information expected to be derived frorn the research.)The California coastal ecosystem is highly impacted by fecal pollution from sewage outfalls, agricultural runoff, and urban stormwater runoff. In the past, water quality monitoring has focused on toxins and bacterial coliform counts. However, this methodology does not address the public health threat of zoonotic protozoal parasites or allow for the identification of pollution sources.Cryptosporidium species are pathogenic protozoal parasites that are shed by both humans and animals, are endemic in the California livestock populations, have a low infective dose, and have environmentally stable oocysts that can be spread via contaminated water. The diarrheal disease cryptosporidiosis is especially threatening to the immunocompromised population such as AIDs patients. Cryptosporidium has been documented in wildlife along the California coast but no studies have yet investigated the magnitude and sources of this pathogen in the marine environment.Bivalves, such as clams and mussels, concentrate Cryptosporidium oocysts, thus acting as indicators of fecal pollution. Bivalves can be efficiently tested for Cryptosporidium using a Real-Time PCR technique that we recently developed. The assay can detect the DNA of a single organism and gives a quantitative result. This method can also be used to differentiate between Cryptosporidium species and genotypes from animal and human sources.Our preliminary studies show that Cryptosporidium DNA is detectable in wild bivalves collected off the California coast. We propose a two year study: the first year will sample bivalves at 20 sites along the coast to map out the hot spots of Cryptosporidium contamination. The second year will target the 10 most highly contaminated sites for further investigation into the probable sources of contamination, as indicated by the Cryptosporidium genotypes and the possible pollution sources located near the collection sites. The results of this study will be very valuable in watershed management and minimizing the impact of fecal pollutants on our coastal ecosystems

Miller, Woutrina

eScholarship - University of California

UC Office of the PresidentCoastal Environmental Quality InitiativeTitleCryptosporidium in Bivalves as Indicators of Fecal Pollution in the California Coastal EcosystemPermalinkhttps://escholarship.org/uc/item/3g8384wwAuthorMiller, WoutrinaPublication Date2004-11-29eScholarship.org Powered by the California Digital LibraryUniversity of California1COASTAL ENVIRONMENTAL QUALITY INITIATIVESTUDENT FELLOWSHIP AND RESEARCH SUPPORTPROJECT COMPLETION REPORTDATE    11/29/04PREPARED BY  Dr. Miller1. Institution:  University of California at Davis2. Title: “Cryptosporidium in Bivalves as Indicators of Fecal Pollution in the California Coastal Ecosystem”3. Graduate Student: Woutrina Miller (formerly Smith), DVM, MPVM, PhDAdvisor: Patricia Conrad, DVM, PhD4. Dates: 1/1/02 - 12/31/035. Hypotheses:1. Cryptosporidium species are present in bivalves collected at sites exposed to fecal contamination, including selected sites near sewage outfalls and agricultural runoff.2. Cryptosporidium genotypes will differ significantly in bivalves collected at sites exposed to human fecal contamination as compared to bivalves collected at sites contaminated with livestock feces.6. Objective:Test mussels collected from up to 20 nearshore marine study sites designated as ‘higher risk’ or ‘lower risk’ for fecal pollution based on the proximity of known sources of livestock runoff and human sewage along the central California coast.Final Progress Report:In order to evaluate mussels as bivalve bioindicators of fecal pollution with Cryptosporidiumspp., we compared molecular and immunologic Cryptosporidium detection methods for use on bivalve tissues in the laboratory before testing the 4800 mussels collected from coastal California study sites.  In our laboratory evaluation of a new real-time TaqMan polymerase chain reaction (PCR) system to amplify Cryptosporidium DNA from mussel hemolymph compared to conventional PCR systems, all PCR assays were able to detect a single Cryptosporidium oocyst spiked into 1 ml hemolymph samples. For gill wash and digestive tissues, the analytic sensitivity of TaqMan PCR was similar to the direct immunofluorescent antibody (DFA) test, detecting 100 or more oocysts.  The use of immunomagnetic separation (IMS) to concentrate oocysts from the tissues before DFA or PCR testing improved the sensitivity limit to 10 oocysts per sample.  In a tank exposure experiment in which mussels filtered C. parvum oocysts from inoculated sea water, IMS concentration followed by DFA analysis detected the most Cryptosporidium-positive mussel samples.  When wild mussels were tested with IMS-DFA and conventional PCR methods, the most Cryptosporidium-positive samples were detected by PCR of hemolymphsamples.  Because our goal was to screen wild mussels for Cryptosporidium spp., conventional PCR with DNA sequence analysis was used to identify Cryptosporidium genotypes in hemolymph from the 4800 wild mussels collected at the ‘higher risk’ and ‘lower risk’ coastal study sites for fecal pollution shown in Figure 1.2a.    b. c.  d.Fig. 1. Mussel collection sites located along the central California coast (a), with close up maps of the (b) northern, (c) middle, and (d) southern regions.  Map numbers represent the core mussel sampling sites, while letters represent subsites within estuarine regions.0 1 2 Kilometers NEWS######Estero BayMorro Bay# sample sitesMussel Collection Sites in the Morro Bay Region8ABCDE9SewerbuoyMonterey BaySalinas River######0 1 2 Kilometers NEWS# sample sitesMussel Collection Sites in the Moss Landing Region4ABCDEElkhorn SloughMossLanding0 1 2 3 4 5 KilometersNEWSPacific OceanTomales BayMussel Collection Sites in the Tomales Bay Region# sample sites####12 ABCBodega BayCALIFORNIABodega HeadTomales BaySanta CruzMoss LandingCarmelGranite CanyonSan SimeonMorro BayPacific Ocean0 20 40 KilometersNEWSMussel Collection Sites Along the Central California Coast###########Site Characterization Lower risk for fecal pathogens Higher risk for human fecal pathogens Higher risk for farm runoff pathogens#123456789Over the 3 year study, 156 batches of 30 mussels per site were tested for CryptosporidiumDNA, with 12% of all mussel batches testing positive.  Within Cryptosporidium-positive mussel batches, 1-4 mussels tested positive, and occasionally more than one genotype was detected in a mussel batch.  Table 1 shows Cryptosporidium mussel batch results from Year 1 of the study.  The fecal risk category, site identification, and Cryptosporidium genotypes detected in mussel hemolymph are indicated for the dry and wet season mussel collections.  Cryptosporidium 3parvum genotype 2 was detected in mussels from a higher risk site for human feces during the dry season, as well as in mussels collected from a higher risk site for livestock feces and from a lower impact site during the wet season.  Cryptosporidium parvum genotype 2 is shed in the feces of humans and many animal species.  At a higher risk site for livestock feces that is located in a nature reserve, C. felis was detected in mussel hemolymph during the dry and wet seasons.  Cryptosporidium felis is shed in the feces of wild and domestic cats and has not been reported previously in bivalve samples.  Additionally, a novel Cryptosporidium-like sequence, designated New-1, was identified at a higher risk site for livestock feces during the dry season.  Novel Cryptosporidium spp. may represent as yet unrecognized genotypes shed in the feces of wildlife or other animals.  The Year 1 Cryptosporidium genotype results were very exciting and gave strong preliminary evidence of fecal contamination present at higher risk and lower risk sites for human and animal fecal exposure along the California coast.Table 1. Mussel testing for Cryptosporidium DNA in Year 1.DRY SEASON WET SEASON4B C. felis C. felis4D - a -9C New-1 b C.parvum gen. 23 nm c -5 nm -9A C.parvum gen. 2 -1 - nm6 nm C.parvum gen. 27 nm -Livestock ImpactedHuman ImpactedLower ImpactFECAL RISK CATEGORY SITE ID2001-2002a - = all mussels in batch Cryptosporidium -negative by PCR.b 'New' indicates a novel Cryptosporidium -like DNA sequence.c nm = no mussels collected.Cryptosporidium genotype results for Years 2 and 3 at the 9 higher risk and lower risk sites for fecal exposure are shown in Table 2.  Mussel batches were collected during the early and late wet and dry seasons for both years.  Cryptosporidium parvum genotype 2 was detected in mussels from a higher risk site for human feces during the early dry season of Year 2, and in mussels at a lower risk site in the early dry season of Year 3.  Novel Cryptosporidium-like DNA sequences, designated New-2 and New-3, were detected in mussels from higher risk sites for livestock feces and human feces during the wet season samplings, and again from a livestock impacted site in the early dry season.  A fourth unique DNA sequence, designated New-4, was detected in mussels from a higher risk site for human feces.  The New-3 Cryptosporidium-like sequences were detected in mussels collected from 2 sites separated by over 200 km.  Table 3 shows the Cryptosporidium genotype results for the estuarine subsites in Tomales Bay (Fig. 1b), Elkhorn Slough (Fig. 1c), and Morro Bay (Fig. 1d).  Table 3 lists the Cryptosporidium genotypes detected in mussels from the 3-4 subsites within each estuary, as well as 1 site outside of the estuary for comparison purposes.  None of the Tomales Bay subsites (2A-2C) tested positive for Cryptosporidium spp. during the study.  However, mussels collected from Elkhorn Slough and Morro Bay contained C. andersoni, shed in the feces of cattle, as well as novel Cryptosporidium spp.  The 4 subsites located within Elkhorn Slough (4A-4D) all tested positive for Cryptosporidium spp., but mussels collected from the site outside the slough (4E) were Cryptosporidium-negative at all timepoints.  In contrast, only 1 of the sites inside Morro Bay (9C) tested Cryptosporidium-positive, and the site located outside Morro Bay (9A) was also 4positive but at a different timepoint.  Detecting Cryptosporidium spp. in mussels within the estuaries but not outside the estuaries is consistent with fecal contamination entering the nearshore environment in the freshwater sources that feed into the estuaries, that are then further diluted in the open ocean outside the estuaries.Table 2. Mussel testing for Cryptosporidium DNA in Years 2-3.LATE DRY SEASONEARLY WET SEASONLATE WET SEASONEARLY DRY SEASONLATE DRY SEASONEARLY WET SEASONLATE WET SEASONEARLY DRY SEASON2A nm a - - - - - - -4A - b New-2 c New-3 - nm - - -9D - - - New-3 - - - -3 - - - C.parvum gen.2 - - - -5 - nm - - - New-4 - -8 - - New-3 - - - - nm1 - - nm - - - - -6 - - nm - - - - C.parvum gen. 27 - - - nm - - - -2003-2004Livestock ImpactedHuman ImpactedLower Impact2002-2003FECAL RISK CATEGORY SITE IDa nm = no mussels collected.b - = all mussels in batch Cryptosporidium -negative by PCR.c 'New' indicates a novel Cryptosporidium -like DNA sequence.Table 3. Mussel testing for Cryptosporidium DNA at estuarine subsites.SITE ID SUBSITE IDEARLY WET SEASONLATE WET SEASONEARLY WET SEASONLATE WET SEASON2 A - a - - -B nmb - - -C nm - - -4 A New-2 New-3 - -B C.andersoni , New-2 New-2 - -C - New-2 - -D - New-3 - -E - - - -9 A - - - New-4B - nm - -C C.andersoni New-2 - -D - - - -E nm - - nm(Tomales Bay)(Elkhorn Slough)(Morro Bay)2002-2003 2003-2004c 'New' indicates a novel Cryptosporidium -like DNA sequence.a - = all mussels in batch Cryptosporidium -negative by PCR.b nm = no mussels collected.This study was the first to evaluate bivalves on the Pacific coast for Cryptosporidium spp.  In an effort to better understand the advantages and limitations of using bivalves as bioindicators in nearshore waters, a risk factor analysis was performed to evaluate whether fecal risk category (higher or lower risk for fecal pollution), bivalve sampling season (early wet = December-February, late wet = March-May,  early dry = June-August, late dry = September-November), freshwater outflow exposure (low, medium, high), recent precipitation (in preceding 1, 7, or 30 days before bivalve collections), water type (estuarine or marine), and bivalve type (resident or transplanted mussels) were associated with detection of Cryptosporidium-positive mussel batches.  Table 4 shows the univariate analysis that evaluated the strength of association for each putative risk factor alone with the odds of detecting Cryptosporidium spp. in a mussel batch.  Fecal risk status was not associated with increased odds of detecting Cryptosporidium spp. in mussel batches.  However, mussel batches sampled in the late wet season, near medium or heavy freshwater outflow, and within 1 or 7 days of a precipitation event were more likely to contain Cryptosporidium spp. than mussel batches sampled at other times.  Water type and bivalve type 5were not significant explanatory variables for Cryptosporidium detection in mussels, suggesting that resident or transplanted mussels could be used to monitor for Cryptosporidium spp. in estuarine or marine environments.Table 4. Univariate logistic regression of risk factors for detection of Cryptosporidium spp. in musselsRisk factor GroupPercent mussel batches positive Odds ratio 95% CIOdds ratio P-valueFecal risk class Lower 8 (n=24) 1.00 - -Higher-Human 9 (n=34) 1.06 0.16-7.12 0.95Higher-Livestock 13 (n=98) 1.68 0.24-11.69 0.60Season Early Wet 9 (n=35) 1.00 - -Late Wet 23 (n=43) 3.23 1.35-7.76 0.01*Early Dry 8 (n=38) 0.91 0.16-5.28 0.92Late Dry 5 (n=40) 0.56 0.13-2.46 0.44Low 2 (n=85) 1.00 - -Medium  19 (n=53) 9.65 2.46-37.88 0.001*High 33 (n=18) 20.75 4.16-103.57 <0.001*No 10 (n=136) 1.00 - -Yes 25 (n=20) 3.15 0.80-12.47 0.10*No 7 (n=97) 1.00 - -Yes 19 (n=59) 2.95 1.23-7.02 0.02*No 4 (n=45) 1.00 - -Yes 14 (n=111) 3.62 0.64-20.51 0.15Water Estuarine 14 (n=90) 1.00 - -Marine 8 (n=66) 0.49 0.16-1.46 0.20Bivalves Resident 14 (n=78) 1.00 - -Transplant 9 (n=78) 0.60 0.19-1.91 0.39* Significant P-values <0.10.Freshwater outflowPrecipitation in past 7 daysPrecipitiation in past 1 dayPrecipitation in past 30 daysTable 5. Logistic regression model of significant risk factors for detection ofCryptosporidium spp. in mussels.Risk factor GroupAdjusted odds ratio 95% CIOdds ratio P-valueLow 1.00 - -Medium 10.84 2.54-46.22 0.001*High 14.85 3.30-66.64 <0.001*No 1.00 - -Yes 2.63 1.07-6.49 0.04** Significant P-values <0.10.Precipitation in past 7 daysFreshwater outflow6After univariate analysis was complete, a multivariable model was created to simultaneously evaluate the risk factors associated with detection of Cryptosporidium spp. in mussel batches (Table 5).  Mussel batches exposed to medium and high freshwater outflow were 10.84 and 14.85 times as likely to contain Cryptosporidium spp. as mussel batches exposed to low freshwater outflow in the day preceding mussel collection.  Similarly, mussel batches collected within 7 days of a precipitation event were 2.63 times as likely to contain Cryptosporidium DNA as mussels collected at other timepoints.  These findings suggest that mussels will be the most useful in monitoring for fecal pollution when outplanted near areas of high freshwater outflow and when collected with a week of a precipitation event.In conclusion, this study evaluated novel approaches for ecosystem monitoring using molecular Cryptosporidium detection techniques on bivalves that had filtered fecal pathogens from nearshore waters along the California coast .  Cryptosporidium spp. were detected in bivalves collected from sites considered at higher risk for livestock fecal contamination, at higher risk for human sewage contamination, and sites more than 5 km from these known fecal sources.  These findings show that Cryptosporidium spp. and fecal contamination are widespread in nearshore ecosystems in California.  The genotypes of Cryptosporidium  detected suggest that both cats and cattle are significant terrestrial sources of fecal loading.  This study demonstratesthe potential for water quality monitoring agencies to use bivalves as bioindicators of fecal contamination, in addition to their current use as bioindicators of pesticide and metal contamination in aquatic ecosystems.MAJOR ACCOMPLISHMENTS:- Development and validation of a real-time TaqMan polymerase chain reaction (PCR) system for quantitative detection of Cryptosporidium DNA in bivalve tissues.- Comparative stidy of the TaqMan PCR system with conventional PCR and direct immunofluorescent antibody (DFA) techniques, with and without immunomagnetic concentration (IMS) for sensitive and specific detection of Cryptosporidium spp. in bivalve tissues.- First evaluation of Cryptosporidium spp. present in wild mussels along the Pacific coast of the United States, as an indicator of fecal pollution in coastal ecosystems.- First report of C. felis and C. andersoni detected in bivalve tissues. - Detection of previously unreported novel Cryptosporidium spp. in wild bivalves.- First report of freshwater outflow and recent precipitation as significant risk factors for detection of Cryptosporidium in wild mussels along the Pacific coast.- This study shows that bivalves can be used as monitoring tools for fecal pathogens in estuarine and marine waters, which is consistent with our upstream studies that have detected Cryptosporidium and Giardia spp. in freshwater clams living in rivers that flowinto the Monterey Bay.77. Changes in income and personnel:No changes in income or personnel were made during the final year of the project.  Dr. Miller is now completing her dissertation and will receive her Ph.D. in December, 2004.8. Changes in scientific goals and anticipated schedule for project completion: No changes in scientific goals were made.  Project objectives were completed.9. a. Publications:1. Conrad, P.A., Miller, M., Kjemtrup, A.J., Smith, W., Gardner, I.A. The human-wildlife-domestic animal interface provides exciting research opportunities for parasitologists. Journal of Parasitology 89(Suppl.): S27-S36. 2003.2. Miller, W.A., Gardner, I.A., Atwill, E.R., Leutenegger, C.M., Miller, M.A., Hedrick, R.P, Melli, A.C., Barnes, N.M.,  Conrad, P.A. Evaluation of Methods for Improved Detection of Cryptosporidium Species in Mussels.  Submitted to Applied and Environmental Microbiology, 11/04.3. Miller, W.A., Miller, M.A., Gardner, I.A., Atwill, E.R.,  Harris, M.,  Ames, J.,  Jessup, D.A., Melli, A.C., Paradies, D.,  Worcester, K.,  Olin, P.,  Barnes, N.M.,  Conrad, P.A. Cryptosporidium Genotypes Detected in California Mussels (Mytilus Species).  In preparation to submit to the International Journal for Parasitology in 12/04.4. Miller, W.A. Cryptosporidium Species in Coastal California Ecosystems. Dissertation, University of California, Davis. 12/2004.b. Conference Presentations:1.  Miller, W.A., Miller, M.A., Gardner, I.A., Atwill, E.R., Harris, M., Ames, J., Jessup, D.A., Worcester, K., Paradies, D., Olin, P., Melli, A.C., Barnes, N.M., Conrad, P.A.  Mussels (Mytilus spp.) as bioindicators of fecal pollution with Cryptosporidium spp. in coastal California ecosystems.  Western Society for Naturalists Conference, Rohnert Park, California, 2004.2.  Miller, W.A., Miller, M.A., Gardner, I.A., Atwill, E.R., Harris, M., Ames, J., Jessup, D.A., Worcester, K., Paradies, D., Melli, A.C., Barnes, N.M., Conrad, P.A.  Cryptosporidiumepidemiology in fecal impacted coastal California ecosystems, using mussels (Mytilusspecies) as bioindicators. International Giardia and Cryptosporidium Congress, Amsterdam, Netherlands, 2004.3.  Conrad, P.A. Exciting Protozoological Discoveries at the Human-Wildlife-Domestic Animal Interface – American Veterinary Medical Association Conference,  Colorado; Society for Tropical Veterinary Medicine, Brazil; Institute for Parasitology,  School of Veterinary Medicine,  Bern, Switzerland, 2003.84.  Conrad, P.A.  Adventures and New Discoveries at the Wildlife-Domestic Animal-Human Interface. Schalm Lectureship, School of Veterinary Medicine, Davis, California, 2003.5.  Smith, W.A., Atwill, E.R., Gardner, I.A., Miller, M.A., Ames, J., Jang, S., Melli, A., Jessup, D.,  Conrad, P.A.  Sea otters, shellfish, and fecal pathogens: can we connect the dots?  Defenders of Wildlife Carnivore Conference, Monterey, California, 2002.6.  Smith, W.A., Miller, M.A., Gardner, I.A., Atwill, E.R., Jang, S., Jessup, D., Leutenegger, C.,  Melli, A., Conrad, P.A.  Shellfish as indicators of fecal borne protozoa and bacteria in the California marine ecosystem.  Wildlife Disease Association Annual Meeting, Humboldt, California, 2002.10. Public relations success story potential: In this study, bivalves collected along the California coast were shown to contain Cryptosporidium parasites for the first time.  The study was undertaken for 2 main reasons: 1) to evaluate mussels as bivalve bioindicators of fecal contamination for water quality and watershed management purposes; and 2) to evaluate the possibility that humans and animals consuming raw bivalve shellfish along the California coast may be ingesting significant doses of fecal pathogens known to cause clinical disease.  Laboratory and field studies demonstrated that mussels can be used as bioindicators of fecal pollution along the California coast and could be incorporated in water quality monitoring programs.  The detection of both host-specific and zoonotic Cryptosporidium genotypes helps to identify contributing sources of fecal contamination in coastal ecosystems, and supports the concept that humans and animals consuming raw bivalve shellfish may be ingesting significant doses of fecal pathogens.

Cryptosporidium in Bivalves as Indicators of Fecal Pollution in the California Coastal Ecosystem

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