Jan. 1, 2023 - Dec. 31, 2024Amount Awarded
Asis Khan, Ph.D.
USDA - ARS
Benjamin M. Rosenthal, SD, Jitender P. Dubey, MV Sc, Ph.D., Mark C. Jenkins, Ph.D.Summary
To understand and mitigate human health risk posed by foodborne pathogens, produce growers and regulators require tools capable of assessing not only their presence, but also their viability. Although being present is a concern, actions to mitigate infection are more urgent if the pathogens are viable. The inability to propagate Cyclospora, either in in vitro or in vivo, hinders viability assessment. Many pathogens repel infiltration of certain dyes only when living. Propidium monoazide (PMA) has proven successful as a basis to establish the viability of other pathogens. Droplet digital PCR (ddPCR) proven very useful for absolute quantification of nucleic acids, without requiring standard curves. Hence, we proposed to develop a viability assay for Cyclospora in produce, combining PMA staining with ddPCR. To accomplish this, we will first optimize conditions and establish performance using Eimeria surrogates that infect poultry. We will then evaluate performance with Cyclospora. Success would afford the industry, and regulators, a rapid, sensitive, specific, and robust assay to diagnose parasite contamination and to test the presence of viable protozoan pathogens.
Many foodborne outbreaks derive from the protozoan pathogen Cyclospora cayetanensis. Although over 20 species in the genus Cyclospora are recognized, C. cayetanensis is the only species known to infect humans (1). Neither in vitro nor in vivo methods enable propagation of Cyclospora, severely limiting development of new control strategies. Access to oocysts is very limited, undermining efforts to understand their maturation and biological processes (2). Thus, there is an urgent need for new tools to detect the presence of viable pathogens in fresh produce, and furthermore, new model to study and understand the biology of this emerging pathogen. Fortunately, a wealth of data from natural and experimental Eimeria infections and comparative genome analysis showed that Eimeria and Cyclospora share conserved gene families, life-history traits, and developmental characteristics. Thus, Eimeria can and should be exploited as surrogates for evaluating methods capable of better detection, diagnosis, and outbreak tracing. Here, we propose to exploit this resemblance to speed development of an in vitro viability assay.
The FDA recently established a molecular detection technique that uses real-time polymerase chain reaction (qPCR) to amplify Cyclospora DNA in produce and clinical samples; this method cannot distinguish dead from live parasites (3). Viability has been assessed for other protozoans by incorporating Propidium Monoazide (PMA) prior to qPCR (4-7). PMA penetrates damaged cell membranes and intercalates in the DNA, inhibiting subsequent PCR amplification. Recently, PMA has been used very successfully in conjunction with droplet digital PCR (ddPCR) to assess and absolutely quantify viable pathogens, without requiring construction of a standard curve each time (8-10). Hence, combining PMA treatment and ddPCR could enable rapid enumeration of viable Cyclospora. Producers, regulators, and public health would benefit.
Objectives: 1) Adapt and validate sensitive biomarkers for risk assessments: We will target at least ten highly-expressed genes to develop biomarkers for ddPCR. After validating the specificity and sensitivity of the ddPCRs, we will develop a multiplex assay for simultaneous detection of 4 genes with the highest specificity and sensitivity. We will then seek to translate these assays to homologs of Cyclospora.
2) Develop quantitative viability assays for Eimeria and Cyclospora, using ddPCR system and PMA: We will evaluate PMA treatment to discriminate viable from nonviable parasites and evaluate ddPCR as a basis for quantifying live parasites. We will vary several treatment conditions to optimize the PMA protocol and will conduct a spiking experiment to evaluate the sensitivity and specificity of ddPCR using experimental and samples from “AFECCT” project. Finally, we will translate the standardized protocol to evaluate live and dead Cyclospora.
These efforts will develop a rapid, sensitive, specific, and robust assay to diagnose parasite contamination and to test the presence of viable protozoan pathogens, particularly Cyclospora by using Eimeria as a surrogate.