Sources and prevalence of Cyclospora cayetanensis in Southeastern US water sources and growing environments

The increased frequency of produce-associated cyclosporiasis outbreaks highlights Cyclospora cayetanensis as an important emerging foodborne pathogen. Most produce associated cyclosporiasis outbreaks have implicated produce grown in areas where the disease is endemic. However, the 2018 detection of C. cayetanensis on US-grown produce and a cyclosporiasis outbreak associated with US-grown produce highlights the need for a better understanding of C. cayetanensis prevalence in US agricultural environments and of the risks of oocyst contamination to domestically grown produce. The aims of the current study are to assess C. cayetanensis prevalence in irrigation water, harvested produce (via spent packing house water as a proxy), on-farm portable toilets, and municipal wastewater influents in the Southeastern Coastal Plain growing region in Georgia. Georgia vegetable growers produce a wide variety of ready-to-eat produce and utilize well water- and surface water-fed irrigation water holding ponds for produce irrigation, similar to other large agricultural regions in the US. Georgia growing regions are also subject to heavy rainfalls, which we have shown contributes to human fecal contamination of irrigation ponds in the region. Additionally, a majority of the farm workers harvesting produce are from Cyclospora-endemic regions, representing a potential shedding source C. cayetanensis oocysts. C. cayetanensis prevalence in the agricultural environment will be assessed by analyzing large-volume (50 L) irrigation water samples on eight farms monthly during non-harvesting periods and twice a month during harvest. At most, one additional irrigation water sample will be collected per month following heavy rainfall. The prevalence of C. cayetanensis from harvested produce will be assessed by analyzing large-volume (up to 50 L) of spent rinse and wash water from packing houses as a proxy measurement. Generic Escherichia coli and the human-specific Bacteroides HF183 fecal marker will be assessed to evaluate the overall and human-specific fecal contamination, respectively, in the water samples. Human sewage samples from municipal wastewater influents and on-farm portable toilets will be analyzed to assess the prevalence of C. cayetanensis shedding in the region generally and on the farm environment, respectively. C. cayetanensis detections will be subsequently analyzed to determine the genotype and sporulation state of detected oocysts, in order to estimate oocyst maturation level and compare the genetic relatedness of oocysts detected in various sample matrices. The typing information will allow for C. cayetanensis contamination to be traced through the growing process. The maturation data will allow for an estimate of the time spent in the environment after shedding from the human host and of potential health risks associated with consumption of contaminated produce, as maturation state determines infectivity of the parasite. Data from this project will aid in the understanding of the domestic prevalence of C. cayetanensis in agricultural environments and provide the information needed to begin answering questions related to domestic C. cayetanensis produce contamination risks and identify best practices for risk reduction. Additionally, the evaluation of the human-specific fecal marker can provide insight into how Georgia vegetable growing practices are potentially preventing environmental and produce contamination.