Surveillance of berries sold on the Norwegian market for parasite contamination using molecular methods.

The risk of foodborne parasite infection linked to the consumption of contaminated fresh produce has long been known. However, despite epidemiological links between the outbreaks and contaminated berries, few studies have assessed the magnitude of parasite contamination on fresh produce sold in Europe. The present study was aimed to address the knowledge gap on parasite contamination of berries sold in Norway. Samples of blueberries, strawberries, and raspberries were analysed by multiplex qPCR for detection of Echinococcus multilocularis, Toxoplasma gondii, and Cyclospora cayetanensis. In addition, a simplex qPCR method was employed for detecting contamination of the berries with Cryptosporidium spp. A total of 820 samples of berries, each of around 30 g (274 samples of blueberries, 276 samples of raspberries, and 270 samples of strawberries), were analysed. We found an overall occurrence of 2.9%, 6.6%, and 8.3% for T. gondii, C. cayetanensis, and Cryptosporidium spp., respectively, whereas E. multilocularis was not detected from any of the samples investigated. Strawberries and raspberries were most often contaminated with Cryptosporidium spp., whereas blueberries were contaminated mostly with C. cayetanensis. Detection of parasite contaminants on fresh berries indicates the need for a system to ensure the parasitological safety of fresh berries.

Contamination of fresh produce sold on the Italian market with Cyclospora cayetanensis and Echinococcus multilocularis.

To investigate the presence of Cyclospora cayetanensis, Toxoplasma gondii and Echinococcus spp. in fresh produce sold in Italy, 324 locally produced 'ready-to-eat' (RTE) mixed-salad packages belonging to three brands and 324 berries packages (blueberries and blackberries imported from Peru and Mexico, respectively, and raspberries grown in Italy) were purchased at retail. Nine individual packages from each of the six types of fresh produce were collected monthly for one year, and with the same produce pooled, this resulted in a total of 72 pools for the whole year. Using microscopy (FLOTAC), a Cyclospora-like oocyst was detected in a blueberry sample and a taeniid egg was detected in a RTE-salad sample. Molecular tools confirmed these to be C. cayetanensis and Echinococcus multilocularis, respectively. Toxoplasma gondii was not detected in any of the samples. This study shows for the first time in Europe that imported berries on the Italian market may be contaminated with C. cayetanensis and RTE salads grown in Italy with E. multilocularis. The results indicate a new epidemiological scenario and highlight that current management of fresh produce, locally produced or imported, does not ensure products are free from parasite contamination.

Removal of Parasite Transmission Stages from Berries Using Washing Procedures Suitable for Consumers.

Due to the delicate nature of berries and the reduced shelf-life once washed, producers usually do not wash berries. Therefore, consumers are expected to wash the berries prior to consumption, and this might be a more effective way of infection prevention. However, the efficacy of consumer berry-washing procedures in removing the parasite contaminants from the berries surface has not been investigated. The aim of the present study was, therefore, to compare the efficacy of three different washing techniques in removing parasite contaminants. Three alternatives to washing berries before consumption were compared on berries artificially contaminated with oo/cysts of Cyclospora cayetanensis, Cryptosporidium parvum, and Giardia duodenalis. The results show that simple washing of berries under the cold tap for 1 min could remove on average at least 80% of the parasites, except for C. cayetanensis, which seems to be stickier than both G. duodenalis and C. parvum. The percent removal was slightly lower for raspberries as compared to blueberries. Although the differences are expected, a relevant result of the study is that washing contaminated berries prior to consumption by the consumer removes a considerable proportion of parasites and thereby lowers the risk of ingesting parasites' transmission stages.

A New Protocol for Molecular Detection of Cyclospora cayetanensis as Contaminants of Berry Fruits.

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 (r 2 = 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 (r 2 = 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.