Objectives:
Objective 1: Develop immunomagnetic separation (Cyclo-IMS) directly from environmental samples using magnetic beads linked with antibodies against a principal oocyst wall protein (COWP2 and/or TA4 antigen-like surface protein) of C. cayetanensis.
Objective 2: Evaluate the value of Cyclo-IMS to concentrate oocysts of C. cayetanensis from environmental samples.
Objective 3: Evaluate improvements to DNA isolation by coupling Cyclo-IMS to extraction methods, previously optimized on Eimeria oocyst surrogates, for C. cayetanensis (including freeze-thaw, bead beating, and osmotic shock to lyse oocysts).
Abstract: Foodborne outbreaks caused by the protozoan pathogen Cyclospora cayetanensis threaten human health and pose financial and reputational threats to produce growers, packers, and grocers. Those seeking to understand and counter these threats lack sufficiently simple and sensitive means to detect parasite contamination. Detecting Cyclospora typically requires microscopic examination of wet smears, staining tests, fluorescence microscopy, or DNA testing for oocysts. Microscopy relies on oocyst morphology or autofluorescence to identify the parasite. However, this method lacks sensitivity and specificity. The validated FDA method, employing qPCR-based detection of Cyclospora spp. can fail when attempting to diagnose a very low number of oocysts in environmental samples and food matrices. Such environmental samples may contain competitors and inhibitors (including bacteria, insects, feces, and other free-living organisms) that impair Cyclospora parasite recovery and detection. Any detection platform would benefit from the means to concentrate and purify oocysts. Here, we propose to develop exactly this capability. We will develop a robust, rapid, specific, efficient, and user-friendly antibody-based method capable to separate and enrich Cyclospora oocysts, demonstrably improving downstream DNA extraction. “Cyclo-IMS” will harness the power of immunomagnetic separation to detect and concentrate oocysts of Cyclospora. Immunomagnetic separation (IMS) recovers a variety of eukaryotic single-cell pathogens, including Giardia lamblia, Toxoplasma, and Cryptosporidium. For Cryptosporidium, this technique has been investigated both in an indirect immunoassay format and in a direct immunoassay format. In clean water matrices, the IMS procedure recovers more than 90% of oocysts, irrespective of oocyst age. Antibodies to cysteine-rich oocyst wall proteins already power commercial IMS kits to detect Cryptosporidium and Toxoplasma. Through comparative genomic analysis, we identified the C. cayetanensis oocyst wall gene (COWP; cyc_01725 www.toxodb.org; WP-2; GenBank: OEH9762.1). Our target molecule was recently affirmed by a DNA-aptamer-based staining system, binding COWP, which shows promise as a basis for detecting Cyclospora. The DNA aptamer study also demonstrated the potential of another protein, the TA4 antigen-like protein, for rapid detection of Cyclospora11. We synthesized and expressed COWP in E. coli, and plan to immunize rabbits to produce polyclonal antisera. Additionally, we will express TA4 antigen-like protein (NCBI reference sequence XP_026191783.1) in E. coli. Once we determine the specificity and sensitivity of purified polyclonal antibodies via immunofluorescence assays (against target and non-target organisms), we will evaluate them for IMS assay sensitivity. We plan to determine the extent to which Cyclo-IMS improves DNA extraction (using methods optimized on Eimeria surrogate oocysts). Cyclo-IMS will simplify oocyst enrichment from complex food matrices, soil, and environmental water samples. Importantly, the antibodies employed in this bead-based kit may also serve as the basis for a fluorescence-based detection assay, greatly aiding risk assessment and environmental sampling. Ultimately, success will be judged by the adoption of such methods by fresh produce growers as well as by regulatory agencies seeking to protect human public health, including the FDA and CDC.