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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U.S. Environmental Protection Agency
                                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 effects—especially
in immunocompromised persons and subpopulations. While serious health outcomes are likely
to be rare in  comparison with  self-limiting illnesses as a result of ambient (recreational) water
exposure, the adverse health impacts of the rare, but more serious illnesses remain an important
public health  challenge.
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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 infections—C.   hominis and  C.  parvum—are 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 coccidian—a diverse group of parasitic protozoa
(WHO, 2006).  There are currently 16 species of Cryptosporidium identified in the literature
(Table II.2.1-1).  However, this taxonomy is likely to change as molecular methods continue to
characterize isolates and 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
hypothesis—a gene that enhances fitness in one host decreases fitness in the other host (Ebert, 1998).
    10 Live vaccines such as Theiler's yellow fever vaccine and Sabin's polio vaccine are examples of attenuated
pathogens that elicit an immune response without inducing disease. However, reemergence of pathogen virulence
after vaccination remains a risk (Ebert, 1998).
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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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V.    REFERENCES

Abbaszadegan, M. 2006. Rotoviruses, In: Waterborne Pathogens, AWWA Manual M48, Second
Edition. American Water Works Association: Denver, CO. Chapter 46.

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

Adam, R.D. 2001. Biology ofGiardia lamblia.  Clinical Microbiology Reviews 14(3): 447-475.

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

Allen, L., Briggle, T., Pfaffenberger, C. 1982. Absorption and excretion of cyanuric acid in long-
distance swimmers. Drug Metabolism 13: 499-516.

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

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

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

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

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

Appelbee, A.J., Thompson, R.C.A., Olson,  M.E.  2005.  Giardia and  Cryptosporidium in
mammalian wildlife - current status and future needs. Trends in Parasitology 21(8): 370-376.

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

Atwill, E.R., Sweitzer, R.A., Pereira, M.G., Gardner, I.A., Vuren, D  V., Boyce, W.M.  1997.
Prevalence of and associated risk factors for  shedding Cryptosporidium  parvum  oocysts and
Giardia  cysts  within feral  pig populations  in  California.  Applied   and  Environmental
Microbiology 63(10): 3946-3949.
February 2009                              50

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

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

Axelsson-Olsson, D., Waldenstrom, J., Broman, T.,  Olsen, B., Holmberg, M. 2005. Protozoan
Acanthamoeba  polyphaga  as a potential reservoir for  Campylobacter jejuni. Applied  and
Environmental Microbiology 71(2): 1987-1992.

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

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

Berk, S.G., Gunderson, J.H., Newsome, A.L., Farone, A.L., Hayes, B.J., Redding, K.S., Uddin,
N., Williams, E.L.,  Johnson, R.A., Farsian, M., Reid, A.,  Skimmyhorn, J., Farone, M.B. 2006.
Occurrence of infected  amoebae in cooling towers compared with natural aquatic environments:
implications for emerging pathogens. Environmental Science and  Technology 40(23): 7440-
7444.

Blankspoor, H. 2006.  Schistosomatidae. In: Waterborne Pathogens,  AWWA Manual M48,
Second Edition. American Water Works Association: Denver, CO. Chapter 35.

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

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

Bolin, C., Brown, C., Rose, J. 2004b. Leptosporidiosis and  other potential zoonoses in water. In:
Waterborne Zoonoses:  Identification, Causes and Control; Cotruvo, J.A, Dufour, A., Rees, G.,
Bartram, J., Carr, R., Cliver, D.O., Craun,  G.F., Payer, R., Gannon, V.P.J. (eds), World Health
Organization (WHO). IWA Publishing: London, UK.  Chapter 20.

Booher, S.L., Cornick, N.A., Moon, H.W. 2002. Persistence of Escherichia coli O157:H7 in
experimentally infected swine. Veterinary Microbiology 89(1): 69-81.
February 2009                              51

-------
U.S. Environmental Protection Agency
Borella, P., Montagna, M.T., Romano-Spica, V., Stampi, S., Stancanelli, G., Triassi, M., Neglia,
R., Marches!, I, Fantuzzi, G., Tato, D., Napoli, C., Quaranta, G., Laurenti, P., Leoni, E., Luca,
G.D., Ossi,  C., Moro, M., D'Alcala, G.R.  2004. Legionella infection risk from domestic hot
water. Emerging Infectious Diseases 10(3): 457-464.

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

Brooks, J.T., Sowers, E.G., Wells, J.G., Greene, K.D., Griffin, P.M., Hoekstra, R.M. Strockbine,
N.A.  2005. Non-0157 Shiga toxin-producing Escherichia coli infections in the United States,
1983-2002. Journal of Infectious Diseases 192(8):  1422-1429.

Brotman,  M.,  Giannella,  R., Aim, P., Bauman, M., AR, A.B., Black, R.  1995.  Consensus
conference statement: Escherichia coli 0157:H7  infections-an emerging national health crisis,
July 11-13, 1994. American Gastroenterological Association 108: 1923-1934.

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

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

Buswell, C.M., Herlihy, Y.M., Lawrence, L.M., McGuiggan, J.T., Marsh, P.O., Keevil, C.W.,
Leach, S.A.  1998. Extended survival and persistence of Campylobacter spp. in water and aquatic
biofilms and their detection by immunofluorescent-antibody and -rRNA staining. Applied and
Environmental Microbiology 64(2): 733-741.

Caccio, M. 2005. Molecular epidemiology of human cryptosporidiosis. Parasitologia 47:  185-
192.

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

Carey, C.M., Lee, H., Trevors, J.T. 2004. Biology, persistence and detection of Cryptosporidium
parvum and  Cryptosporidium hominis oocyst. Water Research 38(4): 818-862.

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

Casemore, D., Wright, S., Coop,  R. 1997. Cryptosporidiosis - human and animal epidemiology.
In: Cryptosporidium and Cryptosporidiosis; L.R. Payer (ed.). CRC Press: New York, New York.
Pp. 65-92.
February 2009                               52

-------
U.S. Environmental Protection Agency
U.S. Centers of Disease Control and Prevention (CDC). 1993a. Update: multistate outbreak of
Escherichia coli  O157:H7 infections from  hamburgers - western United  States, 1992-1993.
Mortality and Morbidity Weekly Report 42(14): 258-263.

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

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

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

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

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

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

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

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

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

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

Chalker,  R.B., Blaser,  MJ.  1988.   A  review of human  salmonellosis:  III. Magnitude of
Salmonella infection in the United States. Reviews of Infectious Diseases 10(1): 111-124.

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

Chan, P.K.S. 2002. Outbreak of avian influenza A(H5N1) virus infection in Hong Kong in 1997.
Clinical Infectious Diseases 34(Suppl 2): S58-S64.
February 2009                               53

-------
U.S. Environmental Protection Agency
Chapman, P.A., Siddons, C.A., Malo, A.T.G., Harkin, M.A. 1997. A 1-year study of Escherichia
coli O157 in cattle, sheep, pigs and poultry. Epidemiology and Infection 119(2): 245-250.

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

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

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

Cieslak, P.R., Noble,  S.J., Maxson, D.J., Empey, L.C., Ravenholt, O., Legarza, G., Turtle,  J.,
Doyle, M.P.,  Barrett,  T.J., Wells, J.G.,  McNamara,  A.M., Griffin, P.M.  1997. Hamburger-
associated Escherichia coli  O157:H7 infection in  Las Vegas:  a  hidden epidemic. American
Journal of Public Health 87(2): 176-180.

Clifford, C.P., Crook, D.W., Conlon, C.P., Praise, A.P., Day, D.G, Peto, T.E. 1990. Impact of
waterborne outbreak  of cryptosporidiosis on  AIDS  and renal  transplant  patients. Lancet
335(8703): 1455-1456.

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

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

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

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

Covert,  T., Meckes, M. 2006. Salmonella. In: Waterborne Pathogens, AWWA Manual  M48,
Second Edition. American Waterworks Association: Denver, CO. Chapter 17.
February 2009                               54

-------
U.S. Environmental Protection Agency
Craun, G., Calderon, R., Craun, M. 2004a. Waterborne outbreaks caused by zoonotic pathogens
in the USA. In: Waterborne Zoonoses: Identification, Causes and Control; Cotruvo, J.A, Dufour,
A.,  Rees, G., Bartram,  1, Carr, R., Cliver, D.O., Craun, G.F., Payer, R., Gannon, V.P.J.  (eds),
World Health Organization (WHO/ IWA Publishing: London, UK. Chapter 8.

Craun,  G.,  Till, D.,  McBride,  G.  2004b.  Epidemiological  studies and surveillance.  In:
Waterborne Zoonoses:  Identification, Causes and Control; Cotruvo, J.A, Dufour, A., Rees, G.,
Bartram, 1, Carr, R., Cliver, D.O., Craun, G.F., Payer, R., Gannon, V.P.J. (eds), World Health
Organization (WHO). IWA Publishing, London, UK. Chapter 10.

Craun, G.F., Calderon,  R.L., Craun, M.F. 2005. Outbreaks associated with recreational water in
the  United States. International Journal of Environmental Health Research 15(4): 243-262.

Craun, M.F., Craun,  G.F., Calderon, R.L., Beach, M.J. 2006. Waterborne outbreaks reported in
the  United States. Journal of Water and Health 4(Suppl 2): 19-30.

Crawford-Miksza, L.K.,  Schnurr, D.P.  1996.  Adenovirus  serotype  evolution is driven by
illegitimate recombination in the hypervariable regions of the hexon protein. Virology 224(2):
357-367.

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

Current, W.L., Garcia,  L.S. 1991. Cryptosporidiosis. Clinical Microbiology Reviews 4(3): 325-
358.

Czajkowska, D., Witkowska-Gwiazdowska, A., Sikorska, I, Boszczyk-Maleszakl,  H., Horoch,
M.  2005. Survival of Escherichia coli serotype 0157:H7 in water and in bottom-shore sediments.
Polish Journal  of Environmental Studies 14(4): 423-430.

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

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

Dieckmann, U. 2002. Adaptive dynamics of pathogen-host interactions. In: Adaptive Dynamics
of Infectious Diseases: In Pursuit of Virulence Management; Dieckmann,  U., Metz, J.A.M.,
Sabelis, M.W., Sigmund, K. (eds), Cambridge University Press: Cambridge,  UK. Chapter 4, pp.
39-59.
February 2009                               55

-------
U.S. Environmental Protection Agency
Domingo, E., Baranowski, E., Ruiz-Jarabo,  C.M., Martin-Hernndez, A.M., Siz,  J.C., Escarmis,
C. 1998. Quasispecies structure and persistence of RNA viruses. Emerging Infectious Diseases
4(4): 521-527.

Donnison, A.M., Ross, C.M. 1999. Animal  and human faecal pollution in New  Zealand rivers.
New Zealand Journal of Marine and Freshwater Research 33: 119-128.

Dubey,  J. 2006.  Toxoplasma gondii. In Waterborne Pathogens, AWWA Manual M48,  Second
Edition. American Water Works Association: Denver, CO. Chapter 36.

Dubey, J.P. 2004. Toxoplasmosis - a waterborne zoonosis. Veterinary Parasitology 126(1-2): 57-
72.

Dufour, A.P., Evans,  O., Behymer, T.D., Cantu, R. 2006. Water ingestion during swimming
activities in a pool: a pilot study. Journal of Water and Health 4(4): 425-430.

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

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

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

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

Eisenberg, J.N.S., Lei, X., Hubbard, A.H.,  Brookhart, M.A., Colford, J.M.  2005. The role of
disease transmission and conferred immunity in outbreaks: analysis of the 1993 Cryptosporidium
outbreak in Milwaukee, Wisconsin. American Journal of Epidemiology  161(1): 62-72.

El Mansoury, S.T.E., Naga, I.F.A.E., Negm, A.Y., Amer, E.E.  2004. Influence  of temperature
and  salinity  on the viability and infectivity of Giardia  lamblia and Cryptosporidia parvum.
Journal of the Egyptian Society of Parasitology 34(1): 161-172.

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

Enriquez, C., Thurston-Enriquez, J. 2006. Adenoviruses.  In: Waterborne Pathogens, AWWA
Manual M48, Second Edition. American Waterworks Association: Denver, CO. Chapter 38.
February 2009                              56

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

Falker, S., Schmidt, M.A., Heusipp, G. 2005. DNA methylation in Yersinia enterocolitica: role
of the DNA adenine methyltransferase in mismatch repair and regulation of virulence factors.
Microbiology 151(Pt7): 2291-2299.

Farthing, MJ.  2000. Clinical aspects  of human cryptosporidiosis.  In: Cryptosporidiosis and
Microsporidiosis; Petry, F. (ed). S. Karger AG: New York, NY. Pp. 50-74.

Farthing, MJ.  1996. Giardiasis. Gastroenterology Clinics of North America 25(3): 493-515.

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

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

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

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

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

Payer, R.,  Morgan, U., Upton, SJ. 2000.  Epidemiology  of  Cryptosporidium:  transmission,
detection and identification. International Journal for Parasitology 30(12-13): 1305-1322.

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

Payer, R., Nerad, T., Rail, W., Lindsay, D.S., Blagburn, B.L. 1991. Studies on cryopreservation
of Cryptosporidium parvum.  International Journal for Parasitology 77(3): 357-361.

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

Payer, R.,  Trout, J.M., Jenkins, M.C. 1998. Infectivity of Cryptosporidium parvum oocysts
stored in water at environmental temperatures. International Journal for Parasitology  84(6):
1165-1169.
February 2009                               57

-------
U.S. Environmental Protection Agency
Payer,  R. 2003.  Cryptosporidium:  from  molecules  to disease. In:  Cryptosporidium: From
Molecules to Disease, Thompson R.C.A., Armson A., Ryan U.M. (eds). Elsevier: Amsterdam,
The Netherlands; pp. 11-18.

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

FDA. 2001.  Draft risk assessment on the public health impact of Vibrio parahaemolyticus in
raw molluscan shellfish. Technical Report.  Center for Food Safety and Applied Nutrition, Food
and Drug Administration, U.S. Department of Health and Human Services.

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

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

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

Flicker, C. 2006b. Yersinia. In: Waterborne Pathogens, AWWA Manual M48, Second Edition.
American Water Works Association: Denver, CO. Chapter 22.

Flicker, C. 2006a. Camplylobacter.  In: Waterborne Pathogens, AWWA Manual M48,  Second
Edition. American Water Works Association: Denver, CO. Chapter 7,

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

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

Gagliardi, J.V., Karns, J.S. 2002. Persistence of Escherichia coli O157:H7 in soil and on plant
roots. Environmental Microbiology 4(2):  89-96.

Ganoza, C.A., Matthias, M.A., Collins-Richards, D., Brouwer, K.C.,  Cunningham, C.B.,  Segura,
E.R., Gilman, R.H., Gotuzzo, E., Vinetz, J.M. 2006. Determining risk for severe leptospirosis by
molecular analysis of environmental surface waters for pathogenic Leptospira. Public Library of
Science Medicine 3(8): 1329-1340.
February 2009                              58

-------
U.S. Environmental Protection Agency
Garcia,  L.  2006c.  Isosprora belli. In: Waterborne Pathogens, AWWA Manual M48, Second
Edition. American Water Works Association: Denver, CO. Chapter 32.

Garcia,  L.  2006b. Blastocystis hominis. In: Waterborne Pathogens, AWWA Manual  M48,
Second Edition. American Water Works Association: Denver, CO. Chapter 27.

Garcia,  L. 2006a. Balantidium coli. In: Waterborne Pathogens, AWWA Manual M48, Second
Edition. American Water Works Association: Denver, CO. Chapter 26.

Garg, A.X., Marshall, I, Salvador!, M., Thiessen-Philbrook, H.R., Macnab,  1,  Suri, R.  S.,
Haynes, R.B., Pope, J., Clark, W.,  Investigators, W.H.S. 2006. A gradient of acute gastroenteritis
was  characterized, to  assess risk of  long-term health sequelae  after drinking bacterial-
contaminated water. Journal of Clinical Epidemiology 59(4): 421-428.

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

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

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

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

Geldreich, E., Standridge, J. 2006a. Klebsiella. In: Waterborne Pathogens, AWWA Manual M48,
Second Edition. American Waterworks Association: Denver, CO. Chapter 13.

Geldreich, E., Standridge, J.  2006b. Serratia. In: Waterborne Pathogens, AWWA Manual M48,
Second Edition. American Waterworks Association: Denver, CO. Chapter 18.

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

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

Gerba, C. 2006b. Hepatitis E virus. In:  Waterborne Pathogens, AWWA Manual M48, Second
Edition. American Water Works Association: Denver, CO. Chapter 43.
February 2009                               59

-------
U.S. Environmental Protection Agency
Gillespie, I.A., O'Brien, S.J., Frost, J.A., Adak, O.K., Horby, P., Swan, A.V., Painter, M.J., Neal,
K.R.,  Campylobacter Sentinel  Surveillance  Scheme  Collaborators.  2002.  A  case-case
comparison  of Campylobacter coli and  Campylobacter jejuni infection: a tool for generating
hypotheses. Emerging Infectious Diseases 8(9): 937-942.

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

Graczyk, T.K., Kacprzak, M., Neczaj, E., Tamang, L., Graczyk, H., Lucy, F E., Girouard, A.S.
2007. Occurrence of Cryptosporidium and Giardia  in sewage sludge and  solid waste landfill
leachate  and  quantitative  comparative  analysis   of  sanitization  treatments  on  pathogen
inactivation. Environmental Research 106(1):  27-33.

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

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

Griffin, D.W., Donaldson, K.A., Paul, J.H., Rose, J.B. 2003. Pathogenic human viruses in coastal
waters. Clinical Microbiology Reviews 16(1): 129-143.

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

Guan,  T.Y., Holley,   R.A.  2003.  Pathogen survival in swine manure  environments  and
transmission of human enteric illness - a review.  Journal of Environmental  Quality 32(2): 383-
392.

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

Hall, N.  2006. Legionella. In: Waterborne Pathogens, AWWA Manual M48, Second Edition.
American Waterworks Association: Denver,  CO. Chapter 14.

Hammermueller, J.,  Kruth,  S.,  Prescott,  J., Gyles, C.  1995. Detection  of toxin genes  in
Escherichia  coli isolated  from  normal  dogs and   dogs  with diarrhea.  Canadian  Journal  of
Veterinary Research 59(4): 265-270.

Hammond, E.G., Barbee, S.J., Inoue, T., Ishida, N., Levinskas, G J., Stevens, M.W., Wheeler,
A.G., Cascieri, T.  1986.  A  review  of toxicology  studies on  cyanurate  and its chlorinated
derivatives. Environmental Health Perspectives 69: 287-292.
February 2009                              60

-------
U.S. Environmental Protection Agency
Hamnes, IS.,  Gjerde, B.K., Forberg, T.,  Robertson,  LJ. 2007. Occurrence  of Giardia and
Cryptosporidium in Norwegian red foxes  (Vulpes vulpes).  Veterinary Parasitology  143(3-4):
347-353.

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

Health Canada. 2006. Guidelines for Canadian Drinking Water Quality: Guideline Technical
Document - Bacterial Waterborne Pathogens - Current and Emerging Organisms of Concern.
Water Quality and Health Bureau, Healthy Environments and Consumer Safety Branch, Health
Canada: Ottawa, Ontario.

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

Hellard, M.E., Sinclair, M.I., Hogg, G.G.,  Fairley, C.K. 2000. Prevalence of enteric pathogens
among community based asymptomatic individuals. Journal of Gastroenterology and Hepatology
15(3): 290-293.

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

Hopkins, R.S., Gaspard, G.B., Williams,  F.P., Karlin, R.J., Cukor, G., Blacklow,  N.R. 1984. A
community waterborne gastroenteritis outbreak: evidence for rotavirus as the agent. American
Journal of Public Health 74(3): 263-265.

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

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

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

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

Hunter, P.R., Nichols, G. 2002. Epidemiology and clinical features of Cryptosporidium infection
in immunocompromised patients.  Clinical Microbiology Reviews 15(1): 145-154.
February 2009                              61

-------
U.S. Environmental Protection Agency
Hunter,  P.R.,  Thompson,  R.C.A.  2005.  The  zoonotic  transmission  of  Giardia  and
Cryptosporidium. International Journal for Parasitology 35(11-12): 1181-1190.

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

Inpankaew, T., Traub, R., Thompson, R.C.A., Sukthana, Y. 2007. Canine parasitic zoonoses in
Bangkok temples. Southeast Asian Journal of Tropical Medicine and Public Health 38(2): 247-
255.

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

Jenkins, M., Higgins, J., Abrahante, J., Kniel, K., O'Brien, C., Trout, J., Lancto, C., Abrahamsen,
M., Payer, R. 2007. Fecundity of Cryptosporidium parvum is correlated with intracellular levels
of the viral symbiont CPV. International Journal for Parasitology 11: 1-5.

Johnson,  D., Enriquez, C., Pepper, I, Davis, T., Gerba, C.P., Rose, J. 1997. Survival of Giardia,
Cryptosporidium, poliovirus and  Salmonella  in marine waters. Water Science and Technology
35(11/12): 261-268.

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

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

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

Katz, A.R., Ansdell, V.E., Effler, P.V., Middleton, C.  & Sasaki,  D.M. 2001. Assessment of the
clinical presentation and treatment of 353 cases of laboratory-confirmed leptospirosis in Hawaii,
1974-1998. Clinical Infectious Diseases 33: 1834-1841.

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

Keene, W. 2006. Entamoeba histolytica.  In: Waterborne Pathogens,  AWWA Manual M48,
Second Edition. American Water Works Association: Denver, CO. Chapter 30.
February 2009                              62

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

Keene, W.E., Sazie, E., Kok, I, Rice, D.H.,  Hancock, D.D., Balan, V.K., Zhao, T., Doyle, M.P.
1997. An outbreak of Escherichia coli O157:H7 infections traced to jerky made from deer meat.
Journal of the American Medical Association 277(15): 1229-1231.

King, B.J., Keegan, A.R., Monis, P.T., Saint, C.P. 2005. Environmental temperature controls
Cryptosporidium  oocyst metabolic rate and associated  retention of infectivity. Applied and
Environmental Microbiology 71(7): 3848-3857.

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

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

LeChevallier, M.W., Giovanni, G.D.D., Clancy,  J.L.,  Bukhari,  Z., Bukhari, S., Rosen, J.  S.,
Sobrinho, J., Frey, M.M. 2003. Comparison of method 1623 and cell culture-PCR for detection
of Cryptosporidium spp. in source waters.  Applied and Environmental Microbiology 69(2): 971-
979.

Lederberg,  J.  1998.  Emerging infections:  an  evolutionary perspective. Emerging  Infectious
Diseases 4(3): 366-371.

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

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

Li, X., Atwill, E.R., Dunbar, L.A., Jones, T., Hook, J., Tate, K.W. 2005. Seasonal temperature
fluctuations induces rapid inactivation of Cryptosporidium parvum. Environmental Science and
Technology 39(12): 4484-4489.

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

Low,  D.A., Weyand, N.J., Mahan, M.J. 2001. Roles of DNA adenine methylation in regulating
bacterial gene expression and virulence. Infection and Immunity 69(12): 7197-7204.

Mackenzie, J.S. 1999. Emerging viral diseases: an Australian perspective. Emerging  Infectious
Diseases 5(1): 1-8.
February 2009                              63

-------
U.S. Environmental Protection Agency
MacKenzie, W.R.,  Hoxie, N.J., Proctor,  M.E., Gradus, M.S., Blair,  K.A.,  Peterson, D.E.,
Kazmierczak, J.J., Addiss, D.G., Fox, K.R., Rose, J.B. 1994. A massive outbreak in Milwaukee
of Cryptosporidium infection transmitted through the public water supply. New England Journal
of Medicine 331(3): 161-167.

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

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

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

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

McCarthy, T.A., Barrett, N.L., Hadler, J.L.,  Salsbury, B., Howard, R.T.,  Dingman, D.  W.,
Brinkman, C.D., Bibb, W.F., Cartter, M.L. 2001. Hemolytic-uremic syndrome and Escherichia
coli O121 at a Lake in Connecticut, 1999. Pediatrics 108(4): E59.

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

McGee, P.,  Bolton, D.J., Sheridan,  J.J., Earley, B., Kelly, G., Leonard, N.  2002. Survival of
Escherichia coli O157:H7 in farm water: its role as a vector in the transmission of the organism
within herds. Journal of Applied Microbiology  93(4): 706-713.

Mead, P.S., Slutsker, L., Dietz, V., McCaig, L.F., Bresee, J.S., Shapiro, C., Griffin, P.M., Tauxe,
R.V.  1999. Food-related  illness and death in  the United States.  Emerging Infectious Diseases
5(5): 607-625.

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

Mendez-Hermida,  F.,  Castro-Hermida,  J.A.,  Ares-Mazas, E., Kehoe, S.C., McGuigan,  K.G.
2005. Effect of batch-process solar disinfection on  survival of Cryptosporidium parvum oocysts
in drinking water. Applied and Environmental Microbiology 71(3): 1653-1654.
February 2009                              64

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

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

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

Moe, C. 2004. What are the criteria for determining whether a disease is zoonotic and water
related? In: Waterborne Zoonoses: Identification, Causes and Control; Cotruvo, J.A, Dufour, A.,
Rees, G., Bartram, J., Carr, R., Cliver, D.O., Craun, G.F., Payer, R., Gannon, V.P.J.  (eds), World
Health Organization (WHO). IWA Publishing: London, UK.  Chapter 3.

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

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

Morse, S.S.  1997. The public health threat of emerging viral disease. Journal of Nutrition 127(5
Suppl): 951S-957S.

Moyer, N., Degnan, A. 2006. Shigella. In: Waterborne Pathogens, AWWA Manual M48, Second
Edition. American Water Works Association: Denver, CO. Chapter 19.

Moyer, N., Standridge, J. 2006. Aeromonas. In: Waterborne Pathogens, AWWA Manual M48,
Second Edition. American Water Works Association: Denver, CO. Chapter 6.

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

Nachamkin, I. 2002. Chronic effects of Campylobacter infection. Microbes and Infection 4(4):
399-403.
February 2009                               65

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

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

Nataro,  J.P., Deng, Y., Cookson,  S., Cravioto, A.,  Savarino,  S.J., Guers,  L.D., Levine, M.M..
Tacket,  C.O. 1995. Heterogeneity  of enteroaggregative Escherichia coli virulence demonstrated
in volunteers. Journal of Infectious Diseases 171(2): 465-468.

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

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

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

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

Ohl, M.E., Miller, S.I. 2001. Salmonella: a model for bacterial pathogenesis. Annual Review of
Medicine 52: 259-274.

Okhuysen, P.C., Chappell,  C.L., Crabb, J.H.,  Sterling, C.R., DuPont, H.L.  1999. Virulence of
three distinct Cryptosporidium parvum isolates for healthy adults. Journal of Infectious Diseases
180(4):  1275-1281.

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

Olsen, S.J., Miller, G., Breuer, T., Kennedy, M.,  Higgins, C.,  Walford, J.,  McKee, G., Fox, K.,
Bibb, W., Mead, P. 2002. A waterborne outbreak of Escherichia  coli  O157:H7 infections and
hemolytic uremic syndrome: implications for rural water systems. Emerging Infectious Diseases
8(4): 370-375.
February 2009                               66

-------
U.S. Environmental Protection Agency
Olson, M., Ralston, B., O'Handley, R., Guselle, N., Appelbee, A. 2003. What is the clinical and
zoonotic  significance  of   cryptosporidiosis   in   domestic  animals   and  wildlife?  In:
Cryptosporidium: From Molecules  to Disease;  Thompson,  R., Armson,  A., Ryan,  U. (eds).
Elsevier: Amsterdam, The Netherlands.

Olson, M.E.,  Goh, 1, Phillips, M., Guselle, N., McAllister,  T.A.  1999. Giardia  Cyst and
Cryptosporidium  oocyst survival in water,  soil, and cattle  feces.  Journal  of  Environmental
Quality 28: 1991-1996.

Olson, M.E., O'Handley, R.M., Ralston, B.J., McAllister, T.A., Thompson, R.C.A. 2004. Update
on Cryptosporidium and Giardia infections in cattle. Trends in Parasitology 20(4): 185-191.

Ortega, Y. 2006. Cyclospora cayetanensis. In: Waterborne Pathogens, AWWA Manual M48,
Second Edition. American Water Works Association: Denver, CO. Chapter 29.

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

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

Ostroff, S.M.,  Kobayashi, J.M., Lewis, J.H. 1989. Infections with Escherichia coli O157:H7 in
Washington State. The first year of statewide disease surveillance.  Journal  of the American
Medical Association 262(3): 355-359.

Palmer, S., Biffin, A. & Group 1990. Cryptosporidiosis in England and Wales:  prevalence and
clinical and epidemiological features. British Medical Journal  30: 774-777'.

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

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

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

Pokorny, N.J., Weir, S.C., Carreno,  R.A., Trevors, J.T., Lee, H. 2002. Influence of temperature
on  Cryptosporidium parvum  oocyst infectivity  in river water samples as detected  by tissue
culture assay. International Journal for Parasitology 88(3): 641-643.

Poly, F., Guerry, P. 2008. Pathogenesis of Campylobacter. Current Opinion in Gastroenterology
24(1): 27-31.
February 2009                               67

-------
U.S. Environmental Protection Agency
Pond, K.  2005a. E. coll O157. In: Water Recreation and Disease: Plausibility of Associated
Infections: Acute Effects, Sequelae and Mortality.  World Health Organization (WHO). IWA
Publishing: London, UK. Chapter 4.

Pond, K.  2005b.  Leptospira.  In:  Water Recreation and Disease:  Plausibility of Associated
Infections: Acute Effects, Sequelae and Mortality.  World Health Organization (WHO). IWA
Publishing: London, UK. Chapter 4.

Rangel, J.M., Sparling, P.H., Crowe, C., Griffin, P.M., Swerdlow, D.L. 2005. Epidemiology of
Escherichia coli O157:H7 outbreaks, United  States,  1982-2002. Emerging Infectious Diseases
11(4): 603-609.

Rendtorff, R.C. 1954. The experimental transmission of human intestinal protozoan parasites. II.
Giardia lamblia cysts given in capsules. American Journal of Tropical Medicine and Hygiene
59(2): 209-220.

Reynolds, K.A. 2006.  Identifying  hazards  of waterborne disease.  Water  Conditioning  and
Purification.

Rice, D.H., Hancock, D.D., Besser,  T.E. 1995. Verotoxigenic E. coli O157 colonisation of wild
deer and range cattle. The Veterinary Record, 524.

Roberts, J.D.,  Silbergeld, E.K.,  Graczyk,  T. 2007.  A probabilistic risk  assessment  of
Cryptosporidium  exposure  among  Baltimore  urban anglers.  Journal  of Toxicology  and
Environmental Health Part A: Current Issues 70(18): 1568-1576.

Roberts, W.G., Green, P.H.,  Ma,  J.,   Carr,  M.,  Ginsberg,  A.M.  1989.  Prevalence  of
cryptosporidiosis in patients undergoing endoscopy: evidence for an asymptomatic carrier state.
American Journal of Medical Sciences 87(5): 537-539.

Robertson, B., Sinclair, M.I., Forbes, A.B., Veitch, M., Kirk, M., Cunliffe, D., Willis, J., Fairley,
C.K. 2002.  Case-control  studies of sporadic cryptosporidiosis  in  Melbourne  and Adelaide,
Australia. Epidemiology and Infection 128(3): 419-431.

Robertson, L.J. 1996. Severe giardiasis and cryptosporidiosis in Scotland, UK. Epidemiology
and Infection 117(3): 551-561.

Robertson, L.J., Campbell,  A.T.,  Smith, H.V. 1992. Survival of Cryptosporidium parvum
oocysts under  various  environmental pressures. Applied and  Environmental Microbiology
58(11): 3494-3500.

Robertson, L.J., Gjerde, B.K. 2006. Fate  of Cryptosporidium  oocysts and Giardia cysts in the
Norwegian aquatic environment overwinter. Microbial Ecology 52(4): 597-602.

Roefer, P., Monscvitz, J., Rexing, D.  1996. The Las Vegas cryptosporidiosis outbreak. American
Waterworks Association September 1996. Pp. 95-106.
February 2009                               68

-------
U.S. Environmental Protection Agency
Rollins, D.M., Colwell, R.R.  1986. Viable but nonculturable stage of Campylobacter jejuni and
its role in survival in the natural aquatic environment. Applied and Environmental Microbiology
42(3): 531-538.

Rose, J.B., Haas, C.N., Regli, S. 1991. Risk assessment and control of waterborne giardiasis.
American Journal of Public Health 81(6): 709-713.

Rosqvist,  R.,   Skurnik,   M.,   Wolf-Watz,  H.  1988.  Increased  virulence  of   Yersinia
pseudotuberculosis by two independent mutations. Nature 334(6182): 522-524.

Runde,  V., Ross, S., Trenschel,  R., Lagemann, E.,  Basu, O.,  Renzing-Kohler, K.,  Schaefer,
U.W., Roggendorf,  M., Holler, E. 2001.  Adenoviral  infection after allogeneic stem  cell
transplantation (SCT):  report on 130 patients from a  single SCT unit involved in a prospective
multi center surveillance study. Bone Marrow Transplant 28(1): 51-57.

Ryan, U.,  Monis, P.,  Enemark, H., Sulaiman, I,  Samarasnghe, B.,  Read, C., Buddie, R.,
Robertson, I,  Zhou,  L.,   Thompson, R.,  Xiao,  L.  2003.  Cryptosporidium  suis n.  sp.
(Apicomplexa: Cryptosporidiidae) in pigs (Sus scrofd). International Journal for Parasitology 90:
769-773.

Samadpour, M.,  Stewart, J., Steingart, K.,  Addy, C., Louderback, J., McGinn, M., Ellington, J.,
Newman, T.  2002.  Laboratory  investigation  of an E. coli O157:H7 outbreak  associated with
swimming in Battle Ground Lake,  Vancouver, Washington. Journal of Environmental Health
64(10):  16-26.

Sargeant, J.M., Gillespie, J.R., Oberst, R.D., Flood, S.J.A.  1999. Prevalence of Escherichia coli
0157:H7 in white-tailed deer sharing rangeland with  cattle. Veterinary Medicine Today: Public
Veterinary Medicine 215(6): 792-794.

Sattar, S., Chauret, C., Springthorpe, V.,  Battigelli,  D., Abbaszadegan, M., LeChevallier, M.
1999. Giardia cyst and Cryptosporidium  oocyst survival in watersheds and factors affecting
inactivation. American Water Works Association Research Foundation (AWWARF): Denver,
CO.

Sattar, S., Springthorpe, V.  2006. Reoviruses.  In: Waterborne Pathogens, AWWA Manual M48,
Second Edition. American Water Works Association: Denver, CO.  Chapter 45.

Schaefer, F. 2006. Giardia lamblia.  In: Waterborne Pathogens,  AWWA Manual M48, Second
Edition. American Water Works Association: Denver, CO.  Chapter 31.

Schaub,  S. 2004. A regulatory  perspective on  zoonotic pathogens in water.  In:  Waterborne
Zoonoses: Identification, Causes and Control; Cotruvo, J.A, Dufour, A., Rees,  G.,  Bartram, J.,
Carr, R., Cliver,  D.O., Craun, G.F., Payer, R., Gannon, V.P.J. (eds), World Health Organization
(WHO). IWA Publishing: London, UK. Chapter 27.
February 2009                               69

-------
U.S. Environmental Protection Agency
Schoeni, J.L., Doyle,  M.P. 1994. Variable colonization of chickens perorally inoculated with
Escherichia coli O157:H7 and subsequent contamination  of eggs. Applied and Environmental
Microbiology 60(8): 2958-2962.

Schwab, K. 2006. Astroviruses.  In:  Waterborne Pathogens, AWWA Manual  M48,  Second
Edition. American Water Works Association: Denver, CO.  Chapter 39.

Schwab, K., Hurst, C. 2006. Human caliciviruses.  In: Waterborne Pathogens, AWWA Manual
M48, Second Edition. American Water Works Association: Denver, CO. Chapter 44.

Selifonova,  O.,  Valle,  F.,  Schellenberger,  V.  2001. Rapid  evolution  of novel traits in
microorganisms. Applied and Environmental Microbiology 67(8): 3645-3649.

Serviene, E., Shapka,  N., Cheng, C., Panavas,  T., Phuangrat, B., Baker, I, Nagy, P.O.  2005.
Genome-wide screen identifies host genes affecting viral  RNA recombination. Proceedings of
the National Academy of Sciences 102(30): 10545-10550.

Shadduck, J.A.,  Greeley, E.  1989. Microsporidia and human infections. Clinical Microbiology
Reviews 2(2): 158-165.

Sischo,  W.M., Atwill, E.R., Lanyon, L.E., George, J. 2000. Cryptosporidia on dairy farms and
the role these farms may have in contaminating  surface water supplies in the northeastern United
States. Preventative Veterinary Medicine 43(4):  253-267.

Smith, A.W., Skilling, D.E.,  Cherry, N., Mead,  J.H., Matson, D.O.  1998. Calicivirus  emergence
from ocean reservoirs: zoonotic and interspecies movements. Emerging Infectious Diseases 4(1):
13-20.

Smith, H., Grimason,  A., Holland,  C.  2006a. Ascaris lumbricoides. In: Waterborne  Pathogens,
AWWA Manual M48,  Second  Edition.  American Water Works Association: Denver,  CO.
Chapter 24.

Smith, H.,  Grimason, A.,  Holland, C. 2006b.  Trichuris trichiura. In: Waterborne  Pathogens,
AWWA Manual M48,  Second  Edition.  American Water Works Association: Denver,  CO.
Chapter 37.

Sobsey, M. 2006. Hepatitis A virus. In: Waterborne Pathogens, AWWA Manual M48,  Second
Edition. American Water Works Association: Denver, CO.  Chapter 42.

Sterling, C., Marshall, M. 2006. Cryptosporidium parvum and Cryptosporidium hominis. In:
Waterborne  Pathogens, AWWA Manual M48,  Second Edition.  American  Water  Works
Association: Denver, CO. Chapter 28.

Stewart, M., Rochelle, P. 2006. Acinetobacter. In: Waterborne Pathogens, AWWA Manual M48,
Second Edition. American Water Works Association: Denver, CO. Chapter 5.
February 2009                              70

-------
U.S. Environmental Protection Agency
Straub, T., Mena, H., Gerba, C. 1994. Viability of Giardia muris and Cryptosporidium parvum
oocysts after aging, pressure, pH manipulations,  and disinfection in mountain reservoir water.
Proceedings of the 94th American Society Microbiology General Meeting, Las Vegas, NV.

Straub, T.M., zu Bentrup, K.H., Orosz-Coghlan, P., Dohnalkova, A., Mayer, B.K., Bartholomew,
R.A., Valdez, C.O., Bruckner-Lea, C.J., Gerba, C.P., Abbaszadegan, M., Nickerson, C.A. 2007.
In vitro cell culture infectivity assay for human noroviruses. Emerging Infectious Diseases 13(3):
396-403.

Sunderland, D., Graczyk, T.K., Tamang, L., Breysse, P.N. 2007. Impact of bathers on levels of
Cryptosporidium parvum oocysts and Giardia lamblia cysts in recreational beach waters. Water
Research 41(15): 3483-3489.

Suresh, K., Smith, H. 2004. Tropical organisms in  Asia/Africa/South America. In: Waterborne
Zoonoses: Identification, Causes and Control. Cotruvo, J.,  Dufour, A., G. Rees, J.B.,  Carr, R.,
Cliver, D., Craun, G., Payer, R.,  Gannon, V. (eds), World Health Organization (WHO). IWA
Publishing: London, UK. Chapter 6.

Swerdlow, D.L., Woodruff,  B.A., Brady, R.C.,  Griffin, P.M.,  Tippen,  S.,  Donnell, H.D.,
Geldreich, E., Payne, B.J., Meyer, A., Wells, J.G., Greene, K.D., Bright, M., Bean, N.H., Blake,
P.A.  1992. A waterborne outbreak in missouri  of Escherichia coli  0157:H7  associated  with
bloody diarrhea and death. Annals of Internal Medicine 117(10): 812-819.

Tarr, P.I.  1995. Escherichia coli O157:H7: clinical, diagnostic, and epidemiological aspects of
human infection. Clinical Infectious Diseases 20(1):  1-10.

Tate, K.W., Atwill, E.R., George,  M.R., McDougald, N.K., Larsen, R.E. 2000. Cryptosporidium
parvum transport  from  cattle fecal  deposits on  California  rangelands. Journal of  Range
Management 53(3): 295-299.

Tezcan-Merdol, D., Ljungstrom, M., Winiecka-Krusnell, J., Linder, E., Engstrand, L., Rhen, M.
2004. Uptake and replication of Salmonella enterica in Acanthamoeba rhysodes. Applied and
Environmental Microbiology 70(6): 3706-3714.

Thomas, K.M., Charron, D.F., Waltner-Toews, D., Schuster, C., Maarouf, A.R., Holt, J.D. 2006.
A role of high impact weather events in waterborne disease outbreaks in Canada, 1975 - 2001.
International Journal of Environmental Health Research 16(3): 167-180.

Thompson R.C.  2000. Giardiasis as a re-emerging infectious disease and its zoonotic potential.
International Journal for Parasitology 30: 1259-1267.

Thompson, R.C.A., Monis, P.T.  2004. Variation in Giardia: implications  for taxonomy and
epidemiology. Advances in Parasitology 58: 69-137.
February 2009                               71

-------
U.S. Environmental Protection Agency
Till, D., McBride, G. 2004. Potential public health risk of Campylobacter and other zoonotic
waterborne infections in New Zealand.  In: Waterborne Zoonoses: Identification,  Causes and
Control; Cotruvo, J.A, Dufour,  A., Rees, G., Bartram, 1, Carr, R., Cliver, D.O., Craun, G.F.,
Payer, R., Gannon, V.P.J. (eds), World Health Organization (WHO). IWA Publishing: London,
UK. Chapter 12.

Toranzos, G., Toro, A., Degnan, A. 2006. Vibrio cholerae. In: Waterborne Pathogens, AWWA
Manual M48, Second Edition. American Water Works Association: Denver, CO. Chapter 21.

Tozzi,  A.E., Niccolini, A., Caprioli, A., Luzzi, I, Montini,  G., Zacchello,  G., Gianviti, A.,
Principato, F., Rizzoni, G.  1994.  A community outbreak of haemolytic-uraemic syndrome  in
children  occurring  in a large area  of northern Italy  over a  period  of several months.
Epidemiology and Infection 113(2): 209-219.

Traub,  R., Wade,  S., Read, C., Thompson, A., Mohammed, H.  2005. Molecular characterization
of potentially zoonotic isolates of  Giardia duodenalis in horses. Veterinary Parasitology  130(3-
4): 317-321.

Traub,  R.J., Monis, P.T.,  Robertson, I,  Irwin,  P.,  Mencke, N., Thompson,  R.C.A. 2004.
Epidemiological and molecular  evidence supports the zoonotic transmission of Giardia among
humans and dogs living in the same community. Parasitology 128(Pt 3): 253-262.

Tupchong, M.,  Simor, A., Dewar,  C.  1999. Beaver fever - a rare cause of reactive arthritis. The
Journal of Rheumatology 26(12): 2701-2702.

Tzipori, S. 2000. Predicting human dose-response relationships from multiple biological models.
Conference transcript from Conference on Predicting Human Dose-response Relationships from
Multiple Biological Models: Issues with  Cryptosporidium parvum,  September 28, 2000, USDA
Center at Riverside, Riverdale, MD.
http://www.foodrisk.org/IRAC/events/2000-09-28/speakers/tzipori.cfm.

Ungar, B.L. 1990. Enzyme-linked immunoassay for detection of Cryptosporidium antigens  in
fecal specimens. Journal of Clinical Microbiology 28(11): 2491-2495.

U.S. Department of Agriculture (USDA). 2001. Risk draft assessment of the public health impact
of Escherichia coll O157:H7 from  ground beef. U.S. Department of Agriculture. September.
http://www.fsis.usda.gov/Science/Risk_Assessments/index.asp.

U.S. Department  of Agriculture (USDA).  2005. Risk Assessment of the Impact of Lethality
Standards on Salmonellosis from  Ready-to-Eat  Meat  and Poultry Products. Final Report. U.S.
Department of  Agriculture, The Food Safety and Inspection  Service, Office  of Public Health
Science, Risk Assessment Division.
http://www.fsis.usda.gov/Science/Risk Assessments/index.asp.
February 2009                              72

-------
U.S. Environmental Protection Agency
U.S. Environmental Protection Agency (USEPA). 1986. Bacteriological Ambient Water Quality
Criteria for  Bacteria.   U.S. Environmental Protection Agency,  Office  of Emergency and
Remedial Response: Washington, DC.

USEPA. 1989. Risk Assessment Guidance for Superfund Volume I. Human Health Evaluation
Manual. U.S. Environmental Protection Agency, Office of Emergency and Remedial Response:
Washington, DC. EPA/540/1-89/002.

USEPA. 1997. Exposure Factors Handbook. U.S. Environmental Protection Agency, Office of
Research and  Development National Center for Environmental  Assessment.  EPA/600/P-
95/002Fa.

USEPA.  1998.  Giardia: Human Health  Criteria  Document. U.S. Environmental Protection
Agency, Office of Water: Washington, DC. EPA-823-R-002.

USEPA.  1999.  Drinking  Water  Criteria  Document for Viruses:  An Addendum.  U.S.
Environmental Protection Agency, Office of Water:  Washington, DC.

USEPA.  200la.  Cryptosporidium: Human Health Criteria Document.  U.S. Environmental
Protection Agency, Office of Water: Washington, DC.

USEPA.  2001b.  Cryptosporidium  and  Giardia in  Water  by  Filtration/EVIS/FA.  U.S.
Environmental Protection Agency, Office of Water:  Washington, DC.

USEPA. 2002. Implementation Guidance for Ambient Water Quality Criteria for Bacteria. U.S.
Environmental Protection Agency, Office of Water:  Washington, DC.

USEPA. 2005a. Occurrence and Exposure Assessment for the Final Long Term 2 Enhanced
Surface Water Treatment  Rule. U.S. Environmental  Protection  Agency, Office of Water:
Washington, DC.

USEPA. 2005b. Appendices to the Occurrence and Exposure Assessment for the Final  Long
Term 2 Enhanced Surface Water Treatment Rule. U.S. Environmental Protection Agency, Office
of Water: Washington, DC.

Visvesvara,  G., Moura,  H. 2006a. Acanthamoeba spp. In: Waterborne Pathogens,  AWWA
Manual M48, Second Edition. American Water Works Association: Denver,  CO. Chapter 23.

Visvesvara, G., Moura, H. 2006b. Balamuthia mandrillaris. In: Waterborne Pathogens, AWWA
Manual M48, Second Edition. American Water Works Association: Denver,  CO. Chapter 25.

Visvesvara, G., Moura, H. 2006c. Naegleriafowleri. In: Waterborne Pathogens, AWWA Manual
M48, Second Edition. American Water Works Association: Denver, CO. Chapter 34.
February 2009                             73

-------
U.S. Environmental Protection Agency
Walker, M., Leddy, K., Hagar, E.  2001. Effects of combined water potential  and temperature
stresses on Cryptosporidium parvum oocysts. Applied and Environmental Microbiology 76(12):
5526-5529.

Wang, G., Doyle, M.P.  1998. Heat shock response enhances acid tolerance of Escherichia coll
O157:H7. Letters in Applied Microbiology 26(1): 31-34.

Whitman, R.L., Nevers, M.B, Byappanahalli, M.N. 2006. Examination of the water shed-wide
distribution of Escherichia along southern Lake Michigan: an integrated approach. Applied and
Environmental Microbiology 72(11): 7301-7310.

WHO. 2003. Human Leptospirosis: Guidance for Diagnosis, Surveillance and Control. Technical
Report. World Health  Organization (WHO)  and the  International  Leptospirosis  Society.
http://whqlibdoc.who.int/hq/2003AVHO CDS CSR EPH 2002.23.pdf.

WHO.  2004.  Waterborne  Zoonoses: Identification,  Causes  and  Control.  World Health
Organization (WHO); Cotruvo, J.A.,  Dufour, A., Rees, G., Bartram, J., Carr, R.,  Cliver, D.O.,
Craun, G.F., Payer, R., Gannon, V.P.J (eds). IWA Publishing: London, UK. ISBN: 1 84339 058
2. http://www.who.int/water_sanitation_health/diseases/zoonoses/en/.

WHO. 2006. Guidelines for Drinking Water Quality: Cryptosporidium. Technical report, World
Health Organization.

Widmer, G., Tchack, L., Chappell, C.L., Tzipori, S. 1998. Sequence polymorphism in  the beta-
tubulin gene reveals  heterogeneous  and  variable population structures in Cryptosporidium
parvum. Applied and Environmental Microbiology 64(11): 4477-4481.

Wills, C. 1996. Fever Black Goddess: The Co-Evolution of People and  Plagues. Addison-Wesley
Publishing Company, Inc.: New York, NY.  P.44.

Wolfe, M.,  Jakubowski, W., Hoff,  J. (editors) 1979.  Managing  the patient with giardiasis:
clinical, diagnostic and  therapeutic aspects.  In: Waterborne Transmission of Giardiasis.  U.S.
Environmental Protection Agency (USEPA): Cincinnati, OH.

Wolfe, M.S. 1992. Giardiasis. Clinical Microbiology Reviews 5(1): 93-100.

Xiao, L.,  Ryan, U.M. 2004. Cryptosporidiosis: an  update in molecular epidemiology. Current
Opinion in Infectious Diseases 17(5):  483-490.

Yamahara, K.M., Layton, B.A.,  Santoro,  A.E., Boehm,  A.B. 2007. Beach  sands along the
California Coast are diffuse sources of fecal bacteria to coastal waters. Environmental Science
and Technology 41: 4515-4521. Supporting information for beach sands along the California
coast are diffuse  sources  of fecal  bacteria to coastal waters. Environmental  Science and
Technology Supplemental to 41: 4515-4521, 17 pp.
February 2009                               74

-------
U.S. Environmental Protection Agency
Yang, S., Benson, S.K., Du, C., Healey, M.C. 2000. Infection of immunosuppressed C57BL/6N
adult  mice  with a single oocyst  of  Cryptosporidium  parvum.  International  Journal  for
Parasitology 86(4): 884-887.

Yarze, J.C., Chase, M.P.  2000. E.  coli O157:H7 - another waterborne outbreak!  American
Journal of Gastroenterology 95(4): 1096.

Yoder, J.S., Blackburn, E.G., Craun, G.F., Hill, V., Levy, D.A., Chen, N., Lee, S.H., Calderon,
R.L.,  Beach,  M.J.  2004. Surveillance  for  waterborne-disease  outbreaks  associated with
recreational water - United States, 2001-2002. Mortality and Morbidity Weekly Report CDC
Surveillance Summaries 53(8): 1-22.

Zhou, L., Singh, A., Jiang, J., Xiao, L. 2003. Molecular surveillance of Cryptosporidium spp. in
raw  wastewater in Milwaukee: implications for  understanding  outbreak  occurrence  and
transmission dynamics. Journal of Clinical Microbiology 41(11): 5254-5257.
February 2009                               75

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U.S. Environmental Protection Agency
                                  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.
February 2009                             A-l

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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


February 2009
A-2

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U.S. Environmental Protection Agency
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
February 2009
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U.S. Environmental Protection Agency
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
A-4

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U.S. Environmental Protection Agency
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
February 2009
A-5

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U.S. Environmental Protection Agency
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
February 2009
A-6

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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
February 2009
A-8

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U.S. Environmental Protection Agency
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
February 2009
A-9

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U.S. Environmental Protection Agency
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
February 2009
A-10

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U.S. Environmental Protection Agency
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
February 2009
A-ll

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U.S. Environmental Protection Agency
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
A-12

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U.S. Environmental Protection Agency
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
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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
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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
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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
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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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    •   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.
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