Cryptosporidiosis: a brief literature review and update regarding Cryptosporidium in feces of Canada geese (Branta canadensis).

Introduction

Cryptosporidium, a protozoan parasite, is an important cause of the human enteric disease cryptosporidiosis. The disease is among the most common causes of diarrhea in patients with AIDS in the United States (Juranek, 1995). In recent years, laboratory-confirmed cases of cryptosporidiosis have become increasingly evident in the United States (Dietz & Roberts, 2000). According to one study, 17 percent to 32 percent of immunocompetent persons in Virginia, Texas, and Wisconsin, as well as immunocompetent Peace Corps volunteers (before travel), had serological evidence of Cryptosporidium infection by young adulthood (Guerrant, 1997).

The parasite has an oocyst stage that is highly resistant to drying and commonly used disinfectants. The infective dose may be as low as 30 oocysts in healthy adults with no evidence of previous infection (DuPont et al., 1995) but is probably less than 30 oocysts for immunocompromised persons. The most common modes of infection are ingestion of contaminated food or water and direct contact with infected persons, animals, or contaminated environmental surfaces (Desselberger, 2000).

Infection with Cryptosporidium is rarely fatal. Most infected persons are asymptomatic or, if symptomatic, recover with no therapeutic treatment. Symptoms may vary from a self-limiting diarrhea in healthy people to severe and occasionally fatal diarrhea in immunocompromised people, particularly among those with late-stage AIDS (Navin & Hardy, 1987). Diarrhea, when present, may be intermittent. Oocysts may be excreted for several weeks after the diarrhea has finally ceased, indicating that such persons could still be infectious.

In 2001, 3,785 cases of cryptosporidiosis were reported in the United States, of which 183 cases were from Ohio (Centers for Disease Control and Prevention [CDC], 2002). It has been estimated, however, that there may be as many as 300,000 cases of human cryptosporidiosis each year in the United States, 90 percent of which may be attributed to use of contaminated water (including filtered and chlorinated municipal supplies) or person-to-person transmission and 10 percent of which may be attributed to foodborne transmission (Mead et al., 1999). It has been estimated that between 65 and 97 percent of natural surface waters, ponds, lakes, rivers, streams, and so forth in the United States are contaminated with Cryptosporidium (Juranek, 1995). Surface water accounts for 62.5 percent of the public water supply in the United States (Solley & Pierce, 1995). As oocysts of Cryptosporidium are not easily killed by chlorination, there is a real potential for contamination of drinking-water systems in many areas in the United States (Ford & Mac Kenzie, 2000).

Between 1991 and 1998, 11 documented waterborne outbreaks of cryptosporidiosis in the United States involving an estimated 408,331 people (Craun, Nwachuku, Calderon, & Craun, 2002). Seven of the outbreaks occurred in community water systems, and two each occurred in non-community and individual water systems. The Milwaukee outbreak, which to date is the largest reported outbreak in the United States, accounted for 403,000 (98.7 percent) of the cases (Mackenzie et al., 1994). While the Milwaukee outbreak was caused by failure of one of the water filtration systems (Mackenzie et al.), contamination of groundwater and inadequate treatment of surface waters were responsible for 55 percent and 27 percent, respectively, of the documented outbreaks (Craun, Nwachuku, Calderon, & Craun, 2002).

In addition to drinking contaminated water, swimming or playing in contaminated surface water bodies was responsible for some of the outbreaks (CDC, 1998b & 1999; Kramer et al., 1998). In the period 1997-1998, nine outbreaks of cryptosporidiosis associated with recreational waters were reported (three each in Wisconsin and Minnesota, and one each in Oregon, Pennsylvania, and Florida) (CDC, 2000). It is important to note that coliforms were inadequate indicator organisms for the presence of Cryptosporidium and Giardia in water (Craun, Berger, & Calderon, 1997; Craun et al., 2002).

A single case of cryptosporidiosis may result in many people becoming infected in a short period of time. Because of the low infective dose and the very large numbers of oocysts typically excreted, it is possible, for example, for one fecal accident to contaminate an entire swimming pool with infectious material. In one outbreak, one child had a fecal accident in three different community swimming pools on three successive days; pool operators were unaware of the accidents until the child's mother reported them two weeks later (CDC, 2000). Two recent outbreaks, one involving 700 cases in Ohio and lasting three months, and another involving 225 people in Nebraska, were traced to infected people swimming in chlorinated waters. The outbreaks went unreported for several months, during which time ill people continued to use the pool (CDC, 2001). These outbreaks, and probably other unrecognized or unreported ones, show the significance of undiagnosed cases of cryptosporidiosis.

At the present time, C. parvum is not a significant cause of foodborne illness in the United States, accounting only for about 0.2 percent of the total foodborne illnesses occurring each year (Mead et al., 1999). Two recent outbreaks of cryptosporidiosis were caused by infected food handlers contaminating food (CDC, 1998a; Quiroz et al., 2000). Oocysts of C. parvum potentially infectious to humans were found in oysters from the Chesapeake Bay area (Fayer et al., 1999), indicating that eating raw or inadequately cooked contaminated shellfish may cause cryptosporidiosis.

While cryptosporidiosis is a reportable disease in the United States, it may be under-diagnosed and under-reported. In a 1999 nation-wide survey of clinical laboratories, only 27 percent of respondents reported including testing for cryptosporidiosis in their routine examination of feces for parasites (Kehl, 2000). For the five-year period 1997-2001, the numbers of reported cryptosporidiosis cases for the area addressed by the present study--three Ohio counties and one Michigan county around Toledo, Ohio, area--were very low and are shown in Table 1. A telephone survey of seven area clinical laboratories revealed that only one (a small hospital) routinely tests for Cryptosporidium in stool samples submitted for parasitology. The other laboratories still are not testing for Cryptosporidium unless the test is specifically requested by the ordering physician, and they are not seeing any increase in the number of physicians' requests. Two of the laboratories, both in Toledo, did indicate that they have increased the efficiency of their reporting of all reportable diseases to the appropriate health department. The authors propose that cryptosporidiosis is under-suspected, under-diagnosed, and under-reported in the Toledo area.

Between October 1999 and February 2001, for an incidence survey and a comparison of diagnostic procedures, the authors obtained 1,404 human stool specimens from two clinical laboratories in the Toledo area. The subjects had diarrheal diseases, and the stools were submitted to the laboratories for parasitology testing, or culturing for enteric pathogenic bacteria, or both. The specimens had not previously been tested for Cryptosporidium. Out of 1,404 samples, nine (0.64 percent) were positive for Cryptosporidium by the enzyme immunoassay (EIA) test (Harrington & Kassa, 2002). The immune status of the patients, or how they acquired the infections, is unknown.

Cryptosporidium has been associated with diseases in many animal species as well as in humans (O'Donoghue, 1995). Currently six to eight Cryptosporidium species are recognized; C. parvum is the most widespread and important cause of cryptosporidiosis in mammals, including in humans (Mosier & Oberst, 2000). Two distinct genotypes of C. parvum have been identified in humans: Genotype 1 (human genotype) observed only in human patients and proposed to be transmitted from humans to humans, and Genotype 2 (bovine genotype), observed in both calves and human patients and proposed to be transmitted from cattle to humans (Peng et al., 1997).

Recently, viable oocysts of Cryptosporidium parvum (bovine genotype) were isolated from feces of Canada geese (Graczyk et al., 1998), and the authors suggested that Canada geese might disseminate infectious oocysts of C. parvum through their feces into public water sources. Drinking water contaminated with animal feces was suggested as a likely source for a recent outbreak of cryptosporidiosis in an English community (Howe et al., 2002), but the study did not identify the animal source.

Most Canada geese populations are migratory, wintering in the United States and migrating to their breeding grounds in Canada during the summer; however, with the availability of suitable habitats such as grassy areas next to bodies of water in urban and suburban areas, increasing numbers of Canada geese have become year-round residents in the United States (U.S. Fish & Wildlife Service, 2002). At the present time, 3.5 million Canada geese are resident in the United States. Over the last 10 years, the number of resident Canada geese populations has increased in the Atlantic and Mississippi flyways by 14 and 6 percent respectively (U.S. Fish & Wildlife Service, 2002). In Ohio, Canada geese nest in all 88 counties, and their numbers have increased from an estimated 18,000 in 1979 to an estimated 120,000 in 2001, a 500 percent increase in just two decades (Ohio Department of Natural Resources, 2002). The increase is attributed to the proliferation in urban and suburban areas of office buildings and apartment complexes with ponds and mowed landscapes. Feces of Canada geese also litter the ground in many public areas such as parks, golf courses, cemeteries, and residential areas. Since Canada geese cohabit such environments, their feces can be a potential source of infection to humans.

In 1999 the authors surveyed the area within a 20-mile radius of Toledo, Ohio, for potential Canada geese breeding sites that were close to a body of water and where people could easily come into contact with the geese (e.g., parks, golf courses, apartment or condominium complexes, cemeteries, campuses of teaching and health care facilities) and identified 24 such sites. Of the 24 sites, 16 had Canada geese. The authors collected fecal samples from Canada geese at the 16 sites and analyzed the samples for two enteric protozoan parasites (Cryptosporidium and Giardia), and for an enteric pathogenic bacterium (Campylobacter). Cryptosporidium was found in feces at 86 percent of the sites (Kassa, Harrington, & Bisesi, 2001). Because of the high prevalence of Cryptosporidium in fecal samples collected in 1999, the authors conducted a follow-up study in 2000 and 2001, and the results are reported here.

Materials and Methods

In 2000 and 2001, the authors re-sampled some of the 1999 sites in Toledo, Ohio, and surrounding communities. The samples were collected between June and October of each year with each site being visited one or more times. Fecal samples were collected on the first day that the geese were observed on the site. In 2000 and 2001, fecal samples were collected from 11 and 10 sites, respectively, including from public parks, golf courses, cemeteries, and health care and teaching facilities. Each site was surveyed for geese droppings, and if droppings were present, 12 recently deposited feces of wet and loose appearance were collected, placed in sealable plastic bags, mixed, and compressed manually into one composite sample. This sample was then analyzed for Cryptosporidium-specific antigen (CSA) with the ProSpect[R] Microplate assay (Alexon-Trend, Inc., Ramsey, Minnesota). This solid-phase enzyme immunoassay (EIA) technique uses monoclonal antibodies with an analytical sensitivity for CSA of approximately 20 nanograms per milliliter (ng/mL). The assays were interpreted visually according to parameters and color charts supplied by the manufacturer of the assay kit.

Results

In 2000 and 2001, Canada geese were observed in 11 and 10, respectively, of the original 16 sites. The remaining sites had dried goose feces indicating that the birds might have been there at other times. The number of birds in each site ranged from a few dozen to several hundred. Two of the sites, in south Toledo, had the densest populations of geese, and the fecal droppings were spread over large areas. Often people--adults, young adolescents, and children--could be seen feeding the birds. In 2000, nine out of 11 sites (81.8 percent) were positive for Cryptosporidium. In 2001, nine out of 10 (90 percent) were positive. Two of the sites that were tested in 2000 were not tested in 2001 since there were no geese at these sites at the time of sample collection. Similarly, one site that was not tested in 2000 was included in 2001. The results are shown in Table 2.

Discussion

In areas where geese congregate, such as parks and golf courses, it is increasingly difficult to avoid contact with their feces. As has been noted before (Feare, Sanders, Blasco, & Bishop, 1999), Canada geese range further from water to feed than other waterfowl and spread their feces over large areas of grass parkland. This use of cultivated grass parkland increases the health risk to people who would never have contact with contaminated surface waters directly for recreational purposes such as swimming, boating, and fishing. After the 1999 phase of the goose feces study, the authors concentrated on Cryptosporidium, knowing that it was rarely reported to health departments in the tri-county area and, unlike Giardia and Campylobacter, not routinely tested for by area laboratories.

Large flocks of Canada geese are seen in the Toledo area between June and October of each year. Most of these birds may be found in groups of a few dozen to several hundred birds in residential and public areas. Over 80 percent of the sites tested in this study were positive for Cryptosporidium. In addition, a site that is negative on one day may not remain negative over time, as flocks of geese move between sites. The authors sampled feces from 11 sites, but the number of sites utilized by Canada geese is far greater. This study involved composite samples at each site and so did not determine the prevalence of enteric pathogens in the flocks. Kostroff & Rollender (2001), however, examined 100 individual goose feces collected at two parks in New City, New York, and found by EIA that 81 percent were positive for Cryptosporidium and 74 percent for Giardia.

The finding of 0.64 percent incidence of Cryptosporidum in human stool specimens of patients within the Toledo area (Harrington & Kassa, 2002) is in line with results from other recent surveys--approximately 0.6 percent in Ontario, Canada (Palmer, Bleicher, Gandhi, & Young, 1997) and, in a wide geographical survey, less than 0.5 percent in adults and 0.9 percent in children (Kehl, 2000). Even though, in general, the rates are lower than those found for Giardia lamblia, in some regions of the United States, notably the Southeast and the Northwest, Cryptosporidium has been found to be more prevalent than Giardia (Kehl, 2000). Low rates should not discourage physicians and laboratories from including Cryptosporidium testing in cases of diarrhea/enteritis. The importance of undiagnosed cases has been well demonstrated in the swimming pool outbreaks referred to earlier (CDC, 2000, 2001). The importance of person-to-person transmission in households, hospitals, and daycare centers has recently been reviewed (Guerrant, 1997). The issue also has been discussed by CDC's Working Group on Waterborne Cryptosporidiosis (1997).

While most cases of human cryptosporidiosis were in the past associated with C. parvum (human genotype), recently the situation has become complicated; zoonotic types of Cryptosporidum have been found in the feces of infected persons. C. meleagridis (bird genotype) has been isolated from stools of immunocompromised people (Guyot et al., 2001; Pedraza-Diaz, Amar, & McLauchlin, 2000) as well as from immunocompetent persons (Xiao et al., 2001; Chalmers et al., 2002). C. felis (cat genotype) has been isolated from feces of infected children (Xiao et al., 2001). C. parvum (zoonotic genotype) has been isolated from infected children (Xiao et al.) and adults (Chalmers et al., 2002). In addition, Chalmers and co-authors report that two isolates from two patients did not match currently known species and genotypes of Cryptosporidium. One study has found C. parvum (deer genotype) in fecal specimens from humans diagnosed with clinical symptoms consistent with cryptosporidiosis (Ong et al., 2002). Thus, more than one genotype or species of Cryptosporidium can cause cryptosporidiosis in humans, and animals may be sources of Cryptosporidium infectious to humans.

Oocyst viability, and the species and genotypes of Cryptosporidium found in the goose feces were not determined in this study. Currently available immunoassays and nonspecific visual staining such as acid-fast stain do not determine viability or distinguish between species and genotypes. A previous study, however, found viable oocysts of C. parvum (zoonotic genotype) in the feces of Canada geese (Graczyk et al., 1998). Further research is required to define the public health importance of Cryptosporidium in feces of Canada geese and other wildlife (Jellison, Hemond, & Schauer, 2002; Perz & LeBlancq, 2001). The authors are working with CDC to determine the species and genotypes of Cryptosporidium in their goose feces collection.

Conclusion and Recommendation

Cryptosporidiosis is emerging as an important waterborne enteric disease in the United States. Small numbers of Cryptosporidium oocysts can cause infection in healthy people. Canada geese, which are becoming increasingly abundant in urban, suburban, and rural areas in the United States, are one source of Cryptosporidium and other enteric human pathogens. Infection could result from direct contact with infected geese or their droppings, or indirectly from environmental surfaces contaminated with feces of infected geese. So far, however, no known cases of human cryptosporidiosis have been associated with exposure to geese feces.

Since cryptosporidiosis is under-suspected and under-diagnosed in the United States, local health departments, in collaboration with state health officials, local hospitals, and laboratory personnel, should develop and implement procedures for the surveillance, detection, reporting, and prevention of cryptosporidiosis. Public health officials should recognize the potential health risks associated with feces of Canada geese. Local health departments should explain to the general public the importance of diagnosing the cause(s) of diarrhea. Swimming pool operators should educate the users of their pools in basic hygiene procedures and should develop policies for preventing potential infection from fecal accidents by children and other pool users.

TABLE 1 Cases of Cryptosporidiosis Reported to Local Health Departments
in Three Ohio Counties and One Michigan County, 1997-2001

County        1997    1998    1999    2000    2001    Total

Lucas, OH      1       1       0       5      20      27
Wood, OH       0       0       0       0       6       6
Ottawa, OH     0       0       0       0       0       0
Monroe, MI     0       0       0       0       0       0

Note: All the counties are located within a 20-mile radius of the City
of Toledo; Toledo, located in Lucas County, is the largest city in the
four counties.

TABLE 2 Results of Testing for Cryptosporidium at Sites with Fecal
Droppings of Canada Geese*

Site                                              2000        2001

Bowling Green State University, Golf Course,
  Bowling Green                                 Positive    Positive
Flower Hospital, Sylvania Township              Positive    Positive
Arlington/Detroit Ave. Pond, Toledo             Positive    Positive
Sylvania Memorial Cemetery, Sylvania Township   NT          Negative
Owens Community College, Toledo                 Negative    Positive
Detwiler Golf Course, Toledo                    Positive    NT
Toledo Botanical Garden, Toledo                 Positive    Positive
La Salle, Monroe, Michigan                      Positive    NT
Woodland Cemetery, Toledo                       Positive    Positive
University of Toledo Scott Park Campus, Toledo  Negative    Positive
Hecklinger Pond, Toledo                         Positive    Positive
Crane Creek Wildlife Refuge, Ottawa County      Positive    Positive

*Results were positive or negative as determined by the monoclonal EIA
method.
NT = not tested.

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Corresponding Author: Hailu Kassa, Department of Public & Allied Health, College of Health & Human Services, Bowling Green State University, Bowling Green, Ohio 43403. E-mail: [e-mail].

Hailu Kassa, Ph.D., M.P.H., M.S.O.H., R.S.

Brian J. Harrington, Ph.D., M.P.H.

Michael S. Bisesi, Ph.D., R.E.H.S., C.I.H.