EPA 822-R-09-002 REVIEW OF ZOONOTIC PATHOGENS IN AMBIENT WATERS U.S. Environmental Protection Agency Office of Water Health and Ecological Criteria Division February 2009 ------- [this page intentionally left blank] ------- U.S. Environmental Protection Agency DISCLAIMER This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. February 2009 ------- [this page intentionally left blank] ------- U.S. Environmental Protection Agency ACKNOWLEDGMENTS Questions concerning this document or its application should be addressed to the EPA Work Assignment Manager: John Ravenscroft USEPA Headquarters Office of Science and Technology, Office of Water 1200 Pennsylvania Avenue, NW Mail Code: 4304T Washington, DC 20460 Phone: 202-566-1101 Email: ravenscroft.john@epa.gov This literature review was managed under EPA Contract EP-C-07-036 to Clancy Environmental Consultants, Inc. The following individuals contributed to the development of the report: Lead writer: Audrey Ichida Leiran Biton Alexis Castrovinci Jennifer Clancy Mary Clark Elizabeth Dederick Kelly Hammerle Walter Jakubowski Margaret McVey Michelle Moser Jeffery Rosen Tina Rouse Jennifer Welham Kerry Williams ICF International ICF International ICF International Clancy Environmental Consultants ICF International ICF International ICF International WaltJay Consulting ICF International ICF International Clancy Environmental Consultants ICF International ICF International ICF International February 2009 in ------- [this page intentionally left blank] ------- U.S. Environmental Protection Agency TABLE OF CONTENTS ACKNOWLEDGMENTS iii TABLES AND FIGURES vii ACRONYMS ix EXECUTIVE SUMMARY 1 I. BACKGROUND AND INTRODUCTION 5 I.I Background: Context and Purpose 5 1.2 Introduction 6 II. KEY WATERBORNE ZOONOTIC PATHOGENS OF CONCERN 8 II. 1 Bacteria 8 II. 1.1 Escherichia coli 8 II. 1.2 Campylobacter 12 II.1.3 Salmonella 14 II. 1.4 Leptospira 17 II.2 Protozoa 19 II.2.1 Cryptosporidium 19 II.2.2 Giardia 26 II.3 Viruses Zoonotic Potential 33 III. PATHOGEN INTERACTIONS WITH THEIR ENVIRONMENT 37 III.l Water Environment Affects Pathogen Survivability andPhenotype 37 III. III. III. III. III. III. III. .1 Pathogenic E. coli Survival in the Environment 38 .2 Campylobacter Survival in the Environment 39 .3 Salmonella Survival in the Environment 39 .4 Leptospira Survival in the Environment 39 .5 Cryptosporidium Survival in the Environment 40 .6 Giardia Survival in the Environment 43 .7 Virus Survival in the Environment 44 III.2 Host Animals Can Influence Pathogen Characteristics and Mechanisms of Rapid Evolution 44 IV. SUMMARY 49 V. REFERENCES 50 APPENDIX A: WATERBORNE PATHOGENS A-l APPENDIX B: LITERATURE SEARCH STRATEGY AND RESULTS B-l APPENDIX C: INCIDENTAL INGESTION OF AMBIENT WATER DURING RECREATIONAL ACTIVITIES C-l February 2009 ------- [this page intentionally left blank] ------- U.S. Environmental Protection Agency TABLES AND FIGURES Table II. 1.1-1. Outbreaks of E. coli Associated with Recreational Waters in the United States 12 Table II.2.1-1. Cryptosporidium Species 20 Table II.2.1-2. Outbreaks of Cryptosporidiosis Associated with Recreational Waters in the United States 27 Table II.2.2-1. Giardia Taxonomy 28 Table II.2.2-2. Giardia Infection Occurrence in U.S. Populations 32 Table II.2.2-3. Outbreaks of Giardiasis Associated with Recreational Waters in the United States 34 Table III.l.1-1. Survival of E. coli 0157 :H7 in Ambient Waters 38 Figure II.2.1-1. Reported Cryptosporidium infections in the United States, 1999 to 2005 26 Figure II.2.2-1. Reported Giardia cases in the 50 states plus Washington DC, 1992 to 2005 32 February 2009 vn ------- [this page intentionally left blank] ------- U.S. Environmental Protection Agency AEC AIDS AWQC BEACH Act CDC CPV CWA DAEC DEC EAggEC EEC EHEC EPA EPEC ETEC GI GLV GBS HC HEV HUS IPSID MALT NA PCR POTW ppt RFLP SPE STEC U.S. U.K. USDA UV VTEC WHO ACRONYMS attaching E. coli Acquired Immune Deficiency Syndrome ambient water quality criteria Beaches Environmental Assessment and Coastal Health Act of 2000 U.S. Centers for Disease Control and Prevention C. parvum virus Clean Water Act diffuse adherent E. coli diarrheagenic E. coli enteroaggregative E. coli effacing E. coli enterohemorrhagic E. coli U.S. Environmental Protection Agency enteropathogenic E. coli enterotoxigenic E. coli gastrointestinal Giardia lamblia virus Guillain-Barre Syndrome hemorrhagic colitis hepatitis E virus hemolytic uremic syndrome immunoproliferative small intestinal disease mucosa-associated lymphoid tissue not available or not applicable polymerase chain reaction Publicly Owned Treatment Works parts per thousand (salinity) restriction fragment length polymorphism serial passage experiment Shiga toxin-producing E. coli United States United Kingdom U.S. Department of Agriculture ultraviolet (light) verocytotoxin-producing E. coli World Health Organization (United Nations) February 2009 IX ------- [this page intentionally left blank] ------- U.S. Environmental Protection Agency EXECUTIVE SUMMARY Introduction The overall goal of the current Clean Water Act (CWA) §304(a) ambient water quality criteria (AWQC) for bacteria in the United States is to provide public health protection from gastrointestinal (GI) illness (gastroenteritis) associated with exposure to fecal contamination during recreational water contact. Water quality criteria are specified throughout the world in terms of concentrations of fecal indicator organisms because fecal matter can be a major source of pathogens in ambient water and because it is not practical or feasible to monitor for the full spectrum of all pathogens that may occur in water. For decades, these fecal indicator organisms have served as surrogates for potential pathogens and subsequent health risks in both recreational and drinking waters. The U.S. Environmental Protection Agency (EPA) currently recommends AWQC for recreational water that encompass all fecal sources that contain the relevant indicator species (enterococci and E. coif). This approach assumes that animal fecal material is as hazardous as human fecal material. There is limited evidence, however, that recreational water contaminated with animal fecal material is less risky to swimmers than recreational water contaminated with human fecal material. In order to evaluate the potential risks posed by animal fecal contamination, EPA is interested in understanding what human illnesses are caused by swimming in waters contaminated with animal fecal material ranging from wildlife sources to agricultural inputs. The animal species of most interest are the warm-blooded animals (mammals and birds) whose fecal material is detected by current indicators. The purpose of this white paper is to provide a summary of information on waterborne zoonotic pathogens that come primarily from warm-blooded animals, and which can be used to conceptualize potential risks from warm-blooded animal feces in ambient (untreated) recreational waters. Approach Seventy pathogens from warm-blooded animals were evaluated for their potential to be both waterborne and zoonotic. Twenty of the 70 pathogens evaluated had all 4 of the following attributes: 1. The pathogen spends part of its lifecycle within one or more warm-blooded animal species. 2. Within the lifecycle of the pathogen, it is probable or conceivable that some life stage will enter water. 3. Transmission of the pathogen from animal source to human is through a water related route. 4. The pathogen causes infection or illness in humans. February 2009 ------- U.S. Environmental Protection Agency Six of the 20 waterborne, zoonotic pathogens from warm-blooded animals were selected for further discussion based on their relevance in the United States. Five were selected based on their potential for outbreaks in ambient (untreated) recreational water and one (Salmonella) was included based on outbreaks in drinking water. Some well-known waterborne pathogens were excluded from analysis because they are not zoonotic. Excluded pathogens include bacteria that are generally found in the environment, free- living protozoa, viruses, and helminthes that have cold-blooded hosts (e.g., snails, copepods). Some common zoonotic pathogens were also excluded because they do not have well- documented waterborne transmission (i.e., primarily transmitted via soil, food, or drinking water). Pathogens interact with the ambient environment, other microorganisms, plants, and with their hosts. The behavior of pathogens in ambient waters is often different from the behavior of indicators in ambient water. The most common environmental factors studied for their impact on pathogen survival in water are pH, salinity, light exposure, and temperature. Additional environmental characteristics that may influence pathogen survival, infectivity, and virulence include the following: ultraviolet (UV) light (duration, intensity), rainfall, runoff, dispersal, suspended solids, turbidity, nutrients, organic content, organic foams, water quality, biological community in water column, water depth, stratification, mixing (e.g., wind and waves), presence of aquatic plants, biofilms, and predation. There is evidence that zoonotic pathogens may change in infectivity, virulence, and the severity of disease caused in humans depending on their previous host environment. There is also evidence that some of these host-factor changes can influence subsequent infection cycles in exposed hosts. The key mechanisms of phenotypic change in pathogens are genetic diversity (coinfection and quasispecies), cryptic genes, mutators, and epigenetic effects. Six Key Waterborne Zoonotic Pathogens Pathogenic E. coli E. coli is an important waterborne bacterial zoonosis because many human pathogenic strains occur in livestock and wildlife feces and can survive in ambient waters. In addition, the potential illnesses caused by pathogenic E. coli can be severe or fatal. Mortality estimates range from 0.08 to 1.9 percent of E. coli O157:H7 infections. Children are more at risk than healthy adults to suffer more severe outcomes. Between 1991 and 2004, 14 outbreaks of E. correlated illness have been associated with ambient recreational waters. E. coli O157:H7 can survive for at least several weeks in animal feces and slurries and has been demonstrated to survive at least 500 days at -20 °C in frozen soil. Campylobacter Campylobacter is a well-known foodborne bacterial pathogen that is commonly associated with poultry and livestock. There are also wildlife hosts. Although few waterborne outbreaks have been reported, there is potential for Campylobacter to cause recreational water-related illness. Normally, infection results in diarrhea that is self-limiting; however, approximately 1 out of 1,000 infections results in Guillain-Barre syndrome, which is a serious nervous system affliction. February 2009 ------- U.S. Environmental Protection Agency Reactive arthritis is also a possible chronic sequela of Campylobacter infection. Campylobacter has been shown to survive in aquatic environments with low temperatures (4°C) up to 4 months. Salmonella Salmonella is also a well-known foodborne bacterial pathogen that is associated with poultry and livestock. There are also wildlife hosts. Elevated levels of Salmonella have been observed in major water bodies that receive discharges of meat processing wastes, raw sewage, farming operations, and effluents from ineffective sewage treatment plants. The clinical symptoms of salmonellosis may include diarrhea, abdominal pain, nausea, chills, and fever. Between 1991 and 2002, 3 waterborne outbreaks were attributed to nontyphoid Salmonella; Salmonella were the etiologic agent in 0.9 percent of 259 recreational waterborne outbreaks occurring from 1971 to 2000. Under suitable environmental conditions, Salmonella can survive for weeks in waters or years in soils. Leptospira Leptospira is an important waterborne bacterial pathogen for most of the world. Because warmer climates favor its survival in the environment, the highest incidence of leptospirosis in the United States is found in Hawaii. The source of infection in humans is usually either direct or indirect contact with the urine of an infected animal. There are numerous symptoms associated with leptospirosis, but the more severe form is known as Weil's disease. In addition, acute infection during pregnancy has been reported to cause abortion and fetal death. From 1971 to 2000, 16 percent of recreational waterborne disease outbreaks in the United States were attributed to Leptospira. In 1998, an outbreak (375 cases) of leptospirosis was reported that was associated with a triathlon in a lake in Illinois. Cryptosporidium Cryptosporidium is a well-known waterborne protozoan pathogen. EPA currently regulates it under the Safe Drinking Water Act because the level of Cryptosporidium in many ambient source waters is sufficiently high that risks to drinking water must be managed. Cryptosporidium infection has been reported in more than 155 mammalian species and numerous reptiles, amphibians, birds, and fish. In watersheds with diverse land-use patterns, it is likely that different sources contribute different proportions to the total contamination load at different times during the year, with the relative contributions depending on a wide range of watershed characteristics. Cryptosporidiosis is primarily characterized by GI symptoms such as profuse, watery diarrhea. Immunocompromised individuals generally experience chronic gastroenteritis, which may last as long as the immune impairment. The largest known outbreak of Cryptosporidiosis occurred in 1993 in Milwaukee, Wisconsin and infected 403,000 individuals. Animal fecal contamination of drinking water was indicated in the outbreak. In the United States, from 1991 to 2004, 6 outbreaks of Cryptosporidiosis have been associated with untreated recreational waters. Excreted Cryptosporidium oocysts can survive for substantial periods in animal wastes and soils. Thus, contaminated runoff can enter ambient water and result in potential human exposures. The majority of oocysts (99 percent) are inactivated by repeated freeze-thaw cycles; therefore, Cryptosporidium may be environmentally limited in parts of the United States during winter months. Between 4 and 20° C, there is very little inactivation of oocysts in different types of agricultural soils. Oocyst survival in various water matrices is highly variable, but survival for longer than 30 days has been demonstrated in several studies. February 2009 ------- U.S. Environmental Protection Agency Giardia Giardia is also a well-known waterborne protozoan pathogen. Although both livestock and humans have been implicated in contaminating water sources with Giardia, humans are responsible for the majority of the contaminations. Zoonotic transfer plays only a minor role in the infection cycles of Giardia, and animal contact is not a major risk factor. There is a wide spectrum of symptoms associated with giardiasis that ranges from asymptomatic infection and acute self-limiting diarrhea to persistent chronic diarrhea, which sometimes fails to respond to treatment. Asymptomatic infection is very common, with 50 to 75 percent of infected persons reporting no symptoms. In the United States, from 1991 to 2004, 7 reported outbreaks of giardiasis were associated with untreated recreational waters. At 4° C, Giardia cysts were infective for 11 weeks in water, 7 weeks in soil, and 1 week in cattle feces. Summary Although the most common waterborne recreational illnesses are probably due to nonzoonotic human viruses, which typically cause short-term gastroenteritis, the waterborne zoonotic pathogens discussed in this report have the potential to cause serious health effectsespecially in immunocompromised persons and subpopulations. While serious health outcomes are likely to be rare in comparison with self-limiting illnesses as a result of ambient (recreational) water exposure, the adverse health impacts of the rare, but more serious illnesses remain an important public health challenge. February 2009 ------- U.S. Environmental Protection Agency I. BACKGROUND AND INTRODUCTION 1.1 Background: Context and Purpose Since the U.S. Environmental Protection Agency (hereafter EPA or the Agency) last published recreational water quality criteria in 1986, there have been significant scientific and technical advances, particularly in the areas of molecular biology, microbiology, and analytical chemistry. EPA believes that these advances need to be considered and evaluated for feasibility and applicability in the development of new or revised CWA §304(a) criteria for recreational water. To this end, EPA has been conducting research and assessing relevant information to provide the scientific and technical foundation for the development of new or revised criteria. The enactment of the Beaches Environmental Assessment and Coastal Health (BEACH) Act of 2000 (which amended the CWA) required EPA to conduct new studies and to issue new or revised criteria for Great Lakes and coastal marine waters. In response to the BEACH Act of 2000, EPA also has engaged a range of stakeholders representing the general public, public interest groups, state and local governments, industry, and municipal wastewater treatment professionals. In March 2007, EPA convened a group of 43 national and international technical, scientific, and implementation experts from academia, numerous state agencies, public interest groups, EPA, and other federal agencies at a formal workshop to discuss the state of the science on recreational water quality research and criteria implementation. Among the input from the individuals attending the workshop were suggestions for incorporating the ability to differentiate sources of fecal contamination and to determine the relative human health risk from these sources into the new or revised criteria. Based on the feedback from the large group of stakeholders, as well as input and recommendations from the scientific community, the Agency has developed a Critical Path Science Plan for Development of New or Revised Recreational Water Quality Criteria. One of the key questions posed in the science plan asks: what is the risk to human health from swimming in water contaminated with human fecal matter as compared to swimming in water contaminated with nonhuman fecal matter? Human and animal feces can both potentially contain pathogens that cause human illness. Some human pathogens are host-specific (i.e., human enteric viruses), while other human pathogens are found in and can be shed by both humans and other animals. Moreover, while all enteric pathogens of humans are infectious to other humans, only a subset of the enteric pathogens of animals is infectious to humans. Understanding which pathogens could be present depending on the source of fecal contamination might allow the Agency to better estimate human health risks from identified sources of fecal matter. EPA's current recommended recreational water quality criteria for microbes treat human health risks from the various sources of fecal contamination as equivalent (USEPA, 1986). These criteria are based on the risk of illness from swimming in waters influenced by sewage treatment plants effluents. Health risks from other sources (e.g., poorly-treated or untreated human waste, nonhuman sources of fecal contamination, mixed sources such as urban stormwater runoff) were not well understood at the time the 1986 criteria were developed, and EPA's approach was to be protective of human health regardless of the source. EPA recognizes, however, that the health February 2009 ------- U.S. Environmental Protection Agency risk from sources other than sewage treatment plants may be different and that the scientific advances over the intervening years may now allow the Agency to better characterize the relative risks to human health from these various sources of fecal contamination. Specifically, the Agency is interested in understanding which human illnesses can be caused by swimming in waters contaminated with nonhuman fecal matter including both wildlife sources and agricultural inputs. The animal species of most interest are the warm-blooded animals (mammals and birds) for which fecal matter is detectable by current fecal indicators. The purpose of this white paper is to provide summary information on waterborne zoonotic pathogens that come primarily from warm-blooded animals, and which can be used to conceptualize potential risks to humans from warm-blooded animal feces in recreational waters. 1.2 Introduction In the development of health protective criteria for recreational waters, pathogen contamination is the central concern, and fecal matter can be a major source of pathogens in ambient water. Current recreational AWQC are designed specifically to protect humans from GI illness (gastroenteritis) associated with exposure to fecal contamination in recreational waters (USEPA, 1986). Because widespread monitoring of recreational waters directly for all disease-causing microorganisms (especially pathogenic bacteria, viruses, and protozoa) remains infeasible, public health and environmental protection agencies have relied on the detection of fecal indicator organisms, which comprise a few groups of nonpathogenic fecal bacteria and some viruses, to indicate the presence and magnitude of fecal material. This approach assumes that waterborne pathogens co-occur with the fecal material. "More specifically, fecal indicator bacteria provide an estimation of the amount of feces, and indirectly, the presence and quantity of fecal pathogens in the water" (NRC, 2004). Therefore, use of bacterial indicators is predicated on the presumption that there are no significant environmental sources of these microorganisms (i.e., nonenteric sources). However, this presumption is not entirely valid; fecal indicator organisms have been demonstrated to have natural reservoirs in the aquatic environment where they can survive for extended periods and even proliferate (e.g., fecal indicator bacteria in U.S. coastal [Yamahara et al., 2007] and Great Lake waters and sand [Whitman et al., 2006]). Historically, EPA has recommended that recreational water quality criteria encompass all fecal sources of pathogens that contain the relevant indicator microbes. This recommendation is based on the regulatory premise that animal-derived (zoonotic) human pathogens in fecally contaminated waters are as hazardous as their human-derived counterparts (Schaub, 2004). This presumption is supported by current research that confirms that there are many waterborne zoonotic bacteria and protozoa common to both humans and animals, especially mammals (WHO, 2004). Research also suggests, however, that there may be some attenuation of infectivity, virulence, and disease severity to humans from animal-derived human pathogens (see Section III.2). In addition, there are many pathogens that could be zoonotic, fecal, and/or waterborne, but these routes of transmission have not yet been conclusively demonstrated. Given the scientific advancements in pathogen characterization since EPA's 1986 AWQC were released, it is appropriate to examine the broadest array of currently known and suspected waterborne, fecal, and zoonotic pathogens and their human health impacts. These include February 2009 ------- U.S. Environmental Protection Agency primarily bacteria, viruses, protozoa, and helminths. There are many zoonotic pathogens and many waterborne pathogens; however, there is a more limited subset of pathogens that are both. For this report, the following attributes were used to select the waterborne, zoonotic pathogens of concern (partially adapted from Bolin et al., 2004a): 1. The pathogen must spend part of its lifecycle within one or more warm-blooded animal species. 2. Within the lifecycle of the pathogen, it is probable or conceivable that some life stage will enter water. 3. Transmission of the pathogen from animal source to human must be through a water related route. There are zoonotic pathogens for which waterborne exposure has not been detected as a significant route of cross-species transmission. This does not exclude the possibility that these zoonotic pathogens could be transmitted via water. 4. The pathogen must cause infection or illness in humans. There are animal pathogens that have waterborne transmission between animals yet are not known to cause illness in humans. There are many waterborne zoonotic pathogens that exhibit all four attributes listed above. See Appendix A, Table A-l, for a summary of waterborne pathogens that meet the above criteria and selected pathogens that meet some, but not all, of the above criteria. The remainder of this paper is organized into two main sections. Section II characterizes six key groups of zoonotic pathogens for which there is evidence of human health risks from recreational exposure via ambient waters. Section III presents an overview of how the key pathogens interact with their environment, which includes both the water environment and the host animals. February 2009 ------- U.S. Environmental Protection Agency II. KEY WATERBORNE ZOONOTIC PATHOGENS OF CONCERN Based on a review of the literature and other information sources (Appendix B), 6 of the 20 pathogens identified as waterborne and zoonotic from warm-blooded animals stood out as having the most evidence for human health impacts due to recreational exposures in the United States. The six pathogens discussed in this paper in more detail are pathogenic E. coli, Campylobacter, Salmonella, Leptospira, Cryptosporidium, and Giardia. This list correlates well with the top five waterborne pathogens for recreational and drinking waters. The top five waterborne pathogens for recreational water are E. coli, Campylobacter, Leptospira, Cryptosporidium, and Giardia, while the top five for drinking water are E. coli, Campylobacter, Salmonella, Cryptosporidium, and Giardia (Craun et al., 2004a). The waterborne zoonosis potential of viruses also is discussed because viruses may be emerging waterborne zoonoses. For each of the six key pathogens, information on strain variation, known zoonoses, route(s) of exposure, illness symptoms, and disease incidence are discussed in the sections that follow. Because incidental ingestion is the primary route of recreational exposure to pathogens, summary information on incidental ingestion is provided in Appendix C. II. 1 Bacteria II. 1.1 Escherichia coli E. coli is an important waterborne zoonosis because human pathogenic strains occur in livestock and wildlife feces and can survive in ambient water. In addition, the potential illnesses caused by pathogenic E. coli can be severe or fatal, especially in immuncompromised persons and subpopulations. Pathogenic E. coli is also a well-documented foodborne pathogen (USDA, 2001). Although pathogenic E. coli have been found in treated wastewater effluents in the United States (Boczek et al., 2007), waterborne outbreaks are not as prevalent as foodborne cases. E. coli Strain Variation Benign strains of E. coli are a part of the normal microbial flora present in the colons and feces of all warm-blooded animals. However, multiple disease causing serotypes of E. coli have been identified in the past few decades. The most well-known serotype is E. coli O157:H7, which frequently occurs in wastewater and feces in developed countries and can result in severe illnesses and death. E. coli O157:H7 (or just O157:H7) is the "poster child" for pathogenic E. coli and is the serotype most extensively investigated (Chart et al., 2000). E. coli is a versatile bacterium and multiple subtypes and strains that are pathogenic have been documented. Although the nomenclature for these strains has not yet stabilized in the literature, M01bak and Scheutz (2004) provided the following helpful summary of the current nomenclature: Diarrheagenic E. coli (DEC) - includes all the strains on this list; February 2009 8 ------- U.S. Environmental Protection Agency Verocytotoxin (Shiga toxin)-producing E. coli (VTEC or STEC) - E. coll that produce verocytotoxin (Shiga toxin) VT1 and/or VT2; o Enterohemorrhagic E. coli (EHEC) - originally defined as serotypes that cause a clinical illness similar to E. coli O157:H7, now used as a term for VTEC that cause hemorrhagic colitis (HC) in humans; Enterotoxigenic E. coli (ETEC) - E. coli that produce enterotoxins that are heat stable (STh, STp) and/or heat labile (LT); Attaching and effacing E. coli (A/EEC) - E. coli that attach to and efface the microvilli of enterocytes, but do not produce high levels of verocytotoxin; o Enteropathogenic E. coli (EPEC) - Subtype of A/EEC, usually of particular serotypes that mostly contain an EPEC adherence factor plasmid and often produce bundle-forming pili; Enteroaggregative E. coli (EAggEC) - E. coli that exhibit a pattern of aggregative adherence to tissue culture; and Diffuse adherent E. coli (DAEC) - E. coli that exhibit a pattern of diffuse adherence to tissue culture. Nataro and Kaper (1998) provide a comprehensive, albeit dated, review of the DEC. E. coli Zoonotic Potential E. coli is part of the normal intestinal flora of humans and warm-blooded animals and can readily spread to humans through contaminated food and water (WHO, 2004). Pathogenic E. coli have been documented in a wide variety of animal species including cattle (Chapman et al., 1997; Rangel et al., 2005), chickens (Schoeni and Doyle, 1994), sheep (Chapman et al., 1997; Kudva et al., 1996), pigs (Booher et al., 2002; Chapman et al., 1997; Feder et al., 2003), deer (Keene et al., 1997; Rice et al., 1995; Sargeant et al., 1999), horses (Chalmers et al., 1997), and dogs (Hammermueller et al., 1995). Ruminants, and cattle in particular, are considered one of the most important animal sources of E. coli O157:H7 and other VTECs. All of the VTECs including EHEC are capable of causing severe disease in humans, are typically shed by healthy cattle and other species, and have documented cases of transmission via humans and water (M01bak and Scheutz, 2004). Chapman et al. (1997) determined that the monthly prevalence of E. coli O157:H7 in cattle ranged from 4.8 to 36.8 percent, which was higher than the prevalence range in poultry, sheep, and pigs. Michel et al. (1999) examined the relationship between 3,001 cases of VTEC and the livestock density in rural areas of Ontario, Canada. Their research indicated that cattle density had a positive and significant association with the number of reported cases of VTEC in humans, suggesting that living near cattle farms may increase a person's risk of contracting VTEC. Current data suggest that the prevalence of E. coli O157:H7 in poultry is low (Chapman et al., 1997), although contact with chickens has resulted in outbreaks. Schoeni and Doyle (1994) inoculated 1-day-old chicks with strains of serotype O157:H7. The chicks shed serotype O157:H7 up to 11 months after inoculation, and O157:H7 was subsequently recovered from their egg shells, but not from the yolks or whites. In an outbreak in northern Italy, the source of exposure was believed to be chickens in 15 cases of hemolytic uremic syndrome (HUS; see more February 2009 ------- U.S. Environmental Protection Agency below) that were caused by serotype O157 and other EHEC serotypes (Tozzi et al., 1994). In this outbreak, a case-controlled study showed an association between contact with chickens and HUS although VTEC was not isolated from any of the chickens. In a study by Chapman et al. (1997), O157:H7 was isolated from 2.2 percent of sheep and 4 percent of pigs, respectively. In a Russian study, O157:H7 was found in sheep, and the incidence was highly variable, ranging from 31 percent in June to 0 percent in November (Kudva et al., 1996). Kudva and colleagues also showed that 80 percent of the O157:H7 isolates had at least two of the Shiga-like toxin types I or II or the attaching-effacing lesion genes. Strains that produce Shiga-like toxins have been documented in humans and in animals (Kudva et al., 1996; M01bak and Scheutz, 2004). E. coli Route of Exposure Waterborne transmission of E. coli O157:H7 has been reported from both recreational water (Ackman, 1997; CDC, 2002; McCarthy et al., 2001; Samadpour et al., 2002; Yoder et al., 2004) and contaminated drinking water (CDC, 2002; Hrudey et al., 2002, 2003; Olsen et al., 2002; Pond, 2004; Swerdlow et al., 1992; Yarze and Chase, 2000). Most studies examining contamination from recreation immersion have suggested that ingestion was the primary route of exposure (Keene et al., 1994). Because E. coli O157:H7 has a relatively low infectious dose, swallowing a small amount of contaminated water may cause illness (Haas et al., 2000; Keene et al., 1994). Immersion in and ingestion of recreational waters has been the route of exposure for strains of E. coli other than EHEC (Yoder et al., 2004). In Connecticut, 11 persons were infected with E. coli O121 in an outbreak associated with swimming in a lake (CDC, 2000). E. coli Illness Symptoms E. coli can cause a relatively wide range of illness symptoms, depending on the strain and the underlying health of the host (Hunter, 2003; M01bak and Scheutz, 2004). Incubation periods vary and can be as short as 8 hours for EAEC infections (Nataro et al., 1995), 14 to 50 hours for ETEC (Dupont et al., 1971), and 3 to 4 days for EHEC, with shorter (1 to 2 days) and longer (5 to 8 days) incubations noted in some outbreaks (Nataro and Kaper, 1998). Approximately 82 to 95 percent of all E. coli cases result in relatively minor illness symptoms including abdominal cramps, vomiting, diarrhea (often bloody), and sometimes fever (Ostroff et al., 1989; Swerdlow et al., 1992). The duration of these symptoms is 4 to 10 days (CDC, 1993a) although, in the Cincinnati outbreak, infants remained hospitalized for 21 to 120 days (Nataro and Kaper, 1998). Symptoms may be more severe for persons with hemorrhagic colitis (HC) or bloody inflammation of the colon (Griffin and Tauxe, 1991). Symptoms commonly associated with human illness from the various DEC include the following: VTEC (or STEC) - Diarrhea, hemorrhagic colitis, HUS; o EHEC - hemorrhagic colitis (HC); February 2009 10 ------- U.S. Environmental Protection Agency ETEC - Acute watery diarrhea; A/EEC - Acute or persistent diarrhea; o EPEC - Acute or persistent diarrhea; EAggEC - Acute watery, often protracted diarrhea; and DAEC - Acute or persistent diarrhea. In rare cases, HC can develop into HUS, which is a severe life-threatening disease that can result in kidney failure and neurological complications such as seizures and strokes (Brotman, 1995). Brooks et al. (2005) conducted a study that suggests E. coli strains that produce Shiga toxin 2 are much more likely to result in HUS than strains that only produce Shiga toxin 1. Approximately 2 to 7 percent of all E. coli O157:H7 infections result in HUS, and HUS is most common among children under 5 years old and the elderly (Griffin and Tauxe, 1991; WHO, 2004). Less than 10 percent of HUS cases turn into a chronic illness such as chronic kidney failure, blindness, or partial paralysis (Tarr, 1995). Approximately 33 percent of persons that contract HUS have abnormal kidney function for several years, sometimes requiring long-term dialysis (WHO, 2004). In very rare cases, HUS can also lead to death. Cases of infection with E. coli O157:H7 from swimming-associated outbreaks, compared to other routes of exposure, have the highest rate of HUS. This difference may be due to the higher proportion of young children participating in swimming and becoming infected during such outbreaks and the higher likelihood of young children developing HUS (Rangel et al., 2005). HUS, non-bloody diarrhea, and HC may occur with O157:H7 infections; other complications may include cholecystitis, colonic perforation, intusssception, pancreatitis, posthemolytic biliary lithiasis, post-infection colonic stricture, rectal prolapse, appendicitis, hepatitis, hemorrhagic cystitis, pulmonary edema, myocardial dysfunction, and neurological abnormalities (Nataro and Kaper, 1998). As noted above, infection with E. coli O157:H7 can also result in death. The U.S. Centers for Disease Control and Prevention (CDC) estimates a death rate of 0.08 percent, although case studies reveal slightly higher rates. For example, the 1993 fast-food E. coli O157:H7 outbreak in Washington, California, Idaho, and Nevada resulted in 4 deaths out of approximately 700 who fell ill, which corresponds to approximately 0.57 percent mortality (Brotman, 1995). Griffin and Tauxe (1991) reviewed 12 outbreaks in the United States between 1982 and 1990 and calculated a mortality rate of 1.9 percent among those with diagnosed E. coli O157:H7 infections. E. coli Illness Incidence The CDC estimates that there are 73,000 cases of E. coli 0157:H7 infections and 61 deaths annually in the United States. Non-0157 Shiga-like toxin serotypes cause approximately 37,000 illnesses per year (Mead et al., 1999).* Craun et al. (2004a) estimated that E. coli was the etiological agent for 30 percent of the outbreaks from zoonotic contamination in untreated recreational waters from 1971 to 2000. E. coli O157:H7 infection associated with recreational exposure was first reported in June 1991 in a lake in Oregon (Keene et al., 1994). From 1991 until 2002, 20 additional outbreaks as a result of exposure to contaminated recreational waters have been reported to the CDC (Rangel et 1 The CDC is currently updating these numbers, but the newest values have not yet been released. February 2009 11 ------- U.S. Environmental Protection Agency al., 2005). Fourteen of the outbreaks occurred as a result of exposure to contaminated lakes or ponds, while seven occurred from exposure to contaminated swimming pools (Rangel et al., 2005). CDC's surveillance system probably captures a small proportion of E. coli O157:H7 outbreaks that occur because many illnesses are not reported to public health officials or the CDC, are not recognized as E. coli infections, or the outbreak is considered of unknown etiology (Cieslak et al., 1997). From 1991 to 2004, 14 outbreaks of E. coli related illness have been associated with untreated recreational waters (Table II. 1.1-1). II. 1.2 Campylobacter Campylobacter Strain Variation Of the 17 species in the genus Campylobacter., C. jejuni., and C. coli are the most important human pathogens. Eight strains of C. jejuni have been DNA sequenced. At least two strains of C. jejuni have been shown to cause illness in ferrets, mice, rabbits, and rats, and several of the strains have been associated with Guillain-Barre syndrome (GBS) (Nachamkin, 2002). Table II.l.l-l. Outbreaks of E. coli Associated with Recreational Waters in the United States Year 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 Number of Cases 80 0 0 166 28 24 8 31 61 0 49 9 0 0 Number of Outbreaks 1 0 0 1 4 2 1 2 5 0 2 1 0 0 Source of Outbreak(s) Lake NA NA Lake Lakes 1 pool, 1 lake Lake 1 pool, 1 lake 1 pool, 3 lakes, 1 ditch water NA Lakes Pool NA NA Source: Data from CDC Surveillance Summaries: Morbidity and Mortality Weekly Report (MMWR) Surveillance for Waterborne-Disease Outbreaks - United States: 1991-1992, 1993-1994, 1995- 1996, 1997-1998, 1999-2000, 2001-2002, 2003-2004 (CDC, 1993b, 1996, 1998, 2000, 2002, 2004, 2006). February 2009 12 ------- U.S. Environmental Protection Agency Campylobacter Zoonotic Potential Because most of the published literature focuses on food contamination from human waste as the primary risk factor for Campylobacter infection, some professionals in the health community have concluded that the zoonotic waterborne route is unlikely to be important (Till and McBride, 2004). Some evidence indicates that the human sources of Campylobacter may obscure the zoonotic risk factors (McBride, 1993). McBride et al. (2002) found that 60 percent of all samples collected from 25 freshwater recreational water sites in New Zealand over 15 months contained at least one species of Campylobacter. This finding led to an inference that 4 percent of all campylobacteriosis cases in New Zealand were due to water contact recreation (McBride et al., 2002). Donnison and Ross (2003) note that nearly all streams near dairy farms in New Zealand contain Campylobacter, and despite generally low concentrations, these streams may help cycle Campylobacter in farm animals and indirectly contribute to the high incidence of human infection in New Zealand. The species and strain of Campylobacter contained in a stream is highly influenced by the path of the stream, with surface waters running through beef- and sheep-grazing pastures typically contaminated with C. coli and C. jejuni (Jones, 2001). Surface waters that have contact with avian species are typically contaminated with C. jejuni, C. lari, C. coli, and avian-sourced urease-positive thermophilic campylobacters (Jones, 2001). The main source of campy lob acters for bathing waters is typically assumed to be sewage effluent, though there is evidence that birds may be primarily responsible for contamination (Jones, 2001). Campylobacter Route of Exposure The main route of exposure to Campylobacter from recreational waters is from incidental ingestion of water during full immersion activities such as swimming. The bacteria may be of anthropogenic or animal sources. From 1991 to 2002, 3 percent of waterborne outbreaks were attributed to Campylobacter (7 outbreaks, 360 cases) (Craun et al., 2006). Craun et al. (2005) summarized and discussed 259 waterborne outbreaks occurring in the United States from 1971 to 2000 and associated only with recreational water. Campylobacter was the etiologic agent for 0.9 percent of the outbreaks. Although waterborne sources of Campylobacter are important, most infections occurred as a result of handling contaminated foods (e.g., raw chicken) and direct person-to-person transmission is rare (Allos, 2001). Campylobacter Illness Symptoms Campylobacter infection can result in a number of symptoms, including loose and watery or bloody diarrhea, dysentery, fever, and severe abdominal cramps (Allos, 2001; Flicker, 2006a). Infections of Campylobacter are symptomatically indistinguishable from those of other bacterial pathogens such as Salmonella (Nachamkin, 2002). Diarrheal symptoms may be frequent at the peak of the illness, typically occurring 8 to 10 times per day. Although the disease usually lasts only one week, some infected individuals experience relapses leading to several weeks of symptoms (Allos, 2001). February 2009 13 ------- U.S. Environmental Protection Agency Some complications may result from Campylobacter infection including cholecystitis, pancreatitis, peritonitis, and massive GI hemorrhage. Some immunocompromised individuals experience bacteremia as a result of campylobacteriosis, and in rare instances, some individuals have experienced meningitis, endocarditis, septic arthritis, osteomyelitis, or neonatal sepsis. Infection rarely results in death, with 1 death per 20,000 infections (Allos, 2001). Several chronic sequelae have been associated with Campylobacter infection including the development of GBS (Fricker, 2006a). GBS is an acute demyelinating disease of the peripheral nervous system that affects 3,000 to 6,000 people in the United States annually, with approximately 1,000 to 2,000 cases preceded by C. jejuni infection (Allos, 1998). The risk of developing GBS after C. jejuni infection, however, is small (approximately 1 case of GBS per 1,000 infections), and many GBS-related C. jejuni infections are asymptomatic (Allos, 2001). Reactive arthritis and depression also have been reported as chronic sequelae following campylobacteriosis (Garg, 2006; Nachamkin, 2002). Recent evidence has associated C. jejuni with a rare form of mucosa-associated lymphoid tissue (MALT) lymphoma called immunoproliferative small intestinal disease (IPSID); campylobacteriosis has not yet been shown as causative agent of IPSID (Poly and Guerry, 2008). In 2000, a municipal water supply in Walkerton Ontario became contaminated with E. coli and C. jejuni. In the outbreak that resulted from that contamination, persons who showed symptoms of acute bacterial gastroenteritis at the time of the outbreak were more likely to exhibit hypertension and reduced kidney function approximately 4 years after infection than those who were asymptomatic at the time of the outbreak (Garg et al., 2005). Campylobacteriosis Incidence Although Campylobacter became a reportable illness in the United States in the early 1980s, it is estimated that only 1 in 38 cases of detected infection actually are reported (Mead et al., 1999). Approximately 2.4 million Campylobacter infections are estimated to occur in the United States every year (Allos, 2001). Between 1996 and 1999, the incidence of campylobacteriosis in the United States declined by 26 percent, from 23.5 to 17.3 cases per 100,000 people (Allos, 2001). According to reports from 1999, Britain experiences approximately 5 times this infection rate (103.7 cases per 100,000 people), though reporting rates may not be comparable (Gillespie et al., 2002). C. jejuni infection is one of the most common causes of gastroenteritis across the world and is frequently responsible for diarrheal illness in travelers (Allos, 2001). Other than an outbreak in 1999, in Florida, where 6 cases of C. jejuni infections resulted from a private swimming pool, no other recreational waterborne outbreaks have been reported to the CDC between 1991 and 2004 (CDC, 2002). II. 1.3 Salmonella Salmonella Strain Variation and Zoonotic Potential Salmonella are non-encapsulated, Gram-negative bacteria of the Enterobacteriaceae family that infect both animals and humans causing a wide range of illnesses (Cohen, 1986; Lightfoot, 2004; February 2009 14 ------- U.S. Environmental Protection Agency Ohl, 2001). There are more than 2,500 serovars/serotypes in the genus Salmonella (Lightfoot, 2004). The genus Salmonella consists of two species, S. enterica and S. bongori. S. enterica is divided into the following six subspecies: S. e. enterica; S. e. salamae; S. e. arizonae; S. e. diarizonae; S. e. houtenae; and S. e. indica (Lightfoot, 2004). The many serovars in the group are closely related to each other by somatic and flagellar antigens, and most strains show diphasic variation of flagellar antigens). Thus, Salmonella can be serotyped by means of somatic and flagellar antigens and further subtyped by antibiotic-sensitivity testing, biochemical reactions, phage- typing, and analysis of the plasmids they carry. All Salmonella serotypes share the ability to invade the host by inducing their own uptake into cells of the intestinal epithelium (Lightfoot, 2004). Although more than 2,500 Salmonella serotypes exist, only 10 account for more than 70 percent of the isolates reported annually in the United States (Cohen, 1986; Lightfoot, 2004). Because the vast majority of Salmonella isolates from humans are of the subspecies S. e. enterica, the CDC recommends that Salmonella strains only be referred to by their genus and serotype (e.g., S. typhi). S. typhimurium and S. enteritidis are the most prevalent serotypes found in the United States (CDC, 2006). The other serotypes have much smaller incidences in the United States; S. enteritidis accounted for 10 percent in 1984, and S. newport, S. infantis, and S. heidelberg accounted for 4.5, 3.0, and 1.0 percent, respectively. Some Salmonella serotypes are highly host-specific, restricted to a single host species and rarely causing disease in other species (Lightfoot, 2004). S. typhi and S. paratyphi are exclusively human pathogens, with no known animal reservoirs, while S. enteriditis and S. typhimurium infect a wide range of animal hosts, including poultry, cattle, and pigs. The serotypes with a wide range of animal hosts can also infect humans, usually via food consumption, and cause self- limited gastroenteritis in humans (WHO, 2004). S. gallinarum and S. pullorum are almost exclusively pathogens of poultry (Cohen, 1986). Human pathogens S. heidelberg and S. litchfield have primarily avian and reptilian reservoirs, respectively (Cohen, 1986). S. abortusovis is specific to sheep (Lightfoot, 2004). Salmonella has also been reported in swine, cattle, rodents, birds, turtles, dogs, and cats (Covert and Meckes, 2006). The CDC estimates that 74,000 cases of salmonellosis per year are associated with exposure to reptiles or amphibians (directly or indirectly) (Lightfoot, 2004). In 1993, an outbreak of S. typhimurium where more than 650 people became ill and that resulted in 7 deaths, was traced to a water-storage tower that allowed access to birds (Covert and Meckes, 2006). Although this outbreak was from drinking water rather than recreational water, it illustrates the potential for waterborne exposure due to birds. February 2009 15 ------- U.S. Environmental Protection Agency Salmonella Route of Exposure Salmonella infections begin with the ingestion of organisms in contaminated food or water (Lightfoot, 2004; Ohl, 2001). Although ingestion or exposure to infected water from recreational swimming is less common, it has been reported worldwide (Cohen, 1986; Lightfoot, 2004). Researchers have observed a reduction in the infectious dose of Salmonella under conditions where the gastric pH is elevated suggesting that gastric acidity may create an initial barrier to infection (Lightfoot, 2004). Salmonella also exhibit an adaptive, acid-tolerance response in low pH conditions thereby allowing them to live in acidic host environments like the stomach (Ohl, 2001). Elevated levels of Salmonella also have been observed in major water bodies that receive discharges of meat processing wastes, raw sewage, and effluents from ineffective sewage treatment plants (Geldreich, 1996). Farming operations with cattle and poultry result in large quantities of fecal products in relatively small areas due to the dense population of animals. Thus, if the animal waste is not discharged into a lagoon or landfill, the stormwater runoff over the animal feedlots will transport massive loads of fecal pollution to the receiving waters of the drainage basin (Lightfoot, 2004). Salmonella Illness Symptoms Illnesses caused by Salmonella range from asymptomatic colonization and mild gastroenteritis to the more serious enteric fever (typhoid), meningitis, and osteomyelitis (Cohen, 1986). Enteric fever and gastroenteritis are the key clinical syndromes associated with a Salmonella infection (Lightfoot, 2004). Different Salmonella serovars cause different clinical symptoms. S. typhi causes enteric fever (typhoid) in humans, and S. typhimurium causes diarrhea in humans and other animal species but a typhoid-like syndrome in mice (Lightfoot, 2004). S. abortusovis is responsible for abortion in ewes. Most Salmonella serovars cause an acute and mild enteritis, but S. blegdam, S. bredeney, S. choleraesuis, S. dublin, S. enteritidis, S. panama, S. typhimurium, and S. virchow may also be invasive and cause pyemic infections localizing in the viscera, meninges, bones, joints, and serous cavities (Lightfoot, 2004; Covert and Meckes, 2006). S. dublin is also particularly associated with different extraintestinal infections in persons with acquired immunodeficiency syndrome (AIDS) (Lightfoot, 2004). Infection with S. typhi or S. paratyphi, which are exclusively human pathogens, results in enteric fever (Ohl, 2001). Clinical symptoms of enteric fever include diarrhea, abdominal pain, fever, and sometimes a maculopapular rash. The pathological sign of enteric fever is mononuclear cell infiltration and hypertrophy of the reticuloendothelial system (Ohl, 2001). Salmonellosis is caused by ingestion of nontyphoidal salmonellae (e.g., S. enteriditis and S. typhimurium) with an incubation period of 8 to 72 hours (Lightfoot, 2004). The estimated inoculum size of nontyphoidal Salmonella required to cause symptomatic disease in healthy adult volunteers is 105 to 1010 organisms (Lightfoot, 2004). The infectious dose varies depending on the age and health of the person, strain differences, and the vector. Most February 2009 16 ------- U.S. Environmental Protection Agency salmonellosis cases are self-limiting, and the affected persons recover without treatment (CDC, 2006; Cohen, 1986). Some infections are more severe, however, particularly in the young, elderly, and people with weakened immune systems, and such infections may become invasive (APHA, 2004; Lightfoot, 2004). The clinical symptoms of salmonellosis may include diarrhea, abdominal pain, nausea, chills, fever, and prostration with the duration of illness ranging approximately 2 to 7 days (APHA, 2004; WHO, 2004). Vomiting may occur as well, but it is rare and usually a sign of invasive disease (APHA, 2004). Organisms that leave the GI tract and invade the rest of the body can cause bacteremia and septicemia, thus spreading the salmonellae to many organs in the body and possibly leading to abscesses, septic arthritis, cholecystitis, endocarditis, meningitis, pericarditis, pneumonia, pyodrema, or pyelonephritis (APHA, 2004). Diarrhea from Salmonella infection is usually self-limiting and does not require treatment unless severe. Overall, there is an estimated 22.1 percent hospitalization rate, and an estimated 0.8 percent fatality rate (USDA, 2005). Mortality from S. typhi and S. paratyphi is estimated to be between 10 to 15 percent without treatment (Ohl, 2001). In severe cases, fluid and electrolyte replacement may be needed. Antibiotics are not recommended to treat Salmonella infections, except where there is evidence of invasion and septicemia, because they do not alleviate the symptoms or reduce the duration of the illness (Kanarat, 2004). Antibiotics may even prolong excretion of Salmonella in the feces. Salmonellosis Incidence Foodborne Salmonella are the estimated cause of approximately 1.4 million foodborne-related illnesses, 15,600 foodborne illness-related hospitalizations, and 550 foodborne-related deaths each year in the United States (Mead et al., 1999). In 2005, 45,322 salmonellosis cases were reported to the CDC through the Public Health Laboratory Information System (CDC, 2007a). Community surveys during outbreaks suggest that the proportion of infections reported is between 1 in 10 and 1 in 100 (Cohen, 1986). Between 1991 and 2002, 3 waterborne outbreaks (833 cases) were attributed to nontyphoid Salmonella (Craun et al., 2006). Craun et al. (2005) reported that Salmonella were the etiologic agent in 0.9 percent of 259 recreational waterborne outbreaks occurring from 1971 to 2000. II. 1.4 Leptospira Leptospira Strain Variation and Zoonotic Potential Leptospira is an aerobic, motile spirochaete, 6 to 20 jim long and 0.1 jim wide. Leptospira occurs worldwide and has become an important recreational zoonosis due to its prolonged survival in water (Pond, 2005). The genotypic classification of Leptospira into two species (L. interrogans and L. biflexd) has been replaced by a phenotypic classification system in which 13 genomospecies are currently defined (L. interrogans., L. noguchii, L. santarosai, L. meyeri, L. wolbachiic, L. biflexac, L. fainei, L. borgpetersenii, L. kirschneri, L. weilii, L. inadai, L. parvac, and L. alexanderf) (Levett, 2001). Of the 28 serovars, several occur in more than one February 2009 17 ------- U.S. Environmental Protection Agency genomospecies. Leptospirosis is probably the most widespread zoonosis in the world (Levett, 2001; Meites et al., 2004). Zoonotic reservoirs include livestock (pigs and cattle), domestic pets (dogs), and wild or feral animals (rats, voles, and mice) (Kanarat, 2004; Levett, 2001). Complete genome sequences of L. interrogcms serovars Copenhagen! and Lai reveal that despite overall genetic similarity there are significant structural differences in their genomes (Nascimento et al., 2004). Nascimento and colleagues analyzed the genomic sequences to gain insight into genes that influence motility, chemotaxis, pathogenicity, and colonization of the pathogen. Leptospira Route of Exposure The source of infection in humans is usually either direct or indirect contact with the urine of an infected animal (Levett, 2001; WHO, 2003). Workers in direct contact with animal reservoirs are at increased risk (e.g., cattle, pig, and dairy farmers, slaughterhouse workers, and veterinarians) (Meites et al., 2004). Recreational exposure from swimming or boating in freshwater lakes is also possible (Levett, 2004; Meites et al., 2004). Outbreaks are often associated with unusual rainfall events or flooding (Bolin et al., 2004b). Leptospira Illness Symptoms Leptospirosis was first described by Adolf Weil in 1886; thus, the more serious form of leptospirosis is still known as Weil's disease (WHO, 2003). Leptospirosis is biphasic with a week-long acute stage followed by approximately 2 weeks of convalescence (Levett, 2001). During the acute stage, antibodies are low and the pathogen is detected mainly in the blood and cerebrospinal fluid, whereas the convalescent stage corresponds with the appearance of antibodies and the presence of pathogens in the urine (Levett, 2001). Anicteric leptospirosis can be mild or acute. The majority of infections are either subclinical or mild, and patients usually do not seek medical attention (Levett, 2001). Icteric leptospirosis is a much more severe disease than anicteric leptospirosis. Icteric leptospirosis accounts for most of the high mortality rate, which ranges between 5 and 15 percent. Between 5 and 10 percent of all patients with leptospirosis have the icteric form of the disease (Levett, 2001). Symptoms associated with leptospirosis include the following: jaundice, anorexia, headache, conjunctival suffusion, chills, vomiting, myalgia, abdominal pain, nausea, cough, hemoptysis, hepatomegaly, lymphadenopathy, diarrhea, rash (usually lasting less than 24 hours), and fever, which can be biphasic and reoccur after 3 to 4 days of remission (Levett, 2001). In some cases, acute infection in pregnancy has been reported to cause abortion and fetal death (Levett, 2001). Uveitis (ocular complications) is recognized as a chronic sequela of leptospirosis in humans and horses. Chronic visual disturbance lasting 20 years or more has been reported (Levett, 2001). In 1994, CDC removed leptospirosis from the notifiable diseases list; however, the Hawaii Department of Health still requires reporting (Katz, 2001; Levett, 2001). February 2009 18 ------- U.S. Environmental Protection Agency Leptospirosis Incidence Levett (2001) summarized information for 28 waterborne outbreaks of leptospirosis worldwide, 22 of which were associated with swimming, 1 with kayaking, and 1 with rafting. Within the United States, the highest incidence of leptospirosis is found in Hawaii (Katz, 2001). Between 1971 and 2000, 16 percent of recreational waterborne disease outbreaks were attributed to Leptospira (Craun et al., 2004a). In 1998, in Illinois, there was an outbreak (375 cases) of leptospirosis associated with a triathalon in a lake (CDC, 2000). In a prospective, population-based study of patients presenting with acute febrile illness, the geographic distribution of human Leptospira isolates mirrored the distribution of Leptospira 16S ribosomal gene sequences in urban and rural water sources (Ganoza et al., 2006). II.2 Protozoa II.2.1 Cryptosporidium Cryptosporidium is a small protozoan parasite that infects the microvillous region of epithelial cells in the digestive and respiratory tract of humans and other mammals, birds, reptiles, and fish. Cryptosporidium does not replicate outside of a host. Environmentally robust oocysts are shed by infected hosts into the environment and can survive in environmental conditions for long periods of time (up to months) until ingested by a new host. In the new host, the life cycle starts again, and multiplication occurs using the biological resources of the host (WHO, 2006). Cryptosporidium exists in the natural environment in the oocyst form and which are resistant to conventional drinking water treatment measures such as chlorination. Cryptosporidium is recognized as a widespread pathogen for the general population, including both immunocompromised and immunocompetent persons (WHO, 2006). Cryptosporidium Life Cycle and Strain Variation Cryptosporidium has a complex life cycle. Each oocyst, which has an environmentally resistant wall, holds four sporozoites. Oocysts enter the environment by passing with the feces of an infected host organism (Payer and Ungar, 1986; Payer et al., 1997). Oocysts are immediately infectious and may remain in the environment for very long periods without losing their infectivity. Oocysts are resistant to environmental conditions and natural decay and can travel passively through the environment until they are ingested by a new host organism. In the GI tract of the new host, 4 sporozoites exit each oocyst (excyst) and may form an infection in the epithelial cells of the small intestine of the host (USEPA, 200la). The sporozoites transform through several life stages including an asexual and a sexual reproduction cycle. Oocysts are the result of the sexual reproduction cycle. Oocysts of the two species of Cryptosporidium responsible for most human infectionsC. hominis and C. parvumare spherical with a diameter of 4 to 6 |im. Thin-walled oocysts may excyst within the same host and start a new life cycle (autoinfection), whereas thick-walled oocysts generally are shed by the host. Autoinfection may lead to a heavily infected epithelium of the small intestine resulting in secretory diarrhea (WHO, 2006). February 2009 19 ------- U.S. Environmental Protection Agency Cryptosporidium is part of phylum Apicomplexa, family Cryptosporidiidae, and has been classified as a member of the group of eimeriid coccidiana diverse group of parasitic protozoa (WHO, 2006). There are currently 16 species of Cryptosporidium identified in the literature (Table II.2.1-1). However, this taxonomy is likely to change as molecular methods continue to characterize isolates and potential new species. Payer (2004a) and Olson et al. (2003) updated the list of Cryptosporidium species that have been reported to infect humans to include C. baileyi, C. canis, C.felis, C. hominis, C. meleagridis, C. muris, and C. parvum. It is important to note that the human form of C. parvum (formerly referred to as H-type or genotype 1) was recently reclassified as a new species, C. hominis (Morgan-Ryan et al., 2002). The cattle form of C. parvum (formerly referred to as C-type or genotype 2) maintains the designation C. parvum. Thus, studies published prior to 2002 should be interpreted carefully keeping in mind that authors who refer to C. parvum may be referring to C. parvum., C. hominis., or both. Of the current 16 species of Cryptosporidium., C. parvum and C. hominis most commonly cause GI illness in humans. Studies with volunteers have demonstrated that a low dose of C. parvum (e.g., 10 oocysts) is sufficient to cause infection in healthy adults although some strains may be more infectious than others (Chappell et al., 1999; DuPont et al., 1995; Okhuysen et al., 2002). The relationship Table II.2.1-1. Cryptosporidium Species Cryptosporidium Species C. andersoni C. baileyi C. bovis C. canis C. felis C. galli C. hominis C. meleagridis C. molnari C. muris C. nasorum C. parvum C. scopthalmi C. serpentis C. suis C. varanii C. wrairi Initially Described Host Species Bos taurus (domestic cattle) Gallus gallus (domestic chicken) Bos taurus (domestic cattle) Canis familiaris (dogs) Fe/;s catis (domestic cat) Gallus gallus (domestic chicken) Homo sapiens (humans, formerly C. parvum genotype 1) Meleagris gallopavo (turkey) Dicentrarchus labrax (fish) Mus musculus (house mouse) Naso lituratus (fish) Mus musculus (house mouse) (formerly genotype 2) Scopthalmi maximus (turbot) Elaphe guttata (corn snake) E. subocularis (rat snake) Sanzinia madagascarensus (Madagascar boa) Sus scrofa (pig) Varanus prasinus (emerald monitor lizard) Cavia porcellus (guinea pig) Source: Adapted from 2007; Morgan- Caccio, 2005; Payer, 2003, 2004a; Payer et al., 1997, 2000; Payer and Xiao, Ryan et al., 2002; Ryan et al., 2003; and Xiao and Ryan, 2004. February 2009 20 ------- U.S. Environmental Protection Agency between the number of oocysts humans are exposed to and the probability of infection is discussed in detail in the subsequent section. Studies of immunosuppressed adult mice have demonstrated that a single viable oocyst can induce C. parvum infections (Okhuysen et al., 2002; Yang et al., 2000). Genetic and molecular studies of C. parvum (including C. hominis) indicate that the species is genetically heterogeneous among isolated strains found in different host species (Xiao and Ryan, 2004). There also is evidence that C. parvum and C. hominis experience recombination and that polymorphisms exist in the C. hominis species (Widmer et al., 1998). Okhuysen et al. (1999) showed that different isolates of C. parvum (including C. hominis) have different levels of infectivity for humans, and therefore the heterogeneity of the species may influence the risk posed to public health. Furthermore, distinct transmission cycles are evident among different genotypes of C. parvum. Multiple genotypes have been shown to circulate among different host species, and mixed infections with genotypically distinct populations have been reported (Widmer et al., 1998). Cryptosporidium Zoonotic Potential Human cases of cryptosporidiosis typically have been associated with different kinds of animal contact, which has led to the widespread belief that the host-range of Cryptosporidium is very broad and many animals can serve as reservoirs for Cryptosporidium. Cryptosporidium infection has been reported in more than 155 mammalian species (Payer, 2004a) as well as numerous reptiles, amphibians, birds, and fish (O'Donoghue, 1995). Several lines of evidence indicate that livestock, primarily cattle and sheep, are a major source of Cryptosporidium contamination of drinking water sources. These include the detection of C. parvum (presumably from animals) in many source waters and elevated levels of Cryptosporidium in watersheds with extensive agricultural activity (Payer, 2004a; WHO, 2006). Also, direct zoonotic transmission of Cryptosporidium infection from livestock to humans has been repeatedly demonstrated (WHO, 2006). During outbreaks of cryptosporidiosis, frequent detection of Cryptosporidium in human stool samples suggests that human sources can also add significantly to the occurrence of oocysts in source waters (Payer, 2004a). Based on data from the western United States, depending on climate and feedlot management systems, the average animal in a cattle feedlot excretes between 28,000 and 140,000 oocysts per day (Atwill et al., 2006). Furthermore, 91 percent of dairy farms studied by Sischo et al. (2000) had Cryptosporidium at their locations, with 15 percent of infant dairy calves shedding oocysts. Nine percent of farm-associated streams contained C. parvum. Therefore, cattle represent a significant reservoir and potential environmental source of C. parvum. Tate et al. (2000) demonstrated that oocysts can be carried by runoff during rain events. Most outbreaks of cryptosporidiosis are caused by C. hominis, which is only transmitted by human hosts. Outbreaks represent only 10 percent of domestic cryptosporidiosis cases, however, and at least one study (Feltus et al., 2006) has shown that the zoonotic C. parvum may be responsible for the majority of sporadic (endemic) cases. Atwill et al. (1997) reported that feral February 2009 21 ------- U.S. Environmental Protection Agency pigs may serve as an environmental reservoir for C. parvum and may represent a potential source of Cryptosporidium contamination of ambient waters. Although it is clear that livestock may be a major contributor to drinking water source contamination, there are few data to support a quantitative estimate of the proportion of this contribution. Cryptosporidium levels in source water are known to vary seasonally (USEPA, 2005a, 2005b), and short-term levels in surface water can be strongly associated with storm events or other weather variables (Payer, 2004a; Naumova et al., 2005; USEPA, 2005a; WHO, 2006). In watersheds with diverse land-use patterns, it is likely that different sources contribute different proportions to the total contamination load at different times during the year, with the relative contributions depending on a wide range of watershed characteristics. Besides animal contamination, the other major source of Cryptosporidium in ambient water is human fecal wastes. Several mechanisms can be responsible for the contamination of source waters and include malfunctioning septic systems, routine or "upset" releases from municipal wastewater treatment facilities, combined sewer system overflows, or human recreational uses (e.g., swimming, camping, and hiking). Due to the large numbers of oocysts excreted by infected individuals (Okhuysen et al., 1999) and oocyst resistance to many conventional wastewater treatment processes, even small releases can be significant. The World Health Organization (WHO) found reports of raw sewage samples containing up to 14,000 oocysts/L (average was up to 5,300 oocysts/L) and treatment plant effluents containing between 17 and 250 oocysts/L (WHO, 2006). Clearly, in water bodies where treatment effluents comprise an appreciable proportion of total flow, their contribution to the total Cryptosporidium load may be substantial. LeChevallier et al. (2003) reported the results of Cryptosporidium monitoring for approximately 600 samples from 6 watersheds located in the United States and Canada. They found that the two watersheds with the highest proportion of agricultural land use had the highest average Cryptosporidium levels2 and that approximately 90 percent of the samples exhibited the bovine (cattle) genotype (C. parvum). These findings implicate livestock, at least in these watersheds, as the major contributor to overall Cryptosporidium levels. This finding is consistent with studies indicating that the prevalence of Cryptosporidium infection in young livestock (i.e., calves and lambs) is very high (Payer, 2004a) and that the oocyst counts in feces of young infected animals ranges from 106 to 108 oocysts per gram (WHO, 2006). McCuin and Clancy (2006) conducted a 15-month occurrence study of Cryptosporidium occurrence in 10 wastewater facilities across the United Sates. Indigenous oocysts were detected in 30 percent of raw influents, 46 percent of primary effluents, 58 percent of secondary effluents and 19 percent of tertiary effluents in the 289 analyzed samples. Zhou et al. (2003) analyzed 179 wastewater treatment plant effluent samples from Milwaukee, Wisconsin using polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) methods to characterize genotypes of detected Cryptosporidium. In contrast with the results observed for source waters by LeChevallier et al. (2003), C. hominis (13.4 percent of samples) was detected more frequently than C. parvum (2.8 percent). This finding suggests that the distribution of Cryptosporidium in 2 These results were obtained using EPA Method 1623 (USEPA, 200 Ib), for which the authors reported an average recovery rate of 72±22 percent. February 2009 22 ------- U.S. Environmental Protection Agency Milwaukee's population was not heavily influenced by agricultural sources, though the relative low levels of C. parvum in wastewater may also have been due to differences in infectivity, excretion, or both of this species relative to C. hominis. As is the case for livestock, the extent to which human wastes contribute to overall Cryptosporidium contamination is highly site-specific, seasonal, and variable. Cryptosporidium Route of Exposure The main route of exposure to illness-causing microorganisms in recreational waters is through accidental ingestion of contaminated water while engaging in full immersion activities such as swimming or bathing. Secondary contact or partial body contact recreation such as wading, canoeing, motor boating, and fishing, in which ingestion is unlikely due to lack of direct water contact with the ears, eyes, mouth, or nose, is not considered to result in significant exposure (USEPA, 2002). A recent pilot study of anglers in the Baltimore area by Roberts et al. (2007), however, suggested that between 1 and 8 of 10 urban anglers could become infected with Cryptosporidium. This small study (56 anglers; a total of 46 fish/hand wash samples) included quantitative risk modeling. No data were reported on microbial water quality at the sampling sites, nor was there any attempt to obtain either health effects data or clinical samples to evaluate infection rates in the study population. The potential for person-to-person secondary transmission is high for Cryptosporidium infections. Based on an analysis of data from the 1993 Milwaukee outbreak, Eisenberg et al. (2005) suggested that 10 percent (95 percent confidence interval: 6 to 21 percent) of the cases of disease were due to person-to-person transmission. There is considerable evidence that direct person-to-person transmission, as well as indirect transmission through contact with contaminated objects, can be a significant route of infection, especially where human population densities are high or personal contact is frequent (USEPA, 200la). Direct transmission is affected by behavioral factors (e.g., frequent travel) and ethnic and dietary differences (USEPA, 200la). Using data from the Milwaukee outbreak, approximately 3 to 5 percent of infected individuals transmit the disease to others (Mackenzie et al., 1995; Osewe et al., 1996). Among children and their caretakers, however, the transmission rate is considerably higher (12 to 22 percent) (Osewe et al., 1996). Cryptosporidium Illness Symptoms Cryptosporidiosis is primarily characterized by GI symptoms such as profuse watery diarrhea; however, diarrheal symptoms are generally not distinguishable from those caused by other common enteric pathogens. Other symptoms reported by individuals afflicted with Cryptosporidiosis include dehydration, fever, anorexia, weight loss, weakness, abdominal cramps, vomiting, lethargy, general malaise, and progressive loss of overall condition (Hunter et al., 2004). The incubation period (time from ingestion to appearance of symptoms) has been reported to range from 2 to 10 days (Arrowood, 1997). Some infections may be asymptomatic. In other words, not all infections will result in illness and observable symptoms. Asymptomatic hosts may still shed oocysts, however. Asymptomatic carriage, as determined by stool surveys, generally occurs at very low rates (less than 1 percent) February 2009 23 ------- U.S. Environmental Protection Agency in industrialized countries (Current and Garcia, 1991), though higher rates have been reported in day care centers. Routine bile endoscopy suggests a higher asymptomatic prevalence; for example, 13 percent of nondiarrheic patients were shown to carry Cryptosporidium oocysts (Roberts et al., 1989). High rates of asymptomatic infection (between 10 and 30 percent) are common in nonindustrialized countries (Current and Garcia, 1991). In more severe illnesses, the parasite may be found in the stomach, colon, liver, or lungs with associated symptoms corresponding to infections in those tissues. However, the presence of the parasite in tissues other than the small intestine does not necessarily indicate infection of host cells in those organs (O'Donoghue, 1995). The level of immunocompetence of the infected person directly relates to the symptoms experienced. Age, concurrent illness/medical treatment, genetic background, pregnancy, and nutritional status all contribute to immune status. Symptoms may be more severe in immunocompromised persons (Carey et al., 2004; Frisby et al., 1997). Such persons include those with AIDS, certain cancer patients undergoing chemotherapy, organ transplant recipients treated with drugs that suppress the immune system, and patients with autoimmune disorders (e.g., lupus). In AIDS patients, Cryptosporidium has been found in the lungs, ears, stomach, bile duct, and pancreas in addition to the small intestine (Farthing, 2000). Clifford et al. (1990) found that cryptosporidiosis affected 10 to 15 percent of the AIDS patients, resulting in death in 50 percent of those cases. Besides the immunocompromised, children and the elderly may also be at higher risk from Cryptosporidium than the general population; however, specific data are not currently available to document the degree to which these individuals are subject to elevated risk. However, previous exposure to Cryptosporidium has been shown to confer some amount of immunity (Chappell et al., 1999). Symptoms of cryptosporidiosis typically last from several days to 2 weeks although, in a small percentage of cases, the symptoms may persist for months or longer. Individuals with either compromised or healthy immune systems may experience illness for long periods. Illness from Cryptosporidium is usually self-limiting, with a median duration of 6 days and a mean duration of 9 days (Dupont et al., 1995; Palmer et al., 1990), although longer durations (mean 19 to 22 days, maximum 100 to 120 days) were reported in a recent Australian survey by Robertson et al. (2002). Relapses were common, with 1 to 5 additional episodes in 40 to 70 percent of patients. Shedding of oocysts may continue after the cessation of the disease symptoms. Both individuals with compromised and with healthy immune systems have been shown to exhibit chronic sequelae. Immunocompromised individuals generally experience chronic gastroenteritis, which may last as long as the immune impairment. Immunocompromised populations include patients undergoing chemotherapy for treatment of neoplasms, persons undergoing immune suppression treatment to prevent rejection of skin or organ transplants, malnourished individuals, persons with concurrent infectious diseases (e.g., measles), the elderly, and persons with AIDS. Chronic illness may manifest itself as a series of intermittent episodes or may be persistent. Individuals with CD4+ cell counts (a key measure of the health of the immune system) less than 100 cells per mm3 of blood are at increased risk of illness from Cryptosporidium, while individuals with less than 50 cells per mm3 are at the greatest risk for February 2009 24 ------- U.S. Environmental Protection Agency severe disease and prolonged carriage of Cryptosporidium (Hunter and Nichols, 2002; Roefer et al., 1996). As noted above, chronic sequelae in immunocompetent patients also have been documented. After resolution of the acute phase in the 2 months following their initial diagnosis, 40.9 percent of patients in one case study reported recurrence of intestinal symptoms (includes both C. hominis and C. parvum) (Hunter et al., 2004). In addition, in individuals infected with C. hominis, other sequelae such as joint pain, eye pain, recurrent headache, dizzy spells, and fatigue were significantly more common than in control subjects. Both C. parvum and C. hominis infections have sometimes resulted in recurrence of GI symptoms, but only C. hominis infections have been related to the other sequelae noted previously. Cryptosporidiosis Incidence Limited information is available on the endemic incidence of Cryptosporidiosis in the United States. Mead et al. (1999) estimated that there are approximately 15 million physician visits annually for diarrhea and that approximately 2 percent of these, or 300,000 cases, are due to Cryptosporidiosis. Mead and colleagues also estimated that of these 300,000 cases, only about 10 percent are attributable to foodborne transmission, with the remainder due to the consumption of contaminated water (from drinking or recreational exposure) or person-to-person contact. Mead et al. (1999) estimated that there are approximately 211 million episodes of gastroenteritis (GI illness) in the United States each year, of which only about 38 million are attributable to known pathogens.3 Prior to 1982, when the CDC implemented routine reporting of Cryptosporidium among ADDS patients, only 13 cases of Cryptosporidiosis had been documented (Ungar, 1990). Subsequently, between 1982 and 1997, more than 1,000 cases of the disease were reported worldwide (Payer et al., 1997). Cases reported by CDC between 1999 and 2005 (for all routes of exposure) are shown in Figure II.2.2-1; however, documented cases underestimate actual Cryptosporidium infection rates because most cases go unreported. Worldwide, infection is widespread, exceeding several million according to Casemore et al. (1997). As noted previously, the largest known outbreak of the disease occurred in 1993 in Milwaukee, Wisconsin (MacKenzie et al., 1994) and infected 403,000 individuals (CDC, 1996). The most recent data from CDC (2007b) for the year 2005 reported 8,269 cases of Cryptosporidium infection nationally, and reported significant fluctuations in incidence between summer and other seasons, peaking in late July and early August. According to CDC (2007b), Cryptosporidium is the leading cause of diarrheal illness outbreaks in recreational (chlorinated and nonchlorinated) water. Although 8,269 cases were reported in the United States in 2005, CDC (2007b) estimates that the total incidence of domestic cases of Cryptosporidium infection exceeds 300,000 each year. 3 Mead et al. (1999) based the estimates on reported cases and estimates for degree of under reporting. For example, in the 1993 Cryptosporidiosis outbreak in Milwaukee, Wisconsin, medical care was sought in only 12 percent of cases (Corso et al., 2003). February 2009 25 ------- U.S. Environmental Protection Agency Cryptosporidium Cases in the United States 9000 1999-2005 2000 2001 2002 Year 2003 2004 2005 Data sources: CDC 2005, 2007 Figure II.2.1-1. Reported Cryptosporidium Infections in the United States, 1999 to 2005 The large increase in the number of cases reported from 2003 to 2005 might have resulted from outbreak-related case reporting (CDC, 2007b); however, that factor is unlikely to account for all of the increase. It is not clear how much, if any, of the increase may be due to changes in reporting patterns and diagnostic testing practices or to a real change in infection and disease (CDC, 2007b). From 1991 to 2004, six outbreaks of cryptosporidiosis have been associated with untreated recreational waters (Table II.2.1-2). II.2.2 Giardia Giardia Life Cycle and Strain Variation Organisms in the genus Giardia are binucleate, flagellated protozoan parasites that exist in trophozoite and cyst forms and are an important cause of waterborne illness worldwide. Both humans and some animal species can carry and transmit Giardia lamblia (also known as G. intestinalis or G. duodenalis), which causes the GI illness giardiasis. G. lamblia is highly infectious and has been shown to cause giardiasis with exposure to as few as 10 cysts (Rendtorff, 1954). Over the course of the Giardia life cycle, the parasite lives both as a trophozoite and as a cyst form. Inside a vertebrate host, the Giardia trophozoites divide by binary fission, attach to the brush border of the small intestinal epithelium, detach, then become rounded and form a cyst wall. The environmentally resistant cyst is excreted with the feces, where it moves passively February 2009 26 ------- U.S. Environmental Protection Agency Table II.2.1-2. Outbreaks of Cryptosporidiosis Associated with Recreational Waters in the United States Year 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 Number of Cases 0 526 174 519 5,487 3,025 369 169 64 1,368 538 936 702 567 Number of Outbreaks 0 2 4 2 3 3 1 8 4 13 4 7 5 7 Source of Outbreak(s) NA Waterslide and wave pool Pools 1 pool, 1 lake 1 pool, 2 waterparks 2 pools, 1 lake Fountain 7 pools, 1 lake 3 pools, 1 fountain 12 pools, 1 lake 3 pools, 1 hot spring 6 pools, 1 lake 4 pools,* 1 lake Pools * In one pool outbreak, both Cryptosporidium and Giardia were detected in water samples, so that outbreak of 63 cases is counted in both the Cryptosporidium and the Giardia data. Source: Data from CDC Surveillance Summaries: Morbidity and Mortality Weekly Report (MMWR) Surveillance for Waterborne-Disease Outbreaks - United States: 1991-1992, 1993-1994, 1995-1996, 1997-1998, 1999-2000, 2001-2002, 2003-2004 (CDC, 1993b, 1996, 1998, 2000, 2002, 2004, 2006). through the environment and may be ingested by another host organism. Inside the digestive track of a new host, active cysts release trophozoites, and repeat their lifecycle (USEPA, 1998). Cysts can be excreted in the stool intermittently for weeks or months, resulting in a protracted period of communicability (CDC, 2007b). Furthermore, cysts can remain viable under typical environmental conditions for periods up to 77 days (Bingham et al., 1979, as cited in USEPA, 1998). Currently, there are five recognized species of Giardia and six generally recognized assemblages of G. lamblia. Each assemblage has a varying degree of host specificity (see Table II.2.2-1). Some assemblages (i.e., Assemblages A and B) act as zoonotic assemblages that can infect most species of mammal, while others (i.e., Assemblages C through F) are more adapted to a particular host species (Appelbee et al., 2005). For example, Assemblage A has been found in human, beaver, cat, lemur, sheep, calf, dog, fox, chinchilla, alpaca, horse, pig, and cow. The role of these animals as a source of human infection, however, remains unclear. Assemblages C and D seem to primarily infect dogs, while Assemblage E infects livestock, and Assemblage F infects only cats (Appelbee et al., 2005). February 2009 27 ------- U.S. Environmental Protection Agency Table II.2.2-1. Giardia Taxonomy Species Giardia lamblia (syn. G. duodenalis, G. intestinalis) G. lamblia G. lamblia G. lamblia G. lamblia G. lamblia G. lamblia G. lamblia G. lamblia G. muris G. microti G. psittaci G. ardeae G. agilis Assemblage A1 A2 B (G. enterica*) C and D (G. canis*) E (G. bovis*) F (G. caff*) G (G. simoni*) Novel I Novel II Host(s) Human, beaver, cat, lemur, sheep, calf, dog, fox, chinchilla, alpaca, horse, pig, cow Human, beaver Human, beaver, guinea pig, dog, monkey, horse (BIV) Dog, coyote, mouse Cow, sheep, alpaca, goat, pig Cat Domestic rat Marsupial (Quenda - bandicoot, mouse, sheep) Marsupial II (Tasmanian devil) Rodents (mice) Vole and muskrat Birds (budgerigars) Birds (heron and ibis) Amphibians (frogs) * Denotes recently proposed new species names (Hunter and Thompson, 2005; Thompson and Monis, 2004). Adapted from: Adam, 2001; Appelbee et al., 2005; Hamnes et al., 2007; Olson et al., 2004; and Traub et al.,2005. Giardia Zoonotic Potential Cross-species transmission of Giardia is known to occur, and there are many known species and variants (or assemblages) of the Giardia parasite. Of all of the animal host species suspected of being a significant zoonotic source of human giardiasis by waterborne transmission, the evidence presently available suggests that the beaver (Dykes et al., 1980) and muskrat (USEPA, 1998) are the most likely candidates. The role of these animals as a source of human infection, however, remains controversial. Both of these aquatic mammals can be infected with isolates of Giardia from humans, but each has also been shown to harbor strains of Giardia that are phenotypically distinct from those found in humans. Thus, it is possible that the beaver harbors two types of Giardia. One type may be highly adapted to this animal and rarely, if ever, transmitted to humans. The other type may be one acquired by the beaver from human sources, which can multiply in the beaver and in turn be transmitted via water back to humans (USEPA, 1998). The role that livestock play as zoonotic reservoirs of Giardia infection also remains controversial. G. lamblia may be maintained independently through transmission cycles involving wildlife and livestock, though it is unclear how these cycles may interact in zoonotic transfer (Hunter and Thompson, 2005). While both livestock and humans have been implicated February 2009 28 ------- U.S. Environmental Protection Agency in contaminating water sources with Giardia, humans are responsible for the majority of the contaminations. Hunter and Thompson (2005) examined case studies for the zoonotic potential of Giardia and found only one case having a significant association with animal contact. They concluded that zoonotic transfer plays only a minor role in the infection cycles of Giardia and that animal contact is not a major risk factor. They authors did not, however, rule out the importance of zoonotic transfer indirectly through water sources. Both Traub et al. (2004) and Inpankaew (2007) have shown that, in some communities with inadequate sanitation, zoonotic transfer is evident between humans and dogs; however, it is unclear which species is the primary reservoir. Thompson (2007) suggested that data gaps regarding zoonotic transfer for Giardia can be filled using molecular epidemiological studies. Molecular genotyping of parasite isolates from susceptible hosts in localized foci of transmission or longitudinal surveillance with genotyping might help address such data gaps on zoonotic transfer of Giardia. Giardia Routes of Exposure Giardia is transmitted via fecal-oral exposure and causes both endemic and epidemic cases of giardiasis. It is frequently spread person-to-person, especially among children or among persons with poor access to or practice of sanitation. The main route of exposure to Giardia from recreational immersion in water is from incidental ingestion of water during full immersion activities such as swimming. Although inhalation of aerosolized water that contains cysts is theoretically possible, cysts are not known to be infectious in lung tissue. Dermal absorption is not known to be a route of exposure to Giardia cysts in environmental waters. Giardia Illness Symptoms Giardia is responsible for a number of health effects including acute symptoms that occur during and after the infection. However, Giardia has also been implicated in a number of chronic sequelae. There are a wide variety of symptoms associated with giardiasis that range from asymptomatic infection and acute self-limiting diarrhea to persistent chronic diarrhea, which sometimes fails to respond to treatment. Giardia produces a broad spectrum of GI symptoms including one or more of the following symptoms: diarrhea, bloating, weight loss, malabsorption, steatorrhea (fatty stool), pale greasy and malodorous stools, flatulence, abdominal cramps, nausea and vomiting, fatigue, anorexia, and chills (CDC, 2000; Hellard et al., 2000; Hopkins and Juranek, 1991; Thompson, 2000). Fever may occur at the beginning of the infection (Ortega and Adam, 1997). Lactose intolerance is frequently present during infection and may persist even after Giardia has cleared from the stool (Wolfe, 1992). Chronic giardiasis appears to be infrequent, but when it occurs, may persist for years (USEPA, 1998). Case reports also indicate that giardiasis can be associated with the development of reactive arthritis (Tupchong et al., 1999). Illness durations vary, lasting only 3 to 4 days for some individuals and several months for others. Most infections resolve spontaneously, and the acute stage lasts from 1 to 4 weeks February 2009 29 ------- U.S. Environmental Protection Agency (USEPA, 1998), but individuals with compromised immune systems may have more serious and prolonged infection (APHA, 2004). Immunodeficiency with varying degrees of hypogammaglobulinemia or agammaglobulinemia is the most commonly reported form of immunodeficiency associated with chronic giardiasis (Farthing, 1996). However, giardiasis is one of the few potentially treatable causes of diarrhea in persons with AIDS, and chronic giardiasis does not appear to be a major clinical problem in persons with HIV infections or AIDS (Farthing, 1996; USEPA, 1999). Asymptomatic infection is very common, with 50 to 75 percent of infected persons reporting no symptoms (Mintz, 1993)especially in children and in persons with prior infections (CDC, 2007b). In a study at the Swiss Tropical Institute, only 27 percent of 158 patients who had Giardia cysts in their feces exhibited symptoms (Degremont et al., 1981). Although persons with asymptomatic Giardia infection are not likely to seek medical treatment and be diagnosed, they can serve as carriers of infection. Hospitalizations and deaths due to giardiasis are relatively rare. The CDC estimates that giardiasis causes approximately 10 deaths and 5,000 hospitalizations annually in the United States (Mead et al., 1999). Blood volume depletion or dehydration is the most frequently listed codiagnosis on hospital admission. Among children under 5 years of age who had severe giardiasis, almost 19 percent also were diagnosed with failure to thrive (Lengerich et al., 1994). Additionally, in the United States and Scotland, more severe cases of giardiasis (i.e., hospitalized patients) seem to occur primarily in children under the age of 5 (Lengerich et al., 1994; Robertson, 1996). Age has been shown to significantly affect recovery time; in Scotland, the median length of stay in the hospital for giardiasis was significantly longer for persons older than 70 years than for other age groups (11 days compared to 3 days) (Robertson, 1996). Infants and young children may have increased susceptibility to giardiasis due to immunological factors that increase sensitivity and behavioral factors that increase exposure. Chronic giardiasis patients often experience recurrent, persistent, brief episodes of loose, foul smelling stools that may be yellowish and frothy in appearance and frequently accompanied by distension of the bowel, foul flatus, anorexia, nausea, and uneasiness in the epigastrium (Wolfe, 1979). In some cases, these symptoms may persist for years; however, in the majority of cases, the parasite and symptoms disappear spontaneously. Among 65 cases of giardiasis encountered in an urban private practice outpatient setting, the mean duration of symptoms was reported to be 1.9 years, and in 38 patients (58 percent) who exhibited chronic symptoms for 6 months or longer, the mean duration of symptoms was 3.3 years (USEPA, 1998). Corsi et al. (1998) evaluated ocular manifestations in 141 Italian children with current and past giardiasis and 300 children without giardiasis. Retinal changes were diagnosed in 20 percent of the children with giardiasis (mean age was 4.7 years) and in none of the children without giardiasis. These findings suggest that asymptomatic, nonprogressive retinal lesions may occur in young children with giardiasis. The risk of retinal lesions did not seem to be related to the severity of infection, its duration, or use of metronidazole to treat the infection, and may reflect a genetic predisposition to retinal lesions (Corsi et al., 1998). February 2009 30 ------- U.S. Environmental Protection Agency There is usually no extra-intestinal invasion when Giardia trophozoites infect the small intestine, but reactive arthritis may occur, and in severe giardiasis, duodenum and jejunal mucosal cells may be damaged (APHA, 2004). Giardiasis Incidence Giardia lamblia is the most common intestinal parasite identified by public health laboratories in the United States (Kappus et al., 1994; Rose et al., 1991). CDC estimates that there are approximately 2 million illnesses annually in the United States due to Giardia (Mead et al., 1999). As noted previously, while all age groups are affected by giardiasis, the highest incidence is in children (USEPA, 1998). High risk groups for giardiasis include infants and young children, travelers to developing countries, the immunocompromised, and persons who consume untreated water from lakes, streams, and shallow wells (USEPA, 1998). Communities with unfiltered surface water systems experienced a waterborne outbreak rate that was 8 times greater than communities where surface water was both filtered and disinfected (USEPA, 1998). Data therefore indicate that filtering water to remove microorganisms substantially reduces risk of giardiasis. As with all pathogens, underreporting limits estimates of the true incidence of giardiasis. The ratio of reported cases of giardiasis to actual cases is not known. Mead et al. (1999) used a 38- fold multiplier to estimate incidence for nonbloody diarrhea outcomes (based on Salmonella data) and a 20-fold multiplier for bloody diarrhea outcomes (based on E. coli data). Applying the 38-fold multiplier to the 20,075 giardiasis cases that were reported in 2005 (CDC, 2007b) results in an estimated incidence of approximately 762,800 total cases in 2005. However, broader estimates are also supported. For example, an estimated 1 to 5 percent of cases of salmonellosis are reported to CDC through passive surveillance (Chalker and Blaser, 1988). If the 1 to 5 percent reported cases is applied to the giardiasis data, then the giardiasis disease burden in the United States in 2006 could have been 401,500 to 2,007,500 cases (135.4 to 677.0 cases per 100,000 population).4 The true burden of giardiasis in the United States is likely to fall between these two estimates (CDC, 2007b). Using data from 1992 to 1997, the number of states reporting occurrence of giardiasis increased from 23 to 43, while the annual count of giardiasis cases rose from 12,793 to 27,778, nationally (CDC, 2000). Between 1996 and 2001, however, the number of reported cases of Giardia infection in the United States (50 states plus Washington, DC) decreased gradually from 27,778 to 19,659 (CDC, 2007b). The cause of this decrease is unknown. Giardiasis became a nationally notifiable disease in 2002, and the number of cases increased from 2001 to 2002, then stabilized, averaging approximately 20,200 cases per year (CDC, 2007b). See Figure II.2.2-1 and Table II.2.2-2 for reported cases of Giardia infection in the United States from 1992 to 2005. 4 Based on U.S. Census data for 2006, the U.S. population was estimated to be 296,528,800. February 2009 31 ------- U.S. Environmental Protection Agency Giardia Cases in the United States 1992-2005 30,000 n 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 Year Data sources: CDC 2000, 2005, 2007 Figure II.2.2-1. Reported Giardia Cases in the 50 States plus Washington DC, 1992 to 2005 The increase in Giardia cases observed for 2002 might reflect increased reporting after the designation of giardiasis as a nationally notifiable disease starting in 2002. Outbreak-related cases made up 1.6 to 11.6 percent of the total number of cases reported annually for 1999 to 2002. Although the number of states reporting cases increased from 42 to 46 during that time, the number of states reporting more than 15 cases per 100,000 population decreased from 10 in 1998 to 5 in 2002. Transmission of giardiasis occurs throughout the United States, with increased diagnosis or reporting of cases per 100,000 population occurring in northern states than Table II.2.2-2. Giardia Infection Occurrence in U.S. Populations Population Description General Homosexual males (in New York) Small children Occurrence 2-5% infection Prevalence in stool samples (infection) 4-12% depending on year and state 30% of the population has seropositivity for Giardia (indicates current or past infection) In national survey, 7.2% (of 216,675) stool specimens were positive for Giardia in 1987 and 5.6 percent (of 178,786) were positive in 1991 (infection) 18% found positive for Giardia (infection) (compared with 2% among other patients, but giardiasis is not a major clinical problem in persons with HIV or AIDS) 7% are asymptomatically infected with Giardia Reference(s) USEPA, 1998 USEPA, 1998 Frost and Craun, 1998 USEPA, 1999 Faubert et al., 2000; Kean, 1979; USEPA, 1999 Frost and Craun, 1998 February 2009 32 ------- U.S. Environmental Protection Agency in southern states (CDC, 2005). For 2002, among states reporting cases, the incidence of giardiasis ranged from less than 0.1 cases (Texas) to 23.5 cases (Vermont) per 100,000 population. Vermont reported the greatest number of cases per 100,000 population for each of the 5 years of the reporting period. Giardiasis occurs most frequently in the early summer through early fall, with increases in transmission during the summer (CDC, 2007b). This increased transmission coincides with the summer recreation season, which includes increased use of recreational (including community) swimming facilities. Given that a single person can shed millions of cysts and yet remain asymptomatic, transmission in these facilities is likely to be an important mechanism for increased incidence during the summertime (CDC, 2005). People at recreational beaches have been shown to disturb sediment, leading to resuspension of cysts and an increase in exposure to Giardia, with higher densities of bathers leading to higher turbidity and correspondingly higher cyst concentrations (Graczyk et al., 2007; Sunderland et al., 2007). Giardiasis is found most frequently in children 9 years old and younger and in adults aged 35 to 44 years. These groups correspond to young children and their caretakers, who are at increased risk of infection (CDC, 2007b). In 2005, 54.3 percent of reported cases occurred in males compared to only 43.9 percent in females, while 1.8 percent of reports did not record gender (CDC, 2007b). According to CDC (2007b), the discrepancy between cases in males versus females might be attributable in part to increased risk of infection during sexual contact between men although the discrepancy was found in nearly every age group. A review of the data presented in Table II.2.2-2 clearly indicates that Giardia infection in the United States is common and widespread. From 1991 to 2004, seven outbreaks of giardiasis have been associated with untreated recreational waters (Table II.2.2-3). II.3 Viruses Zoonotic Potential A great deal of public and animal health policy is based on the premise of the host specificity of viruses. That is, it has long been assumed that each virus has a distinct and limited range of host species that it can infect.5 Viruses also are restricted to particular tissues of the host's body that they can infect (i.e., tropism), which affects both the mode of transmission and the disease that the virus infection may cause (Cliver and Moe, 2004). Most waterborne viruses are thought to be transmitted by a fecal-oral route (i.e., the virus is shed via the intestines and infects upon ingestion) that requires a tropism that includes the lining of the GI tract. Viruses that infect via the intestine and cause GI illness (e.g., enteroviruses) may also have secondary tropisms in other 5 In this case, a distinction is being made between the important and much studied hypothesis that viral evolution accelerates when viruses "jump species" in the case of emerging diseases and the regular maintenance of multi-host adaptation that is recognized in other pathogens. Although rapid viral evolution facilitated by cross- species transmission is a recognized public health concern for viruses that have airborne transmission (e.g. influenza and severe acute respiratory syndrome [SARS] -associated coronavirus), cross-species transmission has not been a traditional concern for waterborne, fecally transmitted viruses. February 2009 33 ------- U.S. Environmental Protection Agency Table II.2.2-3. Outbreaks of Giardiasis Associated with Recreational Waters in the United States Year 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 Number of Cases 34 0 61 80 0 77 0 0 18 0 0 2 212 9 Number of Outbreaks 4 0 3 1 0 1 0 0 1 0 0 1 2 1 Source of Outbreak(s) 3 pools, 1 lake NA 2 lakes, 1 river Pool NA Pool NA NA Pond NA NA River Pools* Lake * In one pool outbreak both Cryptosporidium and Giardia were detected in water samples, so that outbreak of 63 cases is counted in both the Cryptosporidium and the Giardia data. Source: Data from CDC Surveillance Summaries: Morbidity and Mortality Weekly Report (MMWR) Surveillance for Waterborne-Disease Outbreaks - United States: 1991-1992, 1993-1994, 1995-1996, 1997-1998, 1999-2000, 2001-2002, 2003-2004 (CDC, 1993b, 1996, 1998, 2000, 2002, 2004, 2006). tissues (e.g., neurological tissues). While much virus infectivity research has been conducted in vitro using cultured animal cells that at least partially reflect the host specificity (but not the tropisms) of viruses, many important enteric waterborne viruses of humans (e.g., noroviruses) remain difficult to detect or quantify in cultured cells (NRC, 2004; Straub et al., 2007). For this reason, it remains unclear whether in vitro infectivity is relevant to the in vivo host ranges of viruses. Although no confirmed examples of waterborne viral zoonoses have been reported, several viruses (e.g., swine hepatitis E virus [HEV]) are potentially transmissible between species, and water may serve as a vehicle for their transmission under some circumstances. Thus, assessment of the prospect of waterborne viral zoonoses is ongoing (Cliver and Moe, 2004). Cliver and Moe (2004) consider the criteria for determining whether a virus can function as a waterborne zoonosis to include the following: 1. Animal reservoir: Does the agent regularly infect at least one animal species, independent of exposure to humans? 2. Transmission to humans: Are humans who are in contact with the alleged animal reservoir more frequently infected with this virus than people who are not? February 2009 34 ------- U.S. Environmental Protection Agency 3. Shedding: Is the candidate virus shed by the reservoir animal species in ways that might lead to contamination of water? 4. Stability: Is the candidate virus stable enough in the water vehicle to permit transmission by this pathway? Despite the potential for rapid evolution in viruses, ongoing monitoring and research activities are important for public health protection, even though viruses are not currently known to have the attributes outlined in the introduction of this paper. An overview of information related to zoonotic potential for rotavirus, HEV, and adenovirus follows. Rotaviruses Rotaviruses (groups A, B, and C) have been documented in humans and animals, and interspecies transmission including human infection by a bovine strain has been reported (Abbaszadegan, 2006). The primary route of exposure for rotavirus is the fecal-oral route although exposure through other routes also has been reported to a lesser extent. Rotavirus is stable in the environment, and its transmission can occur through ingestion of contaminated water or food. Rotavirus illness typically results in vomiting and watery diarrhea for 3 to 8 days. Fever and abdominal pain occur frequently as well. According to the CDC, this virus is the most common cause of severe diarrhea in children. Symptoms tend to be less severe for adults. Approximately 70,000 children in the United States are treated in the hospital for rotavirus each year (Glass, 2006). A community waterborne outbreak of rotavirus gastroenteritis occurred in Colorado in 1981 (Hopkins et al., 1984). The outbreak was attributed to sewage contamination of the water supply and a failure of chlorination treatment. Hepatitis E HEV may be zoonotic (Cliver and Moe, 2004; USEPA, 1999). In pigs and rats, this virus is very similar to human HEV. Experimental studies have indicated that human strains can infect pigs, and porcine strains can infect primates (Cliver and Moe, 2004). In developing countries, the seroprevalence of HEV infection can be as high as 60 percent. The most common route of exposure for HEV is ingestion of contaminated food or water. Transmission via person-to-person contact is less common. Typical symptoms of HEV illness may include jaundice, fatigue, abdominal pain, loss of appetite, nausea, and vomiting. Pregnant women who contract hepatitis E are at high risk of severe illness and death. For this sensitive subpopulation, mortality can be as high as 20 percent in developing countries, but mortality is rare in developed countries (Craun et al., 2004a). HEV is uncommon in the United States and the CDC does not track incidence rates. In an EPA study of sporadic human HEV, nearly 50 percent of the infected persons had traveled to endemic areas in other countries or received blood transfusions (USEPA, 1999). February 2009 35 ------- U.S. Environmental Protection Agency Adenovirus Adenovirus may be zoonotic (Mwenda et al., 2005). Mwenda and colleagues identified enteric adenovirus in captive olive baboons, vervet monkeys, and the yellow baboons in Kenya. These findings suggest there may be a possibility of zoonotic transmission of adenoviruses from nonhuman primates to humans in Kenya. Adenovirus can enter a susceptible host by the nose, mouth, or eye membranes. Water may play a meaningful role in the transmission for many human adenovirus serotypes, including the enteric adenovirus that is transmitted via the fecal- oral route (Heerden et al., 2005). Heerden et al. (2005) detected human adenovirus in 4 of 51 (7.8 percent) samples of river water and 9 of 51 (17.7 percent) samples of dam water. Human adenoviruses may cause of a wide spectrum of acute and chronic diseases, including keratoconjunctivitis, upper respiratory tract infections, pneumonia, gastroenteritis, cystitis, and encephalitis (Gray et al., 2005). Molecular studies have recently shown adenoviruses to be associated with bronchopulmonary dysplasia (Couroucli et al., 2000) and chronic obstructive pulmonary disease (Hogg, 2001). Immunocompromised persons, including people with AIDS, bone marrow transplant patients, pregnant women, and children are more susceptible to adenovirus infections (Baldwin et al., 2000; Crawford-Miksza and Schnurr, 1996). Infections in these populations may result in severe illness and death (Chakrabarti et al., 2002; Runde et al., 2001). U.S. surveillance for adenovirus is relatively incomplete (Gray et al., 2005) and well-documented incidence rates are not available, especially for waterborne outbreaks. February 2009 36 ------- U.S. Environmental Protection Agency III. PATHOGEN INTERACTIONS WITH THEIR ENVIRONMENT Pathogens interact with the ambient environment, other microorganisms, plants, and with their hosts. This section provides a summary of how pathogens respond to various environmental parameters. Information on interactions with other microorganisms and animal manure are briefly covered in this overview followed by sections that describe how the water environment affects pathogen survivability and phenotype and how host animals can influence pathogen characteristics and mechanisms of rapid evolution. The behavior of pathogens in ambient waters is often different from the behavior of indicators in ambient water. Although there are important waterborne amoebic pathogens, they are not associated with animal fecal material. However, important bacterial pathogens that are associated with fecal material (e.g., Salmonella, Campylobacter) interact with free-living amoebae in ways that could impact recreational water quality. The most notable, Legionella, infects and replicates in free-living amoebae and is considered more of a risk in drinking water than in recreational waters (Borella et al., 2004; Marrie et al., 2001). Some human bacterial pathogens are even thought to have evolved in association with amoebae (Berk et al., 2006). Tezcan-Merdol et al. (2004) investigated the uptake and replication of salmonellae in amoebae. Three different serovars of Salmonella enterica (Dublin, Enteritidis, and Typhimurium) were evaluated for internalization by 5 different isolates of axenic Acanthamoeba species. The Dublin serovar was internalized more efficiently than the other two serovars, and the Acanthamoeba rhysodes isolate was more efficient than the other four isolates. The researchers concluded that Acanthamoeba species can differentiate Salmonella serovars and that internalization of the bacteria produces cytotoxic effects mediated by defined bacterial virulence loci. Axelsson-Olsson et al. (2005) studied the infection of Acanthamoeba polyphaga by four different Campylobacter jejuni strains. The infecting bacterial cells were observed to be actively moving in amebic vacuoles and survived longer when cocultured with amoebae than when cultured alone. They indicated that free-living amoebae may serve as a nonvertebrate reservoir for Campylobacter jejuni. Guan and Holley (2003) examined E. coli O157:H7, Salmonella., Campylobacter, Yersinia, Cryptosporidium, and Giardia in animal manure. Of those pathogens considered, E. coli O157:H7 was the most persistent in cattle manure regardless of the temperature and manure form (solid or slurry) while Campylobacter and Giardia were weakest survivors in manure. The authors concluded that holding manure at 25° C for 90 days will render it free from the pathogens considered. This has indirect implications for ambient water quality because the conditions experienced by pathogens after excretion but before introduction into ambient water contribute to the overall likelihood the pathogen will be infectious by the time it potentially can reach a human host through recreational contact. III.1 Water Environment Affects Pathogen Survivability and Phenotype The most common environmental factors studied for their impact on pathogen survival in water are pH, salinity, light exposure, and temperature. Additional environmental characteristics that may influence pathogen survival, infectivity, and virulence include: UV light (duration, intensity), rainfall, runoff, dispersal, suspended solids, turbidity, nutrients, organic content, February 2009 37 ------- U.S. Environmental Protection Agency organic foams, water quality, biological community in water column, water depth, stratification, mixing (e.g., wind and waves), presence of aquatic plants, biofilms, and predation. Thomas et al. (2006) examined extreme rainfall and spring snowmelt in association with 92 Canadian waterborne disease outbreaks between 1975 and 2001. Accumulated rainfall, air temperature, and peak stream flow were used to determine the relationship between high impact weather events and the occurrence of waterborne disease outbreaks. For rainfall events greater than the 93rd percentile, there was a greater than 2-fold increase in the odds of an outbreak compared to rainfall events less than the 93rd percentile. For each degree-day above 0° C, the relative odds of an outbreak increased by a factor of 1.007. The odds ratio is small on a per degree-day scale, but is notable over longer timeframes. For example, over a 42-day period, a 5° C increase in the maximum daily air temperature would result in over a 4-fold increase (1.007(5x42) = 4.33) in the relative odds of an outbreak. Stream flow and stream flow peaks did not show a difference between cases (outbreaks) and controls (no outbreaks), but there was a considerable lack of data on stream flow. An overview of the available information for the six key waterborne zoonotic pathogens follows. Information concerning these factors is limited for most waterborne pathogens. Thus, in order to develop effective control strategies, additional research may be necessary. III.1.1 Pathogenic E. coli Survival in the Environment Pathogenic E. coli can be expected to survive outside of a host animal anywhere from a few days to several weeks, depending upon environmental conditions. Pathogenic E. coli seem to interact with the environment in a similar fashion to nonpathogenic E. coli and do not lose their virulence during prolonged survival in the environment. Survival of E. coli O157:H7 in surface waters was found to be longer at lower temperatures (Czajkowska et al., 2005) and was 2 to 3 times longer in river and lake sediments at the same temperatures (see Table III. 1.1-1). Table IIL1.1-1. Survival of E. coli O157:H7 in Ambient Waters Water matrix Surface waters (lakes and rivers) Surface waters (lakes and rivers) Sediments (same lakes and rivers) Sediments (same lakes and rivers) River water with feces Unfiltered lake water Filtered tap water Unfiltered lake water °C 6 24 6 24 15 8 15 25 Days to Decrease 99% 4-11 2-8 10-20 5-8 7.5 91 >91 14-28 99.99% 8-22 5-10 25-39 10-30 14.5 NA NA NA Reference Czajkowska et al., 2005 McGee etal., 2002 Wang and Doyle, 1998 February 2009 38 ------- U.S. Environmental Protection Agency Although direct contamination of ambient water due to domestic animals or livestock wading in water is possible, most contamination of water is due to runoff from pastures and fields after land application of manure. E. coli O157:H7 can survive for at least several weeks in animal feces and slurries (Avery et al., 2005) and has been demonstrated to survive at least 500 days at -20° C in frozen soil (Gagliardi and Karns, 2002). III. 1.2 Campylobacter Survival in the Environment Campylobacter has been shown to survive in aquatic environments with low temperatures (4° C) between 8 days (Buswell et al., 1998) and 4 months (Rollins and Colwell, 1986). Buswell et al. (1998) found that survival times of Campylobacter isolates differed by 2- to 4-fold depending on the combination of temperature and oxygenation tested. The mean survival times in sterile microcosms were 202 hours at 4° C, 176 hours at 10° C, 43 hours at 22° C, and 22 hours at 37° C. The survival times were considerably longer in the presence of the autochthonous water microflora (two strains tested survived 700 and 360 hours at 4° C). Aerobic conditions decreased the survival of one strain 30 percent and increased the persistence of another strain by more than 3-fold. Within biofilms, the pathogen persisted up to the termination of the experiments after 28 and 42 days of incubation at 30 and 4° C, respectively. Rollins and Colwell (1986) found that Campylobacter incubated in filter-sterilized stream water was recoverable after 4 months at 4°C. Incubation at 25°C resulted in a decline to the nonculturable state within 28 days, and at 37°C, the nonculturable state was reached in 10 days. Direct counting methods indicate that the nonculturable but viable state of Campylobacter is significant. III.1.3 Salmonella Survival in the Environment Salmonella are also found frequently in sewage, soil, and various surface waters. The greatest source of the bacteria is fecal contamination. Under suitable environmental conditions, Salmonella can survive for weeks in waters or years in soils (Lightfoot, 2004). Salmonella grow at temperatures ranging from 10 to 43° C, but some serovars have suppressed growth at temperatures above 40° C (Covert and Meckes, 2006). Salmonella can grow at pH 4-8 and at water activities above 0.93. Under some conditions, Salmonella may proliferate below 4 ° C and survive below pH 4 (Lightfoot, 2004). III.1.4 Leptospira Survival in the Environment Leptospira survive longer in the environment in warm, humid conditions. Leptospirosis is seasonal, which relates to the pathogen's survival in the environment. In temperate regions, there is a peak during summer or fall, whereas in warm-climate regions, the rainy season is associated with peaks because dessication decreases pathogen survival (Levett, 2001). Ganoza et al. (2006) compared levels of Leptospira in urban and rural environmental surface waters in the Peruvian Amazon region of Iquitos. The concentration of pathogenic Leptospira was higher in urban than rural water sources and rats were the indicated zoonosis. February 2009 39 ------- U.S. Environmental Protection Agency III.1.5 Cryptosporidium Survival in the Environment As noted previously, because Cryptosporidium oocysts are extremely resistant to environmental or engineered degradation, the survival of Cryptosporidium under a variety of environmental and drinking water treatment conditions has been evaluated by many investigators. While the majority of these studies have considered the effects of physical antagonism (e.g., freezing, heating, UV exposure), studies have also been conducted to consider the role of microbial antagonists (microbial predation), chemical antagonists (such as disinfection), and aging. This section focuses primarily on aspects of physical antagonists in the environment because they are most pertinent to the topic of this paper. Robertson et al. (1992) evaluated the sensitivity of C. parvum oocysts to a variety of environmental pressures such as freezing, dessication, and water treatment processes, as well as in physical environments commonly associated with oocysts. Approximately 97 percent of the test oocysts were inactivated after 18 days at 22° C, suggesting that the levels of viable oocysts in surface waters might be influenced by seasonal temperature variations. After 2 hours of drying oocysts at room temperature, only 3 percent of oocysts were still viable, and after 4 hours, no oocysts were viable. When stored at 4° C, the percentage of oocysts remaining viable in stool samples decreased steadily with time. (In the study, the relationship between oocyst viability and time varied with individual.) After 176 days in tap water, river water, or cow feces, there was a statistically significant increase in the proportion of dead oocysts in test samples. Seawater was even more lethal to oocysts, with a statistically significant increase in dead oocysts by 35 days of exposure to the test conditions. C. parvum oocyst viability is sensitive to a wide range of typical environmental conditions while remaining relatively insensitive to some water treatment processes. Robertson et al. (1992) also emphasized that oocyst viability depends on the amount of time to which oocysts are exposed to a physical or chemical stress in the environment. Temperature has a significant effect on oocyst survival, with (unfrozen) colder waters promoting the highest survival rates (USEPA, 200la). Warm and boiling water completely neutralizes oocysts, and the temperature of the water determines the time required for the treatment to become effective (Anderson, 1985). Cryopreservation studies conducted by Payer et al. (1991, 1997) indicate that oocyst survival depends on the temperature and duration of freezing conditions, implying that C. parvum oocysts are not necessarily rendered noninfectious by being frozen per se. In another study, Payer and Nerad (1996) demonstrated that the infectivity of C. parvum oocysts after freezing is dependent on the temperature and duration of freezing. In general, shorter freezing times are required to neutralize infectivity when lower freezing temperatures are employed (e.g., 1 hour at -70° C versus 168 hours at -15° C to completely neutralize infectivity) (Payer and Nerad, 1996). Temperature stability studies also were conducted by Sattar et al. (1999) who evaluated the freeze/thaw susceptibility of various preparations of oocysts including highly purified preparations as well as infected calf feces. The results of this study indicated that oocyst stability under freezing conditions is at least partially dependent upon the surrounding matrix, with fecal material conferring a cryopreservative effect on oocysts. In the absence of freezing conditions, colder water temperatures tended to promote the survival of most microorganisms. In water, C. February 2009 40 ------- U.S. Environmental Protection Agency parvum may survive outside of mammalian hosts for several months or more depending upon water temperature (Straub et al., 1994). Payer et al. (1998) investigated the effect of water temperatures ranging from -10 to 35° C and a few higher temperatures on oocyst infectivity. As water temperature was increased from -10 to 20° C, oocysts remained infectious for longer exposure times. For example, oocysts retained their infectivity for only 1 week when suspended in water held at -10°C but remained infectious for up to 24 weeks in 20° C water. As water temperatures were increased above 20° C, oocysts retained their infectivity for shorter exposure times (Payer et al., 1998). Under conditions of high water temperatures, higher than typically found in surface waters, Payer (1994) indicated that all evidence of C. parvum infectivity was lost within 60 seconds when temperatures exceeded 72° C or when temperatures of at least 64° C were maintained for 2 minutes. Holding oocysts to 45°C for 5 to 20 minutes was effective in completely neutralizing infectivity (Anderson, 1985). Anderson (1986) examined the infectivity (determined in infant mice) of oocysts from calf fecal samples that had been dried in a barn (< 60 percent humidity) in either winter or summer months. In summer temperatures (i.e., 18 to 29° C), oocysts completely lost infectivity in 1 to 4 days. Experiments conducted in winter, with air temperatures ranging from - 1 to 10° C, demonstrated a complete loss of infectivity within 2 to 4 days. Control samples kept moist and refrigerated retained infectivity for up to 2 to 3 weeks. Limited studies have been conducted on the effects of physical shear on oocyst viability; these studies have attempted to assess the potentially abrasive effects of oocyst contact with sand and gravel particles or through fast-flowing waters. Parker and Smith (1993) found that after shaking with sand for 5 minutes, 90 minutes, and 2 hours, the number of non-viable oocysts increased significantly to 50 percent, 99.7 percent, and 100 percent, respectively. When chlorination followed 5 minutes of sand shaking, the observed non-viable oocysts increased to 68 percent. When oocysts were on an orbital shaker at 60 and 120 rpm, oocyst viability declined linearly over the course of an hour, with approximately 50 percent loss of viability noted at 20 minutes (Sattar et al., 1999). These authors also showed that oocysts subjected to 2000 psi (13.9 Mpa) for 1 minute had little reduction in viability. Sattar et al. (1999) also evaluated the effects of microbial predation on oocyst survival. They observed that oocysts incubated in dialysis cassettes that were suspended in natural waters exhibited significantly longer survival times when bacterial populations were excluded from the suspension water. The observation implies that microbial predation may play an important role in reducing oocyst survival in ambient (natural) waters. Nasser et al. (2007) examined the effect of sunlight and salinity on the die-off of C. parvum. Experiments were carried out for 7 days in tap and seawater and sunlight and dark conditions. Oocyst die-off was greatest when exposed to seawater and sunlight (0.44 log/day); oocysts in tap water in the dark, exposed to sunlight, and oocysts in seawater in the dark had die-off rates of 0.1, 0.22, and 0.19 log, respectively. At the end of the 7-day study period, a 3 log reduction in infectivity was measured in the sunlight- and seawater-exposed oocysts. Cryptosporidium oocysts retain substantial infectivity for several months at salinity levels corresponding to estuarine coastal waters (Payer, 2004a) and for several weeks in seawater (WHO, 2006). These February 2009 41 ------- U.S. Environmental Protection Agency studies indicate that Cryptosporidium can thus pose a serious health hazard to humans by direct and indirect contact in recreational waters. King et al. (2005) measured oocyst inactivation rates in reagent-grade and environmental waters over a range of temperatures. Oocysts incubated at 4 and 15° C remained infective over a 12- week holding period. A 4 logic reduction in infectivity was observed for both 20 and 25°C incubation treatments at 12 and 8 weeks, respectively, for all water types examined. This is a faster rate of inactivation for oocysts than had been previously reported. Inactivation at higher temperatures is likely a function of increased oocyst metabolic activity. Li et al. (2005) measured the inactivation rate of bovine C. parvum oocysts subjected to temperature regimes designed to mimic the diurnal oscillations of ambient temperature in bovine feces exposed to sunlight in commercial cattle operations in California. No infectious oocysts were observed after 1- to 5-day cycles of 40, 50, 60, and 70° C. The loss of infectivity was primarily due to partial or complete in vitro excystation during the first 24-hour diurnal cycle and secondarily to thermal inactivation of the remaining intact or partial oocysts. The results suggest that as ambient conditions generate internal fecal temperatures greater than or equal to 40° C, rapid inactivation occurs at a rate equal to or greater than 3.27 logic reduction per day for C. parvum oocysts deposited in the feces of cattle. Mendez-Hermida et al. (2005) reported on batch-process solar disinfection of .C. parvum oocysts in water. Oocyst suspensions were exposed to simulated sunlight (830 W/m2) at 40° C. Viability assays and infectivity tests indicated that exposures of 6 and 12 hours reduced oocyst infectivity from 100 percent to 7.5 percent and 0 percent, respectively. The behavior of lake inflows is important in determining pathogen transport and distribution. Inflows that are warmer than the lake water will move over the surface of the lake, whereas inflows that are colder than the lake will sink beneath the surface layer where they will flow along the bottom towards the deepest point (Brookes et al., 2004). The fate of pathogens in lakes is determined by factors such as settling and inactivation by temperature, UV, and predation by other microorganisms. Brookes and colleagues found that inactivation of Cryptosporidium by UV light can be rapid or slow, depending on the depth of the oocysts in the water column and the extinction coefficient for UV light. Pokorny et al. (2002) investigated the effects of temperature on oocysts stored in the dark in filter-sterilized and nonfilter-sterilized river water. They reported that as the temperature was increased from 4 to 23° C, the infectivity of oocysts decreased; no infectious oocysts were detected after 1 week at -20° C. Excreted Cryptosporidium oocysts can survive for substantial periods in animal wastes and soils. Thus, contaminated runoff can enter ambient water and result in potential human exposures. The numbers of oocysts excreted by infected young animals may be especially large, between 1 and 10 million oocysts per gram of feces (WHO, 2006). Oocyst survival for 4 weeks or more has been documented in concentrated animal wastes, particularly at low temperatures (4° C) (WHO, 2006). In the environment, the vast majority of oocysts (99 percent) are inactivated by repeated freeze-thaw cycles independent of the number of times the soil was frozen (Payer, 2004a). February 2009 42 ------- U.S. Environmental Protection Agency Therefore, Cryptosporidium may be environmentally limited in parts of the United States during the winter season. There is evidence that in warmer ambient temperatures, between 4 and 20° C, very little inactivation of oocysts occurs in different types of agricultural soils (Jenkins et al., 2002; WHO, 2006). Factors that are known to reduce oocyst survival in soils include drying and basic pH (Payer, 2004a). Oocyst survival in various water matrices is highly variable, but survival for longer than 30 days has been demonstrated in several studies. Approximately 4 to 5 percent of oocysts in tap water and river water samples survived after approximately 6 months, with approximately 37 percent inactivation after 2 days exposure to tap water (USEPA, 2005a, 2005b). Walker et al. (2001) tested oocyst degradation (as indicated by microscopic examination) in response to the combined stresses of water potential (sodium chloride solute potential), above- freezing temperatures (4 and 30° C), and a subfreezing temperature (-14° C) for different freeze thaw cycles (-14 to 10° C). The degradation coefficients were estimated using multiplicative error and exponential decay models. Increased water potential increased the rate of C. parvum population degradation for all temperature conditions investigated. The effects of water potential were roughly four times those noted for freezing alone. The difference between the effects of freeze-thaw cycling and simple freezing may be caused by mechanical damage to the oocyst wall. The authors conclude that water potential conditions encountered under field conditions are likely to lead to more rapid degradation of oocyst populations than might be expected from studies of degradation in calf feces, distilled water with antibiotics, and reverse osmosis water at low temperatures. III.1.6 Giardia Survival in the Environment Studies of the effect of temperature and other environmental factors on Giardia cyst survival were summarized in EPA's Giardia Human Health Criteria Document (USEPA, 1998). Survival was determined based on dye inclusion/exclusion, excystation, or animal infectivity studies. Some studies used Giardia muris cysts, whereas others used Giardia lamblia cysts. Overall, the studies on the effect of temperature indicated that survivability decreased as temperature increased and that while some cysts could survive a single freeze-thaw cycle, repeated freeze- thaws as might be expected in the environment would likely inactivate cysts (USEPA, 1998). Johnson et al. (1997) investigated the survival of cysts in marine waters and determined that viability was reduced 99.9 percent in 3 hours in marine waters exposed to sunlight (as reported in USEPA, 1998). They also found that 77 hours were required to get 99.9 percent inactivation in the dark and that cysts survived longer at a salinity of 28 mg/L than at 35 mg/L. Because different waters were used in the experiments, however, these investigators could not rule out the effect of factors other than salinity. Olson et al. (1999) found that Giardia cysts were noninfective in water, feces, and soil following 1 week of freezing to -4° C and within 2 weeks at 25° C. At 4° C, Giardia cysts were infective for 11 weeks in water, 7 weeks in soil, and 1 week in cattle feces. Robertson and Gjerde (2006) tested survival of Giardia cysts (as well as Cryptosporidium oocysts) during winter in an aquatic environment (approximately 1 to 7° C) in Norway. Three conditions were compared, distilled water, river water, and submersion of a filter chamber containing cysts into the river. The rate of decline in viability was similar under all three conditions, and no Giardia cysts with apparently February 2009 43 ------- U.S. Environmental Protection Agency viable morphology could be detected after 1 month. Boiling Giardia for 1 minute reduces viability (as determined by flurogenic dyes) to less than 1 percent and renders them noninfectious (as determined by animal infectivity) (El Mansoury et al., 2004). Storage at 4° C and -4° C for up to 7 days preserves Giardia cyst viability and infectivity. Storage at 30 ppt (parts per thousand) salinity for 4 weeks decreased viability to 30 percent, but all animals were infected. Storage at 50 ppt salinity for 4 weeks decreased viability to 5 percent and 80 percent of animals were infected. Storage at 50 ppt salinity for 4 weeks resulted in zero viability (El Mansoury et al., 2004). III.1.7 Virus Survival in the Environment Viruses are more stable at lower temperatures; however, different viruses have different stabilities under similar environmental conditions. For example, astroviruses survive longer than poliovirus and adenovirus, but shorter than rotavirus and hepatitis A virus (Sobsey, 2006). For poliovirus and parvovirus, 90 percent inactivation was observed after 1 to 3 days at 28° C, and 90 percent inactivation was observed after 10 days at 6° C (Griffin et al., 2003). Parvoviruses are the most heat stable enteric viruses known (Gerba, 2006a). Enterovirus nucleic acid is detectable for 60 or more days, whereas infectious virus is detectable for 51 days or less (Griffin et al., 2003). Below 5° C, enteroviruses can survive for years (Gerba, 2006b). Limited data suggests that adenoviruses survive longer in water than enteroviruses and hepatitis A virus (Enriquez and Thurston-Enriquez, 2006). Pesaro et al., (1995) evaluated the persistence of five animal viruses, representing picorna-, rota-, parvo-, adeno-, and herpesviruses, and the coliphage f2, using a filter sandwich technique that mimics the environment in various states of manure. Depending on ambient temperature, pH, and type of animal waste, 90 percent reduction of virus titer varied, ranging from less than 1 week for herpesvirus to more than 6 months for rotavirus. Virus inactivation was faster in liquid cattle manure, a mixture of urine and water (pH > 8.0), than in semiliquid wastes that consisted of mixtures of feces, urine, water, and bedding materials (pH < 8.0). The authors conclude that viruses contained in manure may persist for prolonged periods of time if stored under nonaerated conditions and this may lead to environmental contamination with viruses. III.2 Host Animals Can Influence Pathogen Characteristics and Mechanisms of Rapid Evolution There is evidence that zoonotic pathogens may change in infectivity, virulence, and the severity of disease caused in humans depending on their previous host environment. There is also evidence that some of these host-factor changes can influence subsequent infection cycles in exposed hosts. For example, in laboratory experiments using nutritionally deficient mice (selenium-deficient or vitamin E-deficient), Morse (1997) reported that a normally avirulent (mild) coxsackievirus B3 isolate gave rise to a virulent variant, albeit through an unknown mechanism. The virulent variant resembled other known virulent genotypes and was stable upon subsequent infection of other mice with adequate nutrition. In another demonstration of host influence on pathogen evolution, yeast with deletions in genes that normally suppress viral RNA recombination became "hotbeds" for viral recombination. The host (in this case yeast) genes could affect viral recombinant accumulation by up to 80-fold (Serviene et al., 2005). The key February 2009 44 ------- U.S. Environmental Protection Agency mechanisms of phenotypic change in pathogens are genetic diversity (coinfection and quasispecies), cryptic genes, mutators, and epigenetic effects, and are summarized as follows. Genetic diversity (co-infection and quasispecies): The pathogen population within a single host can be comprised of several or even numerous genetic variants of the same zoonotic pathogen. This variation can occur when the infectivity is such that several genotypes coinfect the host simultaneously, then "compete" or "cooperate" as infection progresses. The fluctuations in genotype prevalence depend on host-pathogen interactions as some genotypes gain dominance and others become rare. High genetic diversity also can occur when pathogens have high mutation rates. This latter phenomenon, referred to as "quasispecies," occurs mainly in RNA viruses because viral RNA replicase enzymes lack proofreading functions during replication, which leads to high mutation rates (Domingo et al., 1998; Morse, 1997). For example, caliciviruses may have as many as 1 to 10 mutations per template copy (Smith et al., 1998), and within-person HEV sequence diversity has been documented to range from 0.11 to 3.4 percent (Grandadam et al., 2004). For retroviruses, every virus particle may be genetically different from every other particle. In fact, the cooperativity6 of many virus particles' genetic material may be necessary to complete the infection process (Lederberg, 1998). Cryptic genes: Meiotropy is the ability of an organism to gain a phenotypic trait through mutation (Brubaker, 1991). One example of a cryptic gene is the yopA gene in Yersinia pseudotuberculosis. A stretch of deoxyadenosine nucleotides (DNA base A) can spontaneously change in length between 8 and 9 bases, which can result in the absence or presence of the gene product. In Y. pseudotuberculosis., the absence of the gene product results in increased virulence.7 In Y. pestis (plague), the gene yopA is not normally expressed; however, molecular manipulation that causes expression of the yopA gene product reduces the virulence of Y. pestis. Thus, it is possible that endemic hosts of Y. pestis could harbor a strain of lower virulence, which by one mutation could become hypervirulent and potentially cause an outbreak of plague (Rosqvist et al., 1988). Another example is in Salmonella, in which flagellar antigens can be expressed or silenced in a reversible manner by inversion of a segment of DNA that moves the promoter from one locus to another (Lederberg, 1998). This type of genetic rearrangement may serve as a mechanism of phase variation for antigenic factors in the bacteria. More standard transcriptional control of the gene expression might allow for low levels of transcriptional leakage and small quantities of antigen to be produced. Prior trace levels of antigens could be sufficient to promote host immunity. Bacteria carry site-specific recombinases that are capable of scrambling bacterial genomes in order to suppress and unsuppress genes. Mutators: Several genes have been identified that influence the mutation rate of bacteria. For example, bacterial cells that carry the MutDS protein, which binds to DNA polymerase, accumulate a broad spectrum of base substitutions and frameshift mutations (the mutation 6 The hypothesis that swarms of viral genotypes cooperate to comprise an "average phenotype" differs from quorum sensing, which is the coordination of bacterial cells via cell-to-cell communication with small signaling molecules to form biofilms and other group-related phenotypes. 7 In this case, the presence of the gene leads to decreased virulence because the gene product facilitates chronic infection, which is less severe than acute infection. February 2009 45 ------- U.S. Environmental Protection Agency frequency can be 20- to 4,000-fold) (Selifonova et al., 2001). Mutator strains can pass genetic material to other bacteria and thus transfer the mutator trait. Therefore, the ability of bacteria to evolve rapidly is heritable. Epigenetic effects: Epigenetic effects are reversible, heritable changes in gene regulation that occur without a change in DNA sequence. Genomic imprinting through DNA methylation is one type of epigenetic effect that has been documented for pathogen regulation of virulence (Low et al., 2001). The zoonotic bacteria E. coll, S. enterica (serovar Typhimurium), Vibrio cholerae, Y. pseudotuberculosis, Y. enterocolitica, Helicobacter pylori, and Campylobacter are a few notable pathogens for which DNA methylases are known to regulate gene expression (Falker et al., 2005; Low et al., 2001). Although DNA methylase promoters are known to be responsive to in vivo growth conditions, the degree to which host factors influence pathogen DNA methylation and whether those methylation patterns provide a memory system for subsequent generations of pathogens are not known. All of these mechanisms can contribute to rapid evolution of zoonotic pathogens.8 Rapid evolution is most often observed in new hosts, where new stresses are thought to lead to strong selection and rapid evolution (Ebert, 1998). Evolution in new host environments can lead to a reduction of virulence when the original host is encountered again. Serial passage experiments (SPEs) often result in attenuation of pathogenicity in one host along with an escalation of pathogenicity in the new host.9 Attenuation can be so severe that it can even lead to an altered host range (complete loss of ability to infect the original host species).10 In SPEs when only one or a few pathogens are transferred at each passage, however, genetic drift can result in decreased genetic variability and lead to a failure to adapt to new hosts and a decline in overall pathogen fitness. Text Box III.2-1 summarizes some feeding studies done in humans and pigs with zoonotic parasites. The examples help illustrate how changes in laboratory isolates due to passage through hosts should be considered during experimental design. Although SPEs are useful for studying pathogen evolution, the trends observed in SPEs are not necessarily broadly applicable to pathogen evolution as a whole (Ebert, 1998). In fact, the observation that virulence increases in SPEs in new hosts seems contrary to the classic expectation that pathogens and hosts evolve over time in ways that render infections benign (Dieckmann, 2002; Wills, 1996). Currently, evolutionary biologists estimate the evolutionary success or failure (i.e., "fitness") of pathogens by their rate of spread through a given host population. Low virulence can lead to missed opportunities to spread due to low pathogen numbers. High virulence can lead to host death and a subsequent lack of spread. Those hypotheses are supported by the observation that intermediate levels of pathogen virulence can be stable. Thus, the dynamic between pathogen and host drives pathogen (and host) evolution For the purpose of this paper, rapid changes are phenotype (or genotype or epigenetic) changes that occur within one or a few passages of a zoonotic pathogen through a particular host species. 9 The negative correlation in the fitness of a pathogen in different hosts is the antagonistic pleiotropy hypothesisa gene that enhances fitness in one host decreases fitness in the other host (Ebert, 1998). 10 Live vaccines such as Theiler's yellow fever vaccine and Sabin's polio vaccine are examples of attenuated pathogens that elicit an immune response without inducing disease. However, reemergence of pathogen virulence after vaccination remains a risk (Ebert, 1998). February 2009 46 ------- U.S. Environmental Protection Agency and is influenced by numerous factors including the infection status of the host population as a whole (Dieckmann, 2002). Because pathogens can evolve quickly within one host, it is reasonable to suspect that zoonotic pathogen pools that were propagated in animals would be different from pathogen pools that came from human sources. However, the extent to which known waterborne zoonotic pathogens are attenuated or gain enhanced virulence in humans when passing through animal hosts remains unknown. Even if general trends could be characterized, it is unlikely that differences could be quantified adequately in the near-term to predict differences in human infectivity and virulence for pathogens passing through different host animals. Another mechanism by which virulence and fecundity of a microorganism may be affected was recently reported by Jenkins et al. (2007) for C. parvum. These investigators infected dairy calves with oocysts from either C. parvum Beltsville (B) or C. parvum Iowa (I). Calves given the B isolate excreted 5-fold more oocysts than those receiving the I isolate. Quantitative reverse transcriptase-PCR indicated that the B isolate contained a 3-fold greater number of the symbiont C. parvum virus (CPV) than the I isolate. They concluded that CPV may have a role in fecundity and possibly virulence of C. parvum. The authors indicated that their results contrasted to those found by Miller et al. (1988) in which there was an inverse relationship between presence of a Giardia lamblia virus (GLV) and parasite growth. Miller et al. (1988) used virus-free isolate that they infected with various numbers of GLV, and Jenkins et al. (2007) noted that virus-free cysts may be more susceptible to the effects of virus infection. Jenkins et al. (2007) also indicated that C. hominis and C. parvum, the two species most often associated with human infections, are the only species reported thus far to be infected by CPV. February 2009 47 ------- U.S. Environmental Protection Agency Text Box III.2-1. Examples of Feeding Studies with Zoonotic Parasites For the widespread waterborne zoonotic protozoa Cryptosporidium and Giardia, human volunteers (immunocompetent adults) have been exposed to measured doses of various strains of Cryptosporidium and to a single Giardia isolate to evaluate their dose-response relationships. However, there are uncertainties associated with characterizing dose even in clinical settings. Microbes can exhibit variability in infectivity, virulence, or environmental survival within strains; even for "pure isolates," batches can differ. For microorganisms that cannot survive freezer storage or do not maintain their genetic integrity in freezer storage or tissue culture, in vivo passage in animals is required to maintain stocks. Even if the starting inoculum for a dose-response study is clonal, mutations will occur that may impact pathogen characteristics. When the starting inoculum is not clonal, which is most often the case, the subpopulation ratios within an individual host can differ from other hosts receiving the same inoculum. The subpopulation ratios also may vary as infection progresses, so collecting pathogens from a host on one day may not yield the same pool of pathogens as collecting on another day. In addition, some pathogens are not amenable to storage or maintenance in laboratory settings under any conditions and must be continuously collected from the field for each experiment. Although the challenges of properly characterizing and controlling pathogen variability in experimental research settings are considerable, the challenges are even greater for epidemiological studies. Cryptosporidium in Humans Data on the infectivity of Cryptosporidium in humans are available from studies conducted at the University of Texas, Houston, by DuPont, Chappell, Okhuysen, and colleagues. These studies all involved healthy adult volunteers ingesting different numbers of Cryptosporidium oocysts. Subjects were then evaluated for Cryptosporidium in stool samples and for diarrheal and other Gl illness symptoms. Infectivity was estimated for five C. parvum isolates: TAMU (collected from a veterinary student); Iowa (derived from a calf); UCP (derived from a calf); Moredun, (collected from a red deer calf); 16W (from a calf); and TU502 (an isolate collected from an infected child and propagated in gnotobiotic piglets [Chappell et al., 2006]). Cryptosporidium from animal sources were found to be infectious in humans. No attempt was made to evaluate differences in potential infectivity due to repeated passage in animal hosts. However, the UCP isolate was less infectious in humans than the other isolates, and EPA's Science Policy Council speculated that this could be due to prolonged maintenance in calves. In the risk assessment that was conducted in support of EPA's Long Term 2 Enhanced Surface Water Treatment Rule (LT2) Economic Analysis, a dose-response relationship was chosen that weighs the UCP data less than the other isolates. Cryptosporidium in Animals Akiyoshi et al. (2003) investigated mixed infections of Type 1 (C. hominis) and Type 2 (C. parvum) in gnotobiotic piglets. In all the time intervals tested, Type 2 displaced Type 1, even if Type 1 was permitted to become established before inoculation with Type 2. This result raises significant questions regarding the relative perpetuation and survival of the two genotypes in mammalian hosts. The same researchers noted that field technicians very readily became infected with C. hominis, but that cross-contamination of C. hominis with C. parvum in the animals used to maintain stocks resulted in C. parvum overwhelming C. hominis (Tzipori, 2000). These observations suggest that discovering the mechanisms by which C. hominis is maintained in natural setting when C. parvum is also present should improve understanding of risks to humans. Giardia in Humans Rendtorff (1954) conducted a controlled, clinical study of male prison volunteers who were fed Giardia cysts obtained from a human source. Giardia cysts of known numbers varying from 1 to 106 were placed into gelatin capsules along with a small amount of saline. The capsules were given to the volunteers along with 4 to 6 ounces of water. Control subjects were given sterile saline in the same manner. A dose of 10 cysts was found to be sufficient to produce human infection, as determined by observing the presence of Giardia in fecal smears (Rendtorff, 1954). However, because cyst viability could not be determined prior to administration to volunteers, the failure to elicit infection in the five men treated with a dose that was calculated to contain only one cyst may have been due to dosing with inactive cysts. The Giardia Assemblages used in these studies are not known but presumably are A or B because these are the Assemblages known to infect humans. February 2009 48 ------- U.S. Environmental Protection Agency IV. SUMMARY Contamination of recreational waters with feces from warm-blooded animals poses a risk of zoonotic infection of humans with some of the pathogens in those waters. Although the risk and severity of human illness due to contamination with animal feces and zoonotic pathogens is most likely lower than the risk and severity of illness from treated or untreated human sewage, currently available data are insufficient to quantify the differences. At present, the six most important zoonotic waterborne pathogens are the following: Pathogenic E. coli; Salmonella; Campylobacter; Leptospira; Cryptosporidium; and Giardia. All of these waterborne pathogens are likely to cause more severe symptoms in children and immunocompromised individuals and subpopulations than in the remainder of the population. Of these six, pathogenic E. coli has the most potential for severe adverse health effects that can even be fatal. Potential debilitating chronic sequelae such as Guillain-Barre Syndrome and reactive arthritis have been associated with Campylobacter infections. Although the most common recreational illnesses are probably due to human viruses causing short-term GI, the waterborne zoonotic pathogens discussed in this report have the potential to cause serious health effects. 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Beach sands along the California Coast are diffuse sources of fecal bacteria to coastal waters. Environmental Science and Technology 41: 4515-4521. Supporting information for beach sands along the California coast are diffuse sources of fecal bacteria to coastal waters. Environmental Science and Technology Supplemental to 41: 4515-4521, 17 pp. February 2009 74 ------- U.S. Environmental Protection Agency Yang, S., Benson, S.K., Du, C., Healey, M.C. 2000. Infection of immunosuppressed C57BL/6N adult mice with a single oocyst of Cryptosporidium parvum. International Journal for Parasitology 86(4): 884-887. Yarze, J.C., Chase, M.P. 2000. E. coli O157:H7 - another waterborne outbreak! American Journal of Gastroenterology 95(4): 1096. Yoder, J.S., Blackburn, E.G., Craun, G.F., Hill, V., Levy, D.A., Chen, N., Lee, S.H., Calderon, R.L., Beach, M.J. 2004. Surveillance for waterborne-disease outbreaks associated with recreational water - United States, 2001-2002. Mortality and Morbidity Weekly Report CDC Surveillance Summaries 53(8): 1-22. Zhou, L., Singh, A., Jiang, J., Xiao, L. 2003. Molecular surveillance of Cryptosporidium spp. in raw wastewater in Milwaukee: implications for understanding outbreak occurrence and transmission dynamics. Journal of Clinical Microbiology 41(11): 5254-5257. February 2009 75 ------- U.S. Environmental Protection Agency APPENDIX A WATERBORNE PATHOGENS As described in Section 1.2 of this paper, four attributes were used to select the waterborne zoonotic pathogens of concern for recreational uses of ambient waters (partially adapted from Bolin et al., 2004a). Table A-l lists all the pathogens that were evaluated for potential inclusion in this paper. Information provided in Table A-l includes whether the pathogen is considered waterborne, the species that are zoonotic hosts, whether the zoonotic hosts are warm-blooded, what illnesses the pathogen causes in humans, and the importance of considering the pathogen as EPA decides whether animal sources of fecal material should be considered differently from human sources for CWA §304(a) AWQC. February 2009 A-l ------- U.S. Environmental Protection Agency Table A-l. Known and Potential Zoonotic Waterborne Pathogens Type Bacteria Bacteria Bacteria Bacteria Pathogen Acinetobacter Aeromonas Campylobacter Cyanobacteria Waterborne Yes (generally in environment) Yes (generally in environment) Yes Yes (generally in environment) Zoonotic Host None None Poultry, cattle, sheep, and wild birds None Zoonotic Host is Warm- blooded NA NA Yes NA Illnesses and Symptoms in Humans (less common symptoms) Septicemia, meningitis, endocarditis, brain abscesses, pneumonia, empyema, urinary tract infections, eye infections, and skin and wound infections Gastroenteritis (septicemia) Diarrhea, abdominal pain, malaise, fever, nausea, and vomiting (typhoid- like syndrome, febrile convulsions, meningeal arthritis, reactive arthritis, and GBS) Rash and gastroenteritis U.S. Outbreaks Hospital settings None reported Mainly foodborne and drinking water Drinking water and recreational water Importance to consider if animal fecal sources are discounted in AWQC No No Important No Reference(s) Stewart and Rochelle, 2006 Lightfoot, 2004; Moyer and Standridge, 2006 Allos, 1998; 2001; APHA, 2006; Fricker, 2006a Fredericksen et al., 2006 February 2009 A-2 ------- U.S. Environmental Protection Agency Type Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Pathogen Verotoxin-producing E.coli (VTEC) - includes enterohemorrhagicE. coli (EHEC), including O1 57:H7 Enterotoxigenic E. coli (ETEC) Attaching and effacing £. coli (A/EEC) EnteropathogenicE. coli (EPEC) Enteroaggregative E. coli (EAggEC) Diffuse adherent E. coli (DAEC) Enteroinvasive E. coli (EIEC) Waterborne Yes Yes Yes Yes Yes Yes Yes Zoonotic Host Cattle, chicken, sheep, pigs, horses, dogs, and deer Same as VTEC Same as VTEC Same as VTEC Same as VTEC Same as VTEC None Zoonotic Host is Warm- blooded Yes Yes Yes Yes Yes Yes NA Illnesses and Symptoms in Humans (less common symptoms) Diarrhea (bloody), severe abdominal cramping, headache, hemorrhagic colitis, and hemolytic uremic syndrome Acute, watery diarrhea Acute or persistent diarrhea Acute or persistent diarrhea Acute, watery, and often protracted diarrhea Acute or persistent diarrhea Acute, often inflammatory diarrhea; dysentery U.S. Outbreaks Food borne and waterborne (drinking water and recreational water) Waterborne No waterborne reported Waterborne No waterborne reported No waterborne reported No waterborne reported Importance to consider if animal fecal sources are discounted in AWQC Important Possibly important Possibly important Possibly important Possibly important Possibly important No Reference(s) APHA, 2004; Degnan and Standridge, 2006; M0lbak and Scheutz, 2004 Hunter, 2003; M0lbak and Scheutz, 2004 M0lbak and Scheutz, 2004 Lee et al, 2002; M0lbak and Scheutz, 2004 M0lbak and Scheutz, 2004 M0lbakand Scheutz, 2004 M0lbak and Scheutz, 2004 February 2009 A-3 ------- U.S. Environmental Protection Agency Type Bacteria Bacteria Bacteria Bacteria Bacteria Pathogen Flavobacterium Helicobacter pylori Klebsiella Legionella Leptospira Waterborne Yes Possibly Yes (generally in environment) Yes Yes Zoonotic Host None Weak evidence for ferrets, racoons, swine, sheep, rodents, and primates Warm-blooded animals None Rats, dogs, raccoons, swine, and cattle Zoonotic Host is Warm- blooded NA Yes Yes NA Yes Illnesses and Symptoms in Humans (less common symptoms) Gastroenteritis, meningitis, pneumonia, endocarditis, and septicemia Gastric disorders, pepticand duodenal ulcer disease, lymphoma of the digestive tract, and ademenocarcinoma of the stomach Infections in respiratory system, genitourinary tract, nose, and throat, (meningitis and septicemia) Legionellosis, pneumonia, Legionnaire's disease, and Pontiac fever Leptospirosis (Weil's disease) U.S. Outbreaks Rare waterborne (stagnation in drinking water), hospital settings more common No waterborne reported Hospital settings Hospitals, pools, and spas Recreational waterborne over 50% of cases in Hawaii Importance to consider if animal fecal sources are discounted in AWQC No No No No Important Reference(s) Geldreich and Degnan, 2006a Baker and Degnan, 2006; Health Canada, 2006 Geldreich and Standridge, 2006 Hall, 2006 Levett, 2001 February 2009 A-4 ------- U.S. Environmental Protection Agency Type Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Pathogen Listeria monocytogenes Mycobacterium avium complex (MAC) and ssp. Paratuberculosis (MAP) Pseudomonas Salmonella Serratia Shigella Staphylococcus Waterborne No Yes (generally in environment) Yes (generally in environment) Yes Yes (generally in environment) Yes Yes Zoonotic Host Domestic and wild animals Possibly sheep, cattle, goats, and birds None Poultry, swine, cattle, rodents, wild birds, turtles, dogs, and cats None None (except primate colonies) Skin of warm- blooded hosts Zoonotic Host is Warm- blooded Yes Yes NA Yes NA NA Yes Illnesses and Symptoms in Humans (less common symptoms) Listeriosis, meningoencephaliti s, fever, and abortion Respiratory infection, fever, and Crohn's disease Dermatitis Gastroenteritis (enteric fever and septicemia) Opportunistic infection (cystitis) Shigellosis, acute gastroenteritis, dysentary, fever, nausea, vomiting, and cramps Cellulitis, pustules, boil, carbuncles, and impetigo (diarrhea and vomiting) U.S. Outbreaks Food borne No recreational waterborne reported Recreational waterborne Mainly foodborne and drinking water Hospital settings Recreational and drinking water Hospital settings, pools, and spas Importance to consider if animal fecal sources are discounted in AWQC No Possibly important No Important No No No Reference(s) APHA, 2004 Bolin et al., 2004b; Carr and Bartram, 2004; LeChevallier, 2006 Geldreich and Degnan, 2006b APHA, 2004; Covert and Meckes, 2006 Geldreich and Standridge, 2006a APHA, 2004; Cliverand Payer, 2004; Moyer and Degnan, 2006 Geldreich and Standridge, 2006b February 2009 A-5 ------- U.S. Environmental Protection Agency Type Bacteria Bacteria Bacteria Protozoa Protozoa Protozoa Protozoa Protozoa Pathogen Vibrio cholerae Vibrio parahaemolyticus Yersinia Acanthamoeba Ascaris lumbricoides Balamuthia mandrillaris Balantidium coli Blastocystis hominis Waterborne Yes Yes (generally in environment) Yes Yes (free- living) Yes Yes (free- living) Yes Yes Zoonotic Host Cope pods, zooplankton Molluscan shellfish Pigs None None Primates, sheep, dogs, and horses Primates and pigs Primates, cattle, sheep, pigs, horses, dogs, chickens, wild birds, alpacas, llamas, koalas, and wombats Zoonotic Host is Warm- blooded No No Yes NA NA Yes Yes Yes Illnesses and Symptoms in Humans (less common symptoms) Profuse, watery diarrhea; vomiting Acute gastroenteritis (septicemia) Yersiniosis; acute, febrile diarrhea Granulomatus ameobic encephalitis Ascariasis and roundworm infection Granulomatus ameobic encephalitis Severe dysentery Diarrhea, cramps, nausea, fever, vomiting, and abdominal pain U.S. Outbreaks None recently Food borne Mainly foodborne None reported Foodborne None reported None reported None reported Importance to consider if animal fecal sources are discounted in AWQC No No Possibly important No No No No No Reference(s) APHA, 2004; Toranzos et al., 2006 FDA, 2001 APHA, 2004; Fricker, 2006b Fayer, 2004b; Visvesvara and Moura, 2006a APHA, 2004; Smith et al., 2006a Visvesvara and Moura, 2006b Garcia, 2006a Garcia, 2006b February 2009 A-6 ------- U.S. Environmental Protection Agency Type Protozoa Protozoa Protozoa Protozoa Pathogen Cryptosporidium parvum and Cryptosporidium hominis Cyclospora cayetanensis Entamoeba histolytica Giardia intestinalis (also known as G. duodenalis and G. lamblia) Waterborne Yes Yes Yes Yes Zoonotic Host Cattle, sheep, goats, pigs, horses, cats, dogs, wild animals, birds, reptiles, and fish None Potentially primates, dogs, cats, pigs, rats, and possibly cattle Beavers, cats, lemurs, sheep, calves, dogs, foxes, chinchillas, alpacas, horses, pigs, cows, and muskrats Zoonotic Host is Warm- blooded Yes NA Yes Yes Illnesses and Symptoms in Humans (less common symptoms) Cryptosporidiosis, profuse watery diarrhea, malaise, fever, anorexia, nausea, and vomiting Watery diarrhea, malaise, fever, anorexia, nausea, and vomiting Amoebiasis, dysentery, and diarrhea Giardiasis; diarrhea (chronic); steatorrhea; abdominal cramps; bloating; frequent loose, pale, greasy stools; fatigue; and malabsorption U.S. Outbreaks Recreational and drinking water Food borne and waterborne (drinking water and recreational water) None recently Recreational and drinking water Importance to consider if animal fecal sources are discounted in AWQC Important No No Important Reference(s) APHA, 2004; CDC, 2007b; Olson, et al., 2003; Sterling and Marshall, 2006 APHA, 2004; Cliverand Payer, 2004; Cross and Sherchand, 2004; Ortega, 2006 APHA, 2004; Payer, 2004b; Keene, 2006 APHA, 2004; Appelbee et al., 2005; Schaefer, 2006 February 2009 A-7 ------- U.S. Environmental Protection Agency Type Protozoa Protozoa Protozoa Virus Virus Virus Pathogen Isosprora belli Microsporidia (Enterocytozoon bieneusi, Encephalitozoon cuniculi, E. intestinalis) Naegleria fowleri Adenoviruses Astrovi ruses Avian influenza (H5N1) Waterborne Yes Yes Yes (free- living) Yes Yes Unknown Zoonotic Host None Cattle, pigs, cats, rabbits, and sheep None None None Birds Zoonotic Host is Warm- blooded NA Yes NA NA NA Yes Illnesses and Symptoms in Humans (less common symptoms) Foul smelling, foaming diarrhea (months to years), abdominal colic, and fever Diarrhea Primary amebic meningoencephaliti s Acute gastroenteritis and respiratory disease Gastroenteritis Mild upper respiratory illness to severe pneumonia and multiple organ failure U.S. Outbreaks None reported None reported Mainly ambient recreational waters Waterborne Waterborne None reported Importance to consider if animal fecal sources are discounted in AWQC No Possibly important No No No No Reference(s) Garcia, 2006c Bolin et al., 2004b; Call, 2006; Shadduckand Greely, 1989 Payer, 2004b; Visvesvara and Moura, 2006c Enriquez and Thurston- Enriquez, 2006; Griffin et al., 2003; Reynolds, 2006 Reynolds, 2006; Schwab, 2006 Chan, 2002; Suresh and Smith, 2004 February 2009 A-8 ------- U.S. Environmental Protection Agency Type Virus Virus Virus Virus Virus Virus Virus Pathogen Coronaviruses (e.g., severe acute respiratory syndrome (SARS)-CoV) Enteroviruses (e.g., coxsackie) Hendra virus Hepatitis A virus Hepatitis E virus Human caliciviruses (norovirus, sapovirus) Nipah virus Waterborne Potentially (aerosolized waste water) Yes Unknown Yes Yes Yes Unknown Zoonotic Host Possibly civets and other wild animals None Horses None Possibly pigs, chickens, and rats (close viral relatives to human form) None Pigs, bats, and flying foxes Zoonotic Host is Warm- blooded NA NA Yes NA NA NA Yes Illnesses and Symptoms in Humans (less common symptoms) Fever, dry cough, dyspnoea, and myalgia (diarrhea) Gastroenteritis, exanthema, diarrhea, fever, pharyngeal lesions, myocarditis, respiratory disease, and pneumonia Severe respiratory disease and meningoencephaliti s Hepatitis - acute inflammation of the liver Hepatitis - acute inflammation of the liver (fatality in pregnant women) Diarrhea and vomiting Acute and febrile encephalitis U.S. Outbreaks None reported Recreational and drinking water None reported None recently Rare in United States (common in other countries) Very common (waterborne and foodborne) None reported Importance to consider if animal fecal sources are discounted in AWQC No No No No No No No Reference(s) Moe, 2004; Reynolds, 2006; Suresh and Smith, 2004 APHA, 2004; Gerba, 2006a; Griffin, 2003; Reynolds, 2006 MacKenzie, 1999; Suresh and Smith, 2004 Sobsey, 2006 Oliver and Moe, 2004; Craun, 2004a; Gerba, 2006b Reynolds, 2006; Schwab and Hurst, 2006 Harcourt, 2005; Suresh and Smith, 2004 February 2009 A-9 ------- U.S. Environmental Protection Agency Type Virus Virus Virus Helminths Helminths Helminths Pathogen Parechoviruses Reoviruses Rotaviruses Ancylostoma braziliense Angiostrongylus Ascaris lumbricoides Waterborne Yes Yes Yes Mainly soil Yes Mainly soil (drinking water possible) Zoonotic Host None None None Dogs or cats Mollusks None Zoonotic Host is Warm- blooded NA NA NA Yes No NA Illnesses and Symptoms in Humans (less common symptoms) Pericarditis, herpangina, and respiratory disease Mostly mild or subclinical (rarely biliary atresia, juvenile onset diabetes, fever, rash, respiratory disease, and diarrhea Diarrhea Larval migration leads to creeping eruption Meningitis Ascaris pneumonia (lung hemorrhage) U.S. Outbreaks None reported (probably occurs, but unidentified) None reported (probably occurs, but unidentified) Very common waterborne None reported (most important hookworm in humans - soil route) None reported None reported Importance to consider if animal fecal sources are discounted in AWQC No No No No No No Reference(s) Gerba, 2006a Sattar and Springthrope, 2006 Abbaszadegan, 2006; Reynolds, 2006 Endo and Morishima, 2004 Endo and Morishima, 2004 Endo and Morishima, 2004; Smith et al., 2006 February 2009 A-10 ------- U.S. Environmental Protection Agency Type Helminths Helminths Helminths Helminths Helminths Helminths Helminths Pathogen Ascaris suum avian schistosomes (includes S. mansonni) Baylisascaris procyonis Dracunculus medinensis Echinococcus Fasciola hepatica Pseudophyllid cestodes Waterborne Mainly soil Yes Mainly soil Yes Yes Yes Yes Zoonotic Host Pigs Snails (birds are infected but not sources) Raccoons Crustaceans Foxes, dogs Snails (herbivores and humans infected) Copepods, fish Zoonotic Host is Warm- blooded Yes Yes Yes No Yes Yes No Illnesses and Symptoms in Humans (less common symptoms) Ascaris pneumonia (lung hemorrhage) Cercarial dermatitis (swimmer's itch) Larval migration may damage to the visceral and ocular systems (migration to brain) Connective tissue migration, emerging in lower limb blister Cystic hydatid disease, alveolar hydatid disease, polycystic hydatid disease, and polycystic hydatid disease Fascioliasis Cutaneous or mucocutaneous invasion U.S. Outbreaks None reported None reported None reported None reported None reported None reported None reported Importance to consider if animal fecal sources are discounted in AWQC No Low importance No No Low importance Low importance No Reference(s) Endo and Morishima, 2004 Endo and Morishima, 2004 Endo and Morishima, 2004 Endo and Morishima, 2004 Endo and Morishima, 2004 Endo and Morishima, 2004 Endo and Morishima, 2004 February 2009 A-ll ------- U.S. Environmental Protection Agency Type Helminths Helminths Helminths Helminths Helminths Pathogen Schistosomes Strongyloides Taenia solium (pork tapeworm) Toxocara canis Toxocara cati Waterborne Yes Mainly soil Yes Yes Yes Zoonotic Host Snails (many animals can be infected, but are not sources) Dogs Pigs Dogs Cats Zoonotic Host is Warm- blooded No Yes Yes Yes Yes Illnesses and Symptoms in Humans (less common symptoms) Schistosomiasis (chronic infection - liver fibrosis, portal hypertension) Eosinophilia Cysticercosis and myositis Toxocariasis (larval migration leads to hemorrhage and granulomatous lesions in the central nervous system) Toxocariasis (larval migration leads to hemorrhage and granulomatous lesions in the central nervous system) U.S. Outbreaks None reported None reported None reported None reported None reported Importance to consider if animal fecal sources are discounted in AWQC No No Low importance Low importance Low importance Reference(s) APHA, 2004; Blankespoor, 2006; Endo and Morishima, 2004 Endo and Morishima, 2004; Pardo, 2006 Endo and Morishima, 2004 Endo and Morishima, 2004 Endo and Morishima, 2004 February 2009 A-12 ------- U.S. Environmental Protection Agency Type Helminths Helminths Helminths Prion Pathogen Toxoplasma gondii Trichinella spiralis Trichuris trichiuria Bovine Spongiform Encephalopathy (BSE) Waterborne Yes Mainly soil Mainly soil Unknown Zoonotic Host Cats Variety of animals Monkeys, pigs, dogs, cats, and chicken Cattle Zoonotic Host is Warm- blooded Yes Yes Yes Yes Illnesses and Symptoms in Humans (less common symptoms) Toxoplasmosis, (in fetally exposed children - mental retardation, loss of vision, hearing impairment, and mortality) Trichinosis Trichuris dysentary syndrome, chronic diarrhea, anemia, and growth retardation Variant Creutzfeldt- Jakob disease U.S. Outbreaks None reported in recreational water Food borne (mainly consumption of undercooked pork) Food borne Food borne Importance to consider if animal fecal sources are discounted in AWQC Potentially important No No No Reference(s) APHA, 2004; Dubey, 2004, 2006 Endo and Morishima, 2004 Endo and Morishima, 2004; Smith et al., 2006b Brown et al., 2006 NA = Not applicable February 2009 A-13 ------- U.S. Environmental Protection Agency APPENDIX B LITERATURE SEARCH STRATEGY AND RESULTS The literature search strategy consisted of a number of combined approaches. Search terms and a synopsis of information needed were given to a professional librarian to search the online DIALOG databases. To supplement the DIALOG searches, individual authors used free search engines on the internet to find articles pertaining to specific information needed. Experts that participated in EPA's Experts Scientific Workshop on Critical Research Needs for the Development of New or Revised Recreational Water Quality Criteria11 were contacted by email and requested to contribute literature they felt was important. The titles of literature cited in specific reports, books, review articles, and conference proceedings were evaluated for relevance. B.I Initial Literature Search Strategy Conducted by Professional Librarian Selection of DIALOG data base files used for this search: File 155:MEDLINE(R) 1950-2007/Nov 30 (c) format only 2007 Dialog File 266:FEDRIP 2007/Sep Comp & dist by NTIS, Intl Copyright All Rights Res File 144:Pascal 1973-2007/Nov W3 (c) 2007 INIST/CNRS File 110:WasteInfo 1974-2002/Jul (c) 2002 AEA Techn Env. File 245:WATERNET(TM) 1971-2007Jul (c) 2007 American Water Works Association File 117:Water Resources Abstracts 1966-2007/Aug (c) 2007 CSA. File 5:Biosis Previews(R) 1926-2007/Nov W4 (c) 2007 The Thomson Corporation File 40:Enviroline(R) 1975-2007/Oct (c) 2007 Congressional Information Service File 143:Biol. & Agric. Index 1983-2007/Oct (c) 2007 The HW Wilson Co File 6:NTIS 1964-2007/Dec W3 (c) 2007 NTIS, Intl Cpyrght All Rights Res File 72:EMBASE 1993-2007/Dec 04 (c) 2007 Elsevier B.V. Main search strategy used for this search: Fecal set AND Composition set AND (Pathogen or Waterborne sets): 1985-present SI 240528 FECAL OR FECES OR FAECAL OR FAECES OR DUNG OR SCAT OR EXCREMENT OR MANURE OR STOOLS) S2 1686966 COMPOSITION OR COMPOSED OR ANIMAL(IN)HUMAN()(TRANSMISSION OR CONTAMINATION) 11 Report from this workshop: http://www.epa.gov/waterscience/criteria/recreation/. February 2009 B-l ------- U.S. Environmental Protection Agency S3 3842348 PATHOGEN? ? OR INDICATOR? ? OR RISK OR RISKS S4 983 WATERBORNEO (PATHOGEN? ? OR ZOONO? OR ILLNESS?) S6 1140 SI AND S2 AND (S3 OR S4) S719961011 PY=1920:1984 S8 1037 S6 NOT S7 (limited to 1985-present; foreign language OK) S9 704 RD S8 (unique items, deduped) Dates: 1985-present Language: No restrictions Retrieve: Titles and year Format: MS Word Interested in international and domestic journals and government reports. Descriptions of these files are available at http://library.dialog.com/bluesheets/. Search terms: Waterborne pathogen* Waterborne illness* Waterborne zoon* Emerging waterborne zoonotic pathogen* Antibiotic resistan* AND zoonotic pathogen* Transmission rate waterborne pathogen* Evolution zoon* pathogen* Human fecal composition micro* Enterohemorrhagic E. coli symptoms Salmonella symptoms Shigella symptoms Campylobacter symptoms Listeria symptoms Cryptosporidium symptoms Giardia symptoms norovirus symptoms rotavirus symptoms hepatitis E symptoms waterborne dermal infection* waterborne eye infection* waterborne ear infection* waterborne respiratory infection* What we want from the literature search: Fecal composition of species that harbor zoonotic pathogens (species of livestock and wild animals Fecal composition of humans - pathogens and indicators The extent to which strains found in animals can be transmitted to humans February 2009 B-2 ------- U.S. Environmental Protection Agency An evaluation of the extent to which the information identified can be used to support the differentiation of risk from animal and human sources of fecal contamination Which organisms are of substantial public health concern that occur in ambient waters and are pathogenic to humans Which of these organisms are also present in animal populations The extent to which these organisms found in animals can be transmitted to humans The potential outcomes of human infection and disease from animal sources Specific pathogens: E. coli O157-H7, Salmonella., Shigella, Campy lobacter, Listeria, Cryptosporidiiim, Giardia, norovirus, rotavirus, Hepatitis E, emerging pathogens. For each: o Describe illness symptoms (range asymptomatic to severe) o Describe route of exposure from recreational immersion in water including, inhalation, skin and mucus, eyes, ears o Incidence (morbidity and mortality data, through recent time) o Zoonotic potential - which animal species Variations in strains that effect infectivity, severity of symptoms, environmental survival and treatability Short summary review of emerging pathogen mechanisms (not too deep into molecular mechanisms of evolution) Anything in the water matrix that affects survivability and infectivity, and virulence Pathogen:indicator ratios B.2 Summary of Literature Search Results This process resulted in a total order of 535 documents (primarily peer reviewed scientific articles), of which a total of 332 (62 percent) were received during the expedited writing process, not all of which could be reviewed. There are many more papers in the peer-reviewed literature, and this by no means represents all of them. 319 citations were included in the white paper. B.3 Supplemental Free Online Search Engines The following terms were searched on Google (http://www.google.com/): Search Topic E. coli + deer Adenoviruses + zoonotic Hepatitis E+ condition + symptoms Rotavirus + illness + symptoms Approximate # Titles Reviewed 10 30 30 20 # Titles of Interest 1 3 2 1 February 2009 B-3 ------- U.S. Environmental Protection Agency The following terms were searched on Google Scholar (http://scholar.google.com/): Search Topic Adenoviruses + zoonotic Rotavirus + illness + symptoms Giardiasis + swimming Giardiasis + surveillance Giardia + symptoms Giardia + cyst Giardia + beach Cryptosporidium + hominis Cryptosporidium + beach Cryptosporidium + incidental ingestion Cryptosporidium + review Cryptosporidium + exposure factors Cryptosporidium + exposure Cryptosporidium + symptoms Cryptosporidium + risk Campylobacter + chronic Campylobacter + illness Campylobacter + infection Campylobacter + symptoms Campylobacter + water Campylobacter + pathogenesis Salmonella + transmission Salmonella + transmission in water Salmonella + antibiotic resistance Salmonella + pathogenesis Shigella + outbreak Shigellosis Approximate # Titles Reviewed 10 30 200 200 100 100 200 100 150 250 100 150 50 50 50 50 50 50 50 15 5 20 11 20 3 23 35 # Titles of Interest 1 3 5 3 5 2 1 1 1 4 2 3 2 3 0 3 3 4 1 2 1 8 2 3 1 4 4 The following terms were searched on PubMed (http://www.ncbi.nlm.nih.gov/sites/entrez): Search Topic E. co/; O157:H7 + survival + environment Salmonella + survival + environment Shigella + survival + environment Campylobacter + survival + environment Norwalk Virus + survival + environment Rotavirus + survival + environment Hepatitis E + survival + environment # Titles Reviewed 204 376 65 57 2 54 3 # Titles of Interest 23 4 10 17 Yes 17 Yes February 2009 B-4 ------- U.S. Environmental Protection Agency The following terms were searched on Scirus (http://www.scirus.com/): Search Topic Helicobacter + environmental survival Leptospira + CID Leptospira + environmental survival Leptosporidiosis + symptoms Approximate # Titles Reviewed 30 10 30 30 # Titles of Interest 1 1 2 0 The following terms were searched on the CDC website (http://www.cdc.gov/): Search Topic Legionella + EID Leptospira + EID Avian influenza + EID H5N1 +EID SARS + EID Hendra virus + EID Nipah virus + EID Strongyloides + EID BSE + EID Approximate # Titles Reviewed 10 10 10 20 30 20 20 10 10 # Titles of Interest 1 1 1 0 2 0 2 1 1 B.4 Experts Contacted The flowing experts in the field were contacted directly by email and asked to suggest references: Nicholas Ashbolt, USEPA Thomas Atherholt, New Jersey Department of Environmental Protection Michael Beach, Centers for Disease Control and Prevention Bart Bibler, Florida Department of Health Alexandria Boehm, Stanford University, California Rebecca Calderon, USEPA Jennifer Clancy, Clancy Environmental Consultants Jack Colford, University of California, Berkeley Elizabeth Doyle, USEPA Alfred Dufour, USEPA Lee Dunbar, Connecticut Department of Environmental Protection Lora Fleming, University of Miami School of Medicine and Rosenstiel School of Marine and Atmospheric Sciences, Florida Charles Hagedorn, Virginia Tech Joel Hansel, USEPA February 2009 B-5 ------- U.S. Environmental Protection Agency Lawrence Honeybourne, Orange County Health Care Agency, Santa Ana, California Donna Francy, U.S. Geological Survey Roger Fuji oka, University of Hawaii, Manoa Toni Glymph, Wisconsin Department of Natural Resources Mark Gold, Heal the Bay, California Paul Hunter, University of East Anglia, U.K. Dennis Juranek, Centers for Disease Control and Prevention (retired) David Kay, University of Wales, U.K. Sharon Kluender, Wisconsin State Laboratory of Hygiene Erin Lipp, University of Georgia Graham McBride, National Institute of Water and Atmospheric Research, New Zealand Charles McGee, Orange County Sanitation District, California Samuel Myoda, Delaware Department of Natural Resources Charles Noss, USEPA Robin Oshiro, USEPA James Pendergast, USEPA Mark Pfister, Lake County Health Department, Illinois John Ravenscroft, USEPA Stephen Schaub, USEPA Mark Sobsey, University of North Carolina, Chapel Hill Jeffrey Seller, Seller Environmental, California Michael Tate, Kansas Department of Health and Environment Peter Teunis, RIVM (National Institute of Public Health and the Environment), Netherlands Gary Toranzos, University of Puerto Rico, Rio Piedras Timothy Wade, USEPA John Wathen, USEPA Stephen Weisberg, Southern California Coastal Water Research Project David Whiting, Florida Department of Environmental Protection Richard Zepp, USEPA B.4 Previously Cited References The following specific reports were obtained and the titles of the references cited in the reports were reviewed for relevance: NRC. (2004) Indicators for Waterborne Pathogens. The National Academies Press USEPA. (2007) Report of the Experts Scientific Workshop on Critical Research Needs for the Development of New or Revised Recreational Water Quality Criteria http://www.epa.gov/waterscience/criteria/recreation WHO. (2004) Waterborne Zoonoses. http://www.who.int/water sanitation health/ diseases/zoonoses.pdf References cited by the Natural Resources Defense Council reviewers of the EPA Critical Path Science Plan USEPA Internal draft Adenovirus criteria document USEPA Internal draft pathogenic E. coli criteria document February 2009 B-6 ------- U.S. Environmental Protection Agency Boehm et al. (2008) A sea change ahead for recreational water quality criteria, (peer review in progress) In addition, Clancy Environmental Consultants, Inc., ICF International, Seller Environmental, WaltJay Consulting, and EPA's Health and Ecological Criteria Division all maintain extensive literature databases and reference lists from previously completed projects. All of those in house resources were also sources of literature. February 2009 B-7 ------- U.S. Environmental Protection Agency APPENDIX C INCIDENTAL INGESTION OF AMBIENT WATER DURING RECREATIONAL ACTIVITIES There is a paucity of data concerning rates of incidental ingestion of surface water during recreational activities. Most of the available estimates address exposures during swimming in swimming pools, which may not necessarily be representative of typical "incidental" exposures in ambient waters. Dufour et al. (2006) reviewed early estimates of swimming-related water ingestion and concluded that incidental ingestion ranged from 10 to 50 mL per hour. None of the early estimates, however, were based on actual studies of water ingestion. EPA's Risk Assessment Guidance for Superfund (USEPA, 1989) recommended a value of 50 mL per hour for ingestion during water recreation, citing an early version of EPA's Exposure Factors Handbook (EFH). The latest version of the EFH (USEPA, 1997) contains no recommendation concerning recreational water intake. Hammond et al. (1986), in their assessment of the potential toxicity of swimming pool disinfectants, estimated that a 70-kg adult might ingest "1 to 2 cups" of water, meaning approximately 500 mL. However, this is a "ballpark" estimate and is not supported by observational data. Allen et al. (1982) estimated water ingestion by competitive swimmers by measuring urinary excretion of isocyanuric acid, an unmetabolized compound used to stabilize chlorine levels in swimming pools. The average estimated water intake among the five swimmers that were studied was 161 mL per hour. The investigators also determined that (1) essentially all of the ingested isocyanuric acid appeared in urine within 24 hours, thus negating concern for elimination by other pathways; and (2) dermal absorption of the tracer compound was insignificant compared to the ingestion intake. Using methods similar to those used by Allen et al. (1982), Dufour et al. (2006) estimated water intake in 12 adults and 41 "nonadults" engaged in less vigorous water recreation at a community swimming pool. Based on the amounts of isocyanurate excreted in urine, they estimated that 45 minutes of water recreation resulted in average water intakes of 37 mL (49 mL/hr) for nonadults and 16 mL (21 mL/hr) for adults. The exposures measured by Dufour et al. (2006) are perhaps more likely to be representative of typical "incidental" exposures than those of the competitive swimmers measured by Allen et al. (1982), suggesting the lower values may provide a better basis for estimating incidental exposures. A larger follow-up study of 549 participants was subsequently conducted at several public and private outdoor swimming pools (Evans et al., 2006). Participants were requested to engage in active swimming for between 45 and 60 minutes. The overall average incidental ingestion rate was 32 mL/hr, with a range of 1 to 280 mL/hr. Adults averaged 24 mL/hr, and children averaged 47 mL/hr. Children (ages not specified) swallowed approximately twice as much water as adults. The follow-up study also showed that males ingested more than females and that adult men ingested more than adult women. February 2009 C-l ------- U.S. Environmental Protection Agency The small number of studies that are available measured water intake in only a few subjects and characterized water ingestion during either very active swimming or poorly defined water recreational activities. In addition, the incidental ingestion data that are available are for "clean" pool water and may not represent incidental ingestion for surface waters, which may provoke stronger avoidance behaviors due to the perception that surface waters are nonpotable. February 2009 C-2 ------- |