Detection of Parisites in Produce Wash Water

November 2014

Cyclosporiasis is a parasitic foodborne disease that has been associated in the U.S. with the consumption of fresh produce. Since the parasite was first described in 1992, multiple large outbreaks have occurred. In the 1990s, outbreaks were primarily linked to the consumption of imported berries. Regulatory changes for imported berries resulted in an initial decrease in the number and frequency of outbreaks. However, major multistate outbreaks have recently occurred.

In 2013, more than 640 cases of cyclosporiasis were reported in two outbreaks that spanned 25 states. The first outbreak occurred primarily in Iowa and Nebraska, where infections were associated with the consumption of imported salad mixes. A second large outbreak occurred in Texas and was associated with consumption of cilantro. In both outbreaks, fresh produce had been imported from Mexico. This past summer, another major outbreak affecting more than 310 people occurred in the U.S. As in 2013, infections were associated with the consumption of cilantro imported from Mexico.

Cyclospora cayetanensis is a parasite that has unique features. It has an environmentally resistant cyst wall, requires time in the environment before it forms oocysts and becomes infectious to humans, and does not have an animal reservoir. These characteristics make it important to determine where or when produce may become contaminated in order to develop strategies for its control. It is conceivable that contamination could occur during production, harvesting, or packaging. At various pre-harvest stages, produce can come in contact with contaminated soil or water. Contamination could also result from Cyclospora shed by infected people handling produce.

Testing methods to detect Cyclospora in environmental samples can be challenging. Surface and well waters are typically analyzed using the EPA methods; however, there is very limited information regarding the validation of methodologies for testing produce wash waters for parasites.

We tested produce wash water containing chlorine or peroxyacetic acid for the presence of Cyclospora and Cryptosporidium. To simulate water containing vegetable debris, carrots were grated and added to the wash water to give a turbidity of 20 to 60 NTUs. Water was then filtered using water-permeable bags with 300 ┬Ám pore size. Wash waters were then inoculated with Cryptosporidium or Cyclospora at concentrations of 10 or 50 oocysts per 10 L.

Wash water was subsequently processed using two methods: filtration (Envirocheck capsules) or flocculation using calcium carbonate. Detection of the oocysts present in the concentrates was done using immunomagnetic separation and immunofluorescence for Cryptosporidium oocysts and autofluorescence for Cyclospora. All samples were evaluated using specific nested PCR, targeting the 18 rRNA gene for both parasites.

Both methods could detect 10 and 50 oocysts. Flocculation recovered oocysts of both parasites consistently from low- and high-turbidity chlorinated rinse water but not from rinse water containing peroxyacetic acid, which has a low pH. Filtration, although less sensitive and less consistent compared with flocculation, could be used to test water at various pH values but the cost per test is significantly higher than that for flocculation. Another drawback of filtration is that filters quickly clog, thus limiting the volume of water that can be tested for each sample. Hence, the efficacy of methods for testing water containing high organic matter for the presence of parasites can vary and perhaps be more challenging than testing surface or well water used for irrigation and drinking purposes.