are  one or more combinations of virulence factors that are necessary for establishing diarrhea in
the models. Aeromonas isolates from the UCMR survey of finished drinking water, which is being conducted
in 2003,  also will be tested for the above virulence factor genes.
           The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


       Effects of pH and Temperature on the Kinetics of Aeromonas
                hydrophila Inactivation With Combined Chlorine

                          Kwanrawee Sirikanchana and Benito J. Marinas
               Department of Civil and Environmental Engineering, University of Illinois
                                 at Vrbana-Champaign, Vrbana, IL

                                         Poster Abstract

    Aeromonas hydrophila is a waterborne  opportunistic pathogen commonly  detected in  natural  water
sources (e.g., surface water and groundwater) and also in drinking water, even in the presence of detectable
levels of chlorine residual. There is a need to elucidate the mechanisms responsible for the survival of A. hy-
drophila during and/or after the disinfection process. Previous studies have shown that the inactivation kinetics
of A. hydrophila with monochloramine is independent of monochloramine concentration in the range of 0.01 -
10 mg/L as C12 at constant pH  and constant temperature. However, water quality parameters including pH and
temperature could affect the inactivation kinetics of A. hydrophila. The main objective of this study is to char-
acterize the effects of pH and temperature on the inactivation kinetics of A. hydrophila (ATCC 7966) with
combined chlorine. Experiments are performed in batch reactors with the temperature controlled at target val-
ues in the range of 1-30 °C with a recirculating water bath. The solution pH is maintained constant at target
values  in the range of 6-10 with phosphate and borate buffers. Disinfectant concentrations range from 0.01 to
10 mg/L.

    The kinetics of A.  hydrophila inactivation with monochloramine under all conditions investigated was
characterized by an initial lag  phase followed by pseudo-first order inactivation. The temperature effect was
found to be consistent with Arrhenius law at each pH. Experiments performed at pH 6, 8, and  10 for each tem-
perature revealed strong pH dependence. As the pH decreased from pH 10 to 6, the inactivation kinetics was
faster due to both a shorter lag-phase and a faster rate of post-lag phase inactivation. In general,  the concentra-
tion of monochloramine used did not affect the kinetics of A. hydrophila inactivation with this disinfectant,
thereby confirming the validity of the CT concept (a given value of the product of disinfectant concentration
and contact time resulted in a given degree of inactivation independently of the disinfectant concentration
used) at each pH and temperature tested for the concentration range investigated.
           The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


                     The U.S. Environmental Protection Agency's
              Research on Microorganisms in Drinking Water Workshop

                             Marriott Kingsgate Conference Hotel
                                     151  Goodman Drive
                                    Cincinnati, OH  45219

                                      August 5-7,  2003

                                         AGENDA

Tuesday, August 5, 2003

10:00 - 10:10 a.m.     Welcome
                     Hugh McKinnon, Director, National Risk Management Research Laboratory

10:10 - 10:25 a.m.     Introductory Remarks
                     J. Paul Gilman, Assistant Administrator and Agency Science Advisor
                     Office of Research and Development

10:25 - 10:50 a.m.     Overview of the U.S. EPA's Drinking Water Research Program
                     Fred Hauchman, Drinking Water National Program Manager, NHEERL

10:50 — 11:15 a.m.     Overview Presentation From the U.S. EPA's Office of Ground Water and Drinking
                     Water
                     Gregory Carroll, Chief, Technical Support Center, OW/OGWDW

11:15 - 11:35 a.m.     Overview of Water Security Research and Technical Support Activities
                     Jonathan Herrmann, Water Security Team Leader, and Hiba Shukairy, Technical
                     Support Center, OW/OGWDW

11:35 — 12:00 noon    Overview of Regional  Concerns for Microorganisms and Drinking Water
                     Bruce Macler, EPA Region 9 (San Francisco)

12:00-1:00 p.m.      Lunch

1:00 - 4:15 p.m.       Topic Area 1:  Research Supporting Office of Water's Ground
                     Water/Source Water  Regulatory Activities
                     Moderators: Stig Regli, OW/OGWDW, and Pat Murphy, NCEA

                     1:00 - 1:30 p.m.    Safe Drinking Water Act (SDWA) Requirements and Microbial
                                       Research Needs (Surface Water, Ground Water, and Distribu-
                                       tion Systems)
                                       Presented by Stig Regli, OW/OGWDW

                     1:30 — 1:50 p.m.    Microbial Dose-Response Modeling:  A Predictive Bayesian
                                       Approach (EIMS #54468)
                                       Presented by Jeff Swartout, NCEA

                     1:50 — 2:10 p.m.    The Use of Randomized Trials of In-Home Drinking Water
                                       Treatment To Study Endemic Water Borne Disease
                                       Presented by Timothy J. Wade, NHEERL
          The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
2:10-2:30 p.m.       Break

                      2:30 - 2:50 p.m.    Screening Models To Predict Probability of Contamination by
                                         Pathogenic Viruses to Drinking Water Aquifers
                                         Presented by Bart Faulkner, NRMRL

                      2:50 — 3:10 p.m.    Integrated Approach for the Control of Cryptosporidiumpar-
                                         vum Oocysts and Disinfection By-Products in Drinking Water
                                         Treated With Ozone and Chloramines
                                         Presented by Benito Marinas, University of Illinois at Urbana-
                                         Champaign

                      3:10 — 3:30 p.m.    Prevalence and Distribution of Genotypes of Cryptosporidium
                                         parvum in United States Feedlot Cattle
                                         Presented by Robert Atwill, University of California-Davis

                      3:30 - 4:15 p.m.    Panel Discussion

4:15-5:45 p.m.       Poster Session I
                      All posters will be set up during both Poster Sessions with one-half of the posters
                      staffed at each session.

5:45 p.m.              Adjournment

Wednesday, August 6, 2003

8:15 - 5:40 p.m.       Topic Area 2: Research Supporting Office of Water's Contaminant Candidate
                      List  (CCL)
                      Moderator:  Cynthia Nolt-Helms, NCER

                      8:15 — 8:50 a.m.    CCL and National Drinking Water Advisory Council
                                         (NDWAC) Process
                                         Presented by Tom Carpenter, OW/OGWDW

                      8:50 - 9:15 a.m.    The Roles of Pathogen Risk Assessment in the Contaminant
                                         Candidate List Process (EIMS #22389)
                                         Presented by Glenn Rice, NCEA

                      9:15 - 9:40 a.m.    Overview:  CCL Pathogens Research at NRMRL
                                         Presented by Don Reasoner, NRMRL

9:40-10:00 a.m.      Break

10:00-11:50 a.m.      Topic Area 2.1:  CCL Protozoa
                      Moderators: Carrie Moulton, OW/OGWDW, and Alan Lindquist, NERL

                      10:00 - 10:20 a.m.  Detection of Cyclospora cayetanensis and Microsporidial
                                         Species Using  Quantitative Fluorogenic 5' Nuclease PCR
                                         Assays (EIMS #56083)
                                         Presented by Frank Schaefer, NERL
          The Office of Research and Development's National Center for Environmental Research

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                Research on Microorganisms in Drinking Water Progress Review Workshop
                      10:20-10:40 a.m.  Development of Detection and Viability Methods for
                                         Wateborne Microsporidia Species Known to Infect Humans
                                         Presented by Rebecca Hoffman, University of Wisconsin at
                                         Madison

                      10:40 — 11:00 a.m.  Development and Evaluation of Procedures for Detection of
                                         Infectious Microsporidia in Source Waters
                                         Presented by Paul Rochelle, Metropolitan Water District of
                                         Southern California

                      11:00 - 11:20 a.m.  Development and Evaluation of Methods for the Concentration,
                                         Separation, Detection, and Viability/Infectivity of Three
                                         Protozoa From Large Volumes of Water
                                         Presented by Saul Tzipori, Tufts University

                      11:20 - 11:50 a.m.  Panel Discussion

11:50-12:50 p.m.     Lunch

12:50 - 2:20 p.m.      Poster Session II
                      All posters will be set up during both Poster Sessions with one-half of the posters
                      staffed at each session.

2:20-3:50 p.m.       Topic Area 2.2:  CCL Viruses
                      Moderators: Robin Oshiro, OW/OST, and Betsy Hilborn, NHEERL

                      2:20 - 2:40 p.m.    Use of PCR-Based Methods for Virus Occurrence Studies
                                         (EIMS #56084)
                                         Presented by Shay Fout, NERL

                      2:40 - 3:00 p.m.    Norwalk Virus Dose Response and Host Susceptibility
                                         Presented by Peter Teunis, National Institute of Public Health
                                         and Environment,  The Netherlands

                      3:00 - 3:20 p.m.    Development of a Rapid, Quantitative Method for the Detection
                                         of Infective Coxsackie and Echo Viruses in Drinking Water
                                         Presented by Marylynn Yates, University of California-
                                         Riverside

                      3:20 - 3:50 p.m.    Panel Discussion

3:50-4:10 p.m.       Break

4:10-5:40 p.m.       Topic Area 2.3:  CCL Bacteria
                      Moderators: Jim Sinclair, OW/TSC, and Don Reasoner, NRMRL

                      4:10 — 4:30 p.m.    Disinfection of Helicobacterpylori and Aeromonas Species
                                         Presented by Don  Reasoner, NRMRL

                      4:30 - 4:50 p.m.    Genomic and Physiological Diversity ofMycobacterium avium
                                         Complex
                                         Presented by Gerard Cangelosi, Seattle Biomedical Research
                                         Institute
           The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
                      4:50 — 5:10 p.m.    Mycobacterium avium Complex (MAC) in Drinking Water:
                                        Detection, Distribution, and Routes of Exposure
                                        Presented by Phanida Prommasith, Harvard School of Public
                                        Health

                      5:10-5:40 p.m.    Panel Discussion

5:40 p.m.              Adjournment

Thursday, August 7, 2003

8:00-9:00 a.m.        Topic Area 3: Distribution Systems and Biofilms
                      Moderators: Lisa Almodovar, OW/OST, and Mark Meckes, NRMRL

                      8:00 — 8:20 a.m.    The Effect of Chlorine, Chloramine, and Mixed Oxidants on
                                        Biofilms in a Simulated Water Distribution System
                                        Presented by Mark Meckes, NRMRL

                      8:20 - 8:40 a.m.    Molecular Characterization of Drinking Water Microbial
                                        Communities
                                        Presented by Jorge Santo Domingo, NRMRL

                      8:40 - 9:00 a.m.    Panel Discussion

9:00-9:20 a.m.        Break

9:20 - 12:30 p.m.       Topic Area 4: Cross-Cutting Research and  Emerging Topics
                      Moderators: Keya Sen, OGWDW/TSC, and Rebecca Calderon, NHEERL

                      9:20 - 9:40 a.m.    The Application of Mass  Spectrometry to the Study of
                                        Microorganisms (EIMS #18338)
                                        Presented by Jody Shoemaker, NERL

                      9:40 - 10:00 a.m.   Cyanobacteria and Their  Toxins (EIMS #54617)
                                        Presented by Elizabeth Hilborn, NHEERL

                      10:00 - 10:20 a.m.  Transport of Chemical and Microbial Contaminants From
                                        Known Wastewater Discharges: Potential Chemical Indicators
                                        of Human Fecal Contamination (EIMS #18337)
                                        Presented by Susan Glassmeyer, NERL

                      10:20 - 10:40 a.m.  High Throughput DNA-Based Tools To Study Water Microbial
                                        Communities
                                        Presented by Jorge Santo Domingo, NRMRL

10:40 - 11:00 a.m.      Break

                      11:00 — 11:20 a.m.  Detection of Emerging Microbial Contaminants in Source
                                        and Finished Drinking Water Using DNA Microarrays
                                        Presented by Timothy Straub, Pacific Northwest National
                                        Laboratory
          The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop


                      11:20 — 11:40 a.m. Mammalian Cell Response to Pathogens
                                       Presented by Sam Hayes, NRMRL

                      11:40-12:30 p.m. Panel Discussion

12:30 p.m.            Adjournment of Public Workshop

12:20-1:30 p.m.       Lunch

1:30 - 4:00 p.m.        EPA-Only Discussion Session

4:00 p.m.              Adjournment of EPA-Only Discussion Session
                           U.S. Environmental Protection Agency
                                Organization Abbreviations
ORD, Office of Research and Development

    ORD Laboratories and Centers:

       NHEERL - National Health and Environmental Effects Laboratory
       NERL - National Exposure Research Laboratory
       NCEA - National Center for Environmental Assessment
       NRMRL - National Risk Management Research Laboratory
       NCER - National Center for Environmental Research

OW. Office of Water

    OW Offices:

       OGWDW - Office of Ground Water and Drinking Water
       OST - Office of Science and Technology
       TSC - Technical Support Center
          The Office of Research and Development's National Center for Environmental Research

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_ Research on Microorganisms in Drinking Water Progress Review Workshop _


                     The U.S. Environmental Protection Agency's
              Research on Microorganisms in Drinking Water Workshop

                             Marriott Kingsgate Conference Hotel
                                     151 Goodman Drive
                                    Cincinnati, OH  45219

                                      August 5-7, 2003

                            POSTER TITLES AND SESSIONS

                     Poster Session I: Tuesday, August 5, 4: 15 - 5:45 p.m.
                     Poster Session II: Wednesday, August 6, 12:50 - 2:20 p.m.
                     All posters will be set up during both poster sessions.

Poster Session     Topic Area 1: Research Supporting Office of Water's Ground Water/Source Water
                  Regulatory Activities

       I          Microbial Drinking Water Contaminants: Endemic and Epidemic Waterborne
                  Gastrointestinal Disease Risks in the United States
                  Presented by Rebecca L.  Calderon, NHEERL

       II         Evaluating Microbial Indicators and Health Risks Associated With Bank Filtration
                  Presented by Twila Kunde, Lovelace Clinic Foundation

       I          A Prospective Epidemiological Study of Gastrointestinal Health Effects Associated With
                  Consumption of Conventionally Treated Groundwater
                  Presented by Stuart Hooper, Emory University

       II         Using Neural Networks To Create New Indices and Classification Schemes
                  Presented by Gail Brion, University of Kentucky

                  Topic Area 2: Research Supporting Office of Water's Contaminant Candidate List
                  Topic Area 2.2: CCL Viruses

       II         Dose-Response Assessments for NLV and Coxsackievirus in Drinking Water (EIMS
                  #22389)
                  Presented by Brenda Boutin, NCEA

       I          Methods Used To Analyze a Norovirus Outbreak (EIMS #56084)
                  Presented by Jennifer Cashdollar, NERL

       II         Development of a Molecular Method To Identify Astrovirus in Water (EIMS #56080)
                  Presented by Ann C. Grimm, NERL

       I          Detection and Occurrence of Human Caliciviruses in Drinking Water
                  Presented by Gwy-Am Shin, University of North Carolina at Chapel Hill
          The Office of Research and Development's National Center for Environmental Research

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        Research on Microorganisms in Drinking Water Progress Review Workshop
           Topic Area 2.3:  CCL Bacteria

I          Sensitivity of Three Encephalitozoon Species to Chlorine and Chloramine Treatment as
           Detected by an In Vitro Microwell Plate Assay
           Presented by Cliff Johnson, NRMRL

II          Inactivation ofAeromonas by Chlorine and Monochloramine
           Presented by L.A. DeMaria (Laura Boczek), NRMRL

I          Mycobacterium paratuberculosis and Nontuberculous Mycobacteria in Potable Water
           (EIMS #18289 & #18341)
           Presented by Terry Covert, NERL, and Stacy Pfaller, NERL

II          Detection of Helicobacter pylori Using a Highly Variable Locus Upstream of the 16S
           Ribosomal RNA  Gene
           Presented by Manoucher Shahamat, University of Maryland

I          Using Real-Time PCR To Detect Toxigenic Strains ofMicrocystis aeruginosa
           Presented by Carrie Moulton, Technical Support Center, OW/OGWDW

II          Role of Adaptive Response in the Kinetics of Mycobacterium avium Inactivation With
           Monochloramine
           Presented by Benito Marinas, University of Illinois at Urbana-Champaign

           Topic Area 3: Distribution Systems and Biofilms

II          Phylogenetic Analysis of Prokaryotic and Eukaryotic Microorganisms in a Drinking
           Water Distribution System Simulator
           Presented by Margaret M. Williams, NRMRL

I          Identification and Characterization ofAeromonas Isolates From Drinking Water Distribu-
           tion Systems
           Presented by Jennifer Birkenhauer, NERL

II          Pathogenicity of Biofilm Bacteria (EIMS #18286)
           Presented by Dennis Lye, NERL

           Topic Area 4: Cross-Cutting Research and Emerging Topics

II          Effectiveness of UV Irradiation for Pathogen Inactivation in Surface Waters
           Presented by Gwy-Am Shin, University of North Carolina at  Chapel Hill

I          Survey of U.S. Public Health Laboratories:  Microbial Pathogens on the Candidate
           Contaminant List (EIMS #54616)
           Presented by Elizabeth D. Hilborn, NHEERL

II          Comparative Diversity of Fecal Bacteria in  Agriculturally Significant Animals To
           Identify Alternative Targets for Microbial Source Tracking
           Presented by Joyce M. Simpson, NRMRL

I          Developing Dynamic Infection Transmission Models for Microbial Risk Assessment
           (MRA) Applications (EIMS #18473)
           Presented by Pat Murphy, NCEA
   The Office of Research and Development's National Center for Environmental Research

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        Research on Microorganisms in Drinking Water Progress Review Workshop
II          Virulence Factors ofAeromonas: A Molecular Genetic Characterization
           Presented by Keya Sen, Technical Support Center, Office of Ground Water and Drinking
           Water

I          Effects of pH and Temperature on the Kinetics ofAeromonas hydrophila Inactivation
           With Combined Chlorine
           Presented by Benito Marinas, University of Illinois at Urbana-Champaign
   The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
                 The U.S. Environmental Protection Agency's
        Research on Microorganisms  in  Drinking Water Workshop

                           Marriott Kingsgate Conference Hotel
                                   151 Goodman Drive
                                  Cincinnati, OH  45219

                                    August 5-7, 2003

                                    Participants List
Noreen Adcock
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Division
  (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7724
Fax: (513)569-7328
E-mail: adcock.noreen@epa.gov

Lisa Almodovar
U.S. Environmental Protection Agency
Office of Water
Ariel Rios Building (4304T)
1200 Pennsylvania Avenue, NW
Washington, DC  20460
Telephone: (202) 566-1085
Fax: (202)566-1139
E-mail: almodovar.lisa@epa.gov

Rob Atwill
University of California, Davis
Department of Population Health
  and Reproduction
School of Veterinary Medicine
18830 Road 112
Tulare, CA 93274
Telephone: (559)688-1731
Fax: (559)686-4231
E-mail: ratwill@vmtrc.ucdavis.edu
Tom Behymer
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-564)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7392
Fax:  (513)569-7757
E-mail: behymer.thomas@epa.gov

Jennifer Birkenhauer
U.S. Environmental Protection Agency
Oak Ridge Institute for Science and Education
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7012
Fax:  (513)569-7191
E-mail: birkenhauer.jennifer@epa.gov

Ben Blaney
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
  Laboratory (MS-235)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7852
Fax:  (513)569-7680
E-mail: blaney.ben@epa.gov
          The Office of Research and Development's National Center for Environmental Research

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                Research on Microorganisms in Drinking Water Progress Review Workshop
Laura Boczek
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7282
Fax: (513)569-7328
E-mail: boczek.laura@epa.gov

Brenda Boutin
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
  Assessment (MS-190)
26 W Martin Luther King Drive
Cincinnati, OH 45230
Telephone:  (513)569-7532
Fax: (513)569-7916
E-mail: boutin.brenda@epa.gov

Susan Boutros
Environmental Associates Limited
24 Oak Brook Drive
Ithaca, NY  14850
Telephone:  (607) 272-8902
Fax: (607)256-7092
E-mail: susanboutros@eal-labs.com
Nichole Brinkman
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-320)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7315
E-mail:  brinkman.nichole@epa.gov

Gail Brion
University of Kentucky
Department of Civil Engineering
161 Raymond Building
Lexington, KY 40506-0281
Telephone:  (859) 257-4467
Fax: (859)257-4404
E-mail:  gbrion@engr.uky.edu
Rebecca Calderon
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental
  Effects Research Laboratory
Human Studies Division (MS-58C)
Research Triangle Park, NC 27516
Telephone: (919) 966-6200
Fax: (919)966-6212
E-mail:  calderon.rebecca@epa.gov

Gerard Cangelosi
Seattle Biomedical Research Institute
4 Nickerson Street
Seattle, WA  98117
Telephone: (206) 284-8846
Fax: (206)284-0313
E-mail:  gcang@sbri.org

Tom Carpenter
U.S. Environmental Protection Agency
Office of Ground Water and Drinking
  Water
Ariel Rios Building (4607M)
1200 Pennsylvania Avenue, NW
Washington, DC  20460
Telephone: (202) 564-4885
Fax: (202)564-3760
E-mail:  carpenter.thomas@epa.gov

Gregory Carroll
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7948
Fax: (513)569-7191
E-mail:  carroll.gregory@epa.gov

Jennifer Cashdollar
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Microbiological and Chemical Exposure
  Assessment Research Division
Biohazard Assessment Research Branch (MS-320)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7142
Fax: (513)569-7117
E-mail:  cashdollar.jennifer@epa.gov
          The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
Hongtu Chen
Harvard Medical School
Harvard School of Public Health
Building I, Room G.29
665 Huntington Avenue
Boston, MA 02115
Telephone:  (617) 432-0738
Fax: (617)432-3349
E-mail:  hongtuchen@hotmail.com

John Cicmanec
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
  Laboratory (MS-G75)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7481
Fax: (513)569-7585
E-mail:  cicmanec.john@epa.gov

Terry Covert
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-314)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7318
Fax: (513)487-2512
E-mail:  covert.terry@epa.gov

Armah de la Cruz
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-314)
26 W Martin Luther King Drive
Cincinnati, OH 45268-1314
Telephone:  (513)569-7224
Fax: (513)569-7170
E-mail:  delacruz.armah@epa.gov

Clyde Dempsey
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
  Laboratory (MS-235)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7842
E-mail:  dempsey.clyde@epa.gov
Jorge Santo Domingo
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7085
Fax: (513)569-7328
E-mail: santodomingo.jorge@epa.gov

Vivian Doyle
U.S. Environmental Protection Agency
Environmental Accountability Division
Region 4
61 Forsyth Street, SW
Atlanta, GA  30303
Telephone:  (404) 562-9942
Fax: (404)562-9439
E-mail: doyle.vivian@epa.gov

David Dziewulski
New York State Department of Health
Bureau of Water Supply Protection
Flanigan Square, Room 400
547 River Street
Troy, NY 12180
Telephone:  (518)402-7650
Fax: (518)402-7659
E-mail: dmdl4@health.state.ny.us

Bart Faulkner
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Ecosystem and Subsurface Protection Branch
919 Kerr Research Drive
Ada, OK  74820
Telephone:  (580) 436-8530
Fax: (580)436-8703
E-mail: faulkner.bart@epa.gov

Mary Ann Feige
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7944
Fax: (513)569-7191
E-mail: feige.maryann@epa.gov
          The Office of Research and Development's National Center for Environmental Research

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                Research on Microorganisms in Drinking Water Progress Review Workshop
Shay Fout
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Microbiological and Chemical Exposure
  Assessment Research Division
Biohazard Assessment Research Branch (MS-320)
26 W Martin Luther King Drive
Cincinnati, OH  45268-1320
Telephone:  (513)569-7387
Fax: (513)569-7117
E-mail: fout.shay@epa.gov

Christy Frietch
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Treatment Technology Evaluation Branch
  (MS-B-24)
26 W Martin Luther King Drive
Cincinnati, OH  45268
Telephone:  (513)569-7001
Fax: (513)569-7172
E-mail: frietch.christy@epa.gov

Paul Gilman
U.S. Environmental Protection Agency
Office of Research and Development
Ariel Rios Building (8101R)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone:  (202) 564-6620
Fax: (202)565-2910
E-mail: gilman.paul@epa.gov

Susan Glassmeyer
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Microbiological and Chemical Exposure
  Assessment Research Division (MS-564)
26 W Martin Luther King Drive
Cincinnati, OH  45268
Telephone:  (513)569-7526
Fax: (513)569-7757
E-mail: glassmeyer.susan@epa.gov
Ann Grimm
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-320)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7397
Fax: (513)569-7117
E-mail: grimm.ann@epa.gov

Sally Gutierrez
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
  (MS-689)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7118
Fax: (513)569-7658
E-mail: gutierrez.sally@epa.gov

Jafrul Hasan
U.S. Environmental Protection Agency
Office of Water
Office of Science and Technology
Health and Ecological Criteria Division
Ariel Rios Building (4304T)
1200 Pennsylvania Avenue, NW, Room 7233Q
Washington, DC  20460
Telephone:  (202) 566-1322
Fax: (202)566-1140
E-mail: hasan.jafrul@epa.gov

Fred Hauchman
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects
  Research Laboratory (MS-B105-01)
Research Triangle Park, NC  27711
Telephone:  (919) 541-3893
Fax: (919)685-3247
E-mail: hauchman.fred@epa.gov
           The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
Richard Haugland
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Microbiological and Chemical Exposure
  Assessment Research Division
Microbial Exposure Research Branch  (MS-314)
26 W Martin Luther King Drive
Cincinnati, OH  45268
Telephone:  (513)569-7135
Fax: (513)487-2512
E-mail: haugland.rich@epa.gov

Sam Hayes
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
  Laboratory (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH  45268
Telephone:  (513)569-7514
Fax: (513)569-7328
E-mail: hayes.sam@epa.gov

Roland Hemmett
U.S. Environmental Protection Agency
Region 2 (MS-100)
8790 Woodbridge Avenue
Edison, NJ  08837
Telephone:  (732) 321-6754
E-mail: hemmett.roland@epa.gov

Jonathan Herrmann
U.S. Environmental Protection Agency
National Homeland Security
  Research Center (MS-163)
26 W Martin Luther King Drive
Cincinnati, OH  45268
Telephone:  (513)569-7839
Fax: (513)487-2555
E-mail: herrmann.jonathan@epa.gov

Elizabeth Hilborn
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects
  Research Laboratory (MS-58C)
Research Triangle Park, NC 27711
Telephone:  (919) 966-0658
Fax: (919)966-0655
E-mail: hilborn.e@epa.gov
Rebecca Hoffman
Wisconsin State Laboratory of Hygiene
Environmental Health Division
2601 Agriculture Drive
Madison, WI  53718
Telephone: (608) 224-6260
Fax: (608)224-6213
E-mail:  beckyh@mail.slh.wisc.edu

Stuart Hooper
Emory University
Rollins School of Public Health
Department of International Health
1518 Clifton Road, NE, Room 762
Atlanta, GA 30322
Telephone: (404) 712-8355
Fax: (404)727-4590
E-mail:  shooper@emory.edu

Fu-Chih Hsu
Scientific Methods, Inc.
12441 Beckley Street
Granger, IN 46530
Telephone: (574) 277-4078
E-mail:  fuchih@scientificmethods.com

Jay Hua
U.S. Environmental Protection Agency
Region 7
Water, Wetlands, and Pesticides Division
Water Resource Protection Branch
90 IN 5th  Street
Kansas City, KS 66101
Telephone: (913)551-7748
Fax: (913)551-9748
E-mail:  hua.jay@epa.gov

Maggie Javdan
U.S. Environmental Protection Agency
Office of Research and Development
Office of Science Policy
Ariel Rios  Building (8103R)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 564-5264
E-mail:  javdan.maggie@epa.gov
          The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
Patrick Jjemba
University of Cincinnati
Department of Biological Sciences
PO Box ML006
Cincinnati, OH 45221-0006
Telephone: (513)521-9757
Fax:  (513)556-5299
E-mail: jjembap@email.uc.edu

Cathy Kelty
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Division
  (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7080
Fax:  (513)569-7328
E-mail: kelty.catherine@epa.gov

Twila Kunde
Lovelace Clinic Foundation
Environmental Health Research Division
2309 Renard Place, SE, Suite 103
Albuquerque, NM 87106
Telephone: (505) 262-3472
Fax:  (505)262-7598
E-mail: twila@lcfresearch.org

Robert Lange
U.S. Environmental Protection Agency
Region 3
Office of Water
Safe Drinking Water Act Branch (MS-3WP32)
1650 Arch Street
Philadelphia, PA 19103-2029
Telephone: (215) 814-5459
Fax:  (215)814-2302
E-mail: lange.robert@epa.gov

Jim Larkin
Scientific Methods, Inc.
12441 Beckley Street
Granger, IN  46530
Telephone: (574) 277-4078
Fax:  (574)243-1148
E-mail: jim@scientificmethods.com
Alan Lindquist
U.S. Environmental Protection Agency
National Homeland Security Research
  Center (MS-163)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7192
Fax: (513)487-2555
E-mail: lindquist.alan@epa.gov

Srinivasa Lingireddy
University of Kentucky
Department of Civil Engineering
354H, Raymond Building
Lexington, KY 40506
Telephone:  (859) 257-5243
Fax: (859)257-4404
E-mail: lreddy@engr.uky.edu

Suzanne List
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7957
Fax: (513)569-7116
E-mail: list.suzanne@epa.gov

Dennis Lye
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-314)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7870
Fax: (513)569-7170
E-mail: lye.dennis@epa.gov

Benito Marinas
University of Illinois at Urbana-Champaign
Department of Civil and Environmental
  Engineering
3230 Newmark Civil Engineering Laboratory
205 N Mathews Avenue
Urbana, IL  61801
Telephone:  (217) 333-6961
Fax: (217)333-6968
E-mail: marinas@uiuc.edu
          The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
Dennis McChesney
U.S. Environmental Protection Agency
Region 2
Division of Environmental Science
  and Assessment
Monitoring and Assessment Branch (MS-220)
2890 Woodbridge Avenue
Edison, NJ 08837-3602
Telephone: (732) 321-6729
Fax: (732)321-6616
E-mail: mcchesney.dennis@epa.gov

Hugh McKinnon
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
  Laboratory (MS-235)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7418
E-mail: mckinnon.hugh@epa.gov

Mark Meckes
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone: (513)569-7348
Fax: (513)569-7328
E-mail: meckes.mark@epa.gov

Bruce Mintz
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research
  Laboratory (MS-D305-01)
109 TW Alexander Drive
Research Triangle Park, NC 27709
Telephone: (919) 541-0272
Fax: (919)541-7588
E-mail: mintz.bruce@epa.gov

Debbie Moll
Centers for Disease Control and Prevention
Health Studies Branch (MS-E-23)
1600 Clifton Road, NE
Atlanta, GA 30333
Telephone: (404) 498-1364
Fax: (404)498-1355
E-mail: dmoll@cdc.gov
Carrie Moulton
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7919
Fax: (513)569-7191
E-mail: moulton.carrie@epa.gov

Patricia Murphy
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
  Assessment (MS-272)
2890 Woodbridge Avenue
Edison, NJ  08837
Telephone:  (732) 906-6830
Fax: (732)906-6845
E-mail: murphy.patricia@epa.gov

Cynthia Nolt-Helms
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
  Research
Ariel Rios Building (8722R)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone:  (202) 564-6763
Fax: (202)565-2446
E-mail: nolt-helms.cynthia@epa.gov

Nena Nwachuku
U.S. Environmental Protection Agency
Office of Water
Office of Science and Technology
Ariel Rios Building (4304T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone:  (202)566-1116
Fax: (202)566-1140
E-mail: nwachuku.nena@epa.gov

Robin Oshiro
U.S. Environmental Protection Agency
Office of Water
Ariel Rios Building (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone:  (202) 566-1075
Fax: (202)566-1053
E-mail: oshiro.robin@epa.gov
          The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
James Owens
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Microbiological and Chemical Exposure
  Assessment Research Division (MS-593)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7235
Fax: (513)569-7464
E-mail: owens.jim@epa.gov

Angela Page
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
  Research
Ariel Rios Building (8722R)
1200 Pennsylvania Avenue, NW
Washington, DC  20460
Telephone:  (202)564-5172
Fax: (202)565-2446
E-mail: page.angelad@epa.gov

Linda Papa
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
  Assessment (MS-190)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7587
Fax: (513)569-7916
E-mail: papa.lynn@epa.gov

Latisha Parker
U.S. Environmental Protection Agency
Office of Science and Technology
Ariel Rios Building (4304T)
1200 Pennsylvania Avenue, NW
Washington, DC  20460
Telephone:  (202)566-1118
Fax: (202)566-1139
E-mail: parker.latisha@epa.gov
W. Bruce Peirano
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources
  Division (MS-690)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7540
E-mail: peirano.bruce@epa.gov

Joyce Perdek
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
  Laboratory (MS-104)
2890 Woodbridge Avenue
Edison, NJ  08837
Telephone:  (732) 321-4380
Fax:  (732)321-6640
E-mail: perdek.joyce@epa.gov

Dan Petersen
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research
  Laboratory (MS-G75)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7831
E-mail: petersen.dan@epa.gov

Stacy Pfaller
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-314)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7893
Fax:  (513)487-2512
E-mail: pfaller.stacy@epa.gov

Phanida  Prommasith
Harvard School of Public Health
Department of Environmental Health
665 Huntington Avenue, HSPH 1, Room G-28
Boston, MA 02115
Telephone:  (617) 432-3615
Fax:  (617)432-3349
E-mail: pprommas@hsph.harvard.edu
          The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
Bebe Raupe
BNA, Inc.
PO Box 498769
Cincinnati, OH 45249
Telephone:  (513)677-2870
Fax:  (513)677-3278
E-mail: braupe@bna.com

Donald Reasoner
U.S.  Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch (MS-387)
26 W Martin Luther King Drive.
Cincinnati, OH 45268
Telephone:  (513)569-7234
Fax:  (513)569-7328
E-mail: reasoner.donald@epa.gov

Chrissy Reckelhoff
Association of Schools of Public Health
6368 Dry Ridge Road
Cincinnati, OH 45252
Telephone:  (513)385-1050
Fax:  (202)296-1252
E-mail: reckelhoff.chrissy@epa.gov

Stig Regli
U.S.  Environmental Protection Agency
Office of Ground Water and Drinking
  Water
Ariel Rios Building (4607M)
1200 Pennsylvania Avenue, NW
Washington, DC  20460
Telephone:  (202) 564-5270
Fax:  (202)564-3760
E-mail: regli.stig@epa.gov

Jon Reid
U.S.  Environmental Protection Agency
Office of Research and Development
National Center for Environmental Assessment
4556 Winton Road
Cincinnati, OH 45232
Telephone:  (513)967-3231
E-mail: reid.jon@epa.gov
Randy Revetta
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch
  (MS-G75)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7129
E-mail: revetta.randy@epa.gov

Glenn Rice
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
  Assessment (MS-190)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7813
Fax: (513)569-7916
E-mail: rice.glenn@epa.gov

BK Robertson
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7763
E-mail: robertson.boakai@epa.gov

Paul Rochelle
Metropolitan Water District of Southern California
Water Quality Laboratory
700 Moreno Avenue
La Verne, CA 91750
Telephone:  (909)392-5155
Fax: (909)392-5246
E-mail: prochelle@mwdh2o.com

Pam Rodgers
Battelle Memorial Institute
505 King Avenue
Columbus, OH 43201
Telephone:  (614) 424-4624
Fax: (614)424-3667
E-mail: rodgersp@battelle.org
          The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
Mary Rothermich
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
  Assessment (MS-190)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7869
Fax: (513)569-7916
E-mail: rothermich.mary@epamail.epa.gov

Frank Schaefer
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory (MS-320)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7222
Fax: (513)569-7117
E-mail: schaefer.frank@epa.gov

Rita Schoeny
U.S. Environmental Protection Agency
Office of Water
Ariel Rios Building (430IT)
1200 Pennsylvania Avenue, NW
Washington, DC  20460
Telephone:  (202)566-1127
Fax: (202)566-0441
E-mail: schoeny.rita@epa.gov

Pati Schultz
U.S. Environmental Protection Agency
Office of External Affairs (MS-284)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7966
Fax: (513)569-7770
E-mail: schultz.patricia@epa.gov

Keya Sen
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7026
Fax: (513)569-7191
E-mail: sen.keya@epa.gov
Lois Shadix
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7864
E-mail:  shadix.lois@epa.gov

Manou Shahamat
University of Maryland Biotechnology Institute
Center of Marine Biotechnology
70 IE Pratt Street
Baltimore, MD 21202
Telephone:  (410)234-8881
Fax: (410)234-8896
E-mail:  shahamat@umbi.umd.edu

Gwy-Am Shin
University of North Carolina, Chapel Hill
Department of Environmental Sciences
  and Engineering
CB# 7431, ESE, SPH, UNC-CH
Chapel Hill, NC 27599-7431
Telephone:  (919) 966-0793
Fax: (919)966-4711
E-mail:  gwyam@isis.unc.edu

Jody Shoemaker
U.S. Environmental Protection Agency
Office of Research and Development
National Exposure Research Laboratory
Microbiological and Chemical Exposure
  Assessment Research Division  (MS-564)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7298
Fax: (513)569-7757
E-mail:  shoemaker.jody@epa.gov

Hiba Shukairy
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7943
Fax: (513)569-7191
E-mail:  shukairy.hiba@epa.gov
          The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
Joyce Simpson
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch
  (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7016
Fax: (513)569-7328
E-mail: simpson.joyce@epamail.epa.gov

Jim Sinclair
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Technical Support Center (MS-140)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7970
Fax: (513)569-7191
E-mail: sinclair.james@epa.gov

Mano Sivaganesan
U.S. Environmental Protection Agency
National Risk Management Research Laboratory
Water Supply and Water Resources
  Division (MS-690)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7638
Fax: (513)569-7658
E-mail: sigaganesan.mano@epa.gov

Barbara Smith
U.S. Environmental Protection Agency
Region 9
75 Hawthorne Street (MS-PMD1)
San Francisco, CA  94105
Telephone:  (415) 972-3735
Fax: (415)972-3735
E-mail: (415)947-3558

Stuart Smith
Ground Water Science
372 W Wyandot Avenue
Upper Sandusky, OH 43351
Telephone:  (419) 209-0298
Fax: (419)209-0336
E-mail: stusmith@udata.com
Timothy Straub
Battelle Memorial Institute - Pacific Northwest
  National Laboratory
Environmental Microbiology
PO Box 999, MSIN P7-50
902 Battelle Boulevard
Richland,WA 99352
Telephone:  (509) 372-1953
Fax: (509)376-1321
E-mail: timothy.straub@pnl.gov

Jeff Swartout
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
  Assessment (MS-190)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7811
Fax: (513)569-7916
E-mail: swartout.jeff@epa.gov

Ruth Sykes
U.S. Environmental Protection Agency
Region 2
Division of Environmental Science
  and Assessment
Laboratory Branch (MS-230)
2890 Woodbridge Avenue
Edison, NJ 08837
Telephone:  (732) 906-6961
Fax: (732)906-6165
E-mail: sykes.ruth@epa.gov

Peter Teunis
RIVM-IMA
National Institute of Public Health
  and Environment
Antonie van Leeuwenhoeklaan 9
PO Box 1
Bilthoven, 3720BA
The Netherlands
Telephone:  31-30-274-2937
Fax: 31-30-274-4456
E-mail: peter.teunis@rivm.nl
          The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
Marilyn Thornton
U.S. Environmental Protection Agency
Region 4
Science and Ecosystem Support Division
980 College Station Road
Athens, GA 30605-2720
Telephone: (706) 355-8553
Fax: (706)355-8803
E-mail: thornton.marilyn@epa.gov

Saul Tzipori
Tufts University School of Veterinary Medicine
Department of Biomedical Sciences
200 Westboro Road, Building 20
North Grafton, MA 01536
Telephone: (508) 839-7955
Fax: (508)839-7911
E-mail: saul.tzipori@tufts.edu

Steve Via
American Water Works Association
1401 New York Avenue, NW, Suite 640
Washington, DC 20005
Telephone: (202) 628-8303
Fax: (202)628-2846
E-mail: svia@awwa.org

Tim Wade
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects
  Research Laboratory
Human Studies Division
Epidemiology and Biomarkers Branch (MS-58C)
Research Triangle Park, NC  27711
Telephone: (919) 966-8900
Fax: (919)966-0655
E-mail: wade.tim@epa.gov

Barb Walton
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects
  Research Laboratory (MS-B305-02)
Research Triangle Park, NC  27711
Telephone: (919) 541-7776
E-mail: walton.barb@epa.gov
Dan Williams
U.S. Environmental Protection Agency
Office of Research and Development
National Water Supply and Water Resources
  Division
Treatment Technology Evaluation Branch
  (MS-B24)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7237
Fax: (513)509-7172
E-mail: williams.daniel@epa.gov

Margaret Williams
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7243
Fax: (513)569-7328
E-mail: williams.margaret@epa.gov

Karen White
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Research Laboratory
Water Supply and Water Resources Division
Microbial Contaminants Control Branch (MS-387)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7248
Fax: (513)569-7328
E-mail: white.karenm@epa.gov

Karen Wirth
U.S. Environmental Protection Agency
Office of Ground Water and Drinking
  Water
Ariel Rios Building (4607M)
1200 Pennsylvania Avenue, NW
Washington, DC  20460
Telephone:  (202) 564-5246
Fax: (202)564-3760
E-mail: wirth.karen@epa.gov
          The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
Michael Wright
U.S. Environmental Protection Agency
Office of Research and Development
National Center for Environmental
  Assessment (MS-190)
26 W Martin Luther King Drive
Cincinnati, OH 45268
Telephone:  (513)569-7922
Fax: (513)569-7916
E-mail: wright.michael@epa.gov
Marylynn Yates
University of California, Riverside
Associate Executive Vice Chancellor
4108HinderakerHall
Riverside, CA 92521
Telephone: (909) 787-2358
Fax: (909)787-4362
E-mail:  marylynn.yates@ucr.edu
                                     Remote Participants
Kerri Alderisio
New York City Department of Environmental
   Protection
465 Columbus Avenue
Valhalla, NY  10595
Telephone: (914) 773-4423
E-mail:
kalderisio@dep.nyc.gov@westchestergov.com

Paul Berger
U.S. Environmental Protection Agency
Office of Water
Office of Ground Water and Drinking Water
Ariel Rios Building (4607M)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone: (202) 564-5253
E-mail:  berger.paul@epa.gov

Abel Euresti
U.S. Environmental Protection Agency
Region 6 Houston Laboratory
Environmental Services Branch
10625 Fallstone Road
Houston, TX  77099
Telephone: (281)983-2162
Fax: (281)983-2248
E-mail:  euresti.abel@epa.gov

JeffGratz
U.S. Environmental Protection Agency
Region 2
290 Broadway, 28th Floor
New York, NY 10007-1866
Telephone: (212) 637-3554
E-mail:  gratz.jeff@epa.gov
Ariel Iglesias
U.S.Environmental Protection Agency
Region 2
2890 Woodbridge Avenue (MS-215)
Edison, NJ  08837
Telephone:  (732) 452-6426
E-mail:  iglesias.ariel@epa.gov

Paul Kutzy
Westchester County Department of Health
145 Huguenot Street
New Rochelle, NY 10801
Telephone:  (914)813-5156
E-mail:  pjk3@westchestergov.com

Michael Lowy
U.S. Environmental Protection Agency
Region 2
290 Broadway, 24th Floor
New York, NY  10007-1866
Telephone:  (212) 637-3830
E-mail:  lowy.michael@epa.gov

Cindy Mack
U.S. Environmental Protection Agency
Office of Water
Office of Wastewater Management
Ariel Rios Building (4707M)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone:  (202) 564-6280
E-mail:  mack.cindy-y@epa.gov
          The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
Bruce Macler
U.S. Environmental Protection Agency
Region 9
75 Hawthorne Street (WTR-6)
San Francisco, CA 94105
Telephone:  (415) 972-3569
Fax: (415)947-3549
E-mail:  macler.bruce@epa.gov

Cristina Maldonado
U.S. Environmental Protection Agency
Region 2, CEPD
CEPD Centre Europa 417
San Juan, PR 00907
Telephone:  (787) 977-5827
Fax: (787)289-7102
E-mail:  maldonado.cristina@epa.gov

Jorge Martinez
U.S. Environmental Protection Agency
Region 2, CEPD
Centro Europa 417
San Juan, PR 00907
Telephone: (787) 977-5827
Fax: (787)289-7102
E-mail:  martinez.jorge@epa.gov

Yves Mikol
New York City Department of Environmental
   Protection
465 Columbus Avenue
Valhalla, NY  10595
Telephone:  (914) 773-4426
E-mail:  ymikol@dep.nyc.gov

Renee Morris
U.S. Environmental Protection Agency
Office of Water
Office of Wastewater Management
Ariel Rios Building (4707M)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Telephone:  (202) 564-8037
E-mail:  morris.renee@epa.gov

Robert Poon
U.S. Environmental Protection Agency
Region 2
290 Broadway, 24th Floor
New York, NY 10007-1866
Telephone:  (212) 637-3821
E-mail:  poon.robert@epa.gov
Rebecca Quinones
U.S. Environmental Protection Agency
Region 6
Sample Management
10625 Fallstone Road
Houston, TX  77099
Telephone:  (281)983-2168
Fax: (281)983-2248
E-mail:  quinones.rebecca@epa.gov

Anne Seeley
New York City Department of Environmental
   Protection
59-17 Junction Boulevard, 20th Floor
Flushing, NY  10595
Telephone:  (718)595-5346
E-mail:  aseeley@dep.nyc.gov

Erin Shutak
New York City Department of Health
2 Lafayette, 11th Floor CN 56
New York, NY 10007
Telephone:  (212) 676-1542
E-mail:  eshutak@health.nyc.gov

Sandra Spence
U.S. Environmental Protection Agency
Region 8 Laboratory
16194 W 45th Drive
Golden, CO 80403
Telephone:  (303)312-7754
Fax: (303)312-7800
E-mail:  spence.sandra@epa.gov

Alysia Tani
U.S. Environmental Protection Agency
Region 8 Laboratory
16194 W 45th Drive
Golden, CO 80403
Telephone:  (303)312-7809
Fax: (303)312-7800
E-mail:  tani.alysia@epa.gov

Paul Zambratto
U.S. Environmental Protection Agency
Region 2
290 Broadway, 28th Floor
New York, NY 10007-1866
Telephone:  (212) 637-4012
E-mail:  zambratto.paul@epa.gov
          The Office of Research and Development's National Center for Environmental Research

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Appendix 1: Presentations of Regional Research
 Needs and Office of Water Regulatory Activities
             and Research Needs

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       Regional Concerns foi
 Microoganisms in Drinking Water
              Bruce Macler
          Drinking Water Office
               Region 9

              415 972-3569

    Regions Mostly Need Help
         With Applications
The here and now of regional operations
offices day-to-day activities require
 • Information to prioritize work
 • Tools to make decisions
 • Tools for regulatory monitoring and compliance
Some of these are being worked on, some are
not

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   Information to Prioritize Work
Regions and State counterparts are swamped
 • Have to aggressively prioritize
 • Ask: What is the magnitude of the problem to be
  solved? (that is, beyond the political...)
For microbial pathogens, mostly epidemiology
 • What is the extent of microbial disease from
  drinking water?
 • Do we have a microbial problem on beaches?

       Microbial Epidemiology
    Questions for Drinking Water
 Do we have a national public health problem
 from undisinfected wells?
  • >50% of public wells not disinfected
  • But, is there a problem?
 Is there remaining microbial disease from
 treated water?
 Answers useful for reg implementation

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      Microbial Epidemiology
       Questions for Beaches
What is the extent of waterborne illness from
bathing exposures?
 • Is freshwater exposure different from marine?
 • Can we confirm the significance of bather-to-
  bather contamination?
 • Are some exposure situations more problematic?
Are the current beach criteria accurate?

     Microbial Tools to Make
               Decisions
Drinking water decisions primarily involve
determining "fecal contamination"
Beach decisions need information on sources
of fecal contamination
 • Is it pathogenic to humans?
 • Or, is it "false positive" and non-pathogenic?

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       Drinking Water Tools
Need confidence in vigor of current fecal
indicators
 • Do they adequately represent range of
  pathogens? (apparently, no)
 • Need an approach that is more definitive
Need suitable surrogates to determine
adequacy of disinfection treatment
 • Chlorine, UV, ozone, etc

       Beach Microbial Needs
Biggest problem is to determine when a
bathing beach may be contaminated with
microbials pathogenic to humans
Current fecal indicators can be positive for
apparently non-pathogenic situations
 • Birds versus people
Indicators may not match risk

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   Monitoring and Compliance
               Needs
Cheaper
Easier
More definitive
For drinking water, Cryptosporidium parvum
For bathing beaches, approach for human
pathogens.

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    SDWA Requirements &
   Microbial Research Needs
(Surface Water, Ground Water,
    & Distribution Systems)
             Stig Regli
          OGWDW/USEPA
             8/5/2003
    EPA Regulation Setting
 Requirements Under SDWA

Must publish MCLGs for contaminants that
 - may have adverse health effects
 - occur in public water systems at frequencies &
  levels of public health concern
 - provide meaningful opportunity for health risk
  reduction for persons served by PWS
MCLGs shall be set at levels at which no
known health effects occur and which
allows an adequate margin of safety

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   EPA Requirements Under
          SDWA (cont'd)
Must promulgate MCLs as close to the
MCLG as is "feasible"
 - "feasible" means with use of "best available
   technology" (taking costs into consideration)
Must promulgate treatment technique
requirement if not economically or
technically feasible to monitor
Must perform regulatory impact analysis
(RIA) for each regulation
  SDWA  - Risk Assessment

For each regulation specify to extent practicable:
 - Estimates of public health effects for each population
 - Expected, upper, and lower bound risk estimates for
  each population
 - Each significant uncertainty identified in risk
  assessment
 - Peer reviewed studies that support estimates above &
  methodologies used to reconcile data inconsistencies

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 SDWA - Health Risk Reduction

    & Cost Analysis (HERCA)

1  Quantifiable & nonquanitifiable reduction of
  health risk
  - Above also for reductions in co-occurring contaminants
1  Quantifiable and nonquantifiable costs
•  Incremental costs & benefits associated with each
  alternative standard
•  Effects of contaminant on general population and
  groups within general population
  - Children, pregnant women, elderly, individuals with
    serious illness, or subpopulations at greater risk
•  Any increase in health risk that may occur
 SDWA - Feasible technologies

 List feasible technologies, treatment
 techniques, and other means for achieving
 compliance
 List any technology, treatment technique, or
 other means affordable for small systems:
  ->3300 to 10,000
  - >500 to 3300 people
  - >25 to 500 people

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    Research Inputs into DW
             Regulations
Health Effects & Assessment
TIMCLGs
TIEpidemiology
Analytical Methods
71 Feasibility of monitoring
               Drinking Water
               Regulations
               MCL/Treatment Technique
 Occurrence/Exposure
  Treatment Technologies
  71 Effectiveness, costs
Long Term 2 Enhanced Surface
  Water Treatment Rule (LT2)
 SDWA requires EPA to promulgate LT2
 with Stage 2 Disinfection Byproducts Rule
 Goal: provide equivalent level of protection
 for all systems using surface water
 Covers: 5500 systems,  174 million people
 Status: propose 2003, promulgate 2004

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         LT2 Components
 Systems monitor Cryptosporidium in source water
 to determine if more treatment is needed
 - Small systems monitor E.coli;if levels are low they can
   avoid monitoring Cryptosporidium
 Tool box of treatment options to achieve different
 Cryptosporidium removal credits
 Unfiltered systems must provide at least 2 log
 inactivation of Cryptosporidium
 Finished water reservoirs must be covered or
 disinfected (4 log virus inactivation)
 Source water treatment level reevaluated six years
 after first round of monitoring
       LT2 Research Issues
What treatment strategies are available for small
systems & how can they be evaluated?
What are appropriate indicators to determine
source water pathogen risk?
What are appropriate indicators for assessing
effectiveness of surface water treatment?
What proportion of the total waterborne pathogen
risk is linked to source/treatment issues?
Does control for Giardia & Cryptospridium
adequately control for other pathogens?

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  Ground Water Rule (GWR)

SDWA requires EPA to promulgate GWR before
Stage 2 DBPR promulgation
GWR Goal: identify GW systems vulnerable to
fecal contamination & require remedial action for
such systems
Covers: 154,000 systems serving 118 million
people
Status: proposed FRN 5/10/00, promulgate 03/04
        GWR Components
Periodic sanitary surveys
Hydrogeologic sensitivity assessments
Source water monitoring (E.coli, enterococci, or
coliphage) for systems if:
 - Sensitive hydrogeologic setting
 - Total coliform hit in distribution system
Corrective action if significant deficiency or fecal
indicator is positive
Compliance monitoring for disinfected systems
(show > 4 log virus inactivation)

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            GWR Issues


What are the most appropriate indicator(s) for
vulnerability to fecal contamination?
What tools are available to make indicator
monitoring more cost effective (e.g, micro arrays,
molecular techniques)?
How does the sensitivity of naturally occurring vs.
lab-adapted viruses to different disinfectants
compare?
What proportion of the total waterborne pathogen
risk is linked to source/treatment related GW
issues?
 Revised TCR & Distribution
  System (DS) Requirements
SDWA requires 6 year review of all rules
including TCR
TCR revision to be coupled with
development of DS requirements
DS Goal: protect public health from
distribution system contamination
Status: now in problem definition phase;
proposed FRN anticipated 2006
                                                           7

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               DS Issues

How do we assess public health risk associated
with: cross connections, intrusion, contamination
following repair or replacement, biofilms,
nitrification, uncovered storage & water age?
Which DS deficiencies pose greatest risk to public
health?
What are appropriate indicators of DS
deficiencies?
How effective is current technology for reducing
the most important potential DS health risks?
What proportion of the total waterborne pathogen
risk is linked to distribution system issues?
              TCR Issues
What are the most appropriate monitoring
strategies for routine monitoring? After a TC-
positive?
 - Location, frequency, sample volume
What control and prevention strategies are
effective?
What are the most appropriate approaches for
indicating distribution system risk?
 - Microbes: TC, E.coli, etc.
 - Non-microbial: hydraulics, disinfectant residual, etc.?

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        National Estimate

SDWA requires EPA & CDC to develop
national estimate of waterborne disease
Status: Approaches for generating estimate
are in development (OGWDW, ORD, CDC)
 - FRN indicating estimates & uncertainties using
  different approaches to be published 2004
 - Much more data will be needed to address
  uncertainty of estimates (beyond 2004)
   National Estimate - Issues
What percent of national incidence of GI illness is
associated with drinking water?
 - Which estimation methodologies are most reliable?
What percent of waterborne illness is associated
with source/treatment issues versus DS issues?
Can ongoing estimates of national drinking
waterborne disease be used as a benchmark for
evaluating benefits of drinking water regulations?

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       The Contaminant Candidate
       List: Determining the Need
        for Future Drinking Water
                Standards

       US Environmental Protection Agency
          Research on Microorganisms in
            Drinking Water Workshop
               August 5-7, 2003
                            Tom Carpenter
                           CCL Team Lead
                    Office of Ground Water and
                            Drinking Water
 Overview of Presentation
Statutory Requirements for the CCL
NRC recommendations for future CCLs
NDWAC Schedule
Overview of the Methods and Issues
Efforts Underway

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                SDWA CCL Schedule
Research & Occurrence (UCMR) Data
 Collection for CCL2 contaminants
 CCL1
  Feb
  1998
 Reg Determinations
     2003
     (Actual)
                    CCL3
                    Feb 08
        t
02
04
06
    Reg Determinations
       Aug 2001        CCL2
                    Feb 03
                                 08
                          Reg
                         Determ'
                          CCL2
                          8/06
10

                                Reg
                               Determ'
                                CCL3
                                8/11
                                      Research & Occurrence
                                      DataCollection for CCL3
            Future CCL Development

       Same SDWA requirements as CCL1 and similar
       components needed to develop future CCLs
       -  Develop list
       -  Initiate the regulatory determination process
       National Academy of Sciences Panel Reports
       -  Last report of trilogy recommends strategies for future CCLs
       Evaluate NRC Report "Classifying Drinking Water
       Contaminants for Regulatory Consideration"
       Provides Recommendations
       -  Extensive process for identifying and narrowing contaminant
          universe
       -  Recommendations for data quantity and quality
       -  Significant quantitative aspect
       -  Validation of recommended approach using case example

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    NRC Recommendations  for the CCL
  "Universe"

      (-100,000)

The universe of potential
drinking water
contaminants includes:
Naturally occurring
substances
Emerging waterborne
pathogens
Food-borne pathogens
Animal pathogens
Chemical agents
Byproducts and
degradates of chemical
agents
Radionuclides
Biological toxins
     STEP ONE

Screening Criteria and
Expert Judgement
     PCCL
       (>1,000)
     STEP TWO

Classification Tool and
Expert Judgement
                       PCCL Includes:
                       Contaminants that
                       occur, or have the
                       potential to occur in
                       drinking water AND
                       cause, or may cause,
                       adverse health effects
      NRC Recommendations (cont'd)
  Strongly recommends a classification approach that should
  not sacrifice complexity for transparency
  -  allows for complex decision process that scores and weights classification attributes
     of contaminants based on pattern recognition
  -  calibrate and validate using existing contaminants as training sets

  Evaluate new molecular/genetic methods to identify
  new/emerging microbiological contaminants as part of new
  approach
  -  base evaluation of microbes on similarities of virulence, physical, and/or genetic
     attributes (Virulence Factor Activity Relationships)
  -  relies on new genomic and molecular analytical methods and indicators
  -  WAR is long-term goal — need to identify interim products as proof of concept

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Methodology Issues NDWAC is discussing

 Prototype Classification Approach

  - How did NRC arrive at this approach
     . evaluation of several prioritization approaches
     . common characteristics were selection of contaminant pool,
      determination of exposure and toxicity, what was the prioritization
      method
     . most examples used for chemicals not/pathogen
  - NRC Panel started at the beginning
     . had limited resources
     . wrestled with how the attributes of exposure and toxicity inter-relate
      to one another
     . NRC recommendations are not a complete road map, there is more
      work to do
   Methodology issues discussions (cont'd)
 The universe of potential contaminants
 Occur in Drinking water
                                    Adverse
                                  Health Effects
        Potential
        Adverse
      Health Effects
 Potential to
  Occur in
Drinking water

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   Methodology issues discussions (cont'd)

  . The universe to the PCCL
    -  Intersection of the major characteristics identify
      contaminants to carry to the PCCL
    -  Seek expert judgement on the screening  process
    -  Screening criteria need to be connected to the data
      sources
    -  Identify data elements to capture and compile the
      universe from these evaluations
    -  Develop screening criteria
      . NDWAC discussing  guidelines and concept
      . generate from the data sources to identify potential and
        known criteria
      . apply "automated" screening
Virulence Factor Activity Relationships
Indicators
Genetic Elements
Surface Proteins
Attachment Factors
Metabolic Pathways
Other Virulence Attributes
Classification
Outcomes
Virulence
Potency
    » Classifies pathogens
    » Will not be fully developed for CCL2
    • Relies on molecular technologies and gene sequencing
    • Research and analytical capabilities are improving
    • EPA research efforts are underway for microbes (i.e., Aeromonas)
    » NRC strongly recommends interagency participation (e.g. participation
     in National Science and Technology Council's Biotechnology Research Group)
    » Next steps are to identify and coordinate range of research needs

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  Methodology issues discussions (cont'd)

VFARs:  A new process for pathogens
   Genomics and proteomics are rapidly emerging
   technologies and should provide new indicators
   Identify pilot and prototype projects
    . Literature reviews, State of the science, available data
     sources
    . Develop model systems to test "virulence" of the potential
     pathogens
    . Develop interagency partnerships
    . Identify/develop/modify analytical methods for VFAR indicators
              NDWAC Charge

Discuss, evaluate, and provide advice on
methodologies, activities, and analysis needed to
implement the NRCs recommendations on an
expanded approach for the CCL listing process. This
may include advice on:
 -  an overall implementation strategy
 -  classification attributes and criteria
 -  pilot projects to validate new classification approaches
 -  proof of concept activities to support VFAR analysis
 -  communication issues
 -  additional issues not addressed in the NRC report

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            CCL NDWAC Work Group


    Request NDWAC advice to assist EPA in developing
    methodologies that can be used for future CCLs
    NDWAC Work Group with 3 Activity Groups
     - CCL may need parallel paths for pathogens and chemical
       contaminants
     - One microbiological/pathogen and one chemical subworkgroup
     - Both technical  activity group should include classification/information
       technology expertise
    Convened Work Group September 2002
    6 plenary meetings to date
    2 remaining meetings through the Fall of 2003
     Novel approach:  comparative genetics
Identify virulence genes from genomes by location
      •Many virulence-associated genes cluster together (pathogenicity islands)
      •Genes flanking virulence genes may be co-expressed and have related
      functions www.tigr.org select genome browser

Identify virulence genes from genomes by expression
      •Transcriptosomes (grouping of genes according to their transcription
      regulation patterns) may identify virulence-associated genes

Identify virulence genes from genomes by primary sequence
      •GC-content, flanking IS sequences or repeats, can identify recently acquired
      genes. Surface-exposed genes are generally more AT-rich which makes
      them prone to mutation www.cbs.dtu.dk/services/GenomeAtlas

Identify virulence genes by multiple sequences
      •substitution rates (Ka/Ks) identify genes under strong selection
      •polymorphisms within (surface expressed) genes indicate avoidance of
      immune response
                                                                                    7

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           Microarrays:  promises, outcome
  "Applications of functional genomics of food microorganisms:
  novel risk assessment procedures" (Curr. Opin.Biotech. 1999,10:511)

  Applications  of MA to food pathogens (published as of May 2002)
Detection/
differentiation
Diversity/
conservation

 Gene
 regulation
•detection of bacterial virulence genes by microarray performs
better than PCR
•direct detection of £. co//on chicken carcasses
•differentiate Staph. spp. by low-density microarray of PCR products
•identification of diversity in gene content of C.jejuni
•identification of homologs conserved within pathogenic
Salmonellas
•gene regulation under Fe-limitation (in Pasteurella) proves complex
and pleiotrophic. How to differentiate primary from secondary,
down-stream effects?
•gene expression during acid-adaptation of E. coli was studied
•sarA and agrof S. aureus regulate known virulence genes, also
many others
                 Genomic Data Searches

                 VFAR Discovery Phases

          Phase I September-October, 2002
           41 Limited virulence factor keyword search of GenBank
           41 Basic Local Alignment Search Tool (BLAST) alignments
          Phase II October-November, 2002
           41 Comprehensive keyword search of GenBank
           4> Comparison of other available genomic databases
          Phase III November-December 2002
           4> Keyword search of whole genomes
           4> Selective virulence factor sequence alignments against other
             whole genomes
           vp BLAST alignment of complete virus genomes

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            Bioinformatics Pilot
           Assessement of VFAR
  Literature about E. coli
Select representative
      genes
   Select probes]
                       Alignment
                     [Primer design]
 Check for sequence
      similarity
Select representative
      probes
            Download sequences
             Investigators:
            Syed Hashham,
             James Cloe
              Joan Rose
   Gene Combinations Associated with
     Water-borne Pathogen Virulence
                       waterborn
                       properties
                  waterborn,
                  pathogenic
     waterborn,
     non-pathogenic
            virulence
            properties
                   Investigators
                   T Wassenaar
                   J. Gamieldien
          Subtract: all genes from non-pathogenic, non-
                 i organisms (minimal gene set)

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   For Additional Information or Questions
     Tom Carpenter
      202-564-4885
    202-564-3760 (fax)
carpenter.thomas@epa.gov
                    Jitendra Saxena
                     202-564-5241
                   202-564-3760 (fax)
                Saxena.Jitendra@epa.gov
                                                         10

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Appendix 2: Additional NCER STAR Drinking
      Water Grant Microbial Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


     Experimental  Infection of Healthy Adults with a Cryptosporidium
                              Genotype 1  Isolate (TU502)

               Cynthia Chappell1, P. Okhuysen2, R. Lunger1, D. Akiyoshi3, and S. Tzipori3
     Center for Infectious Diseases, The University of Texas Health Science Center at Houston, School
     of Public Health, Houston, TX; 2Department of Internal Medicine, The University of Texas Health
        Science Center at Houston Medical School, Houston, TX; 3Division of Infectious Diseases,
                  School of Veterinary Medicine, Tufts University, North Grofton, MA

    Cryptosporidium parvum causes  diarrheal illness worldwide. Molecular studies have  identified two dis-
tinct genotypes with different transmission cycles. Genotype 1 (Gl) strains are primarily  a human-to-human
transmission, and genotype 2 (G2) strains are zoonotic. Previous dose-response studies in  healthy adults em-
ployed five genotype 2 isolates, which varied widely in infectivity, yielding ID50's between 9 and 1042 oo-
cysts. This study is the first report of experimental Gl infections in healthy adults. The Gl isolate (TU502)
used in this study originated from a human case and was amplified in gnotobiotic piglets.  A single dose (10,
30, 100, or 500 oocysts) of TU502 was administered to 16 volunteers, which were monitored for 6 weeks.
Results showed that the TU502 ID50 was similar to the most infectious of the G2 isolates. The onset of diarrhea
and oocyst shedding following TU502 challenge were similar to the G2 isolates; however, the duration of diar-
rhea and oocyst shedding showed important differences. The typical 4-7 days of diarrhea seen with G2 isolates
was  prolonged in TU502 volunteers, lasting up to 22 days. Further, 83  percent of volunteers challenged with
the G2 isolates cleared their oocysts by <14 days as compared to 60 percent of volunteers receiving TU502.
Two subjects shed for 24 and 35 days, respectively. Total oocysts shed per person ranged from 5 X 106 to 1 X
1010, the latter occurring in the volunteer with the longest episode of diarrhea and oocyst shedding. These data
suggest that the Gl isolate, TU502, was highly infectious in healthy adults and was associated with a longer
diarrheal illness followed by an extended period of oocyst shedding. These characteristics  suggest a high risk
of infection from environmental sources and a risk of secondary transmission from contact with symptomatic
and asymptomatic oocyst shedders. These findings are consistent with the high proportion of Gl isolates  asso-
ciated with outbreaks of human cryptosporidiosis. This work was  supported, in part, by  EPA STAR Grant
#R-82918001 and NIH GCRC Grant #RR-02558.
           The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


             Experimental Challenge of Healthy Adult Volunteers
                       With Cryptosporidium muris Oocysts

                     Cynthia Chappell, P. Okhuysen , R. Lunger , and S. Tzipori
    1 Center for Infectious Diseases, The University of Texas Health Science Center at Houston, School
    of Public Health, Houston, TX; 2Department of Internal Medicine, The University of Texas Health
        Science Center at Houston Medical School, Houston, TX; 3Division of Infectious Diseases,
                  School of Veterinary Medicine, Tufts University, North Grofton, MA

    Cryptosporidium muris has long thought to be a pathogen of animals, but not humans. However, a recent
study indicated that infections with C.  muris  might occasionally occur in immunocompromised persons. In
addition, probable C. muris cases were reported in two children but were not confirmed. Detection of cases is
complicated by the fact that monoclonal antibodies commonly used in Cryptosporidium assays do not recog-
nize C. muris and might yield false negative results. The purpose of this study was to determine if C. muris
oocysts are infectious for healthy  adults. Six serologically negative, healthy volunteers were challenged with
105 C. muris oocysts and monitored for infection and illness for a minimum of 6 weeks. All six volunteers be-
came infected, but only one developed a diarrheal illness, which lasted for 4 days. In contrast, previous studies
have shown that C. parvum isolates were associated with illness attack rates of 52-86 percent. Interestingly, the
duration  of C. muris oocyst  shedding was longer (40-45 days or more) than with C. parvum (3-12 days, de-
pending  on the isolate tested). In some volunteers, the study period was extended due to continued oocyst
shedding. These data indicate that healthy adults are susceptible to infection with C. muris oocysts and in some
cases (17 percent) might experience a diarrheal illness. Further, the longer period of oocyst shedding might be
important for secondary transmission. These findings support the need for dose-response studies to more fully
describe the risk of C muris  infectivity and illness in the community  setting.
           The Office of Research and Development's National Center for Environmental Research

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Appendix 3: STAR Grant Presentation Abstracts
  and Agenda From the USEPA/USGS Meeting
     on Cryptosporidium Removal by Bank
       Filtration, September 9-10, 2003

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 William Blanford



(Abstract Not Provided)

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


      Study of Particle and  Pathogen  Removal  During Bank Filtration
                                       of River Waters

      Edward J. Bouwer1, Charles R. O'Melia1, W. Joshua Weiss1, Kellogg J. Schwab2, Binh T. Le2,
                                        and Ramon Aboytes3
        1 Johns Hopkins University, Baltimore, MD; 2Johns Hopkins University Bloomberg School
          of Public Health, Baltimore, MD;3American Water Belleville Laboratory, Belleville, IL

Project Goals and Objectives

    The overall objective of this research project is to evaluate the merits of riverbank filtration (RBF) for re-
moving/controlling pathogens in drinking water and to investigate the potential for using removal of particles
and other water quality indicators as surrogates for pathogen removal in riverbank systems.

Approach

    This research consists of: (1) field  studies to document actual changes in pathogen and particle concentra-
tions  from rivers of similar source quality in the context of variations in subsurface travel distances, pumping
rates, porous medium properties, and residence times;  and (2) parallel laboratory column studies with aquifer
media to provide insights into process  mechanisms and the relationship between pathogens and potential sur-
rogate parameters upon transport through riverbank media under a variety of physical and chemical conditions.

Preliminary Findings

    Field monitoring results from the three study sites indicate that RBF serves as a consistently significant
barrier to the transport of microorganisms from the river water sources (see Table 1). Cryptosporidium oocysts
were  detected in 11 out of 16 sampling rounds in the Ohio River,  12 out of 16 sampling rounds in the Wabash
River, and 8 out of 16 sampling rounds in the Missouri River, but never in any of the corresponding well wa-
ters. Similarly, Giardia cysts were detected in 4 of the sampling rounds in the Ohio River, 2 sampling rounds
in the Wabash River, and 3 sampling rounds in the Missouri River, but never in any of the corresponding well
waters. With the exception of Bacillus  and bacteriophage  4>X174, all of the monitored organisms  were always
below the detection limit in the well waters, with corresponding reductions of average concentrations ranging
from  0.8 logs to more than 6 logs.

    Preliminary design of column experiments has been accomplished. Riverbed sediment was collected from
the Potomac River in western Maryland to serve as representative  riverbank media for the column studies. The
sediment was  dried and sieved to remove fine and coarse material but otherwise  was not manipulated  or
cleaned prior to packing into glass columns. Preliminary experiments used 2.5 cm diameter, 30 cm long col-
umns. Breakthrough curves for bacteriophage MS2, bacteriophage PRD1, and Polio virus indicate that under
several ionic strength conditions, polio  virus is removed by a substantially greater extent than MS2, suggesting
that the phage might be useful as a conservative indicator for the transport of the human virus through river-
bank  media.

Significance of Findings

    The field monitoring data support the use of RBF at controlling the transport of potentially harmful micro-
organisms from river water sources. Reductions in average bacteria concentrations are often well in excess of 2
logs.  This is significant because bacteria have been proposed as potential surrogate parameters for the protozo-
ans (which are more difficult to measure accurately in the field due to their low and variable concentrations in
natural systems).
           The Office of Research and Development's National Center for Environmental Research

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                  Research on Microorganisms in Drinking Water Progress Review Workshop
 Next Steps

     Future column studies are intended to examine the relationship between the protozoans and the other mi-
 crobes  in aquifer media, as well as other potential surrogate/indicator parameters (including latex micro-
 spheres, natural river water particles, and turbidity measurements).

 Table 1.  Field monitoring results: January 2002 through July 2003 (log removals given in brackets). Aver-
            ages were calculated as the sum of counts divided by the sum of volumes  sampled over all sam-
            pling rounds; detection limit was calculated as 1 divided by the sum of the volumes.
Ohio
River
Well #9

Well #2

8.7 xlO4

1.7 xlO2
[2.7]
8.0 xlO2
[2.0]
                       7.6 xlO2
                         <2.0
1.3 x 10b

  <5.0

  <5.0
 1.5xl04
                                               <1.0xl0
<1.0xl0
4.6x10'
  <2.0
1.7x10

   1.1
  [3.2]
  <2.0
                                                                               3
 2.7 x 10"2     7.3 x 10"2
                                    <1.2 x 10"3   <1.2 x 10"3
<2.4 x 10"3   <2.4 x 10"3
Wabash    2.8 xlO5    2.2 x 103     6.1 x 106     5.7 x 105    3.6 xlO1    2.4 x 103    1.7 xlO"2     1.0 x 10"1
 River
Collec-    3.5xl03      <1.1        <5.0         <5.0        <1.1        <1.1
torWell      [1.9]        [>3.3]       [>6.1]       [>5.1]       [>1.5]       [>3.3]        [>1.1]        [>1.9]
Well #3   <2.0xl02      <2.0        <5.0         <5.0        <2.0        <2.0      <2.5 x 10'3   <2.5 x 10'3
                                    <1.3 x 10"3   <1.3 x 10"3
Missouri
River
Well #4
Well #5

4.2 xlO5
6.3 x 104
[0.8]
l.lxlO3
[2.6]
8.9 xlO2
<1.1
[2.9]
<2.5
[>2.6]
6.1 xlO5
<1.0xl01
<1.0xl01
[>4.8]
3.1xl04 3.4 xlO1
<1.0xl01 <1.1
<1.0xl01 <2.5
[>3.5] [>1.1]
2.6 x 103 2.4 x 1Q-2 6.3 x 10'2
<1.1 3.0] [>1.0] [>1.4]
             The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


                Evaluating Microbial Indicators and Health Risks
                            Associated  With Bank Filtration

                                           Floyd J. Frost
                            Lovelace Clinic Foundation, Albuquerque, NM

    The purpose of the proposed project is to compare serological responses to Cryptosporidium antigens in
users of bank-filtered water (one community with only bank filtration and disinfection and one community
with bank filtration, conventional filtration, and disinfection) with the responses of similar people residing in
an area that uses disinfected but unfiltered high-quality groundwater. The hypothesis is that, if bank filtration
completely removes  Cryptosporidium oocysts, the serological responses of the three populations should be
similar. The specific goals of the study are to: (1) identify approaches to collecting sera from similar popula-
tions in different geographic locations so that rates of serological responses can be compared; (2) pilot test the
approach in three different geographical locations by collecting sera from cities that use bank filtration and
nearby cities that use high-quality groundwater for a drinking water source; (3) analyze the sera for serological
responses to Cryptosporidium and Giardia antigens and compare the frequency and intensity of responses be-
tween the bank filtration cities and the groundwater cities; and (4) compare serological responses in the same
cities at times when bank filtration efficacy is likely to be optimal and when it is likely to be least effective.

    Sera from 50 people from each of three communities (users of bank filtered and chlorinated, bank filtered
plus direct filtration plus ozonation, and chlorinated groundwater) will be collected at baseline and at five fol-
lowup blood draws. A questionnaire on risk factors will be collected at each blood draw. Sera will be tested for
the presence of antibody responses to two Cryptosporidium antigens (15/17-kDa and 27-kDa) and for serologi-
cal changes (seroconversion). The baseline level of serological responses as well as the rates of seroconversion
will be compared for each population (50 baseline and 250 periods for estimating rates of seroconversion).
Comparisons will adjust for collected risk factor  data from each individual. For purposes of extrapolating these
results to other locations,  a  series of source and finished water quality indicators will be measured for each
water source.

    No results are available  at this time. Analysis of sera will take place once all sera are collected. Then, all
sera from a subject will be run on the same Western blot to reduce variations between blots. Blood draws will
continue every 4 months. Data entry protocols will be developed and implemented for data entry of the ques-
tionnaires. Sample analysis will commence once all of the samples are collected, because the analysis of each
subject's samples will be on  the same Western blot. The distribution systems analysis also will commence.
This abstract also was presented as a poster at the Research on Microorganisms in Drinking Water Workshop
in Cincinnati, OH, August 5-7, 2003.
           The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


 Application of a Multipath Microsphere Tracer Test To Understanding
        Transport of Bacteria and Protozoa at a Bank Filtration Site

                       Rick Langford1, Dirk Schulze-Makuch1, and Suresh Pillai2
                University of Texas, El Paso, TX; 2Texas A&M University, Kingsville, TX

    The objective of this research study is to determine whether bank filtration is effective in removing micro-
bial pathogens in an arid environment. The study site uses the Rio Grande that experiences significant annual
fluctuations in both water quantity and quality. A well-characterized site with numerous monitoring wells has
been established. The pumping well is 17 m from the stream bank. The water table during the experiment was
2 m below the land and the stream surface. The aquifer is composed of medium and fine-grained sand compris-
ing two flow units. Observation wells are screened over 1 or 1.5 m intervals. The average hydraulic conductiv-
ity was about 2x10"  m/s based on a test analysis. However, the responses indicated that sediment heteroge-
neities affected the hydraulic behavior at the field site.

    A 427-hour tracer test using bromide and fluorescent microspheres provides initial results that are relevant
to the transport of pathogens through the subsurface under riverbank filtration conditions. Bromide was in-
jected into an observation well at the channel margin. Differently colored fluorescent microspheres (0.25 jam,
1  jam, 6 |_un, and 10 jam) were injected into the stream bottom and into two observation wells. Conclusions
from the tracer test include the following:

•   Both bromide and microspheres continued to be observed throughout the 18 days of the  experiment.
•   The bromide recovery in the pumping well and in the deeper observation wells showed early and late
    peaks with long tails indicating that the geological medium at the field site behaves like a double-porosity
    medium, allowing the tracer to move relatively quickly through the higher conductivity units while being
    significantly retarded in the low hydraulic conductivity units.
•   Some wells showed consistently higher concentrations of bromide.
•   The 1 |_m micospheres were abundant in the observation wells and allowed tracing of flowpaths. These
    showed multiple peaks similar to the bromide results.  This indicates highly preferential transport paths in
    the sediment.
•   Microspheres from the three injection sites had distinctly different transport paths and rates.
•   Both bromide and microspheres  appeared in the stream soon  after injection, moving apparently against a
    2 m head difference.
•   The 6 |_m and 10 |_m microspheres were observed in low concentrations and were episodically detected in
    the stream and in two widely spaced observation wells.

The significance of these results is that:

•   Inorganic microspheres might mimic the episodic occurrence of microorganisms in wells.
•   Even in this relatively homogeneous aquifer, preferential transport within the aquifer results in highly di-
    vergent transport paths and rates. Microspheres from one of the injection sites traveled essentially perpen-
    dicular to the expected transport direction.
•   Even small variations in the sand grain size can effectively compartmentalize the aquifer.

    The next steps of this project will include field studies to observe the migration and persistence of selected
organisms (Escherichia coli, enterococci, coliphages, cysts, oocysts, and enteroviruses) in the pumping well
and observation wells under different pumping rates. Continued combined chemical sampling, along with the
microbial sampling, will document whether the changes in water chemistry alter the behavior of the organisms.
           The Office of Research and Development's National Center for Environmental Research

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                Research on Microorganisms in Drinking Water Progress Review Workshop
          Pathogenic Microbe Removal During  Riverbank Filtration

         Joseph N. Ryan1, Yumiko Abe1, Rula Abu-Dalo1, Menachem Elimelech2, Garrett Miller2,
                      Zachary Kuznar2, Ronald W. Harvey3 and David W. Metge3
           University of Colorado at Boulder, Boulder, CO; 2Yale University, New Haven, CT;
                                  U.S. Geological Survey, Reston, VA

Project Goals and Objectives

    Our incomplete understanding of processes and properties affecting the transport of pathogenic microbe
transport during riverbank filtration is currently limiting our ability to predict the effectiveness of this water
treatment option. We are conducting  a series of fundamental experiments designed to better understand the
effects of microbe size, physical and geochemical heterogeneity of the porous media, and high pumping rates
on the transport of Cryptosporidium parvum oocysts in alluvial valley aquifers used for riverbank filtration.

    Our major objective for this research is to develop a model of oocyst transport in porous media that can
accommodate the physical and geochemical heterogeneity present in alluvial valley aquifer used for riverbank
filtration. Our goal is that this model can be used to predict the oocyst removal  during riverbank filtration. To
do this, we are in the process of providing: (1) improved characterization of the properties of C. parvum oo-
cysts related to transport in porous media; (2) improved understanding of the mechanisms of oocyst removal in
porous media; (3) including special features of riverbank filtration in alluvial valley aquifers; and (4) incor-
porating the improved characterization and mechanistic understanding into a two-dimensional model of mi-
crobe transport during riverbank filtration.

Approach

    The experiments are being conducted in a stagnation point flow apparatus, flow-through columns, an inter-
mediate-scale two-dimensional aquifer tank (5 m length, 0.5 m height, 10 cm width). The stagnation point flow
experiments are being used to examine the effects of the surface charge heterogeneity and the DLVO secon-
dary minimum on oocyst attachment and release. The flow-through column experiments are being  used to ex-
plore the effects of grain-scale heterogeneities  on oocyst transport and to provide data for modeling of the
intermediate-scale tank experiments. The tank experiments are being used to examine random physical and
geochemical heterogeneities above the grain scale. The porous media being used in the column and tank ex-
periments are designed to simulate the complex and variable stratigraphies and geochemical gradients encoun-
tered in alluvial valley aquifers.

    The following tasks are being conducted to achieve the project objectives and goals: (1) stagnation point
flow and column experiments to test the effect of microbe size on attachment  to porous media, velocity en-
hancement, and straining; (2) column and tank experiments  to test the effect of grain size on attachment to po-
rous media, velocity enhancement, and straining;  (3) stagnation point flow, column, and  tank experiments to
test the effect of geochemical heterogeneity on attachment to porous media; and (4) stagnation point flow and
column experiments to test the effect of flow rate on oocyst deposition in  the DLVO secondary minimum and
release.

Preliminary Findings

    We have conducted column experiments and a tank experiment examining the effects of physical and geo-
chemical heterogeneity on oocyst transport. The tank was filled with a heterogeneous porous medium consist-
ing of sands of 5 grain sizes and 12 ferric oxyhydroxide surface coverages. Oocyst transport in each of the
porous media was tested in column experiments. Oocysts (formalin-inactivated) and polystyrene latex micro-
spheres (4.6 |am diameter) were injected into the tank and monitored over 5 days. Physical heterogeneity (the
difference in hydraulic conductivity as a result of grain size) was much more important than geochemical het-
           The Office of Research and Development's National Center for Environmental Research

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                Research on Microorganisms in Drinking Water Progress Review Workshop
erogeneity in controlling oocyst transport. Micro spheres broke through at the same time as the oocysts, but the
microspheres were removed much more rapidly than the oocysts.

    In column experiments, we showed that straining is contributing to the removal of oocysts in fine-grained
sands. Straining was demonstrated by comparing the transport of polystyrene latex microspheres (0.32 to
4.1  |am) to oocyst transport. Removal was consistent for microspheres from 0.32 to 1.9 jam and increased sig-
nificantly for 4.1 |am microspheres (see Figure  1).

    Stagnation point flow experiments have been conducted to explore the effect of ionic strength on oocyst
deposition. The dynamics of oocyst deposition in these experiments—the oocysts come into contact with the
glass deposition surface, but do not remain in the position of first contact—clearly indicate that the oocysts are
depositing in the secondary minimum of the DLVO potential energy profile.

Significance of Findings

    The physical and geochemical heterogeneity experiments show that oocyst transport modeling can focus
on variations in the hydraulic conductivity of porous media. Geochemical heterogeneity is far less important
because oocysts deposit equally well on unfavorable and favorable surfaces. The straining and secondary mini-
mum experiments show that: (1) straining must be considered in oocyst transport modeling; (2) oocyst deposi-
tion depends on solution ionic strength; and (3) oocyst deposition is reversible.

Next Steps

    The next steps in this research project will focus on column experiments to explore the effects of microbe
size, grain size, and flow rate on oocyst deposition, release, and velocity enhancement.
     Figure 1.
               0.00
                                    1000         2000         3000
                                          Time  (seconds)
                                                                     4000
Breakthrough of carboxyl-modified polystyrene  latex microspheres  through  a  column
filled with quartz sand (d50 of 0.210 mm) as a function of time. Microsphere concentration
normalized to the influent microsphere concentration (C/C0). Diameters of the micro-
spheres shown above the breakthrough curves. The experiments were conducted  at a pH
of 5.6-5.8, an ionic strength of 1 mM, and a flow rate of 2 mL min"1.
           The Office of Research and Development's National Center for Environmental Research

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              Research on Microorganisms in Drinking Water Progress Review Workshop
                              NCER Calendar of Events

                                   September 2003

Title:        The U.S. Environmental Protection Agency / U.S. Geological Survey Meeting on
             Cryptosporidium Removal by Bank Filtration

Date:        Tuesday, September 9 - Wednesday, September 10, 2003

Location:    U.S. Geological Survey National Center
             Main Auditorium
             12210 Sunrise Valley Drive
             Reston, VA 20192

             See Logistics below for information regarding hotel reservations at the
             Sheraton Reston Hotel, Reston, VA.
             Please make your reservations by Wednesday, August 27, 2003.

Contact:     Angela Page, (202) 564-5172 or page.angelad@epa.gov
             Philip Berger (202) 564-5255 or berger.philip@epa.gov and
             Tina Conley (202) 564-3209 or conley.tina@epa.gov

Purpose:     The U.S. Environmental Protection Agency (EPA) Office of Research and
             Development, National Center for Environmental Research (NCER) and the
             EPA's Office of Water together with the U.S. Geological Survey (USGS) are
             sponsoring a meeting to discuss the research being conducted on
             cryptosporidium removal by bank filtration. The USEPA/USGS Meeting on
             Cryptosporidium Removal by Bank Filtration will be held on September 9-10,
             2003 at the USGS facility in Reston, VA.  The meeting will consist of a series of
             plenary sessions where EPA, NCER's Science To Achieve Results (STAR) grant-
             ees, USGS, U.S. Department of Agriculture, university and state researchers will
             present their research. This public meeting is open to all who are interested in
             hearing about the research in this exciting area.

             Please mark your calendars now and make your hotel reservations by August 27.

Registration: Fill out the Registration Form at:
             http://www.scgcorp.eom/epachildhealth2003/registration.asphttp://www.scgcorp.
             com/USEPA_USGS/registration.asp
         The Office of Research and Development's National Center for Environmental Research

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	Research on Microorganisms in Drinking Water Progress Review Workshop	


    The U.S. Environmental Protection Agency/U.S. Geological Survey Meeting
                   on Cryptosporidium Removal by Bank Filtration

                           U.S. Geological Survey National Center
                                 12201 Sunrise Valley Drive
                                     Reston, VA  20192

                                   September 9-10, 2003

                                         AGENDA

Tuesday, September 9,2003

10:00 - 10:15 a.m.    Welcome
                    James LaBaugh, USGS Office of Ground Water

10:15 - 10:30 a.m.    Overview of the U.S. EPA's Office of Research and Development and The Science
                    To Achieve Results (STAR) Program
                    Cynthia Nolt-Helms, EPA, Office of Research and Development

10:30 — 10:45 a.m.    Overview Presentation From the U.S. EPA's Office of Ground Water and Drinking
                    Water
                    Dan Schmelling, EPA, Office of Ground Water and Drinking Water

10:45 - 11:00 a.m.    Overview of the U.S. EPA's Water Security Program
                    Regan Murray, EPA, Homeland  Security Research Center

11:00 - 2:00 p.m.     Field Studies of Cryptosporidium, Surrogate and Indicator Transport in
                    Saturated Porous Media
                    Moderator: Glenn Patterson, USGS, Reston, VA

                    11:00 - 11:25 a.m.  Initial Results From the Rio Grande Bank Filtration Site
                                      Presented by Rick Langford, University of Texas, El Paso, TX,
                                      STAR Grant

                    11:25 - 11:50 a.m.  Using Riverbank Filtration To Improve Water Quality
                                      Presented by Ed Bouwer, The Johns Hopkins University,
                                      Baltimore, MD, STAR Grant

11:50-12:50 p.m.    Lunch

                    12:50 - 1:15 p.m.   Bank Filtration Studies at the City of Lincoln, Nebraska
                                      Wellfield
                                      Research conducted by Jason Vogel, USGS; Presented by
                                      Philip Berger, EPA,  Washington, DC

                    1:15-1:40 p.m.    Assessment of the Microbial Removal Capabilities of Riverbank
                                      Filtration
                                      Presented by Robin Collins, New England Water Treatment
                                      Technology Assistance Center at the University of New
                                      Hampshire, Durham,  NH, Funded by EPA
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


                      1:40-2:00 p.m.    General Discussion

2:00-2:15 p.m.       Break

2:15 - 3:45 p.m.       Laboratory and Simulation Studies of Cryptosporidium, Surrogate and Indicator
                      Transport in Saturated Porous Media
                      Moderator: Ingrid Verstraeten, USGS, Baltimore, MD

                      2:15 - 2:40 p.m.    Effect of Heterogeneity on Transport of Cryptosporidium
                                        parvum in Saturated Porous Media
                                        Presented by Joe Ryan, University of Colorado, Boulder, CO,
                                        STAR Grant

                      2:40 - 3:05 p.m.    Cryptosporidium Transport in Porous Media
                                        Presented by Scott Bradford, USDA Salinity Laboratory,
                                        Riverside, CA

                      3:05 — 3:30 p.m.    Streamline-Based Simulation of Cryptosporidium Transport
                                        in Riverbank Filtration
                                        Presented by Reed Maxwell, Lawrence Livermore National
                                        Laboratory, Livermore, CA

                      3:30-3:45 p.m.    General Discussion

3:45-4:00 p.m.       Break

4:00 - 5:00 p.m.       Estimating Cryptosporidium Removal and Health Effects
                      Moderator: John Grace, Maryland Department of the Environment, Baltimore, MD

                      4:00 — 4:25 p.m.    Cryptosporidium Removal at the Louisville, Kentucky Wellfield
                                        Presented by Steve Hubbs, Louisville Water Company,
                                        Louisville, KY

                      4:25 - 4:50 p.m.    Serological Monitoring of Pathogen Occurrence
                                        Presented by Floyd Frost, Lovelace Respiratory Research
                                        Institute, Albuquerque, NM, STAR Grant

                      4:50-5:00 p.m.    General Discussion

5:00 p.m.             Adjournment


Wednesday, September 10,2003

9:00 - 11:45 a.m.      Ground Water Flow, Heat Flow, and Environmental Tracer Studies at Bank
                      Filtration Sites
                      Moderator: Tom Grubbs, EPA, Washington, DC

                      9:00 - 9:25 a.m.    Infiltration Rate Variability and Ground Water Flow at the
                                        Cincinnati Wellfield
                                        Presented by Bill Gollnitz, Greater Cincinnati Water Works,
                                        Cincinnati, OH
           The Office of Research and Development's National Center for Environmental Research

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               Research on Microorganisms in Drinking Water Progress Review Workshop
                     9:25 — 9:50 a.m.    Application of Different Tracers To Evaluate the Flow Regime
                                        at Riverbank Filtration Sites in Berlin Germany
                                        Presented by Gudrun Massmann, Free University of Berlin,
                                        Berlin, Germany

                     9:50 - 10:15 a.m.   Diatom Proteins as a Surface Water Indicator in Ground Water
                                        Presented by Tim Reilly, USGS, Trenton, NJ

10:15-10:30 a.m.    Break

                     10:30 - 10:55 a.m.  Inclined Well Studies at the Cincinnati Wellfield
                                        Presented by Bruce Whitteberry, Greater Cincinnati Water
                                        Works, Cincinnati, OH
                     10:55 - 11:20 a.m.  Heat as a Tracer at Sonoma County Bank Filtration Site
                                        Presented by Jim Constantz, USGS, Menlo Park, CA (via
                                        PlaceWare)

                     11:20 - 11:45 a.m.  General Discussion

11:45- 12:45 p.m.    Lunch

12:45- 3:55 p.m.

                     Moderator: Mike Finn, EPA, Washington, DC

                     12:45 — 1:10 p.m.   Cryptosporidium Transport in Soil-Aquifer Treatment
                                        Presented by William Blanford, Louisiana State University,
                                        Baton Rouge, LA, STAR Grant (to University of Arizona)

                     1:10 — 1:35 p.m.    Cryptosporidium Transport in Unsaturated Flow
                                        Presented by Christophe Darnault, Environmental Engineering
                                             &
                                        Technology, Inc., Newport News, VA

                     1:35 - 2:00 p.m.    Comparison of Batch and Flow Experimental Data on Retention
                                        of Manure-Borne Cryptosporidium parvum Oocysts in Soils
                                        Presented by Yakov Pachepsky, USDA, Beltsville, MD

2:00-2:15 p.m.       Break

                     2:15 - 2:40 p.m.    Release of Cryptosporidium and Giardia Dairy Manure Due
                                        to Flowing Water
                                        Presented by Scott Bradford, USDA Salinity Laboratory,
                                        Riverside, CA

                     2:40 - 3:05 p.m.    Unsaturated Zone Processes in the Sonoma County Recharge
                                        Basins
                                        Presented by Jim Constantz, USGS, Menlo Park, CA (via
                                        PlaceWare)

                     3:05 — 3:30 p.m.    One-Dimensional Variably Saturated Microbial Transport
                                        Simulations
                                        Presented by Bart Faulkner, EPA, Ada, OK
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	Research on Microorganisms in Drinking Water Progress Review Workshop	


                     3:30-3:55 p.m.    General Discussion

3:55-4:10 p.m.      Break

4:10 - 4:50 p.m.      Cryptosporidium Removal by Bank Filtration Summary, Regulations and Caveats
                     Moderator: Ronald Payer, U.S. Department of Agriculture, Beltsville, MD

                     4:10 - 4:35 p.m.    Cryptosporidium Removal by Bank Filtration Summary,
                                        Regulations, and Caveats
                                        Presented by Philip Berger, EPA, Washington, DC

                     4:35 - 4:50 p.m.    General Discussion and Closing Remarks

4:50 p.m.            Adjournment
           The Office of Research and Development's National Center for Environmental Research

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