Cyclospora prevalence in irrigation water in fresh produce growing regions

Cyclospora cayetanensis is an intestinal parasite that has been causing foodborne outbreaks in the United States almost every year since the 1990s. Most foodborne outbreaks are attributed to imported fruits and vegetables. Contamination is thought to be caused by contaminated irrigation water. Cases of cyclosporiasis are not able to be linked to outbreaks because of the lack of validated molecular typing tools. Studies determining the diversity of Cyclospora cayetanensis should be performed. From January 2017 to December 2018, samples from 3 wastewater treatment plants and 4 canals were collected in Arizona and Texas for a project funded by the Center of Produce Safety (CPS). There have been approximately 106 positive samples for C. cayetanensis using qPCR detection techniques. To determine diversity, the 18S region of C. cayetanensis will be targeted deep amplicon sequencing. 18S is part of the ribosomal RNA, one of the basic components of eukaryotic cells and is one of the most frequently used genes for diversity studies. After, sequences will be compared to determine the diversity between samples. The results could aid in understanding diversity of C. cayetanensis and aid in validating molecular typing tools that will help in outbreak investigations in the future.

Cyclospora cayetanensis has been implicated in recent outbreaks. Outbreaks have been associated with imported produce and water, more recently to produce grown in the United States. Information on the sources of the outbreaks and occurrence of this pathogen is still very limited due to lack of validated methods and genomic data available on environmental samples. Recently, the whole genome sequence of C. cayetanensis was obtained by clinical samples. Gathering genomic information on C. cayetanensis present in environmental sources will assist in linking cases of cyclosporiasis and will expedite the tracking and control of future outbreaks. C. cayetanensis only infects humans and it is only transmitted through the fecal-oral route. Being able to compare sequences of C. cayetanensis can aid in determining the source of contamination, which will improve food industry control practices. Our specific objectives are to: a) determine the most efficient DNA extraction method for C. cayetanenesis in environmental water samples, b) develop a method for purification and concentration of C. cayetanensis oocysts in environmental samples using a FACS machine, c) perform Next Generation Sequencing (NGS) on samples with presence of C. cayetanensis and d) determine the diversity of C. cayetanensis present in Arizona and Texas using Next Generation Sequencing. This data will allow us to validate methods that will lead to an improvement in outbreak’s investigation and control practices. This study will help gather information on the best available practices to collect genetic information from C. cayetanensis in environmental samples, a practice that has not been very well studied, per our knowledge. Gathering information in environmental samples is essential for understanding and improving outbreaks’ surveillance, as well as improving control practices in food and water that are in risk of contamination of this pathogen.

1. Determine the prevalence of Cyclospora cayetanensis in irrigation waters in Arizona using the new BAM Chapter 19b method used for produce, with slight modifications. This will allow a determination of any risk from C. cayetanensis and to identify areas of potential risk.

This project showed a 3% prevalence of C. cayetanensis overall in the sampled canal water of the Yuma Valley growing region, with six positive samples out of the 196 collected (Table 1). The six samples positive for C. cayetanensis were found during February and December 2019, and January 2020; samples collected in all other months were negative. Four of the six positive samples were collected from unlined canals (~4%, 4/103) and the other two samples were from lined canals (~2%, 2/93) (Table 1). The values for gene copies/well ranged from less than 1 to 1.68 x 100. There was no statistically significant difference between lined and unlined canals for C. cayetanensis gene copies/liter (Table 1). The value of gene copies/liter is an estimation of what could be found per liter of agricultural water since only less than 1 mL was used for DNA extraction from sample volumes ranging from 5–15 mL. Determining the concentration of C. cayetanensis oocysts from agricultural water still presents challenges due to the high levels of sediment and organic material found in samples when collecting large volumes of up to 100 liters of agricultural water. For canal water positive for C. cayetanensis, there was a statistically significant difference between unlined and lined canals for total dissolved solids (TDS), and statistical differences between positive overall and lined canals for conductivity, salinity and TDS (Table 2). In addition, there was a significant difference between positive and negative canals for air, water, salinity, TDS and pH (data not shown). These findings show an overall lower prevalence from the previous (2017–2018) Cyclospora project, where ~13% (15/119) samples were positive for C. cayetanensis. The three-year average from the combined studies shows a ~7% (21/315) prevalence of C. cayetanensis. These findings suggest that the risk of fresh produce contamination by C. cayetanensis from agricultural water in this produce growing region seems to be relatively low, given that no C. cayetanensis outbreak has been associated with fresh produce grown in the Yuma Valley and given the volume of raw agricultural commodity irrigated with these agricultural waters. The recommendation is for irrigation districts and growers to continue to implement and practice current water quality safety measures, good agricultural practices (GAPs) and good management practices (GMPs) to continue to reduce the potential contamination of fresh produce with agricultural water contaminated with C. cayetanensis oocysts. Since we know humans infected with C. cayetanensis can transmit the oocysts into the environment, it is recommended that agricultural workers continue to practice hygiene and sanitation practices to minimize the potential of C. cayetanensis oocysts contamination of fresh produce. Stakeholders should continue to monitor human wastewater and nearby encampments, and both wild and domesticated animal intrusion into agricultural water, which can act as physical transporters of C. cayetanensis oocysts after coming across human waste or garbage contaminated with human feces.