Methods for the detection of diverse parasites on packaged salads based on (viable) oocysts

This project will involve an approach for managing and monitoring produce safety to reduce the risk of foodborne illness from consumption of packaged salads: detecting human parasites on the surfaces of leafy greens in a rapid, accurate, and affordable manner. A novel test will simultaneously detect four key parasites that can be associated with produce-borne disease. Applying the newly validated tests in parallel with previously established methods for testing packaged salads will provide a realistic evaluation for the suitability of the developed assays for routine screening by the produce industry. Additional methods will determine the viability of these parasites and whether they are likely to cause disease to produce consumers if they are detected.

Contamination of packaged salads with disease causing microorganisms constitutes a significant health risk for produce consumers. Protozoan pathogens are targeted because they are implicated in produce-borne illness outbreaks, optimal methods for their detection in produce are lacking, and their prevalence in developed countries such as the United States is likely to rise due to increasing demands for imported vegetables as well as climate variability projections. Two key issues that challenge the produce and packaging industry are 1) detecting human pathogens on produce in a rapid, accurate, and affordable manner; and 2) using that information to estimate health risks to the consumer. The overarching goal of this proposal is to address the first challenge by 1) developing a simple one-step procedure for routine screening of protozoan (oo)cysts on produce, and 2) comparing novel molecular techniques and established microscopy methods for further quantification of viable protozoan pathogens. A qualitative multiplex-PCR combined with a restriction fragment length polymorphism (RFLP) assay is proposed for routine monitoring to simultaneously detect and differentiate Cryptosporidium spp., Giardia spp., Cyclospora cayetanensis, and Toxoplasma gondii. In addition – and to generate quantitative data on the viability of these pathogens – we will use a variety of molecular methods based on quantitative PCR (qPCR) and compare results with traditional bright-field and epifluorescence microscopy for T. gondii and C. cayetanensis oocysts, and immunofluorescence microscopy for Cryptosporidium and Giardia (oo)cysts. Molecular method optimization will be performed through systematic laboratory spiking experiments in cooperation with our industrial cooperator. This project foresees the active participation of the industry cooperator, Fresh Express, who will provide produce for spiking experiments and background about growing, processing and packaging steps. Cooperators will be invited to participate in quarterly research meetings. Produce-related data and names of cooperators will be made anonymous in publications arising from the project.

To optimize methods to detect protozoan pathogen (oo)cysts on produce

• The novel multiplex PCR test developed in this project can be immediately adopted by the produce industry to screen leafy greens for the presence of protozoan parasites. 

• The multiplex PCR test can be optimized for application to other produce commodities, such as berries and cilantro, which are at high risk for contamination with parasites. 

• Applying viability assays in field surveillance investigations is key to estimating contamination due to live versus dead parasites, and will aid accurate modeling efforts to predict the risk of illness to consumers. 

• Addressing food safety concerns due to viable Cyclospora cayetanensis contamination is especially challenging due to the difficulty encountered in obtaining oocysts for research purposes. Unlike the other three protozoan parasites targeted in this study, Cyclospora is a human-specific pathogen and oocysts can only be obtained from diarrheic stool samples of patients. While our research group succeeded in procuring oocysts for development of a screening assay, our collaborator at CDC (Dr. Michael Arrowood) could not verify the viability state of the parasites. A collaborative framework among produce industry partners, academic institutions, state-level public health laboratories, and the CDC is strongly recommended for strategizing surveillance and sample collection approaches that would maximize resources for advancing research on this important emerging infectious disease agent.