Abstract
Cyclospora cayetanensis is a coccidian parasite that is associated with foodborne outbreaks of gastrointestinal illnesses. Raspberries have been implicated as a vehicle of infection in some of these outbreaks. Most of the molecular techniques used for the detection of parasites commonly use the 18s rRNA as a target gene, which is highly conserved. The conserved nature of the 18s rRNA gene among coccidia means that there is potential for cross-reactivity from primers intended to target this gene in C. cayetanensis with the same gene in related coccidia. This provides an additional challenge in developing a specific detection method. The aim of this study is to develop a new, more specific assay to detect C. cayetanensis in berry fruits. This new assay, targeting the internal transcribed spacer 1 (ITS-1) region, was tested on three different berry matrices: raspberries, blueberries, and strawberries. The new assay showed good efficiency (102%), linearity (r2 = 0.999), repeatability (standard deviation of Cq 0.2 (95% CI: 0.2, 0.3) and specificity for Cyclospora, with no cross-reactivity with related coccidia (Toxoplasma gondii, Eimeria mitis, Cystoisospora canis, and Cryptosporidium parvum) when tested in vitro. The method development was initially conducted using Cyclospora DNA only. After it was confirmed to have an acceptable performance, the method was evaluated using the oocysts of C. cayetanensis. The method was also improved by incorporating an internal control as a duplex in order to monitor PCR inhibition due to sample matrix components. The duplex assay also showed a good efficiency (100%) and linearity (r2 = 0.99). The results showed that the new assay has potential for standard use in food testing laboratories. Furthermore, results regarding important factors related to assay robustness are discussed.
Highlights
Cyclospora cayetanensis is a coccidian parasite that has been associated with extensive foodborne outbreaks of gastrointestinal disease
According to Centers for Disease Control and Prevention (CDC), 1065 laboratory-confirmed cases of cyclosporiasis from 40 states were recorded in spring/summer outbreaks in 2017, 384 laboratory-confirmed cases in 2016, 546 in 2015, and 304 in 20141
The results of experiment conducted to select an appropriate concentration of Alconox for the washing of blueberries indicated that there was no meaningful difference in Cq values for the two concentrations
Summary
Cyclospora cayetanensis is a coccidian parasite that has been associated with extensive foodborne outbreaks of gastrointestinal disease. The symptoms of cyclosporiasis include watery diarrhoea, nausea, loss of appetite, cramping, bloating, increased gas, weight loss, fatigue, and, less commonly, vomiting and low-grade fever. According to Centers for Disease Control and Prevention (CDC), 1065 laboratory-confirmed cases of cyclosporiasis from 40 states were recorded in spring/summer outbreaks in 2017, 384 laboratory-confirmed cases in 2016, 546 in 2015, and 304 in 20141. The number of cases showed a dramatic increase in 2018; as of 1 October, 2018, 2,299 laboratory-confirmed cases of cyclosporiasis had been reported from 33 states. Previous outbreaks of cyclosporiasis have been commonly associated with imported contaminated raspberries, cilantro, basil, mesclun lettuce, and snow peas (Chacin-Bonilla, 2017)
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