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Discovery Recovery Methods and Detection of food borne parasites

In 2013, more than 640 individuals acquired Cyclospora infection in the US. Illness was associated with consumption of imported salad mixes and cilantro that were commercially processed and rinsed. To date, there is no protocol that would allow testing of large volumes of water to detect small number of parasites in produce wash‐waters. Cryptosporidium parvum and Cyclospora cayetanensis are protozoan parasites causing gastrointestinal illness in humans and animals both immunocompetent and immunocompromised hosts. Cryptosporidium parvum are spherical, 4–5μm in size, it can be acquired when ingesting foods and drinking, or accidentally ingesting, water contaminated with feces containing Cryptosporidium oocysts. The illnesses are characterized by acute diarrhea, stomach cramps, nausea, vomiting, and weight loss. Cyclospora cayetanensis are spherical as well, it is 8–10μm in size, it can also be acquired by person‐to‐person transmission and when in contact with infected animals; it is also spread via the fecal‐oral route (the host must ingest something that had been contaminated with feces that contain infectious oocysts). For oocysts to be termed infectious, the oocyst has to be sporulated (produce or form spores). The related disease Cyclosporiasis is acquired by ingestion of water or foods contaminated with human feces containing Cyclospora oocysts. Water samples were collected from a wash site in the southern area of Georgia every other day. The objective of this study was to compare two methods for oocyst recovery and detection from carrot wash‐water. The carrots were grated and washed with the water sample. The carrot wash‐water was spiked with ten and fifty oocysts of Cryptosporidium and Cyclospora. Two types of produce wash solutions fresh water and chlorinated water used by the fresh produce industry were evaluated ten liters of wash water were flocculated or filtered and then concentrated by centrifuging. Cryptosporidium oocysts from the concentrated water sample was treated with immunomagnetic separation (IMS) and detected by immunofluorescence assay (IFA). Cyclospora oocysts were concentrated by discontinuous sucrose gradients and examined by Nomarski microscopy. All samples were further examined by nested Polymerase Chain Reaction (PCR) specific for each parasite. Though both methods effectively recovered parasites, our trial also suggests that filtration and flocculation does not completely remove Cryptosporidium and Cyclospora oocysts in the wash water. In conclusion, these methods can significantly contribute to environmental testing during outbreak investigations and surveillance studies.