Cryptosporidium and Giardia in commercial and non-commercial oysters (Crassostrea gigas) and water from the Oosterschelde, the Netherlands

International Journal of Food Microbiology 113 (2007) 189 – 194 www.elsevier.com/locate/ijfoodmicro

Cryptosporidium and Giardia in commercial and non-commercial oysters (Crassostrea gigas) and water from the Oosterschelde, the Netherlands Franciska M. Schets ⁎, Harold H.J.L. van den Berg, George B. Engels, Willemijn J. Lodder, Ana Maria de Roda Husman National Institute for Public Health and the Environment, Microbiological Laboratory for Health Protection, P.O. Box 1, 3720 BA Bilthoven, The Netherlands Received 2 December 2005; received in revised form 16 February 2006; accepted 13 June 2006

Abstract The intestinal parasites Cryptosporidium and Giardia cause gastro-enteritis in humans and can be transmitted via contaminated water. Oysters are filter feeders that have been demonstrated to accumulate pathogens such as Salmonella, Vibrio, norovirus and Cryptosporidium from contaminated water and cause foodborne infections. Oysters are economically important shellfish that are generally consumed raw. Commercial and non-commercial oysters (Crassostrea gigas) and oyster culture water from the Oosterschelde, the Netherlands, were examined for the presence of Cryptosporidium oocysts and Giardia cysts. Nine of 133 (6.7%) oysters from two non-commercial harvesting sites contained Cryptosporidium, Giardia or both. Six of 46 (13.0%) commercial oysters harboured Cryptosporidium or Giardia in their intestines. Data on the viability of (oo)cysts recovered from Oosterschelde oysters were not obtained, however viable (oo)cysts were detected in surface waters that enter the Oosterschelde oyster harvesting areas. The detection of Cryptosporidium and Giardia in oysters destined for human consumption has implications for public health only when human pathogenic (oo)cysts that have preserved infectivity during their stay in a marine environment are present. Our data suggest that consumption of raw oysters from the Oosterschelde may occasionally lead to cases of gastro-intestinal illness. © 2006 Elsevier B.V. All rights reserved. Keywords: Cryptosporidium; Giardia; Shellfish; Oysters; Water; Detection

1. Introduction The intestinal parasites Cryptosporidium and Giardia cause gastro-enteritis in humans. Giardia infections are usually selflimiting with clearance within two to four weeks. They may be asymptomatic and chronic infections do occur, but when diagnosed, Giardia infections can be effectively treated (Marshall et al., 1997). In otherwise healthy individuals, symptoms of Cryptosporidium infections generally persist for one to two weeks but in immunocompromised persons infections can be chronic with diarrhoea being severe and life threatening (Arrowood, 1997). Waterborne transmission of Cryptosporidium oocysts and Giardia cysts is associated with consumption of contaminated drinking water and recreation in contaminated surface water or swimming pools (Fayer et al., 2000; Thompson, 2004). Many ⁎ Corresponding author. Fax: +31 30 2744434. E-mail address: [email protected] (F.M. Schets). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.06.031

water related outbreaks of cryptosporidiosis have been reported over the past years (Fayer, 2004; Fayer et al., 2004). Foodborne infections have been reported as the result of consumption of contaminated school milk (Gelletlie et al., 1997), fresh-pressed apple cider (Millard et al., 1994) and chicken salad (BesserWiek et al., 1996), whilst a survey in Norway demonstrated the presence of Cryptosporidium and Giardia on commercially available fruits and vegetables (Robertson and Gjerde, 2001). Bivalve shellfish are filter feeders that filter large volumes of seawater from which particles are extracted and concentrated. In the case of either human or animal faecal contamination of the seawater, waterborne pathogenic bacteria, viruses or protozoa like Cryptosporidium or Giardia are accumulated in the shellfish tissues. Raw consumption of these contaminated shellfish, which is preferred, can make them vectors of disease. Salmonella, Shigella, hepatitis A virus, norovirus and Vibrio have been frequently detected in shellfish and are known causes of many foodborne infections worldwide (Potasman et al., 2002). Consumption of raw oysters was linked to outbreaks of

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Vibrio parahaemolyticus infections (Wechsler et al., 1999; Lozano-León et al., 2003) and outbreaks of norovirus gastroenteritis (Doyle et al., 2004). Cryptosporidium oocysts have been detected in various filter feeding molluscan shellfish destined for human consumption, like mussels, cockles and clams (Freire-Santos et al., 2000; Gomez-Bautista et al., 2000; Gómez-Couso et al., 2003). Cryptosporidium oocysts were detected both in commercial (Fayer et al., 1999) and non-commercial (Fayer et al., 1998) oysters (Crassostrea virginica) from the Chesapeake Bay, Maryland, United States, whereas Giardia cysts were detected in clams from this site (Graczyk et al., 1999). A survey of oysters from commercial harvesting sites along the Atlantic coast in the Unites States and Canada showed a wide-spread but low prevalence of contamination with Cryptosporidium (Fayer et al., 2003). Gómez-Couso et al. (2003) demonstrated the presence of viable Cryptosporidium oocysts in oysters from commercial sites along the Spanish Atlantic coast (Galicia). Although Cryptosporidium and Giardia have been detected in shellfish, they have not been identified as the cause of shellfish related outbreaks of human cryptosporidiosis or giardiasis to date (Potasman et al., 2002). In the Netherlands, large oyster harvesting areas are located in the Oosterschelde. Effluent from several sewage treatment plants (STP) can enter this branch of the North Sea indirectly. Water flow patterns have shown that these effluents may reach and thus contaminate the oyster harvesting sites. Previous studies have shown that Cryptosporidium and Giardia may be detected in marine water close to primary sewage outfalls (Johnson et al., 1995). Cryptosporidium oocysts can survive in seawater long enough to be concentrated by filter feeders such as oysters. Robertson et al. (1992) demonstrated that Cryptosporidium oocysts immersed in natural seawater remained viable for 35 days, whereas Freire-Santos et al. (1999) showed that oocysts stored in artificial seawater with salinity of 35 ppt (parts per thousand) preserved viability and infectivity for 40 days.

Oocysts stored at 10 °C in artificial seawater (30 ppt) preserved infectivity for neonatal BALB/c mice for up to 12 weeks, when stored at 20 °C, infectivity was preserved for two weeks (Fayer et al., 1998). We examined the parasite load of both commercial and noncommercial oysters (Crassostrea gigas) harvested from the Oosterschelde and determined the presence of (viable) Cryptosporidium oocysts and Giardia cysts in sewage effluent, surface waters transporting the effluent to the Oosterschelde and the Oosterschelde water itself. 2. Materials and methods 2.1. Oyster collection and processing Oysters (C. gigas) were collected monthly from December 2000 to January 2002 from non-commercial oyster beds at the points of entry of effluents from the STP Tholen (10,000 population equivalents) and St. Maartensdijk (35,000 population equivalents) into the Oosterschelde (Fig. 1). Oysters destined for human consumption were obtained from an oyster farm in Yerseke, in small baskets ready for transport to the consumers. Whenever available, six oysters from each site were examined. The oyster shells were opened and the digestive tract and gills were carefully excised. From each individual oyster, the gill tissue was washed in a final volume of 40 ml Phosphate Buffered Saline (PBS, 0.01 M, pH 7.2) by horizontal agitation in a wrist action shaker for 10–15 min at 600 rpm. The gill tissue was discarded and the washing was sieved through a 70 μm mesh nylon cell strainer. The sieved fraction was centrifuged for 10 min at 1170 ×g, followed by aspiration of the supernatant and resuspension of the pellet in 100–200 μl PBS. The digestive tracts were homogenised by thoroughly squeezing and rubbing of the tissue in 2.5 ml PBS in a small plastic bag, followed by sieving through a 100 μm mesh nylon cell strainer.

Fig. 1. Situation of sewage treatment plants St. Maartensdijk (1) and Tholen (3), Oosterschelde sampling sites Pumping Engine (PE) Noord (2) and Lock Bergesdiep (4), surface water sampling sites Schelde Rijn canal (5) and ditch PE Noord (6), and commercial (★) and non-commercial ( ) oyster harvesting sites in the Oosterschelde, the Netherlands.

F.M. Schets et al. / International Journal of Food Microbiology 113 (2007) 189–194 Table 1 Cryptosporidium and Giardia in oysters from non-commercial (Lock Bergsediep and Pumping Engine (PE) Noord) and commercial (Yerseke) oyster harvesting sites in the Oosterschelde Sample site

Number Number positive examined Cryptosporidium Giardia Both Total Total %

Lock 65 Bergsediep PE Noord 68 Yerseke 46 Total 179 a b

1

1a

0

2

3.1

4 4 9

1a 2 4

2b 0 2

7 6 15

10.3 13.0 11.9

Presumptive Giardia cysts. Presumptive Giardia cysts in one of two.

2.2. Detection of Cryptosporidium oocysts and Giardia cysts in oyster tissue Concentrated gill washings and sieved digestive tract homogenates were pipetted onto multichamber slides (8 wells per slide; LabTek II, Life Technologies, Paisley, Scotland), 100 μl per well, one or two wells for gill washings, eight wells for digestive tract homogenates. Slides were dried with a medium warm hair dryer in about 1 h. After drying, slides were stained with fluorescein-isothiocyanate-labelled monoclonal antibodies: 75 μl of a 1:5 dilution of a Cryptosporidium/Giardia staining reagent (Cellabs, Brookvale, Australia) was added to each well. Slides were incubated at 37 °C for 45–60 min, followed by drying, removal of the chambers, mounting in Dabco-glycerol (2.0 g Dabco (1,4 diazabicyclo (2.2.2) octane, Sigma D-2522), 60 ml glycerol, 40 ml PBS) and sealing with colourless nail polish. The slides were stored at 2–8 °C in the dark and examined for the presence of Cryptosporidium oocysts and Giardia cysts with an epifluorescence microscope (Zeiss Axioskop) at magnification ×250. Presumptive (oo)cysts were confirmed by Nomarski Differential Interference Contrast (DIC) microscopy at magnification ×1000. 2.3. Water sampling and processing On most days of oyster collection, water samples were taken from the Oosterschelde off Pumping Engine (PE) Noord (STP St. Maartensdijk) and Lock Bergsediep (STP Tholen). The water temperature was recorded on site. Escherichia coli was enumerated according to ISO 9308-1 (Anonymous, 2000) using the Rapid Test. In November and December 2001 additional water samples for the detection of Cryptosporidium and Giardia were taken from the Oosterschelde off PE Noord and Lock Bergsediep (100 l), the effluent from STP St. Maartensdijk and Tholen (10 l) and from the surface waters transporting the effluent to the Oosterschelde (ditch PE Noord and Schelde Rijn canal (Fig. 1); 25 l). The samples were transported to the laboratory at ambient temperatures and processed within 24 h. Water samples were concentrated by using Envirochek HV filtration capsules (Pall Gelman Laboratory, Ann Arbor, USA) as described in ISO/DIS 15553 (Anonymous, 2004). Concen-

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trated samples were purified by IMS using the Dynal GCCombo system (Dynal Biotech ASA, Oslo, Norway) according to the manufacturers' instructions. The purified concentrate was transferred to a Dynal Spot-On slide, fixed with methanol and stained with 50 μl of a 1:5 dilution of the Cellabs Cryptosporidium/Giardia staining reagent for 30–45 min at 37 °C. Ten μl of a propidium iodide (PI) solution (1 mg/ml in PBS) was subsequently added and incubated for 2 min at room temperature. Slides were washed with PBS carefully and dried, mounted, sealed and examined as described above. 3. Results A total of 179 oysters from the Oosterschelde were examined for the presence of Cryptosporidium and Giardia (Table 1). Nine of 133 (6.7%) oysters from 2 non-commercial oyster harvesting sites in the Oosterschelde contained Cryptosporidium, Giardia or both. Six of 46 (13.0%) oysters meant for human consumption harboured Cryptosporidium oocysts or Giardia cysts in their intestines. All gill washings except one were negative. Positive gills originated from an oyster from the Oosterschelde off PE Noord and contained presumptive Giardia cysts, so-called since the detected cysts could not be unambiguously confirmed by DIC microscopy. In addition, three digestive tract homogenates contained presumptive Giardia cysts. Cryptosporidium oocysts and Giardia cysts were detected in declining concentrations in effluent samples from STP St. Maartensdijk and STP Tholen, through samples from surface waters transporting these effluents to the Oosterschelde, to samples from the Oosterschelde off PE Noord (Table 2). (Oo) cysts were not detected in any of the Oosterschelde samples taken off Lock Bergsediep. PI exclusion was recorded for all (oo)cysts detected in the concentrated water samples (Table 2). E. coli counts in the water samples varied between sites and months of sampling (Table 3), but were generally highest in the Oosterschelde of PE Noord. The majority of the positive oysters were found in the warmer months, although low water temperatures at the oyster harvesting site did not necessarily indicate the absence of contaminated oysters. The salinity of the

Table 2 The total concentration and the percentage propidium iodide (PI) positive (dead) Cryptosporidium oocysts and Giardia cysts in effluent from sewage treatment plant (STP) St. Maartensdijk and STP Tholen, surface water (ditch Pumping Engine (PE) Noord and Schelde Rijn canal) and the Oosterschelde (PE Noord and Lock Bergsediep) in November and December 2001 Sample site

STP St. Maartensdijk Ditch PE Noord PE Noord STP Tholen Schelde Rijn canal Lock Bergsediep n.a.: not applicable.

Cryptosporidium

Giardia

Nov. 2001

Dec. 2001

Nov. 2001

Dec. 2001

n/l

n/l

n/l

n/l

% PI+

% PI+

% PI+

% PI+

7.9

95

4.8

90

12.6

42

2.7

67

1.0 0.9 8.8 0.04 b0.04

82 76 93 0 n.a.

4.9 0.6 6.2 0.4 b0.01

46 25 81 88 n.a.

1.0 0.6 20.0 0.4 b0.04

14 35 72 33 n.a.

b0.04 b0.01 5.6 0.04 b0.01

n.a. n.a. 48 100 n.a.

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Table 3 Water temperature, average rainfall, Escherichia coli counts and number of Cryptosporidium and/or Giardia positive oysters observed at three different sampling sites in the Oosterschelde per month from December 2000 up to and including December 2001 Month

Dec. 2000 Jan. 2001 Feb. 2001 Mar. 2001 Apr. 2001 May 2001 Jun. 2001 Jul. 2001 Aug. 2001 Sep. 2001 Oct. 2001 Nov. 2001 Dec. 2001

Average a Average Lock water rainfall b Bergsediep temp. (mm) E. Positive (°C) coli oysters (n/l)

Pumping Harvesting Engine Noord area Yerseke E. coli (n/l)

Positive E. oysters coli (n/l)

Positive oysters

6.9

76

36

0

579

0

98

0

1.7

66

21

0

n.d.

0

14

1

5.6

79

28

0

82

0

7

0

6.5

70

7

0

n.d.

0

n.d.

1

8.1

75

n.d.

0

n.d.

1c

n.d.

0

10.6

34

n.d.

0

n.d.

0

n.d.

0

16.1

50

8

0

4

1

n.d.

0

19.6

71

n.d.

1

4

3

n.d.

3

22.1

108

n.d.

0

12

0

n.d.

0

14.8

177

21

1

38

2

17

1

14.9

55

8

0

8

0

n.d.

0

8.3

90

495

0

6050 0

n.d.

0

4.6

81

350

0

2400 0

n.d.

0

n.d.: not done. a Average of temperature at the three Oosterschelde sites. b www.knmi.nl. c Positive gill washing.

Oosterschelde water is continuously monitored by the National Institute for Coastal and Marine Management (Middelburg, the Netherlands), who kindly provided the data. Salinity is stable throughout the year within a range of 28–31 ppt. 4. Discussion Cryptosporidium oocysts and Giardia cysts were detected in both commercial and non-commercial C. gigas from the Oosterschelde. An average of approximately 12% of all oysters examined was positive. This is probably an underestimation of the true fraction of Oosterschelde oysters contaminated with these parasites, as a result of methodological shortcomings. Microscope slides for Cryptosporidium and Giardia detection were difficult to read due to the presence of thick layers of homogenised oyster tissue. Therefore (oo)cysts may have gone undetected because of masking. Fayer et al. (2002) observed that only 50% of gill washings seeded with 500 Cryptosporidium oocysts tested positive with an immunofluorescence assay, indicating a low sensitivity of this detection method. These results indicate that in our study we may have missed more than

50% of oysters contaminated with 500 oocysts or less because the digestive tract homogenates we analysed were more difficult to examine than gill washings. Although others recently reported the successful use of IMS for isolation of Cryptosporidium from oyster tissues (MacRae et al., 2005), IMS, done according to the manufacturers' instructions for concentrated water samples, did not perform well on our tissue homogenates and we refrained from using the method after several experiments. The obtained E. coli counts in water, although limited, do not indicate poorer water quality at the oyster harvesting areas than at the non-commercial oyster collection sites. However, bathing water quality monitoring demonstrated that the water quality at two official bathing sites close to the oyster harvesting site in Yerseke did not comply with mandatory and guideline values for faecal indicators during the 2001 bathing season (Anonymous, 2002) as required by the European Bathing Water Directive 76/160/EEC (Anonymous, 1976). Elevated levels of faecal indicators in the water in the oyster harvesting areas suggest higher levels of faecal contamination and may thus explain the high number of commercial oysters contaminated with Cryptosporidium and Giardia. Commercial oysters were however smaller than non-commercial oysters, with a smaller digestive tract, resulting in microscope slides that were easier to examine and in which masking of (oo)cysts may have played a less profound role. We did not observe a direct correlation between E. coli counts in water samples and the presence of Cryptosporidium and/or Giardia in oysters harvested from these waters. These findings confirm similar observations by Fayer et al. (1998). However, the Oosterschelde site at which Cryptosporidium and Giardia were detected in November and December 2001 (PE Noord) generally had the highest E. coli counts the sampling year through and yielded the highest number of positive oysters, indicating a link between faecal contamination of seawater and contamination of oysters with protozoan parasites. Freire-Santos et al. (2000) demonstrated a correlation between the faecal coliform contamination level of oyster tissues and the detection of Cryptosporidium in oysters; however, in their study, GómezCouso et al. (2003) did not observe such a correlation. E. coli numbers exceeding the European Union standard for live bivalve molluscs intended for immediate human consumption (b 230 E. coli per 100 g oyster tissue (Anonymous, 1991)) were detected in oysters from the Oosterschelde harvesting area Yerseke and Lock Bergsediep in September 2001 (LodderVerschoor et al., 2005). This coincided with the detection of Cryptosporidium and Giardia in oysters harvested from these sites. In September 2001 E. coli numbers in oysters from the Oosterschelde off PE Noord did not exceed the standards, but Cryptosporidium and Giardia were detected. September 2001 was an extremely wet month, with precipitation over two times as high as normal, which may have resulted in increased faecal contamination of surface water due to runoff from agricultural land and sewage outfall. Fayer et al. (2002) could relate the detection of Cryptosporidium in oysters to heavy rainfall, whereas Miller et al. (2005) found more Cryptosporidium positive mussels once they were collected within a week following a

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rainfall event. Heavy rainfall does not explain the elevated numbers of positive oysters found at all sampling sites in July 2001, in which precipitation was normal. Gómez-Couso et al. (2003) suggested an increase in the population due to tourists as an explanation for the observed increased levels of contamination by Cryptosporidium in oysters from harvesting sites along the Galician coast in Spain. The Oosterschelde is a popular area for water recreation attracting many tourists during the summer months. July 2001 was a hot and dry month, conditions that may have invited extra people to the region. However, the total number of positive oysters in our study may have been too small to observe clear correlation between the presence of Cryptosporidium and/or Giardia in oysters and other (environmental) factors. Oysters are economically important shellfish that are harvested commercially and preferentially consumed raw. The detection of Cryptosporidium and Giardia in oysters destined for human consumption has implications for public health only when human pathogenic (oo)cysts that have preserved infectivity during their stay in a marine environment are present in concentrations high enough to infect consumers. Human feeding trials have shown that the infectious dose for Cryptosporidium varies among isolates, but may be as low as 9 oocysts (Okhuysen et al., 1999). It has been shown that Cryptosporidium oocysts in seawater preserve viability (Robertson et al., 1992) and infectivity (Fayer et al., 1998; Freire-Santos et al., 1999) for several weeks, which is long enough for accumulation in oyster tissues. Freire-Santos et al. (2002) demonstrated that oocysts recovered from artificially contaminated oysters 31 days after exposure, were able to infect Swiss CD1 mice. Effluent from STP St. Maartensdijk and STP Tholen may reach the oyster harvesting areas in the Oosterschelde quicker than oocysts die-off, indicating that infectious Cryptosporidium oocysts that enter the Oosterschelde can be accumulated by oysters destined for human consumption. A variable fraction of Cryptosporidium oocysts and Giardia cysts from effluent, surface water and the Oosterschelde was PI positive (i.e. dead). The PI negative fraction, ranging from 5 to 100% for Cryptosporidium and from 0 to 86% for Giardia, may be viable, but will also include empty (oo)cysts. However, assuming that not all PI negative (oo)cysts are empty shells, it is likely that viable (oo)cysts enter the Oosterschelde and reach oyster harvesting areas. Molecular detection and typing of Cryptosporidium and Giardia in Oosterschelde oyster homogenates that were positive by fluorescence microscopy failed due to the presence of unknown and non-removable components that inhibited the PCRs. In future research recently published protocols that enabled PCR detection of Cryptosporidium and Giardia in shellfish (Gómez-Couso et al., 2004) will be used for processing of oyster tissue samples. Gómez-Couso et al. (2003) have demonstrated the presence of Cryptosporidium oocysts in commercial oysters from the Galician coast in Spain, but also from other European Union countries like Italy, the United Kingdom and Ireland. Our study adds information on the presence of both Cryptosporidium and Giardia in both commercial and non-commercial oysters from another European Union country, the Netherlands. The data

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suggest that oysters from the Oosterschelde, once eaten raw, may occasionally lead to cases of gastro-intestinal illness due to Cryptosporidium or Giardia. Acknowledgements This research was performed by the order and for the account of the Food Inspectorate of the Ministry of Health, Welfare and Sport (at present: Food and Consumer Product Safety Authority (VWA)), within the framework of project 244920 as a spin off project of project QLRT-1999-0634 which received financial support from the European Union. The authors thank the employees of the Ministry of Agriculture, Nature and Food Quality for oyster harvesting, H. van Pelt-Heerschap (Institute for Fisheries Research, IJmuiden, the Netherlands) for providing E. coli counts in oysters and F. Lodder-Verschoor for assisting oyster processing. References Anonymous, 1976. Guideline 76/160/EEG of the Council of 8 December 1975 Concerning the Quality of Bathing Water, PB L 31 of 5.2. Anonymous, 1991. Council Directive 91/492/EEC of 15 July 1991 laying down the health conditions for the production and the placing on the market of live bivalve molluscs. Official Journal of the European Communities. L 268, 1–14. Anonymous, 2000. Water Quality — Detection and Enumeration of Escherichia coli and Coliform Bacteria — Part 1: Membrane Filtration Method, ISO 9308-1. International Organization for Standardization, Geneva. Anonymous, 2002. Kwaliteit van het zwemwater (badseizoen 2001) (Quality of the Bathing Water (Bathing Season 2001)). Office for official publications of the European Communities, Luxembourg. ISBN 92-894-3195-4. (In Dutch). Anonymous, 2004. Water Quality — Isolation and Identification of Cryptosporidium Oocysts and Giardia Cysts from Water, ISO/DIS 15553, 2004-0512. International Organisation for Standardisation, Geneva. Arrowood, M.J., 1997. Diagnosis. In: Fayer, R. (Ed.), Cryptosporidium and Cryptosporidiosis. CRC Press, Boca Raton, USA, pp. 43–64. Besser-Wiek, J.W., Forfang, J., Hedberg, C.W., Korlath, J.A., Osterholm, M.T., Sterling, C.R., Garcia, L., 1996. Foodborne outbreak of diarrheal illness associated with Cryptosporidium parvum — Minnesota, 1995. Morbidity and Mortality Weekly Report 45, 783–784. Doyle, A., Barataud, D., Gallay, A., Thiolet, J.M., Le Guyager, S., Kohli, E., Vaillant, V., 2004. Norovirus foodborne outbreaks associated with the consumption of oysters from the Etang de Thau, France, December 2002. Euro Surveillance 9, 24–26. Fayer, R., 2004. Cryptosporidium: a water-borne zoonotic parasite. Veterinary Parasitology 126, 37–56. Fayer, R., Graczyk, T.K., Lewis, E.J., Trout, J.M., Farley, C.A., 1998. Survival of infectious Cryptosporidium parvum oocysts in seawater and Eastern oysters (Crassostrea virginica) in the Chesapeake Bay. Applied and Environmental Microbiology 64, 1070–1074. Fayer, R., Lewis, E.J., Trout, J.M., Graczyk, T.K., Jenkins, M.C., Higgins, J., Xiao, L., Lal, A.A., 1999. Cryptosporidium parvum in oysters form commercial harvesting sites in the Chesapeake Bay. Emerging Infectious Diseases 5, 706–710. Fayer, R., Morgan, U., Upton, S.J., 2000. Epidemiology of Cryptosporidium: transmission, detection and identification. International Journal for Parasitology 30, 1305–1322. Fayer, R., Trout, J.M., Lewis, E.J., Xiao, L., Lal, A., Jenkins, M.C., Graczyk, T.K., 2002. Temporal variability of Cryptosporidium in the Chesapeake Bay. Parasitology Research 88, 998–1003. Fayer, R., Trout, J.M., Lewis, E.J., Santin, M., Zhou, L., Lal, A.A., Xiao, L., 2003. Contamination of Atlantic coast commercial shellfish with Cryptosporidium. Parasitology Research 89, 141–145.

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