### Appendix C.2: Review of Zoonotic Pathogens in Ambient Waters, 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

December 31 , 2007[this page intentionally left blank]U.S. Environmental Protection Agency

December 2007 i 

## D ISCLAIMER

This document has not y et been reviewed in accordance with U.S Environmental Protection Agency policy and approved for publication and distribution.

Mention of commercial products, trade names, or services in this document or in the references and/or endnotes cited in this doc ument does not convey, and should not be interpreted as conveying official EPA approval, endorsement, or recommendation.[this page intentionally left blank]U.S. Environmental Protection Agency

December 2007 iii 

## A CKNOWLEDGMENTS

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 liter ature review was managed under EPA Contract EP

- C

- 07

- 036 to Clancy Environmental Consulting, Inc. The following individuals contributed to the development of the report:

Lead writer: Audrey Ichida ICF International Leiran Biton ICF International Alexis Ca strovinci ICF International Jennifer Clancy Clancy Environmental Consultants Mary Clark ICF International Elizabeth Dederick ICF International Kelly Hammerle ICF International Walter Jakubowski WaltJay Consulting Margaret McVey ICF International Michelle M oser ICF International Jeffery Rosen Clancy Environmental Consultants Tina Rouse ICF International Jennifer Welham ICF International Kerry Williams ICF International[this page intentionally left blank]U.S. Environmental Protecti on Agency

December 2007 v 

## T ABLE OF 

## C ONTENTS

A CKNOWLEDGMENTS

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

T ABLES AND F IGURES

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

A CRONYMS

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

E XECUTIVE S UMMARY

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

I. B ACKGROUND AND I NTRODUCTION

................................ ................................ ................................ .................... 5

I.1 Background: Context and Purpose

................................ ................................ ................................ ............ 5 I.2 Introduction

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

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

................................ ................................ ............... 7

II.1 Ba cteria

................................ ................................ ................................ ................................ ...................... 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

................................ ................................ ................................ ................................ ................... 18 II.2.1 Cry

ptosporidium

................................ ................................ ................................ .......................... 18 II.2.2 Giardia

................................ ................................ ................................ ................................ ........ 27 II.3 Viruses Zoonotic Potential

................................ ................................ ................................ ....................... 34

III. P ATHOGEN I NTERACTIONS WITH THE IR E NVIRONMENT

................................ ................................ ................. 36

III.1 Water Environment Affects Pathogen Survivability and Phenotype

................................ ....................... 37 III.1.1 Pathogenic E. coli Survival in the Environment

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

................................ ................................ .............. 38 III.1.3 Salmone lla Survival in the Environment

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

................................ ................................ ...................... 39 III.1.5 Cryptosporidium Survival in the Env ironment

................................ ................................ ............ 39 III.1.6 Giardia Survival in the Environment

................................ ................................ .......................... 43 III.1.7 Virus Survival in the Environment

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

................ 44

IV. S UMMARY

................................ ................................ ................................ ................................ ........................... 48

V. R EFERENCES

................................ ................................ ................................ ................................ ...................... 48

A PPENDIX A : W ATERBORNE P ATHOGENS

................................ ................................ ................................ .............. A

- 1

A PPENDIX B : L ITERATURE S EARCH S TRATEGY

................................ ................................ ................................ ..... B

- 1

A PPENDIX C: I NCIDENTAL I NGESTION OF A MBIENT W ATER D URING R ECREATIONAL A CTIVITIES

................... C

- 1[this page intentionally left blank]U.S. Environmental Protection Agency

December 2007 vii 

## T ABLES AND 

## F IGURES

Table II.1.1

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

........................ 11

Table II.2.1

- 1 Cryptosporidium Species

................................ ................................ ................................ ............. 19

Table II.2.1

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

...... 27

Table II.2.2

- 1 Giardia T axonomy

................................ ................................ ................................ ....................... 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.

.................. 33

Table III.1.1

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

................................ ................................ ......... 38

Figure II.2.1

- 1 Reported Cryptosporidium infections in the United States, 1999 to 2005.

................................ . 25
Figure II.2.2

- 1 Reported Giardia cases in the 50 states plus Washington DC, 1992 to 2005.

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

December 2007 ix 

## A CRONYMS

AEC a ttaching E. coli AIDS Acquired Immune Deficiency Syndrome AWQC a mbient w ater q uality c riteria BEACH Act Beaches Environmental Assessment and Coastal Health Act of 2000 CDC Centers for Disease Control and Prevention CPV C. parvum virus CWA Clean Water Act DAEC d iffuse adherent E. coli DEC d iarrheagenic E. coli EAggEC e nteroaggregative E. coli EEC e ffacing E. coli EHEC e nterohemorrhagic E. coli EPA United States Environmental Protection Agency EPEC e nteropathogenic E. coli ETEC e nterotoxigenic E. coli GI g astrointestinal GLV Giardia lamblia virus GBS Guillain

- Barré Syndrome HC hemorrhagic colitis HEV h epatiti s E virus HUS hemolytic uremic syndrome IPSID i mmunoproliferative small intestinal disease MALT m ucosa

- associated lymph oid tissue PCR p olymerase chain reaction POTW Publicly Owned Treatment Works RFLP r estriction fragment length polymorphi sm SPE s erial pa ssage experiment STEC S higa toxin

- producing E. coli USDA United States Department of Agriculture UV ultraviolet VTEC v erocytotoxin

- producing E. coli WHO World Health Organization (United Nations)[this page intentionally left blank]U.S. Environmental Protection Agency

December 2007 1



## E XECUTIV E 

## S UMMARY

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 ) assoc iated with exposure to fecal contamination during recreation al water contact . W ater 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 ambi ent 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. coli ). This approac h 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 fec al material. Refer to the companion paper in this series ― Review of Published Studies to Characterize Relative Risks f rom Different Sources of Fecal Contamination in Recreational Water ,‖ for a discussion of the differing risks from animal versus human fec al 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 a gricultural 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 zoonoti c pathogens that come primarily from warm

- blooded animals. This information can be used to conceptualize potential risks from warm

- blooded animal feces in recreational waters.

Approach

Seventy pathogens from warm

- blooded animals were evaluated for the ir 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 c ause s infection or illness in humans.U.S. Environmental Protection Agency

December 2007 2 Six o f the 20 water

borne

, zoonotic path ogens 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 helminthe s that have cold

- blooded hosts (e.g., snails and copepods). Some common zoonotic pathogens were also excluded because they did not have well

documented waterborne transmission ( i.e., primarily transmitted via soil, food, or drinking water).

Pathogens int eract 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. See the companion paper in this series ―Review of Fec al Indicator Organism Behavior in Ambient Waters and Alternative Indicators for Tropical Regions‖ for a review of indicator behavior. The most common environmental factors studied for their impact on pathogen survival in water are pH, salinity, light expo sure, and temperature. Additional environmental characteristics that may influence pathogen surviv al, infectivity, and virulence include: ultraviolet (UV) light (duration, i ntensity) , r ainfall , r unoff , d ispersal

, suspended solids

, turbidity

, nutrients

, o rganic 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 in fectivity, 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 e ffects.

Six Key Waterborne Zoonotic Pathogens

Pathogenic E. coli E. coli is an important waterborne zoonosis because human pa thogenic strains occur in livestock and wildlife feces and survive in ambient water. 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 infection s. Children are more at risk than healthy adults to suffer more severe outcomes. Between 1991 and 2004, 14 outbreaks of E. coli

- related illness have been associated with ambient recreational waters. E. coli O157:H7 can survive for at least several weeks in animal feces and slurries and has been demonstrated to survive at least 500 days at

- 20 C in frozen soi l.

Campylobacter Campylobacter is a well

- known foodborne pathogen that is associated with poultry and livestock. There are also wildlife hosts. Al though few waterborne outbreaks have been detected,U.S. Environmental Protection Agency

December 2007 3 there is potential for Campylobacter to cause recreational water

- related illness. Normally

, infection results in diarrhea that is self limiting, but approximately 1 out of 1,000 infections results in Gui llain

- Barré syndrome, a serious nervous system affliction. Reactive arthritis is also a possible chronic sequela of Campylobacter infection. Campylobacter has been shown to survive in aquatic environment s with low temperatures (4 °C ) up to 4 months.

Salmo nella Salmonella is a well

- known foodborne 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 m eat processing wastes, ra w sewage, farming operations, and effluents from ineffective sewage treatment plants

. The c linical symptoms of s almonellosis may include diarrhea, abdominal pain, nausea, chills, and fever. Between 1991 and 2002, 3 waterborne outbreaks were attributed to non typhoid Salmonella

, and 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.

Le ptospira Leptospira is an important waterborne 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 human s is usually either direct or indirect contact with the urine of an infected animal. There are numerous symptoms associated with leptospirosis, but the more sever e form is known as Weil ’ s disease . In addition, acute infection during pregnancy has been re ported to cause abortion and fetal death. F rom 1971 to 2000, 16 percent of recreational waterborne disease outbreaks in the United States were attributed to Leptospira

. In 1998, there was an outbreak (375 cases) of leptosp irosis associated with a triath l on in a lake in Illinois

.
Cryptosporidium Cryptosporidium is a well

- known waterborne pathogen. EPA currently regulates it under the Safe Drinking Water Act because the level of Cryptosporidium in many ambient source waters is high enough that risks to drinking water must be managed. C ryptosporidium 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 contribut e 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 gastrointestinal symptoms s uch 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 inf ected 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 Cryptospo ridium oocysts can survive for substantial periods in animal wastes and soils. Thus, contaminated runoff can enter ambient water resulting in potential human exposures. The majority of oocysts (99 percent) are inactivated by repeated freeze

- thaw cycles ; t herefore, Cryptosporidium may be environmentally limited in parts of the United States during the winterU.S. Environmental Protection Agency

December 2007 4 season. B etween 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.

Giardia Giardia is a well

- known waterborne pathogen. Although b oth livestock and humans have been implicated in contaminating water sources with Giardia , humans are responsible for the majority of the contaminations. Z oonotic 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 gia rdiasis 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

s ymp toms. In the United States, from 1991 to 2004, 7 outbreaks of giardiasis have been 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 fece s.

Summary

Althou gh the most common recreational illnesses are probably due to nonzoonotic human viruses , which caus e short

- term gastrointestinal illness, the waterborne zoonotic pathogens discussed in this report have the potential to cause serious health effects. Even t hough serious health outcomes are likely to be rare in comparison with self

- limiting illnesses, the public health impact s of the rare, but more serious illnesses should not be overlooked.U.S. Environ mental Protection Agency

December 2007 5 

## I. B ACKGROUND AND 

## I NTRODUCTION

I.1 Background: Context and Purpose

Si nce 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, microbiolo gy, 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 re search 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 CW A) 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 p ublic, public interest gr oups, s tate and local government s , 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 agencie s, p ublic 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 determin e 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 in put 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 t o human health from swimming in water contaminated with human fecal matter as compared to swimming in water contaminated with non human fecal matter? Human and animal feces can both potentially contain pathogens that cause human illness. S ome human pathog ens 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 pa thogens 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 risk s from identified sources of fecal
matter

.

EPA’s current recommended recreational water quality criteria for microbes treat human health risk s from the various sources of fecal contamination as equivalent . These criteria are based on the risk o f illness from swimming in waters influenced by sewage treatment pla nts effluents. Health risks from other sources (e.g., poorly

- treated or untreated human waste, non human sources of fecal contamination

, and mixed sources such as urban stormwater runoff) were not well understood at the time the 1986 criteria w ere develope d

, and EPA’s approach was to be protective of human health regardless of the source. EPA recognizes , however, that the healthU.S. Environ mental Protection Agency

December 2007 6 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 contam inated with non human 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 detect able by current fecal indicators.

The purpos e of this white paper is to provide summary information on waterborne zoonotic pathogens that come primarily from warm

- blooded animals. This information can be used to conceptualize potential risks to humans from warm

- blooded animal feces in recreational waters.

I.2 Introduction

In the development of health protective criteria for recreational waters
, pathogen contamination is the central concern, and fecal matter can be a major source of pathogens in ambient water. Current recreational AWQC are designed s pecifically 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 (esp ecially 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 non pathogenic fecal bacteria and some viru ses , 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, th e 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.,

non enteric sources).

Historic ally, 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 human s and an imal s , especially mammal s ( WHO, 2004 )

. R esearch 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 path ogens 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 t o examine the broadest array of currently known and suspected waterborne, fecal, and zoonotic pathogens and their human health impacts. These include primarily bacteria, viruses, protozoa, and helminths. There are many zoonotic pathogens and
many waterbor ne pathogens ; however

, there is a more limited subset of pathogens that are both.U.S. Environ mental Protection Agency

December 2007 7 For this report the following attributes were used to select the waterborne

, zoonotic pathogens of concern (partially adapted from Bolin et al., 2004 a ):

1. The pathogen must s

pend 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 wat er. 4. The pathogen must c ause 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 attr ibutes listed above. See Appendix A, Table A

- 1, for a summary of waterborne pathogens that meet the above criteria and selected pathogens that meet some, but not all of the above criteria

.
The remainder of this paper is organized in to 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, whi ch includes both the water environment and the host animals.



## II. K EY 

## W

ATERBORNE 

## Z OONOTIC 

## P ATHOGENS OF 

## C ONCERN

Based on a review of the literature and other information sources (Appendix B), six of th e 20 pathogens identified as waterborne and zoonotic from w arm

- 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 Gia rdia ( Craun et al., 2004a)

. The top five waterborne pathogens 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 zoonose s

.

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

. Incidental ingestion is the pri mary route of recreational exposure to pathogens , therefore information on incidental ingestion is summarized in Appendix C.U.S. Environ mental Protection Agency

December 2007 8 II.1 Bacteria

II.1.1 Escherichia coli

E. coli is an important waterborne zoonosis because human pathogenic strains occur in livestock and w ildlife feces and survive in ambient water. In addition, the potential illnesses caused by pathogenic E. coli can be severe or fatal. Pathogenic E. coli is 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., 200 7 ), w aterborne outbreaks are not as prevalent as foodborne cases. Refer to the companion paper in this series ― Review of Published Studies to Characterize Relative Risks f rom Different Sources of Fecal Contamination in Recreational Water

, ‖ Table IV.2.1 ― Select Waterborne Disease Outbreaks Associated with Recreational Water in the United States Reported by the CDC (1999 to 2004) ‖ for further information.

E. coli Strain V ariation

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, m ultiple disease causing serotypes of E. coli have been identified in the past few decades. The most well known serotype is E. coli O157:H7 . It frequently occurs in wastewater and feces in developed countries and can result in severe illnesses and death

. E. coli O157:H7 (also referred to as just O157:H7) is the ―poster child‖ for pathogenic E. coli and is the strain most ext ensively investigated (Chart et al., 2000)

.

E. coli is a versatile bacterium and multiple subtypes and strains that are pathogenic have been documented. Although t he nomenclature for these strains has not yet stabilized in the literature

, Mølbak and Scheu tz (2004) provide a helpful summary of the current nomenclature:

Diarrheagenic E. coli (DEC)

– includes all the strains on this list Verocytotoxin (Shiga toxin)

- producing E. coli (VTEC or STEC)

– E. coli that produce verocytotoxin (Shiga toxin) VT1 and/or VT2

o Enterohemorrhagic E. coli (EHEC)

– originally 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 produc e 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 Diffuse adherent E. coli (DAEC)

– E. coli that exhibit a pattern of diffuse adherence to tissue culture

Nataro and Kaper (1998) provide a comprehensive review of the d iarrheagenic Escherichia coli

.U.S. Environ mental Protection Agency

December 2007 9 E. coli Zoonotic P otential

E. coli is part of the nor mal intestinal flora of humans and warm

- blooded animals

, and it can readily be 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 (Kudva et al.

, 1996; Chapman et al. , 1997), pigs (Chapman et al. , 1997; Booher et al.

, 2002; Feder et al.

, 2003), deer (Keene et al., 1997; Rice et al.

, 1995; Sargeant et al

. , 1999), hor ses (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. A ll 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 ( Mølbak and Scheutz

, 2004 ). Chapman et al. (1997) determined that the monthly prevalence of E. coli O157:H7 in cattle range d 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 relation ship between 3

, 001 cases of VTEC and the livestock density in rural areas of Ontario, Canada. This 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 th e 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 ) 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 Chapma n et al. ( 1997), O157:H7 was isolated from 2.2 percent of sheep and 4 percent of pigs. In a Russian study, O 157:H7 was found in sheep

, and the incidence was highly variable , ranging from 31 percent in June to zero percent in November (Kudva et al

. , 1996). Kudva et al. (1996) 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 S hi ga

like toxins have been documented in humans and in a nimal s (Kudva et al.

, 1996; Mølbak and Scheutz

, 2004 ).

E. coli R oute of Exposure

Water borne transmission of E. coli O157:H7 has been reported from both recreational water (Ackman

, 1997; CDC, 2002 ; Yoder et al

. , 2004; Samadpour et al

. , 2002; McCarthy et a l

. , 2001) and contaminated drinking water ( Swerdlow et al., 1992; CDC, 2002 ; Olse n et al

. , 2002; Yarze and Chase

, 2000 ; Hrudey et al., 2002, 2003; Pond, 2004 ). Most studies examiningU.S. Environ mental Protection Agency

December 2007 10 contamination from recreation immersion have suggested that ingestion wa s the primary route of exposure ( Keene et al., 1994 ). Because E. coli O 157:H7 has a relatively low infectious dose, swallowing a small amount of contaminated water may cause illness (Keene et al., 1994 ; Haas et al., 2000 ).

Immersion in and ingestion of r ecreational 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 S ympt oms

E. coli can cause a relatively wide range of illness symptoms , depending on the strain and the underlying health of the host ( Hunter, 2003; Mølbak 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 r esult 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, 1993 a ) although

, in the Cinci nnati outbreak

, infants remained hospitalized for 21 to 120 days (Nataro and Kaper, 1998 )

. S ymptoms may be more severe for persons with hemorrhagic colitis or bloody inflammation of the colo n (Griffin and Tauxe

, 1991 ).

Symptoms associated with illness fr om the various E. coli strains include:

VTEC (or STEC)

– Diarrhea, hemorrhagic colitis, HUS

o EHEC

– hemorrhagic colitis (HC) in humans ETEC

– Acute watery diarrhea A/EEC

– Acute or persistent diarrhea

o EPEC

– Acute or persistent diarrhea EAggEC

– Acute wat ery, often protracted diarrhea DAEC

– Acute or persistent diarrhea

In rare cases, hemorrhagic colitis can develop into HUS , which is a severe life

- threatening disease that can result in kidney failure and neurological complications such as seizures and
st rok es ( Brotman

, 1995). Brooks et al. (2005) conducted a study that suggests E. coli s trains that produce Shiga toxin 2 are much more likely to result in HUS than strains t hat 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 pa ralysis ( Tarr, 1995 ). Approximately 33 percent of persons that contract HUS have abnormal kidney function for several years, a few of which require 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 . T his difference may be due to the higher proportion of young children participating in swimming and becoming infected during such outbre aks and theU.S. Environ mental Protection Agency

December 2007 11 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, pos themolytic biliary lithiasis, post

- infection colonic stricture, rectal prolapse, appendicitis, hepatitis, hemorrhagic cystitis, pulmonary edema, myocardial dysfunction, and neurological abnormalities (Nataro and Kaper, 1998).

I nfection with E. coli O157:H 7 can also result in death

. The Centers for Disea se 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 death s out of approximately 700 who fell ill , which corresponds to approximately 0.57 percent mortality ( Brotman, 1995 ). Griffin and Tauxe (1991)

re viewed 12 outbreaks in the United States between 1982 and 1990 and calcul ated a mortality rate of 1.9 percent among those with reported 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

- O1 57 S higa

- like toxin serotypes cause approximately 37,000 illnesses per year (Mead et al., 1999). 1 Craun et al. (2004 a ) estimate d that E. coli was the etiological agent for 30 percent of the outbreaks from zoonotic contamination in untreated recreational water s fro m 1971 to 2000

.

E. coli O157:H7 infection associated with recreational exposure was first reported in June 1991 in a lake in Orego n (Keene et al., 1994). From 1991 until 2002, 20 additional outbreaks as a result of exposure to contaminated recreational w aters have been reported to the CDC (Rangel et al., 2005). Fourteen of the outbreaks occurred as a result of exposure to contaminated lakes or ponds

, and seven occurred from exposure to contaminated swimming pools (Rangel et al., 2005).

CDC ’ s surveillanc e 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 or are not recognized as E. coli infections or the outbreak is considered of unknown etiolog y (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).

Table II.1.1

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

Year Number of Cases Number of Outbreaks Sources

1991 80 1 Lake

1992 0 0 NA

1993 0 0 NA

1994 166 1 Lake

1 The CDC is currently updating these num bers, but the newest values have not yet been released.U.S. Environ mental Protection Agency

December 2007 12 Year Number of Cases Number of Outbreaks Sources

1995 28 4 Lakes

1996 24 2 1 pool, 1 lake

1997 8 1 Lake

1998 31 2 1 pool, 1 lake

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

2000 0 0 NA

2001 49 2 Lakes

2002 9 1 Pool

2003 0 0 NA

2004 0 0 NA
Source: Data from CDC Surveillance Summaries: Morbidity and Mortality Weekly Report (MMWR)

Surveillance for Waterborne

- Disease Outbreaks

- United States: 1991

- 1992, 1993

- 1994, 1995

- 19 96, 1997

- 1998, 1999

- 2000, 2001

- 2002, 2003

- 2004 (CDC, 1993 b , 1996, 1998, 2000, 2002, 2004, 2006).

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. E ight 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

- Barré syndrome ( GBS ) (Nachamkin

, 2002)

.
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 zoonot ic waterborne route is unlikely to be important (Till and McBride

, 2004). S ome 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 r ecreation (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 indirec tly 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 typ ically contaminated with C. coli and C. jejuni (Jones

, 2001). Surface waters that have contact withU.S. Environ mental Protection Agency

December 2007 13 avian species are typically contaminated with C. jejuni

, C. lari

, C. coli , and avian

- sourced

urease

- positive thermophilic campylobacters (Jones

, 2001). Th e main source of campylobacters for bathing waters is typically assumed to be sewage effluent al though there is evidence that birds may be primarily responsible for contamination (Jones

, 2001).

Campylobacter Route of Exposure

The main route of exposure t o Campylobacter from recreational water s 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 et iologic agent fo r 0.9 percent of the outbreaks. Even though waterborne sources of Campylobacter are important,

m ost 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 ( Fricker, 2006 a ; Allos, 2001 )

. Infections of Campylobacter ar e symptomatically indistinguishable from those of other bacterial pathogens such as Salmonella (Nachamkin

, 2002). Diarrheal symptoms may be frequent at the peak of the illness, occurring 8 to 10 times per day . Al though the disease usually lasts only one week, some infected individuals experience relapses leading to several weeks of symptoms (Allos

, 2001).

Some complications may result from Campylobacter infection including cholecystitis, pancreatitis, peritonitis, and massive gastrointestinal hemorrhage. Some immunocompromised individuals experience bacteremia as a result of campylobacteriosis, and in rare instances, some individuals have experience d meningitis, endocarditis, septic arthritis, osteomyelitis, or neonatal sepsis. Infection rarely results i n death, with 1 death per 20,000 infections (Allos

, 2001).

Several chronic sequel a e have been associated with Campylobacter infection including the development of GBS ( Fricker, 2006 a ). GBS is an acute demyelinating disease of the peripheral nervous syst em 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)

. T he risk of developing GBS after C. jejuni infection , however, is small ( approximately 1 case of GB S 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 (Nachamkin

, 2002; Garg

, 2006). Recent evidence h as 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).U.S. Environ mental Protection Agency

December 2007 14 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 a cute 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)

.
Campylobacter iosis Incidence

Alt hough Campylobacter became a reportable il lness 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). Ap proximately 2.4 million Campylobacter infections are estimated to occur in the United States
every ye ar ( 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 population (Allos

, 2001). According to reports from 1999, Britain experiences approximately 5 ti mes this infection rate (103 .7 cases per 100,000 population ) al 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 199 1 and 2004 (CDC, 2002).

II.1.3 Salmonella

Salmonella Strain Variation and Zoonotic Potential

Salmonella are non

- encapsulated, gram

- negative b acteria of the Enterobacteriaceae family that infect both animals and humans causing a wide range of illnesses (Cohen, 1986; Ohl, 2001; Lightfoot

, 2004). 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 six subspecies : S. e

. e nter ica ; S. e

. s alamae ; S. e

. a rizonae ; 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 flage llar antigens ( Lightfoot , 2004). 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 Salmon ella 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 repor ted annually in the Un ited States (Cohen, 1986; Lightfoot, 2004). Because the vast majority of Salmonella isolates from humans are of the subspecies S. enterica enterica , the CDC recommends that Salmonella strains only be referred to by their genus and

se rotype, e.g., S. typhi

.U.S. Environ mental Protection Agency

December 2007 15 S. typhimurium and S. enteritidis are the most prevalent ser otypes 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. n ewport , 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

caus ing 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. T he serotypes with a wide range of animal hosts can also infect humans, usually via food consumption, and cause self

limited enteritis 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 primari ly avian and reptilian reservoirs, respectively (Cohen, 1986). S. abortusovis is specific to sheep ( Lightfoot, 2 004). Salmonella has also been reported in swine, cattle, rodents, birds, turtles

, dogs, and cats ( Covert and Meckes, 2006 ). The CD C 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 7 deaths occurred was traced t o a water

- storage tower that allowed access to birds (Covert and Meckes, 2006). Although this outbreak was from drinking rather than recreational water
, it illustrates the potential for waterborne exposure due to birds.

Salmonella Route of Exposure

Salm onella infections begin with the ingestion of organisms in contaminated food or water ( Ohl, 2001; Lightfoot

, 2004). Ingestion or exposure to infected water from recreational swimming is less common, but it has been reported worldwi de (Cohen, 1 986; Lightfo ot
, 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 envi ronments like the stomach (Ohl

, 2001).

Elevated levels of Salmonella also have been observed in major water bodies that receive discharges of m eat 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 anim als

. 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 Illnes s Symptoms

I llnesses caused by Salmonella range from asymptomatic colonization and mild gastroenteritis to the more serious enteric fever (typhoid) , meningitis, and osteomyelitis (Cohen, 1986). EntericU.S. Environ mental Protection Agency

December 2007 16 fever and gastroenteritis are the key clinical syndr omes 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 ( Lightfoot, 2004). 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 (Ohl

, 2001). The pathological sign of enteric fever is mononuclear cell infiltration and hypertrophy of the reticuloendothelial system (Ohl

, 2001).

Salmonellosis i s caused by ingestion of non typhoidal salmonellae (e.g., S. enteriditis and S. typhimurium ) with an incubati o n period of 8 to 72 hours ( Lightfoot, 2004). The estimated inoculum size of non typhoidal Salmonella required to cause symptomatic disease in healt hy adult volunteers is 10 5 to 10 10 organisms ( Lightfoot, 2004). The infectious dose varies depending on the age and health of the person , strain differences, and the vector. Most

s almonellosis cases are self

- limiting

, and the affected persons recover wit hout treatment (CDC, 2006; Cohen, 1986)

. S ome infections are more severe , however, particularly in the young, elderly, and people with weakened immune systems and may become invasive ( APHA, 2004 ; Lightfoot, 2004 )

.

The c linical symptoms of s almonellosis may include diarrhea, abdominal pain, nausea, chills, fever, and prostration with the duration of illness ranging approximately 2 to 7 days (WHO, 2004; APHA, 2004 ). V omiting may occur as well, but it is rare and usually a sign of invasive disease ( APHA, 2004 ). Organisms that leave the gastrointestinal 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, endoc arditis, meningitis, pericarditis, pneumonia, pyodrema, or pyelonephritis (APHA, 2004).

Diarrhea from Salmonella infection is usually self

- limiting and does not require treatment unless it is severe. Overall t here is an estimated 22.1 percent hospitaliza tion rate, and an est imated 0.8 percent fatality rate (USDA, 2005). M ortality from S. typhi and S. paratyphi is estimated to be betwee n 10 to 15 percent without treatment (Ohl, 2001). In severe cases, fluid and electrolyte replacement may be needed. Ant ibiotics are not recommended to treat Salmonella infections, except where there is evidence of invasion and s epticemia, because they do not alleviate the symptoms or reduce the duration of the illness ( Kanarat , 2004) . Antibiotics may even prolong excretio n of Salmonella in the feces ( Kanarat, 2004).

Salmonell osis IncidenceU.S. Environ mental Protection Agency

December 2007 17 Foodborne Salmonella are the estimated cause of approximately 1.4 million foodborne

- related illnesses, 15, 6 00 foodborne illness

- related hospitalizations, and 5 5 0 foodborne

- related de aths each year in the United States (Mead et al., 1999). In 2005

, 45,322 s almonellosis cases were reported to the CDC through the Public Health Laboratory Information System (CDC, 2007 a ). 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 non typhoid 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 μm long and 0.1 μm 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. biflexa ) has been replaced by a phenotypic classification system in which 1 3 genomospecies are currently defined ( L. interrogans, L. noguchii, L. santarosai, L. meyeri, L. wolbachiic, L. biflexac, L. fainei, L. borgpetersenii, L. kirschneri , L. weilii, L. inadai, L. parvac, and L. alexanderi ) ( Levett, 2001). Of the 28 serovars

, several occur in more than one genomospecies (Levett, 2001). 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 ) ( Levett, 2001; Kanarat, 2004).

Complete genome sequences of L. interrogans serovars Copenhageni and Lai genome reveal that despite overall genetic similarity there are significant structural diff erences in their genomes ( Nascimento et al., 2004). Nascimento et al., 2004 analyzed the genomic sequences to gain insight into genes that influence m otility

, 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, slaugh terhouse workers, and veterinarians ) ( Meites et al. , 2004). Recreational exposure from swimming or boating in freshwater lakes is also possible (Meites et al. , 2004, Levett, 2004). Outbreaks are often associated with unusual rainfall events or flooding ( Bolin et al., 2004 b ).

Leptospira Illness SymptomsU.S. Environ mental Protection Agency

December 2007 18 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 f ollowed 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 t he convalescent stage corresponds with the appearance of antibo dies 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 leptospir osis 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 (Levett, 2001)

. Between 5 and 10 percent of all patients with leptospirosis have th e icteric form of the disease (Levett, 2001)

.

S ymptoms associated with leptospirosis include: jaundice, anorexia, headache, conjunctival suffusion, chills, vomiting, myalgia, abdominal pain, nausea, cough, hemoptysis, hepatomegaly, lymphadenopathy, diarrh ea, rash (usually lasting less than 24 hours), and fever (may 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 ; h owever, the Hawaii Department of Health still requires reporting ( Katz, 2001; Levett, 2001).

Leptospirosis Incidence

Levett (2001) summarized information for 28 waterborne outbreaks of leptospirosis worldwide, 22 of which were associated with swimming, o ne with kayaking, and one 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 distri bution 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 micr ovillous 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 en vironment. These oocysts can survive in environmental conditions for long periods of time (up to months) until ingested by a new host. In the new host, the lifeU.S. Environ mental Protection Agency

December 2007 19 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 waterborne oocysts are resistant to conventional drinking water treatment measures such as chlorination. Cryptosporidium is recognized as a widespread pathogen for the gen eral population including both immunodeficient and immunocompetent humans (WHO

, 2006).

Cryptosporidium Life Cycle and Strain Variation

Cryptospor idium has a complex life cycle but the sporulated oocyst is the only stage that can survive in the environmen t to infect susceptible hosts. Each oocyst, which has an environmentally resistant wall, holds four sporozoites. O oc yst s enter the environment by passing with the feces of an infected host organism (Fayer and Ungar

, 1986 ; Fayer et al.

, 1997). Oocysts

are immediately infectious and may remain in the 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, 200 1 a ). The sporozoites transform through several life stages in cluding an asexual and a sexual reproduction cycle. Oocysts are the result of the sexual reproduction cycle. Oocysts of the two species of Cryptosporidium responsible for most human infections, C. hominis and C. parvum

, are spherical with a diameter of 4 to 6 μm. Thin

- walled oocysts may excyst within the same host and start a new life cycle (autoinfection), whereas thick

- walled oocysts generally are shed by the host. Autoinfection may lead to a heavily infected epithelium of the small intestine resultin g in secretory diarrhea (WHO

, 2006).

Cryptosporidium is part of phylum Apicomplexa, family Cryptosporidiidae, and has been classified as a member of the group of eimeriid coccidia, a diverse group of parasitic protozoa (WHO

, 2006). There are currently 16 species of Cryptosporidium identified in the literatur e (Table II.2.1

- 1) . However, this taxonomy is likely to change as molecular methods continue to characterize isolates and potential new species. Fayer (2004 a ) 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 o r 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

. Studies published prior to 2002 should be in terpreted 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 usually cause illness in humans.

Table II.2.1

- 1. Cryptosp oridium Species Cryptosporidium Species Initially Described Host Species
C. andersoni Bos taurus ( domestic cattle) C. baileyi Gallus gallus (domestic chicken)

C. bovis Bos taurus (domestic cattle)

C. canis Canis familiaris (dogs)U.S. Environ mental Protection Agency

December 2007 20 Cryptosporidium Species Initially Described Host Species

C. felis Felis catis (domestic cat) C. galli Gallus gallus (domestic chicken)

C. hominis Homo sapiens (humans, formerly C. parvum genotype 1) C. meleagridis Meleagris gallopavo (turkey)

C. molnari Dicentrarchus labrax (fish)

C. muris Mus musculus (ho use mouse)

C. nasorum Naso lituratus (fish) C. parvum Mus musculus (house mouse) (formerly genotype 2) C. scopthalmi Scopthalmi maximus (turbot) C. serpentis Elaphe guttata (corn snake) E. subocularis (rat snake) Sanzinia madagascarensus (Madagascar bo a)

C. suis Sus scrofa (pig)

C. varanii Varanus prasinus (e merald monitor lizard )

C. wrairi Cavia porcellus (guinea pig)

Source: Adapted from Cacciò, 2005; Fayer

, 2003, 2004 a ; Fayer et al., 1997, 2000; Morgan

- Ryan et al.,

2002; Ryan et al., 2003; Xiao a nd Ryan, 2004 ; and Fayer and Xiao, 2007

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 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 demon strated 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 strai ns 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 diffe rent 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 di fferent 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

Hum an cases of cryptosporidiosis typically have been associated with different kinds of animal contact, which has led to the belief that the host

- range of Cryptosporidium is very broad and many animals can serve as reservoirs for Cryptosporidium

. Cryptospori dium infection has beenU.S. Environ mental Protection Agency

December 2007 21 reported in more than 155 mammalian species (Fayer

, 2004 a ) as well as numerous reptiles, amphibians, birds, and fish (O’Donoghue

, 1995).

A number of lines of evidence indicate that livestock, primarily cattle and sheep, are a maj or source of Cryptosporidium contamination of drinking water source s. 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 (Fayer , 2004 a ; WHO

, 2006). Also, direct zoonotic transmission of Cryptosporidium infection from livestock to humans has been repeatedly demonstrated (WHO

, 2006). During o utbreaks of cryptosporidiosis, frequent detection of Cryptosporidium in human stool sample s suggests that human sources can also add significantly to the occurrence of oocysts in source waters (Fayer, 2004 a )

.
Based on data from the western United States, depending on climate and feedlot management systems, the average animal in a cattle feedlo t 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 perc ent of farm

- associated streams contained C. parvum (Sischo et al., 2000). 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 ra in 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 th at the zoonotic C. parvum may be responsible for the majority of sporadic cases. Atwill et al. (1997) reported that feral pigs may serve as an environmental reservoir for C. parvum and may represent a potential source of Cryptosporidium contamination of r ecreational 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 wate r are known to vary seasonally (USEPA

, 2005 a, 2005b ), and short

- term levels in surface water can be strongly associated with storm events or other weather variables (Fayer

, 2004 a ; Naumova et al.

, 2005 ; USEPA , 2005 a ; 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 ani mal contamination, t he other major source of Cryptosporidium in ambient water is human fecal wastes. Several mechanisms can be responsible for the contamination of source waters: malfunctioning septic systems, routine or ―upset‖ releases from municipal w astewater 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 c onventional 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/LU.S. Environ mental Protection Agency

December 2007 22 (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 2 watersheds with the highest proportion of agricultural land use had the highest average Cryp tosporidium levels 2 and that approximately 90 percent of the samples exhibited the bovine (cattle) genotype ( C. parvum ). These findings implicate livestock, at least in these watersheds, as the major contributor to overall Cryptosporidium levels. This fi nding is consistent with studies indicating that the prevalence of Cryptosporidium infection in young livestock ( i.e., calves and lambs) is very high (Fayer

, 2004 a ) and that the oocyst counts in feces of young infected animals ranges from 10 6 to 10 8 oocyst s 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 prima ry effluents, 58 percent of secondary effluents and 19 percent of tertiary effluents in the 289 samples analyzed. Zhou et al. (2003 ) analyzed 179 wastewater treatment plant effluent samples from Milwaukee, Wisconsin using polymerase chain reaction (PCR) r estriction 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 fre quently than C. parvum (2.8 percent). This finding suggest s that the distribution of Cryptosporidium in Milwaukee’s population was not heavily influenced by agricultural sources although the relative low levels of C. parvum in wastewater may also have bee n 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 varia ble.

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. Se condary contact or partial body contact recreation such as wading,

canoe ing, 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 res ult in significant expo sure (US EPA

, 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/h and 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 popu lation.

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.

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

December 2007 23 (2005) suggest 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 eviden ce that direct
person

- to

- person transmission, as well as indirect transmission through contact with contaminated objects, can be a significant rout e of infection, especially where human population densities are high or personal contact is frequent (USEPA

, 2001a ). Direct transmission is affected by behavioral factors (e.g., frequent travel) and ethnic and dietary differences (USEPA

, 2001a ). Using da ta 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 ). A mong children and their caretakers, however, the transmission rate is considerably higher (1 2 to 22 percent ) ( Osewe et al.

, 1996).

Cryptosporidium Illness Symptoms

Cryptosporidiosis is primarily characterized by gastrointestinal symptoms such as profuse watery diarrhea; however, diarrheal symptoms are generally not distinguishable from those c aused 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 ov erall co ndition (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).

S ome infections may be asymptomatic. In other words, not all infections will re sult in illness and observable symptoms. A symptomatic hosts may still shed oocysts , however . Asymptomatic carriage, as determined by stool surveys, generally occurs at very low rates (less than 1 percent ) in industrialized countries ( Current and Garcia

, 1991) although high er rates have been reported in day care centers . Routine bile endoscopy suggests a higher asymptomatic prevalence; for example, 13 percent of non diarrheic patients were shown to carry Cryptosporidium oocysts ( Roberts et al.

, 1989). Hig h rates of asymptomatic infection (between 10 and 30 percent) are common in non industrialized countries (Current and Garcia

, 1991).

In more severe illnesses, the parasite may be found in the stomach, colon, liver, or lungs with associated symptoms corre sponding 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 infec ted 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, ear s , 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 notU.S. Environ mental Protection Agency

December 2007 24 currently available to document the degree to which these individuals are subject to elevated risk. Previous exposure to Cryptosporidium does confer some 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 additiona l 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. I mmunocompromised populations include patients undergoing chemotherapy for treatment of neoplasms, persons undergoing immune suppression treatment t o 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 mm 3 of blood are at increased risk of illness from Cryp tosporidium

, and individuals with less than 50 cells per mm 3 are at the greate st risk for

sever e disease and prolonged carriage of Cryptosporidium (Hunter and Nichols

, 2002 ; Roefer et al.

, 1996).

Sequelae in immunocompetent patients also have been documented. After resolution of the acute phase in the two months following their in itial 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 infection s have sometimes resulted in recurrence of GI symptoms, but only C. hominis infections have been rela ted 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 v isits annually for diarrhea and that approximately 2 percent of these, or 300,000 cases, are due to cryptosporidiosis. Mead and et al. (1999) also estimated that of these 300,000 cases, only about 10 percent are attributable to foodborne transmission, wit h 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 gastroenteritisU.S. Environ mental Protection Agency

December 2007 25 (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 AIDS patients, only 13 cases of cryptosporidiosis had been documented (Ungar

, 1990). Subsequently, between 1982 and 1997, more than 1,000 cases of the disease were reported worldwide (Fayer 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 under estimate actual Cr y p tosporidium infection rates because most cases go unreported.

Figure II.2.1

- 1. Reported Cryptosporidium infections in the United States, 1999 to 2005.

Worldwide, infection is widespread, exceeding several million according to Cas emore et al. (1997). 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 (2007 b ) for the year 2005 reports 8,269 cases of Cryptosporidium infection nationally, and reports significant fluctuations in incidence between summer and other seasons, peaking in late July and early August. According to CDC (2007 b ), Cryptosporidium is the leading cause of diarrheal illness outbreaks in recreational (chlorinated and nonchlorinated)
water. Alt hough 8,269 cases were reported in the United States in 2005 , CDC (2007 b ) estimates that the total incidence of domestic cases of Cryptosporidium infection exceed s 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).U.S. Environ mental Protection Agency

December 2007 26 The large increase in the number of cases reported from 2003 to 2005 might have resulted from outbreak

- related case reporting (CDC

, 2007 b ) ; 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

, 2007 b ).

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

- 2 ).U.S. Environ mental Protection Agency

December 2007 27 Table II.2.1

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

Year Number of Cases Number of Outbreaks Sources

1991 0 0 NA

1992 526 2 Waterslide and wave pool

1993 174 4 Pools

1994 519 2 1 pool, 1 lake

1995 5,487 3 1 pool, 2 waterparks

1996 3,025 3 2 pools, 1 lake

1997 369 1 Fountain

1998 169 8 7 pools, 1 lake

1999 64 4 3 pools, 1 fountain

2000 1,368 13 12 pools, 1 lake

2001 538 4 3 pools, 1 hot spring

2002 936 7 6 pools, 1 l ake

2003 702 5 4 pools,* 1 lake

2004 567 7 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 Sur veillance 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, 1993 b , 1996, 1998, 2000, 2002, 2004, 2006).

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 ani mal species can carry and transmit Giardia lamblia (also known as G. intestinalis or G. duodenalis ), which causes the gastrointestinal 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 intes tinal epithelium, detach, then become rounded and form a cyst wall. The environmentally resistant cyst is excreted with the feces, where it moves passively through the environment and may be ingested by another host organism. Inside the digestive track o f 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

, 2007 b ). Furthermore, cysts can rem ain viable under typicalU.S. Environ mental Protection Agency

December 2007 28 environmental conditions for periods up to 77 days (Bingham et al.

, 1979

, as cited in USEPA

, 1998).

Currently, there are 5 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 ) . S ome 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 particular host s pecies (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. As semblages C and D seem to primarily infect dogs, while Assemblage E infects livestock

, and Assemblage F infects only cats ( Appelbee et al.

, 2005 ).

Table II.2.2

- 1. Giardia Taxonomy Species Assemblage Hosts
Giardia lamblia (syn. G. duodenalis

, G. intest inalis ) A1 Human, beaver, cat, lemur, sheep, calf, dog, fox, chinchilla, alpaca, horse, pig, cow

G. lamblia A2 Human, beaver
G. lamblia B

( G. enterica *) Human, beaver, guinea pig, dog, monkey, horse

(B IV)
G. lamblia C and D

( G. canis *) Dog, coyote, mou se

G. lamblia E

( G. bovis *) Cow, sheep, alpaca, goat, pig

G. lamblia F

( G. cati *) Cat

G. lamblia G

( G. simoni

*) Domestic rat

G. lamblia Novel I Marsupial (Quenda

- bandicoot, mouse, sheep)
G. lamblia Novel II Marsupial II (Tasmanian devil)
G. muris Rodents (mice)
G. microti Vole and muskrat G. psittaci Birds (budgerigars)
G. ardeae Birds (heron and ibis) G. agilis Amphibians (frogs)

Adapted from : Adam, 2001; Appelbee et al., 2005; Olson et al., 2004; Traub et al., 2005; and Hamnes et al., 20 07. * Denotes recently proposed new species names (Hunter and Thompson, 2005; Thompson and Monis, 2004)

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 ofU.S. Environ mental Protection Agency

December 2007 29 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, 19 98) 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 strai ns of Giardia that are phenotypically distinct from those found in humans. 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 ac quired 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 remains controversial. G. lamblia may be maintained independently through transmission cycles involving wildlife and livestock al though it is unclear how these cycles may interact in zoonotic transfer (Hunter and Thompson

, 2005). Although b oth livestock and humans have been implicated in contam inating water sources with Giardia , humans are responsible for the majority of the contaminations (Hunter and Thompson

, 2005). Hunter and Thompson (2005) examined case studies for the zoonotic potential of Giardia and found only one case having a signific ant 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. These authors did not rule out the importance of zoonotic transfer i ndirectly through water sources, however. Both Traub et al. (2004) and Inpankaew (2007) have show n that

, in some communities with inadequate sanitation, zoonotic transfer is evident between humans and dogs ; however, it is unclear which species is the prin cipal reservoir.

Thompson (2007) suggest ed that data gaps regarding zoonotic transfer for Giardia can be filled using molecular epidemiological studies . Molecular genotyp ing of parasite isolates from susceptible hosts in localized foci of transmission or longitudinal surveillance with genotyping might provide the needed data on zoonotic transfer

.

Giardia Routes of Exposure

Giardia is transmitted via fecal

- oral exposure and causes both endemic and epidemic cases of giardiasis. It is frequently spre ad 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 activitie s such as swimming.

Inhalation of aerosolized water that contains cysts is theoretically possible, but 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 water s.

Giardia Illness Symptoms

Giardia is responsible for a number of health effects including acute symptoms that occur during and after the infection, and Giardia has been implicated in a number of chronic sequelae. There is a wide spectrum of symptoms a ssociated with giardiasis that ranges from asymptomaticU.S. Environ mental Protection Agency

December 2007 3 0 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 (C DC, 2000; Hellard et al.

, 2000; Hopkins a nd 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, it may persist for y ears (US EPA

, 1998). Case reports also indicate that giardiasis might 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 months for others. Most infections resolve spontaneously

, and the acute stage l asts from 1 to 4 weeks (US EPA

, 1998)

, but individuals with compromised immune systems may have more serious and prolonged inf ection ( APHA, 2004 ). Immunodeficiency with varying degrees of hypogammaglobulinemia or agammaglobulinem i a is the most commonly reported form of immunodeficiency associated with chronic giardiasis (Farthing

, 1996). However, giardiasis is one of the few po tentially 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
symp toms (Mintz, 1993 ), and is especially common in children and in people with prior infections (CDC

, 2007 b ). In a study at the Swiss Tropical Institute, only 27 percent of 158 patients who had Giardia cysts in th eir 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 v olume depletion or dehydration is the most frequently listed codiagnosis on hospit al admission . A mong 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., ho spitalized 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 sign ificantly 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 an d behavioral factors that increase exposure (Robertson

, 1996).

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 diste nsion of the bowel, fou l 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,U.S. Environ mental Protection Agency

December 2007 31 the parasite and symptoms disappear spontaneously. Among 65 c ases 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, non progressive retinal lesions may occur in young children with giardiasis. The risk of retinal lesions did not seem to be related to the

se verity of infection, its duration, or use of metronidazole to treat the infect ion, and may reflect a genetic predisposition to retinal lesions (Corsi et al.

, 1998).

There is usually no extra

- intestinal invasion when Giardia trophozoites infect the small i ntestine, 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). Although all age groups are affected by giardiasis, the highe st incidence is in children (US EPA

, 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

, 19 98).

Communities with unfiltered surface water systems experienced a waterborne outbreak rate that

was 8 times greater than communities where surface water was bo th filtered and disinfected (US EPA

, 1998). Data therefore indicate that filtering water to r emove microorganisms substantially reduces risk of giardiasis.

As with all pathogens, underreporting limits estimate s of the true incidence of giardiasis. T he 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 non bloody 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 re ported in 2005 (CDC, 2007 b ) results in an estimated incidence of approximately 762,800 total cases in 2005 . However, broader estimates are also supported. For example, a n estimated 1 to 5 percent of cases of

sa lmonellosis are reported to CDC through pass ive surveillance ( Chalker and Blaser

, 1988 ). If the 1 to 5 percent reported cases is applied to the giardiasis data, then the giardiasis disease bur den in the United States in 2006 could have been 401,500 to 2,007,500 cases (135.4 to 677.0 cases per 100,0 00 population )

. 4 The true burden of giardiasis in the United States is likely to fall between these two estimates ( CDC

, 2007 b ).

4 Based on US Census data for 2006, the US population was e stimated to be 296,528,800U.S. Environ mental Protection Agency

December 2007 32 Using data from 1992 to 1997, the number of states reporting occurrence of giardiasis increased from 23 to 43

, while the annu al 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 19,6 59 (CDC, 2007 b )

. The cause of this decrease is unknown

. G iardiasis became a nationally notifiable disease in 2002 , and t he number of cases increased from 2001 to 2002, then stabilized, averaging approximately 20,200 cases per year (CDC

, 2007 b ). See Figu re II.2.2

- 1 and Table II.2.2

- 2 for reported cases of Giardia infection in the United States from 1992 to 2005.

Figure II.2.2

- 1. Reported Giardia cases in the 50 states plus Washington DC, 1992 to 2005

Table II.2.2

- 2. Giardia Infection Occurrence in U. S. Populations Population D escription Occurrence Reference

General 2

- 5% infection USEPA, 1998 Prevalence in stool samples (infection) 4

- 12% depending on year and state USEPA, 1998

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

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) USEPA, 1999

Homosexual males (in New York) 18% found posi tive for Giardia (infection) (compared with

2% among other patients, but giardiasis is not a major clinical problem in persons with HIV or AIDS) Faubert et al., 2000; Kean, 1979; USEPA, 1999
Small children 7% are asymptomatically infected with Giardia Fr ost and Craun, 1998U.S. Environ mental Protection Agency

December 2007 33 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. O utbreak

- related

cases made up 1.6 to 11.6 percent of the tota l number of cases reported annually for 1999 to 2002. Although the number of states reporting cases increased from 42 to 46 during th at 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 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 frequent ly in the early summer through early fall, with increases in transmission during the summer (CDC

, 2007 b ). This increase d 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 be asymptomatic, transmission in these facilities is likely to be an important mechanism for increased incidence during the summertime (CDC

, 2005). People at recreational beaches h ave 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 (Sunderland et al.

, 2007; Graczyk et al

. , 2007).

Giardiasis is found most frequently in children 9 years old and younger and in adults 35 to 44
years old . These groups correspond to young children and their caretakers, who are at increased risk of infection (CDC

, 2007 b ). In 2005, 54.3 percen t of reported cases occurred in males compared to only 43.9 percent in females (1.8 percent of reports did not record gender ) ( CDC

, 2007 b ). According to CDC (2007 b ), the discrepancy between cases in males versus females might be attributable in part to in creased risk of infection during sexual contact between men al though 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 widespre ad.

From 1991 to 2004, seven outbreaks of giardiasis have been associated with untreated recreational waters (Table II.2.2

- 3).

Table II.2.2

- 3 Outbreaks of Giardiasis Associated with Recreational Waters in the United States.

Year Number of Cases Number of Outbreaks Sources

1991 34 4 3 pools, 1 lake

1992 0 0 NA

1993 61 3 2 lakes, 1 river

1994 80 1 P ool

1995 0 0 NA

1996 77 1 PoolU.S. Environ mental Protection Agency

December 2007 34 Year Number of Cases Number of Outbreaks Sources

1997 0 0 NA

1998 0 0 NA

1999 18 1 Pond

2000 0 0 NA

2001 0 0 NA

2002 2 1 River

2003 212 2 Pools*

2004 9 1 Lake

* in one pool outbreak both Cryptosporidium and Giardia were detected in water samples, so that outbreak of 63 cases is counted in both the Cryptosporidium and the Giardia data. Source: Data from CDC Surveillance Summaries: Morbidity and Mortality Weekly Report (MMWR)

Surveillance for Waterborne

- Disease Outbreaks

- United States: 1991

- 1992, 1993

- 1994, 1995

- 1996, 1997

- 1998, 1999

- 2000, 2001

- 2002, 2003

- 2004 (CDC, 1993 b , 1996, 1998, 2000, 2002, 2004, 2006).

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 hos t 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 mode (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. V iruses that infect via the intestine and cause GI illness (e.g., enteroviruses) also may have secondary tropisms in oth er 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 waterborn e viruses of humans (e.g., noroviruses) are difficult to detect or quantify in cultured cells (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, assessm ent 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:

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 synd rome [SARS]

- associated coronavirus), cross

- species transmission has not been a traditional concern for waterborne, fecally transmitted viruses.U.S. Environ mental Protection Agency

December 2007 35 1. Animal reservoir: Does the agent regularly i nfect 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? 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?

Due to the potential for rapid evolution in viruses, ongo ing monitoring and research should be continued even though viruses are not 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.

Rota viruses

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 fec al

- 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 frequently occur 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 U nited States are treated in th e 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 cont amination of the water supply and a failure of chlorination treatment.

Hepatitis E

HEV may be zoonotic (US EPA

, 1999 ; Cliver and Moe, 2004 ). In pigs and rats, this virus is very similar to human HEV . Experimental studies have indicated that human strain s 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 w ater. 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 illn ess and death. For this sensitive subpopulation

, mortality can be as high as 2 0 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 incidenc e rates. In an EPA study of sporadic human HEV, nearly 50 percent of the infected persons had traveled to endemic a reas in other countries or received blood transfusions (USEPA, 1999).U.S. Environ mental Protection Agency

December 2007 36 Adenovirus

Adenovirus may be zoonotic (Mwenda et al., 2005). Mwenda et al. (2005) 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

non human primates to humans in Kenya (M wenda et al.

, 2005). 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 di seases including keratoconjunctivitis, upper respiratory tract infections, pneumonia, gastroenteritis, cystitis, and encephalitis (Gra y et al., 2005). Molecular studies have recently shown adenoviruses to be associated with bronchopulmonary dysplasia (Cou roucli 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 (Crawford

- Miksz a and Schnurr

, 1996 ; Baldwin et al., 2000 ). Infections in these populations may result in severe illness and death (Runde et al., 2001; Chakrabarti et al., 2002). U.S. surveillance for adenovirus is relatively incomplete (Gray et al.

, 2005), and well

- doc umented incidence rates are not available

, especially for waterborne outbreaks.



## III. P ATHOGEN 

## I NTERACTIONS WITH T HEIR 

## E NVIRONMENT

Pathogens interact with the ambient environment , other microorganisms, plants, and with their hosts. This section provides a s ummary 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. See the companion paper in this series ―Review of Fecal Indicator Organism Behavior in Ambient Waters and Alternative Indicators for Tropical Regions‖ for a review of indicator behavior.

Although there are important waterborne amoebic pathogens, they are not associated wi th animal fecal material. However, important b acterial pathogens that are associated with fecal material ( Salmonella and Campylobacter ) interact with free

- living amoebae in ways that could impact recreational water quality. The most notable, Legionella

, infect s and replicate s in free

- living amoebae and is considered more of a risk in drinking water than in recreational waters (Marrie et al

. , 2001; Borella et al., 2004) . Some human bacterial pathogens are even thought to have evolved in association with a moebae (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 byU.S. Environ mental Protection Agency

December 2007 37 5 diffe rent isolates of axenic Acanthamoeba species. Th e Dublin serovar was internalized more efficiently than the other two serovars

, and the Acanthamoeba rhysodes isolate was more efficient tha n the other 4 isolates. The researchers concluded that Acanthamoeb a speci es 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 differe nt 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 nonver tebrate 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 cat tle manure regardless of the temperature and manure form (solid or slurry) and Campylobacter and Giardia were weakest survivors in manure. The authors conclude that holding manure at 25 °C for 90 days will render it free from the pathogens considered. Thi s 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 temper ature. Additional environmental characteristics that may influence pathogen surviv al, infectivity, and virulence include: UV light (duration, i ntensity) , r ainfall , r unoff , d ispersal

, 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

. 6

Thomas et al. (2006) examined extreme rainfall and spring snowmelt in association wit h 92 Canadian waterborne disease outbreaks between 1975 and 2001. A ccumulated rainfall, air temperature, and peak stream flow were used to determine the relationship between high impact weather events and the occurrence of wate rborne disease outbreaks. F or rainfall events greater than the 93 rd percentile

, there was a greater than 2

- fold increase in the odds of an outbreak compared to rainfall events less than the 93 rd percentile. For each degree

- day above 0 °C

, the relative odds of an outbreak increased b y 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 (5*42) = 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.

6 The influence of the above factors on bacterial indicators is discussed in the companion paper in this series

― Review of Fecal Indicator Organism Behavior in Ambient Waters and Alternative Indicators for Tropical Regions ‖U.S. Environ mental Protection Agency

December 2007 38 An overview of the available information for the six key waterborne zoonotic pathogens follows. I nformation concerning these factors is limited for most waterborne pathogens. To develop effective control strategies, it is clear that further research is warranted.

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 t o non pathogenic 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 III.1.1

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

Water matrix °C Days to D ecrease Reference 99% 99.99% Surface waters (lakes and rivers) 6 4

– 11 8

– 22

Czajkowska et al., 2005 Surface waters (lakes and rivers) 24 2

– 8 5

– 10 Sediments (same lakes and rivers) 6 10

– 20 25

– 39 Sediments (same lakes and rivers) 24 5

– 8 10

– 30
River water with feces 15 7.5 14.5 McGee et al., 2002

Unfiltered lake water 8 91 NA Wang and Doyle, 1998 Filtered tap water 15 >91 NA
Unfiltered lake water 25 14

- 28 NA

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 ( Gagliard i and Ka rns, 2002)

.

III.1.2 Campylobacter Survival in the Environment

Campylobacter has been shown to survive in aquatic environment s with low temperatures (4 °C ) between 8 days (Buswell et al.

, 1998) and 4 months (Rollins and Colwell

, 1986)

.

Buswell et al. (1998) fou nd 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 h ours at 37°C. The s urvival times were considerably longer in the presence of the autochthonous water microflora ( two strains tested survived 700 and 360 h ours at 4°C)

. Aerobic conditions decreased the survival of one strain 30 percent and increased the p ersistence of another strain by more than 3

- fold. W ithin biofilms

, the pathogen persisted up to the termination of the experiments after 28 and 42 days of incubation at 30 and 4°C, respectively.U.S. Environ mental Protection Agency

December 2007 39 Rollins and Colwell ( 1986 ) found that Campylobacter incuba ted 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, s oil, 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 gro w at temperatures ranging from 10 to 43 oC , but some serovars have suppressed growth at temperatures above 40 oC (Cov ert and Meckes, 2006). Salmonella can grow at pH 4

– 8 and at water activities above 0.93. U nder some conditions, Salmonella may proliferate below 4 oC and survive below pH 4 ( Lightfoot , 2004).

III.1.4 Leptospira Survival in the Environment

Leptospira survive lo nger 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

a ssociated 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 patho genic Leptospira was higher in urban than rural water sources and rats were the indicated zoonosis.

III.1.5 Cryptosporidium Survival in the Environment

Because Cryptosporidium oocysts are remarkably resistant to environmental or engineered degradation, the surv ival of Cryptosporidium under a variety of environmental and drinking water treatment conditions has been evaluated by a number of investigators. While the majority of these studies have considered the effects of physical antagonism (e.g., freezing, heati ng, and 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 will focus primarily on aspects of physical antagonists in the environment because they are most pertinent to the topic of this white 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 tempera ture 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 steadi ly with time. (In the study, the relationship between oocyst viabilityU.S. Environ mental Protection Agency

December 2007 40 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 samp les. 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 rema ining relatively insensitive to some water treatment processes. Robertson et al. (1992) also emphasize d that oocyst viability depend s 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

, 200 1 a ). Warm and boiling water completely neutralizes oocysts, and the temperature of the water determines the time required for the treat ment to become effective (Anderson

, 1985). Cryopreservation studies conducted by Fayer et al. (1991) and Fayer et al. (1997 ) indicate that oocyst survival depends on the temperature and duration of freezing conditions, implying that C. parvum oocysts are not necessarily rendered noninfectious by being frozen per se

. In another study, Fayer and Nerad (1996) demonstrated that the infectivity of C. parvum oocysts after freezing is dependent on the temperature and duration of freezing. In general, shorter fr eezing times are required to neutralize infectivity when lower freezing temperatures are employed (e.g., 1 hour at

- 70 °C v ersus 168 hours at

- 15 °C to completely neutralize infectivity) (Fayer and Nerad, 1996).

Temperature stability studies also were condu cted 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 t his study indicated that oocyst stability under freezing co nditions 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 tend ed to promote the survival of most microorganis ms. In water, C.
parvum may survive outside of mammalian hosts for several months or more depending upon water temperature (Straub et al., 1994).

Fayer 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 rem ained infectious for up to 24 weeks in 20 °C water (Fayer et al., 1998 ). As water temperatures were increased above 20 °C , oocysts retained their infectivity for shorter exposure times (Fayer et al., 1998 ). Under conditions of high water temperatures , high er than typically found in surface waters

, Fayer (1994) indicated that all evidence of C. parvum infectivity was lost within 60 seconds when temperatures exceeded 72 °C or when temperatures of at least 64 °C were maintained for 2 minutes

.
Holding oocysts to 45 °C for 5 to 20 minutes was effective in completely neutralizing infectivity (Anderson, 1985). Anderson (1986) examined the 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

-U.S. Environ mental Protection Agency

December 2007 41 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 line arly 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 (13.9Mpa) for 1 minute had little reduction in viability.

Sattar et a l. (1999) 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 exclu ded from the suspension water . The observation implies that microbial predation may play an important role in reducing oocyst survival in natural waters.

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

- off of C. parvum

. Expe riments 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 t he 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 (Fayer

, 2004 a ) and for several weeks in seawater (WHO

, 2006). These studies indicate that Cryptosporidium can thus pose a serious health hazard to humans by direct and indire ct contact in recreational waters

.
King et al. (2005) measure d oocyst inactivation rates in reagent

- grade and environmental waters over a range of temperatures. O ocysts incubated at 4 and 15 °C remained infective over a 12

- week holding period. A 4 log 10 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 previously reported. I nactivation at higher temperature s 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 l oss of infectivity was primarily due to partial or complete in vitro excystation during the fir st 24

- h our 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

,U.S. Environ mental Protection Agency

December 2007 42 rapid inactivation occurs at a rate equal to or greater than 3.27 log 10 reduction per day for C.
parvum oocysts deposited in the feces of cattle.

Méndez

- Hermida et al. (2005) report ed on batch

- process solar disinfection of

. C. parvum oocysts in water. Oocyst suspensions were exposed to simulated sunlight (830 W /m 2 ) at 40 °C

. Viability
assays and infectivity te sts indicated that exposures of 6 and 12 h ours reduced oocyst infectivity from 100 percent to 7.5 percent and 0 percent , respectively.

The behavior of lake inflows is importa nt 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 bot tom 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 predatio n by other microorganisms. Brookes et al. (2004) found that inactivation of Cr yptosporidium 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 non filter

- sterilized river water . They reported that as the temperature was increased from 4 to 23 °C , the infectivity of oocysts decreased ; n o infectious oocysts were detected after 1 week at

- 20 °C

.

Excreted Cryptosporidium oocysts can s urvive for substantial periods in animal wastes and soils. Thus, contaminated runoff can enter ambient water, resulting in potential human exposures. The numbers of oocysts excreted by infected young animals may be especially large, between 1 and 10 mill ion 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 inacti vated by repeated freeze

- thaw cycles independent o f the number of times the soil was frozen (Fayer

, 2004 a )

. 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

, there is very little inactivation of oocysts in different types of agricultural soils (Jenkins et al.

, 2002; WHO

, 2006). Factors that are known to reduce oocyst survival in soils include dryi ng and basic pH (Faye r , 2004 a ). 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 approximat ely 6 months, with approximately 37 percent inactivation after 2 days exposure to tap water (USEPA

, 2005 a, 2005b ).

Walker et al. (2001 ) tested oocyst degradation (as indicated by microscopic examination) in response to the combined stresses of water pot ential ( 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 exponenti al 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 ofU.S. Environ mental Protection Agency

December 2007 43 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 po pulations 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 ( US EPA

, 1998). Survival was determined based on dye inclusion/exclusion, excystation

, or animal infectivity studies. Some studies used Giardia muris cysts
, w hereas 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 ( US EPA

, 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 (a s reported in US EPA

, 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, th e se 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 c ysts were infective for 11 weeks in water, 7 weeks in soil, and 1 week in cattle fece s. 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 N orway. 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 viable morphology could be detected after 1 month (Robertson and Gjerde, 2006)

. B oili ng Giardia for 1 minute reduces viability (as determined by flurogenic dyes) to less than 1 percent and renders them non infectious (as determined by animal infecti vity) (El Mans oury et al., 2004)

. Storage at 4 °C and

- 4 °C for up to 7 days preserves Giardia cyst viability and infectivity (El Mansoury et al., 2004)

. Storage at 30 ppt 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 stabl e at lower temperatures; however, different viruses have different stabilities under similar environmental conditions. For example, astroviruses survive longer than poli o virus 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 2 8 °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, 200 6a). Enterovirus nucleic acid is detectableU.S. Environ mental Protection Agency

December 2007 44 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 su rvive longer in water than enteroviruses and hepatitis A vir us (Enriquez and Thurston

- Enriquez, 20 06 ).

Pesaro et al., (1995) evaluated the persistence of five animal viruses, representing picorna

- , rota

- , parvo

- , adeno

- , and herpesviruses, and the colipha ge 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 c onclude 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 Animal s Can Influence Pathogen Characteristics and Mechanisms of R apid 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 in fluence 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 g ave 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 mechanisms of phenotypic change in pathogens are , genetic diversity (coinfection and quasispecies), cryptic genes, mutators, and epigenetic e ffects

, are summarized as follows

.
Genetic diversity (co

- infection and quasispecies): The patho gen 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, ca liciviruses 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 rang e from 0.11 to 3.4 percent (Grandadam et al.

, 2004). For retroviruses, every virus particle may b e genetically different from every other particle. In fact, theU.S. Environ mental Protection Agency

December 2007 45 cooperativity 7 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. 8 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 p lague (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). T his ty pe 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 antig en 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 gene s have been identified that influence the mutation rate of bacteria. For example, bacterial cells that carry the MutD5 protein, which binds to DNA polymerase, accumulate a broad spectrum of base substitutions and frameshift mutations (the mutation frequen cy 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 e ffects 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. coli, S

. enterica (serovar Typhimurium), Vibrio cholerae

, Y. pseu dotuberculosis

, Y. enterocolitica

, Helicobacter pylori , and Campylobacter are a few notable pathogens for which DNA methylases are known to regulate gene
exp ression (Fälker 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

.

7 The hypothesis that swarms of viral genotypes cooperate to comprise an ―average phenotype‖ differs from quorum sensing, which is the coordinat ion of bacterial cells via cell

- to

- cell communication with small signaling molecules to form biofilms and other group

- related phenotypes.

8 In this case, the presence of the gene leads to decreased virulence because the gene product facilitates chronic inf ection, which is less severe than acute infection.U.S. Environ mental Protection Agency

December 2007 46 All of the se mechanisms can contribute to rapid evolution of zoonotic pathogens. 9 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 a long with an escalation of pathogenicity in the new host. 10 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). 11 I n SPEs when only one or a few pathogen s are transferr ed 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

zoo notic 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

n ecessarily 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 in fections 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. L ow virulence can lead to missed oppor tunities to spread due to low pathogen numbers . H igh 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 betw een pathogen and host drives pathogen (and host) evolution 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 t o 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 human s when pass ing 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 pas sing 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 Be ltsville (B) or C. parvum Iowa (I). Calves given the B isolate excreted 5

- fold more oocysts than those receiving the I isolate. Quantitative reverse

9 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.

10 The nega tive 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).

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

December 2007 47 Text Box III.2

- 1. Examples of Feeding Studies with Zoonotic Parasites

For the widespread waterborne zoonotic protozoa Cryptosporidium and Giardia , human volunteers (immunoc ompetent 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 integri ty 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 inocul um 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 eac h 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 i nfectivity 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 Cryptospori dium oocysts. Subjects were then evaluated for Cryptosporidium in stool samples and for diarrheal and other GI illness symptoms. Infectivity was estimated for five C. parvum isolates : TAMU (collected from a veterinary student); Iowa (derived from a calf ); UCP (derived from a calf); Moredun, (collected from a red deer calf); 16W (from a calf); and TU502 (an isolate collected from an infected child and propagated in gnotobiotic piglets [Chappell et al.

, 2006]). Cryptosporidium from animal sources were fou nd 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 wei ghs 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 disco vering the mechanisms by which C. hominis is maintained in natural setting when C. parvum is also present should improve understanding of risks to humans.

Giardia in Humans Rendtorff (1954) conducted a controlled, clinical study of male prison volunteers who were fed Giardia cysts obtained from a human source. Giardia cysts of known numbers varying from 1 to 10 6 were placed into gelatin capsules along with a small amount of saline. The capsules were given to the volunteers along with 4 to 6 ounces of wat er. 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 viab ility could not be determined prior to administration to volunteers, the failure to elicit infection in the five men treated with a dose that was calculated to contain only one cyst may have been due to dosing with inactive cysts. The Giardia Assemblages used in these studies are not known but presumably are A or B because these are the Assemblages known to infect humans

.U.S. Environ mental Protection Agency

December 2007 48 transcriptase

- PCR indicated that the B isolate contained a 3

- fold greater number of the symb iont 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 inve rse 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 suscepti ble to the effects of virus infection. Jenkins et al. (2007) also indi cated 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.



## IV. S UMMARY

Contamination o f 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 pathoge ns 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 (see companion paper entitled

― Review of Published Studies to Characterize Relative Risks from Different Sources of Fecal Contamination in Recreational Water ‖). The six most important zoonotic waterborne pathogens at the present time are:

Pathogenic E. coli Salmonella Campylobacter Leptospira Cryptosporidium Giardia

All of these are li kely to cause more severe symptoms in children and immune suppressed individuals than in the remainder of the population. Of these five, pathogenic E. coli has the most potential for severe acute effects that can even be fatal. Potential debilitating chr onic sequelae such as Guillain

- Barré 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 gastrointestinal illness, the wat erborne zoonotic pathogens discussed in this report have the potential to cause serious health effects. Even though serious health outcomes are likely to be rare in comparison with self

- limiting illnesses, the public health impact of the rare, but more se rious illnesses should not be overlooked.



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December 2007 A

- 1 

## A PPENDIX 

## A

W ATERBORNE 

## P

ATHOGENS

As described i n Section I.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., 2004 a ) . Table A

- 1 lists all the pathogens that were evaluated for i nclusion in this
paper

. Information provided in Table A

- 1 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 impor tance 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.U.S. Environmental Protection Agency

December 2007 A

- 2 Table A

- 1: Known and Potential Zoonotic Waterborne Pathogens

Type Pathogen Waterborne Zo onotic Host Zoonotic Host is Warm

blooded Illnesses and

Symptoms in Humans (less

common symptoms) U.S.

Outbreaks Importance

to consider if animal fecal

sources are discounted in AWQC References

Bacteria Acinetobacter Yes (generally in environment) No ne NA Septicemia, meningitis, endocarditis, brain abscesses, pneumonia, empyema, urinary tract infections, eye infections, and skin and wound infections Hospital

settings No Stewart and Rochelle, 2006

Bacteria Aeromonas Yes (generally in environment) No ne NA G astroenteritis (septicemia) None

reported No Moyer and Standridge, 2006; Lightfoot, 2004

Bacteria Camp

ylobacter Yes Poultry, cattle, sheep, and wild birds Yes Diarrhea, abdominal pain, malaise, fever,
nausea, and vomiting (typhoid

like syndrome, febrile c onvulsions, meningeal arthritis, reactive arthritis, and GBS) Mainly

foodborne and drinking water Important Fricker, 2006a; APHA, 2006; Allos, 1998, 2001

Bacteria Cyanobacteria Yes (generally in environment) No ne NA Rash and gastroenteritis Drinking

water and recreational water No Fred e ricksen et al. , 2006U.S. Environmental Protection Agency

December 2007 A

- 3 Type Pathogen Waterborne Zo onotic Host Zoonotic Host is Warm

blooded Illnesses and

Symptoms in Humans (less

common symptoms) U.S.

Outbreaks Importance

to consider if animal fecal

sources are discounted in AWQC References

Bacteria V erotoxin

- producing E.coli

(VTEC)

– includes enteroh emorrhagic E. coli (EHEC), including O157:H7 Yes Cattle, chicken, sheep, pigs, horses, dogs, and deer Yes Diarrhea (bloody), severe abdominal cramping, headache, hemorrhagic colitis, and hemolytic uremic syndrome Foodborne

and waterborne ( drinking water and recreational water ) Important Mølbak and Scheutz, 2004; APHA, 2004; Degnan and Standridge, 2006

Bacteria E nterotoxigenic E. coli

(ETEC) Ye s Same as VTEC Yes Acute

, watery diarrhea Waterborne Possibly important Hunter, 2003;

Mølbak and Scheutz, 2004 Bacteria A ttaching and effacing E.

coli (A/EEC) Yes Same as VTEC Yes Acute or persistent diarrhea No

waterborne reported Possibly

important Møl bak and

Scheutz, 2004

Bacteria E nteropathogenic E. coli

(EPEC) Yes Same as VTEC Yes Acute or persistent diarrhea W aterborne Possibly important Lee et al, 2002;

Mølbak and Scheutz, 2004 Bacteria E nteroaggregative E. coli

(EAggEC) Yes Same as VTEC Yes Acu te

, watery, and often protracted diarrhea No

waterborne reported Possibly

important Mølbak and

Scheutz, 2004

Bacteria D iffuse adherent E. coli

(DAEC) Yes Same as VTEC Yes Acute or persistent diarrhea No

waterborne reported Possibly

important Mølbak and

Sc heutz, 2004

Bacteria E nteroinvasive E. coli

(EIEC) Yes No ne NA Acute, often inflammatory diarrhea; dysentery No

waterborne reported No Mølbak and Scheutz, 2004U.S. Environmental Protection Agency

December 2007 A

- 4 Type Pathogen Waterborne Zo onotic Host Zoonotic Host is Warm

blooded Illnesses and

Symptoms in Humans (less

common symptoms) U.S.

Outbreaks Importance

to consider if animal fecal

sources are discounted in AWQC References

Bacteria Flavobacterium Yes No ne NA Gastroenteritis, meningitis, pneumonia, endocarditis, and septicemia Rare

waterborne (stagnation in drinking water), hospital settings more

common No Geldreich and Degnan, 2006a

Bacteria Helicobacter pylori Possibly Weak evidence for ferrets,
racoons, swine, sheep, rodents, and primates Yes Gastric disorders, pe pticand duodenal ulcer disease, lymphoma of the digestive tract, and ademenocarcinoma of the stomach No

waterborne reported No Baker and Degnan, 2006; Health Canada, 2006

Bacteria Klebsiella Yes (generally in environment) Warm

- blooded

animals Yes Infectio ns in respiratory system, genitourinary tract, nose, and throat, (meningitis and septicemia) Hospital

settings No Geldreich and Standridge, 2006

Bacteria Legionella Yes No ne NA Legionellosis, pneumonia, Legionnaire ’ s disease, and Pontiac fever Hospitals,

pools, and spas No Hall, 2006

Bacteria Leptospira Yes Rats, dogs,
raccoons, swine, and cattle Yes Leptospirosis (Weil’s disease) Recreational

waterborne over 50% of

cases in Hawaii Important Levett, 2001U.S. Environmental Protection Agency

December 2007 A

- 5 Type Pathogen Waterborne Zo onotic Host Zoonotic Host is Warm

blooded Illnesses and

Symptoms in Humans (less

common symptoms) U.S.

Outbreaks Importance

to consider if animal fecal

sources are discounted in AWQC References

Bacteria Listeria monocytogenes No Domestic and

w ild animals Yes Listeriosis, meningoencephaliti s, fever, and abortion Foodborne No APHA, 2004

Bacteria Mycobacterium avium

complex (MAC) and ssp. Paratuberculosis (MAP) Yes (generally in environment) Possibly

sheep, cattle, goats , and birds Yes Respirator y infection, fever, and Crohn’s disease No

recreational waterborne reported Possibly

important Carr and

Bartram, 2004; Bolin et al., 2004b ; LeChevallier

, 2006 Bacteria Pseudomonas Yes (generally in environment) No ne NA Dermatitis Recreational waterborne N o Geldreich and Degnan, 2006b

Bacteria Salmonella Yes Poult r y, swine, cattle, rodents, wild birds, turtles, dogs, and cats Yes Gastroenteritis (enteric fever and septicemia) Mainly

foodborne and drinking water Important APHA, 2004; Covert and Meckes, 20 06

Bacteria Serratia Yes (generally in environment) No ne NA Opportunisitic infection (cystitis) Hospital

settings No Geldreich and Standridge, 2006a

Bacteria Shigella Yes No ne (except primate colonies) NA Shigellosis, acute gastroenteritis, dysentary, fe ver, nausea, vomiting, and cramps Recreational

and drinking water No Cliver and Fayer, 2004; APHA, 2004 ; Moyer and Degnan, 2006

Bacteria Staphylococcus Yes Skin of warm blooded hosts Yes Cellulitis, pustules, boil, carbuncles, and impetigo (diarrhea and

v omiting) Hospital

settings

, pools, and spas No Geldreich and Standridge, 2006bU.S. Environmental Protection Agency

December 2007 A

- 6 Type Pathogen Waterborne Zo onotic Host Zoonotic Host is Warm

blooded Illnesses and

Symptoms in Humans (less

common symptoms) U.S.

Outbreaks Importance

to consider if animal fecal

sources are discounted in AWQC References

Bacteria Vibrio cholerae Yes Copepods, zooplankton No Profuse

, watery diarrhea ; vomiting None

recently No APHA, 2004; Toranzos et al., 2006

Bacteria Vibrio parahaemolyticus Ye s (generally in environment) Molluscan

shellfish No Acute gastroenteritis (septicemia) Foodborne No FDA, 2001

Bacteria Yersinia Yes Pig s Yes Yersiniosis ; acute

, febrile diarrhea Mainly

foodborne Possibly

important APHA, 2004;

Fricker, 2006b
Protozoa Acan thamoeba Yes (free

living) No ne NA Granulomatus ameobic encephalitis None

reported No Fayer, 2004 b ; Visvesvara and Moura, 2006a

Protozoa Ascaris lumbricoides Yes No ne NA Ascariasis and roundworm infection Foodborne No APHA, 2004; Smith et al., 2006a

Prot ozoa Balamuthia mandrillaris Yes (free

living) Primates,

sheep, dogs, and horses Yes Granulomatus ameobic encephalitis None

reported No Visvesvara and Moura, 2006b

Protozoa Balantidium coli Yes Primates and pigs Yes Severe dysentery None reported No Garci a, 2006a

Protozoa Blastocystis hominis Yes Primates, cattle, sheep, pigs, horses, dogs, chickens, wild birds, alpacas, llamas, koalas, and wombats Yes Diarrhea, cramps, nausea, fever, vomiting, and abdominal pain None

reported No Garcia, 2006bU.S. Environmental Protection Agency

December 2007 A

- 7 Type Pathogen Waterborne Zo onotic Host Zoonotic Host is Warm

blooded Illnesses and

Symptoms in Humans (less

common symptoms) U.S.

Outbreaks Importance

to consider if animal fecal

sources are discounted in AWQC References

Protozoa C ryptosporidium parvum

and Cryptosporidium hominis Yes Cattle, sheep, goats, pigs, horses, cats, dogs, wild animals, birds, reptiles, and fish Yes Cryptosporidiosis, profuse watery diarrhea, malaise, fever, anorexia,
nausea, and vomiting Recreational

and dr inking water Important Olson, et al., 2003; APHA

, 2004; Sterling and Marshall, 2006; CDC

, 2007b

Protozoa Cyclospora cayetanensis Yes No ne NA Watery diarrhea, malaise, fever, anorexia, nausea, and vomiting Foodborne

and waterborne ( drinking water and

recre ational water ) No Ortega, 2006; Cross and Sherchand, 2004; Cliver and Fayer, 2004; APHA, 2004 Protozoa Entamoeba histolytica Yes Potentially primates, dogs, cats, pigs, rats, and possibly cattle Yes Amoebiasis, dysentery

, and diarrhea None

recently No Kee ne, 2006; APHA

, 2004; Fayer, 2004 b

Protozoa Giardia intestinalis (also

known as G. duodenalis and G. lamblia ) Yes Beaver s , cat s , lemur s , sheep, cal ves , dog s , fox es , chinchilla s , alpaca s , horse s , pig s , cow s , and muskrat s Yes Giardiasis ; diarrhea (chronic) ; steatorrhea ; abdominal cramps ; bloating ; frequent loose, pale

, greasy stools ; fatigue ; and malabsorption Recreational

and drinking water Important APHA, 2004; Appelbee et al., 2005; Schaefer, 2006U.S. Environmental Protection Agency

December 2007 A

- 8 Type Pathogen Waterborne Zo onotic Host Zoonotic Host is Warm

blooded Illnesses and

Symptoms in Humans (less

common symptoms) U.S.

Outbreaks Importance

to consider if animal fecal

sources are discounted in AWQC References

Protozoa Isosprora belli Yes No ne NA Foul smelling, foami ng diarrhea (months to years), abdominal colic, and fever None

reported No Garcia, 2006c

Protozoa Microsp oridia

(Enterocytozoon bieneusi, Encephalitozoon cuniculi, E. intestinalis) Yes Cattle, pigs, cats , rabbits, and sheep Yes Diarrhea None reported Poss ibly

important Shadduck and

Greely, 1989; Bolin et al., 2004b; Cali, 2006

Protozoa Naegleria fowleri Yes (free

living) No ne NA Primary amebic meningoencephaliti

s Mainly

ambient recreational waters No Fayer, 2004 b ; Visvesvara and Moura, 2006c

Protozoa Tox

oplasma gondii Yes Cats Yes Toxoplasmosis, (in fetally exposed children

- mental retardation, loss of vision, hearing impairment, and mortality) None

reported in recreational water Potentially

important Dubey, 2004;

Dubey, 2006; APHA, 2004

Virus Adenoviru ses Yes No ne NA Acute gastroenteritis and

r espiratory disease Waterborne No Reynolds, 2006; Enriquez and Thurston

Enriquez, 2006; Griffin et al., 2003
Virus Astroviruses Yes No ne NA Gastroenteritis Waterborne No Reynolds, 2006; Schwab, 2006U.S. Environmental Protection Agency

December 2007 A

- 9 Type Pathogen Waterborne Zo onotic Host Zoonotic Host is Warm

blooded Illnesses and

Symptoms in Humans (less

common symptoms) U.S.

Outbreaks Importance

to consider if animal fecal

sources are discounted in AWQC References

Virus Avian i nfluenza (H5N1) Unknown Birds Yes Mild upper respiratory illness to severe pneumonia and multiple organ failure None reported No Chan, 2002; Suresh and Smith, 2004

Virus C oronaviruses (e.g.,

severe acute respiratory syndrome (SARS)

- CoV) Potentially (aero solized wastewater) Possibly civets

and other wild animals NA Fever, dry cough, dyspnoea, and myalgia (diarrhea) None

reported No Reynolds, 2006; Suresh and Smith, 2004; Moe, 2004 Virus Enteroviruses (e.g.,

coxsackie) Yes No ne NA Gastroenteritis, exanthem a, diarrhea, fever, pharyngeal lesions, myocarditis, respiratory disease, and pneumonia Recreational

and drinking water No Griffin, 2003; APHA, 2004; Reynolds, 2006; Gerba, 2006a

Virus Hendra virus Unknown Horses Yes Severe respiratory disease and meningo encephaliti

s None

reported No MacKenzie, 1999; Suresh and Smith, 2004

Virus Hepatitis A virus Yes No ne NA Hepatitis

- acute inflammation of the liver None

recently No Sobsey, 2006

Virus Hepatitis E virus Yes Possibly pigs, chicken s , and rat s (close viral relatives to human form) NA Hepatitis

- acute inflammation of the liver (fatality in pregnant women) Rare in

United States (common in other countries) No Cliver and Moe, 2004; Craun, 2004a; Gerba, 2006bU.S. Environmental Protection Agency

December 2007 A

- 10 Type Pathogen Waterborne Zo onotic Host Zoonotic Host is Warm

blooded Illnesses and

Symptoms in Humans (less

common symptoms) U.S.

Outbreaks Importance

to consider if animal fecal

sources are discounted in AWQC References

Virus Human caliciviruses

(norovirus, sapovirus) Ye s No ne NA Diarrhea and vomiting Very

common (waterborne and foodborne) No Schwab and Hurst, 2006; Reynolds, 2006

Virus Nipah virus Unknown Pigs, bats, and flying foxes Yes Acute and febrile encephalitis None

reported No Harcourt, 2005; Suresh and Smith, 2 004

Virus Parechoviruses Yes No ne NA Pericarditis, herpangina, and respiratory disease None

reported (probably occurs, but unidentified) No Gerba, 2006a

Virus Reoviruses Yes No ne NA Mostly mild or subclinical ( rarely biliary atresia, juvenile onset diabe tes, feve r , rash, respiratory disease, and diarrhea None

reported (probably occurs, but unidentified) No Sattar and Springthrope, 2006

Virus Rotaviruses Yes No ne NA Diarrhea Very

common waterborne No Abbaszadegan, 2006; Reynolds, 2006

Helminths Ancylosto ma braziliense Mainly soil Dog s or cat s Yes Larval migration leads to creeping eruption None

reported (most important hookworm in humans

- soil route) No Endo and Morishima, 2004U.S. Environmental Protection Agency

December 2007 A

- 11 Type Pathogen Waterborne Zo onotic Host Zoonotic Host is Warm

blooded Illnesses and

Symptoms in Humans (less

common symptoms) U.S.

Outbreaks Importance

to consider if animal fecal

sources are discounted in AWQC References

Helminths Angiostrongylus Yes Mollus ks No Meningitis None reported No Endo a nd Morishima, 2004 Helminths Ascaris lumbricoides Mainly soil (drinking water possible) No ne NA Ascaris pneumonia (lung hemorrhage) None

reported No Endo and Morishima, 2004 ; Smith et al., 2006 Helminths Ascaris suum Mainly soil Pig s Yes Ascaris pneumoni a (lung hemorrhage) None

reported No Endo and Morishima, 2004 Helminths avian schistosomes

(includes S. mansonni) Yes Snails (birds are infected but not sources) Yes Cercarial dermatitis (swimmer’s itch) None

reported Low

importance Endo and

Morishima, 2 004

Helminths Baylisascaris procyonis Mainly soil Raccoon s Yes L arval migration may damage to the visceral and ocular systems (migration to brain) None reported No Endo and Morishima, 2004

Helminths Dracunculus medinensis Yes Crustacean s No Connective ti ssue migration, emerging in lower limb blister None

reported No Endo and Morishima, 2004

Helminths Echinococcus Yes Fox es , dog s Yes Cystic hydatid disease, alveolar hydatid disease, polycystic hydatid disease, and polycystic hydatid disease None

reported Low

importance Endo and

Morishima, 2004U.S. Environmental Protection Agency

December 2007 A

- 12 Type Pathogen Waterborne Zo onotic Host Zoonotic Host is Warm

blooded Illnesses and

Symptoms in Humans (less

common symptoms) U.S.

Outbreaks Importance

to consider if animal fecal

sources are discounted in AWQC References

Helminths Fasciola hepatica Yes Snail s (herbivores and humans infected) Yes Fascioliasis None reported Low

importance Endo and

Morishima, 2004

Helminths Pseudophyllid cestodes Yes Copepods, fish No Cutaneous or

muc ocutaneous invasion None

reported No Endo and Morishima, 2004 Helminths Schistosomes Yes Snail s (many animals can be infected, but are not sources) No Schistosomiasis (chronic infection

- liver fibrosis, portal hypertension) None

reported No Endo and Mori shima, 2004; APHA, 2004; Blankespoor, 2006 Helminths Strongyloides Mainly soil Dog s Yes Eosinophilia None reported No Endo and Morishima, 2004; Pardo, 2006

Helminths Taenia solium (pork

tapeworm) Yes Pig s Yes Cysticercosis and myositis None

reported Low

importance Endo and

Morishima, 2004 Helminths Toxocara canis Yes Dogs Yes Toxocariasis ( larval migration leads to hemorrhage and granulomatous lesions in the central nervous system) None

reported Low

importance Endo and

Morishima, 2004U.S. Environmental Protection Agency

December 2007 A

- 13 Type Pathogen Waterborne Zo onotic Host Zoonotic Host is Warm

blooded Illnesses and

Symptoms in Humans (less

common symptoms) U.S.

Outbreaks Importance

to consider if animal fecal

sources are discounted in AWQC References

Helminths Toxocara cati Yes Cats Yes Toxocariasis (larval migration leads to hemorrhage and granulomatous lesions in the central nervous system) None

reported Low

importance Endo and

Morishima, 2004

Helminths Trichinella spiralis Mainly soil Variety of animals Yes Trichino sis Foodborne (mainly consumption of undercooked pork) No Endo and Morishima, 2004

Helminths Trichuris trichiuria Mainly soil Monkey s , pigs, dogs, cats, and chicken Yes Trichuris dysentary syndrome, chronic diarrhea, anemia, and growth retardation Foodbo rne No Endo and Morishima, 2004; Smith et al., 2006b

Prion Bovine Spongiform

Encephalopathy (BSE) Unknown Cattle Yes V ariant Creutzfeldt

Jakob disease Foodborne No Brown et al., 2006

NA = Not applicable[this page intentionally left blank]U.S. Environmental Protection Agency

December 2007 B

- 1 

## A PPENDI X 

## B

L ITERATURE 

## S EARCH 

## S TRATEGY

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 th e 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 Criteria 12 were contacted by email and requested to contribute literature they felt was important. The titles of literature cited in specific reports, books, review articles, and conference proceedings were evaluated for relevance.

B.1 Initial Literature Search Strategy Conducted by Professional Librarian

Selection of DIALOG data base files used for this search:

File 155:MEDLINE(R) 1950

- 2007/Nov 30

(c) format only 2007 Dialog File 266:FEDRIP 2007/Sep

Comp & dist by NTIS, Intl Copyright All Rights Res File 144:Pascal 1973

- 2007/Nov W3

(c) 2007 INIST/CNRS File 110:WasteInfo 1974

- 2002/Jul

(c) 2002 AEA Techn Env. File 245:WATERNET(TM) 1971

- 2007Jul

(c) 2007 American W ater 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.

M ain search strategy used for this search: Fecal set AND Composition set AND (Pathogen or Waterborne sets): 1985

- present

S1 240528 FECAL OR FECES OR FAECAL OR FAECES OR DUNG OR SCAT OR EXCREMENT

OR MANURE OR STOOLS) S2 1686966 COMPOSITION OR COMPOSED OR ANIMAL(1N)HUMAN()(TRANSMISSION OR

CONTAMINATION)

12 Report from this workshop: http://www.epa.gov /waterscience/criteria/recreation/U.S. Environmental Protection Agency

December 2007 B

- 2 S3 3842348 PATHOGEN? ? OR INDICATOR? ? OR RISK OR RISKS S4 983 WATERBORNE()(PATHOGEN? ? OR ZOONO? OR ILLNESS?) S6 1140 S1 AND S2 AND (S3 OR S4) S719961011 PY=1920:1984 S8 1037 S6 NOT S7 (limited to 1985

- present; foreign language OK) S9 704 RD S8 (unique items , deduped )

Dates: 1985

- present Language: No restrictions Retrieve: Titles and year Format: MS Word Interested in international and domestic journals and gov ernment reports.

Descriptions of these files are available at http://library.dialog.com/bluesheets/

.

Search Terms: Waterborne pathogen* Waterborne illness* Waterborne zoon* Emerging waterborne zoonot ic 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 Liste ria 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 lit erature search : Fecal composition of species that harbor zoonotic pathogens (species of livestock and wild animals Fecal composition of humans

– pathogens and indicators
T he extent to which strains found in animals can be transmitted to humansU.S. Environmental Protection Agency

December 2007 B

- 3 A n evaluat ion 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
W hich organisms are of substantial public health concern that occur in ambient waters and are path ogenic to humans
W hich of these organisms are also present in animal populations
T he extent to which these organisms found in animals can be transmitted to humans
T he potential outcomes of human infection and disease from animal sources Specific pathogens: E. coli 0157

- H7, Salmonella

, Shigella

, Campylobacter

, Listeria

, Cryptosporidium

, Giardia , norovirus, rotavirus, Hepatitis E , emerging pathogens. For each:

o Describe illness symptoms (range asymptomatic to severe)

o Describe route of exposure from recreationa l 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
V ariations in strains that effect infectivity, severity of symptoms, environmenta l survival and treatability S hort summary review of emerging pathogen mechanisms (not too deep into molecular mechanisms of evolution) A nything in the water matrix that affects survivability and infectivity, and virulence
P athogen:indicator ratios

B.2 Su mmary of Literature Search Results

This process resulted in a total order of 535 documents (primarily peer reviewed scientific articles), of which a total o f 332 ( 6 2 percent ) were received during the expedited writing process, not all of which could be re viewed. 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 Approximate # Titles R eviewed # Titles of Interest

E. coli + deer 10 1

Adenoviruses + zoonotic 30 3 Hepatitis E+ condition+ symptoms 30 2 Rotavirus + illness + symptoms 20 1U.S. Environmental Protection Agency

December 2007 B

- 4 The following terms were searched on Google Scholar ( http://scholar.google.com/ ):

Search Topic Approximate # Titles R eviewed # Titles of Interest

Adenoviruses + zoonotic 10 1 Rotavirus + illness + symptoms 30 3 Giardiasis + Swimming 200 5
Giardiasis + surveillance 200 3
Giardia + symptoms 100 5
Giardia + cyst 100 2
Giardia + beach 200 1 Cryptosporidium + hominis 100 1 Cryptosporidium + beach 150 1 Cryptosporidium + incidental ingestion 250 4 Cryptospo ridium + review 100 2 Cryptosporidium + exposure factors 150 3 Cryptosporidium + exposure 50 2 Cryptosporidium + symptoms 50 3 Cryptosporidium + risk 50 0 Campylobacter + chronic 50 3 Campylobacter + illness 50 3 Campylobacter + infection 50 4 Camp ylobacter + symptoms 50 1 Campylobacter + water 15 2 Campylobacter + pathogenesis 5 1
Salmonella + transmission 20 8
Salmonella + transmission in water 11 2
Salmonella + antibiotic resistance 20 3
Salmonella + pathogenesis 3 1 Shigella + outbreak 23 4 Shigellosis 35 4

The following terms were searched on PubMed ( http://www.ncbi.nlm.nih.gov/sites/entrez ):

Search Topic # Titles Reviewed # Titles of Interest

E. coli O157

- H7 + survival + envi ronment 204 23

Salmonella + survival + environment 376 4 Shigella + survival + environment 65 10 Campylobacter + survival + environment 57 17

Norwalk Virus + survival + environment 2 Yes

Rotavirus + survival + environment 54 17 Hepatitis E + survival + environment 3 YesU.S. Environmental Protection Agency

December 2007 B

- 5 The following terms were searched on Scirus ( http://www.scirus.com/ ):

Search Topic Approximate # Titles Reviewed # Titles of Interest

Helicobacter + environmental survival 30 1 Leptospira + CID 10 1 Leptospira + environmental survival 30 2 Leptosporidiosis + symptoms 30 0

The following terms were searched on the CDC website ( http://www.cdc.gov/ ):

Search Topic Approximate # Titles Reviewed # Titles of Interest

Legionella + EID 10 1 Leptospira + EID 10 1

Avian influenza + EID 10 1

H5N1 + EID 20 0

SARS + EID 30 2

Hendra virus + EID 20 0 Nipah virus + EID 20 2 Strongyloides + EID 10 1

BSE + EID 10 1

B.4 Experts Contacted

The flowing experts in the field were 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 Departme nt 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 De partment of Environmental Protection Lora Fleming, University of Miami School of Medicine and Rosenstiel School of Marine and Atmospheric Sciences, Florida Charles Hagedorn, Virginia Tech Joel Hansel, USEPAU.S. Environmental Protection Agency

December 2007 B

- 6 Lawrence Honeybourne, Orange County Health Care A gency, Santa Ana, California Donna Francy, U.S. Geological Survey Roger Fujioka, 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 Jura nek, 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 Ravenscr oft, USEPA Stephen Schaub, USEPA Mark Sobsey, University of North Carolina, Chapel Hill Jeffrey Soller, Soller Environmental, California Michael Tate, Kansas Department of Health and Environment Peter Teunis, RIVM (National Institute of Public Health and t he 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 Ri chard Zepp, USEPA

B.4 Previously Cited References

The following s pecific reports were obtained and the titles of the references cited in the reports were reviewed for relevance :

National Research Council. (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 E. coli criteria documentU.S. Environmental Protection Agency

December 2007 B

- 7 Boehm et al. (2008) A sea ch ange ahead for recreational water quality criteria . (peer review in progress)

In addition, Clancy Environmental Consultants, ICF International, Soller Environmental, WaltJay Consulting, and EPA’s Health and Ecological Criteria Division all maintain extens ive literature databases and reference lists from previously completed projects. All of those in house resources were also sources of literature.[this page intentionally left blank]U.S. Environmental Protection Agency

December 2007 C

- 1 

## A PPENDIX 

## C

I NCIDENTAL 

## I NGESTION OF 

## A MBIENT 

## W ATER 

## D URING 

## R ECREATIO NAL 

## A CTIVITIES

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 represen tative 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 m L per hour. N one of the early estimates , however, were based on actual studies of water ingestion. EPA’s Risk Assessment Guidance for Superfund (USEPA

, 1989 ) recommend ed 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, estimate d 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 5 swimmers that were studied was 161 mL per hour. The investigators also determined that : (1) essentially all of the ingested isocya nuric 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 ― non adults‖ 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 rec reation resulted in average water intakes of 37 mL (49 mL/hr) for non adults 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 co mpetitive 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 pub lic 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 avera ged 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.U.S. Environmental Protection Agency

December 2007 C

- 2 The small number of studies that are available measure d water intake in only a few subjects and characterize d water ingestion during either very active swimming or poorly defined water recreational activities

. In addition, the incidental ingestion data th at are available are for ―clean‖ pool water and may not represent incidental ingestion for surface waters, which may provoke stronger avoidance behaviors due to the perception that surface waters are non potable.