Determination of the Recovery Efficiency of Cryptosporidium Oocysts and Giardia Cysts from Seeded Bivalve Mollusks
National Institute for Public Health and the Environment, Laboratory for Zoonoses and Environmental Microbiology, P.O. Box 1, 3720 BA Bilthoven, The Netherlands. . Journal of food protection
(Impact Factor: 1.85).
01/2013; 76(1):93-8. DOI: 10.4315/0362-028X.JFP-12-326
The intestinal parasites Cryptosporidium and Giardia are transmitted by water and food and cause human gastroenteritis. Filter-feeding bivalve mollusks, such as oysters and mussels, filter large volumes of water and thus concentrate such pathogens, which makes these bivalves potential vectors of disease. To assess the risk of infection from consumption of contaminated bivalves, parasite numbers and parasite recovery data are required. A modified immunomagnetic separation (IMS) procedure was used to determine Cryptosporidium oocyst and Giardia cyst numbers in individually homogenized oysters (Crassostrea gigas) and mussels (Mytilus edulis). About 12% of the commercial bivalves were positive, with low (oo)cyst numbers per specimen. The recovery efficiency of the IMS procedure was systematically evaluated. Experiments included seeding of homogenized bivalves and whole animals with 100 to 1,000 (oo)cysts. Both seeding procedures yielded highly variable recovery rates. Median Cryptosporidium recoveries were 7.9 to 21% in oysters and 62% in mussels. Median Giardia recoveries were 10 to 25% in oysters and 110% in mussels. Giardia recovery was significantly higher than Cryptosporidium recovery. (Oo)cysts were less efficiently recovered from seeded whole animals than from seeded homogenates, with median Cryptosporidium recoveries of 5.3% in oysters and 45% in mussels and median Giardia recoveries of 4.0% in oysters and 82% in mussels. Both bivalve homogenate seeding and whole animal seeding yielded higher (oo)cyst recovery in mussels than in oysters, likely because of the presence of less shellfish tissue in IMS when analyzing the smaller mussels compared with the larger oysters, resulting in more efficient (oo)cyst extraction. The data generated in this study may be used in the quantitative assessment of the risk of infection with Cryptosporidium or Giardia associated with the consumption of raw bivalve mollusks. This information may be used for making risk management decisions.
Available from: Piotr Rzymski
- "The majority of studies of either freshwater or marine bivalves have concentrated only on Cryptosporidium detection (Palos Ladeiro et al., 2013; Willis et al., 2013), rarely on Giardia (Graczyk et al., 1999) or on both the previous mentioned protozoan parasites or combined with human-infectious microsporidia (Graczyk et al., 2004, 2008; Lucy et al., 2008; Schets et al., 2013). Therefore, in these investigations usually one (mainly IFA test) or different combinations of two or three specific diagnostic methods (i.e. "
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ABSTRACT: The presence of environmentally robust dispersive stages of intestinal protozoan parasites in waters represents an important public health threat since these pathogens have caused numerous outbreaks related to either drinking or recreational waters. The purpose of this study was to evaluate the presence of Cryptosporidium oocysts, Giardia and Blastocystis cysts, and microsporidian spores in mussels collected from municipal reservoir, Lake Malta (Poland, Europe). Two species of freshwater bivalves (Anodonta anatina and Unio tumidus) were tested for the enteropathogens. A direct wet smear and smears stained with chromotrope 2R, Ziehl-Neelsen and iron hematoxylin made from each pellet of the hemolymph, gills and gastrointestinal homogenates of mussels were examined microscopically. In the study the immunofluorescence antibody test kit MERIFLUOR Cryptosporidium/Giardia was also used for all bivalve samples. None of investigated parasites were found in U. tumidus. In A. anatina, Cryptosporidium oocysts and Blastocystis cysts were detected in 15.4 and 5.1% of mussel samples, respectively. The present results indicate contamination of Lake Malta with Cryptosporidium and Blastocystis, which is important from the point of view of public health threats because of different human uses of studied reservoir.
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ABSTRACT: Cattle feces are the environmental vehicle for the zoonotic Cryptosporidium oocysts, but there are drawbacks associated with reliability of the existing methods for the detection of oocysts in the feces. Quantification of the immunomagnetic bead separation (IMS) coupled with real-time TaqMan PCR (qPCR) was accomplished by comparing the fluorescence signals obtained from the calf fecal samples of Cryptosporidium parvum oocysts with those obtained from standard dilutions of C. parvum oocysts. TaqMan qPCR assays were developed for the detection of C. parvum based on 18S rDNA gene. This IMS-qPCR assay allowed a reliable quantification of C. parvum oocysts over seven orders of magnitude with a baseline sensitivity of 8.7 oocysts. The newly developed IMS-qPCR technique proved specific as confirmed by negative reactivity against a wide panel of non-parvum Cryptosporidium oocysts. As a field application, experimentally infected calves (15 infected and 9 non-infected) were screened for oocysts shedding on 16, 18, and 21 days postinfection. Acid-fast staining microscopy of infected calves revealed oocysts in the feces of 11, 7, and 4 calves, respectively, compared to 15, 15, and 12 in case of screening by IMS-qPCR. Taken together, the proposed IMS-qPCR method significantly improved the diagnostic capacity for C. parvum infection in calves, making the technique a useful, sensitive, reliable, and time-saving.
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