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•Ag water, wastewater, and portable toilets were tested for C. cayetanensis.•C. cayetanensisqPCR detections from wastewater were confirmed by sequencing.•qPCR detections from water couldn’t be confirmed, suggesting false detection.•A single method may not be able to discern C. cayetanensisfrom other Eimeriidae.

Human norovirus (HuNoV) is an important cause of acute gastroenteritis and can be transmitted by water exposures, but its persistence in water is not well understood. Loss of HuNoV infectivity in surface water was compared with persistence of intact HuNoV capsids and genome segments. Surface water from a freshwater creek was filter-sterilized, inoculated with HuNoV (GII.4) purified from stool, and incubated at 15 or 20 °C. We measured HuNoV infectivity via the human intestinal enteroid system and HuNoV persistence via reverse transcription-quantitative polymerase chain reaction assays without (genome segment persistence) or with (intact viral capsid persistence) enzymatic pretreatment to digest naked RNA. For infectious HuNoV, results ranged from no significant decay to a decay rate constant (“k”) of 2.2 day–1. In one creek water sample, genome damage was likely a dominant inactivation mechanism. In other samples from the same creek, loss of HuNoV infectivity could not be attributed to genome damage or capsid cleavage. The range in kand the difference in the inactivation mechanism observed in water from the same site could not be explained, but variable constituents in the environmental matrix could have contributed. Thus, a single kmay be insufficient for modeling virus inactivation in surface waters.

The recent increase of reported cyclosporiasis outbreaks associated with fresh produce has highlighted the need for understanding environmental transmission of Cyclospora cayetanensisin agricultural settings and facilities. Conducting such environmental investigations necessitates robust sample collection and analytical methods to detect C. cayetanensisin water samples. This study evaluated three sample collection methods for recovery of C. cayetanensisoocysts from water samples during seeded recovery experiments. Two filtration-based methods, dead-end ultrafiltration (DEUF) and USEPA Method 1623.1, were evaluated for oocyst recovery from irrigation water. A non-filter-based method, continuous flow centrifugation (CFC), was evaluated separately for recovery from creek water and spent produce wash water. Median C. cayetanensisrecovery efficiencies were 17% for DEUF and 16–22% for Method 1623.1. The DEUF method proved to be more robust than Method 1623.1, as the recovery efficiencies were less variable and the DEUF ultrafilters were capable of filtering larger volumes of high-turbidity water without clogging. Median C. cayetanensisrecovery efficiencies for CFC were 28% for wash water and 63% for creek water, making it a viable option for processing water with high turbidity or organic matter. The data from this study demonstrate the capability of DEUF and CFC as filter-based and non-filter-based options, respectively, for the recovery of C. cayetanensisoocysts from environmental and agricultural waters.