Cryptosporidium parvum in oysters from commercial harvesting sites in the Chesapeake Bay.

Oocysts of Cryptosporidium parvum, a zoonotic waterborne pathogen, can be removed by bivalve molluscs from contaminated water and retained on gills and in hemolymph. We identified oocysts of C. parvum in oysters from seven sites in the Chesapeake Bay area. These findings document the presence of C. parvum infectious for humans in oysters intended for human consumption.

Oocysts of Cryptosporidium parvum from human feces can enter surface waters through wastewater, leaky septic tanks, or recreational activities. Oocysts from other mammals, including wildlife, pets, and livestock (especially neonatal ruminants) can enter surface waters either directly or through runoff. Oysters can remove C. parvum oocysts from artificially contaminated water and retain them in hemocytes, on gills, and within the body for at least 1 month (1). Oocysts retained for 1 week by oysters were still infectious, as determined by testing in mice (1). Oocysts of C. parvum were found in oysters collected from tributaries of the Chesapeake Bay, at six sites selected for proximity to wastewater outfalls and cattle farms where high levels of fecal contamination might be expected (2). We examined oysters at sites where oysters are harvested for human consumption to determine if C. parvum oocysts were present. Oocysts recovered from these oysters were examined to determine the possible sources of contamination through oocyst genotyping and to determine if the oocysts were infectious.

The Study

From 43 commercial oyster harvesting sites where the Maryland Department of Natural Resources makes routine annual collections, seven were selected to test for the presence of C. parvum oocysts (Table). Approximately 30 oysters were examined from each site on three occasions (Table). From each oyster, 3 to 5 ml of hemolymph was aspirated from the adductor muscle. All gill tissue from each oyster was excised and washed in 5 ml of PBS. For examination by immunofluorescence microscopy, 200 [micro]l of hemolymph and gill washing from each oyster was air dried overnight. Slides were stained with Merifluor fluorescein-labeled anti-Giardia and anti-Cryptosporidium monoclonal antibodies (Merifluor; Meridian Diagnostics, Cincinnati, OH) and examined with an epifluorescence microscope equipped with a fluorescein isothiocyanate-Texas Red dual wavelength filter. Specimens were considered positive when round bodies 4.5 to 5.5 [micro]m in diameter with distinct green fluorescing walls were identified.

Table. Identification of Cryptosporidium parvum oocysts recovered from oysters in the Chesapeake Bay

                       Bay
                     location            Fall 1997
                     or river                         Mice
Site   Location       system      IFA    PCR(a)   infectivity

A      Mt. Vernon    Wicomico    28(c)     ND        Neg(d)
        Wharf
B      Wetipquin     Nanticoke    29       BT         Neg
C      Halfway       Fishing      29       HT         Neg
        Mark          Bay
D      Beacon        Potomac      26       BT         ND
E      Holland       Patuxent     28       BT         Pos
        Point
F      Back Cove     Tangier      ND       ND         ND
                      Sound
       Old Woman's   Tangier      ND       ND         ND
       Leg            Sound

                            Winter 1998           Fall 1998
                                        Mice
Site   Location      IFA   PCR(a)   infectivity   IFA   Cp11

A      Mt. Vernon    15     BT          Pos        4     Pos
        Wharf
B      Wetipquin      3     Neg         Neg        8     Pos
C      Halfway        0     BT &        Neg        1     Pos
        Mark                HT
D      Beacon         ND    ND          ND         2     Pos
E      Holland        ND    ND          ND         1     Pos
         Point
F      Back Cove      2     BT          Neg        6     ND

       Old Woman's    0     BT          Neg        0     ND
       Leg

                           Fall 1998
                         Mice
Site   Location      infectivity   Water(b)

A      Mt. Vernon        Neg          ND
        Wharf
B      Wetipquin         Neg          79
C      Halfway           Neg          ND
        Mark
D      Beacon            Pos          10
E      Holland           Neg          31
        Point
F      Back Cove         Neg           8

       Old Woman's       Neg          ND
       Leg

(a) Polymerase chain reaction and restriction fragment-length polymorphism (PCR-RFLP) on small subunit rRNA gene, 18s.

(b) Number of oocysts recovered per liter of filtered bay water.

(c) Number of oysters found positive for oocysts out of 30 oysters examined from each site.

(d) Neg indicates that PCR using Cp11 primers failed to detect Cryptosporidium DNA in the DNA extracted from the ilea of mice that were intubated with pooled hemolymph and gill washings from oysters. HT, human genotype; BT, bovine genotype; ND, not done; IFA, immunofluorescent assay; PCR, polymerase chain reaction.

Hemolymph and gill washings from six oysters were pooled, resulting in five aliquots from each collection site. Pooled aliquots were tested for infectivity in mice and examined by polymerase chain reaction (PCR) for the presence of C. parvum-specific DNA.

Three hundred to 400 [micro]l of each of the five aliquots of pooled hemolymph and gill washings from each site was administered to each of four 7-to 10-day-old BALB/c mice by gastric intubation. Mice were necropsied 96 hours postinoculation, and 1 cm of terminal ileum was placed in DNA extraction buffer to obtain total DNA as described (3). Mouse ileum DNA (100-1,000 ng/ reaction) was analyzed for Cryptosporidium DNA by PCR, using CP11-P5 and CP11-P6 primers (3). The PCR products were analyzed by polyacrylamide gel electrophoresis and ethidium bromide staining, followed by image capture on a charge coupled device camera.

Pooled hemolymph and gill washings shipped to the Centers for Disease Control and Prevention within 1 week of collection were rinsed three times by repeatedly suspending in 10 ml sterile distilled water and centrifuging at 1,500 X g for 10 min. Supernate was decanted, and pelleted specimens were stored at 4 [degrees] C until subjected to five freeze-thaw cycles, followed by phenol-chloroform extraction to extract DNA. Purified DNA was dissolved in 50 [micro]l distilled water and stored at -20 [degrees] C until PCR analysis.

A small subunit rRNA gene-based, nested PCR and restriction fragment-length polymorphism (RFLP) technique developed for species- and genotype-specific diagnosis of Cryptosporidium (4,5) was used to characterize oocysts from oysters.

To confirm PCR-RFLP results, all positive secondary PCR products were sequenced. Samples collected in the fall of 1998 were also assayed by nested CP11 PCR (Figure).

[Figure ILLUSTRATION OMITTED]

At least 50 L of water from each site was filtered by the membrane disk (393-mm diameter, 3-1am pore size, white SSWP [Millipore Corp., Bedford, MA]) method (7). After filtration, an elution protocol (Method 1622) was followed (7). To test the recovery efficiency of this method, 10-L samples of bay water were processed as above, except that four samples were spiked with [10.sup.5] and four others with [10.sup.6] purified C. parvum oocysts. The concentration of oocysts in Chesapeake Bay water (Table) was adjusted for the recovery efficiency of the membrane disk method.

Findings

During three collection periods, oocysts corresponding in size and shape to those of C. parvum and labeled with fluoresceinated anti-Cryptosporidium antibody were detected in oysters collected at six of seven sites (Table). These findings were confirmed in all but one case by positive PCR results for the 18s rRNA gene. Specimens from the one site at which oocysts were not detected by microscopy were found positive by PCR. The presence of oocysts in oysters obtained at the last collection period was confirmed by PCR for the CP11 gene sequence (GenBank accession no. AF124243).

PCR-RFLP testing for the 18s rRNA gene identified two genotypes of C. parvum in hemolymph and gill washings from oysters. All 18s PCR-positive specimens with the exception of those collected from Fishing Bay were bovine genotype. Of two specimens from that location, one contained human genotype alone and the other contained both genotypes.

Eighty aliquots of pooled hemolymph and gill washings were tested for infectivity in mice by PCR of mouse ileum (Table). Oocysts from sites E (2 of 5 aliquots), A (2 of 5 aliquots), and D (5 of 5 aliquots) were found to be infectious at all three collection periods. No other aliquots had positive PCR findings.

The CP11 gene nested PCR was performed on pooled samples, collected in October 1998 from five oyster beds; although no amplicons were observed for the outer primer set P5/P6, all beds were positive by nested PCR (Figure). The nested amplicons from specimens from sites A and C were sequenced and found to have a 99% homology with the C. parvum CP11 sequence (data not shown). As a control for the quality of DNA extracted from the oyster tissues, we used a PCR assay for the small subunit of the ribosomal RNA gene of Crossostrea virginica (Figure, Panel B). The amplification of this gene fragment from the oysters negative for Cryptosporidium by CP11 gene PCR (e.g., Figure, Panel A, sample 2) indicates that PCR-inhibitory substances were successfully removed by using our nucleic acid purification protocol. Accordingly, sample 2 can be considered a true negative for the presence of Cryptosporidium oocysts.

The mean recovery efficiency of the membrane disk filtration method was 71.1%, CV=13.3%. Oocysts were detected in water samples collected in fall 1998 from sites B, D, E, and F. The concentration of oocysts at each site ranged from 8 to 79 oocysts/L (Table), with a mean of 32 oocysts/L.

Conclusions

C. parvum oocysts were found in oysters collected from all seven commercial oyster harvesting sites sampled in the Chesapeake Bay. These findings confirm those of previous studies, in which oysters (1) and clams (8) acquired Cryptosporidium oocysts from artificially contaminated aquarium water, and oysters (2) and mussels (9) acquired oocysts in nature. Collectively, these findings establish that bivalve molluscs can effectively remove and retain oocysts of Cryptosporidium from feces-contaminated estuarine waters.

PCR-RFLP testing for the 18s rRNA gene identified two genotypes of C. parvum in hemolymph and gill washings from oysters. Although many species of migratory and residential waterfowl, as well as amphibians, reptiles, and numerous mammals, inhabit the drainage area of sites from which oysters were collected, only the human and bovine genotypes of C. parvum were recovered from the oysters.

Results from infectivity studies indicate that only three sites of 16 tested over three collection periods yielded oocysts that produced detectable infections in mice. Based on positive IFA and PCR findings, 16 collections contained C. parvum bovine genotype oocysts. The low rate of infectivity for mice may reflect the small number of oocysts that were administered to each mouse of a lack of infectivity due to age or unknown environmental effects.

Neither the age of the oocysts nor how long they may have been on land, in the water, or retained by the oysters could be determined. In a previous study, oysters retained oocysts for at least 1 month after exposure and the oocysts infected mice when tested 1 week after exposure (1). In this study, salinity values and water temperatures during the three successive collection periods (based on data recorded at site A) were 9.0, 6.0, and 15.0 ppt and 12.0, 9.0, and 16.0 [degrees] C, respectively. Oocysts suspended in 10 and 20 ppt artificial seawater at 20 [degrees] C retained infectivity for mice when held for 12 and 8 weeks, respectively (2). Therefore, freshly deposited oocysts at these sites could have retained infectivity for 2 to 3 months.

At all sites sampled, examination of gill washings and hemolymph by both IFA microscopy and PCR revealed the presence of C. parvum oocysts. This finding indicates that water at these sites contained human or animal feces when oysters were filtering and that oocysts excreted in those feces were acquired by the oysters. Because oocysts of this species are infectious for humans but can be rendered noninfectious by heating to temperatures above 72 [degrees] C (10), we recommend that oysters be cooked before being eaten, especially by persons with any type of immunodeficiency. Oocysts can also be rendered noninfectious by freezing at -20 [degrees] C for 24 hours (11), but because viral or bacterial pathogens might also be acquired by oysters from water contaminated with feces and can survive freezing, we recommend cooking rather than freezing.

Acknowledgments

We thank John Collier for assisting in the collection of oysters and Colleen Carpenter and Anjeli Sonstegard for technical assistance.

This study was funded in part by Maryland SeaGrant, R/ F-88 and funding from CDC's Food Safety Initiative.

References

(1.) Fayer R, Farley CA, Lewis EJ, Trout JM, Graczyk TK. Potential role of the Eastern oyster, Crassostrea virginica, in the epidemiology of Cryptosporidium parvum. Appl Environ Microbiol 1997;63:2086-8.

(2.) Fayer R, Graczyk TK, Lewis EJ, Trout JM, Farley CA. Survival of infectious Cryptosporidiumparvum oocysts in seawater and Eastern oysters (Crassostrea virginica) in the Chesapeake Bay. Appl Environ Microbiol 1998;64:1070-4.

(3.) Jenkins MC, Trout J, Fayer R. Development and application of an improved semiquantitative technique for detecting low-level Cryptosporidium parvum infections in mouse tissue using polymerase chain reaction. J Parasitol 1998;84:182-6.

(4.) Xiao L, Escalante L, Yang C, Sulaiman I, Escalante AA, Montali J, et al. Phylogenetic analysis of Cryptosporidium parasites based on the small subunit ribosomal RNA gene locus. Appl Environ Microbiol 1999;65:111578-83.

(5.) Xiao L, Morgan U, Limor J, Escalante AA, Arrowood M, Shulaw W, et al. Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Appl Environ Microbiol 1999;65:3386-96.

(6.) Littlewood DT, Ford SE, Fong D. Small subunit rRNA gene sequence of Crassostrea virginica (Gmelin) and a comparison with similar sequences from other bivalve molluscs. Nucleic Acids Res 1991;19:6048.

(7.) U.S. Environmental Protection Agency. Method 1622: Cryptosporidium in water by filtration/IM/FA. Washington: The Agency; 1997. EPA 821-R-97-023:51.

(8.) Graczyk TK, Fayer R, Cranfield MR, Conn DB. Recovery of waterborne Cryptosporidium parvum oocysts by freshwater benthic clams (Corbicula fluminea). Appl Environ Microbiol 1998;64:427-30.

(9.) Graczyk TK, Fayer R, Lewis EJ, Trout JM, Farley CA. Cryptosporidium oocysts in Bent mussels (Ischadium recurvum) in the Chesapeake Bay. Parasitol Res 1999;85:30-4.

(10.) Harp JA, Fayer R, Pesch BA, Jackkson GJ. Effect of pasteurization on infectivity of Cryptosporidium parvum oocysts in water and milk. Appl Environ Microbiol 1996;62:2866-8.

(11.) Fayer R, Nerad T. Effects of low temperatures on viability of Cryptosporidium parvum oocysts. Appl Environ Microbiol 1996;62:1431-3.

Dr. Fayer is a senior scientist at the U.S. Department of Agriculture's Agricultural Research Service, Beltsville, Maryland. His primary area of research is zoonotic diseases with an emphasis on foodborne and waterborne parasitic protozoa.

Ronald Fayer,(*) Earl J. Lewis,([dagger]) James M. Trout,(*) Thaddeus K. Graczyk,([double dagger]) Mark C. Jenkins,(*) James Higgins,(*) Lihua Xiao,([sections]) and Altaf A. Lal([sections])

(*) U.S. Department of Agriculture, Beltsville, Maryland, USA; ([dagger]) National Oceanic and Atmospheric Administration, Oxford, Maryland, USA; ([double dagger]) Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland, USA; and ([sections]) Centers for Disease Control and Prevention, Atlanta, Georgia, USA

Address for correspondence: Ronald Fayer, USDA, ARS, IDRL, 10300 Baltimore Avenue, Building 1040, Beltsville, MD 20705, USA; fax: [phone]; e-mail: [e-mail].