EPA 822-R-09-002



## REVIEW OF ZOONOTIC PATHOGENS

IN AMBIENT WATERS

U.S. Environmental Protection Agency

O ffice of Water Health and Ecological Criteria Division

February 2009[this page intentionally left blank]U.S. Environmental Protection Agency

February 2009 i 

## D ISCLAIMER

This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Mention of trade names or commercial products does not

constitute endorsement or recommendation for use.[this page intentionally left blank]U.S. Environmental Protection Agency

February 2009 iii 

## A CKNOWLEDGMENTS

Questions concerning this document or its app lication should be addressed to the EPA Work Assignment Manager:

John Ravenscroft USEPA Headquarters Office of Science and Technology, Office of Water 1200 Pennsylvania Avenue, NW

Mail Code: 4304T Washington, DC 20460

Phone: 202-566-1101

Email: ravenscroft.john@epa.gov

This literature review was managed under EPA Contract EP-C-07-036 to Clancy Environmental Consultants, Inc. The following individuals c ontributed to the development of the report:

Lead writer: Audrey Ichida ICF International

Leiran Biton ICF International

Alexis Castrovinci ICF International Jennifer Clancy Clancy Environmental Consultants Mary Clark ICF International

Elizabeth Dederick ICF International Kelly Hammerle ICF International

Walter Jakubowski WaltJay Consulting Margaret McVey ICF International

Michelle Moser ICF International Jeffery Rosen Clancy Environmental Consultants

Tina Rouse ICF International

Jennifer Welham ICF International Kerry Williams ICF International[this page intentionally left blank]U.S. Environmental Protection Agency

February 2009 v 

## T ABLE OF 

## C ONTENTS

A CKNOWLEDGMENTS

.............................................................................................................................. ................... iii

T ABLES AND F IGURES

.............................................................................................................................. ................... vii

A CRONYMS

.............................................................................................................................. ..................................... ix

E XECUTIVE S UMMARY

.............................................................................................................................. ................... 1

I. B ACKGROUND AND I NTRODUCTION .................................................................................................................... 5

I.1 Background: Cont ext and Purpose .............................................................................................. .............. 5

I.2 Introduc tion

.................................................................................................................. .............................. 6

II. K EY W ATERBORNE Z OONOTIC P ATHOGENS OF C ONCERN ............................................................................... 8

II.1 Bacter ia ...................................................................................................................... ................................ 8

II.1.1 Escherichia coli

............................................................................................................................. 8

II.1.2 Campylobacter ............................................................................................................................ 1 2

II.1.3 Salmonella

.............................................................................................................................. ..... 14

II.1.4 Leptospira

.............................................................................................................................. ...... 17

II.2 Protozoa ...................................................................................................................... ............................. 19

II.2.1 Cryptosporidium .......................................................................................................................... 19

II.2.2 Giardia

.............................................................................................................................. .......... 26

II.3 Viruses Zoonotic Potential .................................................................................................... ................... 33

III. P ATHOGEN I NTERACTIONS WITH THEIR E NVIRONMENT ................................................................................. 37

III.1 Water Environment Affects Pathogen Survivability a nd Phenotype ....................................................... 37

III.1.1 Pathogenic E. coli Survival in th e Environment .......................................................................... 38

III.1.2 Campylobacter Survival in the Environment .............................................................................. 39

III.1.3 Salmonella Survival in th e Environment ..................................................................................... 39

III.1.4 Leptospira Survival in the Environment ...................................................................................... 39

III.1.5 Cryptosporidium Survival in th e Environment ............................................................................ 40

III.1.6 Giardia Survival in th e Environment .......................................................................................... 43

III.1.7 Virus Survival in the Envi ronment ..................................................................................... ......... 44

III.2 Host Animals Can Influence Pathogen Character istics and Mechanisms of Rapid Evolution ................ 44

IV. S UMMARY

.............................................................................................................................. ............................. 49

V. R EFERENCES

.............................................................................................................................. ........................ 50

A PPENDIX A: W ATERBORNE P ATHOGENS .............................................................................................................. A-1

A PPENDIX B: L ITERATURE S EARCH S TRATEGY AND R ESULTS ............................................................................. B-1

A PPENDIX C: I NCIDENTAL I NGESTION OF A MBIENT W ATER D URING R ECREATIONAL A CTIVITIES ................... C-1[this page intentionally left blank]U.S. Environmental Protection Agency

February 2009 vii 

## T

ABLES AND 

## F IGURES

Table II.1.1-1. Outbreaks of E. coli Associated with Recreational Wa ters in the Unite d States. ........................ 12

Table II.2.1-1.

Table II.2.1-2.

Table II.2.2-1.

Table II.2.2-2.

Table II.2.2-3. Cryptosporidium Species ............................................................................................................. 20

Outbreaks of Cryptosporidiosis Associated with Recreational Waters in the United States. ...... 27

Giardia Taxonomy ..................................................................................................................... . 28

Giardia Infection Occurrence in U.S. Populations ...................................................................... 32

Outbreaks of Giardiasis Associated w ith Recreational Waters in the United States. .................. 34

Table III.1.1-1. Survival of E. coli O157:H7 in Ambi ent Waters ......................................................................... 38

Figure II.2.1-1.

Figure II.2.2-1. Reported Cryptosporidium infections in the United St ates, 1999 to 2005. ................................. 26

Reported Giardia cases in the 50 states plus Wa shington DC, 1992 to 2005. ............................ 32[this page intentionally left blank]U.S. Environmental Protection Agency

February 2009 ix 

## A CRONYMS

AEC attaching E. coli

AIDS Acquired Immune Deficiency Syndrome AWQC ambient water quality criteria

BEACH Act Beaches Environmental Assessm ent and Coastal Health Act of 2000

CDC U.S. Centers for Disease Control and Prevention

CPV C. parvum virus

CWA Clean Water Act

DAEC diffuse adherent E. coli

DEC diarrheagenic E. coli EAggEC enteroaggregative E. coli

EEC effacing E. coli

EHEC enterohemorrhagic E. coli

EPA U.S. Environmental Protection Agency

EPEC enteropathogenic E. coli

ETEC enterotoxigenic E. coli

GI gastrointestinal

GLV Giardia lamblia virus

GBS Guillain-Barré Syndrome

HC hemorrhagic colitis

HEV hepatitis E virus

HUS hemolytic uremic syndrome

IPSID immunoproliferative small intestinal disease

MALT mucosa-associated lymphoid tissue

NA not available or not applicable

PCR polymerase chain reaction

POTW Publicly Owned Treatment Works

ppt parts per thousand (salinity)

RFLP restriction fragment length polymorphism

SPE serial passage experiment

STEC Shiga toxin-producing E. coli

U.S. United States

U.K. United Kingdom

USDA U.S. Department of Agriculture

UV ultraviolet (light)

VTEC verocytotoxin-producing E. coli

WHO World Health Organi zation (United Nations)[this page intentionally left blank]U.S. Environmental Protection Agency

February 2009 1 

## E XECUTIVE 

## S UMMARY

Introduction

The overall goal of the current Clean Water Ac t (CWA) §304(a) ambient water quality criteria (AWQC) for bacteria in the United States is to provide public health protection from gastrointestinal (GI) illn ess (gastroenteritis) associated w ith exposure to fecal contamination during recreational water contact. Water quality criteria are specified throughout the world in

terms of concentrations of fecal indicator organisms because f ecal matter can be a major source of pathogens in ambient water a nd because it is not practical or feasible to monitor for the full spectrum of all pathogens that may occur in water. For decades, these fecal indicator organisms have served as surrogates for pot ential pathogens and subsequent health risks in both recreational and drinking waters.

The U.S. Environmental Protection Agency (EPA) currently recommends AWQC for recreational water that encompass all fecal sources that contain the relevant indicator species (enterococci and E. coli ). This approach assumes that anim al fecal material is as hazardous as

human fecal material. There is limited evidence , however, that recreational water contaminated

with animal fecal material is le ss risky to swimmers than recr eational water contaminated with

human fecal material.

In order to evaluate the potential risks posed by animal fecal contamination, EPA is interested in understanding what human illnesses are caused by swimming in waters contaminated with animal fecal material ranging from wildlife source s to agricultural inputs. The animal species of

most interest are the warm-bl ooded animals (mammals and bird s) whose fecal material is detected by current indicators.

The purpose of this white paper is to provide a summary of in formation on waterborne zoonotic pathogens that come primarily from warm-bl ooded animals, and whic h can be used to conceptualize potential risks from warm-bl ooded animal feces in ambient (untreated)

recreational waters.

Approach

Seventy pathogens from warm-blooded animals we re evaluated for their potential to be both waterborne and zoonotic. Twenty of the 70 pa thogens evaluated had all 4 of the following

attributes:

1. The pathogen spends part of its lifecycle within one or more warm-blooded animal species.

2. Within the lifecycle of the pathogen, it is probable or conceivable that some life stage will

enter water.
3. Transmission of the pathogen from animal s ource to human is th rough a water related

route.
4. The pathogen causes infection or illness in humans.U.S. Environmental Protection Agency

February 2009 2 Six of the 20 waterborne, zoonot ic pathogens from warm-bloode d animals were selected for

further discussion based on their re levance in the United States. Five were selected based on their potential for outbreaks in ambient (untreated) recreational water and one ( Salmonella ) was included based on outbreaks in drinking water.

Some well-known waterborne pathogens were ex cluded from analysis because they are not zoonotic. Excluded pathogens include bacteria thatare generally found in the environment, freeliving protozoa, viruses, and helminthes that have cold-blooded hosts (e.g., snails, copepods). Some common zoonotic pathogens were also excluded because they do not have welldocumented waterborne transmission (i.e., prim arily transmitted via soil, food, or drinking water).

Pathogens interact with the ambient environment, other microorganisms, plants, and with their hosts. The behavior of pathogens in ambient waters is often different from the behavior of

indicators in ambient water. The most common environmental factors studied for their impact on pathogen survival in water are pH, salinity, light exposure, and temperature. Additional environmental characteristics that may influenc e pathogen survival, infectivity, and virulence include the following: ultraviole t (UV) light (durati on, intensity), rainfall, runoff, dispersal, suspended solids, turbidity, nutrients, organic c ontent, organic foams, water quality, biological community in water column, water depth, stra tification, mixing (e.g., wind and waves), presence of aquatic plants, biofilms, and predation.

There is evidence that zoonotic pathogens may cha nge in infectivity, virule nce, and the severity of disease caused in humans depending on thei r previous host environm ent. There is also evidence that some of these host-factor change s can influence subsequent infection cycles in exposed hosts. The key mechanisms of phenotypi c change in pathogens are genetic diversity (coinfection and quasispecies), cryptic genes, mutators, and epigenetic effects.

Six Key Waterborne Zoonotic Pathogens

Pathogenic E. coli

E. coli is an important waterborne bacterial z oonosis because many human pathogenic strains

occur in livestock and wildlife f eces and can survive in ambient waters. In addition, the potential illnesses caused by pathogenic E. coli can be severe or fatal. Mortality estimates range from 0.08 to 1.9 percent of E. coli O157:H7 infections. Children are mo re at risk than healthy adults

to suffer more severe outcomes. Between 1991 and 2004, 14 outbreaks of E. coli -related illness

have been associated with ambient recreational waters. E. coli O157:H7 can survive for at least

several weeks in animal feces and slurries and ha s been demonstrated to survive at least 500 days

at -20 ° C in frozen soil.

Campylobacter Campylobacter is a well-known foodborne ba cterial pathogen that is commonly associated with poultry and livestock. There are also wildlife hosts. Although few waterborne outbreaks have been reported, there is potential for Campylobacter to cause recreational water-related illness. Normally, infection results in diarrhea that is self-limiting; however, approximately 1 out of 1,000 infections results in Guillain-Barré syndrome, which is a serious nervous system affliction.U.S. Environmental Protection Agency

February 2009 3 Reactive arthritis is also a possible chronic sequela of Campylobacter infection. Campylobacter has been shown to survive in aquatic environmen ts with low temperatures (4°C) up to 4 months.

Salmonella

Salmonella is also a well-known foodborne bacterial pa thogen that is associated with poultry and

livestock. There are also wildli fe hosts. Elevated levels of Salmonella have been observed in major water bodies that receive discharges of meat processing wastes, raw sewage, farming operations, and effluents from ineffective sewage treatment plants. The clinical symptoms of salmonellosis may include diarrhea, abdominal pa in, nausea, chills, and fever. Between 1991 and 2002, 3 waterborne outbreaks we re attributed to nontyphoid Salmonella ; Salmonella were the etiologic agent in 0.9 percent of 259 recreational waterborne outbrea ks occurring from 1971 to 2000. Under suitable environmental conditions, Salmonella can survive for weeks in waters or years in soils.

Leptospira Leptospira is an important waterborne bacterial pa thogen for most of the world. Because

warmer climates favor its survival in the enviro nment, the highest incide nce of leptospirosis in

the United States is found in Hawaii. The source of infection in humans is usually either direct

or indirect contact with the urine of an in fected animal. There are numerous symptoms associated with leptospirosis, but the more seve re form is known as Weil’s disease. In addition, acute infection during pregnancy has been reported to cause abortion and fe tal death. From 1971 to 2000, 16 percent of recreationa l waterborne disease outbreaks in the United States were

attributed to Leptospira. In 1998, an outbreak (375 cases) of leptospirosis was reported that was

associated with a triathlon in a lake in Illinois.

Cryptosporidium Cryptosporidium is a well-known waterborne protozoan pathogen. EPA currently regulates it under the Safe Drinking Water Act because the level of Cryptosporidium in many ambient source waters is sufficiently high that ri sks to drinking water must be managed. Cryptosporidium infection has been reported in more than 155 mammalian species and numerous reptiles, amphibians, birds, and fish. In watersheds with diverse land-use pa tterns, it is likely that

different sources contribute diffe rent proportions to the total co ntamination load at different times during the year, with the relative contri butions depending on a wide range of watershed characteristics. Cryptosporidiosis is primarily characterized by GI symptoms such as profuse, watery diarrhea. Immunocompromised individuals generally experience ch ronic gastroenteritis, which may last as long as the immune im pairment. The largest known outbreak of cryptosporidiosis occurred in 1993 in Milwaukee, Wisconsin and infected 403,000 individuals. Animal fecal contamination of drinking water wa s indicated in the outbreak. In the United States, from 1991 to 2004, 6 outbreaks of cryptospor idiosis have been associated with untreated

recreational waters. Excreted Cryptosporidium oocysts can survive for substantial periods in animal wastes and soils. Thus, contaminated runoff can enter ambient water and result in potential human exposures. The majority of ooc ysts (99 percent) are inactivated by repeated freeze-thaw cycles; therefore, Cryptosporidium may be environmentally limited in parts of the United States during winter months. Between 4 a nd 20° C, there is very little inactivation of oocysts in different types of agricultural soils. Oocyst survival in various water matrices is highly variable, but survival for longer than 30 da ys has been demonstrated in several studies.U.S. Environmental Protection Agency

February 2009 4 Giardia

Giardia is also a well-known wate rborne protozoan pathogen. Although both livestock and humans have been implicated in contaminating water sources with Giardia , humans are responsible for the majority of the contaminations. Zoonotic transfer plays only a minor role in the infection cycles of Giardia , and animal contact is not a majo r risk factor. There is a wide spectrum of symptoms associated with giardiasis that ranges from asymptomatic infection and acute self-limiting diarrhea to persistent chroni c diarrhea, which sometimes fails to respond to treatment. Asymptomatic infection is very common, with 50 to 75 percent of infected persons reporting no symptoms. In the United Stat es, from 1991 to 2004, 7 reported outbreaks of giardiasis were associated with untr eated recreational waters. At 4° C, Giardia cysts were infective for 11 weeks in water, 7 weeks in soil, and 1 week in cattle feces.

Summary

Although the most common waterborne recreationa l illnesses are probabl y due to nonzoonotic human viruses, which typically cause short-term gastroenteri tis, the waterborne zoonotic pathogens discussed in this report have the pote ntial to cause serious health effects—especially in immunocompromised persons and subpopulations. While serious health outcomes are likely to be rare in comparison with self-limiting illnesses as a result of ambient (recreational) water exposure, the adverse health impacts of the rare, but more serious illnesses remain an important public health challenge.U.S. Environmental Protection Agency

February 2009 5 

## I. B ACKGROUND AND 

## I NTRODUCTION

I.1 Background: Context and Purpose

Since the U.S. Environmental Protection Agency (hereafter EPA or the Agency) last published recreational water quality criteria in 1986, there ha ve been significant scie ntific and technical advances, particularly in the ar eas of molecular biology, microbiol ogy, and analytical chemistry. EPA believes that these advances need to be considered and evaluated for feasibility and applicability in the development of new or revised CWA §304(a) criteria for recreational water. To this end, EPA has been conducting research and assessing relevant info rmation to provide the scientific and technical foundation for the development of new or revised criteria. The

enactment of the Beaches Environmental Assess ment and Coastal Health (BEACH) Act of 2000 (which amended the CWA) required EPA to conduc t new studies and to issue new or revised

criteria for Great Lakes and coastal marine waters.

In response to the BEACH Act of 2000, EPA al so has engaged a range of stakeholders representing the general public, public interest groups, state and local governments, industry, and municipal wastewater treatmen t professionals. In March 2007, EPA convened a group of 43

national and international tec hnical, scientific, and implementation experts from academia, numerous state agencies, public interest groups, EPA, and other federal agencies at a formal workshop to discuss the state of the science on r ecreational water quality research and criteria implementation. Among the input from the indivi duals attending the workshop were suggestions for incorporating the ability to differentiate sour ces of fecal contamination and to determine the

relative human health risk from these sour ces into the new or revised criteria.

Based on the feedback from the large group of stakeholders, as well as input and

recommendations from the scientific community, the Agency has developed a Critical Path Science Plan for Development of New or R evised Recreational Water Quality Criteria . One of the key questions posed in the science plan as ks: what is the risk to human health from swimming in water contaminated with human f ecal matter as compared to swimming in water contaminated with nonhuman fecal matter? Human and animal feces can both potentially contain pathogens that cause human illness. Some human pathogens ar e host-specific (i.e., human enteric viruses), while other human pa thogens are found in and can be shed by both humans and other animals. Moreover, while all enteric pathogens of huma ns are infectious to other humans, only a subset of the enteric pat hogens of animals is infectious to humans. Understanding which pathogens could be present depending on the source of fecal contamination might allow the Agency to better estimate human health risks from identified sources of fecal

matter.

EPA’s current recommended recreational water qua lity criteria for microbes treat human health

risks from the various sources of fecal contam ination as equivalent (USEPA, 1986). These

criteria are based on the risk of illness from sw imming in waters influenced by sewage treatment plants effluents. Health risk s from other sources (e.g., poorly-tr eated or untreated human waste, nonhuman sources of fecal contamination, mixed sour ces such as urban stormwater runoff) were

not well understood at the time the 1986 criteria were developed, a nd EPA’s approach was to be protective of human health rega rdless of the source. EPA rec ognizes, however, th at the healthU.S. Environmental Protection Agency

February 2009 6 risk from sources other than sewage treatment plants may be different and that the scientific

advances over the interv ening years may now allow the Agency to better characterize the relative

risks to human health from these various sour ces of fecal contamination. Specifically, the Agency is interested in understanding which human illnesses can be caused by swimming in

waters contaminated with nonhuman fecal matter in cluding both wildlife sources and agricultural inputs. The animal species of most interest are the warm-blooded animals (mammals and birds) for which fecal matter is detectable by current fecal indicators.

The purpose of this white paper is to provide summary information on waterborne zoonotic pathogens that come primarily from warm-bl ooded animals, and whic h can be used to conceptualize potential risks to humans from warm -blooded animal feces in recreational waters.

I.2 Introduction

In the development of health protective criteria for recreational waters, pathogen contamination is the central concern, and fecal matter can be a major source of pathogens in ambient water. Current recreational AWQC are designed specifically to protect humans from GI illness (gastroenteritis) associated with exposure to f ecal contamination in recreational waters (USEPA, 1986). Because widespread monito ring of recreational waters dire ctly for all disease-causing microorganisms (especially pathogenic bacteria, vi ruses, and protozoa) re mains infeasible, public health and environmental protection agencies ha ve relied on the detecti on of fecal indicator organisms, which comprise a few groups of nonpat hogenic fecal bacteria and some viruses, to indicate the presence and magnit ude of fecal material. This a pproach assumes that waterborne pathogens co-occur with the fecal material. “Mor e specifically, fecal indi cator bacteria provide an estimation of the amount of feces, and indir ectly, the presence and quantity of fecal pathogens in the water” (NRC, 2004). Therefore, use of bacterial indicators is predicated on the presumption that there are no significant environm ental sources of these microorganisms (i.e., nonenteric sources). However, this presumption is not entirely valid; fe cal indicator organisms

have been demonstrated to have natural reserv oirs in the aquatic environment where they can survive for extended periods and even proliferate (e.g., fecal indicator bacteria in U.S. coastal [Yamahara et al., 2007] and Great Lake wa ters and sand [Whitman et al., 2006]).

Historically, EPA has recommended that recreationa l water quality criteria encompass all fecal sources of pathogens that contain the relevant indicator microbes. This recommendation is based on the regulatory premise that animal-der ived (zoonotic) human pathogens in fecally

contaminated waters are as hazardous as their human-derived counterparts (Schaub, 2004). This presumption is supported by current research th at confirms that ther e are many waterborne zoonotic bacteria and protozoa common to both humans and animals, especially mammals (WHO, 2004). Research also suggests, howev er, that there may be some attenuation of infectivity, virulence, and diseas e severity to humans from animal-derived human pathogens (see Section III.2). In addition, there are many pa thogens that could be zoonotic, fecal, and/or waterborne, but these routes of transmission have not yet been conclusively demonstrated.

Given the scientific advancements in pathoge n characterization sinc e EPA’s 1986 AWQC were released, it is appropriate to examine the br oadest array of curren tly known and suspected waterborne, fecal, and zoonotic pathogens and their human health impacts. These includeU.S. Environmental Protection Agency

February 2009 7 primarily bacteria, viruses, protozoa, and helm inths. There are many zoonotic pathogens and many waterborne pathogens; however, there is a mo re limited subset of pathogens that are both. For this report, the following attributes were used to select the waterbor ne, zoonotic pathogens of concern (partially adapted fr om Bolin et al., 2004a):

1. The pathogen must spend part of its lifecycl e within one or more warm-blooded animal species.

2. Within the lifecycle of the pathogen, it is probable or conceivable that some life stage will

enter water.
3. Transmission of the pathogen from animal source to human must be through a water

related route. There are zoonotic pathogens for whic h waterborne exposure has not been detected as a significant route of cross-spec ies transmission. This does not exclude the possibility that these zoonotic pathogens could be transmitted via water.

4. The pathogen must cause infect ion or illness in humans. There are animal pathogens that

have waterborne transmission between anim als yet are not known to cause illness in

humans.

There are many waterborne zoonotic pathogens that exhibit all four attrib utes listed above. See Appendix A, Table A-1, for a summary of waterborne pathogens that meet the above criteria and selected pathogens that meet some, but not all, of the above criteria.

The remainder of this paper is organized into tw o main sections. Section II characterizes six key groups of zoonotic pathogens for which there is ev idence of human health risks from recreational exposure via ambient waters. Section III presents an overview of how the key pathogens interact with their environment, which includes both the water environment and the host animals.U.S. Environmental Protection Agency

February 2009 8 

## II. K EY 

## W ATERBORNE 

## Z OONOTIC 

## P ATHOGENS OF 

## C ONCERN

Based on a review of the literature and other information sources (Appendix B), 6 of the 20 pathogens identified as waterborne and zoonotic from warm-blooded animals stood out as having the most evidence for human health impacts due to recreational exposures in the United States. The six pathogens discusse d in this paper in more detail are pathogenic E. coli

, Campylobacter

, Salmonella

, Leptospira

, Cryptosporidium , and Giardia . This list correlates well with the top five waterborne pathogens for recreati onal and drinking waters. The top five waterborne pathogens

for recreational water are E. coli

, Campylobacter

, Leptospira

, Cryptosporidium , and Giardia

, while the top five for drinking water are E. coli

, Campylobacter, Salmonella

, Cryptosporidium

, and Giardia (Craun et al., 2004a)

. The waterborne zoonosis potential of viruses also is discussed because viruses may be emerging waterborne zoonoses.

For each of the six key pathogens, information on strain variation, known zoonoses, route(s) of exposure, illness symptoms, and disease incidence are discussed in the sections that follow. Because incidental ingestion is the primary route of recreational exposure to pathogens, summary information on incidental in gestion is provided in Appendix C.

II.1 Bacteria

II.1.1 Escherichia coli

E. coli is an important waterborne zoonosis becaus e human pathogenic strain s occur in livestock

and wildlife feces and can survive in ambient wate r. In addition, the potential illnesses caused by pathogenic E. coli can be severe or fatal, especially in immunc ompromised persons and subpopulations. Pathogenic E. coli is also a well-documente d foodborne pathogen (USDA, 2001). Although pathogenic E. coli have been found in treated wastewater effluents in the United States (Boczek et al., 2007), waterborne outbreaks are not as prevalent as foodborne

cases.

E. coli Strain Variation

Benign strains of E. coli are a part of the normal microbial fl ora present in the colons and feces of all warm-blooded animals. However, multiple disease ca using serotypes of E. coli have been identified in the past few decades. The most well-known serotype is E. coli O157:H7, which frequently occurs in wastewater and feces in developed countries and can result in severe

illnesses and death. E. coli O157:H7 (or just O157:H7) is th e “poster child” for pathogenic E.

coli and is the serotype most extensivel y investigated (Chart et al., 2000).

E. coli is a versatile bacterium and multiple subtypes and strains that are pathogenic have been documented. Although the nomenclature for these stra ins has not yet stabilized in the l iterature, Mølbak and Scheutz (2004) provided the fo llowing helpful summary of the current

nomenclature:

• Diarrheagenic E. coli (DEC) – includes all the strains on this list;U.S. Environmental Protection Agency

February 2009 9 • Verocytotoxin (Shiga toxin)-producing E. coli (VTEC or STEC) – E. coli that produce verocytotoxin (Shiga toxin) VT1 and/or VT2;

o Enterohemorrhagic E. coli (EHEC) – originally define d as serotypes that cause a

clinical illness similar to E. coli O157:H7, now used as a term for VTEC that cause hemorrhagic colitis (HC) in humans;

• Enterotoxigenic E. coli (ETEC) – E. coli that produce enterotoxins that are heat stable (STh, STp) and/or heat labile (LT);

• Attaching and effacing E. coli (A/EEC) – E. coli that attach to and efface the microvilli of enterocytes, but do not produce hi gh levels of verocytotoxin;

o Enteropathogenic E. coli (EPEC) – Subtype of A/EE C, usually of particular serotypes that mostly contain an EPEC adherence factor plasmid and often produce bundle-forming pili;

• Enteroaggregative E. coli (EAggEC) – E. coli that exhibit a pattern of aggregative adherence to tissue culture; and

• Diffuse adherent E. coli (DAEC) – E. coli that exhibit a pattern of diffuse adherence to

tissue culture.

Nataro and Kaper (1998) provi de a comprehensive, albeit dated, review of the DEC.

E. coli Zoonotic Potential

E. coli is part of the normal intestinal flora of hum ans and warm-blooded animals and can readily spread to humans through contaminated food and water (WHO, 2004). Pathogenic E. coli have been documented in a wide variety of animal species including cattle (Chapman et al., 1997; Rangel et al., 2005), chickens (Schoeni and Doyl e, 1994), sheep (Chapman et al., 1997; Kudva et al., 1996), pigs (Booher et al., 2002; Chapman et al., 1997; Feder et al., 2003), deer (Keene et al., 1997; Rice et al., 1995; Sarg eant et al., 1999), horses (Cha lmers et al., 1997), and dogs (Hammermueller et al., 1995).

Ruminants, and cattle in particular, are consider ed one of the most important animal sources of

E. coli O157:H7 and other VTECs. All of the V TECs including EHEC are capable of causing severe disease in humans, are typically shed by healthy cattl e and other species, and have documented cases of transmission via humans and water (Mølbak and Scheutz, 2004). Chapman et al. (1997) determined that the monthly prevalence of E. coli O157:H7 in cattle ranged from 4.8 to 36.8 percent, which was higher than the prevalence range in poultry, sheep, and pigs. Michel et al. (1999) examined the relationshi p between 3,001 cases of V TEC and the livestock density in rural areas of Ontario, Canada. Thei r research indicated that cattle density had a positive and significant association with the numbe r of reported cases of VTEC in humans, suggesting that living near cattle farms may incr ease a person’s risk of contracting VTEC.

Current data suggest that the prevalence of E. coli O157:H7 in poultry is low (Chapman et al., 1997), although contact with chickens has resulte d in outbreaks. Schoe ni and Doyle (1994) inoculated 1-day-old chicks w ith strains of serotype O157:H 7. The chicks shed serotype O157:H7 up to 11 months after inoculation, and O 157:H7 was subsequently recovered from their egg shells, but not from the yolks or whites. In an outbreak in norther n Italy, the source of exposure was believed to be chicke ns in 15 cases of hemolytic ur emic syndrome (HUS; see moreU.S. Environmental Protection Agency

February 2009 10 below) that were caused by serotype O157 and ot her EHEC serotypes (Tozzi et al., 1994). In this outbreak, a case-controlled study showed an association between contact with chickens and HUS although VTEC was not isolated from any of the chickens.

In a study by Chapman et al. (1997), O157:H7 was isolated from 2.2 percent of sheep and 4 percent of pigs, respect ively. In a Russian study, O157:H7 wa s found in sheep, and the incidence was highly variable, ranging from 31 percent in June to 0 percent in November (Kudva et al., 1996). Kudva and colleagues also showed that 80 percent of the O157:H7 isolates had at least two of the Shiga-like toxin types I or II or the attaching- effacing lesion genes. Strains that produce Shiga-like toxins have been documented in humans and in animals (Kudva et al., 1996; Mølbak and Scheutz, 2004).

E. coli Route of Exposure

Waterborne transmission of E. coli O157:H7 has been reported from both recreational water (Ackman, 1997; CDC, 2002; McCart hy et al., 2001; Samadpour et al., 2002; Yoder et al., 2004) and contaminated drinking water (CDC, 2002; Hrudey et al., 2002, 2003; Olsen et al., 2002; Pond, 2004; Swerdlow et al., 19 92; Yarze and Chase, 2000). Most studies examining contamination from recreation immersion have sugge sted that ingestion wa s the primary route of exposure (Keene et al., 1994). Because E. coli O157:H7 has a relatively low infectious dose, swallowing a small amount of contaminated water may cause illness (Haas et al., 2000; Keene et al., 1994).

Immersion in and ingestion of recreational waters has been the route of exposure for strains of E.

coli other than EHEC (Yoder et al ., 2004). In Connecticut, 11 persons were infected with E. coli O121 in an outbreak associated with swimming in a lake (CDC, 2000).

E. coli Illness Symptoms

E. coli can cause a relatively wide range of illness symptoms, depending on the strain and the underlying health of the host (Hunter, 2003; Mølb ak and Scheutz, 2004). Incubation periods vary and can be as short as 8 hours for EAEC in fections (Nataro et al., 1995), 14 to 50 hours for ETEC (Dupont et al., 1971), and 3 to 4 days for EH EC, with shorter (1 to 2 days) and longer (5 to 8 days) incubations noted in some outbreaks (Nataro and Kaper, 1998). Approximately 82 to 95 percent of all E. coli cases result in relatively minor illness symptoms including abdominal cramps, vomiting, diarrhea (often bloody), and someti mes fever (Ostroff et al., 1989; Swerdlow et al., 1992). The duration of these symptoms is 4 to 10 days (C DC, 1993a) although, in the Cincinnati outbreak, infants remained hospitalized for 21 to 120 days (Nataro and Kaper, 1998). Symptoms may be more severe for persons with hemorrhagic coli tis (HC) or bloody inflammation of the colon (Griffin and Tauxe, 1991).

Symptoms commonly associated with human illness from the various DEC include the following:

• VTEC (or STEC) – Diarrhea, hemorrhagic colitis, HUS;

o EHEC – hemorrhagic colitis (HC);U.S. Environmental Protection Agency

February 2009 11 • ETEC – Acute watery diarrhea;

• A/EEC – Acute or persistent diarrhea;

o EPEC – Acute or pe rsistent diarrhea;

• EAggEC – Acute watery, often protracted diarrhea; and

• DAEC – Acute or persistent diarrhea.

In rare cases, HC can develop into HUS, which is a severe life-threatening disease that can result in kidney failure and neurological complications such as seizur es and strokes (Brotman, 1995). Brooks et al. (2005) conducte d a study that suggests E. coli strains that produce Shiga toxin 2 are much more likely to result in HUS than strains that only produce Shiga toxin 1. Approximately 2 to 7 percent of all E. coli O157:H7 infections result in HUS , and HUS is most common among children under 5 years old and the elderly (Griffin and Tauxe, 1991; WHO, 2004). Less than 10 percent of HUS cases turn into a chronic illness such as chroni c kidney failure, blindness, or partial paralysis (Tarr, 1995). Approximately 33 percent of pers ons that contract HUS have abnormal kidney function for several years, so metimes requiring long-term dialysis (WHO, 2004). In very rare cases, HUS can also lead to death. Cases of infection with E. coli O157:H7 from swimming-associated outbreaks, compared to other routes of expos ure, have the highest rate of HUS. This difference may be due to the higher proportion of young children participating in swimming and becoming infected during su ch outbreaks and the higher likelihood of young children developing HUS (Rangel et al., 2005). HUS, non-bloody diarrhea, and HC may occur with O157:H7 infections; ot her complications may include cholecystitis, colonic perforation, intusssception, pancreatitis, posthemolytic biliary lithiasis, post-infection colonic stricture, rectal prolapse, appendicitis, hepati tis, hemorrhagic cystitis, pulmonary edema, myocardial dysfunction, and neurological abnormalities (Nataro and Kaper, 1998).

As noted above, infection with E. coli O157:H7 can also result in death. The U.S. Centers for Disease Control and Prevention (CDC) estimates a death rate of 0.08 percent, although case studies reveal slightly higher rate s. For example, the 1993 fast-food E. coli O157:H7 outbreak in Washington, California, Idaho, a nd Nevada resulted in 4 deaths out of approximately 700 who fell ill, which corresponds to approximately 0.57 percent mortality (Brotman, 1995). Griffin and Tauxe (1991) reviewed 12 outbr eaks in the United States be tween 1982 and 1990 and calculated a mortality rate of 1.9 percen t among those with diagnosed E. coli O157:H7 infections.

E. coli Illness Incidence

The CDC estimates that there are 73,000 cases of E. coli 0157:H7 infections and 61 deaths annually in the United States. Non-O157 Shiga-like toxin sero types cause approximately 37,000 illnesses per year (Mead et al., 1999). 1 Craun et al. (2004a) estimated that E. coli was the etiological agent for 30 percent of the outbrea ks from zoonotic contamination in untreated

recreational waters from 1971 to 2000.

E. coli O157:H7 infection associated with recreational exposure was first reported in June 1991 in a lake in Oregon (Keene et al., 1994). From 1991 until 2002, 20 additional outbreaks as a result of exposure to contaminated recreational wa ters have been reported to the CDC (Rangel et

1 The CDC is currently updating th ese numbers, but the newest values have not yet been released.U.S. Environmental Protection Agency

February 2009 12 al., 2005). Fourteen of the outbreak s occurred as a result of exposure to contaminated lakes or ponds, while seven occurred from exposure to c ontaminated swimming pools (Rangel et al., 2005).

CDC’s surveillance system probably captures a small proportion of E. coli O157:H7 outbreaks that occur because many illnesses are not reported to public health officials or the CDC, are not recognized as E. coli infections, or the outbreak is consid ered of unknown etiology (Cieslak et al., 1997).

From 1991 to 2004, 14 outbreaks of E. coli related illness have been associated with untreated recreational waters (Table II.1.1-1).

II.1.2 Campylobacter

Campylobacter Strain Variation

Of the 17 species in the genus Campylobacter

, C. jejuni , and C. coli are the most important human pathogens. Eight strains of C. jejuni have been DNA sequenced. At least two strains of C. jejuni have been shown to cause illness in ferrets, mice, rabbits, and rats, and several of the

strains have been associated with Guilla in-Barré syndrome (GBS) (Nachamkin, 2002).

Table II.1.1-1. Outbreaks of E. coli Associated with Recreational Waters in the United States

Year Number of Cases Number of Outbreaks Source of Outbreak(s)

1991 80 1 Lake

1992 0 0 NA

1993 0 0 NA

1994 166 1 Lake

1995 28 4 Lakes

1996 24 2 1 pool, 1 lake

1997 8 1 Lake

1998 31 2 1 pool, 1 lake

1999 61 5 1 pool, 3 lakes, 1 ditch water

2000 0 0 NA

2001 49 2 Lakes

2002 9 1 Pool

2003 0 0 NA

2004 0 0 NA

Source: Data from CDC Surveillance Summaries: Morbidity and Mortality Weekly Report (MMWR)

Surveillance for Waterborne-Disease Outbreaks - United States: 1991-1992, 1993-1994, 1995- 1996, 1997-1998, 1999-2000, 2001-2002, 2003-2004 (CDC, 1993b, 1996, 1998, 2000, 2002, 2004, 2006).U.S. Environmental Protection Agency

February 2009 13 Campylobacter Zoonotic Potential

Because most of the published literature focuse s on food contamination from human waste as the primary risk factor for Campylobacter infection, some professionals in the health community

have concluded that the zoonotic waterborne route is unlikely to be important (Till and McBride, 2004). Some evidence indicate s that the human sources of Campylobacter may obscure the zoonotic risk factors (McBride, 1993).

McBride et al. (2002) found that 60 percent of all samples collected from 25 freshwater

recreational water sites in New Zealand over 15 months contained at least one species of Campylobacter . This finding led to an inference that 4 percent of all campylobacteriosis cases in

New Zealand were due to water contact recreation (McBride et al., 2002). Donnison and Ross (2003) note that nearly a ll streams near dairy farm s in New Zealand contain Campylobacter , and despite generally low concentrations, these streams may help cycle Campylobacter in farm animals and indirectly contribu te to the high incidence of human infection in New Zealand.

The species and strain of Campylobacter contained in a stream is highly influenced by the path of the stream, with surface waters running thro ugh beef- and sheep-grazing pastures typically

contaminated with C. coli and C. jejuni (Jones, 2001). Surface waters that have contact with avian species are typically contaminated with C. jejuni

, C. lari

, C. coli , and avian-sourced urease-positive thermophilic campylobacters (Jon es, 2001). The main source of campylobacters for bathing waters is typically a ssumed to be sewage effluent, t hough there is evidence that birds may be primarily responsible for contamination (Jones, 2001).

Campylobacter Route of Exposure

The main route of exposure to Campylobacter from recreational waters is from incidental ingestion of water during full im mersion activities such as swim ming. The bacteria may be of anthropogenic or animal sources. From 1991 to 2002, 3 percent of waterborne outbreaks were

attributed to Campylobacter (7 outbreaks, 360 cases) (Craun et al., 2006). Craun et al. (2005)

summarized and discussed 259 waterborne outbreak s occurring in the Un ited States from 1971 to 2000 and associated only w ith recreational water. Campylobacter was the etiologic agent for 0.9 percent of the outbreaks. Although waterborne sources of Campylobacter are important, most

infections occurred as a result of handling contaminated f oods (e.g., raw chicken) and direct person-to-person transmissi on is rare (Allos, 2001).

Campylobacter Illness Symptoms

Campylobacter infection can result in a number of symptoms, including loose and watery or bloody diarrhea, dysentery, fever, and severe ab dominal cramps (Allos, 2001; Fricker, 2006a).

Infections of Campylobacter are symptomatically indistinguisha ble from those of other bacterial pathogens such as Salmonella (Nachamkin, 2002). Diarrheal sy mptoms may be frequent at the peak of the illness, typically occurring 8 to 10 times per day. Although the disease usually lasts only one week, some infected individuals expe rience relapses leading to several weeks of symptoms (Allos, 2001).U.S. Environmental Protection Agency

February 2009 14 Some complications may result from Campylobacter infection including cholecystitis, pancreatitis, peritonitis, and massive GI hemo rrhage. Some immunocompromised individuals experience bacteremia as a result of campylobacter iosis, and in rare instances, some individuals have experienced meningitis, endocarditis, septic arthritis, osteomyelitis, or neonatal sepsis. Infection rarely results in death, with 1 death per 20,000 infections (Allos, 2001).

Several chronic sequelae have been associated with Campylobacter infection including the development of GBS (Fricker, 2006 a). GBS is an acute demyelin ating disease of the peripheral nervous system that affects 3,000 to 6,000 pe ople in the United Stat es annually, with approximately 1,000 to 2,000 cases preceded by C. jejuni infection (Allos, 1998). The risk of developing GBS after C. jejuni infection, however, is small (a pproximately 1 case of GBS per 1,000 infections), and many GBS-related C. jejuni infections are asymptomatic (Allos, 2001). Reactive arthritis and depression also have been reported as chronic sequelae following campylobacteriosis (Garg, 2006; Nachamkin, 20 02). Recent evidence has associated C. jejuni

with a rare form of mucosa-associated lymphoid tissue (MALT) lymphoma called immunoproliferative small intestinal disease (IPSID); campyloba cteriosis has not yet been shown as causative agent of IPSID (Poly and Guerry, 2008).

In 2000, a municipal water supply in Walker ton Ontario became contaminated with E. coli and C. jejuni . In the outbreak that resulted from that contamination, persons who showed symptoms of acute bacterial gastroenteritis at the time of the outbreak were more likely to exhibit hypertension and reduced kidney function approxima tely 4 years after inf ection than those who were asymptomatic at the time of the outbreak (Garg et al., 2005).

Campylobacteriosis Incidence

Although Campylobacter became a reportable illness in the United States in the early 1980s, it is estimated that only 1 in 38 cases of detected infection actually ar e reported (Mead et al., 1999). Approximately 2.4 million Campylobacter infections are estimated to occur in the United States every year (Allos, 2001). Between 1996 and 1999, the incidence of campylobacteriosis in the United States declined by 26 percent, from 23.5 to 17.3 cases per 100,000 people (Allos, 2001). According to reports from 1999, Br itain experiences approximately 5 times this infection rate (103.7 cases per 100,000 people), though reporting rates may not be comp arable (Gillespie et al., 2002). C. jejuni infection is one of the most common cau ses of gastroenteritis across the world and is frequently responsible for diar rheal illness in travelers (Allos, 2001).

Other than an outbreak in 1999, in Florida, where 6 cases of C. jejuni infections resulted from a private swimming pool, no other recreational wate rborne outbreaks have been reported to the CDC between 1991 and 2004 (CDC, 2002).

II.1.3 Salmonella

Salmonella Strain Variation and Zoonotic Potential

Salmonella are non-encapsulated, Gram-negative bacteria of the Enterobacteriaceae family that

infect both animals and humans causing a wide range of illn esses (Cohen, 1986; Lightfoot, 2004;U.S. Environmental Protection Agency

February 2009 15 Ohl, 2001). There are more than 2,5 00 serovars/serotypes in the genus Salmonella (Lightfoot, 2004).

The genus Salmonella consists of two species, S. enterica and S. bongori. S. enterica is divided into the following six subspecies: S. e. enterica ; S. e. salamae ; S. e. arizonae ; S. e. diarizonae ; S.

e. houtenae ; and S. e. indica (Lightfoot, 2004). The many serova rs in the group are closely related to each other by somatic and flagellar an tigens, and most strains show diphasic variation of flagellar antigens). Thus, Salmonella can be serotyped by means of somatic and flagellar antigens and further subtyped by antibiotic-sens itivity testing, biochemical reactions, phagetyping, and analysis of the plasmids they carry. All Salmonella serotypes share the ability to invade the host by inducing their own uptake into cells of the intestinal epithelium (Lightfoot, 2004). Although more than 2,500 Salmonella serotypes exist, only 10 account for more than 70 percent of the isolates reported annually in the United States (Cohen, 1986; Lightfoot, 2004). Because the vast majority of Salmonella isolates from humans are of the subspecies S. e.

enterica , the CDC recommends that Salmonella strains only be referred to by their genus and serotype (e.g., S. typhi ).

S. typhimurium and S. enteritidis are the most prevalent serot ypes found in the United States (CDC, 2006). The other serotypes have much smaller incidences in the United States; S.

enteritidis accounted for 10 pe rcent in 1984, and S. newport

, S. infantis , and S

. heidelberg accounted for 4.5, 3.0, and 1.0 percent, respectively.

Some Salmonella serotypes are highly host-sp ecific, restricted to a single host species and rarely causing disease in other sp ecies (Lightfoot, 2004). S. typhi and S. paratyphi are exclusively human pathogens, with no known animal reservoirs, while S. enteriditis and S. typhimurium infect a wide range of animal hosts, including p oultry, cattle, and pigs. The serotypes with a wide range of animal hosts can also infect humans, usually via food consumption, and cause selflimited gastroenteritis in humans (WHO, 2004). S. gallinarum and S. pullorum are almost exclusively pathogens of poultr y (Cohen, 1986). Human pathogens S. heidelberg and S. litchfield have primarily avian an d reptilian reservoirs, respectively (Cohen, 1986). S. abortusovis is specific to sheep (Lightfoot, 2004). Salmonella has also been reported in swine, cattle, rodents, birds, turtles, dogs, and cats (Covert and Meck es, 2006). The CDC estimates that 74,000 cases of salmonellosis per year are associated with expos ure to reptiles or am phibians (directly or indirectly) (Lightfoot, 2004).

In 1993, an outbreak of S. typhimurium where more than 650 people became ill and that resulted in 7 deaths, was traced to a wa ter-storage tower that allowed acce ss to birds (Covert and Meckes, 2006). Although this outbreak was from drinking water rather than recreational water, it illustrates the potential for waterborne exposure due to birds.U.S. Environmental Protection Agency

February 2009 16 Salmonella Route of Exposure

Salmonella infections begin with the ingestion of organisms in contaminated food or water (Lightfoot, 2004; Ohl, 2001). Although ingestion or exposure to infected water from recreational swimming is less common, it has been report ed worldwide (Cohen, 1986; Lightfoot, 2004).

Researchers have observed a reducti on in the infectious dose of Salmonella under conditions where the gastric pH is elevated suggesting that gastric acidity may create an initial barrier to infection (Lightfoot, 2004). Salmonella also exhibit an adaptive, ac id-tolerance re sponse in low pH conditions thereby allowing them to live in acidic host environments like the stomach (Ohl, 2001).

Elevated levels of Salmonella also have been observed in major water bodies that receive discharges of meat processing wastes, raw se wage, and effluents from ineffective sewage treatment plants (Geldreich, 1996). Farming ope rations with cattle and poultry result in large quantities of fecal products in relatively sma ll areas due to the dense population of animals. Thus, if the animal waste is not discharged in to a lagoon or landfill, the stormwater runoff over the animal feedlots will transport massive loads of fecal pollution to the receiving waters of the drainage basin (Lightfoot, 2004).

Salmonella Illness Symptoms

Illnesses caused by Salmonella range from asymptomatic colonization and mild gastroenteritis to the more serious enteric fever (typhoid), meni ngitis, and osteomyelitis (Cohen, 1986). Enteric fever and gastroenteritis are the key cl inical syndromes associated with a Salmonella infection (Lightfoot, 2004).

Different Salmonella serovars cause different clinical symptoms. S. typhi causes enteric fever (typhoid) in humans, and S. typhimurium causes diarrhea in humans a nd other animal species but a typhoid-like syndrome in mice (Lightfoot, 2004). S. abortusovis is responsible for abortion in

ewes. Most Salmonella serovars cause an acute and mild enteritis, but S. blegdam

, S. bredeney

, S. choleraesuis

, S. dublin

, S. enteritidis

, S. panama

, S. typhimurium , and S. virchow may also be invasive and cause pyemic infections localizing in the viscera, meninges, bones, joints, and serous cavities (Lightfoot, 2004; Covert and Meckes, 2006). S. dublin is also particularly

associated with different extrai ntestinal infections in persons with acquired immunodeficiency syndrome (AIDS) (Lightfoot, 2004).

Infection with S. typhi or S. paratyphi , which are exclusively human pathogens, results in enteric fever (Ohl, 2001). Clinical sympto ms of enteric fever include di arrhea, abdominal pain, fever, and sometimes a maculopapular rash. The pathologi cal sign of enteric fever is mononuclear cell infiltration and hypertrophy of the re ticuloendothelial system (Ohl, 2001).

Salmonellosis is caused by ingestio n of nontyphoidal salmonellae (e.g., S. enteriditis and S. typhimurium ) with an incubation period of 8 to 72 hours (Lightfoot, 2004). The estimated inoculum size of nontyphoidal Salmonella required to cause symptomatic disease in healthy

adult volunteers is 10 5 to 10 10 organisms (Lightfoot, 2004). The infectious dose varies depending on the age and health of the person, strain differences, and the vector. MostU.S. Environmental Protection Agency

February 2009 17 salmonellosis cases are self-limiti ng, and the affected persons r ecover without treatment (CDC, 2006; Cohen, 1986). Some infections are more severe, however, particularly in the young, elderly, and people with weakened immune systems, and such infections may become invasive (APHA, 2004; Lightfoot, 2004).

The clinical symptoms of salmonellosis may incl ude diarrhea, abdominal pain, nausea, chills, fever, and prostration with the duration of illn ess ranging approximately 2 to 7 days (APHA, 2004; WHO, 2004). Vomiting may occur as well, but it is rare and usually a sign of invasive disease (APHA, 2004). Organisms that leave the GI tract and invade the rest of the body can cause bacteremia and septicemia , thus spreading the salmonellae to many organs in the body and possibly leading to abscesses, septic arthritis, cholecystitis, endocarditis, meningitis, pericarditis, pneumonia, pyodrema, or pyelonephritis (APHA, 2004).

Diarrhea from Salmonella infection is usually self-limiting and does not require treatment unless severe. Overall, there is an estimated 22.1 per cent hospitalization rate , and an estimated 0.8 percent fatality rate (U SDA, 2005). Mortality from S. typhi and S. paratyphi is estimated to be between 10 to 15 percent without treatment (Ohl, 2001). In severe cases, fluid and electrolyte replacement may be needed. Antibio tics are not recommended to treat Salmonella infections, except where there is evidence of invasion and septicemia, because they do not alleviate the symptoms or reduce the duration of the illness (Kanarat, 2004). Antibiotics may even prolong

excretion of Salmonella in the feces.

Salmonellosis Incidence

Foodborne Salmonella are the estimated cause of approxi mately 1.4 million foodborne-related illnesses, 15,600 foodborne illness-related hospi talizations, and 550 foodborne-related deaths each year in the United States (Mead et al., 1999). In 2005, 45,322 salmonellosis cases were reported to the CDC through the Public Health Laboratory Information System (CDC, 2007a). Community surveys during outbreaks suggest that the proportion of infe ctions reported is between 1 in 10 and 1 in 100 (Cohen, 1986).

Between 1991 and 2002, 3 waterborne outbreaks (833 cases) were attributed to nontyphoid

Salmonella (Craun et al., 2006). Craun et al. (2005) reported that Salmonella were the etiologic agent in 0.9 percent of 259 recr eational waterborne outbreaks occurring from 1971 to 2000.

II.1.4 Leptospira

Leptospira Strain Variation and Zoonotic Potential

Leptospira is an aerobic, motile spirochaete, 6 to 20 μm long and 0.1 μm wide. Leptospira occurs worldwide and has become an importa nt recreational zoonosis due to its prolonged survival in water (Pond, 2005). The genotypic classification of Leptospira into two species ( L. interrogans and L. biflexa ) has been replaced by a phenotypic classification system in which 13 genomospecies are currently defined ( L. interrogans

, L. noguchii

, L. santarosai

, L. meyeri

, L.

wolbachiic

, L. biflexac

, L. fainei

, L. borgpetersenii

, L. kirschneri

, L. weilii

, L. inadai

, L. parvac

, and L. alexanderi ) (Levett, 2001). Of the 28 serovars, several occur in more than oneU.S. Environmental Protection Agency

February 2009 18 genomospecies. Leptospirosis is probably the mo st widespread zoonosis in the world (Levett, 2001; Meites et al., 2004). Zoonotic reservoirs incl ude livestock (pigs and cattle), domestic pets (dogs), and wild or feral animals (rats, vol es, and mice) (Kanarat , 2004; Levett, 2001).

Complete genome sequences of L. interrogans serovars Copenhageni and Lai reveal that despite overall genetic similarity there are significa nt structural differenc es in their genomes (Nascimento et al., 2004). Nascimento and coll eagues analyzed the genomic sequences to gain insight into genes that influence motility, chem otaxis, pathogenicity, and colonization of the pathogen.

Leptospira Route of Exposure

The source of infection in humans is usually either direct or indirect contact with the urine of an infected animal (Levett, 2001; WHO, 2003). Worker s in direct contact with animal reservoirs are at increased risk (e.g., cattle, pig, and dairy farmers, slaughterhouse workers, and veterinarians) (Meites et al ., 2004). Recreational exposure from swimming or boating in freshwater lakes is also possible (Levett, 200 4; Meites et al., 2004). Outbreaks are often associated with unusual rainfall events or flooding (Bolin et al., 2004b).

Leptospira Illness Symptoms

Leptospirosis was first descri bed by Adolf Weil in 1886; thus , the more serious form of leptospirosis is still known as Weil’s disease (WHO, 2003). Leptospirosis is biphasic with a week-long acute stage followed by approximately 2 weeks of convalescence (Levett, 2001). During the acute stage, antibodies are low and th e pathogen is detected mainly in the blood and cerebrospinal fluid, whereas the convalescent stage corresponds with the appearance of antibodies and the presence of path ogens in the urine (Levett, 2001).

Anicteric leptospirosis can be mild or acute. The majority of infections are either subclinical or mild, and patients usually do not seek medical atte ntion (Levett, 2001). Icte ric leptospirosis is a

much more severe disease than an icteric leptospirosis. Icteric leptospirosis accounts for most of the high mortality rate, which ranges between 5 and 15 percent. Between 5 and 10 percent of all patients with leptospirosis have the icteric form of the disease (Levett, 2001).

Symptoms associated with leptospirosis include the following: jaundi ce, anorexia, headache, conjunctival suffusion, chills, vomiting, myalgia, abdominal pain, nausea, cough, hemoptysis, hepatomegaly, lymphadenopathy, diarrhea, rash (usually lasting less than 24 hours), and fever, which can be biphasic and reoccur after 3 to 4 days of remission (Levett, 2001).

In some cases, acute infection in pregnancy has been reported to cause abortion and fetal death (Levett, 2001). Uveitis (ocular complications) is recognized as a chronic sequela of leptospirosis

in humans and horses. Chronic visual disturba nce lasting 20 years or more has been reported (Levett, 2001). In 1994, CDC removed leptospirosis from the notifiable diseases list; however, the Hawaii Department of Health still re quires reporting (Katz, 2001; Levett, 2001).U.S. Environmental Protection Agency

February 2009 19 Leptospirosis Incidence

Levett (2001) summarized information for 28 waterb orne outbreaks of lept ospirosis worldwide, 22 of which were associated with swimming, 1 w ith kayaking, and 1 with rafting. Within the United States, the highest incidenc e of leptospirosis is found in Hawaii (Katz, 2001). Between 1971 and 2000, 16 percent of recreational waterbor ne disease outbreaks were attributed to Leptospira (Craun et al., 2004a). In 1998, in Illinois, there was an outbreak (375 cases) of leptospirosis associated with a triathalon in a lake (CDC, 2000).

In a prospective, population-based study of patient s presenting with acute febrile illness, the geographic distribution of human Leptospira isolates mirrored the distribution of Leptospira 16S ribosomal gene sequences in urban and rural water sources (Ganoza et al., 2006).

II.2 Protozoa

II.2.1 Cryptosporidium

Cryptosporidium is a small protozoan parasi te that infects the microvill ous region of epithelial cells in the digestive and respirat ory tract of humans and other mammals, birds, reptiles, and fish. Cryptosporidium does not replicate outside of a host. Environmentally robust oocysts are shed by infected hosts into the environment and can survive in environmental conditions for long periods of time (up to months) unt il ingested by a new host. In th e new host, the life cycle starts again, and multiplication occurs using the bi ological resources of the host (WHO, 2006). Cryptosporidium exists in the natural environment in the oocyst form and which are resistant to conventional drinking water treatmen t measures such as chlorination. Cryptosporidium is recognized as a widespread pathogen for the general population, including both immunocompromised and immunocompetent persons (WHO, 2006).

Cryptosporidium Life Cycle and Strain Variation

Cryptosporidium has a complex life cycle. Each oocyst, which has an environmentally resistant wall, holds four sporozoites. Oocysts enter the environment by passing with the feces of an infected host organism (Fayer and Ungar, 1986; Fayer et al., 1997). Oocysts are immediately infectious and may remain in the environmen t for very long period s without losing their infectivity. Oocysts are resistant to environmen tal conditions and natural decay and can travel passively through the environment until they are ingested by a new host organism. In the GI tract of the new host, 4 sporozoi tes exit each oocyst (excyst) and may form an infection in the epithelial cells of the small in testine of the host (USEPA, 2001a). The sporozoites transform through several life stages including an asexual and a sexual reproduction cycle. Oocysts are the result of the sexual reproduction cycle. Oocysts of the two species of Cryptosporidium responsible for most human infections— C. hominis and C. parvum —are spherical with a diameter of 4 to 6 μm. Thin-walled oocysts may excyst within the same host and start a new life cycle (autoinfection), whereas thick-walled oocysts generally are shed by the host. Autoinfection may lead to a heavily infected epithelium of the small intestine resulting in secretory diarrhea (WHO, 2006).U.S. Environmental Protection Agency

February 2009 20 Cryptosporidium is part of phylum Apicomplexa, fa mily Cryptosporidiidae, and has been classified as a member of the group of eimeriid coccidian—a diverse group of parasitic protozoa (WHO, 2006). There are currently 16 species of Cryptosporidium identified in the literature (Table II.2.1-1). However, this taxonomy is likely to change as molecular methods continue to characterize isolates and potenti al new species. Fayer (2004a) and Olson et al. (2003) updated

the list of Cryptosporidium species that have been reported to infect humans to include C. baileyi

, C. canis

, C. felis

, C. hominis

, C. meleagridis

, C. muris , and C. parvum . It is important to

note that the human form of C. parvum (formerly referred to as H-type or genotype 1) was recently reclassified as a new species, C. hominis (Morgan-Ryan et al., 2002). The cattle form of C. parvum (formerly referred to as C-type or genotype 2) maintains the designation C. parvum

. Thus, studies published prior to 2002 should be interpreted carefully keeping in mind that

authors who refer to C. parvum may be referring to C. parvum

, C. hominis , or both. Of the current 16 species of Cryptosporidium

, C. parvum and C. hominis most commonly cause GI

illness in humans.

Studies with volunteers have de monstrated that a low dose of C. parvum (e.g., 10 oocysts) is sufficient to cause infection in healthy adults a lthough some strains may be more infectious than others (Chappell et al., 1999; DuP ont et al., 1995; Okhuysen et al., 2002). The relationship

Table II.2.1-1. Cryptosporidium Species

Cryptosporidium Species Initially Described Host Species

C. andersoni Bos taurus (domestic cattle)

C. baileyi Gallus gallus (domestic chicken)

C. bovis Bos taurus (domestic cattle)

C. canis Canis familiaris (dogs)

C. felis Felis catis (domestic cat)

C. galli Gallus gallus (domestic chicken)

C. hominis Homo sapiens (humans, formerly C. parvum genotype 1)

C. meleagridis Meleagris gallopavo (turkey)

C. molnari Dicentrarchus labrax (fish)

C. muris Mus musculus (house mouse)

C. nasorum Naso lituratus (fish)

C. parvum Mus musculus (house mouse) (formerly genotype 2)

C. scopthalmi Scopthalmi maximus (turbot)

C. serpentis Elaphe guttata (corn snake)

E. subocularis (rat snake) Sanzinia madagascarensus (Madagascar boa)

C. suis Sus scrofa (pig)

C. varanii Varanus prasinus (emerald monitor lizard)

C. wrairi Cavia porcellus (guinea pig)

Source: Adapted from Cacciò, 2005; Fayer, 2003, 2004a; Fayer et al., 1997, 2000; Fayer and Xiao, 2007; Morgan-Ryan et al., 2002; Ryan et al., 2003; and Xiao and Ryan, 2004.U.S. Environmental Protection Agency

February 2009 21 between the number of oocysts humans are expos ed to and the probability of infection is discussed in detail in the subsequent secti on. Studies of immunosuppressed adult mice have demonstrated that a single viable oocyst can induce C. parvum infections (Okhuysen et al., 2002; Yang et al., 2000).

Genetic and molecular studies of C. parvum (including C. hominis ) indicate that the species is genetically heterogeneous among isolated strains found in different host species (Xiao and Ryan, 2004). There also is evidence that C. parvum and C. hominis experience recombination and that polymorphisms exist in the C. hominis species (Widmer et al., 1998). Okhuysen et al. (1999)

showed that different isolates of C. parvum (including C. hominis ) have different levels of infectivity for humans, and therefore the hetero geneity of the species may influence the risk posed to public health. Furthe rmore, distinct transmission cy cles are evident among different genotypes of C. parvum . Multiple genotypes have been shown to circulate among different host species, and mixed infections with genotypica lly distinct populations have been reported (Widmer et al., 1998).

Cryptosporidium Zoonotic Potential

Human cases of cryptosporidiosis ty pically have been associated w ith different kinds of animal contact, which has led to the widespr ead belief that the host-range of Cryptosporidium is very broad and many animals can serve as reservoirs for Cryptosporidium

. Cryptosporidium infection has been reported in more than 155 mammalia n species (Fayer, 2004a) as well as numerous reptiles, amphibians, birds, and fish (O’Donoghue, 1995).

Several lines of evidence indicate that livestoc k, primarily cattle and shee p, are a major source of Cryptosporidium contamination of drinking water source s. These include the detection of C.

parvum (presumably from animals) in many sour ce waters and elevated levels of Cryptosporidium in watersheds with extensive agricu ltural activity (Fayer , 2004a; WHO, 2006). Also, direct zoonotic transmission of Cryptosporidium infection from livestock to humans has been repeatedly demonstrated (WHO, 2006). During outbreaks of cryptosporidiosis, frequent

detection of Cryptosporidium in human stool samples suggests that human sources can also add significantly to the occurr ence of oocysts in source waters (Fayer, 2004a).

Based on data from the western United States, depending on climate and feedlot management systems, the average animal in a cattle f eedlot excretes between 28,000 and 140,000 oocysts per day (Atwill et al., 2006). Furthermore, 91 percent of dairy farms studied by Sischo et al. (2000)

had Cryptosporidium at their locations, with 15 percent of infant dairy calve s shedding oocysts. Nine percent of farm-associated streams contained C. parvum . Therefore, cattle represent a significant reservoir and potenti al environmental source of C. parvum . Tate et al. (2000) demonstrated that oocysts can be carried by runoff during rain events.

Most outbreaks of cryptosporidiosis are caused by C. hominis , which is only transmitted by human hosts. Outbreaks represent only 10 percen t of domestic cryptospori diosis cases, however, and at least one study (Feltus et al., 2006) has shown that the zoonotic C. parvum may be responsible for the majority of sporadic (endemic) cases. Atwill et al. (1997) reported that feralU.S. Environmental Protection Agency

February 2009 22 pigs may serve as an environmental reservoir for C. parvum and may represent a potential source

of Cryptosporidium contamination of ambient waters.

Although it is clear that livestock may be a major contri butor to drinking water source contamination, there are few data to support a quantitative estimate of the proportion of this

contribution. Cryptosporidium levels in source water are known to vary seasonally (USEPA, 2005a, 2005b), and short-term levels in surface wa ter can be strongly associated with storm events or other weather variables (Fayer, 2004a; Naumova et al., 2005; USEPA, 2005a; WHO, 2006). In watersheds with diverse land-use patterns, it is likely that different so urces contribute different proportions to the total contamination lo ad at different times dur ing the year, with the relative contributions depe nding on a wide range of wa tershed characteristics.

Besides animal contamination, the other major source of Cryptosporidium in ambient water is human fecal wastes. Several mechanisms can be responsible for the contamination of source waters and include malfunctioning septic systems, routine or “upset” releases from municipal

wastewater treatment f acilities, combined sewer system ove rflows, or human recreational uses (e.g., swimming, camping, and hiking). Due to th e large numbers of ooc ysts excreted by infected individuals (Okhuysen et al., 1999) and oocyst resistance to many conventional wastewater treatment processes, even small re leases can be significant. The World Health Organization (WHO) found reports of raw sewage samples containing up to 14,000 oocysts/L (average was up to 5,300 oocysts/L ) and treatment plant effluent s containing between 17 and 250 oocysts/L (WHO, 2006). Clearly , in water bodies where treatment effluents comprise an appreciable proportion of total flow , their contribution to the total Cryptosporidium load may be

substantial.

LeChevallier et al. (2003) reported the results of Cryptosporidium monitoring for approximately 600 samples from 6 watersheds located in the Un ited States and Canada. They found that the two watersheds with the highest proportion of agricultural land use ha d the highest average Cryptosporidium levels 2 and that approximately 90 percent of the samples exhibited the bovine (cattle) genotype ( C. parvum ). These findings implicate livestock, at least in these watersheds, as the major contributor to overall Cryptosporidium levels. This finding is consistent with studies indicating that the prevalence of Cryptosporidium infection in young livestock (i.e., calves and lambs) is very high (Fayer, 2004a ) and that the oocyst counts in feces of young infected animals ranges from 10 6 to 10 8 oocysts per gram (WHO, 2006).

McCuin and Clancy (2006) conducted a 15-month occurrence study of Cryptosporidium

occurrence in 10 wastewater facilities across the United Sates. Indigenous oocysts were detected in 30 percent of raw influents, 46 percent of prim ary effluents, 58 percent of secondary effluents and 19 percent of tertiary effluents in the 289 anal yzed samples. Zhou et al. (2003) analyzed 179 wastewater treatment plant effluent samples fr om Milwaukee, Wisconsin using polymerase chain reaction (PCR) restriction fragment length po lymorphism (RFLP) methods to characterize genotypes of detected Cryptosporidium . In contrast with the results observed for source waters by LeChevallier et al. (2003), C. hominis (13.4 percent of samples) wa s detected more frequently

than C. parvum (2.8 percent). This finding s uggests that the distribution of Cryptosporidium in

2 These results were obtained using EPA Method 1623 (USEPA, 2001b), for which the authors reported an average recovery rate of 72±22 percent.U.S. Environmental Protection Agency

February 2009 23 Milwaukee’s population was not heavily influen ced by agricultural sources, though the relative

low levels of C. parvum in wastewater may also have been due to differences in infectivity, excretion, or both of this species relative to C. hominis . As is the case for livestock, the extent to

which human wastes contribute to overall Cryptosporidium contamination is highly site-specific, seasonal, and variable.

Cryptosporidium Route of Exposure

The main route of exposure to illness-causing mi croorganisms in recreational waters is through accidental ingestion of contaminated water while engaging in full immersion activities such as swimming or bathing. Secondary contact or partial body contac t recreation such as wading, canoeing, motor boating, and fishing, in which ingest ion is unlikely due to lack of direct water contact with the ears, eyes, mouth, or nose, is not considered to result in signifi cant exposure (USEPA, 2002). A recent pilot study of angler s in the Baltimore area by Roberts et al. (2007), however, suggested that between 1 and 8 of 10 urban anglers could become infected with Cryptosporidium . This small study (56 anglers; a total of 46 fish/hand wash samples) included quantitative risk modeling. No data were reported on microbial water quality at the sampling sites, nor was there any attempt to obtain either h ealth effects data or clin ical samples to evaluate

infection rates in the study population.

The potential for person-to-person secondary transmission is high for Cryptosporidium infections. Based on an analysis of data from the 1993 Milwa ukee outbreak, Eisenberg et al. (2005) suggested that 10 percent (95 percent confidence in terval: 6 to 21 percen t) of the cases of disease were due to person-to-person transmission. There is considerable evidence that direct person-to-person transmission, as well as i ndirect transmission through contact with contaminated objects, can be a significant route of infection, especially where human population densities are high or personal contact is freq uent (USEPA, 2001a). Direct transmission is affected by behavioral factors (e .g., frequent travel) a nd ethnic and dietary differences (USEPA, 2001a). Using data from the Milwaukee outbrea k, approximately 3 to 5 percent of infected individuals transmit the disease to others (Mackenzie et al., 1995; Osewe et al., 1996). Among children and their caretakers, how ever, the transmission rate is considerably higher (12 to 22 percent) (Osewe et al., 1996).

Cryptosporidium Illness Symptoms

Cryptosporidiosis is primarily characterized by GI symptoms such as profuse watery diarrhea; however, diarrheal symptoms are generally no t distinguishable from those caused by other common enteric pathogens. Other symptoms reported by individuals afflicted with cryptosporidiosis include dehydr ation, fever, anorexia, weight loss, weakness, abdominal cramps, vomiting, lethargy, general malaise, and pr ogressive loss of overa ll condition (Hunter et al., 2004). The incubation period (time from inge stion to appearance of symptoms) has been reported to range from 2 to 10 days (Arrowood, 1997).

Some infections may be asymptomatic. In other words, not all infections will result in illness and observable symptoms. Asymptomatic hosts ma y still shed oocysts, how ever. Asymptomatic carriage, as determined by stool su rveys, generally occurs at very low rates (less than 1 percent)U.S. Environmental Protection Agency

February 2009 24 in industrialized countries (Curre nt and Garcia, 1991), though highe r rates have been reported in day care centers. Routine bile endoscopy su ggests a higher asymptomatic prevalence; for example, 13 percent of nondiarrheic patients were shown to carry Cryptosporidium oocysts (Roberts et al., 1989). High rates of asymptomatic infection (bet ween 10 and 30 percent) are

common in nonindustrialized countries (Current and Garcia, 1991).

In more severe illnesses, the parasite may be found in the stomach, colon, liver, or lungs with associated symptoms corresponding to infections in those tissues. However, the presence of the parasite in tissues other than the small intest ine does not necessarily i ndicate infection of host cells in those organs (O’Donoghue, 1995).

The level of immunocompetence of the infected person directly relates to the symptoms experienced. Age, concurrent illness/medical treatment, genetic background, pregnancy, and

nutritional status all contribute to immune status. Sympto ms may be more severe in immunocompromised persons (Carey et al., 2004; Frisby et al., 1997). Such persons include those with AIDS, certain cance r patients undergoing chemotherapy, organ transplant recipients treated with drugs that suppre ss the immune system, and patients with autoimmune disorders (e.g., lupus). In AIDS patients, Cryptosporidium has been found in the lungs, ears, stomach, bile duct, and pancreas in addition to the small intestine (Farthing, 2000) . Clifford et al. (1990) found that cryptosporidiosis affected 10 to 15 percent of the AIDS patients, resulting in death in 50 percent of those cases. Beside s the immunocompromised, children and the elderly may also be at higher risk from Cryptosporidium than the general population; how ever, specific data are not currently available to document the degree to whic h these individuals are subj ect to elevated risk. However, previous exposure to Cryptosporidium has been shown to c onfer some amount of immunity (Chappell et al., 1999).

Symptoms of cryptosporidiosis t ypically last from several days to 2 weeks alt hough, in a small percentage of cases, the symptoms may persist for months or longe r. Individuals with either compromised or healthy immune systems may expe rience illness for long periods. Illness from Cryptosporidium is usually self-limiting, with a median duration of 6 days and a mean duration of 9 days (Dupont et al., 1995; Palmer et al ., 1990), although longer durations (mean 19 to 22 days, maximum 100 to 120 days) were reported in a recent Australian survey by Robertson et al. (2002). Relapses were common, with 1 to 5 additio nal episodes in 40 to 70 percent of patients. Shedding of oocysts may continue after th e cessation of the disease symptoms.

Both individuals with compromised and with healthy immune systems have been shown to exhibit chronic sequelae. Immunocompromised individuals generally experience chronic gastroenteritis, which may last as long as the immune impairment. Immunocompromised populations include patients unde rgoing chemotherapy for treat ment of neoplasms, persons undergoing immune suppression treat ment to prevent rejection of skin or organ transplants, malnourished individuals, persons with concurrent in fectious diseases (e.g., measles), the elderly, and persons with AIDS. Chronic illness may mani fest itself as a series of intermittent episodes or may be persistent. Individu als with CD4+ cell counts (a key measure of the health of the immune system) less than 100 cells per mm 3 of blood are at increased risk of illness from Cryptosporidium , while individuals with less than 50 cells per mm 3 are at the greatest risk forU.S. Environmental Protection Agency

February 2009 25 severe disease and prolonged carriage of Cryptosporidium (Hunter and Nichols, 2002; Roefer et al., 1996).

As noted above, chronic sequelae in immunocompe tent patients also have been documented. After resolution of the acute phase in the 2 mont hs following their initial diagnosis, 40.9 percent of patients in one case study reported recurrence of intes tinal symptoms (includes both C.

hominis and C. parvum ) (Hunter et al., 2004). In addi tion, in individuals infected with C.

hominis , other sequelae such as join t pain, eye pain, recurrent headache, dizzy spells, and fatigue were significantly more common than in control subjects. Both C. parvum and C. hominis

infections have sometimes resulted in recurrence of GI symptoms, but only C. hominis infections

have been related to the ot her sequelae noted previously.

Cryptosporidiosis Incidence

Limited information is available on the endemic incidence of cryptospori diosis in the United States. Mead et al. (1999) estimated that th ere are approximately 15 million physician visits annually for diarrhea and that approximately 2 percent of these, or 300,000 cases, are due to cryptosporidiosis. Mead and co lleagues also estimated that of these 300,000 cases, only about 10 percent are attributab le to foodborne transmission, with th e remainder due to the consumption of contaminated water (from dr inking or recreational exposure) or person-to-person contact. Mead et al. (1999) estimated that there are a pproximately 211 million episodes of gastroenteritis (GI illness) in the United States each year, of which only about 38 million are attributable to known pathogens. 3

Prior to 1982, when the CDC impl emented routine reporting of Cryptosporidium among AIDS patients, only 13 cases of cryptosporidiosis had been documented (Ungar, 1990). Subsequently, between 1982 and 1997, more than 1,000 cases of the disease we re reported worldwide (Fayer et al., 1997). Cases reported by CDC between 1999 and 2005 (for a ll routes of exposure) are shown in Figure II.2.2-1; however, documented cases underestimate actual Cryptosporidium infection rates because most cases go unreported.

Worldwide, infection is widespread, exceeding se veral million according to Casemore et al. (1997). As noted previously, the largest known outbreak of the disease occurred in 1993 in Milwaukee, Wisconsin (MacKenzie et al., 1994) and infected 403,000 individuals (CDC, 1996). The most recent data from CDC (2007b) for the year 2005 reported 8,269 cases of Cryptosporidium infection nationally, and re ported significant fluctuations in incidence between summer and other seasons, peaking in late July and early August. According to CDC (2007b), Cryptosporidium is the leading cause of diarrheal illness outbreaks in recreational (chlorinated and nonchlorinated) water. Although 8,269 cases were reported in the United States in 2005, CDC (2007b) estimates that the tota l incidence of domestic cases of Cryptosporidium infection exceeds 300,000 each year.

3 Mead et al. (1999) based the estimates on reported cas es and estimates for degree of under reporting. For example, in the 1993 crypto sporidiosis outbreak in Milwaukee, Wisconsin, medical care was sought in only 12 percent of cases (Corso et al., 2003).U.S. Environmental Protection Agency

February 2009 26 Figure II.2.1-1. Reported Cryptosporidium Infections in the United States, 1999 to 2005

The large increase in the number of cases reported from 2003 to 2005 might have resulted from outbreak-related case reporting (CDC , 2007b); however, that factor is unlikely to account for all of the increase. It is not clear how much, if any, of the increase may be due to changes in reporting patterns and diagnostic testing practices or to a real change in infection and disease (CDC, 2007b).

From 1991 to 2004, six outbreaks of cryptosporidiosis have been associated with untreated recreational waters (Table II.2.1-2).

II.2.2 Giardia

Giardia Life Cycle and Strain Variation

Organisms in the genus Giardia are binucleate, flagellated prot ozoan parasites that exist in trophozoite and cyst forms and are an important cause of waterborne illness worldwide. Both humans and some animal species can carry and transmit Giardia lamblia (also known as G.

intestinalis orG. duodenalis ), which causes the GI illness giardiasis. G. lamblia is highly

infectious and has been shown to cause giardiasis with exposure to as few as 10 cysts (Rendtorff, 1954).

Over the course of the Giardia life cycle, the parasite lives both as a trophozoite and as a cyst form. Inside a vertebrate host, the Giardia trophozoites divide by bina ry fission, attach to the

brush border of the small intest inal epithelium, detach, then become rounded and form a cyst wall. The environmentally resistant cyst is ex creted with the feces, where it moves passively 

### Cryptosporidium Cases in the United States

9,000 

### 1999-2005

8,000ses 7,000am C 6,000iu 5,000idro
4,000psot

3,000pyr 2,000C

1,000

0

1999 2000 2001 2002 2003 2004 2005

Year Data sources: CDC 2005, 2007U.S. Environmental Protection Agency

February 2009 27 Table II.2.1-2. Outbreaks of Cryp tosporidiosis Associated w i th Recreati onal W a ters in the

United States

Year Number o f Cases Number o f O u tbre aks Source of O u tbre ak(s)

1991 0 0 NA

1992 526 2 Wate rslid e an d wave po ol

1993 174 4 Pools

1994 519 2 1 pool, 1 lake

1995 5,487 3 1 pool, 2 wat e rpa r ks

1996 3,025 3 2 pool s, 1 lake

1997 369 1 Fountain

1998 169 8 7 pool s, 1 lake

1999 64 4 3 pool s, 1 fountain

2000 1,368 13 12 pool s, 1 la ke

2001 538 4 3 pool s, 1 hot sprin g

2002 936 7 6 pool s, 1 lake

2003 702 5 4 pool s,* 1 lake

2004 567 7 Pools

* In one p ool o u tbrea k , both Cr ypt o spo r id

ium and Gia r dia were det ected i n wate r sa mple s, so that

outbre a k of 6 3 ca se s is co unted in both the Cr ypt o sp oridium and t he Giardia data.

Source: Data from CDC Su rveilla nce Su mma ri es: Mo rbidity and Mo rtalit y Wee k ly Re port (MMWR)
Surveillan c e for Wate rbo r ne-Disea s e Outbre aks - United State s : 1991 -19 9 2

, 1993-1 994,

1995 -19 96, 1997 -19 98, 1999 -20 00, 2001 -20 02, 2003 -20 04 (CDC, 199 3b, 1996, 19 98, 2000, 20 02, 2 004, 200 6).

through the environment and may be ingested by another host organism. Inside the digestive track of a new host, active cysts release trophozo ites, and repeat their lifecycle (USEPA, 1998). Cysts can be excreted in the stool intermittently for weeks or months, resulting in a protracted period of communicability (CDC, 2007b). Furthermore, cysts can remain viable under typical environmental conditions for periods up to 77 da ys (Bingham et al., 1979, as cited in USEPA, 1998).

Currently, there are five recognized species of Giardia and six generally recognized assemblages

of G. lamblia . Each assemblage has a varying degree of host specificity (see Table II.2.2-1). Some assemblages (i.e., Assemblage s A and B) act as zoonotic asse mblages that can infect most species of mammal, while others (i.e., Asse mblages C through F) are more adapted to a particular host species (Appelbee et al., 2005). For example, Assemblage A has been found in human, beaver, cat, lemur, sheep, calf, dog, fox, chinchilla, alpaca, horse, pig, and cow. The role

of these animals as a source of human infecti on, however, remains uncle ar. Assemblages C and D seem to primarily infect dogs, while Assemblage E infects livestock, an d Assemblage F infects only cats (Appelbee et al., 2005).U.S. Environmental Protection Agency

February 2009 28 Table II.2.2-1. Giardia Taxonomy

Species Ass e mblage Hos t(s )

Giardi a lam b lia (sy n

. G. duo d enali s

, G. intes t inalis ) A1 Huma n, beav er, cat, lemu r, shee p, calf, dog, fox, chin chilla, alpa ca, horse , pig, cow

G. lam b lia A2 Huma n, beav er

G. lam b lia B ( G. enteri c a *) Huma n, beav er, guine a pig

, dog, monke y , horse (B IV)

G. lam b lia C and D ( G. ca nis *) Dog, coyote, mouse

G. lam b lia E ( G. bovi s *) Co w, she ep, alpa ca, goat, pig

G. lam b lia F ( G. c a ti *) Cat

G. lam b lia G ( G. sim oni *) D o m e s t i c r a t

G. lam b lia Novel I Marsupi al (Q uend a – ban d i coot, mou s e, shee p)

G. lam b lia Novel II Marsupi al II (Tasm ania n d e vil)

G. m u ris Rod ents (mice)

G. m i croti Vole and mu skrat

G. ps ittac i Birds (bud ge rigars)

G. arde ae Birds (he r on and ibi s )

G. agilis Amphibia n s (f rog s )

* Denote s re cently pro p o s ed ne w spe c i e s nam es (H unter an d Th ompson, 200 5; Thomp s on and Moni s, 2004 ).

Adapted from : Adam, 2001 ; Appelbee et al., 2005; Ha mnes et al., 2 007; Ol son et al., 2004; an d Tra ub et al., 2005.

Giardia Zoonotic Potential

Cross-species transmission of Giardia is known to occur, and there are many known species and variants (or assemblages) of the Giardia parasite. Of all of the an imal host species suspected of being a significant zoonotic sour ce of human giardiasis by water borne transmission, the evidence presently available suggests that the beaver (Dykes et al., 198 0) and muskrat (USEPA, 1998) are the most likely candidates. The role of these animals as a source of human infection, however, remains controversial. Both of these aquatic mammals can be infected with isolates of Giardia from humans, but each has also be en shown to harbor strains of Giardia that are phenotypically distinct from those found in humans. Thus, it is possible that the beaver harbors two types of

Giardia . One type may be highly adapted to this animal and rarely, if ever, transmitted to humans. The other type may be one acquired by the beaver from human sources, which can multiply in the beaver and in turn be tran smitted via water back to humans (USEPA, 1998).

The role that livestock play as zoonotic reservoirs of Giardia infection also remains

controversial. G. lamblia may be maintained independently through transmission cycles involving wildlife and livestock, though it is unclear how these cycles may interact in zoonotic transfer (Hunter and Thompson, 2005). While both livestock and humans have been implicatedU.S. Environmental Protection Agency

February 2009 29 in contaminating water sources with Giardia , humans are responsible for the majority of the contaminations. Hunter and Thompson (2005) ex amined case studies for the zoonotic potential

of Giardia and found only one case having a significant association with animal contact. They concluded that zoonotic transfer plays only a minor role in the infection cycles of Giardia and that animal contact is not a major risk factor . They authors did not , however, rule out the importance of zoonotic transfer i ndirectly through wate r sources. Both Traub et al. (2004) and Inpankaew (2007) have shown that, in some co mmunities with inadequate sanitation, zoonotic transfer is evident between humans and dogs; howeve r, it is unclear which species is the primary

reservoir.

Thompson (2007) suggested that data ga ps regarding zoonotic transfer for Giardia can be filled using molecular epidemiological studies. Mol ecular genotyping of parasite isolates from susceptible hosts in localized fo ci of transmission or longitudina l surveillance with genotyping might help address such data gaps on zoonotic transfer of Giardia

.

Giardia Routes of Exposure

Giardia is transmitted via fecal-oral exposure and cau ses both endemic and epidemic cases of giardiasis. It is frequently spread person-to-person, especially among children or among persons with poor access to or practice of sanita tion. The main route of exposure to Giardia from

recreational immersion in water is from incide ntal ingestion of wate r during full immersion

activities such as swimming.

Although inhalation of aerosol ized water that contai ns cysts is theoretically possible, cysts are not known to be infectious in lung tissue. De rmal absorption is not known to be a route of

exposure to Giardia cysts in environmental waters.

Giardia Illness Symptoms

Giardia is responsible for a number of health effect s including acute sympto ms that occur during and after the infection. However, Giardia has also been implicated in a number of chronic sequelae. There are a wide variety of symptoms associated with giardiasis that range from asymptomatic infection and acute self-limiting di arrhea to persistent chronic diarrhea, which sometimes fails to respond to treatment.

Giardia produces a broad spectrum of GI sympto ms including one or more of the following symptoms: diarrhea, bloating, we ight loss, malabsorption, steato rrhea (fatty stool), pale greasy and malodorous stools, flatul ence, abdominal cramps, nausea and vomiting, fatigue, anorexia, and chills (CDC, 2000; Hellard et al., 2000; Hopkins and Juranek, 1991; Thompson, 2000). Fever may occur at the beginning of the infectio n (Ortega and Adam, 1997) . Lactose intolerance is frequently present during infection and may persist even after Giardia has cleared from the stool (Wolfe, 1992). Chronic giardi asis appears to be infrequent, but when it occurs, may persist for years (USEPA, 1998). Case reports also indicat e that giardiasis can be associated with the development of reactive arthritis (Tupchong et al., 1999). Illness durations vary, lasting only 3 to 4 days for some indi viduals and several months for

others. Most infections reso lve spontaneously, and the acute stage lasts from 1 to 4 weeksU.S. Environmental Protection Agency

February 2009 30 (USEPA, 1998), but individuals w ith compromised immune systems may have more serious and prolonged infection (APHA, 2004). Immunodeficiency w ith varying degrees of hypogammaglobulinemia or agammaglobulinemia is the most commonly reported form of immunodeficiency associated with chronic giardi asis (Farthing, 1996). Ho wever, giardiasis is one of the few potentially treatable causes of diarrhea in persons with AIDS, and chronic giardiasis does not appear to be a major clinical problem in persons with HIV infections or AIDS (Farthing, 1996; USEPA, 1999).

Asymptomatic infection is very common, with 50 to 75 percent of infected persons reporting no symptoms (Mintz, 1993)—especially in children and in persons with pr ior infections (CDC, 2007b). In a study at the Swiss Tropical Institute, only 27 pe rcent of 158 patients who had

Giardia cysts in their feces exhibited symptoms (Degremont et al., 1981). Although persons with asymptomatic Giardia infection are not likely to seek medical treatment and be diagnosed, they can serve as carriers of infection.

Hospitalizations and deaths due to giardiasis are relatively rare. The CDC estimates that giardiasis causes approximately 10 deaths and 5,000 hospitalizations annually in the United States (Mead et al., 1999). Blood volume depletion or dehydration is the mo st frequently listed codiagnosis on hospital admission. Among child ren under 5 years of age who had severe giardiasis, almost 19 percent also were diagnosed with failure to thrive (Lengerich et al., 1994). Additionally, in the United States and Scotland, more severe cases of giardiasis (i .e., hospitalized patients) seem to occur primarily in childre n under the age of 5 (Lengerich et al., 1994; Robertson, 1996). Age has been shown to signifi cantly affect recovery time; in Scotland, the median length of stay in the hospital for giardias is was significantly longer for persons older than 70 years than for other age groups (11 days compared to 3 days) (Robertson, 1996). Infants and young children may have increased susceptibility to giardiasis due to imm unological factors that increase sensitivity and behavioral factors that increase exposure.

Chronic giardiasis patients often experience recu rrent, persistent, brief ep isodes of loose, foul smelling stools that may be yellowish and frothy in appearance and frequently accompanied by distension of the bowel, foul flatus, anorexia, na usea, and uneasiness in the epigastrium (Wolfe, 1979). In some cases, these symptoms may persist for years; however, in the majority of cases, the parasite and symptoms disa ppear spontaneously. Among 65 cas es of giardiasis encountered in an urban private practice outp atient setting, the mean duration of symptoms was reported to be 1.9 years, and in 38 patients (58 percent) who exhibited chronic symptoms for 6 months or longer, the mean duration of sy mptoms was 3.3 years (USEPA, 1998).

Corsi et al. (1998) evaluated ocul ar manifestations in 141 Italian children with cu rrent and past giardiasis and 300 children without giardiasis. Retinal changes we re diagnosed in 20 percent of the children with giardiasis (mean age was 4.7 years) and in none of the children without giardiasis. These findings sugge st that asymptomatic, nonprogressi ve retinal lesions may occur in young children with giardiasis. The risk of reti nal lesions did not seem to be related to the severity of infection, its duration, or use of metronidazole to tr eat the infection, and may reflect a genetic predisposition to retina l lesions (Corsi et al., 1998).U.S. Environmental Protection Agency

February 2009 31 There is usually no extra-intestinal invasion when Giardia trophozoites infect th e small intestine, but reactive arthritis may occur, and in severe giardiasis, d uodenum and jejunal mucosal cells may be damaged (APHA, 2004).

Giardiasis Incidence

Giardia lamblia is the most common intestinal parasite identified by public health laboratories in the United States (Kappus et al., 1994; Rose et al., 1991). CDC estimates that there are approximately 2 million illnesses annually in the United States due to Giardia (Mead et al., 1999). As noted previously, while all age groups ar e affected by giardiasis, the highest incidence is in children (USEPA, 1998). High risk gr oups for giardiasis include infants and young children, travelers to developi ng countries, the immunocompromised, and persons who consume untreated water from lakes, streams, and shallow wells (USEPA, 1998).

Communities with unfiltered surface water systems experienced a waterborne outbreak rate that was 8 times greater than communities where su rface water was both filtered and disinfected (USEPA, 1998). Data therefore indicate that filtering water to remove microorganisms substantially reduces ri sk of giardiasis.

As with all pathogens, underreporti ng limits estimates of the true incidence of giardiasis. The ratio of reported cases of giardiasis to actual cas es is not known. Mead et al. (1999) used a 38- fold multiplier to estimate incidence for nonbloody diarrhea outcomes (based on Salmonella data) and a 20-fold multiplier fo r bloody diarrhea outcomes (based on E. coli data). Applying the 38-fold multiplier to the 20,075 giardiasis cases that were reported in 2005 (CDC, 2007b) results in an estimated incidence of appr oximately 762,800 total cases in 2005. However, broader estimates are also supported. For exampl e, an estimated 1 to 5 percent of cases of salmonellosis are reported to CD C through passive surveillance (Chalker and Blaser, 1988). If the 1 to 5 percent reported cases is applied to the giardiasis data, then the giardiasis disease burden in the United States in 2006 could have been 401,500 to 2,007,500 cases (135.4 to 677.0 cases per 100,000 population). 4 The true burden of giardiasis in the United States is likely to fall between these two estimates (CDC, 2007b).

Using data from 1992 to 1997, the number of states reporting occurrence of giardiasis increased from 23 to 43, while the annual count of giardi asis cases rose from 12,793 to 27,778, nationally (CDC, 2000). Between 1996 and 2001, however, the number of re ported cases of Giardia infection in the United States (50 states plus Washington, DC) decreased gradually from 27,778 to 19,659 (CDC, 2007b). The cause of this decrease is unknown. Giardiasis became a nationally notifiable disease in 2002, and th e number of cases increased from 2001 to 2002, then stabilized, averaging approximately 20,200 cases per year (CDC, 2007b). See Figure II.2.2-1 and Table II.2.2-2 for reported cases of Giardia infection in the United States from 1992 to 2005.

4 Based on U.S. Census data for 2006, the U.S. population was estimated to be 296,528,800.U.S. Environmental Protection Agency

February 2009 32 Figure II.2.2-1. Reported Giardia Cases in the 50 States p lus Washington DC, 1992 to 2005

The increase in Giardia cases observed for 2002 might refl ect increased reporting after the designation of giardiasis as a nationally notifiable disease starting in 2002. Outbreak-related cases made up 1.6 to 11.6 percent of the total number of case s reported annually for 1999 to 2002. Although the number of states reporting cases increased from 42 to 46 during that time, the number of states reporting more than 15 cases per 100,000 population decreased from 10 in 1998 to 5 in 2002. Transmission of giardiasis occurs throughout the United States, with increased diagnosis or reporting of cases per 100, 000 population occurring in northern states than

Table II.2.2-2. Giardia Infection Occurrence in U.S. Populations

Population Description Occurrence Reference(s)

General 2-5% infection USEPA, 1998

Prevalence in stool samples (infection) 4-12% depending on year and state USEPA, 1998

30% of the population has seropositivity for Giardia (indicates current or past infection) Frost and Craun, 1998

In national survey, 7.2% (of 216,675) stool specimens USEPA, 1999 were positive for Giardia in 1987 and 5.6 percent (of 178,786) were positive in 1991 (infection)

Homosexual males 18% found positive for Giardia (infection) (compared with Faubert et al., 2000; (in New York) 2% among other patients, but giardiasis is not a major clinical problem in persons with HIV or AIDS) Kean, 1979; USEPA,

1999

Small children 7% are asymptomatically infected with Giardia Frost and Craun, 1998 

### Giardia Cases in the United States 1992-2005

30,000)C Ds

25,000ul pse

20,000att50 S

15,000(sesa

10,000a Cid

5,000ariG

0

1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

Year Data sources: CDC 2000, 2005, 2007U.S. Environmental Protection Agency

February 2009 33 in southern states (CDC, 2005) . For 2002, among states reporti ng cases, the incidence of giardiasis ranged from less than 0.1 cases (Texas) to 23.5 cases (Vermont) per 100,000 population. Vermont reported the greatest numbe r of cases per 100,000 population for each of the 5 years of the reporting period.

Giardiasis occurs most frequen tly in the early summer through early fall, with increases in transmission during the summer (CDC, 2007b). This increased transmission coincides with the summer recreation season, which includes increased use of recreational (including community) swimming facilities. Given that a single person can shed millions of cysts and yet remain asymptomatic, transmission in these facilities is likely to be an important mechanism for increased incidence during the summertime (CDC , 2005). People at recreational beaches have been shown to disturb sediment, leading to resusp ension of cysts and an increase in exposure to

Giardia , with higher densities of bathers leading to higher turbid ity and correspondingly higher cyst concentrations (Graczyk et al., 2007; Sunderla nd et al., 2007).

Giardiasis is found most frequently in children 9 years old and younger and in adults aged 35 to 44 years. These groups correspond to young children and their caretakers, who are at increased risk of infection (CDC, 2007b). In 2005, 54.3 per cent of reported cases occurred in males compared to only 43.9 per cent in females, while 1.8 percent of reports did no t record gender (CDC, 2007b). According to CD C (2007b), the discrepancy betw een cases in males versus females might be attributable in part to increas ed risk of infection du ring sexual contact between men although the discrepancy was found in nearly every age group.

A review of the data presented in Table II.2.2-2 clearl y indicates that Giardia infection in the United States is common and widespread.

From 1991 to 2004, seven outbr eaks of giardiasis have been associated with untreated recreational waters (Table II.2.2-3).

II.3 Viruses Zoonotic Potential

A great deal of public and animal health policy is based on the pr emise of the host specificity of viruses. That is, it has long been assumed that each virus has a distinct and limited range of host species that it can infect. 5 Viruses also are restricted to pa rticular tissues of the host’s body that they can infect (i.e., tropism), which affects bot h the mode of transmission and the disease that the virus infection may cause (Cliver and Moe, 2004). Most waterborne viruses are thought to be transmitted by a fecal-oral route (i.e., the vi rus is shed via the in testines and infects upon ingestion) that requires a tropism that includes th e lining of the GI tract. Viruses that infect via the intestine and cause GI illness (e.g., enteroviru ses) may also have secondary tropisms in other

5 In this case, a distinction is being made between the important and much studied hypothesis that viral evolution accelerates when viruses “jump species” in the case of emerging dis eases and the regular maintenance of multi-host adaptation that is recognized in other pathogen s. Although rapid viral evolution facilitated by crossspecies transmission is a recognized public health concern for viruses that have airborne transmission (e.g. influenza and severe acute respiratory syndrome [SARS]-associated coronavirus), cros s-species transmission has not been a traditional concern for waterborne, fecally transmitted viruses.U.S. Environmental Protection Agency

February 2009 34 Table II.2.2-3. Outbreaks of Giardiasis Associated with Recreational Waters in the United

States

Year Number of Cases Number of Outbreaks Source of Outbreak(s)

1991 34 4 3 pools, 1 lake

1992 0 0 NA

1993 61 3 2 lakes, 1 river

1994 80 1 Pool

1995 0 0 NA

1996 77 1 Pool

1997 0 0 NA

1998 0 0 NA

1999 18 1 Pond

2000 0 0 NA

2001 0 0 NA

2002 2 1 River

2003 212 2 Pools*

2004 9 1 Lake

* In one pool outbreak both Cryptosporidium and Giardia were detected in water samples, so that
outbreak of 63 cases is counted in both the Cryptosporidium and the Giardia data.

Source: Data from CDC Surveillance Summaries: Morbidity and Mortality Weekly Report (MMWR) Surveillance for Waterborne-Disease Outbreaks - United States: 1991-1992, 1993-1994, 1995-1996, 1997-1998, 1999-2000, 2001-2002, 2003-2004 (CDC, 1993b, 1996, 1998, 2000, 2002, 2004, 2006).

tissues (e.g., neurological tissues ). While much virus infectiv ity research has been conducted in

vitro using cultured animal cells that at least partially reflect the host specificity (but not the tropisms) of viruses, many important enteric wa terborne viruses of humans (e.g., noroviruses) remain difficult to detect or quant ify in cultured cells (NRC, 2004; St raub et al., 200 7). For this reason, it remains unclear whether in vitro infectivity is relevant to the in vivo host ranges of viruses. Although no confirmed examples of wa terborne viral zoonoses have been reported, several viruses (e.g., swine he patitis E virus [HEV]) are pot entially transmi ssible between species, and water may serve as a vehicle for their transmission under some circumstances. Thus, assessment of the prospect of waterbor ne viral zoonoses is ongoing (Cliver and Moe, 2004).

Cliver and Moe (2004) consider the criteria for determining whet her a virus can function as a

waterborne zoonosis to include the following:

1. Animal reservoir: Does the agent regularly infect at least one animal species, independent of exposure to humans?

2. Transmission to humans: Are humans who are in contact with the alleged animal reservoir more frequently infected w ith this virus than people who are not?U.S. Environmental Protection Agency

February 2009 35 3. Shedding: Is the candidate virus shed by the reser voir animal species in ways that might

lead to contamination of water?

4. Stability: Is the candidate virus stable enough in the water vehicle to permit transmission by this pathway?

Despite the potential for rapid evolution in viruses, ongoing mon itoring and research activities are important for public health protection, even though viruses are not currently kn own to have

the attributes outlined in the intr oduction of this paper. An overview of information related to zoonotic potential for rotavirus, HEV, and adenovirus follows.

Rotaviruses

Rotaviruses (groups A, B, and C) have been documented in humans and animals, and interspecies transmission including human in fection by a bovine strain has been reported (Abbaszadegan, 2006). The primary route of expos ure for rotavirus is the fecal-oral route although exposure through ot her routes also has been reported to a lesser extent. Rotavirus is stable in the environment, and its transmissi on can occur through inge stion of contaminated water or food. Rotavirus illness typically results in vomiting and watery diarrhea for 3 to 8 days. Fever and abdominal pain occur fre quently as well. According to th e CDC, this virus is the most common cause of severe diarrhea in children. Sy mptoms tend to be less severe for adults. Approximately 70,000 children in the United States ar e treated in the hospita l for rotavirus each year (Glass, 2006). A community waterborne outbr eak of rotavirus gastroenteritis occurred in Colorado in 1981 (Hopkins et al., 1984). The outb reak was attributed to sewage contamination of the water supply and a failu re of chlorination treatment.

Hepatitis E

HEV may be zoonotic (Cliver and Mo e, 2004; USEPA, 1999). In pigs and rats, this virus is very similar to human HEV. Experimental studies have indicated that human st rains can infect pigs, and porcine strains can infect primates (Cliver and Moe, 2004). In developing countries, the seroprevalence of HEV infection can be as high as 60 percent. The most common route of exposure for HEV is ingestion of contaminated food or water. Transmi ssion via person-to-person contact is less common. Typical symptoms of HEV illness may include jaundice, fatigue, abdominal pain, loss of appetite, nausea, and vomiting. Pregnant women who contract hepatitis E are at high risk of severe illness and death. For this sensitive subpopulation, mortality can be as high as 20 percent in developing countries, but mortality is rare in developed countries (Craun et al., 2004a). HEV is uncommon in the United States and the CDC does not track incidence rates. In an EPA study of sporadic human HEV, nearly 50 percent of the infected persons had

traveled to endemic areas in other countries or received bl ood transfusions (USEPA, 1999).U.S. Environmental Protection Agency

February 2009 36 Adenovirus

Adenovirus may be zoonotic (Mwe nda et al., 2005). Mwenda and colleagues identified enteric adenovirus in captive olive baboons, vervet monkeys, and the yellow baboons in Kenya. These findings suggest there may be a possibility of zoonotic tran smission of adenoviruses from nonhuman primates to humans in Kenya. Adenovirus can enter a susceptible host by the nose, mouth, or eye membranes. Water may play a meaningful role in the transmission for many human adenovirus serotypes, including the enteri c adenovirus that is tr ansmitted via the fecaloral route (Heerden et al., 2005). Heerden et al . (2005) detected human adenovirus in 4 of 51 (7.8 percent) samples of river water and 9 of 51 (17.7 percent) samples of dam water.

Human adenoviruses may cause of a wide spectr um of acute and chronic diseases, including keratoconjunctivitis, upper respirat ory tract infections, pneumonia, gastroenteritis, cystitis, and encephalitis (Gray et al., 2005). Molecular studi es have recently shown adenoviruses to be associated with bronchopulmonary dysplasia (C ouroucli et al., 2000) a nd chronic obstructive pulmonary disease (Hogg, 2001).

Immunocompromised persons, including people w ith AIDS, bone marrow transplant patients, pregnant women, and children are more susceptible to adenovirus infectio ns (Baldwin et al., 2000; Crawford-Miksza and Schnurr, 1996). Infectio ns in these populations may result in severe illness and death (Chakrabarti et al., 2002; Runde et al., 2001). U.S. surveillance for adenovirus is relatively incomplete (Gray et al., 2005) and well-documented in cidence rates are not available, especially fo r waterborne outbreaks.U.S. Environmental Protection Agency

February 2009 37 

## III. P ATHOGEN 

## I NTERACTIONS WITH THEIR 

## E NVIRONMENT

Pathogens interact with the ambient environment, other microorganisms, plants, and with their hosts. This section provides a summary of how pathogens respond to various environmental parameters. Information on in teractions with other microorganisms and animal manure are briefly covered in this overview followed by sections that desc ribe how the water environment affects pathogen survivability and phenotype and how host animals can influence pathogen characteristics and mechanisms of rapid evolution. The behavior of pathoge ns in ambient waters

is often different from the behavior of indicators in ambient water.

Although there are important waterborne amoebic pathogens, they are not associat ed with animal fecal material. However, important bacterial pat hogens that are associated with fecal material (e.g., Salmonella

, Campylobacter ) interact with free-living amoeb ae in ways that could impact recreational water quality. The most notable, Legionella, infects and replicates in free-living

amoebae and is considered more of a risk in dr inking water than in recreational waters (Borella et al., 2004; Marrie et al., 2001). Some human b acterial pathogens are even thought to have evolved in association with amoebae (Berk et al., 2006). Tezcan-Merdol et al. (2004) investigated the uptake and repli cation of salmonellae in amoebae. Three different serovars of

Salmonella enterica (Dublin, Enteritidis, and Typhimurium) were evaluated for internalization by 5 different isolates of axenic Acanthamoeba species. The Dublin serovar was internalized more efficiently than the other two serovars, and the Acanthamoeba rhysodes isolate was more

efficient than the other four isolates. The researchers concluded that Acanthamoeba species can

differentiate Salmonella serovars and that internalization of the bacteria produces cytotoxic effects mediated by defined bact erial virulence loci. Axelsson-Olsson et al . (2005) studied the

infection of Acanthamoeba polyphaga by four different Campylobacter jejuni strains. The infecting bacterial cells were observed to be act ively moving in amebic vacuoles and survived longer when cocultured with amoebae than when cultured alone. They indicated that free-living amoebae may serve as a nonve rtebrate reservoir for Campylobacter jejuni

.

Guan and Holley (2003) examined E. coli O157:H7, Salmonella

, Campylobacter

, Yersinia

, Cryptosporidium , and Giardia in animal manure. Of those pathogens considered, E. coli O157:H7 was the most persistent in cattle manure regardless of the temperature and manure form (solid or slurry) while Campylobacter and Giardia were weakest survivors in manure. The authors concluded that holding manure at 25° C for 90 days wi ll render it free from the pathogens considered. This has indirect imp lications for ambient water quality because the conditions experienced by pathogens after excretion but before introduction into ambient water

contribute to the overall likelihood the pathogen will be infectious by the time it potentially can reach a human host through recreational contact.

III.1 Water Environment Affects Pathogen Survivability and Phenotype

The most common environmental factors studied for their impact on pathoge n survival in water are pH, salinity, light exposure, and temperature. Additional e nvironmental characteristics that may influence pathogen survival, infectivity, and virulence include: UV light (duration, intensity), rainfall, runoff, dispersal, suspended solids, turbidity, nutrients, organic content,U.S. Environmental Protection Agency

February 2009 38 organic foams, water quality, biol ogical community in water colu mn, water depth, stratification, mixing (e.g., wind and waves), presence of aquatic plants, biofilms, and predation.

Thomas et al. (2006) examined extreme rainfa ll and spring snowmelt in association with 92

Canadian waterborne disease outbreaks betw een 1975 and 2001. Accumulated rainfall, air temperature, and peak stream flow were used to determine the relations hip between high impact

weather events and the occurrence of waterborne disease outbreaks. For rainfall events greater

than the 93 rd percentile, there was a grea ter than 2-fold increase in the odds of an outbreak compared to rainfall events less than the 93 rd percentile. For each degree-day above 0° C, the

relative odds of an outbreak in creased by a factor of 1.007. The odds ratio is small on a per degree-day scale, but is notabl e over longer timeframes. For example, over a 42-day period, a 5° C increase in the maximum daily air temperat ure would result in over a 4-fold increase (1.007 (5×42) = 4.33) in the relative odds of an outbrea k. Stream flow and stream flow peaks did not show a difference between cases (outbreaks) and controls (no outbr eaks), but there was a

considerable lack of data on stream flow.

An overview of the available information for the six key waterborne zoonotic pathogens follows. Information concerning these factors is limited for mo st waterborne pathogens. Thus, in order to develop effective control strategies, a dditional research may be necessary.

III.1.1 Pathogenic E. coli Survival in the Environment

Pathogenic E. coli can be expected to survive outside of a host animal anywhere from a few days to several weeks, depending upon environmental conditions. Pathogenic E. coli seem to interact with the environment in a similar fashion to nonpathogenic E. coli and do not lose their virulence during prolonged survival in th e environment. Survival of E. coli O157:H7 in surface waters was found to be longer at lower temperatures (Czajkowska et al., 2005) and was 2 to 3 times longer in river and lake sediments at the sa me temperatures (see Table III.1.1-1).

Table III.1.1-1. Survival of E. coli O157:H7 in Ambient Waters

Water matrix °C Days to Decrease Reference 99% 99.99%

Surface waters (lakes and rivers) 6 4-11 8-22

Czajkowska et al., 2005 Surface waters (lakes and rivers) 24 2-8 5-10

Sediments (same lakes and rivers) 6 10-20 25-39

Sediments (same lakes and rivers) 24 5-8 10-30

River water with feces 15 7.5 14.5 McGee et al., 2002

Unfiltered lake water 8 91 NA

Wang and Doyle, 1998 Filtered tap water 15 >91 NA

Unfiltered lake water 25 14-28 NAU.S. Environmental Protection Agency

February 2009 39 Although direct contamination of ambient water due to domestic an imals or livestock wading in water is possible, most contamination of water is due to runoff from pastures and fields after land application of manure. E. coli O157:H7 can survive for at least several weeks in animal feces and slurries (Avery et al., 2005) and has been demonstrated to survive at least 500 days at -20 ° C in frozen soil (Gagliardi and Karns, 2002).

III.1.2 Campylobacter Survival in the Environment

Campylobacter has been shown to survive in aquatic e nvironments with low temperatures (4° C) between 8 days (Buswell et al., 1998) and 4 months (Rollins and Colwell, 1986).

Buswell et al. (1998) found that survival times of Campylobacter isolates differed by 2- to 4-fold depending on the combination of temperature a nd oxygenation tested. The mean survival times in sterile microcosms were 202 hours at 4° C, 176 hours at 10° C, 43 hours at 22° C, and

22 hours at 37° C. The survival times were considerably longer in the presence of the autochthonous water microflora (two strains tested survived 700 and 360 hours at 4° C).

Aerobic conditions decreased the survival of one strain 30 percent and increased the persistence of another strain by more than 3-fold. With in biofilms, the pathogen persisted up to the termination of the experiments after 28 and 42 days of incubation at 30 and 4° C, respectively.

Rollins and Colwell (1986) found that Campylobacter incubated in filter-s terilized stream water

was recoverable after 4 months at 4°C. Inc ubation at 25°C resulted in a decline to the nonculturable state within 28 days, and at 37°C, th e nonculturable state was reached in 10 days. Direct counting methods indicate that the nonculturable but viable state of Campylobacter is significant.

III.1.3 Salmonella Survival in the Environment

Salmonella are also found frequently in sewage, soil, and various surface waters. The greatest

source of the bacteria is fecal contamina tion. Under suitable environmental conditions,

Salmonella can survive for weeks in waters or years in soils (Lightfoot, 2004). Salmonella grow at temperatures ranging from 10 to 43o C, but some serovars have suppressed growth at temperatures above 40o C (Covert and Meckes, 2006). Salmonella can grow at pH 4–8 and at water activities above 0.93. Under some conditions, Salmonella may proliferate below 4 o C and survive below pH 4 (Lightfoot, 2004).

III.1.4 Leptospira Survival in the Environment

Leptospira survive longer in the environment in warm, humid conditions. Leptospirosis is seasonal, which relates to the pathogen’s survival in the environment. In temperate regions, there is a peak during summer or fall, whereas in warm-climate regions, the rainy season is associated with peaks because dessication de creases pathogen survival (Levett, 2001).

Ganoza et al. (2006) compared levels of Leptospira in urban and rural environmental surface waters in the Peruvian Amazon region of I quitos. The concentration of pathogenic Leptospira was higher in urban than rural water sour ces and rats were the indicated zoonosis.U.S. Environmental Protection Agency

February 2009 40 III.1.5 Cryptosporidium Survival in the Environment

As noted previously, because Cryptosporidium oocysts are extremely resistant to environmental or engineered degrada tion, the survival of Cryptosporidium under a variety of environmental and drinking water treatment conditions has been evaluated by many investigators. While the majority of these studies have considered the effects of physical antagonism (e.g., freezing, heating, UV exposure), studies have also been conducted to consider th e role of microbial antagonists (microbial predation), chemical anta gonists (such as disinfection), and aging. This section focuses primarily on aspects of physical antagonists in the environment because they are most pertinent to the topic of this paper.

Robertson et al. (1992) ev aluated the sensitivity of C. parvum oocysts to a variety of environmental pressures such as freezing, dessication, and water treat ment processes, as well as in physical environments commonly associated w ith oocysts. Approximately 97 percent of the test oocysts were inactivated after 18 days at 22 ° C, suggesting that the levels of viable oocysts in surface waters might be in fluenced by seasonal temperature variations. After 2 hours of drying oocysts at room temperature, only 3 percent of oocysts were still viable, and after 4 hours, no oocysts were viable. When stored at 4° C, th e percentage of oocysts re maining viable in stool samples decreased steadily with time. (In th e study, the relationship between oocyst viability and time varied with individual.) After 176 days in tap water, ri ver water, or cow feces, there was a statistically significant increase in the proportion of dead oocysts in test samples.

Seawater was even more lethal to oocysts, with a statistically significant increase in dead oocysts by 35 days of exposure to the test conditions. C. parvum oocyst viability is sensitive to a wide range of typical environmental conditions while remaining relatively insensitive to some water treatment processes. Robertson et al. (1992) also emphasized that oocyst viability depends on the amount of time to which oocysts are exposed to a physical or chemical stress in the

environment.

Temperature has a significant effect on oocyst surv ival, with (unfrozen) colder waters promoting the highest survival rates (USEPA, 2001a). Wa rm and boiling water completely neutralizes oocysts, and the temperature of the water dete rmines the time required for the treatment to become effective (Anderson, 1985). Cryopreserva tion studies conducted by Fayer et al. (1991, 1997) indicate that oocyst survival depends on the temperature and duration of freezing conditions, implying that C. parvum oocysts are not necessarily rendered noninfectious by being

frozen per se . In another study, Fayer and Nerad (1996) demonstrated that the infectivity of C.

parvum oocysts after freezing is dependent on the te mperature and duration of freezing. In general, shorter freezing times are required to neutralize infectivity when lower freezing temperatures are employed (e.g., 1 hour at -70° C versus 168 hours at -15° C to completely neutralize infectivity) (Fayer and Nerad, 1996).

Temperature stability studies al so were conducted by Sattar et al. (1999) who evaluated the freeze/thaw susceptibility of va rious preparations of oocys ts including highly purified preparations as well as infected calf feces. The re sults of this study indicated that oocyst stability under freezing conditions is at least partially de pendent upon the surrounding matrix, with fecal material conferring a cryopreservative effect on oocysts. In the absence of freezing conditions, colder water temperatures tended to promote the survival of most microorganisms. In water, C.U.S. Environmental Protection Agency

February 2009 41 parvum may survive outside of mammalian hosts fo r several months or more depending upon water temperature (Straub et al., 1994).

Fayer et al. (1998) investigated th e effect of water temperatures ranging from -10 to 35° C and a few higher temperatures on oocyst infectivity. As water temperat ure was increased from -10 to 20° C, oocysts remained infectious for longer exposure times. For example, oocysts retained their infectivity for only 1 week when suspended in water held at -10°C but remained infectious for up to 24 weeks in 20° C water. As water temperatures were increased above 20° C, oocysts retained their infectivity for shorter exposure times (Fayer et al., 1998). Under conditions of high water temperatures, higher than typically found in surface waters, Fayer (1994) indicated

that all evidence of C. parvum infectivity was lost within 60 seconds when temperatures exceeded 72° C or when temperatures of at least 64° C were maintained for 2 minutes.

Holding oocysts to 45°C for 5 to 20 minutes was effective in completely neutralizing infectivity (Anderson, 1985). Anderson (1986) examined the in fectivity (determined in infant mice) of oocysts from calf fecal samples that had been drie d in a barn (< 60 percen t humidity) in either winter or summer months. In summer temperatures (i.e., 18 to 29° C), oocysts completely lost infectivity in 1 to 4 days. Experiments conducted in winter, with air temp eratures ranging from - 1 to 10° C, demonstrated a complete loss of infec tivity within 2 to 4 days. Control samples kept moist and refrigerated retained in fectivity for up to 2 to 3 weeks.

Limited studies have been conducted on the eff ects of physical shear on oocyst viability; these studies have attempted to assess the potentially abrasive effects of oocyst contact with sand and gravel particles or through fast -flowing waters. Parker and Smith (1993) found that after shaking with sand for 5 minutes, 90 minutes, and 2 hours, the number of non-viab le oocysts increased significantly to 50 percent, 99.7 percent, and 100 percent, respectively. When chlorination followed 5 minutes of sand shaking, the observed non-viable oocysts increased to 68 percent. When oocysts were on an orbital shaker at 60 and 120 rpm, oocyst viab ility declined linearly over the course of an hour, with approximately 50 percent loss of viability noted at 20 minutes (Sattar et al., 1999). These au thors also showed that oocysts subjected to 2000 psi (13.9 Mpa)

for 1 minute had little reduction in viability.

Sattar et al. (1999) also evaluate d the effects of microbial pred ation on oocyst survival. They observed that oocysts incubated in dialysis casse ttes that were suspended in natural waters exhibited significantly longer survival times when bacterial populations were excluded from the suspension water. The observation implies that mi crobial predation may play an important role in reducing oocyst survival in ambient (natural) waters.

Nasser et al. (2007) examined the effect of sunlight and salinity on the die-off of C. parvum

. Experiments were carried out for 7 days in tap and seawater and sunlight and dark conditions. Oocyst die-off was greatest when exposed to seawater and sunli ght (0.44 log/day); oocysts in tap water in the dark, exposed to sunlight, and oocysts in seawater in the dark had die-off rates of 0.1, 0.22, and 0.19 log, respectively. At the end of the 7-day study period, a 3 log reduction in infectivity was measured in the sunlight- and seawater-exposed oocysts. Cryptosporidium oocysts retain substantial infectivity for seve ral months at salinity levels corresponding to

estuarine coastal waters (Fayer, 2004a) and for several week s in seawater (WHO, 2006). TheseU.S. Environmental Protection Agency

February 2009 42 studies indicate that Cryptosporidium can thus pose a serious health hazard to humans by direct

and indirect contact in recreational waters.

King et al. (2005) measured oocyst inactivation ra tes in reagent-grade an d environmental waters over a range of temperatures. Oocysts incubate d at 4 and 15° C remained infective over a 12- week holding period. A 4 log 10 reduction in infectivity wa s observed for both 20 and 25°C incubation treatments at 12 and 8 weeks, respectiv ely, for all water types examined. This is a faster rate of inactivation for oocysts than had been previously reported. Inactivation at higher temperatures is likely a function of increased oocyst metabolic activity.

Li et al. (2005) measured th e inactivation rate of bovine C. parvum oocysts subjected to temperature regimes designed to mimic the diurna l oscillations of ambien t temperature in bovine feces exposed to sunlight in co mmercial cattle operations in Califor nia. No infectious oocysts

were observed after 1- to 5-da y cycles of 40, 50, 60, and 70° C. The loss of infectivity was primarily due to partial or complete in vitro excystation during the firs t 24-hour diurnal cycle and secondarily to thermal inactivation of the remaining intact or par tial oocysts. The results suggest that as ambient conditions generate internal feca l temperatures greater than or equal to 40° C, rapid inactivation occurs at a rate equal to or greater than 3.27 log 10 reduction per day for C.

parvum oocysts deposited in the feces of cattle.

Méndez-Hermida et al. (2005) reported on batch-process solar disinfection of . C. parvum oocysts in water. Oocyst suspensions were exposed to simulated sunlight (830 W/m 2 ) at 40° C. Viability assays and infectivity tests indicated that exposures of 6 and 12 hours reduced oocyst infectivity from 100 percent to 7.5 per cent and 0 percent, respectively.

The behavior of lake inflows is important in determining pathogen trans port and distribution. Inflows that are warmer than the lake water will move over the surface of the lake, whereas

inflows that are colder than the lake will si nk beneath the surface layer where they will flow along the bottom towards the deepest point (Brookes et al., 2004). Th e fate of pathogens in lakes is determined by factors such as settling and in activation by temperature, UV, and predation by other microorganisms. Brookes and co lleagues found that inactivation of Cryptosporidium by UV light can be rapid or slow, de pending on the depth of the oocysts in the water column and the extinction coefficient for UV light.

Pokorny et al. (2002) investigated the effects of temperature on ooc ysts stored in the dark in

filter-sterilized and nonfilter-steri lized river water. They report ed that as the temperature was increased from 4 to 23° C, the infectivity of oocysts decreased; no infectious oocysts were

detected after 1 week at -20° C.

Excreted Cryptosporidium oocysts can survive for substantial periods in animal wastes and soils. Thus, contaminated runoff can enter ambient water and result in potential human exposures. The numbers of oocysts excreted by infected young animals may be es pecially large, between 1 and 10 million oocysts per gram of feces (WHO, 2006). Oocyst survival for 4 weeks or more has been documented in concentrated animal wastes, particularly at low temperatures (4° C) (WHO, 2006). In the environment, the va st majority of oocysts (99 per cent) are inactivat ed by repeated freeze-thaw cycles independent of the number of times the soil was frozen (Fayer, 2004a).U.S. Environmental Protection Agency

February 2009 43 Therefore, Cryptosporidium may be environmentally limited in parts of the United States during

the winter season. There is evidence that in wa rmer ambient temperatures, between 4 and 20° C, very little inactivation of oocysts occurs in different types of ag ricultural soils (Jenkins et al., 2002; WHO, 2006). Factors that ar e known to reduce oocyst survival in soils include drying and basic pH (Fayer, 2004a). Oocyst survival in various water matr ices is highly variable, but survival for longer than 30 days has been demonstr ated in several studies. Approximately 4 to 5 percent of oocysts in tap water and river water samples survived after approximately 6 months, with approximately 37 percent in activation after 2 days exposu re to tap water (USEPA, 2005a, 2005b).

Walker et al. (2001) tested ooc yst degradation (as indicated by microscopic examination) in response to the combined stresses of water poten tial (sodium chloride so lute potential), abovefreezing temperatures (4 and 30° C), and a subfreezing temperature (-14° C) for different freeze thaw cycles (-14 to 10° C). The degradation coefficients were estimated using multiplicative error and exponential decay mode ls. Increased water potenti al increased the rate of C. parvum population degradation for all temp erature conditions investigated. The effects of water potential were roughly four times those noted for freezing alone. The difference between the effects of freeze-thaw cycling and simple freezing may be caused by mechanical damage to the oocyst wall. The authors conclude that water potenti al conditions encountered under field conditions are likely to lead to more rapid degradation of oocyst popu lations than might be expected from studies of degradation in calf fece s, distilled water with antibioti cs, and reverse osmosis water at low temperatures.

III.1.6 Giardia Survival in the Environment

Studies of the effect of temperatur e and other environmental factors on Giardia cyst survival

were summarized in EPA’s Giardia Human Health Criteria Docume nt (USEPA, 1998). Survival was determined based on dye inclusion/exclusion, excystation, or animal infectivity studies.

Some studies used Giardia muris cysts, whereas others used Giardia lamblia cysts. Overall, the studies on the effect of temperature indicated that survivability decreased as temperature increased and that while some cysts could surv ive a single freeze-thaw cycle, repeated freezethaws as might be expected in the environm ent would likely inactivate cysts (USEPA, 1998). Johnson et al. (1997) inves tigated the survival of cysts in marine waters and determined that viability was reduced 99.9 percent in 3 hours in mari ne waters exposed to sunlight (as reported in USEPA, 1998). They also found that 77 hours were required to ge t 99.9 percent in activation in the dark and that cysts survived longer at a salinity of 28 mg/L than at 35 mg/L. Because

different waters were used in th e experiments, however, these inve stigators could not rule out the effect of factors other than salinity.

Olson et al. (1999) found that Giardia cysts were noninfective in water, feces, and soil following 1 week of freezing to -4° C and within 2 weeks at 25° C. At 4° C, Giardia cysts were infective for 11 weeks in water, 7 weeks in soil, and 1 week in cattle feces. Robe rtson and Gjerde (2006)

tested survival of Giardia cysts (as well as Cryptosporidium oocysts) during winter in an aquatic environment (approximately 1 to 7° C) in Norw ay. Three conditions were compared, distilled water, river water, and submersion of a filter chambe r containing cysts into the river. The rate of decline in viability was simila r under all three conditions, and no Giardia cysts with apparentlyU.S. Environmental Protection Agency

February 2009 44 viable morphology could be detected after 1 month. Boiling Giardia for 1 minute reduces viability (as determined by flurogenic dyes) to less than 1 percent and renders them noninfectious (as determined by animal infectivity ) (El Mansoury et al., 2004) . Storage at 4° C and -4° C for up to 7 days preserves Giardia cyst viability and infect ivity. Storage at 30 ppt (parts per thousand) salinity for 4 weeks decreased viability to 30 percent, but all animals were infected. Storage at 50 ppt salinity for 4 weeks d ecreased viability to 5 percent and 80 percent of animals were infected. Storage at 50 ppt salinity for 4 weeks resulted in zero viability (El Mansoury et al., 2004).

III.1.7 Virus Survival in the Environment

Viruses are more stable at lower temperatur es; however, different vi ruses have different

stabilities under similar environmental conditions. For example, astrovirus es survive longer than poliovirus and adenovirus, but shorter than rota virus and hepatitis A virus (Sobsey, 2006). For poliovirus and parvovirus, 90 percent inactivation was observed after 1 to 3 days at 28° C, and 90 percent inactivation was observe d after 10 days at 6° C (Gri ffin et al., 2003). Parvoviruses

are the most heat stable enteric viruses know n (Gerba, 2006a). Enterovirus nucleic acid is detectable for 60 or more days, whereas infectious virus is detectable for 51 days or less (Griffin et al., 2003). Below 5° C, enteroviruses can survive for years (Gerba , 2006b). Limited data suggests that adenoviruses survive longer in water than enteroviruses and hepatitis A virus (Enriquez and Thurst on-Enriquez, 2006).

Pesaro et al., (1995) evaluated th e persistence of five animal viru ses, representing picorna-, rota-, parvo-, adeno-, and herpesviruses, and the coliphage f2, using a filter sandwich technique that

mimics the environment in various states of manure. Depending on ambient temperature, pH, and type of animal waste, 90 percent reduction of virus titer varied, ranging from less than 1 week for herpesvirus to more than 6 months for ro tavirus. Virus inactivat ion was faster in liquid cattle manure, a mixture of urine and water (pH > 8.0), than in semiliquid wastes that consisted of mixtures of feces, urine, wa ter, and bedding materials (pH < 8.0) . The authors conclude that viruses contained in manure may persist for prol onged periods of time if stored under nonaerated conditions and this may lead to enviro nmental contaminati on with viruses.

III.2 Host Animals Can Influence Pathogen Characteristics and Mechanisms of Rapid

Evolution

There is evidence that zoonotic pathogens may cha nge in infectivity, virule nce, and the severity of disease caused in humans depending on thei r previous host environm ent. There is also evidence that some of these host-factor change s can influence subsequent infection cycles in exposed hosts. For example, in laboratory e xperiments using nutritionally deficient mice (selenium-deficient or vitamin E-deficient), Morse (1997) reported that a normally avirulent (mild) coxsackievirus B3 isolate gave rise to a virulent variant, albeit through an unknown

mechanism. The virulent variant resembled ot her known virulent genoty pes and was stable upon subsequent infection of other mice with adequate nutrition. In another demonstration of host influence on pathogen evolution, yeast with dele tions in genes that normally suppress viral RNA

recombination became “hotbeds” for viral recombin ation. The host (in this case yeast) genes could affect viral recombinant accumulation by up to 80-fold (Serviene et al., 2005). The keyU.S. Environmental Protection Agency

February 2009 45 mechanisms of phenotypic change in pathog ens are genetic diversity (coinfection and quasispecies), cryptic genes, mutators, and epig enetic effects, and are summarized as follows.

Genetic diversity (co-infection and quasispecies): The pathogen populati on within a single host can be comprised of several or even num erous genetic variants of the same zoonotic pathogen. This variation can occur when the infectivity is such that several genotypes coinfect the host simultaneously, then “compete” or “cooperate” as infection progresses. The fluctuations in genotype prevalence depend on host-pathogen interactions as some genotypes gain dominance and others become rare. Hi gh genetic diversity also can occur when pathogens have high mutation rates. This la tter phenomenon, referred to as “quasispecies,” occurs mainly in RNA viruses because vira l RNA replicase enzymes lack proofreading functions during replication, which leads to high mutation rates (Domingo et al., 1998; Morse, 1997). For example, caliciviruses may have as many as 1 to 10 mutations per template copy (Smith et al., 1998), and within-person HEV sequence diversity has been documented to range from 0.11 to 3.4 percent (Grandadam et al., 2004). For retroviruses, every virus particle may be genetically differe nt from every other pa rticle. In fact, the cooperativity 6 of many virus particles’ genetic material may be n ecessary to complete the infection process (Lederberg, 1998).

Cryptic genes : Meiotropy is the ability of an organism to gain a phenotypic trait through mutation (Brubaker, 1991). One example of a cryptic gene is the yopA gene in Yersinia pseudotuberculosis . A stretch of deoxyadenosine nucleotides (DNA base A) can spontaneously change in length between 8 and 9 bases, which can result in the absence or presence of the gene product. In Y. pseudotuberculosis , the absence of the gene product

results in increased virulence. 7 In Y. pestis (plague), the gene yopA is not normally expressed; however, molecular manipulation that causes expression of the yopA gene product reduces the virulence of Y. pestis . Thus, it is possible that endemic hosts of Y. pestis could harbor a strain of lower virulence, which by one mutation could become hypervirulent and potentially cause an outbreak of plague (Rosqvist et al., 1988). Another example is in

Salmonella , in which flagellar antigens can be expre ssed or silenced in a reversible manner by inversion of a segment of DNA that moves the promoter from one locus to another (Lederberg, 1998). This type of genetic rearrangement may se rve as a mechanism of phase variation for antigenic factors in the bacteria. More standard transcriptional control of the gene expression might allow for low levels of transcriptional leakage and small quantities of antigen to be produced. Prior trace levels of antigens could be sufficient to promote host immunity. Bacteria carry site-specific recombinases that are capable of scrambling bacterial genomes in order to suppress and unsuppress genes.

Mutators : Several genes have been identified that influence the mutation rate of bacteria. For example, bacterial cells that carry the MutD5 protein, which binds to DNA polymerase, accumulate a broad spectrum of base substitu tions and frameshift mutations (the mutation

6 The hypothesis that swarms of viral genotypes coop erate to comprise an “average phenotype” differs from quorum sensing, which is the coordination of bacteria l cells via cell-to-cell comm unication with small signaling molecules to form biofilms and other group-related phenotypes.

7 In this case, the presence of the gene leads to decreased virulence because the gene product facilitates chronic infection, which is less se vere than acute infection.U.S. Environmental Protection Agency

February 2009 46 frequency can be 20- to 4,000-fold) (Selifonova et al., 2001). Mutator strains can pass genetic material to other bacteria and thus transf er the mutator trait. Therefore, the ability of bacteria to evolve rapidly is heritable.

Epigenetic effects : Epigenetic effects are reversible, he ritable changes in ge ne regulation that occur without a change in DNA sequence. Ge nomic imprinting through DNA methylation is one type of epigenetic effect that has been documented for pathogen regulation of virulence (Low et al., 2001). Th e zoonotic bacteria E. coli, S. enterica (serovar Typhimurium), Vibrio

cholerae

, Y. pseudotuberculosis

, Y. enterocolitica

, Helicobacter pylori , and Campylobacter are a few notable pathogens for which DNA methylases are known to regulate gene expression (Fälker et al., 2005; Low et al., 2001). Although DNA met hylase promoters are known to be responsive to in vivo growth conditions, the degree to which host factors influence pathogen DNA methylation and whet her those methylation patterns provide a memory system for subsequent gene rations of pathogens are not known.

All of these mechanisms can contribute to rapid evolution of zoonotic pathogens. 8 Rapid

evolution is most often observed in new hosts, where new stresses are thought to lead to strong selection and rapid evolution (E bert, 1998). Evolution in new hos t environments can lead to a

reduction of virulence when the or iginal host is enc ountered again. Serial passage experiments (SPEs) often result in attenua tion of pathogenicity in one hos t along with an escalation of pathogenicity in the new host. 9 Attenuation can be so severe that it can even lead to an altered host range (complete loss of ability to infect the original host species). 10 In SPEs when only one or a few pathogens are transferred at each passage , however, genetic drift can result in decreased genetic variability and lead to a failure to adap t to new hosts and a decline in overall pathogen

fitness. Text Box III.2-1 summarizes some f eeding studies done in humans and pigs with zoonotic parasites. The examples help illustrate how changes in laboratory isolates due to passage through hosts should be consid ered during experimental design.

Although SPEs are useful for studying pathogen evol ution, the trends observed in SPEs are not necessarily broadly applicable to pathogen evol ution as a whole (Ebert, 1998). In fact, the

observation that virulence increases in SPEs in new hosts seems contrary to the classic expectation that pathogens and hos ts evolve over time in ways that render infections benign (Dieckmann, 2002; Wills, 1996). Currently, evolutionary biologists estimate the evolutionary success or failure (i.e., “fitness” ) of pathogens by their rate of spread through a given host population. Low virulence can lead to missed opportunities to spread due to low pathogen numbers. High virulence can lead to host deat h and a subsequent lack of spread. Those hypotheses are supported by the obse rvation that intermediate leve ls of pathogen virulence can be stable. Thus, the dynamic between pathogen and host drives pathogen (and host) evolution

8 For the purpose of this paper, rapid changes are phenotype (or genotype or epigenetic) changes that occur within one or a few passages of a zoonotic pathogen through a particular host species.

9 The negative correlation in the fitness of a pathogen in different hosts is the antagonistic pleiotropy hypothesis—a gene that enhances fitness in one host decreases fitness in the other host (Ebert, 1998).

10 Live vaccines such as Theiler’s yellow fever vaccine and Sabin’s polio vaccine are examples of attenuated pathogens that elicit an immune response without inducing disease. However, reemergence of pathogen virulence after vaccination remains a risk (Ebert,1998).U.S. Environmental Protection Agency

February 2009 47 and is influenced by numerous fact ors including the infection status of the host population as a whole (Dieckmann, 2002).

Because pathogens can evolve quickly within one host, it is reasonable to suspect that zoonotic pathogen pools that were propagated in animals would be different fr om pathogen pools that came from human sources. However, the extent to which known waterborne zoonotic pathogens are attenuated or gain enhanced virulence in humans when passing through animal hosts remains unknown. Even if general trends could be character ized, it is unlikely that differences could be quantified adequately in the near-term to predic t differences in human infectivity and virulence for pathogens passing through different host animals.

Another mechanism by which virulence and fecund ity of a microorganism may be affected was recently reported by Jenkins et al. (2007) for C. parvum. These investigators infected dairy calves with oocysts from either C. parvum Beltsville (B) or C. parvum Iowa (I). Calves given the B isolate excreted 5-fold more oocysts than th ose receiving the I isolate. Quantitative reverse transcriptase-PCR indicated that the B isolate contained a 3-fold greater number of the symbiont C. parvum virus (CPV) than the I isolate. They conc luded that CPV may have a role in fecundity and possibly virulence of C. parvum. The authors indicated that th eir results contrasted to those found by Miller et al. (1988) in which there was an inverse relationship between presence of a

Giardia lamblia virus (GLV) and parasite growth. Mill er et al. (1988) used virus-free isolate that they infected with various numbers of GLV, and Jenkins et al. (2007) noted that virus-free cysts may be more susceptible to the effects of virus infecti on. Jenkins et al. (2007) also

indicated that C. hominis and C. parvum, the two species most ofte n associated with human infections, are the only species reporte d thus far to be infected by CPV.U.S. Environmental Protection Agency

February 2009 48 Text Box III.2-1. Examples of F eeding Studies with Zoonotic Parasites

For the widespread waterborne zoonotic protozoa Cryptosporidium and Giardia , human volunteers (immunocompetent adults) have been exposed to measured doses of various strains of Cryptosporidium and to a single Giardia isolate to evaluate their dose-response relationships. However, there are uncertainties associated with characterizing dose even in clinical settings. Microbes can exhibit variability in infectivity, virulence, or environmental survival within strains; even for “pure isolates,” batches can differ. For microorganisms that cannot survive freez er storage or do not maintain their genetic integrity in freezer storage or tissue culture, in vivo passage in animals is required to maintain stocks. Even if the starting inoculum for a dose-response study is clonal, mutations will occur that may impact pathogen characteristics. When the starting inoculum is not clonal, which is most often the case, the subpopulation ratios within an individual host can differ from other hosts receiving the same inoculum. The subpopulation ratios also may vary as infection progresses, so collecting pathogens from a host on one day may not yield the same pool of pathogens as collecting on another day. In addition, some pathogens are not amenable to storage or maintenance in laboratory settings under any conditions and must be continuously collected from the field for each experiment. Although the challenges of properly characterizing and controlling pathogen variability in experimental research settings are considerable, the challenges are even greater for epidemiological studies.

Cryptosporidium in Humans Data on the infectivity of Cryptosporidium in humans are available from studies conducted at the University of Texas, Houston, by DuPont, Chappell, Okhuysen, and colleagues. These studies all involved healthy adult volunteers ingesting different numbers of Cryptosporidium oocysts. Subjects were then evaluated for Cryptosporidium in stool samples and for diarrheal and other GI illness symptoms. Infectivity was estimated for

five C. parvum isolates: TAMU (collected from a veterinary student); Iowa (derived from a calf); UCP (derived from a calf); Moredun, (collected from a red deer calf); 16W (from a calf); and TU502 (an isolate collected from an infected child and propagated in gnotobiotic piglets [Chappell et al., 2006]). Cryptosporidium from animal sources were found to be infectious in humans. No attempt was made to evaluate differences in potential infectivity due to repeated passage in animal hosts. However, the UCP isolate was less infectious in humans than the other isolates, and EPA’s Science Policy Council speculated that this could be due to prolonged maintenance in calves. In the risk assessment that was conducted in support of EPA’s Long Term 2 Enhanced Surface Water Treatment Rule (LT2) Economic Analysis, a dose-response relationship was chosen that weighs
the UCP data less than the other isolates.

Cryptosporidium in Animals Akiyoshi et al. (2003) investigated mixed infections of Type 1 ( C. hominis ) and Type 2 ( C. parvum ) in gnotobiotic piglets. In all the time intervals tested, Type 2 displaced Type 1, even if Type 1 was permitted to become established before inoculation with Type 2. This result raises significant questions regarding the relative perpetuation and survival of the two genotypes in mammalian hosts. The same researchers noted that field technicians very readily became infected with C. hominis , but that cross-contamination of C. hominis with C.

parvum in the animals used to maintain stocks resulted in C. parvum overwhelming C. hominis (Tzipori, 2000). These observations suggest that discovering the mechanisms by which C. hominis is maintained in natural setting when C. parvum is also present should improve understanding of risks to humans.

Giardia in Humans Rendtorff (1954) conducted a controlled, clinical study of male prison volunteers who were fed Giardia cysts
obtained from a human source. Giardia cysts of known numbers varying from 1 to 10 6 were placed into gelatin capsules along with a small amount of saline. The capsules were given to the volunteers along with 4 to 6 ounces of water. Control subjects were given sterile saline in the same manner. A dose of 10 cysts was found to be sufficient to produce human infection, as determined by observing the presence of Giardia in fecal smears (Rendtorff, 1954). However, because cyst viability could not be determined prior to administration to volunteers, the failure to elicit infection in the five men treated with a dose that was calculated to contain only one cyst may have been due to dosing with inactive cysts. The Giardia Assemblages used in these studies are not known but presumably are A or B because these are the Assemblages known to infect humans.U.S. Environmental Protection Agency

February 2009 49 

## IV. S UMMARY

Contamination of recreational waters with f eces from warm-blooded animals poses a risk of

zoonotic infection of humans with some of the pathogens in thos e waters. Although the risk and severity of human illness due to contamination with animal feces and zoonotic pathogens is most likely lower than the risk and severity of illness from treated or untreated human sewage, currently available data are insufficient to quant ify the differences. At present, the six most important zoonotic waterborne pathogens are the following:

• Pathogenic E. coli ;

• Salmonella ;

• Campylobacter ;

• Leptospira ;

• Cryptosporidium ; and

• Giardia.

All of these waterborne pathoge ns are likely to cause more se vere symptoms in children and immunocompromised individuals and subpopulations than in the remainder of the population. Of these six, pathogenic E. coli has the most potential for severe adverse health effects that can even be fatal. Potential debilitating chroni c sequelae such as Guillain-Barré Syndrome and

reactive arthritis have been associated with Campylobacter infections. A lthough the most

common recreational illnesses are probably due to human viruse s causing short-term GI, the waterborne zoonotic pathogens disc ussed in this report have the pot ential to cause serious health

effects. While serious health outcomes are likel y to be rare in comp arison with self-limiting

illnesses as a result of ambient (recreational) water exposure, the adverse health impacts of the rare, but more serious illnesses remain an important public health challenge.U.S. Environmental Protection Agency

February 2009 50 

## V. R EFERENCES

Abbaszadegan, M. 2006. Rotoviruses, In: Wate rborne Pathogens, AWWA Manual M48, Second

Edition. American Water Works Asso ciation: Denver, CO. Chapter 46.

Ackman, D., Marks, S., Mack, P., Caldwell, M., Root, T., Birkhead, G. 1997. Swimmingassociated haemorrhagic colitis due to Escherichia coli O157:H7 infection: evidence of prolonged contamination of a fresh water la ke. Epidemiology and Infection 119(1): 1-8.

Adam, R.D. 2001. Biology of Giardia lamblia . Clinical Microbiol ogy Reviews 14(3): 447-475.

Akiyoshi, D.E., Mor, S., Tzipori, S. 2003. Rapid displacement of Cryptosporidium parvum type 1 by type 2 in mixed infections in pigl ets. Infection and Im munity 71(10): 5765-5771.

Allen, L., Briggle, T., Pfaffenberger, C. 1982. Abso rption and excretion of cyanuric acid in longdistance swimmers. Drug Metabolism 13: 499-516.

Allos, B.M. 1998. Campylobacter jejuni infection as a cause of the Guillain-Barre syndrome. Infectious Disease Clinics of North America 12(1): 173-184.

Allos, B.M. 2001. Campylobacter jejuni infections: updat e on emerging issues and trends. Clinical Infectious Diseases 32(8): 1201-1206.

Anderson, B.C. 1985. Moist heat inactivation of Cryptosporidium sp. American Journal of Public Health 75(12): 1433-1434.

Anderson, B.C. 1986. Effect of drying on the infectivity of Cryptosporidia -laden calf feces for 3- to 7-day-old mice. American Journal of Veterinary Research 47(10): 2272-2273.

APHA. 2004. Control of Communicable Diseases Manua l, 18th Edition. American Public Health Association Heymann, D. (ed.) Washington, DC.

Appelbee, A.J., Thompson, R.C.A., Olson, M.E. 2005. Giardia and Cryptosporidium in

mammalian wildlife - current stat us and future needs. Trends in Parasitolo gy 21(8): 370-376.

Arrowood, M. 1997. Diagnosis. In: Cryptosporidium and Cryptosporidiosis; Fayer, R. (ed). CRC Press: New York, NY. Chapter 2.

Atwill, E.R., Sweitzer, R.A., Pereira, M.G., Gardner, I.A., Vuren, D V., Boyce, W.M. 1997.

Prevalence of and associated risk factors for shedding Cryptosporidium parvum oocysts and

Giardia cysts within feral pig populations in California. Applied and Environmental Microbiology 63(10): 3946-3949.U.S. Environmental Protection Agency

February 2009 51 Atwill, E.R., Pereira, M.D.G.C., Alonso, L.H., Elmi, C., Epperson, W.B., Smith, R., Riggs, W., Carpenter, L.V., Dargatz, D.A., Hoar, B. 2006. Environmental load of Cryptosporidium parvum oocysts from cattle manure in feedlots from the central and western United States. Journal of Environmental Quality 35(1): 200-206.

Avery, L.M., Killham, K., Jones, D.L. 2005. Survival of E. coli O157:H7 in organic wastes destined for land application. Journal of Applied Microbiology 98(4): 814-822.

Axelsson-Olsson, D., Waldenstrom, J., Broma n, T., Olsen, B., Holmbe rg, M. 2005. Protozoan Acanthamoeba polyphaga as a potential reservoir for Campylobacter jejuni . Applied and

Environmental Micr obiology 71(2): 1987-1992.

Baker, K., Degnan, A. 2006. Helicobacter pylori . In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Works Association: Denver, CO. Chapter 12.

Baldwin, A., Kingman, H., Darville, M., Foot, A.B., Grier, D., Cornish, J.M., Goulden, N., Oakhill, A., Pamphilon, D.H., Steward, C.G., Mark s, D.I. 2000. Outcome and clinical course of 100 patients with adenovirus infection followi ng bone marrow transplantation. Bone Marrow Transplant 26(12): 1333-1338.

Berk, S.G., Gunderson, J.H., Newsome, A.L., Far one, A.L., Hayes, B.J. , Redding, K.S., Uddin, N., Williams, E.L., Johnson, R.A., Farsian, M. , Reid, A., Skimmyhorn, J., Farone, M.B. 2006.

Occurrence of infected am oebae in cooling towers compared with natural aquatic environments: implications for emerging pathogens. Enviro nmental Science and Technology 40(23): 7440-

7444.

Blankspoor, H. 2006. Schistosomatidae. In: Waterborne Pathoge ns, AWWA Manual M48,

Second Edition. American Water Works Association: Denver, CO. Chapter 35.

Boczek, L.A., Rice, E.W., Johnston, B., Johnson, J.R. 2007. Occurrence of antibiotic-resistant uropathogenic Escherichia coli clonal group A in wastewater effluents. Applied and Environmental Microbiology 73(13): 4180-4184.

Bolin, C., Brown, C., Rose, J. 2004a. Emerging z oonotic diseases and wate r. In: Waterborne Zoonoses: Identification, Causes and Control; Cotruvo, J.A, Dufour , A., Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IWA Publishing: London, UK. Chapter 2.

Bolin, C., Brown, C., Rose, J. 2004b. Leptosporidiosi s and other potential z oonoses in water. In: Waterborne Zoonoses: Identification, Causes an d Control; Cotruvo, J.A, Dufour, A., Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F ., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IWA Publis hing: London, UK. Chapter 20.

Booher, S.L., Cornick, N.A., M oon, H.W. 2002. Persistence of Escherichia coli O157:H7 in experimentally infected swine. Ve terinary Microbiology 89(1): 69-81.U.S. Environmental Protection Agency

February 2009 52 Borella, P., Montagna, M.T., Romano-Spica, V., St ampi, S., Stancanelli, G., Triassi, M., Neglia, R., Marchesi, I., Fantuzzi, G., Tato, D., Napoli, C., Quaranta, G., Laurenti, P., Leoni, E., Luca, G.D., Ossi, C., Moro, M., D’Alcala, G.R. 2004. Legionella infection risk from domestic hot water. Emerging Infectious Diseases 10(3): 457-464.

Brookes, J.D., Antenucci, J., Hipsey, M., Burch, M.D., Ashbolt, N.J., Ferguson, C. 2004. Fate and transport of pathogens in lakes and rese rvoirs. Environment International 30(5): 741-759.

Brooks, J.T., Sowers, E.G., Wells, J.G., Greene, K.D., Griffin, P.M ., Hoekstra, R.M. Strockbine, N.A. 2005. Non-O157 Shiga toxin-producing Escherichia coli infections in the United States,

1983-2002. Journal of Infectious Diseases 192(8): 1422-1429.

Brotman, M., Giannella, R., Alm, P., Baum an, M., AR, A.B., Black, R. 1995. Consensus

conference statement: Escherichia coli 0157:H7 infections-an emerging national health crisis, July 11-13, 1994. American Gastroente rological Associ ation 108: 1923-1934.

Brown, P., McShane, L.M., Zanusso, G., Detwile , L. 2006. On the question of sporadic or atypical bovine spongiform encephalopathy and Cr eutzfeldt-Jakob disease. Emerging Infectious Diseases 12(12): 1816-1821.

Brubaker, R.R. 1991. Factors promoting acute and ch ronic diseases caused by yersiniae. Clinical Microbiology Reviews 4(3): 309-324.

Buswell, C.M., Herlihy, Y.M., Lawrence, L.M., McGuiggan, J.T., Marsh, P.D., Keevil, C.W., Leach, S.A. 1998. Extended survival and persistence of Campylobacter spp. in water and aquatic biofilms and their detection by immunofluores cent-antibody and -rRNA staining. Applied and

Environmental Micr obiology 64(2): 733-741.

Caccio, M. 2005. Molecular epidemiology of human cryptosporidiosis. Parasitologia 47: 185-

192.

Cali, A. 2006. Microsporidia. In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Works Association: Denver, CO. Chapter 3.

Carey, C.M., Lee, H., Trevors, J.T. 2004. Biology, persistenc e and detection of Cryptosporidium

parvum and Cryptosporidium hominis oocyst. Water Research 38(4): 818-862.

Carr, R., Bartram, J. 2004. The control envel ope and risk management. In: Waterborne Zoonoses: Identification, Causes and Control; Cotruvo, J.A, Dufour , A., Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IWA Publishing: London, UK. Chapter 5.

Casemore, D., Wright, S., Coop, R. 1997. Cryptosporidiosis - human and animal epidemiology.

In: Cryptosporidium and Cryptosporidiosis; L.R. Fayer (ed.). CRC Press: Ne w York, New York. Pp. 65-92.U.S. Environmental Protection Agency

February 2009 53 U.S. Centers of Disease Control and Preventio n (CDC). 1993a. Update: multistate outbreak of

Escherichia coli O157:H7 infections from hamburge rs - western United States, 1992-1993. Mortality and Morbidity W eekly Report 42(14): 258-263.

CDC. 1993b. Surveillance for waterborne di sease outbreaks - United States, 1991-1992. Morbidity and Mortality Week ly Report 42 (SS-5): 1-22.

CDC. 1996. Surveillance for waterborne-dis ease outbreaks - United States, 1993-1994. Morbidity and Mortality Week ly Report 45 (SS-1): 1-33.

CDC 1998. Surveillance for waterborne-disease outbreaks - United States, 1995-1996. Morbidity and Mortality Weekly Report 47(SS-5): 1-33.

CDC. 2000. Surveillance for waterborne-dis ease outbreaks - United States, 1997-1998. Morbidity and Mortality W eekly Report 49(SS-4): 1-21.

CDC. 2002. Surveillance for waterborne-disea se outbreaks - United States, 1999-2000. Morbidity and Mortality W eekly Report 51(SS-8): 1-47.

CDC. 2004. Surveillance for waterborne-disease out breaks associated with recreational water - United States, 2001-2002, and surveillance for wate rborne-disease outbreaks associated with drinking water: United States, 2001-2002. Morbid ity and Mortality Weekly Report 53 (SS-8).

CDC. 2005. Cryptosporidiosis Surveillance - United States 1999-2002, and Giardiasis Surveillance - United States, 1998-2002. Morbidity and Mortality Weekly Report 54(SS-1).

CDC. 2007a. Summary of notifiable diseases - United States, 2005. Morbidity and Mortality Weekly Report 54(53).

CDC. 2007b. Cryptosporidiosis outbr eaks associated with recreational water use - five states, 2006. Morbidity and Mortality Weekly Report (56)29: 729-732.

Chakrabarti, S., Mautner, V., Osman, H., Collingham, K.E., Fegan, C.D., Klapper, P.E., Moss, P.A.H., Milligan, D.W. 2002. Adenovirus infections following allogeneic stem cell transplantation: incidence a nd outcome in relation to graf t manipulation, immunosuppression, and immune recovery. Blood 100(5): 1619-1627.

Chalker, R.B., Blaser, M.J. 1988. A review of human salmonellosis: III. Magnitude of

Salmonella infection in the United States. Reviews of Infectious Dise ases 10(1): 111-124.

Chalmers, R.M., Salmon, R.L., Willshaw, G.A., Ch easty, T., Looker, N., Davies, I., Wray, C. 1997. Vero-cytotoxin-producing Escherichia coli O157 in a farmer handling horses. Lancet 349(9068): 1816.

Chan, P.K.S. 2002. Outbreak of avian influenza A(H5N1) virus infection in Hong Kong in 1997. Clinical Infectious Diseases 34(Suppl 2): S58-S64.U.S. Environmental Protection Agency

February 2009 54 Chapman, P.A., Siddons, C.A., Malo, A.T.G ., Harkin, M.A. 1997. A 1-year study of Escherichia

coli O157 in cattle, sheep, pigs and poultry. Epidemiology and Inf ection 119(2): 245-250.

Chappell, C.L., Okhuysen, P.C., Sterling, C. R., Wang, C., Jakubowski, W., Dupont, H.L. 1999. Infectivity of Cryptosporidium parvum in healthy adults with pre-existing anti-C. parvum serum immunoglobulin G. American Journal of Tr opical Medicine and Hygiene 60(1): 157-164.

Chappell, C.L., Okhuysen, P.C., Langer-Curry, R ., Widmer, G., Akiyoshi, D.E., Tanriverdi, S., Tzipori, S. 2006. Cryptosporidium hominis : experimental challenge of healthy adults. American Journal of Tropical Medici ne and Hygiene 75(5): 851-857.

Chart, H., Sussman, M., Stewart-Tull, D. 2000. E. coli – friend or foe? Supplement to Journal of Applied Microbiology Society for Applied Microbiology, Symposium Series No. 29.

Cieslak, P.R., Noble, S.J., Maxson, D.J., Empey, L.C., Ravenholt, O., Legarza, G., Tuttle, J., Doyle, M.P., Barrett, T.J., Wells, J.G., Mc Namara, A.M., Griffin, P.M. 1997. Hamburgerassociated Escherichia coli O157:H7 infection in Las Vegas: a hidden epidemic. American

Journal of Public Health 87(2): 176-180.

Clifford, C.P., Crook, D. W., Conlon, C.P., Fraise, A.P., Day, D.G., Peto, T.E. 1990. Impact of

waterborne outbreak of cryptosporidiosis on AIDS and re nal transplant patients. Lancet 335(8703): 1455-1456.

Cliver, D., Fayer, R. 2004. Cate gories of waterborne disease organisms. In: Waterborne Zoonoses: Identification, Causes and Control; Cotruvo, J.A, Dufour , A., Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IWA Publishing, London, UK. Section V.

Cohen, M.L., Tauxe, R.V. 1986. Drug-resistant Salmonella in the United States: an epidemiologic perspective. Science 234(4779): 964-969.

Corsi, A., Nucci, C., Knafelz, D., Bulgarini, D., Iorio, L.D., Polito, A., Risi, F.D., Morini, F.A., Paone, F.M. 1998. Ocular changes associated with Giardia lamblia infection in children. British Journal of Ophthalmology 82(1): 59-62.

Couroucli, X.I., Welty, S.E., Ramsay, P.L., Wearde n, M.E., Fuentes-Garcia, F.J., Ni, J., Jacobs, T.N., Towbin, J.A., Bowles, N.E. 2000. Detection of microorganisms in the tracheal aspirates of preterm infants by polymerase chain reaction: association of ade novirus infection with bronchopulmonary dysplasia. Pedi atric Research 47(2): 225-232.

Covert, T., Meckes, M. 2006. Salmonella . In: Waterborne Pathogens, AWWA Manual M48,

Second Edition. American Water Works A ssociation: Denver, CO. Chapter 17.U.S. Environmental Protection Agency

February 2009 55 Craun, G., Calderon, R., Craun, M. 2004a. Waterbor ne outbreaks caused by zoonotic pathogens

in the USA. In: Waterborne Zoonoses: Identific ation, Causes and Control; Cotruvo, J.A, Dufour, A., Rees, G., Bartram, J., Carr, R., Cliver, D. O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO ) . IWA Publishing: London, UK. Chapter 8.

Craun, G., Till, D., McBride, G. 2004b. Epidem iological studies and surveillance. In: Waterborne Zoonoses: Identification, Causes an d Control; Cotruvo, J.A, Dufour, A., Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F ., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IWA Pub lishing, London, UK. Chapter 10.

Craun, G.F., Calderon, R.L., Craun, M.F. 2005. Outbr eaks associated with recreational water in

the United States. International Journal of Environmental Health Research 15(4): 243-262.

Craun, M.F., Craun, G.F., Calderon, R.L., Beach, M.J. 2006. Waterborne outbreaks reported in

the United States. Journal of Wate r and Health 4(Suppl 2): 19-30.

Crawford-Miksza, L.K., Schnurr, D.P. 1996. Ad enovirus serotype evolution is driven by illegitimate recombination in the hypervariabl e regions of the hexon protein. Virology 224(2):

357-367.

Cross, J., Sherchand, J. 2004. Cyclosporiasis . In: Waterborne Zoonoses : Identification, Causes and Control; Cotruvo, J.A, Dufour, A., Rees, G., Ba rtram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IW A Publishing: London, UK. Chapter 17.

Current, W.L., Garcia, L.S. 1991. Cryptosporidiosi s. Clinical Microbiol ogy Reviews 4(3): 325-

358.

Czajkowska, D., Witkowska-Gwiazdowska, A., Si korska, I., Boszczyk-Ma leszakl, H., Horoch,

M. 2005. Survival of Escherichia coli serotype 0157:H7 in water a nd in bottom-shore sediments. Polish Journal of Environmental Studies 14(4): 423-430.

Degremont, A., Sturchler, D., Wolfen sberger, E., Osterwalder, B. 1981. E tude clinique et therapeutique d’un collectif de 217 patients atte ints de giardiase et d’amibiase intestinales. Schweiz. Med. Wschr. 111: 2039-2046 [article in French, as cited in ICAIR 1984, Final draft for drinking water criteria document on Giardia prepared for Criteria and Standards Division, Office of Drinking Water, USEPA].

Degnan, A., Standridge, J. 2006. Enterohemorrhagic E coli . Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Work s Association: Denver, CO. Chapter 9.

Dieckmann, U. 2002. Adaptive dynamics of pathoge n-host interactions. In: Adaptive Dynamics

of Infectious Diseases: In Pursuit of Virulence Management; Dieckmann, U., Metz, J.A.M., Sabelis, M.W., Sigmund, K. (eds), Cambridge Univ ersity Press: Cambridge, UK. Chapter 4, pp.

39-59.U.S. Environmental Protection Agency

February 2009 56 Domingo, E., Baranowski, E., Ruiz-Jarabo, C.M., Martin-Hernndez, A.M., Siz, J.C., Escarmis, C. 1998. Quasispecies structure and persiste nce of RNA viruses. Emerging Infectious Diseases 4(4): 521-527.

Donnison, A.M., Ross, C.M. 1999. Animal and huma n faecal pollution in New Zealand rivers.

New Zealand Journal of Marine a nd Freshwater Research 33: 119-128.

Dubey, J. 2006. Toxoplasma gondii . In Waterborne Pathogens, AWWA Manual M48, Second

Edition. American Water Works Associ ation: Denver, CO. Chapter 36.

Dubey, J.P. 2004. Toxoplasmosis - a waterborne z oonosis. Veterinary Parasitology 126(1-2): 57-

72.

Dufour, A.P., Evans, O., Behymer, T.D., Ca ntu, R. 2006. Water inges tion during swimming activities in a pool: a pilot study. Journa l of Water and H ealth 4(4): 425-430.

DuPont, H.L., Chappell, C.L., Sterling, C.R., Okhuysen, P.C., Rose, J.B., Jakubowski, W. 1995. The infectivity of Cryptosporidium parvum in healthy volunteers. New England Journal of Medicine 332(13): 855-859.

DuPont, H.L., Formal, S.B., Hornick, R.B., Snyd er, M.J., Libonati, J.P., Sheahan, D.G., LaBrec, E.H., Kalas, J.P. 1971. Pathogenesis of Escherichia coli diarrhea. New England Journal of Medicine 285(1): 1-9.

Dykes, A.C., Juranek, D.D., Lorenz, R.A., Si nclair, S., Jakubowski, W., Davies, R. 1980. Municipal waterborne giardiasis : an epidemiologic investigation, beavers implicated as a possible reservoir. Annals of Internal Medicine 92: 165-170.

Ebert, D. 1998. Experimental evoluti on of parasites. Science 282(5393): 1432-1435.

Eisenberg, J.N.S., Lei, X., Hubbard, A.H., Br ookhart, M.A., Colford, J.M. 2005. The role of

disease transmission and c onferred immunity in outbreak s: analysis of the 1993 Cryptosporidium outbreak in Milwaukee, Wisconsin. American Journal of Epidemiology 161(1): 62-72.

El Mansoury, S.T.E., Naga, I.F.A.E., Negm, A.Y., Amer, E.E. 2004. Influence of temperature and salinity on the viability and infectivity of Giardia lamblia and Cryptosporidia parvum

. Journal of the Egyptian Society of Parasitology 34(1): 161-172.

Endo, T., Morishima, Y. 2004. Major helminth z oonoses in water. Chapter 18 in waterborne zoonoses: identification, causes and control; Cotruvo, J.A, Dufour, A., Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IWA Publishing: London, UK. Chapter 18.

Enriquez, C., Thurston-Enriquez, J. 2006. Ade noviruses. In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Work s Association: Denver, CO. Chapter 38.U.S. Environmental Protection Agency

February 2009 57 Evans, O., Wymer, L., Behymer, T., Dufour, A. 2006. An observational st udy: determination of the volume of water ingested during recreati onal swimming activities. U.S. Environmental Protection Agency, Cincinnati, OH. Poster presentation at the National Beaches Conference, Niagara Falls, New York. October.

Falker, S., Schmidt, M.A., Heusipp, G. 2005. DNA methylation in Yersinia enterocolitica : role of the DNA adenine methyltransferase in mismatch repair and regulation of virulence factors. Microbiology 151(Pt 7): 2291-2299.

Farthing, M.J. 2000. Clinical aspe cts of human cryptosporidiosis

. In: Cryptosporidiosis and Microsporidiosis; Petry, F. (ed). S. Karger AG: New York, NY. Pp. 50-74.

Farthing, M.J. 1996. Giardiasis. Gastroenterol ogy Clinics of North America 25(3): 493-515.

Faubert, G. 2000. Immune response to Giardia duodenalis . Clinical Microbiology Reviews 13(1): 35-54.

Fayer, R., Xiau, L.X. (eds). 2007. Cryptosporidium and Cryptosporidiosis, Second Edition. CRC
Press: Boca Raton, FL.

Fayer, R. 2004a. Cryptosporidium : a water-borne zoonotic parasi te. Veterinary Parasitology 126(1-2): 37-56.

Fayer, R. 2004b. Waterborne zoonotic protozoa . In: Waterborne Zoonoses: Identification, Causes and Control; Cotruvo, J.A, Dufour, A., R ees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds ), World Health Organization (WHO). IWA Publishing: London, UK. Chapter 16.

Fayer, R. 1994. Effect of high temperature on infectivity of Cryptosporidium parvum oocysts in water. Applied and Environmen tal Microbiology 60(8): 2732-2735.

Fayer, R., Morgan, U., Upton, S.J. 2000. Epidemiology of Cryptosporidium : transmission,

detection and identification. Internationa l Journal for Parasitology 30(12-13): 1305-1322.

Fayer, R., Nerad, T. 1996. Effects of low temperatures on viability of Cryptosporidium parvum oocysts. Applied and Environmen tal Microbiology 62(4): 1431-1433.

Fayer, R., Nerad, T., Rall, W., Lindsay, D.S ., Blagburn, B.L. 1991. Studies on cryopreservation

of Cryptosporidium parvum . International Journal for Parasitology 77(3): 357-361.

Fayer, R., Speer, C., Dubey, J. 1997. The general biology of Cryptosporidium . In: Cryptosporidium and Cryptosporidiosis, Fayer, R. (ed). CRC Press: Ne w York, New York.

Fayer, R., Trout, J.M., Jenki ns, M.C. 1998. Infectivity of Cryptosporidium parvum oocysts stored in water at environmental temperatures . International Journal for Parasitology 84(6):

1165-1169.U.S. Environmental Protection Agency

February 2009 58 Fayer, R. 2003. Cryptosporidium : from molecules to disease. In: Cryptosporidium : From Molecules to Disease, Thompson R.C.A., Armson A., Ryan U.M. (eds). Elsevier: Amsterdam, The Netherlands; pp. 11-18.

Fayer, R., Ungar, B.L. 1986. Cryptosporidium spp. and cryptosporidi osis. Microbiological Reviews 50(4): 458-483.

FDA. 2001. Draft risk assessment on the public heal th impact of Vibrio parahaemolyticus in raw molluscan shellfish. Technical Report. Cent er for Food Safety and Applied Nutrition, Food and Drug Administration, U.S. Departme nt of Health and Human Services.

Feder, I., Wallace, F.M., Gray, J.T., Fratamico, P., Fedorka-Cray, P.J., Pearce, R.A., Call, J.E., Perrine, R., Luchansky, J.B. 2003. Isolation of Escherichia coli O157:H7 from intact colon fecal samples of swine. Emerging Infectious Diseases 9(3): 380-383.

Feltus, D.C., Giddings, C.W., Schneck, B.L., Monson, T., Warshaue r, D., McEvoy, J.M. 2006. Evidence supporting zoonotic transmission of Cryptosporidium spp. in Wisconsin. Journal of Clinical Microbiology 44(12): 4303-4308.

Fredricksen, D., Geldreich, E., Karner, D.A. 2006. Cyanobacteria. In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Works Association: Denver, CO. Chapter 8.

Fricker, C. 2006b. Yersinia . In: Waterborne Pathogens, AWWA Manual M48, Second Edition.

American Water Works Associat ion: Denver, CO. Chapter 22.

Fricker, C. 2006a. Camplylobacter . In: Waterborne Pathogens, AWWA Manual M48, Second

Edition. American Water Works Associ ation: Denver, CO. Chapter 7,

Frisby, H.R., Addiss, D.G., Reiser, W.J., Hancock, B., Vergeront, J.M., Hoxi e, N.J., Davis, J.P. 1997. Clinical and epidemiologic features of a ma ssive waterborne outbreak of cryptosporidiosis in persons with HIV infection. Journal of Ac quired Immune Deficiency Syndromes and Human Retrovirology 16(5): 367-373.

Frost, F., Craun, G. 1998. The importance of acquired immunity in the epidemiology of cryptosporidiosis and giardiasis. OECD (Organization for Economic Cooperation and Development) Workshop: Molecular Technologi es for Safe Drinking Water. Interlaken, Switzerland, July 5-8.

Gagliardi, J.V., Karns, J.S. 2002. Persistence of Escherichia coli O157:H7 in soil and on plant

roots. Environmental Mi crobiology 4(2): 89-96.

Ganoza, C.A., Matthias, M.A., Collins-Richards , D., Brouwer, K.C., Cunningham, C.B., Segura, E.R., Gilman, R.H., Gotuzzo, E., Vinetz, J.M. 2006. Determining risk for se vere leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira . Public Library of Science Medicine 3(8): 1329-1340.U.S. Environmental Protection Agency

February 2009 59 Garcia, L. 2006c. Isosprora belli . In: Waterborne Pathogens, AWWA Manual M48, Second

Edition. American Water Works Associ ation: Denver, CO. Chapter 32.

Garcia, L. 2006b. Blastocystis hominis . In: Waterborne Pat hogens, AWWA Manual M48,

Second Edition. American Water Works A ssociation: Denver, CO. Chapter 27.

Garcia, L. 2006a. Balantidium coli . In: Waterborne Pathogens , AWWA Manual M48, Second

Edition. American Water Works Associ ation: Denver, CO. Chapter 26.

Garg, A.X., Marshall, J., Salvadori, M., Thie ssen-Philbrook, H.R., Macnab, J., Suri, R. S., Haynes, R.B., Pope, J., Clark, W., Investigators, W.H.S. 2006. A gradient of acute gastroenteritis was characterized, to assess ri sk of long-term health sequelae after drinking bacterialcontaminated water. Journal of Clinical Epidemiology 59(4): 421-428.

Garg, A.X., Moist, L., Matsell, D., Thiesse n-Philbrook, H.R., Haynes, R.B., Suri, R.S., Salvadori, M., Ray, J., Clark, W.F. 2005. Risk of hypertension and reduced kidney function after acute gastroenteritis from bacteria-contaminated drinking water. Canadian medical Association Journal 173(3): 261-268.

Geldreich, E. 1996. Creating microbial quality in drinking water. In: Microbial Quality of Water Supply in Distribution Systems. Lewis Publishers. Chapter 2.

Geldreich, E., Degnan, A. 2006b. Pseudomonas . In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Works Association: Denver, CO. Chapter 16.

Geldreich, E., Degnan, A. 2006a. Flavobacterium . In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Works Association: Denver, CO. Chapter 11.

Geldreich, E., Standridge, J. 2006a. Klebsiella . In: Waterborne Pat hogens, AWWA Manual M48,

Second Edition. American Water Works A ssociation: Denver, CO. Chapter 13.

Geldreich, E., Standridge, J. 2006b. Serratia . In: Waterborne Pat hogens, AWWA Manual M48,

Second Edition. American Water Works A ssociation: Denver, CO. Chapter 18.

Geldreich, E., Standridge, J. 2006c. Staphylococcus . In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Works Association: Denver, CO. Chapter 20.

Gerba, C. 2006a. Enteroviruses and parechoviru ses. In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Works Association: Denver, CO. Chapter 41.

Gerba, C. 2006b. Hepatitis E virus. In: Wate rborne Pathogens, AWWA Manual M48, Second

Edition. American Water Works Associ ation: Denver, CO. Chapter 43.U.S. Environmental Protection Agency

February 2009 60 Gillespie, I.A., O’Brien, S.J., Frost, J.A., Adak, G. K., Horby, P., Swan, A.V., Painter, M.J., Neal,
K.R., Campylobacter Sentinel Surveillance Sche me Collaborators. 2002. A case-case comparison of Campylobacter coli and Campylobacter jejuni infection: a tool for generating hypotheses. Emerging Infectious Diseases 8(9): 937-942.

Glass, R. I. 2006. New hope for defeating rotavirus. Scientif ic American 294(4): 46-51, 54-5.

Graczyk, T.K., Kacprzak, M., Neczaj, E., Tamang, L., Graczyk, H., Lucy, F E., Girouard, A.S.

2007. Occurrence of Cryptosporidium and Giardia in sewage sludge and solid waste landfill leachate and quantitative comparative analys is of sanitization treatments on pathogen

inactivation. Environmenta l Research 106(1): 27-33.

Grandadam, M., Tebbal, S., Caron, M., Siriwardan a, M., Larouze, B., Koeck, J.L., Buisson, Y., Enouf, V., Nicand, E. 2004. Evidence for hepatitis E virus quasispecies. Journal of General Virology 85(Pt 11): 3189-3194.

Gray, G.C., Setterquist, S F., Jirsa, S.J., DesJard in, L.E., Erdman, D.D. 2005. Emergent strain of human adenovirus endemic in Iowa. Emergi ng Infectious Diseases 11(1): 127-128.

Griffin, D.W., Donaldson, K.A., Paul, J.H., Rose, J.B. 2003. Pathogenic human viruses in coastal

waters. Clinical Microbio logy Reviews 16(1): 129-143.

Griffin, P.M., Tauxe, R.V. 1991. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli , and the associated hemolytic uremic syndrome. Epidemiologic Reviews 13: 60-98.

Guan, T.Y., Holley, R.A. 2003. Pathogen surv ival in swine manur e environments and

transmission of human enteric illness - a review. Journal of Environmental Quality 32(2): 383-

392.

Haas, C.N., Thayyar-Madabusi, A., Rose, J.B., Gerba, C.P. 2000. Development of a doseresponse relationship for Escherichia coli O157:H7. International Jo urnal of Food Microbiology 56(2-3): 153-159.

Hall, N. 2006. Legionella . In: Waterborne Pathogens, AWWA Manual M48, Second Edition.

American Water Works Associat ion: Denver, CO. Chapter 14.

Hammermueller, J., Kruth, S., Prescott, J., Gyles, C. 1995. Detection of toxin genes in

Escherichia coli isolated from normal dogs and dogs with diarrhea. Canadian Journal of Veterinary Research 59(4): 265-270.

Hammond, B.G., Barbee, S.J., Inoue, T., Ishida, N ., Levinskas, G J., Stevens, M.W., Wheeler, A.G., Cascieri, T. 1986. A review of toxico logy studies on cyanurat e and its chlorinated

derivatives. Environmental Hea lth Perspectives 69: 287-292.U.S. Environmental Protection Agency

February 2009 61 Hamnes, I.S., Gjerde, B.K., Forberg, T ., Robertson, L.J. 2007. Occurrence of Giardia and Cryptosporidium in Norwegian red foxes ( Vulpes vulpes ). Veterinary Parasitology 143(3-4):

347-353.

Harcourt, B.H., Lowe, L., Tami n, A., Liu, X., Bankamp, B., Bowd en, N., Rollin, P.E., Comer, J.A., Ksiazek, T.G., Hossain, M.J., Gurley, E.S., Breiman, R.F., Bellini, W.J., Rota, P.A. 2005. Genetic characterization of Nipah virus, Ba ngladesh, 2004. Emerging Infectious Diseases 11(10): 1594-1597.

Health Canada. 2006. Guidelines for Canadian Drinking Water Quality: Guideline Technical

Document - Bacterial Waterbor ne Pathogens - Curr ent and Emerging Organisms of Concern. Water Quality and Health Bureau, Healthy Enviro nments and Consumer Safety Branch, Health Canada: Ottawa, Ontario.

Heerden, J., Ehlers, M.M., Vivier , J.C., Grabow, W.O.K. 2005. Risk assessment of adenoviruses detected in treated drinking water and recreati onal water. Journal of Applied Microbiology 99(4): 926-933.

Hellard, M.E., Sinclair, M.I., Hogg, G.G., Fair ley, C.K. 2000. Prevalence of enteric pathogens among community based asymptoma tic individuals. Journal of Ga stroenterology and Hepatology 15(3): 290-293.

Hogg, J.C. 2001. Role of latent viral infections in chronic obstructive pulmonary disease and asthma. American Journal of Respiratory and Critical Care Medicine 164(10 Pt 2): S71-S75.

Hopkins, R.S., Gaspard, G.B., Williams, F.P., Ka rlin, R.J., Cukor, G., Blacklow, N.R. 1984. A community waterborne gastroenteritis outbreak: evidence for rotavirus as the agent. American

Journal of Public Health 74(3): 263-265.

Hopkins, R.S.,Juranek, D.D. 1991. Acute giardiasis: an improved clinical case definition for epidemiologic studies. American J ournal of Epidemiology 133(4): 402-407.

Hrudey, S., Huck, P., Payment, P., Gillham, R., Hrudey, E. 2002. Walkerton: lessons learned in comparison with waterborne outbreaks in the developed world. Journal of Environmental Engineering and Science 1, 397-470.

Hrudey, S.E., Payment, P., Huck, P.M., Gillham, R.W., Hrudey, E.J. 2003. A fatal waterborne disease epidemic in Walkerton, Ontario: compar ison with other waterbor ne outbreaks in the developed world. Water Scien ce and Technology 47(3) 7-14.

Hunter, P.R. 2003. Drinking water a nd diarrhoeal disease due to Escherichia coli . Journal of Water and Health 1(2) 65-72.

Hunter, P.R., Nichols, G. 2002. Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients. Clin ical Microbiology Reviews 15(1): 145-154.U.S. Environmental Protection Agency

February 2009 62 Hunter, P.R., Thompson, R.C.A. 2005. The zoonotic transmission of Giardia and Cryptosporidium . International Journal for Parasitology 35 (11-12): 1181-1190.

Hunter, P.R., Hughes, S., Woodhouse, S., Raj, N ., Syed, Q., Chalmers, R.M., Verlander, N.Q., Goodacre, J. 2004. Health sequelae of human crypt osporidiosis in imm unocompetent patients. Clinical Infectious Diseases 39(4): 504-510.

Inpankaew, T., Traub, R., Thomps on, R.C.A., Sukthana, Y. 2007. Canine parasitic zoonoses in Bangkok temples. Southeast Asian Journal of Tr opical Medicine and Public Health 38(2): 247-

255.

Jenkins M.B., Bowman, D.D., Foga rty, E.A., Ghiorse, W.C. 2002. Cryptosporidium parvum oocysts inactivation in three soil types at various temperatures and water potentials. Soil Biology and Biochemistry 34: 1101-1109.

Jenkins, M., Higgins, J., Abrahante, J., Kniel, K., O’Brien, C., Trout, J., Lancto, C., Abrahamsen, M., Fayer, R. 2007. Fecundity of Cryptosporidium parvum is correlated with intracellular levels of the viral symbiont CPV. Internatio nal Journal for Para sitology 11: 1-5.

Johnson, D., Enriquez, C., Pepper, I., Davis, T ., Gerba, C.P., Rose, J. 1997. Survival of Giardia

, Cryptosporidium , poliovirus and Salmonella in marine waters. Water Science and Technology 35(11/12): 261-268.

Jones, K. 2001. Campylobacters in water, sewage and the environment. Proceedings of the Society for Applied Microbiology Symposium (30): 68S-79S.

Kanarat, S. 2004. Symptoms, treatments, and h ealth consequences of waterborne zoonotic diseases. In: Waterborne Zoonoses: Identification, Causes and Control; Cotruvo, J.A, Dufour, A., Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IWA Publishing: London, UK. Chapter 9.

Kappus, K.D., Lundgren, R.G., Juranek, D.D., Robe rts, J.M., Spencer, H.C. 1994. Intestinal parasitism in the United States: update on a co ntinuing problem. American Journal of Tropical Medicine and Hygiene 50(6): 705-713.

Katz, A.R., Ansdell, V.E., Effler, P.V., Middl eton, C. & Sasaki, D.M. 2001. Assessment of the clinical presentation and treatment of 353 cases of laboratory-confirmed leptospirosis in Hawaii,

1974–1998. Clinical Infectious Diseases 33: 1834-1841.

Kean, B.H., William, D.C., Luminais, S.K. 1979. Epidemic of amoebiasis and giardiasis in a biased population. Journal of Di gestive Diseases 55(5): 375-378.

Keene, W. 2006. Entamoeba histolytica . In: Waterborne Pathoge ns, AWWA Manual M48,

Second Edition. American Water Works A ssociation: Denver, CO. Chapter 30.U.S. Environmental Protection Agency

February 2009 63 Keene, W.E., McAnulty, J.M., Hoesly, F.C., Williams, L.P., Hedberg, K., Oxman, G.L., Barrett, T.J., Pfaller, M.A., Fleming, D.W. 1994. A swimmi ng-associated outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7 and Shigella sonnei . New England Journal of Medicine 331(9): 579-584.

Keene, W.E., Sazie, E., Kok, J., Rice, D.H., Ha ncock, D.D., Balan, V.K., Zhao, T., Doyle, M.P.

1997. An outbreak of Escherichia coli O157:H7 infections traced to jerky made from deer meat.

Journal of the American Medi cal Association 277(15): 1229-1231.

King, B.J., Keegan, A.R., Monis, P.T., Saint, C.P. 2005. Environmental temperature controls Cryptosporidium oocyst metabolic rate and associated retention of infec tivity. Applied and

Environmental Micr obiology 71(7): 3848-3857.

Kudva, I.T., Hatfield, P.G., Hovde, C.J. 1996. Escherichia coli O157:H7 in microbial flora of sheep. Journal of Clinical Microbiology 34(2): 431-433.

LeChevallier, M. 2006. Mycobacterium avium complex. In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Work s Association: Denver, CO. Chapter 15.

LeChevallier, M.W., Giovanni, G.D.D., Clancy, J. L., Bukhari, Z., Bukhari, S., Rosen, J. S., Sobrinho, J., Frey, M.M. 2003. Comparison of method 1623 and cell culture-PCR for detection

of Cryptosporidium spp. in source waters. Applied and E nvironmental Microb iology 69(2): 971-

979.

Lederberg, J. 1998. Emerging inf ections: an evolutionary pers pective. Emerging Infectious Diseases 4(3): 366-371.

Lengerich, E.J., Addiss, D.G., Juranek, D.D. 1994. Severe giardiasis in the United States. Clinical Infectious Diseases 18(5): 760-763.

Levett, P.N. 2001. Leptospirosis. Clin ical Microbiology Reviews 14(2): 296-326.

Li, X., Atwill, E.R., Dunbar, L.A., Jones, T., Hook, J., Tate, K.W. 2005 . Seasonal temperature

fluctuations induces rapid inactivation of Cryptosporidium parvum . Environmental Science and Technology 39(12): 4484-4489.

Lightfoot, D. 2004. Salmonella and other enteric organism s. In: waterborne Zoonoses: Identification, Causes and Control; Cotruvo, J.A, Dufour, A., Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IWA Publishing: London, UK. Chapter 14.

Low, D.A., Weyand, N.J., Mahan, M.J. 2001. Roles of DNA adenin e methylation in regulating bacterial gene expression and virulen ce. Infection and Im munity 69(12): 7197-7204.

Mackenzie, J.S. 1999. Emerging viral diseases: an Australian perspective. Emerging Infectious Diseases 5(1): 1-8.U.S. Environmental Protection Agency

February 2009 64 MacKenzie, W.R., Hoxie, N.J., Proctor, M.E ., Gradus, M.S., Blair, K.A., Peterson, D.E., Kazmierczak, J.J., Addiss, D.G., Fox, K.R., Rose , J.B. 1994. A massive outbreak in Milwaukee

of Cryptosporidium infection transmitted through the pub lic water supply. New England Journal of Medicine 331(3): 161-167.

MacKenzie, W.R., Schell, W.L., Blair, K.A., Addiss, D.G., Peterson, D.E., Hoxie, N.J., Kazmierczak, J.J., Davis, J.P. 1995. Massive outbreak of waterborne Cryptosporidium infection in Milwaukee, Wisconsin: recurrence of illness and risk of secondary transmission. Clinical Infectious Diseases 21(1): 57-62.

Marrie, T.J., Raoult, D., Scola, B.L., Birtles, R.J., de Carolis, E., & The Canadian CommunityAcquired Pneumonia Study Canadian Commu nity-Acquired Pneumonia Study Group 2001. Legionella -like and other amoebal pathogens as ag ents of community-acquired pneumonia. Emerging Infectious Diseases 7(6): 1026-1029.

McBride, G. 1993. Faecal indicato r density and illness risk to swimmers in coastal waters: a preliminary study for New Zealand. In: Proc eedings of the Annual Conference of the New Zealand Water and Waste Association, Havelock North, 1-3 September 1993.

McBride, G., Till, D., Ryan, D.T., Ball, A., Lewis, D.G., Palmer, D. S., Weinstein, P. 2002. Freshwater microbiology research programme re port, pathogen occurrence and human health risk assessment analysis. Technical Report. Mi nistry for the Environment/Ministry of Health.

New Zealand.

McCarthy, T.A., Barrett, N.L., Hadler, J.L., Sa lsbury, B., Howard, R.T., Dingman, D. W., Brinkman, C.D., Bibb, W.F., Cartter, M. L. 2001. Hemolytic-uremic syndrome and Escherichia

coli O121 at a Lake in Connectic ut, 1999. Pediatrics 108(4): E59.

McCuin, R.M., Clancy, J.L. 2006. Occurrence of Cryptosporidium oocysts in US wastewaters. Journal of Water and Health 4(4): 437-452.

McGee, P., Bolton, D.J., Sheridan, J.J., Earley , B., Kelly, G., Leonard, N. 2002. Survival of

Escherichia coli O157:H7 in farm water: its role as a v ector in the transmission of the organism within herds. Journal of App lied Microbiology 93(4): 706-713.

Mead, P.S., Slutsker, L., Dietz, V., McCaig, L.F., Bresee, J.S., Shapiro, C., Griffin, P.M., Tauxe,

R.V. 1999. Food-related illness an d death in the United States. Emerging Infectious Diseases 5(5): 607-625.

Meites, E., Jay, M.T., Deresinski, S., Shieh, W ., Zaki, S.R., Tompkins, L., Smith, D.S. 2004. Reemerging leptospirosis, California. Emer ging Infectious Diseases 10(3): 406-412.

Mendez-Hermida, F., Castro-Hermida, J.A., Ares-Mazas, E., Kehoe, S.C., McGuigan, K.G. 2005. Effect of batch-process sola r disinfection on survival of Cryptosporidium parvum oocysts in drinking water. Applied and Envi ronmental Microbiology 71(3): 1653-1654.U.S. Environmental Protection Agency

February 2009 65 Michel, P., Wilson, J.B., Martin, S.W., Clarke, R.C., McEwen, S.A., Gyles, C.L. 1999. Temporal and geographical distributions of reported cases of Escherichia coli O157:H7 infection in Ontario. Epidemiology and Infection 122(2): 193-200.

Miller, R.L., Wang, A.L., Wang, C.C. 1988. Identification of Giardia lamblia isolates susceptible and resistant to infection by the double-stranded RNA virus. Experimental Parasitology 66(1): 118-123.

Mintz, E., Hudson-Wragg, M., Mshar, P., Cartter, M., Hadler, J. 1993. Foodborne giardiasis in a corporate office setting. Journal of Infectious Diseases 167: 250-253.

Moe, C. 2004. What are the criteria for determ ining whether a disease is zoonotic and water

related? In: Waterborne Zoonoses : Identification, Causes and Control; Cotruvo, J.A, Dufour, A., Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IWA P ublishing: London, UK. Chapter 3.

Molbak, K., Scheutz, F. 2004. Verocytotoxin-producing Escherichia coli and other diarrhoeagenic E. coli . In: Waterborne Zoonoses: Identific ation, Causes and Control; Cotruvo, J.A, Dufour, A., Rees, G., Bart ram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (W HO). IWA Publishing: London, UK. Chapter 13.

Morgan-Ryan, U.M., Fall, A., Ward, L.A., Hijja wi, N., Sulaiman, I., Fayer, R., Thompson, R.C.A., Olson, M., Lal, A., Xiao, L. 2002. Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens . Journal of Eukaryotic Microbiology 49(6): 433-440.

Morse, S.S. 1997. The public health threat of em erging viral disease. Jo urnal of Nutrition 127(5 Suppl): 951S-957S.

Moyer, N., Degnan, A. 2006. Shigella . In: Waterborne Pathogens, AWWA Manual M48, Second

Edition. American Water Works Associ ation: Denver, CO. Chapter 19.

Moyer, N., Standridge, J. 2006. Aeromonas . In: Waterborne Pathogens, AWWA Manual M48,

Second Edition. American Water Works A ssociation: Denver, CO. Chapter 6.

Mwenda, J.M., Nyachieo, A., Langat, D.K., St eele, D.A. 2005. Serological detection of adenoviruses in non-human primates maintained in a colony in Kenya. East African Medical Journal 82(7): 371-375.

Nachamkin, I. 2002. Chronic effects of Campylobacter infection. Microbes and Infection 4(4):

399-403.U.S. Environmental Protection Agency

February 2009 66 Nascimento, A.L.T.O., Ko, A.I., Martins, E.A. L., Monteiro-Vitorello, C.B., Ho, P.L., Haake, D.A., Verjovski-Almeida, S., Hartskeerl, R.A., Marques, M.V., Oliveira, M.C., Menck, C.F.M., Leite, L.C.C., Carrer, H., Coutinho, L.L., Degrav e, W.M., Dellagostin, O.A., El-Dorry, H., Ferro, E.S., Ferro, M.I.T., Furlan, L.R., Gamberini, M., Giglioti, E.A., Goes-Neto, A., Goldman, G.H., Goldman, M.H.S., Harakava, R., Jeronimo, S. M.B., Junqueira-de-Azevedo, I.L.M., Kimura, E.T., Kuramae, E.E., Lemos, E.G.M., Lemos, M. V.F., Marino, C.L., Nunes, L.R., de Oliveira, R.C., Pereira, G.G., Reis, M.S., Schriefer, A., Siqueira, W.J., Sommer, P., Tsai, S.M., Simpson, A.J.G., Ferro, J.A., Camargo, L.E.A., Kitajima, J.P., Setubal, J.C., Sluys, M.A.V. 2004. Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis. Journa l of Bacteriology 186(7): 2164-2172.

Nasser, A.M., Telser, L., Nitzan, Y. 2007. E ffect of sunlight on the infectivity of Cryptosporidium parvum in seawater. Canadian Journal Microbiology 53: 1101-1105.

Nataro, J.P., Deng, Y., Cookson, S. , Cravioto, A., Savarino, S.J., Guers, L.D., Levine, M.M.. Tacket, C.O. 1995. Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated

in volunteers. Journal of Infec tious Diseases 171(2): 465-468.

Nataro, J.P., Kaper, J.B. 1998. Diarrheagenic Escherichia coli . Clinical Microbiology Reviews 11(1): 142-201.

Naumova, E.N., Christodouleas, J., Hunter, P.R ., Syed, Q. 2005. Effect of precipitation on seasonal variability in cryptosporidiosis recorded by the North West England surveillance system in 1990-1999. Journal of Water and Health 3(2): 185-196.

National Research Council (NRC). 2004. Indicat ors for Waterborne Pathogens. National Academies Press: Washington, DC.

O’Donoghue, P. 1995. Cryptosporidium and cryptosporidiosis in man and animals. International Journal for Parasitology Research 25(2): 139-195.

Ohl, M.E., Miller, S.I. 2001. Salmonella : a model for bacterial pathog enesis. Annual Review of

Medicine 52: 259-274.

Okhuysen, P.C., Chappell, C.L., Crabb, J.H., Sterling, C.R., DuPont, H.L. 1999. Virulence of

three distinct Cryptosporidium parvum isolates for healthy adults. Jo urnal of Infectious Diseases 180(4): 1275-1281.

Okhuysen, P.C., Rich, S.M., Chappell, C.L., Grim es, K.A., Widmer, G., Feng, X., Tzipori, S. 2002. Infectivity of a Cryptosporidium parvum isolate of cervine origin for healthy adults and interferon-gamma knockout mice. Journal of Infectious Diseases 185(9): 1320-1325.

Olsen, S.J., Miller, G., Breuer , T., Kennedy, M., Higgins, C., Wa lford, J., McKee, G., Fox, K., Bibb, W., Mead, P. 2002. A waterborne outbreak of Escherichia coli O157:H7 infections and hemolytic uremic syndrome: implic ations for rural water systems. Emerging Infectious Diseases 8(4): 370-375.U.S. Environmental Protection Agency

February 2009 67 Olson, M., Ralston, B., O’Handley, R., Guselle, N ., Appelbee, A. 2003. What is the clinical and zoonotic significance of cryptosporidiosis in domestic animals and wildlife? In: Cryptosporidium : From Molecules to Disease; Thomps on, R., Armson, A., Ryan, U. (eds). Elsevier: Amsterdam, The Netherlands.

Olson, M.E., Goh, J., Phillips, M., Guselle, N., McAllister, T.A. 1999. Giardia Cyst and Cryptosporidium oocyst survival in water, soil, and cattle feces. Journal of Environmental Quality 28: 1991-1996.

Olson, M.E., O'Handley, R.M., Ralston, B.J., McAllister, T.A., Thompson, R.C.A. 2004. Update

on Cryptosporidium and Giardia infections in cattle. Trends in Parasitology 20(4): 185-191.

Ortega, Y. 2006. Cyclospora cayetanensis . In: Waterborne Pathogens, AWWA Manual M48,

Second Edition. American Water Works A ssociation: Denver, CO. Chapter 29.

Ortega, Y.R., Adam, R.D. 1997. Giardia : overview and update. Clinic al Infectious Diseases 25(3): 545-550.

Osewe, P., Addiss, D.G., Blair, K.A., Hi ghtower, A., Kamb, M.L., Davis, J.P. 1996. Cryptosporidiosis in Wisconsin: a case-cont rol study of post-outbreak transmission. Epidemiology and Infection 117(2): 297-304.

Ostroff, S.M., Kobayashi, J.M., Le wis, J.H. 1989. Infections with Escherichia coli O157:H7 in Washington State. The first year of statewide disease surveill ance. Journal of the American

Medical Associati on 262(3): 355-359.

Palmer, S., Biffin, A. & Group 199 0. Cryptosporidiosis in England and Wales: prevalence and clinical and epidemiological featur es. British Medical Journal 30: 774-777.

Pardo, J., Carranza, C., Muro, A ., Angel-Moreno, A., Martin, A., Martin, T., Hernandez-Cabrera, M., Perez-Arellano, J. 2006. Helminth-related Eosi nophilia in African immigrants, Gran Canaria. Emerging Infectious Di seases 12(10): 1587-1589.

Parker, J., Smith, H.V. 1993. Destruction of oocysts of Cryptosporidium parvum . Water Research 27(4): 729-731.

Pesaro, F., Sorg, I., Metzler, A. 1995. In situ in activation of animal viru ses and a coliphage in nonaerated liquid and semiliquid animal wastes . Applied and Environmental Microbiology 61(1): 92-97.

Pokorny, N.J., Weir, S.C., Carreno, R.A., Trevors, J.T., Lee, H. 2002. Influence of temperature

on Cryptosporidium parvum oocyst infectivity in river water samples as detected by tissue culture assay. Internat ional Journal for Para sitology 88(3): 641-643.

Poly, F., Guerry, P. 2008. Pathogenesis of Campylobacter . Current Opinion in Gastroenterology 24(1): 27-31.U.S. Environmental Protection Agency

February 2009 68 Pond, K. 2005a. E. coli O157. In: Water Recreation and Diseas e: Plausibility of Associated Infections: Acute Effects, Sequelae and Mortality. World Health Organization (WHO). IWA Publishing: London, UK. Chapter 4.

Pond, K. 2005b. Leptospira . In: Water Recreation and Diseas e: Plausibility of Associated Infections: Acute Effects, Sequelae and Mortality. World Health Organization (WHO). IWA Publishing: London, UK. Chapter 4.

Rangel, J.M., Sparling, P.H., Crowe, C., Griffi n, P.M., Swerdlow, D.L. 2005. Epidemiology of

Escherichia coli O157:H7 outbreaks, United States, 1982-2002. Emerging Infectious Diseases 11(4): 603-609.

Rendtorff, R.C. 1954. The experiment al transmission of human intes tinal protozoan parasites. II.

Giardia lamblia cysts given in capsules. American J ournal of Tropical Me dicine and Hygiene 59(2): 209-220.

Reynolds, K.A. 2006. Identifying hazards of waterborne disease. Water Conditioning and

Purification.

Rice, D.H., Hancock, D.D., Be sser, T.E. 1995. Verotoxigenic E. coli O157 colonisation of wild deer and range cattle. Th e Veterinary Record, 524.

Roberts, J.D., Silbergeld, E.K., Graczyk, T. 2007. A probabilistic risk assessment of Cryptosporidium exposure among Baltimore urban angl ers. Journal of Toxicology and Environmental Health Part A: Current Issues 70(18): 1568-1576.

Roberts, W.G., Green, P.H., Ma, J., Carr, M., Ginsberg, A.M. 1989. Prevalence of cryptosporidiosis in patients unde rgoing endoscopy: evidence for an asymptomatic carrier state.

American Journal of Medi cal Sciences 87(5): 537-539.

Robertson, B., Sinclair, M.I., Forbes, A.B., Veitch, M., Kirk, M., Cunliffe, D., Willis, J., Fairley,

C.K. 2002. Case-control studies of sporadic cryptosporidiosis in Melbourne and Adelaide, Australia. Epidemiology and Infection 128(3): 419-431.

Robertson, L.J. 1996. Severe giardiasis and cr yptosporidiosis in Scotland, UK. Epidemiology and Infection 117(3): 551-561.

Robertson, L.J., Campbell, A.T., Smith, H.V. 1992. Survival of Cryptosporidium parvum oocysts under various environmental pressure s. Applied and Environmental Microbiology 58(11): 3494-3500.

Robertson, L.J., Gjerde, B.K. 2006. Fate of Cryptosporidium oocysts and Giardia cysts in the Norwegian aquatic environment over wi nter. Microbial Ecology 52(4): 597-602.

Roefer, P., Monscvitz, J., Rexing, D. 1996. The La s Vegas cryptosporidiosis outbreak. American Water Works Association September 1996. Pp. 95-106.U.S. Environmental Protection Agency

February 2009 69 Rollins, D.M., Colwell, R.R. 1986. Viable but nonculturable stage of Campylobacter jejuni and its role in survival in the natural aquatic en vironment. Applied and Environmental Microbiology 42(3): 531-538.

Rose, J.B., Haas, C.N., Regli, S. 1991. Risk a ssessment and control of waterborne giardiasis.

American Journal of Public Health 81(6): 709-713.

Rosqvist, R., Skurnik, M., Wolf-Wat z, H. 1988. Increased virulence of Yersinia pseudotuberculosis by two independent mutatio ns. Nature 334(6182): 522-524.

Runde, V., Ross, S., Trenschel, R., Lagemann, E., Basu, O., Renzing- Kohler, K., Schaefer, U.W., Roggendorf, M., Holler, E. 2001. Adenovi ral infection after allogeneic stem cell transplantation (SCT): report on 130 patients from a single SCT unit involved in a prospective multi center surveillance study. Bone Marrow Transplant 28(1): 51-57.

Ryan, U., Monis, P., Enemark, H., Sulaiman, I., Samarasnghe, B., Read, C., Buddle, R., Robertson, I., Zhou, L., Thompson, R., Xiao, L. 2003. Cryptosporidium suis n. sp. (Apicomplexa: Cryptosporidiidae) in pigs ( Sus scrofa ). International Journa l for Parasitology 90:

769-773.

Samadpour, M., Stewart, J., Steingart, K., A ddy, C., Louderback, J., McGinn, M., Ellington, J., Newman, T. 2002. Laboratory investigation of an E. coli O157:H7 outbreak associated with swimming in Battle Ground Lake, Vancouver, Wash ington. Journal of Environmental Health 64(10): 16-26.

Sargeant, J.M., Gillespie, J.R., Oberst , R.D., Flood, S.J.A. 1999. Prevalence of Escherichia coli 0157:H7 in white-tailed deer sharing rangeland with cattle. Veterinary Medicine Today: Public Veterinary Medicine 215(6): 792-794.

Sattar, S., Chauret, C., Springt horpe, V., Battigelli, D., Abbas zadegan, M., LeChevallier, M.

1999. Giardia cyst and Cryptosporidium oocyst survival in waters heds and factors affecting

inactivation. American Wate r Works Association Research Foundation (AWWARF): Denver,

CO.

Sattar, S., Springthorpe, V. 2006. Reoviruses. In: Waterborne Pathogens, AWWA Manual M48,

Second Edition. American Water Works A ssociation: Denver, CO. Chapter 45.

Schaefer, F. 2006. Giardia lamblia . In: Waterborne Pathogens, AWWA Manual M48, Second

Edition. American Water Works Associ ation: Denver, CO. Chapter 31.

Schaub, S. 2004. A regulatory perspective on zoono tic pathogens in water. In: Waterborne Zoonoses: Identification, Causes and Control; Cotruvo, J.A, Dufour , A., Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IWA Publishing: London, UK. Chapter 27.U.S. Environmental Protection Agency

February 2009 70 Schoeni, J.L., Doyle, M.P. 1994. Variable colonizat ion of chickens perora lly inoculated with

Escherichia coli O157:H7 and subsequent contaminati on of eggs. Applied and Environmental Microbiology 60(8): 2958-2962.

Schwab, K. 2006. Astroviruses. In: Water borne Pathogens, AWWA Manual M48, Second

Edition. American Water Works Associ ation: Denver, CO. Chapter 39.

Schwab, K., Hurst, C. 2006. Human calicivirus es. In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Works Association: Denver, CO. Chapter 44.

Selifonova, O., Valle, F., Schellenberger, V. 2001. Rapid evolution of novel traits in microorganisms. Applied and Enviro nmental Microbiology 67(8): 3645-3649.

Serviene, E., Shapka, N., Cheng, C., Panavas, T., Phuangrat, B., Baker, J., Nagy, P.D. 2005. Genome-wide screen identifies host genes affec ting viral RNA recombination. Proceedings of the National Academy of Sciences 102(30): 10545-10550.

Shadduck, J.A., Greeley, E. 1989. Microsporidia an d human infections. C linical Microbiology Reviews 2(2): 158-165.

Sischo, W.M., Atwill, E.R., Lanyon, L.E., George, J. 2000. Cryptosporidia on dairy farms and the role these farms may have in contaminati ng surface water supplies in the northeastern United

States. Preventative Veteri nary Medicine 43(4): 253-267.

Smith, A.W., Skilling, D.E., Cherry, N., Mead, J.H., Matson, D.O. 1998. Ca licivirus emergence

from ocean reservoirs: zoonotic a nd interspecies movements. Emer ging Infectious Diseases 4(1):

13-20.

Smith, H., Grimason, A., Holland, C. 2006a. Ascaris lumbricoides . In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Works Association: Denver, CO. Chapter 24.

Smith, H., Grimason, A., Holland, C. 2006b. Trichuris trichiura . In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Works Association: Denver, CO. Chapter 37.

Sobsey, M. 2006. Hepatitis A virus. In: Water borne Pathogens, AWWA Manual M48, Second

Edition. American Water Works Associ ation: Denver, CO. Chapter 42.

Sterling, C., Marshall, M. 2006. Cryptosporidium parvum and Cryptosporidium hominis . In: Waterborne Pathogens, AWWA Manual M 48, Second Edition. American Water Works Association: Denver, CO. Chapter 28.

Stewart, M., Rochelle, P. 2006. Acinetobacter . In: Waterborne Pat hogens, AWWA Manual M48,

Second Edition. American Water Works A ssociation: Denver, CO. Chapter 5.U.S. Environmental Protection Agency

February 2009 71 Straub, T., Mena, H., Gerba, C. 1994. Viability of Giardia muris and Cryptosporidium parvum oocysts after aging, pressure, pH manipulations, and disinfection in mountain reservoir water. Proceedings of the 94th American Society Microbiology General Meeting, Las Vegas, NV.

Straub, T.M., zu Bentrup, K.H., Orosz-Coghlan, P., Dohnalkova, A., Mayer, B.K., Bartholomew, R.A., Valdez, C.O., Bruckner-Lea, C.J., Gerba, C.P., Abbaszadegan, M., Nickerson, C.A. 2007.

In vitro cell culture infectivity assay for human norovi ruses. Emerging Infecti ous Diseases 13(3):

396-403.

Sunderland, D., Graczyk, T.K., Ta mang, L., Breysse, P.N. 2007. Impact of bathers on levels of Cryptosporidium parvum oocysts and Giardia lamblia cysts in recreational beach waters. Water Research 41(15): 3483-3489.

Suresh, K., Smith, H. 2004. Tropical organisms in Asia/Africa/South America. In: Waterborne Zoonoses: Identification, Causes and Control. Cotruvo, J., Dufour, A., G. Rees, J.B., Carr, R., Cliver, D., Craun, G., Fayer, R., Gannon, V. (e ds), World Health Organization (WHO). IWA Publishing: London, UK. Chapter 6.

Swerdlow, D.L., Woodruff, B. A., Brady, R.C., Griffin, P.M ., Tippen, S., Donnell, H.D., Geldreich, E., Payne, B.J., Meyer, A., Wells, J.G ., Greene, K.D., Bright, M., Bean, N.H., Blake,

P.A. 1992. A waterborne out break in missouri of Escherichia coli 0157:H7 associated with bloody diarrhea and death. Annals of Internal Medicine 117(10): 812-819.

Tarr, P.I. 1995. Escherichia coli O157:H7: clinical, diagnostic, and epidemiological aspects of

human infection. Clinical Infect ious Diseases 20(1): 1-10.

Tate, K.W., Atwill, E.R., George, M. R., McDougald, N.K., Larsen, R.E. 2000. Cryptosporidium

parvum transport from cattle fecal deposits on California rangelands. Journal of Range Management 53(3): 295-299.

Tezcan-Merdol, D., Ljungstrom, M., Winiecka-Krusne ll, J., Linder, E., Engstrand, L., Rhen, M. 2004. Uptake and replication of Salmonella enterica in Acanthamoeba rhysodes . Applied and

Environmental Micr obiology 70(6): 3706-3714.

Thomas, K.M., Charron, D.F., Waltner-Toews, D., Schuster, C., Maarouf, A.R., Holt, J.D. 2006. A role of high impact weather events in wa terborne disease outbreak s in Canada, 1975 - 2001.

International Journal of Environmen tal Health Research 16(3): 167-180.

Thompson R.C. 2000. Giardiasis as a re-emerging in fectious disease and its zoonotic potential.

International Journal for Parasitology 30: 1259-1267.

Thompson, R.C.A., Monis, P.T. 2004. Variation in Giardia : implications for taxonomy and epidemiology. Advances in Parasitology 58: 69-137.U.S. Environmental Protection Agency

February 2009 72 Till, D., McBride, G. 2004. Potential public hea lth risk of Campylobact er and other zoonotic

waterborne infections in New Zealand. In: Wa terborne Zoonoses: Identification, Causes and Control; Cotruvo, J.A, Dufour, A., Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IW A Publishing: London, UK. Chapter 12.

Toranzos, G., Toro, A., Degnan, A. 2006. Vibrio cholerae . In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Work s Association: Denver, CO. Chapter 21.

Tozzi, A.E., Niccolini, A., Caprioli, A., Luzzi, I., Montini, G., Zacchello, G., Gianviti, A., Principato, F., Rizzoni, G. 1994. A community outbreak of haemolytic-uraemic syndrome in children occurring in a large ar ea of northern Italy over a period of several months. Epidemiology and Infection 113(2): 209-219.

Traub, R., Wade, S., Read, C., Thompson, A., Mohammed, H. 2005. Molecular characterization of potentially zoonotic isolates of Giardia duodenalis in horses. Veterinary Parasitology 130(3- 4): 317-321.

Traub, R.J., Monis, P.T., Robertson, I., Ir win, P., Mencke, N., Thompson, R.C.A. 2004. Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same co mmunity. Parasitology 128(Pt 3): 253-262.

Tupchong, M., Simor, A., Dewar, C. 1999. Beaver feve r - a rare cause of re active arthritis. The Journal of Rheumatology 26(12): 2701-2702.

Tzipori, S. 2000. Predicting human dose-response re lationships from multip le biological models. Conference transcript from Conference on Predic ting Human Dose-response Relationships from Multiple Biological Models: Issues with Cryptosporidium parvum , September 28, 2000, USDA Center at Riverside, Riverdale, MD. http://www.foodrisk.org/IRAC/event s/2000-09-28/speakers/tzipori.cfm

.

Ungar, B.L. 1990. Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal specimens. Jour nal of Clinical Microbiology 28(11): 2491-2495.

U.S. Department of Agriculture (USDA). 2001. Risk draft assessment of th e public hea lth impact

of Escherichia coli O157:H7 from ground beef'. U.S. Depa rtment of Agriculture. September. http://www.fsis.usda.gov/Scie nce/Risk_Assessments/index.asp

.

U.S. Department of Agriculture (USDA). 2005. Risk Assessment of the Impact of Lethality Standards on Salmonellosis from Ready-to-Eat M eat and Poultry Products. Final Report. U.S. Department of Agriculture, The Food Safety and Inspection Service, Offi ce of Public Health Science, Risk Assessment Division. http://www.fsis.usda.gov/Scie nce/Risk_Assessments/index.asp

.U.S. Environmental Protection Agency

February 2009 73 U.S. Environmental Protection Agency (USEPA ). 1986. Bacteriological Ambient Water Quality

Criteria for Bacteria. U.S. Environmental Protection Agency, Office of Emergency and Remedial Response: Washington, DC.

USEPA. 1989. Risk Assessment Guidance for Supe rfund Volume I. Human Health Evaluation Manual. U.S. Environmental Protection Agency, Office of Emergency and Remedial Response: Washington, DC. EPA/540/1-89/002.

USEPA. 1997. Exposure Factors Handbook. U.S. Environmental Pr otection Agency, Office of Research and Development National Centerfor Environmental Assessment. EPA/600/P95/002Fa.

USEPA. 1998. Giardia : Human Health Criteria Document . U.S. Environmental Protection Agency, Office of Water: Washington, DC. EPA-823-R-002.

USEPA. 1999. Drinking Water Criteria Document for Viru ses: An Addendum. U.S. Environmental Protection Agency, Office of Water: Washington, DC.

USEPA. 2001a. Cryptosporidium : Human Health Criteria Do cument. U.S. Environmental Protection Agency, Office of Water: Washington, DC.

USEPA. 2001b. Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S. Environmental Protection Agency, Office of Water: Washington, DC.

USEPA. 2002. Implementation Guidance for Ambient Water Quality Criteria for Bacteria. U.S. Environmental Protection Agency, O ffice of Water: Washington, DC.

USEPA. 2005a. Occurrence and Exposure Assessm ent for the Final Long Term 2 Enhanced

Surface Water Treatment Rule. U.S. Environm ental Protection Agency, Office of Water: Washington, DC.

USEPA. 2005b. Appendices to the Occurrence a nd Exposure Assessment for the Final Long

Term 2 Enhanced Surface Water Treatment Rule. U.S. Environmental Protection Agency, Office of Water: Washington, DC.

Visvesvara, G., Moura, H. 2006a. Acanthamoeba spp

. In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Work s Association: Denver, CO. Chapter 23.

Visvesvara, G., Moura, H. 2006b. Balamuthia mandrillaris . In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Work s Association: Denver, CO. Chapter 25.

Visvesvara, G., Moura, H. 2006c. Naegleria fowleri . In: Waterborne Pathogens, AWWA Manual M48, Second Edition. American Water Works Association: Denver, CO. Chapter 34.U.S. Environmental Protection Agency

February 2009 74 Walker, M., Leddy, K., Hagar, E. 2001. Effects of combined water potential and temperature

stresses on Cryptosporidium parvum oocysts. Applied and Environmental Microbiology 76(12):

5526-5529.

Wang, G., Doyle, M.P. 1998. Heat shock response enhances acid tolerance of Escherichia coli O157:H7. Letters in Applie d Microbiology 26(1): 31-34.

Whitman, R.L., Nevers, M.B, Byappanahalli, M.N. 2006. Examination of the watershed-wide

distribution of Escherichia along southern Lake Michigan: an integrated approach. Applied and Environmental Microbiology 72(11): 7301-7310.

WHO. 2003. Human Leptospirosis: Guidance for Di agnosis, Surveillance and Control. Technical Report. World Health Organization (WHO) a nd the International Leptospirosis Society. http://whqlibdoc.who.int/hq/ 2003/WHO_CDS_CSR_EPH_2002.23.pdf

.

WHO. 2004. Waterborne Zoonoses : Identification, Causes and Control. World Health Organization (WHO); Cotruvo, J.A., Dufour, A., Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F., Fayer, R., Gannon, V.P.J (eds). IWA Publishing: London, UK. ISBN: 1 84339 058

2. http://www.who.int/water_sanita tion_health/diseases/zoonoses/en/

.

WHO. 2006. Guidelines for Drinking Water Quality: Cryptosporidium. Technical report, World Health Organization.

Widmer, G., Tchack, L., Chappell, C.L., Tzipor i, S. 1998. Sequence polymorphism in the betatubulin gene reveals heterogeneous a nd variable population structures in Cryptosporidium

parvum . Applied and Environmental Microbiology 64(11): 4477-4481.

Wills, C. 1996. Fever Black Goddess: The Co-Evolution of People and Plagues. Addison-Wesley Publishing Company, Inc.: New York, NY. P.44.

Wolfe, M., Jakubowski, W., Hoff, J. (editors ) 1979. Managing the patient with giardiasis: clinical, diagnostic and therapeuti c aspects. In: Waterborne Tran smission of Giardiasis. U.S. Environmental Protection Agency (USEPA): Cincinnati, OH.

Wolfe, M.S. 1992. Giardiasis. Clinical Microbiology Reviews 5(1): 93-100.

Xiao, L., Ryan, U.M. 2004. Cryptos poridiosis: an update in molecular epidemiology. Current Opinion in Infectious Diseases 17(5): 483-490.

Yamahara, K.M., Layton, B.A., Santoro, A.E ., Boehm, A.B. 2007. Beach sands along the

California Coast are diffuse sources of fecal bact eria to coastal waters. Environmental Science and Technology 41: 4515-4521. Supporting information for beach sands along the California

coast are diffuse sources of fecal bacteria to coastal waters. Environmental Science and Technology Supplemental to 41: 4515-4521, 17 pp.U.S. Environmental Protection Agency

February 2009 75 Yang, S., Benson, S.K., Du, C., Healey, M.C. 2000. Infection of immunosuppressed C57BL/6N adult mice with a single oocyst of Cryptosporidium parvum . International Journal for Parasitology 86(4): 884-887.

Yarze, J.C., Chase, M.P. 2000. E. coli O157:H7 - another waterbor ne outbreak! American

Journal of Gastroen terology 95(4): 1096.

Yoder, J.S., Blackburn, B.G., Craun, G.F., Hill, V., Levy, D.A., Chen, N., Lee, S.H., Calderon, R.L., Beach, M.J. 2004. Surveillance for water borne-disease outbreaks associated with recreational water - United States, 2001-2002. Mo rtality and Morbidity Weekly Report CDC Surveillance Summaries 53(8): 1-22.

Zhou, L., Singh, A., Jiang, J., Xiao, L. 2003. Molecular surveillance of Cryptosporidium spp

. in raw wastewater in Milwaukee: implicatio ns for understanding out break occurrence and transmission dynamics. Journal of Clinical Microbio logy 41(11): 5254-5257.U.S. Environmental Protection Agency

February 2009 A-1 

## A PPENDIX 

## A

W ATERBORNE 

## P ATHOGENS

As described in Section I.2 of this paper, four attributes were used to select the waterborne zoonotic pathogens of concern for recreational uses of ambient wa ters (partially adapted from Bolin et al., 2004a). Table A-1 li sts all the pathogens that were evaluated for potential inclusion in this paper. Information provided in Table A-1 includes whether the pathogen is considered waterborne, the species that are zoonotic hos ts, whether the zoonotic hosts are warm-blooded, what illnesses the pathogen causes in humans, and the importance of considering the pathogen as

EPA decides whether animal sources of fecal mate rial should be considered differently from human sources for CWA §304(a) AWQC.U.S. Environmental Protection AgencyFebruary 2009A-2Table A-1. Known and Potential Zoonotic Waterborne PathogensImportanceTypePathogenWaterborneZoonoticHostZoonoticHost isWarmIllnesses andSymptoms inHumans (lessU.S.Outbreaksto considerif animalfecalReference(s)bloodedcommonsymptoms)sources arediscountedin AWQCBacteriaAcinetobacterYesNoneNASepticemia,HospitalNoStewart and(generally inmeningitis,settingsRochelle, 2006environment)endocarditis, brainabscesses,pneumonia,empyema, urinarytract infections, eyeinfections, and skinand woundinfectionsBacteriaAeromonasYes(generally inenvironment)NoneNAGastroenteritis(septicemia)NonereportedNoLightfoot, 2004;Moyer andStandridge,2006BacteriaCampylobacterYesPoultry,cattle,YesDiarrhea,MainlyImportantAllos, 1998;sheep, andabdominal pain,foodborne2001; APHA,wild birdsmalaise, fever,and drinking2006; Fricker,nausea, andwater2006avomiting (typhoidlike syndrome,febrile convulsions,meningeal arthritis,reactive arthritis,and GBS)BacteriaCyanobacteriaYesNoneNARashandDrinkingNoFredericksen et(generally ingastroenteritiswater andal., 2006environment)recreationalwaterU.S. Environmental Protection AgencyFebruary 2009A-3TypePathogenWaterborneZoonoticHostZoonoticHost isWarmbloodedIllnesses andSymptoms inHumans (lesscommonsymptoms)U.S.OutbreaksImportanceto considerif animalfecalsources arediscountedin AWQCReference(s)BacteriaVerotoxin-producingE.coli(VTEC) – includesenterohemorrhagicE. coli(EHEC), including O157:H7YesCattle,chicken,sheep, pigs,horses, dogs,and deerYesDiarrhea (bloody),severe abdominalcramping,headache,hemorrhagic colitis,and hemolyticuremic syndromeFoodborneandwaterborne(drinkingwater andrecreationalwater)ImportantAPHA, 2004;Degnan andStandridge,2006; Mølbakand Scheutz,2004BacteriaEnterotoxigenicE. coli(ETEC)YesSameasVTECYesAcute, waterydiarrheaWaterbornePossiblyimportantHunter, 2003;Mølbak andScheutz, 2004BacteriaAttaching and effacingE.coli(A/EEC)YesSameasVTECYesAcute or persistentdiarrheaNowaterbornereportedPossiblyimportantMølbak andScheutz, 2004BacteriaEnteropathogenicE. coli(EPEC)YesSameasVTECYesAcute or persistentdiarrheaWaterbornePossiblyimportantLee et al, 2002;Mølbak andScheutz, 2004BacteriaEnteroaggregativeE. coli(EAggEC)YesSameasVTECYesAcute, watery, andoften protracteddiarrheaNowaterbornereportedPossiblyimportantMølbak andScheutz, 2004BacteriaDiffuse adherentE. coli(DAEC)YesSameasVTECYesAcute or persistentdiarrheaNowaterbornereportedPossiblyimportantMølbak andScheutz, 2004BacteriaEnteroinvasive(EIEC)E. coliYesNoneNAAcute,ofteninflammatorydiarrhea; dysenteryNowaterbornereportedNoMølbakandScheutz, 2004U.S. Environmental Protection AgencyFebruary 2009A-4TypePathogenWaterborneZoonoticHostZoonoticHost isWarmbloodedIllnesses andSymptoms inHumans (lesscommonsymptoms)U.S.OutbreaksImportanceto considerif animalfecalsources arediscountedin AWQCReference(s)BacteriaFlavobacteriumYesNoneNAGastroenteritis,meningitis,pneumonia,endocarditis, andsepticemiaRarewaterborne(stagnation indrinkingwater),hospitalsettings moreNoGeldreich andDegnan, 2006acommonBacteriaHelicobacter pyloriPossiblyWeakevidence forferrets,racoons,swine, sheep,rodents, andprimatesYesGastric disorders,pepticand duodenalulcer disease,lymphoma of thedigestive tract, andademenocarcinomaof the stomachNowaterbornereportedNoBakerandDegnan, 2006;Health Canada,2006BacteriaKlebsiellaYes(generally inenvironment)Warm-bloodedanimalsYesInfections inrespiratory system,genitourinary tract,nose, and throat,(meningitis andsepticemia)HospitalsettingsNoGeldreich andStandridge,2006BacteriaLegionellaYesNoneNALegionellosis,pneumonia,Legionnaire’sdisease, andPontiac feverHospitals,pools, andspasNoHall,2006BacteriaLeptospiraYesRats,dogs,raccoons,swine, andcattleYesLeptospirosis(Weil’s disease)Recreationalwaterborneover 50% ofcases inImportantLevett, 2001HawaiiU.S. Environmental Protection AgencyFebruary 2009A-5TypePathogenWaterborneZoonoticHostZoonoticHost isWarmbloodedIllnesses andSymptoms inHumans (lesscommonsymptoms)U.S.OutbreaksImportanceto considerif animalfecalsources arediscountedin AWQCReference(s)BacteriaListeria monocytogenesNoDomesticandwild animalsYesListeriosis,meningoencephalitis, fever, andabortionFoodborneNoAPHA, 2004BacteriaMycobacterium aviumcomplex (MAC) and ssp.Paratuberculosis (MAP)Yes(generally inenvironment)Possiblysheep, cattle,goats, andbirdsYesRespiratoryinfection, fever, andCrohn’s diseaseNorecreationalwaterbornereportedPossiblyimportantBolin et al.,2004b; Carrand Bartram,2004;LeChevallier,2006BacteriaPseudomonasYesNoneNADermatitisRecreationalNoGeldreich and(generally inenvironment)waterborneDegnan, 2006bBacteriaSalmonellaYesPoultry,swine,cattle, rodents,wild birds,turtles, dogs,and catsYesGastroenteritis(enteric fever andsepticemia)Mainlyfoodborneand drinkingwaterImportantAPHA, 2004;Covert andMeckes, 2006BacteriaSerratiaYes(generally inenvironment)NoneNAOpportunisiticinfection (cystitis)HospitalsettingsNoGeldreich andStandridge,2006aBacteriaShigellaYesNone(exceptprimatecolonies)NAShigellosis, acutegastroenteritis,dysentary, fever,nausea, vomiting,and crampsRecreationaland drinkingwaterNoAPHA, 2004;Cliver andFayer, 2004;Moyer andDegnan, 2006BacteriaStaphylococcusYesSkin of warmblooded hostsYesCellulitis, pustules,boil, carbuncles,and impetigo(diarrhea andvomiting)Hospitalsettings,pools, andspasNoGeldreich andStandridge,2006bU.S. Environmental Protection AgencyFebruary 2009A-6TypePathogenWaterborneZoonoticHostZoonoticHost isWarmbloodedIllnesses andSymptoms inHumans (lesscommonsymptoms)U.S.OutbreaksImportanceto considerif animalfecalsources arediscountedin AWQCReference(s)BacteriaVibrio choleraeYesCopepods,zooplanktonNoProfuse, waterydiarrhea; vomitingNonerecentlyNoAPHA,2004;Toranzos et al.,2006BacteriaVibrio parahaemolyticusYes(generally inenvironment)MolluscanshellfishNoAcutegastroenteritis(septicemia)FoodborneNoFDA, 2001BacteriaYersiniaYesPigsYesYersiniosis;acute,febrile diarrheaMainlyfoodbornePossiblyimportantAPHA, 2004;Fricker, 2006bProtozoaAcanthamoebaYes (freeliving)NoneNAGranulomatusameobicencephalitisNonereportedNoFayer,2004b;Visvesvara andMoura, 2006aProtozoaAscaris lumbricoidesYesNoneNAAscariasisandroundworminfectionFoodborneNoAPHA, 2004;Smith et al.,2006aProtozoaBalamuthia mandrillarisYes (freeliving)Primates,sheep, dogs,and horsesYesGranulomatusameobicencephalitisNonereportedNoVisvesvara andMoura, 2006bProtozoaBalantidium coliYesPrimatespigsandYesSeveredysenteryNonereportedNoGarcia,2006aProtozoaBlastocystis hominisYesPrimates,cattle, sheep,pigs, horses,dogs,chickens, wildbirds, alpacas,llamas, koalas,and wombatsYesDiarrhea, cramps,nausea, fever,vomiting, andabdominal painNonereportedNoGarcia,2006bU.S. Environmental Protection AgencyFebruary 2009A-7TypePathogenWaterborneZoonoticHostZoonoticHost isWarmbloodedIllnesses andSymptoms inHumans (lesscommonsymptoms)U.S.OutbreaksImportanceto considerif animalfecalsources arediscountedin AWQCReference(s)ProtozoaCryptosporidium parvumandCryptosporidiumhominisYesCattle,sheep,goats, pigs,horses, cats,dogs, wildanimals, birds,reptiles, andfishYesCryptosporidiosis,profuse waterydiarrhea, malaise,fever, anorexia,nausea, andvomitingRecreationaland drinkingwaterImportantAPHA, 2004;CDC, 2007b;Olson, et al.,2003; Sterlingand Marshall,2006ProtozoaCyclospora cayetanensisYesNoneNAWaterydiarrhea,malaise, fever,anorexia, nausea,and vomitingFoodborneandwaterborne(drinkingwater andrecreationalwater)NoAPHA,2004;Cliver andFayer, 2004;Cross andSherchand,2004; Ortega,2006ProtozoaEntamoeba histolyticaYesPotentiallyprimates,dogs, cats,pigs, rats, andpossibly cattleYesAmoebiasis,dysentery, anddiarrheaNonerecentlyNoAPHA,2004;Fayer, 2004b;Keene, 2006ProtozoaGiardia intestinalis(alsoknown asG. duodenalisandG. lamblia)YesBeavers,cats,lemurs, sheep,calves, dogs,foxes,chinchillas,alpacas,horses, pigs,cows, andmuskratsYesGiardiasis; diarrhea(chronic);steatorrhea;abdominal cramps;bloating; frequentloose, pale, greasystools; fatigue; andmalabsorptionRecreationaland drinkingwaterImportantAPHA, 2004;Appelbee et al.,2005;Schaefer, 2006U.S. Environmental Protection AgencyFebruary 2009A-8TypePathogenWaterborneZoonoticHostZoonoticHost isWarmbloodedIllnesses andSymptoms inHumans (lesscommonsymptoms)U.S.OutbreaksImportanceto considerif animalfecalsources arediscountedin AWQCReference(s)ProtozoaIsosprora belliYesNoneNAFoulsmelling,foaming diarrhea(months to years),abdominal colic,and feverNonereportedNoGarcia, 2006cProtozoaMicrosporidia(Enterocytozoon bieneusi,Encephalitozoon cuniculi,E. intestinalis)YesCattle,pigs,cats, rabbits,and sheepYesDiarrheaNonereportedPossiblyimportantBolin et al.,2004b; Cali,2006;Shadduck andGreely, 1989ProtozoaNaegleria fowleriYes (freeliving)NoneNAPrimaryamebicmeningoencephalitisMainlyambientrecreationalNoFayer,2004b;Visvesvara andMoura, 2006cwatersVirusAdenovirusesYesNoneNAAcutegastroenteritis andrespiratory diseaseWaterborneNoEnriquez andThurstonEnriquez, 2006;Griffin et al.,2003;Reynolds, 2006VirusAstrovirusesYesNoneNAGastroenteritisWaterborneNoReynolds,2006; Schwab,2006VirusAvian influenza (H5N1)UnknownBirdsYesMild upperrespiratory illnessto severepneumonia andmultiple organfailureNonereportedNoChan,2002;Suresh andSmith, 2004U.S. Environmental Protection AgencyFebruary 2009A-9TypePathogenWaterborneZoonoticHostZoonoticHost isWarmbloodedIllnesses andSymptoms inHumans (lesscommonsymptoms)U.S.OutbreaksImportanceto considerif animalfecalsources arediscountedin AWQCReference(s)VirusCoronaviruses (e.g.,severe acute respiratorysyndrome (SARS)-CoV)Potentially(aerosolizedwastewater)Possibly civetsand other wildanimalsNAFever, dry cough,dyspnoea, andmyalgia (diarrhea)NonereportedNoMoe,2004;Reynolds,2006; Sureshand Smith,2004VirusEnterovirusescoxsackie)(e.g.,YesNoneNAGastroenteritis,exanthema,diarrhea, fever,pharyngeal lesions,myocarditis,respiratory disease,and pneumoniaRecreationaland drinkingwaterNoAPHA, 2004;Gerba, 2006a;Griffin, 2003;Reynolds, 2006VirusHendra virusUnknownHorsesYesSevere respiratorydisease andmeningoencephalitisNonereportedNoMacKenzie,1999; Sureshand Smith,2004VirusHepatitis A virusYesNoneNAHepatitis - acuteinflammation of theliverNonerecentlyNoSobsey, 2006VirusHepatitis E virusYesPossibly pigs,chickens, andrats (close viralrelatives tohuman form)NAHepatitis - acuteinflammation of theliver (fatality inpregnant women)Rare inUnited States(common inothercountries)NoCliver and Moe,2004; Craun,2004a; Gerba,2006bVirusHuman caliciviruses(norovirus, sapovirus)YesNoneNADiarrheaandvomitingVerycommon(waterborneandNoReynolds,2006; Schwaband Hurst,2006foodborne)VirusNipah virusUnknownPigs, bats, andflying foxesYesAcute and febrileencephalitisNonereportedNoHarcourt, 2005;Suresh andSmith, 2004U.S. Environmental Protection AgencyFebruary 2009A-10TypePathogenWaterborneZoonoticHostZoonoticHost isWarmbloodedIllnesses andSymptoms inHumans (lesscommonsymptoms)U.S.OutbreaksImportanceto considerif animalfecalsources arediscountedin AWQCReference(s)VirusParechovirusesYesNoneNAPericarditis,herpangina, andrespiratory diseaseNonereported(probablyoccurs, butunidentified)NoGerba,2006aVirusReovirusesYesNoneNAMostly mild orsubclinical (rarelybiliary atresia,juvenile onsetdiabetes, fever,rash, respiratorydisease, anddiarrheaNonereported(probablyoccurs, butunidentified)NoSattarandSpringthrope,2006VirusRotavirusesYesNoneNADiarrheaVerycommonwaterborneNoAbbaszadegan,2006;Reynolds, 2006HelminthsAncylostoma brazilienseMainly soilDogs or catsYesLarval migrationleads to creepingeruptionNonereported(mostimportanthookworm inNoEndoandMorishima,2004humans - soilroute)HelminthsAngiostrongylusYesMollusksNoMeningitisNonereportedNoEndoandMorishima,2004HelminthsAscaris lumbricoidesMainly soil(drinkingwaterpossible)NoneNAAscarispneumonia(lung hemorrhage)NonereportedNoEndoandMorishima,2004; Smith etal., 2006U.S. Environmental Protection AgencyFebruary 2009A-11TypePathogenWaterborneZoonoticHostZoonoticHost isWarmbloodedIllnesses andSymptoms inHumans (lesscommonsymptoms)U.S.OutbreaksImportanceto considerif animalfecalsources arediscountedin AWQCReference(s)HelminthsAscaris suumMainly soilPigsYesAscaris pneumonia(lung hemorrhage)NonereportedNoEndoandMorishima,2004Helminthsavian schistosomes(includes S. mansonni)YesSnails(birdsare infectedbut notYesCercarial dermatitis(swimmer’s itch)NonereportedLowimportanceEndo andMorishima,2004sources)HelminthsBaylisascaris procyonisMainly soilRaccoonsYesLarval migrationmay damage to thevisceral and ocularNonereportedNoEndoandMorishima,2004systems (migrationto brain)HelminthsDracunculus medinensisYesCrustaceansNoConnectivetissueNoneNoEndoandmigration,emerging in lowerlimb blisterreportedMorishima,2004HelminthsEchinococcusYesFoxes, dogsYesCystic hydatiddisease, alveolarhydatid disease,polycystic hydatiddisease, andpolycystic hydatiddiseaseNonereportedLowimportanceEndo andMorishima,2004HelminthsFasciola hepaticaYesSnails(herbivoresand humansYesFascioliasisNonereportedLowimportanceEndo andMorishima,2004infected)HelminthsPseudophyllid cestodesYesCopepods, fishNoCutaneous ormucocutaneousinvasionNonereportedNoEndoandMorishima,2004U.S. Environmental Protection AgencyFebruary 2009A-12TypePathogenWaterborneZoonoticHostZoonoticHost isWarmbloodedIllnesses andSymptoms inHumans (lesscommonsymptoms)U.S.OutbreaksImportanceto considerif animalfecalsources arediscountedin AWQCReference(s)HelminthsSchistosomesYesSnails(manyanimals can beinfected, butare notsources)NoSchistosomiasis(chronic infection -liver fibrosis, portalhypertension)NonereportedNoAPHA, 2004;Blankespoor,2006; Endoand Morishima,2004HelminthsStrongyloidesMainly soilDogsYesEosinophiliaNonereportedNoEndoandMorishima,2004; Pardo,2006HelminthsTaenia solium(porktapeworm)YesPigsYesCysticercosisandmyositisNonereportedLowimportanceEndo andMorishima,2004HelminthsToxocara canisYesDogsYesToxocariasis(larvalmigration leads tohemorrhage andgranulomatouslesions in theNonereportedLowimportanceEndo andMorishima,2004central nervoussystem)HelminthsToxocara catiYesCatsYesToxocariasis(larvalmigration leads tohemorrhage andgranulomatouslesions in theNonereportedLowimportanceEndo andMorishima,2004central nervoussystem)U.S. Environmental Protection AgencyFebruary 2009A-13TypePathogenWaterborneZoonoticHostZoonoticHost isWarmbloodedIllnesses andSymptoms inHumans (lesscommonsymptoms)U.S.OutbreaksImportanceto considerif animalfecalsources arediscountedin AWQCReference(s)HelminthsToxoplasma gondiiYesCatsYesToxoplasmosis,(infetally exposedchildren - mentalNonereported inrecreationalPotentiallyimportantAPHA, 2004;Dubey, 2004,2006retardation, loss ofvision, hearingimpairment, andmortality)waterHelminthsTrichinella spiralisMainly soilVariety ofanimalsYesTrichinosisFoodborne(mainlyconsumptionofNoEndo andMorishima,2004undercookedpork)HelminthsTrichuris trichiuriaMainly soilMonkeys, pigs,dogs, cats,and chickenYesTrichuris dysentarysyndrome, chronicdiarrhea, anemia,and growthretardationFoodborneNoEndo andMorishima,2004; Smith etal., 2006bPrionBovine SpongiformEncephalopathy (BSE)UnknownCattleYesVariant CreutzfeldtJakob diseaseFoodborneNoBrown et al.,2006NA = Not applicableU.S. Environmental Protection Agency

February 2009 B-1 

## A PPENDIX 

## B

L ITERATURE 

## S EARCH 

## S TRATEGY AND 

## R ESULTS

The literature search strategy consisted of a number of combined approaches. Search terms and a synopsis of information needed were given to a professional librarian to search the online DIALOG databases. To supplement the DIALOG searches, individual au thors used free search engines on the internet to find ar ticles pertaining to specific in formation needed. Experts that participated in EPA’s Experts Scientific Wo rkshop on Critical Research Needs for the Development of New or Revised R ecreational Water Quality Criteria 11 were contacted by email and requested to contribute literature they felt was important. The titles of literature cited in specific reports, books, review articles, and conference proceedings were evaluated for

relevance.

B.1 Initial Literature Search Strategy Conducted by Professional Librarian

Selection of DIALOG data base files used for this search:

File 155:MEDLINE(R) 1950-2007/Nov 30 (c) format only 2007 Dialog File 266:FEDRIP 2007/Sep Comp & dist by NTIS, Intl Copyright All Rights Res
File 144:Pascal 1973-2007/Nov W3

(c) 2007 INIST/CNRS
File 110:WasteInfo 1974-2002/Jul

(c) 2002 AEA Techn Env.
File 245:WATERNET(TM) 1971-2007Jul
(c) 2007 American Water Works Association File 117:Water Resources Abstracts 1966-2007/Aug

(c) 2007 CSA.
File 5:Biosis Previews(R) 1926-2007/Nov W4 (c) 2007 The Thomson Corporation
File 40:Enviroline(R) 1975-2007/Oct (c) 2007 Congressional Information Service File 143:Biol. & Agric. Index 1983-2007/Oct
(c) 2007 The HW Wilson Co
File 6:NTIS 1964-2007/Dec W3 (c) 2007 NTIS, Intl Cpyrght All Rights Res
File 72:EMBASE 1993-2007/Dec 04
(c) 2007 Elsevier B.V.

Main search strategy used for this search: Fecal set AND Composition set AND (Pathogen or Waterborne sets): 1985-present

**S1 240528 FECAL OR FECES OR FAECAL OR FAECES OR DUNG OR SCAT OR EXCREMENT**

**OR MANURE OR STOOLS)**

**S2 1686966 COMPOSITION OR COMPOSED OR ANIMAL(1N)HUMAN()(TRANSMISSION OR**

**CONTAMINATION)**

11 Report from this workshop: http://www.epa.gov/waterscience/criteria/recreation/

.U.S. Environmental Protection Agency

February 2009 B-2 **S3 3842348 PATHOGEN? ? OR INDICATOR? ? OR RISK OR RISKS**

**S4 983 WATERBORNE()(PATHOGEN? ? OR ZOONO? OR ILLNESS?)**

**S6 1140 S1 AND S2 AND (S3 OR S4)**

**S719961011 PY=1920:1984 S8 1037 S6 NOT S7 (limited to 1985-present; foreign language OK)**  **S9 704 RD S8 (unique items, deduped)**

Dates: 1985-present Language: No restrictions Retrieve: Titles and year

Format: MS Word

Interested in international and domes tic journals and government reports.

Descriptions of these files are available at http://library.dial og.com/bluesheets/

.

Search terms: Waterborne pathogen*

Waterborne illness*

Waterborne zoon* Emerging waterborne zoonotic pathogen* Antibiotic resistan* AND zoonotic pathogen* Transmission rate waterborne pathogen* Evolution zoon* pathogen* Human fecal composition micro*

Enterohemorrhagic E. coli symptoms
Salmonella symptoms Shigella symptoms Campylobacter symptoms

Listeria symptoms Cryptosporidium symptoms
Giardia symptoms norovirus symptoms rotavirus symptoms hepatitis E symptoms

waterborne dermal infection* waterborne eye infection*

waterborne ear infection* waterborne respiratory infection*

What we want from the literature search:

• Fecal composition of species that harbor zoon otic pathogens (species of livestock and

wild animals

• Fecal composition of humans – pathogens and indicators

• The extent to which strains found in an imals can be transmitted to humansU.S. Environmental Protection Agency

February 2009 B-3 • An evaluation of the extent to which the inform ation identified can be used to support the

differentiation of risk from animal a nd human sources of fecal contamination

• Which organisms are of substantial public heal th concern that occur in ambient waters and are pathogenic to humans

• Which of these organisms are also present in animal populations

• The extent to which these organisms found in animals can be transmitted to humans

• The potential outcomes of human infec tion and disease from animal sources

• Specific pathogens: E. coli O157-H7, Salmonella

, Shigella

, Campylobacter

, Listeria

, Cryptosporidium

, Giardia , norovirus, rotavirus, Hepati tis E, emerging pathogens. For

each:

o Describe illness symptoms (range asymptomatic to severe)

o Describe route of exposure from r ecreational immersion in water including, inhalation, skin and mucus, eyes, ears

o Incidence (morbidity and mortality data, through recent time)

o Zoonotic potential – which animal species

• Variations in strains that effect infectivity, severity of symptoms, environmental survival and treatability

• Short summary review of emerging pathogen m echanisms (not too deep into molecular mechanisms of evolution)

• Anything in the water matrix that affects su rvivability and infec tivity, and virulence

• Pathogen:indicator ratios

B.2 Summary of Literature Search Results

This process resulted in a total order of 535 doc uments (primarily peer reviewed scientific articles), of which a total of 332 (62 percent) we re received during the ex pedited writing process,

not all of which could be reviewed. There are ma ny more papers in the peer-reviewed literature, and this by no means represents al l of them. 319 citations were included in the white paper.

B.3 Supplemental Free Online Search Engines

The following terms were searched on Google ( http://www.google.com/ ):

Search Topic Approximate # Titles
Reviewed # Titles of Interest

E. coli + deer 10 1

Adenoviruses + zoonotic 30 3

Hepatitis E+ condition + symptoms 30 2

Rotavirus + illness + symptoms 20 1U.S. Environmental Protection Agency

February 2009 B-4 The following terms were se arched on Google Scholar ( http://scholar.google.com/ ):

Search Topic Approximate # Titles
Reviewed # Titles of Interest

Adenoviruses + zoonotic 10 1

Rotavirus + illness + symptoms 30 3
Giardiasis + swimming 200 5

Giardiasis + surveillance 200 3

Giardia + symptoms 100 5

Giardia + cyst 100 2

Giardia + beach 200 1

Cryptosporidium + hominis 100 1
Cryptosporidium + beach 150 1
Cryptosporidium + incidental ingestion 250 4
Cryptosporidium + review 100 2
Cryptosporidium + exposure factors 150 3

Cryptosporidium + exposure 50 2
Cryptosporidium + symptoms 50 3
Cryptosporidium + risk 50 0
Campylobacter + chronic 50 3
Campylobacter + illness 50 3
Campylobacter + infection 50 4

Campylobacter + symptoms 50 1
Campylobacter + water 15 2
Campylobacter + pathogenesis 5 1

Salmonella + transmission 20 8

Salmonella + transmission in water 11 2

Salmonella + antibiotic resistance 20 3

Salmonella + pathogenesis 3 1
Shigella + outbreak 23 4
Shigellosis 35 4

The following terms were searched on PubMed ( http://www.ncbi.nlm.nih.gov/sites/entrez ):

Search Topic # Titles Reviewed # Titles of Interest

E. coli O157:H7 + survival + environment 204 23

Salmonella + survival + environment 376 4

Shigella + survival + environment 65 10

Campylobacter + survival + environment 57 17

Norwalk Virus + survival + environment 2 Yes

Rotavirus + survival + environment 54 17

Hepatitis E + survival + environment 3 YesU.S. Environmental Protection Agency

February 2009 B-5 The following terms were searched on Scirus ( http://www.scirus.com/ ):

Search Topic Approximate # Titles
Reviewed # Titles of Interest

Helicobacter + environmental survival 30 1

Leptospira + CID 10 1

Leptospira + environmental survival 30 2

Leptosporidiosis + symptoms 30 0

The following terms were searched on the CDC website ( http://www.cdc.gov/ ):

Search Topic Approximate # Titles
Reviewed # Titles of Interest

Legionella + EID 10 1

Leptospira + EID 10 1

Avian influenza + EID 10 1

H5N1 + EID 20 0

SARS + EID 30 2

Hendra virus + EID 20 0

Nipah virus + EID 20 2

Strongyloides + EID 10 1

BSE + EID 10 1

B.4 Experts Contacted

The flowing experts in the field were contact ed directly by email and asked to suggest

references:

Nicholas Ashbolt, USEPA Thomas Atherholt, New Jersey Depa rtment of Environmental Protection Michael Beach, Centers for Disease Control and Prevention Bart Bibler, Florida De partment of Health Alexandria Boehm, Stanfo rd University, California Rebecca Calderon, USEPA Jennifer Clancy, Clancy E nvironmental Consultants Jack Colford, University of California, Berkeley Elizabeth Doyle, USEPA Alfred Dufour, USEPA Lee Dunbar, Connecticut Department of Environmental Protection Lora Fleming, University of Miami School of Medicine and Rosenstiel School of Marine and Atmospheric Sciences, Florida Charles Hagedorn, Virginia Tech Joel Hansel, USEPAU.S. Environmental Protection Agency

February 2009 B-6 Lawrence Honeybourne, Orange County Health Care Agency, Santa Ana, California Donna Francy, U.S. Geological Survey Roger Fujioka, University of Hawaii, Manoa Toni Glymph, Wisconsin Depart ment of Natural Resources Mark Gold, Heal the Bay, California Paul Hunter, University of East Anglia, U.K. Dennis Juranek, Centers for Disease Control and Prevention (retired) David Kay, University of Wales, U.K. Sharon Kluender, Wisconsin State Laboratory of Hygiene Erin Lipp, University of Georgia Graham McBride, National Institute of Wa ter and Atmospheric Research, New Zealand Charles McGee, Orange County Sanitation District, California Samuel Myoda, Delaware Department of Natural Resources Charles Noss, USEPA Robin Oshiro, USEPA James Pendergast, USEPA Mark Pfister, Lake County Health Department, Illinois John Ravenscroft, USEPA Stephen Schaub, USEPA Mark Sobsey, University of North Carolina, Chapel Hill Jeffrey Soller, Soller Environmental, California Michael Tate, Kansas Department of Health and Environment Peter Teunis, RIVM (National Institute of Pub lic Health and the Environment), Netherlands Gary Toranzos, University of Puerto Rico, Rio Piedras Timothy Wade, USEPA John Wathen, USEPA Stephen Weisberg, Southern Californi a Coastal Water Research Project David Whiting, Florida Department of Environmental Protection Richard Zepp, USEPA

B.4 Previously Cited References

The following specific reports were obtained and the titles of the re ferences cited in the reports

were reviewed for relevance:

• NRC. (2004) Indicators fo r Waterborne Pathogens. The National Academies Press

• USEPA. (2007) Report of the Experts Scie ntific Workshop on Criti cal Research Needs for the Development of New or Revised Recreational Water Quality Criteria http://www.epa.gov/waterscience/criteria/recreation

• WHO. (2004) Waterborne Zoonoses. http://www.who.int/wat er_sanitation_health/ diseases/zoonoses.pdf

• References cited by the Natural Resources De fense Council reviewers of the EPA Critical

Path Science Plan

• USEPA Internal draft Adenovirus criteria document

• USEPA Internal draft pathogenic E. coli criteria documentU.S. Environmental Protection Agency

February 2009 B-7 • Boehm et al. (2008) A sea change ahead for recreational water qual ity criteria. (peer review in progress)

In addition, Clancy Environmental Consultants, Inc., ICF International, Soller Environmental, WaltJay Consulting, and EPA’s Health and Ecologica l Criteria Division all maintain extensive

literature databases and reference lists from previously completed projects. All of those in house

resources were also sources of literature.U.S. Environmental Protection Agency

February 2009 C-1 

## A PPENDIX 

## C

I NCIDENTAL 

## I NGESTION OF 

## A MBIENT 

## W ATER 

## D URING


## R ECREATIONAL 

## A CTIVITIES

There is a paucity of data c oncerning rates of incidental i ngestion of surface water during

recreational activities. Most of the availabl e estimates address exposures during swimming in swimming pools, which may not necessarily be repr esentative of typical “incidental” exposures

in ambient waters. Dufour et al. (2006) reviewed early estim ates of swimming-related water ingestion and concluded that inci dental ingestion ranged from 10 to 50 mL per hour. None of the early estimates, however, were based on actu al studies of water ingestion. EPA’s Risk Assessment Guidance for Superfund (USEPA, 1989) recommended a value of 50 mL per hour for ingestion during water recreation, citing an early version of EPA’s Exposure Factors

Handbook (EFH). The latest version of the EFH (USEPA, 1997) contains no recommendation concerning recreational water intake. Hammond et al. (1986), in their assessment of the potential toxicity of swimming pool disinfectants, es timated that a 70-kg adult might ingest “1 to 2 cups” of water, meaning approximately 500 mL. However, this is a “ballpark” estimate and is not supported by observational data.

Allen et al. (1982) estimated water ingestion by competitive swimmers by measuring urinary excretion of isocyanuric acid, an unmetabolized compound used to stabilize chlori ne levels in swimming pools. The average estimated water intake among the five swimmers that were studied was 161 mL per hour. The investigators also determined that (1) essentially all of the ingested isocyanuric acid appeared in urin e within 24 hours, thus negating concern for elimination by other pathways; and (2) derm al absorption of the tracer compound was insignificant compared to the ingestion intake.

Using methods similar to those used by Allen et al. (1982), Dufour et al. (2006) estimated water intake in 12 adults and 41 “nonadults” engaged in less vigorous water re creation at a community swimming pool. Based on the amount s of isocyanurate excreted in urine, they estimated that 45 minutes of water recreation resulted in average water intakes of 37 mL (49 mL/hr) for nonadults and 16 mL (21 mL/hr) for adults. The exposures measured by Dufour et al. (2006) are perhaps more likely to be representative of typical “inc idental” exposures than those of the competitive swimmers measured by Allen et al. (1982), sugge sting the lower values may provide a better basis for estimating inci dental exposures.

A larger follow-up study of 549 participants wa s subsequently conducted at several public and private outdoor swimming pools (Eva ns et al., 2006). Participants were requested to engage in active swimming for between 45 a nd 60 minutes. The overall averag e incidental ingestion rate was 32 mL/hr, with a range of 1 to 280 mL/hr. Adults averaged 24 mL/hr, and children averaged 47 mL/hr. Children (ages not specified) swallowed approximately twice as much water as adults. The follow-up study also showed that male s ingested more than females and that adult men ingested more than adult women.U.S. Environmental Protection Agency

February 2009 C-2 The small number of studies that are available measured water in take in only a few subjects and characterized water ingestion during either ve ry active swimming or poorly defined water

recreational activities. In additi on, the incidental ingestion data th at are available are for “clean” pool water and may not represent incidental ingestion for surface waters, which may provoke stronger avoidance behaviors due to the per ception that surface waters are nonpotable.