Detection of Cyclospora, Cryptosporidium, and Giardia in Ready-to-Eat Packaged Leafy Greens in Ontario, Canada

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Numerous foodborne outbreaks of diarrheal illness associated with the consumption of produce contaminated with protozoan parasites have been reported in North America in recent years. The present study reports on the presence of Cyclospora, Cryptosporidium, and Giardia in precut salads and leafy greens purchased at retail in Ontario, Canada. A total of 544 retail samples were collected between April 2009 and March 2010 and included a variety of salad blends and individual leafy greens. Most of these products were grown in the United States, with some from Canada and Mexico. Parasites were eluted and concentrated before detection by PCR and immunofluorescence microscopy. DNA sequences were aligned with reference sequences in GenBank. Cyclospora spp. were identified by PCR-restriction fragment length polymorphism in nine (1.7 % ) samples and by DNA sequence analysis. Cryptosporidium spp. were identified in 32 (5.9%) samples; 29 were sequenced and aligned with the zoonotic species Cryptosporidium parvum. Giardia duodenalis was identified in 10 (1.8%) samples, and of the 9 samples successfully sequenced, 7 aligned with G. duodenalis assemblage B and 2 with assemblage A, both of which are also zoonotic. The presence of Cryptosporidium oocysts and Giardia cysts was confirmed in some of the PCR-positive samples using microscopy, while Cyclospora -like oocysts were observed in most of the Cyclospora PCR-positive samples. The relatively high prevalence of these parasites in packaged salads and leafy greens establishes a baseline for further studies and suggests a need for additional research with respect to the possible sources of contamination of these foods, the determination of parasite viability and virulence, and means to reduce foodborne transmission to humans.