APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/01/$04.0010 DOI: 10.1128/AEM.67.3.1097–1101.2001

Mar. 2001, p. 1097–1101 Vol. 67, No. 3

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of Cryptosporidium Oocysts inSamples of Raw Surface Water and WastewaterLIHUA XIAO,1* AJAIB SINGH,2 JOSEF LIMOR,1 THADDEUS K. GRACZYK,3

STEVE GRADUS,2 AND ALTAF LAL1

Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 303411; City of MilwaukeePublic Health Laboratories, Milwaukee, Wisconsin 532022; and Departments of Molecular Microbiology and

Immunology and Environmental Health Sciences, School of Hygiene and Public Health,Johns Hopkins University, Baltimore, Maryland 212053

Received 7 September 2000/Accepted 14 December 2000

Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate the human-pathogenic Cryptosporidium parasites from those that do not infect humans and to track the source ofCryptosporidium oocyst contamination in the environment. In this study, we used a small-subunit rRNA-basedPCR-restriction fragment length polymorphism (RFLP) technique to detect and characterize Cryptosporidiumoocysts in 55 samples of raw surface water collected from several areas in the United States and 49 samplesof raw wastewater collected from Milwaukee, Wis. Cryptosporidium parasites were detected in 25 surface watersamples and 12 raw wastewater samples. C. parvum human and bovine genotypes were the dominant Crypto-sporidium parasites in the surface water samples from sites where there was potential contamination byhumans and cattle, whereas C. andersoni was the most common parasite in wastewater. There may begeographic differences in the distribution of Cryptosporidium genotypes in surface water. The PCR-RFLPtechnique can be a useful alternative method for detection and differentiation of Cryptosporidium parasites inwater.

Consumption of contaminated water has been implicated asa major source of Cryptosporidium infection in various out-break investigations and case control studies (22, 24). Surveysconducted in several regions of the United States revealed thepresence of Cryptosporidium oocysts in 67 to 100% of waste-waters, 24 to 100% of surface waters, and 17 to 26.8% ofdrinking waters (11–13, 24). The identity and human-infectivepotential of these waterborne oocysts are not known, althoughit is likely that not all oocysts are from human-infecting Cryp-tosporidium species. Likewise, the source of oocyst contamina-tion is not clear. Farm animal and human sewage dischargesare generally considered the major sources of surface watercontamination with C. parvum (15). Because Cryptosporidiuminfection is common in wildlife, it is conceivable that wildlifecan also be a source of Cryptosporidium oocysts in water (24).

Currently, Cryptosporidium oocysts in environmental sam-ples are identified largely by an immunofluorescent assay afterconcentration by methods such as the ICR method or method1622/1623. Because the immunofluorescent assay detects oo-cysts of most Cryptosporidium spp., the species distribution ofCryptosporidium parasites in environmental samples cannot beassessed. Although many surface water samples contain Cryp-tosporidium oocysts, it is unlikely that all of these oocysts arefrom human-pathogenic species or genotypes, because onlyfive genotypes of Cryptosporidium parasites (the C. parvumhuman, bovine, and dog genotypes, C. meleagridis, and C. felis)have been explicitly found in humans so far (17, 18, 32). In-

formation on the source of C. parvum contamination is neces-sary for effective evaluation and selection of management prac-tices to reduce Cryptosporidium contamination of surface waterand the risk of cryptosporidiosis. Thus, identification of oocyststo species and strain levels is of public health importance.

The existence of host-adapted Cryptosporidium spp. and C.parvum genotypes makes it possible to develop species differ-entiation and genotyping tools to determine whether the Cryp-tosporidium oocysts found in water are from human-infectivespecies and to track the source of Cryptosporidium oocyst con-tamination in water (16, 32). One such tool, the small-subunit(SSU) rRNA-based nested PCR-restriction fragment lengthpolymorphism (RFLP) method, has been successfully used byus to differentiate Cryptosporidium species and C. parvum ge-notypes in clinical samples and storm water (29–31). In thisstudy, we evaluated the use of this technique for detection andcharacterization of Cryptosporidium oocysts in samples of rawsurface water and wastewater.

MATERIALS AND METHODS

Water samples and sample processing. Samples of raw surface water andwastewater were used in this study. Most of the surface water samples werecollected from the Milwaukee region of Lake Michigan, from rivers in Illinois,and from the Maryland portion of the Chesapeake Bay area. A few samples,however, were collected from rivers in Iowa, Missouri, and Texas (see Table 1).These samples were collected during 1999 and the first half of 2000. Samplesfrom the Chesapeake Bay area were collected from sites located near wastewaterdischarges (samples from Choptank River, Severn River, and Miles River) orbeef cattle farms (samples from Wye River, St. George’s Creek, and WicomicoRiver), which were adjacent to the river. The allowable amount of waste dis-charge was 3.6 million gallons per day (MGD) at the Choptank River dischargesite, 7.5 MGD at the Severn River discharge site, and 0.3 MGD at the MilesRiver discharge site. One sample from each of the sites was taken during thespring (May), summer (August), and fall (October) of 1999 to avoid seasonalfluctuations in oocyst contamination. Water samples were always taken down-

* Corresponding author. Mailing address: Division of Parasitic Dis-eases, Centers for Disease Control and Prevention, 4770 Buford High-way, Atlanta, GA 30341. Phone: (770) 488-4840. Fax: (770) 488-4454.E-mail: lax0@cdc.gov.

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stream (less than 1 mile) of cattle operations or wastewater discharges. Almostall surface water samples were collected during base flow. All wastewater sam-ples were collected from a wastewater treatment plant in Milwaukee, Wis.,during the period from April to July 2000.

Surface water samples were taken by filtering water through an Envirocheckfilter (Pall Gelman Laboratory, Ann Arbor, Mich.) or a membrane disk (diam-eter, 393 mm; pore size, 3 mm; Millipore Corp., Bedford, Mass.) using previouslydescribed procedures (4, 7) and method 1623 procedures recommended by theU.S. Environmental Protection Agency (27). Membrane disks were used for riverwater samples from the Chesapeake Bay area, whereas Envirocheck filters wereused for the rest of the surface water samples. In most cases the amount of waterfiltered was 50 liters when membrane disks were used and 10 liters or less whenEnvirocheck filters were used; the exceptions were samples from Lake Michigan,which were taken by filtering 50 to 60 liters of water (except for two samples)through Envirocheck filters. Wastewater was concentrated by centrifuging 10- or50-ml portions of grab samples of raw wastewater at 1,000 3 g for 10 min.Cryptosporidium oocysts in surface water and wastewater were further concen-trated and purified by immunomagnetic separation (IMS) by using magneticbeads coated with an anticryptosporidial monoclonal antibody (Dynal, Inc., LakeSuccess, N.Y.) and the manufacturer’s recommended procedures. Usually, onlyan aliquot of each surface water sample was processed by IMS because of thepresence of excessive sediment.

DNA extraction. IMS concentrates from surface water and wastewater wereused for DNA extraction. They were subjected to five freeze-thaw cycles, incu-bated with 1 mg of proteinase K (Sigma, St. Louis, Mo.) per ml at 56°C for atleast 1 h, and diluted with an equal volume of pure ethanol. Oocyst DNA wasextracted by passing the oocyst-ethanol suspension through QIAamp DNA Miniisolate columns (Qiagen, Valencia, Calif.). DNA was stored at 220°C before itwas used for PCR analysis.

PCR-RFLP analysis. Cryptosporidium oocysts in water samples were identifiedto the species and genotype levels by a previously described PCR-RFLP tech-nique (29, 30), except that a 3-bp correction was made in the sequence of thereverse primer for primary PCR. Each sample was analyzed at least three timesby the PCR-RFLP method by using different volumes of the DNA preparation(0.25, 0.5, and 1 ml) for PCR. Briefly, an approximately 1,325-bp PCR productwas amplified first in a primary PCR by using primers 59-TTCTAGAGCTAATACATGCG-39 and 59-CCCATTTCCTTCGAAACAGGA-39. A total of 35 cy-cles were carried out; each of these consisted of 94°C for 45 s, 55°C for 45 s, and72°C for 1 min. There was also an initial hot start at 94°C for 3 min and a finalextension at 72°C for 7 min. A secondary 826- to 864-bp PCR product (depend-ing on the isolate) was then amplified from 2 ml of the primary PCR mixture byusing primers 59-GGAAGGGTTGTATTTATTAGATAAAG-39 and 59-AAG

GAGTAAGGAACAACCTCCA-39. The cycling conditions were identical tothose used for the primary PCR.

To differentiate Cryptosporidium spp. and C. parvum genotypes, 20 ml of eachsecondary PCR product was digested in a 50-ml (total volume) reaction mixturecontaining 20 U of SspI (New England BioLabs, Beverly, Mass.) 20 U of VspI(GIBCO BRL, Grand Island, N.Y.) and 5 ml of the appropriate restriction bufferat 37°C for 1 h, as described previously (29, 30). Because C. andersoni and C.muris had identical SspI and VspI restriction patterns, they were differentiated bydigesting secondary PCR products with 20 U of DdeI (New England BioLabs) at37°C for 1 h under conditions recommended by the supplier. Digested productswere fractionated on a 2.0% agarose gel and visualized by ethidium bromidestaining. Cryptosporidium spp. and C. parvum genotypes were differentiated bytheir band patterns. All diagnoses were confirmed by RFLP analysis of addi-tional, independent PCR products from the same sample. The accuracy of theRFLP analysis technique was also examined by performing a sequencing analysisof the secondary PCR products with an ABI377 autosequencer (Perkin-Elmer,Foster City, Calif.).

RESULTS

A total of 55 surface water samples and 49 wastewater sam-ples were used in this study. Examination of IMS concentratesby PCR analysis indicated that 25 of the surface water samplesand 12 of the wastewater samples were positive for Cryptospo-ridium. When the surface water samples were examined, noneof the 13 samples from Lake Michigan contained Cryptospo-ridium, but 19 of 21 samples from rivers in the Chesapeake Bayarea were positive for Cryptosporidium (Tables 1 and 2).

Restriction analysis of secondary PCR products with SspIand VspI revealed the presence of C. parvum human and bo-vine genotypes, C. baileyi, and C. andersoni-C. muris in surfacewater samples and C. parvum human, bovine, and dog geno-types, C. felis, C. andersoni-C. muris, and an unknown Crypto-sporidium genotype in wastewater samples (Fig. 1A). C. ander-soni and C. muris were differentiated from each other by DdeIdigestion of the secondary PCR products. The PCR productsof C. andersoni yielded bands at 20, 156, 186, and 470 bp, andthree bands were visible on an agarose gel. In contrast, the

TABLE 1. Cryptosporidium genotypes in samples of surface water from various locations

Sample location Vol filtered(liters)

Totalno. of

samples

No. ofpositivesamples

Species and/or genotypes(s)a

Lake Michigan, Linnwood, Wis. 50.3–60 8 0Lake Michigan, San Benito, Wis. 10 2 0Lake Michigan, Howard, Wis. 50.0–63.1 3 0Aurora, Ill. 10 7 2 C. parvum human genotype and C. baileyi (1),

C. andersoni (1)Decatur, Ill. 10 2 0Iowa City, Iowa 20.8–24.2 2 1 C. andersoni (1)Kansas City, Mo. 4.6–10.0 3 1 C. andersoni (1)St. Louis, Mo. 10 1 1 C. andersoni (1)San Benito, Tex. 3.0–10.0 5 0Potomac River, Washington, D.C. 10 1 1 C. andersoni (1)Chesapeake Bay, Choptank River, Cambridge, Md. 50 3 2 C. parvum human and bovine genotypes (2)Chesapeake Bay, Severn River, Annapolis, Md. 50 3 3 C. parvum human and bovine genotypes (3)Chesapeake Bay, mouth of Severn River, Annapolis

South, Md.50 3 3 C. parvum human and bovine genotypes (3)

Chesapeake Bay, Miles River, St. Michael’s, Md. 50 3 3 C. parvum bovine genotype (2), C. parvumhuman and bovine genotypes (1)

Chesapeake Bay, Wye River, Pintail Point, Md. 50 3 3 C. parvum bovine genotype (3)Chesapeake Bay, St. George’s Creek, Lunderburg, Md. 50 3 3 C. parvum bovine genotype (3)Chesapeake Bay, Wicomico River, Mt. Vernon, Md. 50 3 2 C. parvum bovine genotype (2)

a Numbers in parentheses are numbers of samples positive for each genotype or species.

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PCR products of C. muris yielded bands at 20, 156, 186, 224,and 247 bp, and four bands were visible (Fig. 1B). Sequenceanalysis of all of the PCR products yielded DNA sequencesidentical to those which we previously obtained from humansor animals infected with C. parvum human, bovine, and doggenotypes, C. felis, C. andersoni, C. muris, and C. baileyi (datanot shown). The unknown Cryptosporidium genotype was iden-tical to a Cryptosporidium wildlife genotype (W3) which wepreviously identified in storm water (31).

C. parvum, (both human and bovine genotypes) was thepredominant Cryptosporidium sp. found in surface water; 10

samples contained the C. parvum human genotype, and 19samples contained the C. parvum bovine genotype. C. ander-soni was also detected at a moderate frequency (five samples)in surface water samples. With the exception of one sample,the C. parvum human genotype was found only in surface watersamples from the Chesapeake Bay area, along with the C.parvum bovine genotype (Table 1). In contrast, C. andersoniwas the major Cryptosporidium sp. found in wastewater, occur-ring in eight samples (Table 2). Many surface water and waste-water samples contained more than one Cryptosporidium ge-notype; this was especially true of surface water samples fromrivers in the Chesapeake Bay area (Tables 1 and 2).

DISCUSSION

Numerous attempts have been made to apply PCR tech-niques to detection of Cryptosporidium oocysts in water sam-ples (1–3, 6, 10, 14, 19–21, 23, 25, 26, 28). In most of thesestudies the workers used water seeded with Cryptosporidiumoocysts, and various degrees of success were reported. Onemajor obstacle is the presence of PCR inhibitors in water,which are coextracted with DNA and inhibit PCR amplifica-tion of the target DNA. This has greatly reduced the sensitivityof PCR detection of oocysts in various water samples. ThePCR inhibitors can be removed by IMS (8, 9). This practice ledto successful detection of Cryptosporidium oocysts in watersamples from the 1993 outbreak in Milwaukee, Wis., by aCryptosporidium genus-specific PCR technique (9) and togenotyping of C. parvum parasites in surface and filter backwashwater samples by an integrated cell culture-PCR technique (3).Results of the present study indicate that in conjunction withIMS, the SSU rRNA-based PCR-RFLP technique which wepreviously developed for differentiating Cryptosporidium spp.and C. parvum genotypes in clinical samples has the specificityand sensitivity needed for analysis of Cryptosporidium oocystsin water samples.

Eight Cryptosporidium parasites that commonly occur in hu-mans, farm animals, pets, or wildlife were found in surfacewater and wastewater samples used in this study. The highfrequency of detection of the C. parvum human and bovinegenotypes and C. andersoni (a gastric Cryptosporidium parasiteof juvenile and adult cattle) is congruent with the previoustheory that humans and farm animals are two major sources ofCryptosporidium oocyst contamination in surface water at lo-cations where this type of contamination potentially occurs (15,22). This is in contrast with Cryptosporidium parasites in stormrunoff water from a feral area, in which there is a high fre-quency of Cryptosporidium genotypes from wildlife (31). Theremay be geographic differences in Cryptosporidium oocyst con-

TABLE 2. Cryptosporidium genotypes in samples of raw wastewater from a wastewater treatment plant in Milwaukee

Month Sample vol(ml)

Total no. ofsamples

No. of positivesamples Species and/or genotypea

April 10 13 2 C. andersoni and C. muris (1), C. andersoni and C. parvum bovine genotype (1)May 10 15 2 C. andersoni (1), C. muris (1)June 10 12 6 C. andersoni (3), C. parvum dog genotype (1), C. felis (1), C. parvum human

genotype and C. andersoni (1)July 50 9 2 C. andersoni (1), unknown genotype (1)

a Numbers in parentheses are numbers of samples positive for each genotype or species.

FIG. 1. Genotyping of Cryptosporidium oocysts in water with a SSUrRNA-based PCR-RFLP technique. (A) Differentiation of Cryptospo-ridium spp. and C. parvum genotypes by digestion of the secondaryPCR products with SspI (upper panel) and VspI (lower panel). Lane 1,C. parvum human genotype (sample 574); lane 2, C. parvum bovinegenotype (sample 5F); lanes 3 and 4, C. parvum human and bovinegenotypes (samples 1F and 2F); lane 5, C. andersoni (sample 104); lane6, C. muris (sample 194); lane 7, C. parvum bovine genotype and C.andersoni (sample 163). (B) Differentiation of C. andersoni from C.muris by digestion of the secondary PCR products with DdeI. Lanes 1through 4 and 6 through 8, C. andersoni (samples 104, 99, 98, 641, 192,224, and 225); lane 5, C. muris (sample 194).

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tamination of surface water, because the Cryptosporidium oo-cyst detection rate for river water samples from the Chesa-peake Bay area was much higher than those for samples fromother areas, and no Cryptosporidium oocysts were detected inwater from Lake Michigan. This was expected, because thesampling sites in the Chesapeake Bay area were located nearpotential sources of contamination of water with Cryptospo-ridium oocysts (i.e., wastewater discharges and runoff fromlarge commercial cattle farms) (5). Three of six rivers exam-ined in this region frequently contained C. parvum humangenotype oocysts, a finding congruent with these samplingsites’ locations near wastewater discharges.

Although Cryptosporidium was not detected in surface waterfrom the Milwaukee portion of Lake Michigan and only 10 to50 ml of wastewater was examined for each sample, Cryptospo-ridium oocysts were detected in raw wastewater from Milwau-kee at a moderate frequency. The high rate of detection of C.andersoni in wastewater was probably the result of effluentsfrom cattle slaughterhouses in the city. This hypothesis is sup-ported by the fact that mature cattle are more likely to beinfected with C. andersoni than with the C. parvum bovinegenotype, which was detected in wastewater at a much lowerfrequency. The biggest slaughterhouse in the city processes1,800 beef cattle daily and drains its contents into the citysewage system after satisfying city specifications (amount offat, size of meat chunks, etc.). The slaughterhouse is less than5 miles upstream of the Jones Island wastewater treatmentplant, where we collected composite samples.

Likewise, the C. muris oocysts in wastewater were probablyfrom rodents, which are expected to be present in a wastewaterdistribution system in abundance. Urban runoff may also havebeen a contributing factor in Cryptosporidium oocysts in waste-water, because the C. parvum dog genotype, C. felis, and anunknown Cryptosporidium genotype from wildlife were alsodetected in wastewater at low frequencies. The low rate ofdetection of the C. parvum human genotype in wastewaterfrom a major metropolitan area is surprising, because it islikely that a major component of the wastewater in this area ishuman sewage. However, sampling was done during the periodfrom April to July, when the incidence of human cryptospori-diosis is generally low.

In summary, the results of this study show the usefulness ofthe SSU rRNA-based PCR-RFLP technique for differentiatingCryptosporidium spp. and the C. parvum genotype and fortracking Cryptosporidium contamination sources in water. Ex-tensive genotyping of water samples from various matrices(source water, finished water, wastewater, river storm water,combined sewer overflow) and environmental settings (feral,rural, urban, recreational) is needed in order to obtain a betterunderstanding of the distribution of Cryptosporidium spp. invarious waters, the human infection potential of waterborneCryptosporidium oocysts, and the contributions of humans,farm animals, companion animals, wildlife, and other factors,such as sanitation, wastewater discharge, agriculture, recre-ation, and weather, to Cryptosporidium oocyst contaminationof water in certain settings. Such information would be usefulfor scientific management of watersheds and for source waterprotection.

ACKNOWLEDGMENTS

This work was supported in part by an interagency agreement be-tween the Centers for Disease Control and Prevention and the U.S.Environmental Protection Agency (DW 75937984-01-1) and by Mary-land Sea Grant R/F-88.

We thank Birhane Dashew and Clem Ng for sample preparation andmicroscopy and Daniel Colley and Mary Bartlett for suggestions forimproving the manuscript.

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