Use of Oxidative Stress Responses to Determine the Efficacy of Inactivation Treatments on Cryptosporidium Oocysts.

Cryptosporidium oocysts are known for being very robust, and their prolonged survival in the environment has resulted in outbreaks of cryptosporidiosis associated with the consumption of contaminated water or food. Although inactivation methods used for drinking water treatment, such as UV irradiation, can inactivate Cryptosporidium oocysts, they are not necessarily suitable for use with other environmental matrices, such as food. In order to identify alternative ways to inactivate Cryptosporidium oocysts, improved methods for viability assessment are needed. Here we describe a proof of concept for a novel approach for determining how effective inactivation treatments are at killing pathogens, such as the parasite Cryptosporidium. RNA sequencing was used to identify potential up-regulated target genes induced by oxidative stress, and a reverse transcription quantitative PCR (RT-qPCR) protocol was developed to assess their up-regulation following exposure to different induction treatments. Accordingly, RT-qPCR protocols targeting thioredoxin and Cryptosporidium oocyst wall protein 7 (COWP7) genes were evaluated on mixtures of viable and inactivated oocysts, and on oocysts subjected to various potential inactivation treatments such as freezing and chlorination. The results from the present proof-of-concept experiments indicate that this could be a useful tool in efforts towards assessing potential technologies for inactivating Cryptosporidium in different environmental matrices. Furthermore, this approach could also be used for similar investigations with other pathogens.

Multiplex Quantitative PCR Analysis of Strawberries from Bogotá, Colombia, for Contamination with Three Parasites.

ABSTRACT: Berries are potential vehicles for the transmission of parasites and have been implicated in illness outbreaks in various countries around the world, particularly in the United States. Although data on contamination of fresh produce with foodborne parasites have been obtained from various global regions, data from Colombia are lacking even though South American countries are major producers of fresh produce, which is both consumed nationally and exported. In this study, we used a previously published multiplex quantitative PCR approach to investigate contamination of strawberries purchased in either supermarkets or local markets in 20 localities. Strawberries were washed in a detergent solution after purchase, and the eluate was concentrated and sent to Norway for molecular analysis. Of the 120 strawberry samples analyzed, wash eluate from 6 samples (5%) tested positive for Toxoplasma gondii DNA, and 1 sample (0.83%) was positive for Cyclospora cayetanensis DNA. These results indicate that strawberries for sale in Bogotá, Colombia, may be contaminated with T. gondii and C. cayetanensis and, therefore, could act as transmission vehicles for these parasites. These data also indicate that cat and human fecal contamination of the strawberries has occurred at some point in their production, transportation, or storage. These findings highlight the need for a hazard analysis critical control point investigation of the strawberry production chain and implementation of measures to reduce the risk of strawberry contamination, thereby minimizing the risk of transmission of parasitic infection via these fruits, which are usually consumed raw.

Comparative evaluation of UNEX-based DNA extraction for molecular detection of Cyclospora cayetanensis, Toxoplasma gondii, and Cryptosporidium parvum as contaminants of berries.

The potential public health impact of foodborne parasites (FBP) transmitted via contaminated fresh produces indicates the necessity for robust and reliable laboratory methods for their detection and identification on this infection vehicle. Standardization of methods for detection of common FBP in fresh produce is to be expected and ensuring that the DNA extraction approach is most appropriate for the FBP of interest and for the matrix being analyzed is also important. Therefore, the aim of the present study was to compare the efficacy of two commercially available DNA extraction procedures, the UNEX-based method and DNeasy PowerSoil kit in the detection of three protozoan parasites, C. cayetanensis, C. parvum, and T. gondii, on contaminated berries. Oocysts of each parasite were spiked into the pellets of raspberry and blueberry washes. The spiked pellets were then randomly assigned to DNA extraction using either the PowerSoil or UNEX method, with DNA extraction with both methods performed by two independent analysts. The detection rate when berry washes were spiked with 20 oocysts of C. cayetanensis, T. gondii, and C. parvum was 95%, 85%, and 40%, respectively, when using the PowerSoil kit; whereas the equivalent results using the UNEX method were 55%, 60%, and 5%, respectively. In addition, significantly lower Cq values were achieved for each parasite in the samples spiked with 500 oocysts when the PowerSoil kit was used. Possible reasons for these results are discussed, and include the composition of both the beads and the buffers in each method.

A novel multiplex real-time PCR for the detection of Echinococcus multilocularis, Toxoplasma gondii, and Cyclospora cayetanensis on berries.

Foodborne parasites (FBP) are of major public health importance and warrant appropriate detection and control strategies. Most of the FBP considered for risk-ranking by a panel of experts are potentially transmitted via consumption of contaminated fresh produce, including berries. In this study we focused on the potential of three FBP, namely Echinococcus multilocularis, Toxoplamsa gondii, and Cyclospora cayetanensis, as contaminants of berries. Surveys to assess these parasites as contaminants of fresh produce in general, and berries in particular, are scanty or non-existent mainly due to the lack of optimized laboratory methods for detection. The aim of the present study was to develop and evaluate a novel multiplex qPCR for the simultaneous detection of E. multilocularis, T. gondii, and C. cayetanensis from berry fruits. The efficiency and linearity of each channel in the multiplex qPCR were within the acceptable limits for the range of concentrations tested. Furthermore, the method was shown to have good repeatability (standard deviation ≤0.2 Cq) and intermediate precision (pooled standard deviation of 0.3-0.6 Cq). The limit of detection was estimated to 10 oocysts for Toxoplasma and Cyclospora, and 5 eggs for Echinococcus per 30 g of raspberries or blueberries. In conclusion, evaluation of the present method showed that the newly developed multiplex qPCR is highly specific, precise, and robust method that has potential for application in food-testing laboratories.