Analysis of the presence of Cyclospora in waters of the Mid-Atlantic States and evaluation of removal and inactivation by filtration

The proposed project aims to address concerns surrounding fresh water availability and safety, as more outbreaks of foodborne illness caused by parasites have been linked to water used in produce production. Cyclospora cayetanensis, a parasite, has presented a unique challenge to the scientific community in understanding its persistence, transfer, and detection in the environment. Cases of domestically acquired foodborne illness associated with C. cayetanensis have drastically increased in the past five years. In 2018, the first documented domestic C. cayetanensis outbreak was linked to produce grown in the United States, previously outbreaks had often been associated with produce imported from countries endemic for this organism. Zero valent iron (ZVI), a by-product of the steel industry, is affordable and has been shown to be effective in removing and neutralizing bacterial, viral, and chemical contaminants from water. The efficacy of ZVI on parasites has not been studied but shows great potential in filtration applications. The objectives of this study are to determine the prevalence of C. cayetanensis in potential sources of irrigation water within the Mid-Atlantic region and to assess the effectiveness of ZVI filtration in removing and inactivating protozoa like C. cayetanensis from irrigation water to ensure the safety of produce.

Cyclospora cayetanensis is a protozoan parasite that causes gastrointestinal illness following consumption of contaminated produce or water. Cyclospora is still considered an emerging pathogen by many, due to the limited understanding surrounding the transmission and survival of oocysts in the environment along with difficulties and limitations in detection of oocysts. This project has two main objectives, which will address critical data gaps that affect the produce industry across multiple commodities. The first objective is to provide an understanding of the impact of C. cayetanensis on waters in the Mid-Atlantic region of the United States, a previously unstudied area. In many ways the Delmarva area serves as a model region, centered on a large watershed with active agriculture and within the urban heart of the East Coast. The water sources tested include those that could be used for irrigation of raw agricultural commodities such as surface water (pond, river), reclaimed wastewater, and reclaimed produce wash water. The second objective will elucidate the efficacy of ZVI filtration in the removal and inactivation of parasitic pathogens to improve pre-harvest food safety. This work will also facilitate the development of novel on-farm filtration technology and guidelines for commercial applications to control parasitic pathogens in agricultural water, thus improving the safety of produce and reducing parasitic foodborne illness. The proposed ZVI technology may offer advantages to growers including adaptation to existing filtration systems, feasibility, effective filtration and broad neutralization of numerous biological and chemical hazards with minimal environmental impacts. As the number of C. cayetanensis outbreaks associated with produce continue to increase, knowledge of the organism and novel intervention strategies will be crucial to ensure the safety of produce.

1. Confirmation of the presence of Cyclospora cayetanensis in presumptive positive environmental water samples using the Sanger (dideoxy) sequencing method. 

2. Evaluation of zero-valent iron (ZVI) and sand filtration in the removal and inactivation of C. cayetanensis surrogates in artificial agricultural water.

Research with C. cayetanensis is severely limited by the lack of resources and inability to obtain C. cayetanensis oocysts. Throughout history, under such conditions scientists have garnered vast amounts of data through the use of surrogate microorganisms. In this study, two protozoan surrogates were used. Cryptosporidium is a robust pathogen resistant to chemical disinfectants but smaller in size compared to Cyclospora. Eimeria tenella is a bit larger than Cyclospora but more similar in morphology to Cyclospora. Together these two pathogens provide useful information. 

Phylogenetic analysis of apicomplexan protozoa is complex. The need for additional analysis following PCR detection of presumptive Cyclospora positive environmental samples is clear; however, the pathway to further identification of these samples remains murky. Computational biologies may be able to clarify this issue given more time for analysis. Microbiologists perform multiple steps of analysis on bacterial detection and confirmation from environmental samples, including biochemical tests, culture isolation, and sequencing. It seems that a similar set of multiple steps to analysis is warranted for these complex environmental samples that may be positive for Cyclospora. Alternate means of detection, using multiple primer sets and potential for inclusion of mitochondrial genomes should also be considered. 

Sand biofilters are used for recreational water purification and on some farms. The incorporation of zero-valent iron has previously been shown to increase the removal and inactivation of bacteria and viruses by filtration. The project findings suggest that zero-valent iron can enhance the efficacy of filtration for removing protozoa of varying sizes (5 to 22 microns). Future research should address the inactivation of protozoan oocysts, including feasibility and mechanistic studies. 

NOTE: This project received an extension through January 2022 to complete additional analysis of the samples from the project and to use the funds remaining from the original budget. The Addendum results are presented on pp. 13–20.