EPA 822-R-09-002
REVIEW OF ZOONOTIC PATHOGENS
IN AMBIENT WATERS
U.S. Environmental Protection Agency
Office of Water
Health and Ecological Criteria Division
February 2009
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U.S. Environmental Protection Agency
DISCLAIMER
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.
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U.S. Environmental Protection Agency
ACKNOWLEDGMENTS
Questions concerning this document or its application 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 contributed to the development of the report:
Lead writer: Audrey Ichida
Leiran Biton
Alexis Castrovinci
Jennifer Clancy
Mary Clark
Elizabeth Dederick
Kelly Hammerle
Walter Jakubowski
Margaret McVey
Michelle Moser
Jeffery Rosen
Tina Rouse
Jennifer Welham
Kerry Williams
ICF International
ICF International
ICF International
Clancy Environmental Consultants
ICF International
ICF International
ICF International
WaltJay Consulting
ICF International
ICF International
Clancy Environmental Consultants
ICF International
ICF International
ICF International
February 2009
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TABLE OF CONTENTS
ACKNOWLEDGMENTS iii
TABLES AND FIGURES vii
ACRONYMS ix
EXECUTIVE SUMMARY 1
I. BACKGROUND AND INTRODUCTION 5
I.I Background: Context and Purpose 5
1.2 Introduction 6
II. KEY WATERBORNE ZOONOTIC PATHOGENS OF CONCERN 8
II. 1 Bacteria 8
II. 1.1 Escherichia coli 8
II. 1.2 Campylobacter 12
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. PATHOGEN INTERACTIONS WITH THEIR ENVIRONMENT 37
III.l Water Environment Affects Pathogen Survivability andPhenotype 37
III.
III.
III.
III.
III.
III.
III.
.1 Pathogenic E. coli Survival in the Environment 38
.2 Campylobacter Survival in the Environment 39
.3 Salmonella Survival in the Environment 39
.4 Leptospira Survival in the Environment 39
.5 Cryptosporidium Survival in the Environment 40
.6 Giardia Survival in the Environment 43
.7 Virus Survival in the Environment 44
III.2 Host Animals Can Influence Pathogen Characteristics and Mechanisms of Rapid Evolution 44
IV. SUMMARY 49
V. REFERENCES 50
APPENDIX A: WATERBORNE PATHOGENS A-l
APPENDIX B: LITERATURE SEARCH STRATEGY AND RESULTS B-l
APPENDIX C: INCIDENTAL INGESTION OF AMBIENT WATER DURING RECREATIONAL ACTIVITIES C-l
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TABLES AND FIGURES
Table II. 1.1-1. Outbreaks of E. coli Associated with Recreational Waters in the United States 12
Table II.2.1-1. Cryptosporidium Species 20
Table II.2.1-2. Outbreaks of Cryptosporidiosis Associated with Recreational Waters in the United States 27
Table II.2.2-1. Giardia Taxonomy 28
Table II.2.2-2. Giardia Infection Occurrence in U.S. Populations 32
Table II.2.2-3. Outbreaks of Giardiasis Associated with Recreational Waters in the United States 34
Table III.l.1-1. Survival of E. coli 0157 :H7 in Ambient Waters 38
Figure II.2.1-1. Reported Cryptosporidium infections in the United States, 1999 to 2005 26
Figure II.2.2-1. Reported Giardia cases in the 50 states plus Washington DC, 1992 to 2005 32
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U.S. Environmental Protection Agency
AEC
AIDS
AWQC
BEACH Act
CDC
CPV
CWA
DAEC
DEC
EAggEC
EEC
EHEC
EPA
EPEC
ETEC
GI
GLV
GBS
HC
HEV
HUS
IPSID
MALT
NA
PCR
POTW
ppt
RFLP
SPE
STEC
U.S.
U.K.
USDA
UV
VTEC
WHO
ACRONYMS
attaching E. coli
Acquired Immune Deficiency Syndrome
ambient water quality criteria
Beaches Environmental Assessment and Coastal Health Act of 2000
U.S. Centers for Disease Control and Prevention
C. parvum virus
Clean Water Act
diffuse adherent E. coli
diarrheagenic E. coli
enteroaggregative E. coli
effacing E. coli
enterohemorrhagic E. coli
U.S. Environmental Protection Agency
enteropathogenic E. coli
enterotoxigenic E. coli
gastrointestinal
Giardia lamblia virus
Guillain-Barre Syndrome
hemorrhagic colitis
hepatitis E virus
hemolytic uremic syndrome
immunoproliferative small intestinal disease
mucosa-associated lymphoid tissue
not available or not applicable
polymerase chain reaction
Publicly Owned Treatment Works
parts per thousand (salinity)
restriction fragment length polymorphism
serial passage experiment
Shiga toxin-producing E. coli
United States
United Kingdom
U.S. Department of Agriculture
ultraviolet (light)
verocytotoxin-producing E. coli
World Health Organization (United Nations)
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EXECUTIVE SUMMARY
Introduction
The overall goal of the current Clean Water Act (CWA) §304(a) ambient water quality criteria
(AWQC) for bacteria in the United States is to provide public health protection from
gastrointestinal (GI) illness (gastroenteritis) associated with 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 fecal matter can be a major source
of pathogens in ambient water and 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 potential 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. coif). This approach assumes that animal fecal material is as hazardous as
human fecal material. There is limited evidence, however, that recreational water contaminated
with animal fecal material is less risky to swimmers than recreational 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 sources to agricultural inputs. The animal species of
most interest are the warm-blooded animals (mammals and birds) whose fecal material is
detected by current indicators.
The purpose of this white paper is to provide a summary of information on waterborne zoonotic
pathogens that come primarily from warm-blooded animals, and which can be used to
conceptualize potential risks from warm-blooded animal feces in ambient (untreated)
recreational waters.
Approach
Seventy pathogens from warm-blooded animals were evaluated for their potential to be both
waterborne and zoonotic. Twenty of the 70 pathogens 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 source to human is through a water related
route.
4. The pathogen causes infection or illness in humans.
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Six of the 20 waterborne, zoonotic pathogens from warm-blooded animals were selected for
further discussion based on their relevance 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 excluded from analysis because they are not
zoonotic. Excluded pathogens include bacteria that are generally found in the environment, free-
living 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 well-
documented waterborne transmission (i.e., primarily 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 influence pathogen survival, infectivity, and virulence
include the following: ultraviolet (UV) light (duration, intensity), rainfall, runoff, dispersal,
suspended solids, turbidity, nutrients, organic content, organic foams, water quality, biological
community in water column, water depth, stratification, mixing (e.g., wind and waves), presence
of aquatic plants, biofilms, and predation.
There is evidence that zoonotic pathogens may change in infectivity, virulence, and the severity
of disease caused in humans depending on their previous host environment. There is also
evidence that some of these host-factor changes can influence subsequent infection cycles in
exposed hosts. The key mechanisms of phenotypic 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 zoonosis because many human pathogenic strains
occur in livestock and wildlife feces 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 more at risk than healthy adults
to suffer more severe outcomes. Between 1991 and 2004, 14 outbreaks of E. correlated 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 has been demonstrated to survive at least 500 days
at -20 °C in frozen soil.
Campylobacter
Campylobacter is a well-known foodborne bacterial 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-Barre syndrome, which is a serious nervous system affliction.
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Reactive arthritis is also a possible chronic sequela of Campylobacter infection. Campylobacter
has been shown to survive in aquatic environments with low temperatures (4°C) up to 4 months.
Salmonella
Salmonella is also a well-known foodborne bacterial pathogen that is associated with poultry and
livestock. There are also wildlife 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 pain, nausea, chills, and fever. Between 1991
and 2002, 3 waterborne outbreaks were attributed to nontyphoid Salmonella; Salmonella were
the etiologic agent in 0.9 percent of 259 recreational waterborne outbreaks 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 pathogen for most of the world. Because
warmer climates favor its survival in the environment, the highest incidence 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 infected animal. There are numerous symptoms
associated with leptospirosis, but the more severe form is known as Weil's disease. In addition,
acute infection during pregnancy has been reported to cause abortion and fetal death. From 1971
to 2000, 16 percent of recreational 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 risks 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 patterns, it is likely that
different sources contribute different proportions to the total contamination load at different
times during the year, with the relative contributions depending on a wide range of watershed
characteristics. Cryptosporidiosis is primarily characterized by GI symptoms such as profuse,
watery diarrhea. Immunocompromised individuals generally experience chronic gastroenteritis,
which may last as long as the immune impairment. The largest known outbreak of
Cryptosporidiosis occurred in 1993 in Milwaukee, Wisconsin and infected 403,000 individuals.
Animal fecal contamination of drinking water was indicated in the outbreak. In the United
States, from 1991 to 2004, 6 outbreaks of Cryptosporidiosis 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 oocysts (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 and 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 days has been demonstrated in several studies.
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Giardia
Giardia is also a well-known waterborne 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 major risk factor. There is a wide
spectrum of symptoms associated with giardiasis that ranges from asymptomatic infection and
acute self-limiting diarrhea to persistent chronic 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 States, from 1991 to 2004, 7 reported outbreaks of
giardiasis were associated with untreated 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 recreational illnesses are probably due to nonzoonotic
human viruses, which typically cause short-term gastroenteritis, the waterborne zoonotic
pathogens discussed in this report have the potential to cause serious health effectsespecially
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.
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I. BACKGROUND AND INTRODUCTION
1.1 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 have been significant scientific and technical
advances, particularly in the areas of molecular biology, microbiology, 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 information to provide the
scientific and technical foundation for the development of new or revised criteria. The
enactment of the Beaches Environmental Assessment and Coastal Health (BEACH) Act of 2000
(which amended the CWA) required EPA to conduct 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 also has engaged a range of stakeholders
representing the general public, public interest groups, state and local governments, industry, and
municipal wastewater treatment professionals. In March 2007, EPA convened a group of 43
national and international technical, 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 recreational water quality research and criteria
implementation. Among the input from the individuals attending the workshop were suggestions
for incorporating the ability to differentiate sources of fecal contamination and to determine the
relative human health risk from these sources 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 Revised Recreational Water Quality Criteria. One of
the key questions posed in the science plan asks: what is the risk to human health from
swimming in water contaminated with human fecal 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 are host-specific (i.e.,
human enteric viruses), while other human pathogens are found in and can be shed by both
humans and other animals. Moreover, while all enteric pathogens of humans are infectious to
other humans, only a subset of the enteric pathogens 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 quality criteria for microbes treat human health
risks from the various sources of fecal contamination as equivalent (USEPA, 1986). These
criteria are based on the risk of illness from swimming in waters influenced by sewage treatment
plants effluents. Health risks from other sources (e.g., poorly-treated or untreated human waste,
nonhuman sources of fecal contamination, mixed sources such as urban stormwater runoff) were
not well understood at the time the 1986 criteria were developed, and EPA's approach was to be
protective of human health regardless of the source. EPA recognizes, however, that the health
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risk from sources other than sewage treatment plants may be different and that the scientific
advances over the intervening years may now allow the Agency to better characterize the relative
risks to human health from these various sources 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 including 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-blooded animals, and which can be used to
conceptualize potential risks to humans from warm-blooded animal feces in recreational waters.
1.2 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 fecal contamination in recreational waters (USEPA,
1986). Because widespread monitoring of recreational waters directly for all disease-causing
microorganisms (especially pathogenic bacteria, viruses, and protozoa) remains infeasible, public
health and environmental protection agencies have relied on the detection of fecal indicator
organisms, which comprise a few groups of nonpathogenic fecal bacteria and some viruses, to
indicate the presence and magnitude of fecal material. This approach assumes that waterborne
pathogens co-occur with the fecal material. "More specifically, fecal indicator bacteria provide
an estimation of the amount of feces, and indirectly, 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 environmental sources of these microorganisms (i.e.,
nonenteric sources). However, this presumption is not entirely valid; fecal indicator organisms
have been demonstrated to have natural reservoirs 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 waters and sand [Whitman et al., 2006]).
Historically, EPA has recommended that recreational 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-derived (zoonotic) human pathogens in fecally
contaminated waters are as hazardous as their human-derived counterparts (Schaub, 2004). This
presumption is supported by current research that confirms that there are many waterborne
zoonotic bacteria and protozoa common to both humans and animals, especially mammals
(WHO, 2004). Research also suggests, however, that there may be some attenuation of
infectivity, virulence, and disease severity to humans from animal-derived human pathogens (see
Section III.2). In addition, there are many pathogens that could be zoonotic, fecal, and/or
waterborne, but these routes of transmission have not yet been conclusively demonstrated.
Given the scientific advancements in pathogen characterization since EPA's 1986 AWQC were
released, it is appropriate to examine the broadest array of currently known and suspected
waterborne, fecal, and zoonotic pathogens and their human health impacts. These include
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primarily bacteria, viruses, protozoa, and helminths. There are many zoonotic pathogens and
many waterborne pathogens; however, there is a more limited subset of pathogens that are both.
For this report, the following attributes were used to select the waterborne, zoonotic pathogens of
concern (partially adapted from Bolin et al., 2004a):
1. The pathogen must spend 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 source to human must be through a water
related route. There are zoonotic pathogens for which waterborne exposure has not been
detected as a significant route of cross-species transmission. This does not exclude the
possibility that these zoonotic pathogens could be transmitted via water.
4. The pathogen must cause infection or illness in humans. There are animal pathogens that
have waterborne transmission between animals yet are not known to cause illness in
humans.
There are many waterborne zoonotic pathogens that exhibit all four attributes listed above. See
Appendix A, Table A-l, 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 two main sections. Section II characterizes six key
groups of zoonotic pathogens for which there is evidence 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.
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II. KEY WATERBORNE ZOONOTIC PATHOGENS OF CONCERN
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 discussed 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 recreational 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 ingestion is provided in Appendix C.
II. 1 Bacteria
II. 1.1 Escherichia coli
E. coli is an important waterborne zoonosis because human pathogenic strains occur in livestock
and wildlife feces and can survive in ambient water. In addition, the potential illnesses caused
by pathogenic E. coli can be severe or fatal, especially in immuncompromised persons and
subpopulations. Pathogenic E. coli is also a well-documented 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 flora present in the colons and feces
of all warm-blooded animals. However, multiple disease causing 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 the "poster child" for pathogenic E.
coli and is the serotype most extensively 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 strains has not yet stabilized in the literature,
M01bak and Scheutz (2004) provided the following helpful summary of the current
nomenclature:
Diarrheagenic E. coli (DEC) - includes all the strains on this list;
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Verocytotoxin (Shiga toxin)-producing E. coli (VTEC or STEC) - E. coll that produce
verocytotoxin (Shiga toxin) VT1 and/or VT2;
o Enterohemorrhagic E. coli (EHEC) - originally defined 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 high levels of verocytotoxin;
o Enteropathogenic E. coli (EPEC) - Subtype of A/EEC, 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) provide a comprehensive, albeit dated, review of the DEC.
E. coli Zoonotic Potential
E. coli is part of the normal intestinal flora of humans 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 Doyle, 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; Sargeant et al., 1999), horses (Chalmers et al., 1997), and dogs
(Hammermueller et al., 1995).
Ruminants, and cattle in particular, are considered one of the most important animal sources of
E. coli O157:H7 and other VTECs. All of the VTECs including EHEC are capable of causing
severe disease in humans, are typically shed by healthy cattle and other species, and have
documented cases of transmission via humans and water (M01bak 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 relationship between 3,001 cases of VTEC and the livestock
density in rural areas of Ontario, Canada. Their research indicated that cattle density had a
positive and significant association with the number of reported cases of VTEC in humans,
suggesting that living near cattle farms may increase 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 resulted in outbreaks. Schoeni and Doyle (1994)
inoculated 1-day-old chicks with strains of serotype O157:H7. The chicks shed serotype
O157:H7 up to 11 months after inoculation, and O157:H7 was subsequently recovered from their
egg shells, but not from the yolks or whites. In an outbreak in northern Italy, the source of
exposure was believed to be chickens in 15 cases of hemolytic uremic syndrome (HUS; see more
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below) that were caused by serotype O157 and other 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, respectively. In a Russian study, O157:H7 was 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;
M01bak 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; McCarthy 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., 1992; Yarze and Chase, 2000). Most studies examining
contamination from recreation immersion have suggested that ingestion was 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; M01bak and Scheutz, 2004). Incubation periods
vary and can be as short as 8 hours for EAEC infections (Nataro et al., 1995), 14 to 50 hours for
ETEC (Dupont et al., 1971), and 3 to 4 days for EHEC, 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 sometimes fever (Ostroff et al., 1989; Swerdlow
et al., 1992). The duration of these symptoms is 4 to 10 days (CDC, 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 colitis (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);
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ETEC - Acute watery diarrhea;
A/EEC - Acute or persistent diarrhea;
o EPEC - Acute or persistent 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 seizures and strokes (Brotman, 1995).
Brooks et al. (2005) conducted 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 chronic kidney failure, blindness, or
partial paralysis (Tarr, 1995). Approximately 33 percent of persons that contract HUS have
abnormal kidney function for several years, sometimes 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 exposure, 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 such 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; other complications may include cholecystitis, colonic perforation,
intusssception, pancreatitis, posthemolytic biliary lithiasis, post-infection colonic stricture, rectal
prolapse, appendicitis, hepatitis, 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 rates. For example, the 1993 fast-food E. coli O157:H7 outbreak in
Washington, California, Idaho, and 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 outbreaks in the United States between 1982 and 1990 and calculated
a mortality rate of 1.9 percent 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-0157 Shiga-like toxin serotypes cause approximately 37,000
illnesses per year (Mead et al., 1999).* Craun et al. (2004a) estimated that E. coli was the
etiological agent for 30 percent of the outbreaks 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 waters have been reported to the CDC (Rangel et
1 The CDC is currently updating these numbers, but the newest values have not yet been released.
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al., 2005). Fourteen of the outbreaks occurred as a result of exposure to contaminated lakes or
ponds, while seven occurred from exposure to contaminated 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 considered 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 Guillain-Barre syndrome (GBS) (Nachamkin, 2002).
Table II.l.l-l. Outbreaks of E. coli Associated with Recreational Waters in the United States
Year
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
Number of Cases
80
0
0
166
28
24
8
31
61
0
49
9
0
0
Number of Outbreaks
1
0
0
1
4
2
1
2
5
0
2
1
0
0
Source of Outbreak(s)
Lake
NA
NA
Lake
Lakes
1 pool, 1 lake
Lake
1 pool, 1 lake
1 pool, 3 lakes, 1 ditch water
NA
Lakes
Pool
NA
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).
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Campylobacter Zoonotic Potential
Because most of the published literature focuses 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 indicates 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 all streams near dairy farms in New Zealand contain Campylobacter, and
despite generally low concentrations, these streams may help cycle Campylobacter in farm
animals and indirectly contribute 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 through 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 (Jones, 2001). The main source of campy lob acters
for bathing waters is typically assumed to be sewage effluent, though 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 immersion activities such as swimming. 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 outbreaks occurring in the United States from 1971 to
2000 and associated only with 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 foods (e.g., raw chicken) and direct
person-to-person transmission 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 abdominal cramps (Allos, 2001; Flicker, 2006a).
Infections of Campylobacter are symptomatically indistinguishable from those of other bacterial
pathogens such as Salmonella (Nachamkin, 2002). Diarrheal symptoms 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 experience relapses leading to several weeks of
symptoms (Allos, 2001).
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Some complications may result from Campylobacter infection including cholecystitis,
pancreatitis, peritonitis, and massive GI hemorrhage. Some immunocompromised individuals
experience bacteremia as a result of campylobacteriosis, 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, 2006a). GBS is an acute demyelinating disease of the peripheral
nervous system that affects 3,000 to 6,000 people in the United States 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 (approximately 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, 2002). Recent evidence has associated C. jejuni
with a rare form of mucosa-associated lymphoid tissue (MALT) lymphoma called
immunoproliferative small intestinal disease (IPSID); campylobacteriosis has not yet been shown
as causative agent of IPSID (Poly and Guerry, 2008).
In 2000, a municipal water supply in Walkerton 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 approximately 4 years after infection 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 are 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, Britain experiences approximately 5 times this infection rate
(103.7 cases per 100,000 people), though reporting rates may not be comparable (Gillespie et al.,
2002). C. jejuni infection is one of the most common causes of gastroenteritis across the world
and is frequently responsible for diarrheal 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 waterborne 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 illnesses (Cohen, 1986; Lightfoot, 2004;
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Ohl, 2001). There are more than 2,500 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 serovars in the group are closely
related to each other by somatic and flagellar antigens, 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-sensitivity testing, biochemical reactions, phage-
typing, 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 serotypes found in the United States
(CDC, 2006). The other serotypes have much smaller incidences in the United States; S.
enteritidis accounted for 10 percent 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-specific, restricted to a single host species and rarely
causing disease in other species (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 poultry, cattle, and pigs. The serotypes with a
wide range of animal hosts can also infect humans, usually via food consumption, and cause self-
limited gastroenteritis in humans (WHO, 2004). S. gallinarum and S. pullorum are almost
exclusively pathogens of poultry (Cohen, 1986). Human pathogens S. heidelberg and S. litchfield
have primarily avian and 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 Meckes, 2006). The CDC estimates that 74,000 cases of
salmonellosis per year are associated with exposure to reptiles or amphibians (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 water-storage tower that allowed access 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.
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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 reported worldwide (Cohen, 1986; Lightfoot, 2004).
Researchers have observed a reduction 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, acid-tolerance response 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 sewage, and effluents from ineffective sewage
treatment plants (Geldreich, 1996). Farming operations with cattle and poultry result in large
quantities of fecal products in relatively small areas due to the dense population of animals.
Thus, if the animal waste is not discharged into 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), meningitis, and osteomyelitis (Cohen, 1986). Enteric
fever and gastroenteritis are the key clinical 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 and 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 extraintestinal 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 symptoms of enteric fever include diarrhea, abdominal pain, fever,
and sometimes a maculopapular rash. The pathological sign of enteric fever is mononuclear cell
infiltration and hypertrophy of the reticuloendothelial system (Ohl, 2001).
Salmonellosis is caused by ingestion 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 105 to 1010 organisms (Lightfoot, 2004). The infectious dose varies
depending on the age and health of the person, strain differences, and the vector. Most
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salmonellosis cases are self-limiting, and the affected persons recover 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 include diarrhea, abdominal pain, nausea, chills,
fever, and prostration with the duration of illness 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 percent hospitalization rate, and an estimated 0.8
percent fatality rate (USDA, 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. Antibiotics 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 approximately 1.4 million foodborne-related
illnesses, 15,600 foodborne illness-related hospitalizations, 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 infections 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 recreational 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 jim long and 0.1 jim wide. Leptospira
occurs worldwide and has become an important recreational zoonosis due to its prolonged
survival in water (Pond, 2005). The genotypic classification of Leptospira into two species (L.
interrogans and L. biflexd) 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. alexanderf) (Levett, 2001). Of the 28 serovars, several occur in more than one
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genomospecies. Leptospirosis is probably the most widespread zoonosis in the world (Levett,
2001; Meites et al., 2004). Zoonotic reservoirs include livestock (pigs and cattle), domestic pets
(dogs), and wild or feral animals (rats, voles, and mice) (Kanarat, 2004; Levett, 2001).
Complete genome sequences of L. interrogcms serovars Copenhagen! and Lai reveal that despite
overall genetic similarity there are significant structural differences in their genomes
(Nascimento et al., 2004). Nascimento and colleagues analyzed the genomic sequences to gain
insight into genes that influence motility, chemotaxis, 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). Workers 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, 2004; Meites et al., 2004). Outbreaks are often
associated with unusual rainfall events or flooding (Bolin et al., 2004b).
Leptospira Illness Symptoms
Leptospirosis was first described 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 the pathogen is detected mainly in the blood and
cerebrospinal fluid, whereas the convalescent stage corresponds with the appearance of
antibodies and the presence of pathogens 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 attention (Levett, 2001). Icteric leptospirosis is a
much more severe disease than anicteric 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: jaundice, 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 disturbance 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 requires reporting (Katz, 2001; Levett, 2001).
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Leptospirosis Incidence
Levett (2001) summarized information for 28 waterborne outbreaks of leptospirosis worldwide,
22 of which were associated with swimming, 1 with kayaking, and 1 with rafting. Within the
United States, the highest incidence of leptospirosis is found in Hawaii (Katz, 2001). Between
1971 and 2000, 16 percent of recreational waterborne 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 patients 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 parasite that infects the microvillous region of epithelial
cells in the digestive and respiratory 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) until ingested by a new host. In the new host, the life cycle starts
again, and multiplication occurs using the biological resources of the host (WHO, 2006).
Cryptosporidium exists in the natural environment in the oocyst form and which are resistant to
conventional drinking water treatment 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 (Payer and Ungar, 1986; Payer et al., 1997). Oocysts are immediately
infectious and may remain in the environment for very long periods without losing their
infectivity. Oocysts are resistant to environmental 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 sporozoites exit each oocyst (excyst) and may form an infection in the
epithelial cells of the small intestine of the host (USEPA, 200la). 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 infectionsC. hominis and C. parvumare spherical with a
diameter of 4 to 6 |im. 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).
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Cryptosporidium is part of phylum Apicomplexa, family Cryptosporidiidae, and has been
classified as a member of the group of eimeriid coccidiana 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 potential new species. Payer (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 demonstrated that a low dose of C. parvum (e.g., 10 oocysts) is
sufficient to cause infection in healthy adults although some strains may be more infectious than
others (Chappell et al., 1999; DuPont et al., 1995; Okhuysen et al., 2002). The relationship
Table II.2.1-1. Cryptosporidium Species
Cryptosporidium Species
C. andersoni
C. baileyi
C. bovis
C. canis
C. felis
C. galli
C. hominis
C. meleagridis
C. molnari
C. muris
C. nasorum
C. parvum
C. scopthalmi
C. serpentis
C. suis
C. varanii
C. wrairi
Initially Described Host Species
Bos taurus (domestic cattle)
Gallus gallus (domestic chicken)
Bos taurus (domestic cattle)
Canis familiaris (dogs)
Fe/;s catis (domestic cat)
Gallus gallus (domestic chicken)
Homo sapiens (humans, formerly C. parvum genotype 1)
Meleagris gallopavo (turkey)
Dicentrarchus labrax (fish)
Mus musculus (house mouse)
Naso lituratus (fish)
Mus musculus (house mouse) (formerly genotype 2)
Scopthalmi maximus (turbot)
Elaphe guttata (corn snake)
E. subocularis (rat snake)
Sanzinia madagascarensus (Madagascar boa)
Sus scrofa (pig)
Varanus prasinus (emerald monitor lizard)
Cavia porcellus (guinea pig)
Source: Adapted from
2007; Morgan-
Caccio, 2005; Payer, 2003, 2004a; Payer et al., 1997, 2000; Payer and Xiao,
Ryan et al., 2002; Ryan et al., 2003; and Xiao and Ryan, 2004.
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between the number of oocysts humans are exposed to and the probability of infection is
discussed in detail in the subsequent section. 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 heterogeneity of the species may influence the risk
posed to public health. Furthermore, distinct transmission cycles are evident among different
genotypes of C. parvum. Multiple genotypes have been shown to circulate among different host
species, and mixed infections with genotypically distinct populations have been reported
(Widmer et al., 1998).
Cryptosporidium Zoonotic Potential
Human cases of cryptosporidiosis typically have been associated with different kinds of animal
contact, which has led to the widespread 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 mammalian species (Payer, 2004a) as well as numerous
reptiles, amphibians, birds, and fish (O'Donoghue, 1995).
Several lines of evidence indicate that livestock, primarily cattle and sheep, are a major source of
Cryptosporidium contamination of drinking water sources. These include the detection of C.
parvum (presumably from animals) in many source waters and elevated levels of
Cryptosporidium in watersheds with extensive agricultural activity (Payer, 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 occurrence of oocysts in source waters (Payer, 2004a).
Based on data from the western United States, depending on climate and feedlot management
systems, the average animal in a cattle feedlot 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 calves shedding oocysts.
Nine percent of farm-associated streams contained C. parvum. Therefore, cattle represent a
significant reservoir and potential 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 percent of domestic cryptosporidiosis 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 feral
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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 contributor 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 water can be strongly associated with storm
events or other weather variables (Payer, 2004a; Naumova et al., 2005; USEPA, 2005a; WHO,
2006). In watersheds with diverse land-use patterns, it is likely that different sources contribute
different proportions to the total contamination load at different times during the year, with the
relative contributions depending on a wide range of watershed 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 facilities, combined sewer system overflows, or human recreational uses
(e.g., swimming, camping, and hiking). Due to the large numbers of oocysts excreted by
infected individuals (Okhuysen et al., 1999) and oocyst resistance to many conventional
wastewater treatment processes, even small releases 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 effluents 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 United States and Canada. They found that the
two watersheds with the highest proportion of agricultural land use had the highest average
Cryptosporidium levels2 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 (Payer, 2004a) and that the oocyst counts in feces of young
infected animals ranges from 106 to 108 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 primary effluents, 58 percent of secondary effluents
and 19 percent of tertiary effluents in the 289 analyzed samples. Zhou et al. (2003) analyzed 179
wastewater treatment plant effluent samples from Milwaukee, Wisconsin using polymerase chain
reaction (PCR) restriction fragment length polymorphism (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) was detected more frequently
than C. parvum (2.8 percent). This finding suggests that the distribution of Cryptosporidium in
2 These results were obtained using EPA Method 1623 (USEPA, 200 Ib), for which the authors reported an
average recovery rate of 72±22 percent.
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Milwaukee's population was not heavily influenced 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 microorganisms 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 contact recreation such as wading,
canoeing, motor boating, and fishing, in which ingestion is unlikely due to lack of direct water
contact with the ears, eyes, mouth, or nose, is not considered to result in significant exposure
(USEPA, 2002). A recent pilot study of anglers 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 health effects data or clinical 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 Milwaukee outbreak, Eisenberg et al.
(2005) suggested that 10 percent (95 percent confidence interval: 6 to 21 percent) 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 indirect transmission through contact with
contaminated objects, can be a significant route of infection, especially where human population
densities are high or personal contact is frequent (USEPA, 200la). Direct transmission is
affected by behavioral factors (e.g., frequent travel) and ethnic and dietary differences (USEPA,
200la). Using data from the Milwaukee outbreak, 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, however, 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 not distinguishable from those caused by other
common enteric pathogens. Other symptoms reported by individuals afflicted with
Cryptosporidiosis include dehydration, fever, anorexia, weight loss, weakness, abdominal
cramps, vomiting, lethargy, general malaise, and progressive loss of overall condition (Hunter et
al., 2004). The incubation period (time from ingestion 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 may still shed oocysts, however. Asymptomatic
carriage, as determined by stool surveys, generally occurs at very low rates (less than 1 percent)
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in industrialized countries (Current and Garcia, 1991), though higher rates have been reported in
day care centers. Routine bile endoscopy suggests 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 (between 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 intestine does not necessarily indicate 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. Symptoms may be more severe in
immunocompromised persons (Carey et al., 2004; Frisby et al., 1997). Such persons include
those with AIDS, certain cancer patients undergoing chemotherapy, organ transplant recipients
treated with drugs that suppress 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. Besides the immunocompromised, children and the elderly may also be
at higher risk from Cryptosporidium than the general population; however, specific data are not
currently available to document the degree to which these individuals are subject to elevated risk.
However, previous exposure to Cryptosporidium has been shown to confer some amount of
immunity (Chappell et al., 1999).
Symptoms of cryptosporidiosis typically last from several days to 2 weeks although, in a small
percentage of cases, the symptoms may persist for months or longer. Individuals with either
compromised or healthy immune systems may experience 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 additional episodes in 40 to 70 percent of patients.
Shedding of oocysts may continue after the 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 undergoing chemotherapy for treatment of neoplasms, persons
undergoing immune suppression treatment to prevent rejection of skin or organ transplants,
malnourished individuals, persons with concurrent infectious diseases (e.g., measles), the elderly,
and persons with AIDS. Chronic illness may manifest itself as a series of intermittent episodes
or may be persistent. Individuals with CD4+ cell counts (a key measure of the health of the
immune system) less than 100 cells per mm3 of blood are at increased risk of illness from
Cryptosporidium, while individuals with less than 50 cells per mm3 are at the greatest risk for
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severe disease and prolonged carriage of Cryptosporidium (Hunter and Nichols, 2002; Roefer et
al., 1996).
As noted above, chronic sequelae in immunocompetent patients also have been documented.
After resolution of the acute phase in the 2 months following their initial diagnosis, 40.9 percent
of patients in one case study reported recurrence of intestinal symptoms (includes both C.
hominis and C. parvum) (Hunter et al., 2004). In addition, in individuals infected with C.
hominis, other sequelae such as joint 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 other sequelae noted previously.
Cryptosporidiosis Incidence
Limited information is available on the endemic incidence of Cryptosporidiosis in the United
States. Mead et al. (1999) estimated that there 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 colleagues also estimated that of these 300,000 cases, only about
10 percent are attributable to foodborne transmission, with the remainder due to the consumption
of contaminated water (from drinking or recreational exposure) or person-to-person contact.
Mead et al. (1999) estimated that there are approximately 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 implemented routine reporting of Cryptosporidium among ADDS
patients, only 13 cases of Cryptosporidiosis had been documented (Ungar, 1990). Subsequently,
between 1982 and 1997, more than 1,000 cases of the disease were reported worldwide (Payer et
al., 1997). Cases reported by CDC between 1999 and 2005 (for all 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 several 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 reported 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 total incidence of domestic cases of Cryptosporidium infection
exceeds 300,000 each year.
3 Mead et al. (1999) based the estimates on reported cases and estimates for degree of under reporting. For
example, in the 1993 Cryptosporidiosis outbreak in Milwaukee, Wisconsin, medical care was sought in only 12
percent of cases (Corso et al., 2003).
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Cryptosporidium Cases in the United States
9000 1999-2005
2000
2001
2002
Year
2003 2004 2005
Data sources: CDC 2005, 2007
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 protozoan 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 or G. 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 binary fission, attach to the
brush border of the small intestinal epithelium, detach, then become rounded and form a cyst
wall. The environmentally resistant cyst is excreted with the feces, where it moves passively
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Table II.2.1-2. Outbreaks of Cryptosporidiosis Associated with Recreational Waters in the
United States
Year
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
Number of Cases
0
526
174
519
5,487
3,025
369
169
64
1,368
538
936
702
567
Number of Outbreaks
0
2
4
2
3
3
1
8
4
13
4
7
5
7
Source of Outbreak(s)
NA
Waterslide and wave pool
Pools
1 pool, 1 lake
1 pool, 2 waterparks
2 pools, 1 lake
Fountain
7 pools, 1 lake
3 pools, 1 fountain
12 pools, 1 lake
3 pools, 1 hot spring
6 pools, 1 lake
4 pools,* 1 lake
Pools
* 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).
through the environment and may be ingested by another host organism. Inside the digestive
track of a new host, active cysts release trophozoites, 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 days (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., Assemblages A and B) act as zoonotic assemblages that can infect most
species of mammal, while others (i.e., Assemblages 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 infection, however, remains unclear. Assemblages C and
D seem to primarily infect dogs, while Assemblage E infects livestock, and Assemblage F infects
only cats (Appelbee et al., 2005).
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Table II.2.2-1. Giardia Taxonomy
Species
Giardia lamblia
(syn. G. duodenalis,
G. intestinalis)
G. lamblia
G. lamblia
G. lamblia
G. lamblia
G. lamblia
G. lamblia
G. lamblia
G. lamblia
G. muris
G. microti
G. psittaci
G. ardeae
G. agilis
Assemblage
A1
A2
B (G. enterica*)
C and D
(G. canis*)
E (G. bovis*)
F (G. caff*)
G (G. simoni*)
Novel I
Novel II
Host(s)
Human, beaver, cat, lemur, sheep, calf, dog, fox, chinchilla,
alpaca, horse, pig, cow
Human, beaver
Human, beaver, guinea pig, dog, monkey, horse
(BIV)
Dog, coyote, mouse
Cow, sheep, alpaca, goat, pig
Cat
Domestic rat
Marsupial (Quenda - bandicoot, mouse, sheep)
Marsupial II (Tasmanian devil)
Rodents (mice)
Vole and muskrat
Birds (budgerigars)
Birds (heron and ibis)
Amphibians (frogs)
* Denotes recently proposed new species names (Hunter and Thompson, 2005; Thompson and Monis,
2004).
Adapted from: Adam, 2001; Appelbee et al., 2005; Hamnes et al., 2007; Olson et al., 2004; and Traub 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 animal host species suspected of
being a significant zoonotic source of human giardiasis by waterborne transmission, the evidence
presently available suggests that the beaver (Dykes et al., 1980) 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 been 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 transmitted 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 implicated
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in contaminating water sources with Giardia, humans are responsible for the majority of the
contaminations. Hunter and Thompson (2005) examined 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 indirectly through water sources. Both Traub et al. (2004) and
Inpankaew (2007) have shown that, in some communities with inadequate sanitation, zoonotic
transfer is evident between humans and dogs; however, it is unclear which species is the primary
reservoir.
Thompson (2007) suggested that data gaps regarding zoonotic transfer for Giardia can be filled
using molecular epidemiological studies. Molecular genotyping of parasite isolates from
susceptible hosts in localized foci of transmission or longitudinal 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 causes 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 sanitation. The main route of exposure to Giardia from
recreational immersion in water is from incidental ingestion of water during full immersion
activities such as swimming.
Although inhalation of aerosolized water that contains cysts is theoretically possible, cysts are
not known to be infectious in lung tissue. Dermal 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 effects including acute symptoms 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 diarrhea to persistent chronic diarrhea, which
sometimes fails to respond to treatment.
Giardia produces a broad spectrum of GI symptoms including one or more of the following
symptoms: diarrhea, bloating, weight loss, malabsorption, steatorrhea (fatty stool), pale greasy
and malodorous stools, flatulence, 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 infection (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 giardiasis appears to be infrequent, but when it occurs, may persist
for years (USEPA, 1998). Case reports also indicate 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 individuals and several months for
others. Most infections resolve spontaneously, and the acute stage lasts from 1 to 4 weeks
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(USEPA, 1998), but individuals with compromised immune systems may have more serious and
prolonged infection (APHA, 2004). Immunodeficiency with varying degrees of
hypogammaglobulinemia or agammaglobulinemia is the most commonly reported form of
immunodeficiency associated with chronic giardiasis (Farthing, 1996). However, 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 prior infections (CDC,
2007b). In a study at the Swiss Tropical Institute, only 27 percent 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 most frequently listed
codiagnosis on hospital admission. Among children 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 children under the age of 5 (Lengerich et al., 1994;
Robertson, 1996). Age has been shown to significantly affect recovery time; in Scotland, the
median length of stay in the hospital for giardiasis 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 immunological factors that
increase sensitivity and behavioral factors that increase exposure.
Chronic giardiasis patients often experience recurrent, persistent, brief episodes of loose, foul
smelling stools that may be yellowish and frothy in appearance and frequently accompanied by
distension of the bowel, foul flatus, anorexia, nausea, 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 disappear spontaneously. Among 65 cases of giardiasis encountered
in an urban private practice outpatient 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 symptoms was 3.3 years (USEPA, 1998).
Corsi et al. (1998) evaluated ocular manifestations in 141 Italian children with current and past
giardiasis and 300 children without giardiasis. Retinal changes were 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 suggest that asymptomatic, nonprogressive retinal lesions may occur
in young children with giardiasis. The risk of retinal lesions did not seem to be related to the
severity of infection, its duration, or use of metronidazole to treat the infection, and may reflect a
genetic predisposition to retinal lesions (Corsi et al., 1998).
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There is usually no extra-intestinal invasion when Giardia trophozoites infect the small intestine,
but reactive arthritis may occur, and in severe giardiasis, duodenum 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 are affected by giardiasis, the highest incidence
is in children (USEPA, 1998). High risk groups for giardiasis include infants and young
children, travelers to developing 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 surface water was both filtered and disinfected
(USEPA, 1998). Data therefore indicate that filtering water to remove microorganisms
substantially reduces risk of giardiasis.
As with all pathogens, underreporting limits estimates of the true incidence of giardiasis. The
ratio of reported cases of giardiasis to actual cases 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 for 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 approximately 762,800 total cases in 2005. However,
broader estimates are also supported. For example, an estimated 1 to 5 percent of cases of
salmonellosis are reported to CDC 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 giardiasis cases rose from 12,793 to 27,778, nationally
(CDC, 2000). Between 1996 and 2001, however, the number of reported 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 the 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.
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Giardia Cases in the United States 1992-2005
30,000 n
1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005
Year
Data sources: CDC 2000, 2005, 2007
Figure II.2.2-1. Reported Giardia Cases in the 50 States plus Washington DC, 1992 to 2005
The increase in Giardia cases observed for 2002 might reflect 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 cases 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
General
Homosexual males
(in New York)
Small children
Occurrence
2-5% infection
Prevalence in stool samples (infection) 4-12% depending
on year and state
30% of the population has seropositivity for Giardia
(indicates current or past infection)
In national survey, 7.2% (of 216,675) stool specimens
were positive for Giardia in 1987 and 5.6 percent (of
178,786) were positive in 1991 (infection)
18% found positive for Giardia (infection) (compared with
2% among other patients, but giardiasis is not a major
clinical problem in persons with HIV or AIDS)
7% are asymptomatically infected with Giardia
Reference(s)
USEPA, 1998
USEPA, 1998
Frost and Craun, 1998
USEPA, 1999
Faubert et al., 2000;
Kean, 1979; USEPA,
1999
Frost and Craun, 1998
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in southern states (CDC, 2005). For 2002, among states reporting 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 number of cases per 100,000 population for each of
the 5 years of the reporting period.
Giardiasis occurs most frequently 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 resuspension of cysts and an increase in exposure to
Giardia, with higher densities of bathers leading to higher turbidity and correspondingly higher
cyst concentrations (Graczyk et al., 2007; Sunderland 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 percent of reported cases occurred in males
compared to only 43.9 percent in females, while 1.8 percent of reports did not record gender
(CDC, 2007b). According to CDC (2007b), the discrepancy between cases in males versus
females might be attributable in part to increased risk of infection during 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 clearly indicates that Giardia infection in the
United States is common and widespread.
From 1991 to 2004, seven outbreaks 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 premise 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 particular tissues of the host's body that
they can infect (i.e., tropism), which affects both 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 virus is shed via the intestines and infects upon
ingestion) that requires a tropism that includes the lining of the GI tract. Viruses that infect via
the intestine and cause GI illness (e.g., enteroviruses) 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 diseases and the regular maintenance of
multi-host adaptation that is recognized in other pathogens. Although rapid viral evolution facilitated by cross-
species transmission is a recognized public health concern for viruses that have airborne transmission (e.g. influenza
and severe acute respiratory syndrome [SARS] -associated coronavirus), cross-species transmission has not been a
traditional concern for waterborne, fecally transmitted viruses.
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Table II.2.2-3. Outbreaks of Giardiasis Associated with Recreational Waters in the United
States
Year
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
Number of Cases
34
0
61
80
0
77
0
0
18
0
0
2
212
9
Number of Outbreaks
4
0
3
1
0
1
0
0
1
0
0
1
2
1
Source of Outbreak(s)
3 pools, 1 lake
NA
2 lakes, 1 river
Pool
NA
Pool
NA
NA
Pond
NA
NA
River
Pools*
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 infectivity 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 waterborne viruses of humans (e.g., noroviruses)
remain difficult to detect or quantify in cultured cells (NRC, 2004; Straub et al., 2007). 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 waterborne viral zoonoses have been reported,
several viruses (e.g., swine hepatitis E virus [HEV]) are potentially transmissible between
species, and water may serve as a vehicle for their transmission under some circumstances.
Thus, assessment of the prospect of waterborne viral zoonoses is ongoing (Cliver and Moe,
2004).
Cliver and Moe (2004) consider the criteria for determining whether 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 with this virus than people who are not?
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3. Shedding: Is the candidate virus shed by the reservoir 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 monitoring and research activities
are important for public health protection, even though viruses are not currently known to have
the attributes outlined in the introduction 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 infection by a bovine strain has been reported
(Abbaszadegan, 2006). The primary route of exposure for rotavirus is the fecal-oral route
although exposure through other routes also has been reported to a lesser extent. Rotavirus is
stable in the environment, and its transmission can occur through ingestion of contaminated
water or food. Rotavirus illness typically results in vomiting and watery diarrhea for 3 to 8 days.
Fever and abdominal pain occur frequently as well. According to the CDC, this virus is the most
common cause of severe diarrhea in children. Symptoms tend to be less severe for adults.
Approximately 70,000 children in the United States are treated in the hospital for rotavirus each
year (Glass, 2006). A community waterborne outbreak of rotavirus gastroenteritis occurred in
Colorado in 1981 (Hopkins et al., 1984). The outbreak was attributed to sewage contamination
of the water supply and a failure of chlorination treatment.
Hepatitis E
HEV may be zoonotic (Cliver and Moe, 2004; USEPA, 1999). In pigs and rats, this virus is very
similar to human HEV. Experimental studies have indicated that human strains 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. Transmission 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 blood transfusions (USEPA, 1999).
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Adenovirus
Adenovirus may be zoonotic (Mwenda 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 transmission 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 enteric adenovirus that is transmitted via the fecal-
oral 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 spectrum of acute and chronic diseases, including
keratoconjunctivitis, upper respiratory tract infections, pneumonia, gastroenteritis, cystitis, and
encephalitis (Gray et al., 2005). Molecular studies have recently shown adenoviruses to be
associated with bronchopulmonary dysplasia (Couroucli et al., 2000) and chronic obstructive
pulmonary disease (Hogg, 2001).
Immunocompromised persons, including people with AIDS, bone marrow transplant patients,
pregnant women, and children are more susceptible to adenovirus infections (Baldwin et al.,
2000; Crawford-Miksza and Schnurr, 1996). Infections 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 incidence rates are not
available, especially for waterborne outbreaks.
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III. PATHOGEN INTERACTIONS WITH THEIR ENVIRONMENT
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 interactions with other microorganisms and animal manure are
briefly covered in this overview followed by sections that describe 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 pathogens in ambient waters
is often different from the behavior of indicators in ambient water.
Although there are important waterborne amoebic pathogens, they are not associated with animal
fecal material. However, important bacterial pathogens that are associated with fecal material
(e.g., Salmonella, Campylobacter) interact with free-living amoebae 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 drinking water than in recreational waters (Borella
et al., 2004; Marrie et al., 2001). Some human bacterial pathogens are even thought to have
evolved in association with amoebae (Berk et al., 2006). Tezcan-Merdol et al. (2004)
investigated the uptake and replication 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 bacterial 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 actively 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 nonvertebrate 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 will render it free from the
pathogens considered. This has indirect implications 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 pathogen survival in water
are pH, salinity, light exposure, and temperature. Additional environmental characteristics that
may influence pathogen survival, infectivity, and virulence include: UV light (duration,
intensity), rainfall, runoff, dispersal, suspended solids, turbidity, nutrients, organic content,
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organic foams, water quality, biological community in water column, water depth, stratification,
mixing (e.g., wind and waves), presence of aquatic plants, biofilms, and predation.
Thomas et al. (2006) examined extreme rainfall and spring snowmelt in association with 92
Canadian waterborne disease outbreaks between 1975 and 2001. Accumulated rainfall, air
temperature, and peak stream flow were used to determine the relationship between high impact
weather events and the occurrence of waterborne disease outbreaks. For rainfall events greater
than the 93rd percentile, there was a greater than 2-fold increase in the odds of an outbreak
compared to rainfall events less than the 93rd percentile. For each degree-day above 0° C, the
relative odds of an outbreak increased by a factor of 1.007. The odds ratio is small on a per
degree-day scale, but is notable over longer timeframes. For example, over a 42-day period, a 5°
C increase in the maximum daily air temperature would result in over a 4-fold increase
(1.007(5x42) = 4.33) in the relative odds of an outbreak. Stream flow and stream flow peaks did
not show a difference between cases (outbreaks) and controls (no outbreaks), 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 most waterborne pathogens. Thus, in order to
develop effective control strategies, additional 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 the 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 same temperatures (see Table III. 1.1-1).
Table IIL1.1-1. Survival of E. coli O157:H7 in Ambient Waters
Water matrix
Surface waters (lakes and rivers)
Surface waters (lakes and rivers)
Sediments (same lakes and rivers)
Sediments (same lakes and rivers)
River water with feces
Unfiltered lake water
Filtered tap water
Unfiltered lake water
°C
6
24
6
24
15
8
15
25
Days to Decrease
99%
4-11
2-8
10-20
5-8
7.5
91
>91
14-28
99.99%
8-22
5-10
25-39
10-30
14.5
NA
NA
NA
Reference
Czajkowska et al., 2005
McGee etal., 2002
Wang and Doyle, 1998
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Although direct contamination of ambient water due to domestic animals 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 environments 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 and 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. Within 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-sterilized stream water
was recoverable after 4 months at 4°C. Incubation at 25°C resulted in a decline to the
nonculturable state within 28 days, and at 37°C, the 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 contamination. 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 43° C, but some serovars have suppressed growth at
temperatures above 40° 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 ° 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 decreases 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 Iquitos. The concentration of pathogenic Leptospira
was higher in urban than rural water sources and rats were the indicated zoonosis.
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III.1.5 Cryptosporidium Survival in the Environment
As noted previously, because Cryptosporidium oocysts are extremely resistant to environmental
or engineered degradation, 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 the role of microbial
antagonists (microbial predation), chemical antagonists (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) evaluated the sensitivity of C. parvum oocysts to a variety of
environmental pressures such as freezing, dessication, and water treatment processes, as well as
in physical environments commonly associated with 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 influenced 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, the percentage of oocysts remaining viable in stool
samples decreased steadily with time. (In the study, the relationship between oocyst viability
and time varied with individual.) After 176 days in tap water, river 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 survival, with (unfrozen) colder waters promoting
the highest survival rates (USEPA, 200la). Warm and boiling water completely neutralizes
oocysts, and the temperature of the water determines the time required for the treatment to
become effective (Anderson, 1985). Cryopreservation studies conducted by Payer 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, Payer and Nerad (1996) demonstrated that the infectivity of C.
parvum oocysts after freezing is dependent on the temperature 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) (Payer and Nerad, 1996).
Temperature stability studies also were conducted by Sattar et al. (1999) who evaluated the
freeze/thaw susceptibility of various preparations of oocysts including highly purified
preparations as well as infected calf feces. The results of this study indicated that oocyst stability
under freezing conditions is at least partially dependent 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.
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parvum may survive outside of mammalian hosts for several months or more depending upon
water temperature (Straub et al., 1994).
Payer et al. (1998) investigated the effect of water temperatures ranging from -10 to 35° C and a
few higher temperatures on oocyst infectivity. As water temperature 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 (Payer et al., 1998). Under conditions of
high water temperatures, higher than typically found in surface waters, Payer (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 infectivity (determined in infant mice) of
oocysts from calf fecal samples that had been dried in a barn (< 60 percent 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 temperatures ranging from -
1 to 10° C, demonstrated a complete loss of infectivity within 2 to 4 days. Control samples kept
moist and refrigerated retained infectivity for up to 2 to 3 weeks.
Limited studies have been conducted on the effects 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-viable 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 viability declined linearly
over the course of an hour, with approximately 50 percent loss of viability noted at 20 minutes
(Sattar et al., 1999). These authors also showed that oocysts subjected to 2000 psi (13.9 Mpa)
for 1 minute had little reduction in viability.
Sattar et al. (1999) also evaluated the effects of microbial predation on oocyst survival. They
observed that oocysts incubated in dialysis cassettes that were suspended in natural waters
exhibited significantly longer survival times when bacterial populations were excluded from the
suspension water. The observation implies that microbial 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 sunlight (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 several months at salinity levels corresponding to
estuarine coastal waters (Payer, 2004a) and for several weeks in seawater (WHO, 2006). These
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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 rates in reagent-grade and environmental waters
over a range of temperatures. Oocysts incubated at 4 and 15° C remained infective over a 12-
week holding period. A 4 logic reduction in infectivity was observed for both 20 and 25°C
incubation treatments at 12 and 8 weeks, respectively, 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 the inactivation rate of bovine C. parvum oocysts subjected to
temperature regimes designed to mimic the diurnal oscillations of ambient temperature in bovine
feces exposed to sunlight in commercial cattle operations in California. No infectious oocysts
were observed after 1- to 5-day cycles of 40, 50, 60, and 70° C. The loss of infectivity was
primarily due to partial or complete in vitro excystation during the first 24-hour diurnal cycle and
secondarily to thermal inactivation of the remaining intact or partial oocysts. The results suggest
that as ambient conditions generate internal fecal temperatures greater than or equal to 40° C,
rapid inactivation occurs at a rate equal to or greater than 3.27 logic reduction per day for C.
parvum oocysts deposited in the feces of cattle.
Mendez-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/m2) 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 percent and 0 percent, respectively.
The behavior of lake inflows is important in determining pathogen transport 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 sink beneath the surface layer where they will flow
along the bottom towards the deepest point (Brookes et al., 2004). The fate of pathogens in lakes
is determined by factors such as settling and inactivation by temperature, UV, and predation by
other microorganisms. Brookes and colleagues found that inactivation of Cryptosporidium by
UV light can be rapid or slow, depending 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 oocysts stored in the dark in
filter-sterilized and nonfilter-sterilized river water. They reported 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 especially 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 vast majority of oocysts (99 percent) are inactivated by repeated
freeze-thaw cycles independent of the number of times the soil was frozen (Payer, 2004a).
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Therefore, Cryptosporidium may be environmentally limited in parts of the United States during
the winter season. There is evidence that in warmer ambient temperatures, between 4 and 20° C,
very little inactivation of oocysts occurs in different types of agricultural soils (Jenkins et al.,
2002; WHO, 2006). Factors that are known to reduce oocyst survival in soils include drying and
basic pH (Payer, 2004a). Oocyst survival in various water matrices is highly variable, but
survival for longer than 30 days has been demonstrated 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 inactivation after 2 days exposure to tap water (USEPA, 2005a,
2005b).
Walker et al. (2001) tested oocyst degradation (as indicated by microscopic examination) in
response to the combined stresses of water potential (sodium chloride solute potential), above-
freezing 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 models. Increased water potential increased the rate of C. parvum
population degradation for all temperature 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 potential conditions encountered under field conditions
are likely to lead to more rapid degradation of oocyst populations than might be expected from
studies of degradation in calf feces, distilled water with antibiotics, and reverse osmosis water at
low temperatures.
III.1.6 Giardia Survival in the Environment
Studies of the effect of temperature and other environmental factors on Giardia cyst survival
were summarized in EPA's Giardia Human Health Criteria Document (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 survive a single freeze-thaw cycle, repeated freeze-
thaws as might be expected in the environment would likely inactivate cysts (USEPA, 1998).
Johnson et al. (1997) investigated the survival of cysts in marine waters and determined that
viability was reduced 99.9 percent in 3 hours in marine waters exposed to sunlight (as reported in
USEPA, 1998). They also found that 77 hours were required to get 99.9 percent inactivation 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 the experiments, however, these investigators 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. Robertson 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 Norway. Three conditions were compared, distilled
water, river water, and submersion of a filter chamber containing cysts into the river. The rate of
decline in viability was similar under all three conditions, and no Giardia cysts with apparently
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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 infectivity. 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 decreased 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 temperatures; however, different viruses have different
stabilities under similar environmental conditions. For example, astroviruses survive longer than
poliovirus and adenovirus, but shorter than rotavirus 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 observed after 10 days at 6° C (Griffin et al., 2003). Parvoviruses
are the most heat stable enteric viruses known (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 Thurston-Enriquez, 2006).
Pesaro et al., (1995) evaluated the persistence of five animal viruses, 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 rotavirus. Virus inactivation 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, water, and bedding materials (pH < 8.0). The authors conclude that
viruses contained in manure may persist for prolonged periods of time if stored under nonaerated
conditions and this may lead to environmental contamination with viruses.
III.2 Host Animals Can Influence Pathogen Characteristics and Mechanisms of Rapid
Evolution
There is evidence that zoonotic pathogens may change in infectivity, virulence, and the severity
of disease caused in humans depending on their previous host environment. There is also
evidence that some of these host-factor changes can influence subsequent infection cycles in
exposed hosts. For example, in laboratory experiments 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 other known virulent genotypes and was stable upon
subsequent infection of other mice with adequate nutrition. In another demonstration of host
influence on pathogen evolution, yeast with deletions in genes that normally suppress viral RNA
recombination became "hotbeds" for viral recombination. The host (in this case yeast) genes
could affect viral recombinant accumulation by up to 80-fold (Serviene et al., 2005). The key
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mechanisms of phenotypic change in pathogens are genetic diversity (coinfection and
quasispecies), cryptic genes, mutators, and epigenetic effects, and are summarized as follows.
Genetic diversity (co-infection and quasispecies): The pathogen population within a single
host can be comprised of several or even numerous 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. High genetic diversity also can occur when
pathogens have high mutation rates. This latter phenomenon, referred to as "quasispecies,"
occurs mainly in RNA viruses because viral 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 different from every other particle. In fact, the
cooperativity6 of many virus particles' genetic material may be necessary 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 expressed 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 serve 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 MutDS protein, which binds to DNA polymerase,
accumulate a broad spectrum of base substitutions and frameshift mutations (the mutation
6 The hypothesis that swarms of viral genotypes cooperate to comprise an "average phenotype" differs from
quorum sensing, which is the coordination of bacterial cells via cell-to-cell communication 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 severe than acute infection.
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frequency can be 20- to 4,000-fold) (Selifonova et al., 2001). Mutator strains can pass
genetic material to other bacteria and thus transfer the mutator trait. Therefore, the ability of
bacteria to evolve rapidly is heritable.
Epigenetic effects: Epigenetic effects are reversible, heritable changes in gene regulation that
occur without a change in DNA sequence. Genomic imprinting through DNA methylation is
one type of epigenetic effect that has been documented for pathogen regulation of virulence
(Low et al., 2001). The zoonotic bacteria E. coll, 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 (Falker et al., 2005; Low et al., 2001). Although DNA methylase promoters are
known to be responsive to in vivo growth conditions, the degree to which host factors
influence pathogen DNA methylation and whether those methylation patterns provide a
memory system for subsequent generations 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 (Ebert, 1998). Evolution in new host environments can lead to a
reduction of virulence when the original host is encountered again. Serial passage experiments
(SPEs) often result in attenuation of pathogenicity in one host 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 adapt to new hosts and a decline in overall pathogen
fitness. Text Box III.2-1 summarizes some feeding 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 considered during experimental design.
Although SPEs are useful for studying pathogen evolution, the trends observed in SPEs are not
necessarily broadly applicable to pathogen evolution 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 hosts 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 death and a subsequent lack of spread. Those
hypotheses are supported by the observation that intermediate levels of pathogen virulence can
be stable. Thus, the dynamic between pathogen and host drives pathogen (and host) evolution
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
hypothesisa 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).
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and is influenced by numerous factors 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 from 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 characterized, it is unlikely that differences could be
quantified adequately in the near-term to predict differences in human infectivity and virulence
for pathogens passing through different host animals.
Another mechanism by which virulence and fecundity 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 those 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 concluded that CPV may have a role in fecundity
and possibly virulence of C. parvum. The authors indicated that their 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. Miller 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 infection. Jenkins et al. (2007) also
indicated that C. hominis and C. parvum, the two species most often associated with human
infections, are the only species reported thus far to be infected by CPV.
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Text Box III.2-1. Examples of Feeding 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 freezer 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 Gl 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 106 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.
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IV. SUMMARY
Contamination of recreational waters with feces from warm-blooded animals poses a risk of
zoonotic infection of humans with some of the pathogens in those 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 quantify 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 pathogens are likely to cause more severe 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 chronic sequelae such as Guillain-Barre Syndrome and
reactive arthritis have been associated with Campylobacter infections. Although the most
common recreational illnesses are probably due to human viruses causing short-term GI, the
waterborne zoonotic pathogens discussed in this report have the potential to cause serious health
effects. 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.
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APPENDIX A
WATERBORNE PATHOGENS
As described in Section 1.2 of this paper, four attributes were used to select the waterborne
zoonotic pathogens of concern for recreational uses of ambient waters (partially adapted from
Bolin et al., 2004a). Table A-l lists all the pathogens that were evaluated for potential inclusion
in this paper. Information provided in Table A-l includes whether the pathogen is considered
waterborne, the species that are zoonotic hosts, 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 material should be considered differently from
human sources for CWA §304(a) AWQC.
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U.S. Environmental Protection Agency
Table A-l. Known and Potential Zoonotic Waterborne Pathogens
Type
Bacteria
Bacteria
Bacteria
Bacteria
Pathogen
Acinetobacter
Aeromonas
Campylobacter
Cyanobacteria
Waterborne
Yes
(generally in
environment)
Yes
(generally in
environment)
Yes
Yes
(generally in
environment)
Zoonotic Host
None
None
Poultry, cattle,
sheep, and
wild birds
None
Zoonotic
Host is
Warm-
blooded
NA
NA
Yes
NA
Illnesses and
Symptoms in
Humans (less
common
symptoms)
Septicemia,
meningitis,
endocarditis, brain
abscesses,
pneumonia,
empyema, urinary
tract infections, eye
infections, and skin
and wound
infections
Gastroenteritis
(septicemia)
Diarrhea,
abdominal pain,
malaise, fever,
nausea, and
vomiting (typhoid-
like syndrome,
febrile convulsions,
meningeal arthritis,
reactive arthritis,
and GBS)
Rash and
gastroenteritis
U.S.
Outbreaks
Hospital
settings
None
reported
Mainly
foodborne
and drinking
water
Drinking
water and
recreational
water
Importance
to consider
if animal
fecal
sources are
discounted
in AWQC
No
No
Important
No
Reference(s)
Stewart and
Rochelle, 2006
Lightfoot, 2004;
Moyer and
Standridge,
2006
Allos, 1998;
2001; APHA,
2006; Fricker,
2006a
Fredericksen et
al., 2006
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A-2
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Type
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Pathogen
Verotoxin-producing E.coli
(VTEC) - includes
enterohemorrhagicE. coli
(EHEC), including O1 57:H7
Enterotoxigenic E. coli
(ETEC)
Attaching and effacing £.
coli (A/EEC)
EnteropathogenicE. coli
(EPEC)
Enteroaggregative E. coli
(EAggEC)
Diffuse adherent E. coli
(DAEC)
Enteroinvasive E. coli
(EIEC)
Waterborne
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Zoonotic Host
Cattle,
chicken,
sheep, pigs,
horses, dogs,
and deer
Same as
VTEC
Same as
VTEC
Same as
VTEC
Same as
VTEC
Same as
VTEC
None
Zoonotic
Host is
Warm-
blooded
Yes
Yes
Yes
Yes
Yes
Yes
NA
Illnesses and
Symptoms in
Humans (less
common
symptoms)
Diarrhea (bloody),
severe abdominal
cramping,
headache,
hemorrhagic colitis,
and hemolytic
uremic syndrome
Acute, watery
diarrhea
Acute or persistent
diarrhea
Acute or persistent
diarrhea
Acute, watery, and
often protracted
diarrhea
Acute or persistent
diarrhea
Acute, often
inflammatory
diarrhea; dysentery
U.S.
Outbreaks
Food borne
and
waterborne
(drinking
water and
recreational
water)
Waterborne
No
waterborne
reported
Waterborne
No
waterborne
reported
No
waterborne
reported
No
waterborne
reported
Importance
to consider
if animal
fecal
sources are
discounted
in AWQC
Important
Possibly
important
Possibly
important
Possibly
important
Possibly
important
Possibly
important
No
Reference(s)
APHA, 2004;
Degnan and
Standridge,
2006; M0lbak
and Scheutz,
2004
Hunter, 2003;
M0lbak and
Scheutz, 2004
M0lbak and
Scheutz, 2004
Lee et al, 2002;
M0lbak and
Scheutz, 2004
M0lbak and
Scheutz, 2004
M0lbakand
Scheutz, 2004
M0lbak and
Scheutz, 2004
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Type
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Pathogen
Flavobacterium
Helicobacter pylori
Klebsiella
Legionella
Leptospira
Waterborne
Yes
Possibly
Yes
(generally in
environment)
Yes
Yes
Zoonotic Host
None
Weak
evidence for
ferrets,
racoons,
swine, sheep,
rodents, and
primates
Warm-blooded
animals
None
Rats, dogs,
raccoons,
swine, and
cattle
Zoonotic
Host is
Warm-
blooded
NA
Yes
Yes
NA
Yes
Illnesses and
Symptoms in
Humans (less
common
symptoms)
Gastroenteritis,
meningitis,
pneumonia,
endocarditis, and
septicemia
Gastric disorders,
pepticand duodenal
ulcer disease,
lymphoma of the
digestive tract, and
ademenocarcinoma
of the stomach
Infections in
respiratory system,
genitourinary tract,
nose, and throat,
(meningitis and
septicemia)
Legionellosis,
pneumonia,
Legionnaire's
disease, and
Pontiac fever
Leptospirosis
(Weil's disease)
U.S.
Outbreaks
Rare
waterborne
(stagnation in
drinking
water),
hospital
settings more
common
No
waterborne
reported
Hospital
settings
Hospitals,
pools, and
spas
Recreational
waterborne
over 50% of
cases in
Hawaii
Importance
to consider
if animal
fecal
sources are
discounted
in AWQC
No
No
No
No
Important
Reference(s)
Geldreich and
Degnan, 2006a
Baker and
Degnan, 2006;
Health Canada,
2006
Geldreich and
Standridge,
2006
Hall, 2006
Levett, 2001
February 2009
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Type
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Pathogen
Listeria monocytogenes
Mycobacterium avium
complex (MAC) and ssp.
Paratuberculosis (MAP)
Pseudomonas
Salmonella
Serratia
Shigella
Staphylococcus
Waterborne
No
Yes
(generally in
environment)
Yes
(generally in
environment)
Yes
Yes
(generally in
environment)
Yes
Yes
Zoonotic Host
Domestic and
wild animals
Possibly
sheep, cattle,
goats, and
birds
None
Poultry, swine,
cattle, rodents,
wild birds,
turtles, dogs,
and cats
None
None (except
primate
colonies)
Skin of warm-
blooded hosts
Zoonotic
Host is
Warm-
blooded
Yes
Yes
NA
Yes
NA
NA
Yes
Illnesses and
Symptoms in
Humans (less
common
symptoms)
Listeriosis,
meningoencephaliti
s, fever, and
abortion
Respiratory
infection, fever, and
Crohn's disease
Dermatitis
Gastroenteritis
(enteric fever and
septicemia)
Opportunistic
infection (cystitis)
Shigellosis, acute
gastroenteritis,
dysentary, fever,
nausea, vomiting,
and cramps
Cellulitis, pustules,
boil, carbuncles,
and impetigo
(diarrhea and
vomiting)
U.S.
Outbreaks
Food borne
No
recreational
waterborne
reported
Recreational
waterborne
Mainly
foodborne
and drinking
water
Hospital
settings
Recreational
and drinking
water
Hospital
settings,
pools, and
spas
Importance
to consider
if animal
fecal
sources are
discounted
in AWQC
No
Possibly
important
No
Important
No
No
No
Reference(s)
APHA, 2004
Bolin et al.,
2004b; Carr
and Bartram,
2004;
LeChevallier,
2006
Geldreich and
Degnan, 2006b
APHA, 2004;
Covert and
Meckes, 2006
Geldreich and
Standridge,
2006a
APHA, 2004;
Cliverand
Payer, 2004;
Moyer and
Degnan, 2006
Geldreich and
Standridge,
2006b
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Type
Bacteria
Bacteria
Bacteria
Protozoa
Protozoa
Protozoa
Protozoa
Protozoa
Pathogen
Vibrio cholerae
Vibrio parahaemolyticus
Yersinia
Acanthamoeba
Ascaris lumbricoides
Balamuthia mandrillaris
Balantidium coli
Blastocystis hominis
Waterborne
Yes
Yes
(generally in
environment)
Yes
Yes (free-
living)
Yes
Yes (free-
living)
Yes
Yes
Zoonotic Host
Cope pods,
zooplankton
Molluscan
shellfish
Pigs
None
None
Primates,
sheep, dogs,
and horses
Primates and
pigs
Primates,
cattle, sheep,
pigs, horses,
dogs,
chickens, wild
birds, alpacas,
llamas, koalas,
and wombats
Zoonotic
Host is
Warm-
blooded
No
No
Yes
NA
NA
Yes
Yes
Yes
Illnesses and
Symptoms in
Humans (less
common
symptoms)
Profuse, watery
diarrhea; vomiting
Acute
gastroenteritis
(septicemia)
Yersiniosis; acute,
febrile diarrhea
Granulomatus
ameobic
encephalitis
Ascariasis and
roundworm
infection
Granulomatus
ameobic
encephalitis
Severe dysentery
Diarrhea, cramps,
nausea, fever,
vomiting, and
abdominal pain
U.S.
Outbreaks
None
recently
Food borne
Mainly
foodborne
None
reported
Foodborne
None
reported
None
reported
None
reported
Importance
to consider
if animal
fecal
sources are
discounted
in AWQC
No
No
Possibly
important
No
No
No
No
No
Reference(s)
APHA, 2004;
Toranzos et al.,
2006
FDA, 2001
APHA, 2004;
Fricker, 2006b
Fayer, 2004b;
Visvesvara and
Moura, 2006a
APHA, 2004;
Smith et al.,
2006a
Visvesvara and
Moura, 2006b
Garcia, 2006a
Garcia, 2006b
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U.S. Environmental Protection Agency
Type
Protozoa
Protozoa
Protozoa
Protozoa
Pathogen
Cryptosporidium parvum
and Cryptosporidium
hominis
Cyclospora cayetanensis
Entamoeba histolytica
Giardia intestinalis (also
known as G. duodenalis
and G. lamblia)
Waterborne
Yes
Yes
Yes
Yes
Zoonotic Host
Cattle, sheep,
goats, pigs,
horses, cats,
dogs, wild
animals, birds,
reptiles, and
fish
None
Potentially
primates,
dogs, cats,
pigs, rats, and
possibly cattle
Beavers, cats,
lemurs, sheep,
calves, dogs,
foxes,
chinchillas,
alpacas,
horses, pigs,
cows, and
muskrats
Zoonotic
Host is
Warm-
blooded
Yes
NA
Yes
Yes
Illnesses and
Symptoms in
Humans (less
common
symptoms)
Cryptosporidiosis,
profuse watery
diarrhea, malaise,
fever, anorexia,
nausea, and
vomiting
Watery diarrhea,
malaise, fever,
anorexia, nausea,
and vomiting
Amoebiasis,
dysentery, and
diarrhea
Giardiasis; diarrhea
(chronic);
steatorrhea;
abdominal cramps;
bloating; frequent
loose, pale, greasy
stools; fatigue; and
malabsorption
U.S.
Outbreaks
Recreational
and drinking
water
Food borne
and
waterborne
(drinking
water and
recreational
water)
None
recently
Recreational
and drinking
water
Importance
to consider
if animal
fecal
sources are
discounted
in AWQC
Important
No
No
Important
Reference(s)
APHA, 2004;
CDC, 2007b;
Olson, et al.,
2003; Sterling
and Marshall,
2006
APHA, 2004;
Cliverand
Payer, 2004;
Cross and
Sherchand,
2004; Ortega,
2006
APHA, 2004;
Payer, 2004b;
Keene, 2006
APHA, 2004;
Appelbee et al.,
2005;
Schaefer, 2006
February 2009
A-7
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U.S. Environmental Protection Agency
Type
Protozoa
Protozoa
Protozoa
Virus
Virus
Virus
Pathogen
Isosprora belli
Microsporidia
(Enterocytozoon bieneusi,
Encephalitozoon cuniculi,
E. intestinalis)
Naegleria fowleri
Adenoviruses
Astrovi ruses
Avian influenza (H5N1)
Waterborne
Yes
Yes
Yes (free-
living)
Yes
Yes
Unknown
Zoonotic Host
None
Cattle, pigs,
cats, rabbits,
and sheep
None
None
None
Birds
Zoonotic
Host is
Warm-
blooded
NA
Yes
NA
NA
NA
Yes
Illnesses and
Symptoms in
Humans (less
common
symptoms)
Foul smelling,
foaming diarrhea
(months to years),
abdominal colic,
and fever
Diarrhea
Primary amebic
meningoencephaliti
s
Acute
gastroenteritis and
respiratory disease
Gastroenteritis
Mild upper
respiratory illness
to severe
pneumonia and
multiple organ
failure
U.S.
Outbreaks
None
reported
None
reported
Mainly
ambient
recreational
waters
Waterborne
Waterborne
None
reported
Importance
to consider
if animal
fecal
sources are
discounted
in AWQC
No
Possibly
important
No
No
No
No
Reference(s)
Garcia, 2006c
Bolin et al.,
2004b; Call,
2006;
Shadduckand
Greely, 1989
Payer, 2004b;
Visvesvara and
Moura, 2006c
Enriquez and
Thurston-
Enriquez, 2006;
Griffin et al.,
2003;
Reynolds, 2006
Reynolds,
2006; Schwab,
2006
Chan, 2002;
Suresh and
Smith, 2004
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Type
Virus
Virus
Virus
Virus
Virus
Virus
Virus
Pathogen
Coronaviruses (e.g.,
severe acute respiratory
syndrome (SARS)-CoV)
Enteroviruses (e.g.,
coxsackie)
Hendra virus
Hepatitis A virus
Hepatitis E virus
Human caliciviruses
(norovirus, sapovirus)
Nipah virus
Waterborne
Potentially
(aerosolized
waste water)
Yes
Unknown
Yes
Yes
Yes
Unknown
Zoonotic Host
Possibly civets
and other wild
animals
None
Horses
None
Possibly pigs,
chickens, and
rats (close viral
relatives to
human form)
None
Pigs, bats, and
flying foxes
Zoonotic
Host is
Warm-
blooded
NA
NA
Yes
NA
NA
NA
Yes
Illnesses and
Symptoms in
Humans (less
common
symptoms)
Fever, dry cough,
dyspnoea, and
myalgia (diarrhea)
Gastroenteritis,
exanthema,
diarrhea, fever,
pharyngeal lesions,
myocarditis,
respiratory disease,
and pneumonia
Severe respiratory
disease and
meningoencephaliti
s
Hepatitis - acute
inflammation of the
liver
Hepatitis - acute
inflammation of the
liver (fatality in
pregnant women)
Diarrhea and
vomiting
Acute and febrile
encephalitis
U.S.
Outbreaks
None
reported
Recreational
and drinking
water
None
reported
None
recently
Rare in
United States
(common in
other
countries)
Very
common
(waterborne
and
foodborne)
None
reported
Importance
to consider
if animal
fecal
sources are
discounted
in AWQC
No
No
No
No
No
No
No
Reference(s)
Moe, 2004;
Reynolds,
2006; Suresh
and Smith,
2004
APHA, 2004;
Gerba, 2006a;
Griffin, 2003;
Reynolds, 2006
MacKenzie,
1999; Suresh
and Smith,
2004
Sobsey, 2006
Oliver and Moe,
2004; Craun,
2004a; Gerba,
2006b
Reynolds,
2006; Schwab
and Hurst,
2006
Harcourt, 2005;
Suresh and
Smith, 2004
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Type
Virus
Virus
Virus
Helminths
Helminths
Helminths
Pathogen
Parechoviruses
Reoviruses
Rotaviruses
Ancylostoma braziliense
Angiostrongylus
Ascaris lumbricoides
Waterborne
Yes
Yes
Yes
Mainly soil
Yes
Mainly soil
(drinking
water
possible)
Zoonotic Host
None
None
None
Dogs or cats
Mollusks
None
Zoonotic
Host is
Warm-
blooded
NA
NA
NA
Yes
No
NA
Illnesses and
Symptoms in
Humans (less
common
symptoms)
Pericarditis,
herpangina, and
respiratory disease
Mostly mild or
subclinical (rarely
biliary atresia,
juvenile onset
diabetes, fever,
rash, respiratory
disease, and
diarrhea
Diarrhea
Larval migration
leads to creeping
eruption
Meningitis
Ascaris pneumonia
(lung hemorrhage)
U.S.
Outbreaks
None
reported
(probably
occurs, but
unidentified)
None
reported
(probably
occurs, but
unidentified)
Very
common
waterborne
None
reported
(most
important
hookworm in
humans - soil
route)
None
reported
None
reported
Importance
to consider
if animal
fecal
sources are
discounted
in AWQC
No
No
No
No
No
No
Reference(s)
Gerba, 2006a
Sattar and
Springthrope,
2006
Abbaszadegan,
2006;
Reynolds, 2006
Endo and
Morishima,
2004
Endo and
Morishima,
2004
Endo and
Morishima,
2004; Smith et
al., 2006
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Type
Helminths
Helminths
Helminths
Helminths
Helminths
Helminths
Helminths
Pathogen
Ascaris suum
avian schistosomes
(includes S. mansonni)
Baylisascaris procyonis
Dracunculus medinensis
Echinococcus
Fasciola hepatica
Pseudophyllid cestodes
Waterborne
Mainly soil
Yes
Mainly soil
Yes
Yes
Yes
Yes
Zoonotic Host
Pigs
Snails (birds
are infected
but not
sources)
Raccoons
Crustaceans
Foxes, dogs
Snails
(herbivores
and humans
infected)
Copepods, fish
Zoonotic
Host is
Warm-
blooded
Yes
Yes
Yes
No
Yes
Yes
No
Illnesses and
Symptoms in
Humans (less
common
symptoms)
Ascaris pneumonia
(lung hemorrhage)
Cercarial dermatitis
(swimmer's itch)
Larval migration
may damage to the
visceral and ocular
systems (migration
to brain)
Connective tissue
migration,
emerging in lower
limb blister
Cystic hydatid
disease, alveolar
hydatid disease,
polycystic hydatid
disease, and
polycystic hydatid
disease
Fascioliasis
Cutaneous or
mucocutaneous
invasion
U.S.
Outbreaks
None
reported
None
reported
None
reported
None
reported
None
reported
None
reported
None
reported
Importance
to consider
if animal
fecal
sources are
discounted
in AWQC
No
Low
importance
No
No
Low
importance
Low
importance
No
Reference(s)
Endo and
Morishima,
2004
Endo and
Morishima,
2004
Endo and
Morishima,
2004
Endo and
Morishima,
2004
Endo and
Morishima,
2004
Endo and
Morishima,
2004
Endo and
Morishima,
2004
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Type
Helminths
Helminths
Helminths
Helminths
Helminths
Pathogen
Schistosomes
Strongyloides
Taenia solium (pork
tapeworm)
Toxocara canis
Toxocara cati
Waterborne
Yes
Mainly soil
Yes
Yes
Yes
Zoonotic Host
Snails (many
animals can be
infected, but
are not
sources)
Dogs
Pigs
Dogs
Cats
Zoonotic
Host is
Warm-
blooded
No
Yes
Yes
Yes
Yes
Illnesses and
Symptoms in
Humans (less
common
symptoms)
Schistosomiasis
(chronic infection -
liver fibrosis, portal
hypertension)
Eosinophilia
Cysticercosis and
myositis
Toxocariasis (larval
migration leads to
hemorrhage and
granulomatous
lesions in the
central nervous
system)
Toxocariasis (larval
migration leads to
hemorrhage and
granulomatous
lesions in the
central nervous
system)
U.S.
Outbreaks
None
reported
None
reported
None
reported
None
reported
None
reported
Importance
to consider
if animal
fecal
sources are
discounted
in AWQC
No
No
Low
importance
Low
importance
Low
importance
Reference(s)
APHA, 2004;
Blankespoor,
2006; Endo
and Morishima,
2004
Endo and
Morishima,
2004; Pardo,
2006
Endo and
Morishima,
2004
Endo and
Morishima,
2004
Endo and
Morishima,
2004
February 2009
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Type
Helminths
Helminths
Helminths
Prion
Pathogen
Toxoplasma gondii
Trichinella spiralis
Trichuris trichiuria
Bovine Spongiform
Encephalopathy (BSE)
Waterborne
Yes
Mainly soil
Mainly soil
Unknown
Zoonotic Host
Cats
Variety of
animals
Monkeys, pigs,
dogs, cats,
and chicken
Cattle
Zoonotic
Host is
Warm-
blooded
Yes
Yes
Yes
Yes
Illnesses and
Symptoms in
Humans (less
common
symptoms)
Toxoplasmosis, (in
fetally exposed
children - mental
retardation, loss of
vision, hearing
impairment, and
mortality)
Trichinosis
Trichuris dysentary
syndrome, chronic
diarrhea, anemia,
and growth
retardation
Variant Creutzfeldt-
Jakob disease
U.S.
Outbreaks
None
reported in
recreational
water
Food borne
(mainly
consumption
of
undercooked
pork)
Food borne
Food borne
Importance
to consider
if animal
fecal
sources are
discounted
in AWQC
Potentially
important
No
No
No
Reference(s)
APHA, 2004;
Dubey, 2004,
2006
Endo and
Morishima,
2004
Endo and
Morishima,
2004; Smith et
al., 2006b
Brown et al.,
2006
NA = Not applicable
February 2009
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APPENDIX B
LITERATURE SEARCH STRATEGY AND RESULTS
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 authors used free search
engines on the internet to find articles pertaining to specific information needed. Experts that
participated in EPA's Experts Scientific Workshop on Critical Research Needs for the
Development of New or Revised Recreational Water Quality Criteria11 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.I 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
SI 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(IN)HUMAN()(TRANSMISSION OR
CONTAMINATION)
11 Report from this workshop: http://www.epa.gov/waterscience/criteria/recreation/.
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S3 3842348 PATHOGEN? ? OR INDICATOR? ? OR RISK OR RISKS
S4 983 WATERBORNEO (PATHOGEN? ? OR ZOONO? OR ILLNESS?)
S6 1140 SI 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 domestic journals and government reports.
Descriptions of these files are available at http://library.dialog.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 zoonotic pathogens (species of livestock and
wild animals
Fecal composition of humans - pathogens and indicators
The extent to which strains found in animals can be transmitted to humans
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An evaluation of the extent to which the information identified can be used to support the
differentiation of risk from animal and human sources of fecal contamination
Which organisms are of substantial public health 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 infection and disease from animal sources
Specific pathogens: E. coli O157-H7, Salmonella., Shigella, Campy lobacter, Listeria,
Cryptosporidiiim, Giardia, norovirus, rotavirus, Hepatitis E, emerging pathogens. For
each:
o Describe illness symptoms (range asymptomatic to severe)
o Describe route of exposure from recreational 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 mechanisms (not too deep into molecular
mechanisms of evolution)
Anything in the water matrix that affects survivability and infectivity, and virulence
Pathogen:indicator ratios
B.2 Summary of Literature Search Results
This process resulted in a total order of 535 documents (primarily peer reviewed scientific
articles), of which a total of 332 (62 percent) were received during the expedited writing process,
not all of which could be reviewed. There are many more papers in the peer-reviewed literature,
and this by no means represents all 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
E. coli + deer
Adenoviruses + zoonotic
Hepatitis E+ condition + symptoms
Rotavirus + illness + symptoms
Approximate # Titles
Reviewed
10
30
30
20
# Titles of Interest
1
3
2
1
February 2009
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U.S. Environmental Protection Agency
The following terms were searched on Google Scholar (http://scholar.google.com/):
Search Topic
Adenoviruses + zoonotic
Rotavirus + illness + symptoms
Giardiasis + swimming
Giardiasis + surveillance
Giardia + symptoms
Giardia + cyst
Giardia + beach
Cryptosporidium + hominis
Cryptosporidium + beach
Cryptosporidium + incidental ingestion
Cryptosporidium + review
Cryptosporidium + exposure factors
Cryptosporidium + exposure
Cryptosporidium + symptoms
Cryptosporidium + risk
Campylobacter + chronic
Campylobacter + illness
Campylobacter + infection
Campylobacter + symptoms
Campylobacter + water
Campylobacter + pathogenesis
Salmonella + transmission
Salmonella + transmission in water
Salmonella + antibiotic resistance
Salmonella + pathogenesis
Shigella + outbreak
Shigellosis
Approximate # Titles
Reviewed
10
30
200
200
100
100
200
100
150
250
100
150
50
50
50
50
50
50
50
15
5
20
11
20
3
23
35
# Titles of Interest
1
3
5
3
5
2
1
1
1
4
2
3
2
3
0
3
3
4
1
2
1
8
2
3
1
4
4
The following terms were searched on PubMed (http://www.ncbi.nlm.nih.gov/sites/entrez):
Search Topic
E. co/; O157:H7 + survival + environment
Salmonella + survival + environment
Shigella + survival + environment
Campylobacter + survival + environment
Norwalk Virus + survival + environment
Rotavirus + survival + environment
Hepatitis E + survival + environment
# Titles Reviewed
204
376
65
57
2
54
3
# Titles of Interest
23
4
10
17
Yes
17
Yes
February 2009
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U.S. Environmental Protection Agency
The following terms were searched on Scirus (http://www.scirus.com/):
Search Topic
Helicobacter + environmental survival
Leptospira + CID
Leptospira + environmental survival
Leptosporidiosis + symptoms
Approximate # Titles
Reviewed
30
10
30
30
# Titles of Interest
1
1
2
0
The following terms were searched on the CDC website (http://www.cdc.gov/):
Search Topic
Legionella + EID
Leptospira + EID
Avian influenza + EID
H5N1 +EID
SARS + EID
Hendra virus + EID
Nipah virus + EID
Strongyloides + EID
BSE + EID
Approximate # Titles
Reviewed
10
10
10
20
30
20
20
10
10
# Titles of Interest
1
1
1
0
2
0
2
1
1
B.4 Experts Contacted
The flowing experts in the field were contacted directly by email and asked to suggest
references:
Nicholas Ashbolt, USEPA
Thomas Atherholt, New Jersey Department of Environmental Protection
Michael Beach, Centers for Disease Control and Prevention
Bart Bibler, Florida Department of Health
Alexandria Boehm, Stanford University, California
Rebecca Calderon, USEPA
Jennifer Clancy, Clancy Environmental 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, USEPA
February 2009
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U.S. Environmental Protection Agency
Lawrence Honeybourne, Orange County Health Care Agency, Santa Ana, California
Donna Francy, U.S. Geological Survey
Roger Fuji oka, University of Hawaii, Manoa
Toni Glymph, Wisconsin Department 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 Water 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 Seller, Seller Environmental, California
Michael Tate, Kansas Department of Health and Environment
Peter Teunis, RIVM (National Institute of Public Health and the Environment), Netherlands
Gary Toranzos, University of Puerto Rico, Rio Piedras
Timothy Wade, USEPA
John Wathen, USEPA
Stephen Weisberg, Southern California 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 references cited in the reports
were reviewed for relevance:
NRC. (2004) Indicators for Waterborne Pathogens. The National Academies Press
USEPA. (2007) Report of the Experts Scientific Workshop on Critical 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/water sanitation health/
diseases/zoonoses.pdf
References cited by the Natural Resources Defense Council reviewers of the EPA Critical
Path Science Plan
USEPA Internal draft Adenovirus criteria document
USEPA Internal draft pathogenic E. coli criteria document
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U.S. Environmental Protection Agency
Boehm et al. (2008) A sea change ahead for recreational water quality criteria, (peer
review in progress)
In addition, Clancy Environmental Consultants, Inc., ICF International, Seller Environmental,
WaltJay Consulting, and EPA's Health and Ecological 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.
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APPENDIX C
INCIDENTAL INGESTION OF AMBIENT WATER DURING
RECREATIONAL ACTIVITIES
There is a paucity of data concerning rates of incidental ingestion of surface water during
recreational activities. Most of the available estimates address exposures during swimming in
swimming pools, which may not necessarily be representative of typical "incidental" exposures
in ambient waters. Dufour et al. (2006) reviewed early estimates of swimming-related water
ingestion and concluded that incidental ingestion ranged from 10 to 50 mL per hour. None of the
early estimates, however, were based on actual 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, estimated 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 chlorine 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 urine within 24 hours, thus negating concern for
elimination by other pathways; and (2) dermal 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 recreation at a community
swimming pool. Based on the amounts 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 "incidental" exposures than those of the competitive
swimmers measured by Allen et al. (1982), suggesting the lower values may provide a better
basis for estimating incidental exposures.
A larger follow-up study of 549 participants was subsequently conducted at several public and
private outdoor swimming pools (Evans et al., 2006). Participants were requested to engage in
active swimming for between 45 and 60 minutes. The overall average 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 males ingested more than females and that adult
men ingested more than adult women.
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The small number of studies that are available measured water intake in only a few subjects and
characterized water ingestion during either very active swimming or poorly defined water
recreational activities. In addition, the incidental ingestion data that 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 perception that surface waters are nonpotable.
February 2009 C-2
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