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Applied and Environmental Microbiology, November 2007, p. 6910-6915, Vol. 73, No. 21
0099-2240/07/$08.00+0 doi:10.1128/AEM.01027-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Maximizing Recovery and Detection of Cryptosporidium parvum Oocysts from Spiked Eastern Oyster (Crassostrea virginica) Tissue Samples
Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205,1 Department of Environmental Health Sciences, Division of Environmental Health Engineering, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205,2 Johns Hopkins Center for Water and Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 212053
Received 8 May 2007/ Accepted 31 August 2007
Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.
* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 502-8952. Fax: (410) 955-0105. E-mail: agirouar{at}jhsph.edu
Published ahead of print on 7 September 2007.
Applied and Environmental Microbiology, November 2007, p. 6910-6915, Vol. 73, No. 21
0099-2240/07/$08.00+0 doi:10.1128/AEM.01027-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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