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    • The qPCR MCA is a screening assay designed to simultaneously

    2018-10-22

    The qPCR MCA is a screening assay designed to simultaneously detect the presence or absence of multiple protozoan oocyst species of human health importance in several matrices (Lalonde and Gajadhar, 2011; Lalonde et al., 2013), including leafy greens and berries, and therefore has several advantages over traditional species-specific microscopy methods. Definitive identification of oocysts by microscopy alone requires significant parasitological expertise and follow-up sporulation or molecular identification, and debris from produce washes may resemble oocysts and lead to false-positive results. The qPCR products can be sequenced directly to verify the species detected and provide further information on the zoonotic potential of the contaminated sample (Lalonde et al., 2013), which is information not always available from oocysts detected by microscopy alone. The universal primer set used with the assay may also detect unknown or emerging protozoan oocysts present in produce samples which may or may not be pathogenic for humans, but could indicate fecal contamination or poor hygienic practices during processing. Thus, when combined with the specific processing methods developed here, the qPCR MCA is a useful non-subjective, high-throughput tool for routine surveillance of berries and leafy greens for protozoan oocysts. The processing procedures optimized in this study could also be used together with a variety of other DNA-based protozoan oocyst detection methods depending on the purpose of the testing program.
    Conflict of interest
    Acknowledgments
    Introduction Numerous outbreaks of foodborne illness associated with protozoan Tacrolimus on fresh produce have been reported in recent years. During 2013–2014, over 900 people in the US were infected with Cyclospora cayetanensis in foodborne outbreaks linked to imported cilantro and salad mixes (CDC, 2014; Harvey, 2013). C. cayetanensis has been linked to sporadic outbreaks of gastrointestinal illness associated with the consumption of leafy greens and berry fruits in North America and Europe for over 20years (Dixon, 2015; Ortega and Sanchez, 2010). Toxoplasma gondii has recently been listed as the second most harmful foodborne pathogen in the US based on the disease burden calculated using the disability-adjusted life year (Scallan et al., 2015). Although the risk of T. gondii-contaminated water and meat are well established (Guo et al., 2015; Jones and Dubey, 2010), there are few reports of contamination of leafy greens with T. gondii oocysts (Lass et al., 2012). Waterborne, person-to-person, and animal-to-human transmission routes for Cryptosporidium spp. are commonly recognized, but the parasite is also regularly detected in produce surveys and linked to foodborne outbreaks (Dixon, 2015). Of the three protozoan parasites listed above, C. cayetanensis is the most common pathogen that is associated with contaminated fresh produce. Direct or indirect contamination of produce may occur at many points between the farm and consumer, through contaminated soil, manure, irrigation or wash water, equipment, or handlers (Gamble, 2015). The consumption of raw, pre-washed, ready-to-eat leafy greens is increasing and there is increased risk for transmission of foodborne parasites from these products since most post-harvest control measures such as washing or disinfection are aimed at reducing bacterial contamination and are ineffective in removing or inactivating the infective oocyst stage of coccidia including C. cayetanensis, Cryptosporidium spp., or T. gondii (Dixon, 2015; Gajadhar and Allen, 2004; Macpherson and Bidaisee, 2015). There is no single universally accepted “gold standard” method for the elution, isolation, and detection of multiple species of protozoan oocysts from fresh produce, due to differences in the properties of the various food matrices and parasites of concern. An ISO standard method for the detection and enumeration of Cryptosporidium and Giardia is now available; however, this method involves immunomagnetic separation and immunofluorescent microscopic detection assays and is only genus-specific with no capability for genetic characterization. There are no such assays currently available for C. cayetanensis, so detection is generally performed using standard microscopy and is limited by low sample throughput, and analyst subjectivity and fatigue (Traub and Cuttell, 2015). We have developed and optimized a method for isolation of protozoan oocysts from several types of leafy greens and berry fruits and developed and evaluated a qPCR melt curve analysis (qPCR MCA) assay for use in detecting a variety of protozoan oocysts in produce (Lalonde and Gajadhar, 2016), as well as human fecal samples (Lalonde and Gajadhar, 2011; Lalonde et al., 2013) The objective of this study was to estimate the incidence of C. cayetanensis, T. gondii, and Cryptosporidium spp. contamination in leafy greens available at retail outlets in Canada, using this validated qPCR MCA assay.