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Applied and Environmental Microbiology, June 2007, p. 4069-4070, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.00166-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Risk of Handling as a Route of Exposure to Infectious Waterborne Cryptosporidium parvum Oocysts via Atlantic Blue Crabs (Callinectes sapidus)

Thaddeus K. Graczyk,^1,2,3* Cynthia McOliver,¹ Ellen K. Silbergeld,¹ Leena Tamang,¹ and Jennifer D. Roberts⁴

Department of Environmental Health Sciences, Division of Environmental Health Engineering, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205,¹ Johns Hopkins Water and Public Health Center, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205,² Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205,³ ChemRisk, Inc., San Francisco, California 94105⁴

Received 23 January 2007/ Accepted 16 April 2007

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Commercial Atlantic blue crabs (Callinectes sapidus) were exposed to 2.0 x 10⁴ infectious waterborne oocysts of Cryptosporidium parvum. The study demonstrated that blue crabs can transfer C. parvum oocysts to persons involved in handling or preparing crabs and that they may contaminate other surfaces or products during storage.

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Cryptosporidium parvum is a human enteric pathogen that can be transmitted very efficiently via the fecal-oral route (i.e., autoinfection and person-to-person) and indirectly via contact with contaminated water, including consumption and recreational activities (5). Edible crabs may take up and retain human-virulent bacterial contaminants (1, 10, 11, 13) and organic and inorganic pollutants from ambient water and sediments at levels posing risks to consumers (12). There is no published information on contamination of crabs with Cryptosporidium, a waterborne pathogen commonly reported from coastal waters (3, 6). However, our previous study demonstrated mechanical passage of C. parvum oocysts via handling of fish caught in urban watersheds to the hands of recreational anglers (16). The purposes of the present study were to determine if commercially harvested Atlantic blue crabs (Callinectes sapidus), which are widely consumed, can serve as a vehicle for infectious waterborne oocysts of C. parvum and if the handling of crabs collected from Cryptosporidium-contaminated water can result in oocyst transfer to the handler's hands.

A 120-liter-capacity marine tank was filled with 4 liters of artificial seawater of 12-ppt salinity (9) to which 2.0 x 10⁴ C. parvum oocysts were added. The oocysts were tested and found to be infectious to neonatal BALB/c mice (6). C. parvum oocysts were eluted from 12 blue crabs (C. sapidus) purchased from a local market by sprinkling 0.5 liter of the eluting fluid (4) on the crabs' surfaces and then collecting all the fluid into a single plastic bottle. The crabs were alive and actively ventilating with their mouthpart appendages during the experiments. The crabs were left in the tank for 24 h, and then the oocysts were eluted from the individual surfaces of six randomly selected crabs as described above and the fluid was collected into six corresponding plastic bottles. The oocysts were eluted collectively from the surfaces of the remaining crabs, and the fluid was collected into a single plastic bottle. The crabs were handled by a single person, and the hands of that person were washed in a plastic ziplock bag (16) containing 0.5 liter of eluting fluid (4). The tank water was collected into a plastic container, and the tank was washed with 1 liter of the eluting fluid (4), which was added to the container. The samples were processed by a cellulose acetate membrane filter dissolution method (4), and the recovered material was tested by combined fluorescence in situ hybridization and a direct immunofluorescent antibody assay for C. parvum (7-9).

C. parvum oocysts were detected in the eluting fluid from crabs after the exposure in contaminated water, in the hand wash sample, and in the tank water (Table 1). Overall, 74.8% of the oocysts from the original inoculum were recovered through testing (Table 1). The numbers of C. parvum oocysts recovered individually from six crabs varied from 8.0 x 10² to 3.1 x 10², with a mean of 5.6 x 10². The data presented in Table 1 indicate that (i) on average, a single crab carried on its external surfaces approximately 7.6 x 10² oocysts (i.e., approximately 3.8% of the original inoculum); (ii) all 12 crabs collectively accumulated 9.2 x 10³ oocysts on their shells (i.e., 45.8% of the original inoculum); and (iii) approximately 10.4% of oocysts carried by the crabs ended up on the hands of a person who was handling these crabs during the experiment. The fraction of C. parvum oocysts retained by the crabs (45.8%, i.e., 9.2 x 10³ oocysts) was significantly higher (chi-square test; ² = 16.2, P < 0.001) than the fraction of oocysts that remained in the water (29.0%, i.e., 5.8 x 10³ oocysts) after the experiment (Table 1), thus demonstrating the uptake and retention effects.

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TABLE 1. Results of elution of Atlantic blue crabs exposed to Cryptosporidim parvum oocystsa

The present study raises a serious question concerning the safety of handling blue crabs from waters contaminated with Cryptosporidium, such as from coastal regions receiving wastewater effluents and agricultural runoff, particularly from dairy and beef cattle operations (3, 6). Cryptosporidium-contaminated edible crabs may not cause food-borne cryptosporidiosis via consumption, as the oocysts will most likely be inactivated by adequate steaming or cooking processes; however, handling such crabs will expose the persons involved in the handling and may also contaminate the areas where they are stored. Such epidemiological circumstances have been incriminated in Vibrio cholerae and Vibrio parahaemolyticus outbreaks caused by crabs destined for human consumption (14, 15, 17). Chesapeake Bay blue crabs, a major seafood item harvested from this region, have been shown to contain V. parahaemolyticus (2).

The study emphasizes the great potential for the spread of this pathogen via contamination of the crab storage and preparation areas and crab handlers. It also emphasizes a need for high hygiene standards to be maintained in facilities and restaurants that are cooking live crabs. Minor inattention to hygiene standards in handling edible crabs resulted in V. parahaemolyticus and V. cholerae outbreaks (14, 15, 17). After the harvest, blue crabs are usually stored alive for several days in a moist and low-temperature environment, which preserves the infectivity of potential Cryptosporidium oocysts. The present study indicates that handling and storage of edible crabs harvested from contaminated waters may represent an occupational health risk for cryptosporidiosis. Proper hand washing is effective in the removal of C. parvum oocysts (16).

Environmental pollution of coastal waters has shifted from a local problem to a global concern as agricultural and urban runoff and wastewater effluents intensify due to a steady growth of the human population, which, in turn, drives a higher demand for intensive seafood harvest and production (3). Approximately 60% of disease outbreaks and cases linked to seafood are due to unknown etiological agents with usually unexplained epidemiological circumstances (3). The present study demonstrated the potential of mechanical transmission of C. parvum oocysts from a common commercial seafood item such as blue crabs, which can then potentially result in an enteric disease via contamination rather than the actual crab consumption.

 
The study was supported by the Johns Hopkins Center in Urban Environmental Health (grant P30 ES03819), the Fulbright senior specialist fellowship (grant 2225 to T. K. Graczyk), the Johns Hopkins Faculty Research Innovation Fund, the Johns Hopkins Center for a Livable Future, and the University of the District of Columbia (grant GF4136F4201).

 
* Corresponding author. Mailing address: Johns Hopkins Bloomberg School of Public Health, Department of Environmental Health Sciences, 615 N. Wolfe Street, Baltimore, MD 21205. Phone: (410) 614-4984. Fax: (410) 955-0105. E-mail: tgraczyk{at}jhsph.edu

Published ahead of print on 20 April 2007.

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Applied and Environmental Microbiology, June 2007, p. 4069-4070, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.00166-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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• Xiao, L. (2009). Overview of Cryptosporidium Presentations at the 10th International Workshops on Opportunistic Protists. Eukaryot Cell 8: 429-436 [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Graczyk, T. K. Articles by Roberts, J. D. Search for Related Content PubMed PubMed Citation Articles by Graczyk, T. K. Articles by Roberts, J. D. Agricola Articles by Graczyk, T. K. Articles by Roberts, J. D.