Determination of physical and chemical mechanisms to prevent Cyclospora infection

Cyclospora is a ubiquitous foodborne parasite that causes gastrointestinal illness in humans and is typically acquired through consumption of contaminated water or fresh foods. With the development of more sensitive diagnostic techniques and increased surveillance, the number of cases in the U.S. has continued to rise. Although it is difficult to trace Cyclospora infections to a single product, foodborne infections are primarily from the consumption of produce. In 2018, the first Cyclospora infection tied to domestically grown produce was reported, demonstrating the increased threat to U.S. consumers. While molecular techniques can be applied as a sensitive tool to identify Cyclospora oocysts, the molecular data on the viability/infectivity of oocysts is not available. Currently, the viability of oocysts can only be assessed by analysis of sporulation rates, which must be determined microscopically by a trained investigator. This inability to rapidly determine oocyst viability creates a significant bottleneck for testing new control measures. In addition, the lack of a model system for propagating oocysts make obtaining oocysts a barrier to widespread research. The primary goal of this proposal is to identify new control measures for inactivation of Cyclospora in agricultural water inputs and on the surface of produce.

The number of Cyclospora outbreaks due to contamination of produce has continued to rise over the last decade with 2018 marking the first outbreak associated with domestic produce. This increased risk has led to numerous studies focused on surveillance of Cyclospora in raw sewage, treated wastewater, irrigation water, and on produce. Since 2014, CPS has invested $1.6M in Cyclospora research, with efforts primarily focused on surveillance. Results of this investment confirmed that Cyclospora is endemic in the U.S. and can be recovered from wastewater and irrigation water, which poses a significant risk for produce contamination. Despite this risk, few studies have investigated methods to inactivate Cyclospora due to two bottlenecks: 1) the inability to culture the organism has limited the number of oocysts that can be collected for inactivation studies; and 2) the methods for determining oocyst viability are laborious, requiring a trained parasitologist to microscopically validate the sporulation of oocysts. The objectives of this 2 year project are: 1) systematically evaluate inactivation of Cyclospora oocysts by gamma radiation, ultraviolet radiation (UV), ozonation, and chlorine dioxide gas (ClO2); 2) develop a high-throughput, automated method for determining inactivation of Cyclospora oocysts; and 3) employ the automated method to screen a library of chemical compounds for inactivation of Cyclospora. To meet these objectives, a parallel workflow will be established, where inactivation of Cyclospora by the four primary strategies will be evaluated using manual microscopy as the “gold standard” to determine inactivation, while the high-throughput, automated method is simultaneously developed. By standardizing the methods used to determine inactivation it will be possible to compare the results between the different methods. The inactivation protocol will consist of 1×104 oocysts/treatment in water with the same concentration of natural organic matter (NOM), at a starting pH of 7.0, and constant temperature of 25°C. The efficiency of an inactivation method will be reported as the number of intact oocysts after application of the test method and the % sporulation of oocysts. This will capture both physical disruption of oocysts and inactivation of intact oocysts. Both metrics are important to characterize the efficiency of inactivation and decreasing the overall risk of Cyclospora infection. In year 2, the automated system will be used to evaluate inactivation of Cyclospora oocysts from a library of chemical targets that will be identified (Objective 3), but not tested, in year 1. Using the automated system, inactivation of Cyclospora oocysts will be measured for each of the target compounds with the number of compounds tested determined by the availability of oocysts. This study will identify ≥ 2 methods for inactivation of Cyclospora oocysts to reduce or eliminate the risks associated with agricultural water inputs. In addition, the successful methods will be evaluated for the feasibility of their use on produce and in the produce processing environment, with consideration paid to cost, availability, effectiveness, and regulation. Further, by developing a system for automated determination of oocyst viability, it will be possible for other researchers to engage in further Cyclospora inactivation studies with reduced effort.

1. Validate strategies for inactivation of Cyclospora oocysts, including gamma radiation, UV, ozonation, and chlorine dioxide gas. 

2. Develop an automated method for rapid determination of Cyclospora oocyst viability that would enable screening of antimicrobial libraries. 

3. Utilize the automated method to identify novel antimicrobial compounds and effective delivery systems leading to inactivation of Cyclospora.

During this funding period, we identified at least two methods for oocyst inactivation, with several experiments still underway due to sporadic and late availability of sufficient quantities of oocysts. First, UV-C irradiation was successfully employed to inhibit sporulation across a series of doses and time points. Since we noted a significant difference in the efficacy of UV-C irradiation at nearly every dose tested between the 1- and 3-minute time points, we have applied treatments with finer temporal resolution to oocysts derived from the same USDA batch of Eimeria. By further refining the dosage curve, we will define a minimal UV exposure time required to sufficiently inhibit oocyst development toward minimizing energy inputs in field applications. 

UV-C treatment for sterilization of fresh produce has demonstrated significant benefits, including cosmetic improvement in button mushrooms with the prevention of browning (Wang et al., 2022), maintenance of post-harvest flavor profile in pepino fruits (Zhao et al., 2021), and accumulation of human health-promoting compounds in tomato fruit (Yan, et al., 2020). Thus, the benefits of UV-C inactivation methods are manifold. Upon completion of 222 and 282-nm testing, we expect to maintain the benefits of UV treatment at 254 nm while significantly lowering health risks associated with harmful wavelengths (Schuch & Menck, 2010; Zaffina et al., 2012). 

In addition to UV light, we tested a battery of phytocompounds for their ability to disrupt oocyst development. While most compounds tested to date show little promise, we did develop and validate a largely automated pipeline to assay the disinfectant properties of antimicrobial compounds. While testing various methods to establish a positive control treatment, it was noted that pH modification using 0.1N NaOH was an effective measure in blocking development through sporulation. Interestingly, reducing pH using 1N HCl had no effect, showing that mildly alkalizing, but not acidifying, Eimeria acervulina suspensions can reduce viability. It would be valuable to determine the minimal concentration of NaOH or other alkalizing agents sufficient to prevent foodborne transmission of infective oocysts. One avenue for further exploration could be modifying pH in produce washes prior to further inactivation using UV light. Along these lines, synergism between or among inactivation methods used in this work could lead to new self-contained systems for field implementation.

Infectivity assays can be used to determine the viability of some coccidians in the endogenous phase, or host tissue penetration (Felici et al., 2021). However, since no method for culturing Cyclospora cayetanensis has yet been developed, evaluating the success of inactivation assays currently relies on microscopic examination of treated oocysts. We noticed first-hand that this method is subject to variation depending on the relative experience of individual researchers. To obviate complications involving human error in scoring oocysts, we developed an automated image detection system capable of accurately predicting viability based on sporulated state. Algorithm training sets are still being analyzed to increase the success in sporulation detection, with current a detection accuracy of ~80% on training sets. Accompanied by a largely automated and standardized screening assay, we hope to deliver tools that will alleviate the burden of manual counting and reduce the subjectivity inherent to viability screening. By making this algorithm publicly available, we will bolster the capacity for rapid screening of test compounds across research and production facilities. 

By mid-February 2023, data from the initial ozone screening and gamma ray testing will be available for analysis, potentially increasing the number of validated methods to break the life cycles of food- and waterborne coccidians. Ozonated water was shown to reduce the incidence of Giardia contamination in drinking water (Kondo et al., 2020) and has proven effective to inactivate Cryptosporidium parvum (Morrison et al., 2022). Further, ozone exposure has demonstrated benefits in produce such as strawberries (Piechowiak et al., 2022). However, as exposure time increases, consequences begin to arise, such as a reduction in vitamin content or the development of unwanted cosmetic traits (reviewed in Sachadyn-Król & Agriopoulo, 2020). Thus, our current method relies on decontamination in a matter of minutes. 

The above highlights some significant findings from the current work. As previously stated, a major hindrance to completing experiments has been the availability of test organisms. A coordination between CPS and local health departments in which Cyclospora specimens are received, or with the FDA, could help to promote awareness of the need for viable test organisms to combat the spread of cyclosporiasis.