Quantitative assessment of viable Cryptosporidium parvum load in commercial oysters (Crassostrea virginica) in the Chesapeake Bay

Abstract

The epidemiological importance of increasing reports worldwide on Cryptosporidium contamination of oysters remains unknown in relation to foodborne cryptosporidiosis. Thirty market-size oysters (Crassostrea virginica), collected from each of 53 commercial harvesting sites in Chesapeake Bay, MD, were quantitatively tested in groups of six for Cryptosporidium sp. oocysts by immunofluorescent antibody (IFA). After IFA analysis, the samples were retrospectively retested for viable Cryptosporidium parvum oocysts by combined fluorescent in situ hybridization (FISH) and IFA. The mean cumulative numbers of Cryptosporidium sp. oocysts in six oysters (overall, 42.1+/-4.1) were significantly higher than in the numbers of viable C. parvum oocysts (overall, 28.0+/-2.9). Of 265 oyster groups, 221 (83.4%) contained viable C. parvum oocysts, and overall, from 10-32% (mean, 23%) of the total viable oocysts were identified in the hemolymph as distinct from gill washings. The amount of viable C. parvum oocysts was not related to oyster size or to the level of fecal coliforms at the sampling site. This study demonstrated that, although oysters are frequently contaminated with oocysts, the levels of viable oocysts may be too low to cause infection in healthy individuals. FISH assay for identification can be retrospectively applied to properly stored samples.

First report of Cryptosporidium spp. oocysts in oysters (Crassostrea rhizophorae) and cockles (Tivela mactroides) in Brazil

“…October (2005) Although this protozoan parasite does not reproduce in bivalve tissues, it can accumulate in high quantity, and oocysts retained in mollusk tissues may remain infectious (Fayer et al 1997;Freire-Santos et al 2001;Graczyk et al 2007). …”

Molecular markers and sentinel organisms for environmental monitoring

“…R ecovery of Cryptosporidium oocysts from environmental-matrices remains a technologically complex process, but even more challenging is subsequent species-specific identification and viability assessment of the oocysts (Graczyk et al, 2007b). Microscopy has a low sensitivity and requires a skilled microscopist.…”

“…Microscopy has a low sensitivity and requires a skilled microscopist. Immunofluorescent antibody (IFA) usually overestimates the Cryptosporidium load because IFA cross-reacts with other species of Cryptosporidium not virulent for humans, small unicellular algae, and non-viable oocysts (Graczyk et al, 2007b). PCR-based methods, although very sensitive and specific, do not allow for viability assessment, and are highly sensitive for massive amounts of PCR inhibitors present in environmental samples.…”

“…PCR-based methods, although very sensitive and specific, do not allow for viability assessment, and are highly sensitive for massive amounts of PCR inhibitors present in environmental samples. Vital dyes, e.g., DAPI/PI stains, can be used for viability assessment; however, these stains are not Cryptosporidium-specific and produces positive reactions with any organism that have nucleic acid (Graczyk et al, 2007b). There is currently no consensus on the best method for determining species/genotype of Cryptosporidium.…”

Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization

“…The multiplexed approach has been successfully applied to testing freshly collected environmental samples (19) and animal, i.e., bird, fecal samples (31). The RNA and DNA of pathogens in preserved samples is stable for years (1,20,35) which can potentially allow retrospective analyses. The multiplexed FISH assay (19,31) has not previously been used for archival, longterm formalin-preserved fecal samples, although FISH assay using a single E. bieneusi-specific probe has been used for formalin-fixed, paraffin-embedded duodenal biopsy (3,34).…”