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Agricultural water is a potential source of microbial contamination whereby Escherichia coli, Salmonella, Cryptosporidium, and Cyclospora cayetenensis can enter the food supply. To reduce this risk, effective sanitization of agricultural water may be critical to food safety. As such, it is important to investigate the effects of aqueous peracetic acid (PAA) and chlorine (Cl) on bacteria and protozoa at different treatment times and temperatures in agricultural water with respect to key water characteristics. Multiple concentrations of each sanitizer, ranging from 3 to 200 ppm, were prepared in recently collected agricultural water, the solution was brought to the desired temperature, and the target organisms were added and left for the desired contact time (5 or 10 minutes) when sodium metabisulfite was added to neutralize the sanitizers. Bacterial samples were enumerated on MacConkey or XLT4 agar. Samples with protozoa were added to mammalian cell culture (HCT-8 cells for Cryptosporidium parvum and MDBK cells for Eimeria tenella ). After 48 hours, the infected cells were collected, DNA extracted and infectivity assessed by quantitative PCR (qPCR). Low and high concentrations of sanitizer were effective at eliminating bacteria with Cl being significantly ( P < 0.05) more effective. The greatest reductions in E. coli and Salmonella (3.48 log and 2.5 log cfu/mL, respectively) were observed after 10 minutes of exposure to 10 ppm Cl. Concentrations of sanitizer 50 ppm and lower resulted in insignificant ( P > 0.05) reductions in parasite infectivity of less than 1 log for both organisms. A 200 ppm PAA treatment reduced infectious oocyst populations by 3.8 log for C. parvum and 2.6 log for E. tenella , with Cl being significantly ( P < 0.05) less effective against these organisms., Importance: This research is critical to inform decisions regarding the application and use of sanitizers in pre-harvest agricultural water settings to enhance food safety. Understanding the effectiveness of chlorine (Cl) and peracetic acid (PAA) on bacteria and protozoa will allow for the more efficient and practical use of these sanitizers, thus improving agricultural practices in ways that are beneficial to both growers and consumers., Competing Interests: The authors declare no conflict of interest.

This study addresses the global issue of foodborne illness, specifically focusing on those resulting from the consumption of leafy green vegetables. It explores the rising trend of consuming minimally processed or raw foods and the imperative of maintaining safety standards starting at the preharvest stage to prevent pathogenic bacterial contamination. The study identifies soil and irrigation water as key sources of pathogens and emphasizes the need for strict preventive measures during production and preharvest. It discusses the challenges of postharvest decontamination and highlights the importance of early-stage prevention strategies. The paper also examines advanced pathogen detection methods and food safety practices recommended by USDA and FSMA's PSR, including HACCP and LGMA strategies. Aimed at providing insights for consumers and producers, the study underscores the necessity of effective manufacturing strategies to ensure the safety of leafy greens., Competing Interests: The authors do not have any conflicts of interest. Ukti Bimal Sheth and Md Ariful Haque have contributed to building the concept, acquisition, analysis, and interpretation of data and writing the manuscript; Min Ji Jang contributed to the writing section; Samuel Haruna, Tony V. Johnston, Deokyeong Choe, and Ying Gao provided valuable suggestions and revised the manuscript, Seockmo Ku contributed to building the concept, guiding, providing suggestions, and revising the manuscript critically., (© 2024 The Author(s). Food Science & Nutrition published by Wiley Periodicals LLC.)

Contaminated agricultural water has been implicated in produce-associated outbreaks, including dry bulb onions (Allium cepa). This study was designed to quantify risks associated with contaminated water used to prepare crop protection sprays applied immediately before the onset of field curing of dry bulb onions. Laboratory experiments determining the behavior of Salmonella and Escherichia coli in crop protection chemical solutions were performed to guide selection for field use. Field trials were conducted (2022, 2023) in eastern Oregon (Treasure Valley) using two onion cultivars ('Red Wing' and 'Cometa') inoculated with a rifampicin-resistant E. coli cocktail (3-4 log CFU/100 mL) suspended in fungicide solution or clay suspension, and applied with a backpack sprayer at the end of the growing season. Onions were sampled through the next 4 weeks of field curing and after 1 and 4-5 mos of postharvest storage. In 2022, onions were initially contaminated at a maximum cell density of 48 MPN/onion (Geometric mean (GM): 3.7 MPN/onion). At the end of curing, a single onion (out of 320) tested positive at 2 MPN/onion. In 2022, E. coli was not detected during postharvest storage (n = 160). In 2023, the application of contaminated sprays resulted in a maximum contamination of 275 MPN/onion (GM: 8.6 MPN/onion). At the end of the 2023 curing period, three out of 320 onions (0.9%) had detectable levels of E. coli (1-2 MPN/onion). Three 'Cometa' onions from the same plot that were treated with fungicide were positive for E. coli after 5 months of postharvest storage (2, 11, and 83 MPN/onion). These field trials indicate field curing conditions in the Treasure Valley help mitigate risks associated with contaminated water used for applying crop protection sprays. E. coli was detected on a small percentage of onions at low cell density after curing. The single onion with elevated E. coli populations after postharvest storage had internal damage characteristic of bacterial rot., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: “Joy Waite-Cusic reports financial support was provided by the Center for Produce Safety, U.S. Department of Agriculture’s (USDA) Agricultural Marketing Service, and Washington State Department of Agriculture’s Specialty Crop Block Grant Program. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper”., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Irrigating fresh produce with contaminated water contributes to the burden of foodborne illness. Identifying fecal contamination of irrigation waters and characterizing fecal sources and associated environmental factors can help inform fresh produce safety and health hazard management. Using two previously collected data sets, we developed and evaluated the performance of logistic regression and conditional random forest models for predicting general and human-specific fecal contamination of ponds in southwest Georgia used for fresh produce irrigation. Generic Escherichia coli served as a general fecal indicator, and human-associated Bacteroides (HF183), crAssphage, and F+ coliphage genogroup II were used as indicators of human fecal contamination. Increased rainfall in the previous 7 days and the presence of a building within 152 m (a proxy for proximity to septic systems) were associated with increased odds of human fecal contamination in the training data set. However, the models did not accurately predict the presence of human-associated fecal indicators in a second data set collected from nearby irrigation ponds in different years. Predictive statistical models should be used with caution to assess produce irrigation water quality as models may not reliably predict fecal contamination at other locations and times, even within the same growing region., Competing Interests: The authors declare no competing financial interest.

The application of overhead evaporative cooling (EC) with untreated surface water is common in Washington State to decrease sunburn on apples. While this practice reduces crop and economic losses, applying untreated surface water to produce introduces potential risks of foodborne pathogen contamination. This study examined EC and the survival of inoculated generic Escherichia coli for two apple varieties over three growing seasons. Factors examined included EC treatment (conventional, untreated), canopy location (high, low), and apple variety (Gala, Golden Delicious). Fruit maturity (mature, immature) was also examined for one year. A rifampicin-resistant generic E. coli cocktail was applied to apples at 7.3 ± 0.4 log CFU/apple, and apples were enumerated for E. coli levels at 0, 2, 10, 18, 34, 42, 58, 82, 106, and 154 h postinoculation. Log-linear, Weibull, and Biphasic models were utilized to characterize the die-off pattern of E. coli. For most treatments, inoculated E. coli decreased by almost or over 6 log CFU/apple over the study duration; however, E. coli remained detectable at low levels on apples at 154 h postinoculation. EC treatment and canopy location did not significantly impact daily linear E. coli die-off rates. Apple variety and maturity demonstrated small but statistically significant impacts on daily linear die-off rates. Nonlinear models (i.e., biphasic and Weibull) best captured E. coli die-off on apples, showing a rapid initial decrease followed by a slower decline over time. Overall, results demonstrate that EC, a useful fruit quality practice, did not impact the survival of inoculated generic E. coli on apple surfaces. The study provides valuable insights for the apple industry regarding E. coli die-off rates on in-field apples, guiding practical decisions to mitigate risk., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Agricultural water is commonly treated with chlorine-based disinfectants, which are impacted by water quality. Understanding how water quality influences disinfectants such as chlorine dioxide (ClO 2 ) against pathogenic bacteria is important for creating efficacious sanitation regimens. In this study, the minimum inhibitory concentration (MIC) of ClO 2 needed to achieve a 3-Log reduction against Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes was compared across agricultural water samples. Sterile ddH 2 O served as a control to compare with environmental samples from Salinas Valley, CA, and laboratory standards. To test different dosages and water qualities, stock ClO 2 was diluted in 24-well plates with target concentrations of 10, 5, 2.5, and 1.25 mg/L. Well plates were inoculated with pathogens and treated with sanitizer for 5 min. Following treatment, surviving pathogens were enumerated using viable cell counts. The results demonstrate that groundwater samples had the highest water quality of the environmental samples and required the lowest concentration of disinfectant to achieve 3-Log reduction against both bacteria, with MIC between 1.4 and 2.0 mg/L. Open-source samples had lower water quality and required a higher concentration of ClO 2 for 3-Log reduction, with MIC between 2.8 and 5.8 mg/L for both pathogens. There was no correlation between pH, turbidity, or conductivity/TDS and reduction for either STEC or L. monocytogenes , suggesting no individual water metric was driving reduction. A lower dosage was required to achieve 3-Log reduction against STEC, while L. monocytogenes required greater concentrations to achieve the same level of reduction. Overall, these results help guide growers in using ClO 2 as a broad-spectrum disinfectant and demonstrate its efficacy in reaching 3-Log reduction across agricultural water samples., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Van Blair, Lacombe, Harvey and Wu.)

Aims: The survival of inoculated Escherichia coli on Fuji apples in Washington State orchards was studied, considering evaporative cooling, canopy location, year, and region, with the examination of sunlight exposure and inoculation levels in year 2., Methods and Results: Rifampicin-resistant E. coli was applied to Fuji apples. Initial concentrations for the high-inoculation study were 7.4 ± 0.3 log10 CFU per apple and 3.4 ± 0.3 log10 CFU per apple for the low-inoculation study. Enumeration of E. coli was conducted at 0, 2, 10, 18, 34, 42, 58, 82, 106, and 154 h after inoculation. Results were analyzed using Tukey's honest significance difference test and a log-linear model. Log-linear, Weibull, and biphasic models characterized E. coli die-off patterns for high and low inoculations. The application of evaporative overhead cooling water did not significantly influence E. coli survival on Fuji apples; inoculation level and sunlight exposure were significant factors in a log-linear model. Escherichia coli decreased by 5.5 ± 1.3 and 3.3 ± 0.4 log10 CFU per apple for high and low-inoculated apples, respectively, by 154 h. The biphasic model best explained the die-off pattern for high and low-inoculated Fuji apples., Conclusions: Overhead evaporative cooling, a useful fruit quality practice, did not impact the survival of generic E. coli on Fuji apple surfaces. The significant impact of sunlight exposure and inoculation levels on die-off highlights the importance of ultraviolet radiation in risk reduction and the need for various inoculum concentrations in preharvest field studies., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Two U.S. outbreaks of salmonellosis in 2020 and 2021 were epidemiologically linked to red onions. The 2020 outbreak investigation implicated the production of agricultural water as a likely contamination source. Field trials were designed to investigate the prevalence and survival of Escherichia coli (surrogate for Salmonella) on dry bulb onions after the application of contaminated irrigation water at the end of the growing period. Irrigation water was inoculated at 3 log most probable number (MPN)/100 mL (2022 and 2023) or 5 log MPN/100 mL (2023, drip only) with a cocktail of rifampin-resistant E. coli and applied with the final irrigation (0.4 acre-inch/0.4 ha-cm) to onions. Onion bulbs (40 or 80) were sampled immediately after irrigation and throughout field curing (4 weeks) and E. coli was enumerated using an MPN method. For drip irrigation, at 3 log MPN/100 mL E. coli was detected on 13% of onions at 24 h but not detected at 0 h; at 5 log MPN/100 mL for drip irrigation applied to saturated soil, E. coli was detected in 63% of onions at 0 h. Prevalence significantly (P < 0.05), decreased after 7 d of curing with cell densities of 1-1,400 MPN/onion. At the end of field curing in 2023, 1/80 of onions had detectable E. coli (2.04 MPN/onion). E. coli was detected in a significantly smaller percentage of onions (2022: 13%; 2023: 68%) after a contaminated drip irrigation event compared to overhead irrigation (98-100%; P < 0.05). After overhead irrigation, E. coli was detected in onions (1-1,000 MPN/onion) on day 0. Prevalence decreased significantly (P < 0.05) after 7 d of field curing in both years (2022: 15%; 2023: 7%). E. coli was not detected on Calibra onions (80/year) at the end of field curing in either year but was detected at <12 MPN/onion in 2.5-3.75% of onions (n = 80) for other cultivars. These data confirm limited contamination risk associated with drip irrigation water quality and begin to quantify contamination risks associated with overhead irrigation of dry bulb onions., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: “Joy Waite-Cusic reports financial support was provided by the Center for Produce Safety, U.S. Department of Agriculture’s (USDA) Agricultural Marketing Service, and Washington State Department of Agriculture’s Specialty Crop Block Grant Program. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.”., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cyclospora cayetanensis is a coccidian parasite of the phylum Apicomplexa that causes cyclosporiasis, a human-specific gastrointestinal disease. Unlike most enteric pathogens, C. cayetanensis does not infect via direct fecal-oral transmission between humans because shed oocysts must be exposed to environmental triggers prior to becoming infectious. The development of specific and sensitive detection methods for C. cayetanensis is crucial to effectively address data gaps and provide regulatory support during outbreak investigations. In this study, new more specific molecular markers for the detection of C. cayetanensis were developed based on updated genomic databases of Apicomplexa mitochondrial sequences. Novel alternative reagents and supplies, as well as optimization protocols, were tested in spiked produce and agricultural water samples. The selected Mit1C primers and probe combined showed at least 13 mismatches to other related species. The new optimized qualitative real-time PCR assay with modifications to sample processing and replacement of discontinued items produced results comparable to the previously validated methods. In conclusion, the new optimized qualitative Mit1C real-time PCR assay demonstrated an increase in its specificity in comparison to other detection methods previously published, while it showed to be robust and as sensitive as the previously validated method at the FDA. This study has also expanded the array of PCR reagents that can be used to detect C. cayetanensis in produce and agricultural water samples and provided several improvements to the method for detection in agricultural water including replacements for discontinued items and a new dialysis filter for water filtration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Fresh produce can be contaminated by enteric pathogens throughout crop production, including through contact with contaminated agricultural water. The most common outbreaks and recalls in fresh produce are due to contamination by Salmonella enterica and Shiga toxin-producing E. coli (STEC). Thus, the objectives of this study were to investigate the prevalence of markers for STEC ( wzy, hly , fliC , eaeA , rfbE , stx -I, stx -II) and Salmonella ( invA ) in surface water sources ( n  = 8) from produce farms in Southwest Georgia and to determine correlations among the prevalence of virulence markers for STEC, water nutrient profile, and environmental factors. Water samples (500 mL) from eight irrigation ponds were collected from February to December 2021 ( n  = 88). Polymerase chain reaction (PCR) was used to screen for Salmonella and STEC genes, and Salmonella samples were confirmed by culture-based methods. Positive samples for Salmonella were further serotyped. Particularly, Salmonella was detected in 6/88 (6.81%) water samples from all ponds, and the following 4 serotypes were detected: Saintpaul 3/6 (50%), Montevideo 1/6 (16.66%), Mississippi 1/6 (16.66%), and Bareilly 1/6 (16.66%). Salmonella isolates were only found in the summer months (May-Aug.). The most prevalent STEC genes were hly 77/88 (87.50%) and stx -I 75/88 (85.22%), followed by fliC 54/88 (61.63%), stx -II 41/88 (46.59%), rfbE 31/88 (35.22%), and eaeA 28/88 (31.81%). The wzy gene was not detected in any of the samples. Based on a logistic regression analysis, the odds of codetection for STEC virulence markers ( stx -I, stx -II, and eaeA ) were negatively correlated with calcium and relative humidity ( p  < 0.05). A conditional forest analysis was performed to assess predictive performance (AUC = 0.921), and the top predictors included humidity, nitrate, calcium, and solar radiation. Overall, information from this research adds to a growing body of knowledge regarding the risk that surface water sources pose to produce grown in subtropical environmental conditions and emphasizes the importance of understanding the use of abiotic factors as a holistic approach to understanding the microbial quality of water., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chevez, Dunn, da Silva and Rodrigues.)

To mitigate excess of nitrate-N (NO 3 - -N) derived from agricultural activity, constructed wetlands (CWs) are created to simulate natural removal mechanisms. Irrigated agricultural drainage water is commonly characterized by an organic carbon/nitrogen (C/N) imbalance, thus, C limitation constrains heterotrophic denitrification, the main biotic process implicated in NO 3 - -N removal in wetlands. We studied a pilot plant with three series (169 m 2 ) of hybrid CWs over the first two years of functioning to examine: i) the effect of adding different C-rich substrates (natural soil vs. biochar) to gravel on NO 3 - -N removal in a subsurface flow (Phase I), ii) the role of a second phase with a horizontal surface flow (Phase II) as a source of dissolved organic C (DOC), and its effect in a consecutive horizontal subsurface flow (Phase III) on NO 3 - -N removal, and iii) the contribution of each phase to global NO 3 - -N removal. Our results showed that the addition of a C-rich substrate to gravel had a positive effect on NO 3 - -N removal in Phase I, with mean efficiencies of 40% and 17% for soil and biochar addition, respectively, compared to only gravel (0.75%). In Phase II, the algae growth turned into a DOC concentration increase, but it did not enhance NO 3 - -N removal in Phase III. In series with C-rich substrate addition, the largest contribution to NO 3 - -N removal was found in Phase I. However, in series with only gravel, Phase II was the most effective on NO 3 - -N removal. Contribution of Phase III to NO 3 - -N removal was almost negligible., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A broad understanding of community member food safety priorities in the fresh produce supply chain does not currently exist. This information is essential to improve food safety knowledge and practices effectively and efficiently throughout the fresh produce industry; therefore, the goal of this study was to identify and rank community produce safety priorities in the United States. Survey questions were designed and approved by food safety experts for participants to rank 24 fresh produce safety priorities. The anonymous survey was distributed online via Qualtrics™ to fresh produce community members from November 2020 to May 2021. A score was calculated for each priority by summing weighted ranking scores across responses. Descriptive statistics and logistic regression were used to determine frequencies and distribution of response and identify factors (e.g., role in produce safety, size/location of organization/operation) that influenced rankings. A total of 281 respondents represented fourteen different roles in the fresh produce industry, with most identified as growers (39.5%). Produce operations were distributed across the U.S. and annual produce sales ranged from below $25,000 to over $5,000,000. Health and hygiene, training, postharvest sanitation, traceability, and harvest sanitation were ranked as the top five food safety priorities. These findings provide insight into community member priorities in fresh produce safety and can be used to inform intervention efforts, ranging from specialized training for produce growers and packers, industry-driven research projects, and gaps in risk communication strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ultraviolet-C (UV-C) light-assisted water treatment systems are an increasingly investigated alternative to chemical sanitizers for agricultural surface water decontamination. However, the relatively high concentration of particulate matter in surface water is a major challenge to expanding its application in the production of fresh produce. The objective of this project was to test the efficacy of two commercial UV-C devices to reduce the microbial risk of agricultural water in order to develop a web application to assist growers in decision-making related to the on-farm implementation of UV-C technologies for agricultural water treatment. An on-farm study using three agricultural water sources was performed to determine the microbial reduction efficacy of a low power, low flow (LP/LF; 1-9 gallons per minute (GPM), 1.34-gallon capacity) and a high powered, high flow (HP/HF; 1-110 GPM, 4.75-gallon capacity) device at flow rates of 6, 7, and 9 GPM. A threshold of 30% UVT for the HP/HF device was observed, wherein lower water transmissibility significantly impacted microbial inactivation. Although less effective at lower %UVT, the LP/LF device costs less to install, maintain, and operate. The observations were used to design an online tool for growers to calculate the predicted reduction of generic Escherichia coli using either device based on the %UVT of their water source. However, because this study utilized an exploratory and proof-of-concept approach, the experimental flow rates were limited to reflect the capacities of the smaller unit (9 GPM) for direct comparison to the larger unit. Thus, the preliminary model and tool are largely limited to the experimental conditions. Yet, these results of this study demonstrate the utility of UV-C light in reducing the microbial risk of agricultural water, and future studies using different UV-C devices and higher flow rates will expand the use of the decision-making tool., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cyclospora cayetanensis is a coccidian parasite that causes diarrheal illness outbreaks worldwide. The development of new laboratory methods for detection of C. cayetanensis is of critical importance because of the high potential for environmental samples to be contaminated with a myriad of microorganisms, adversely impacting the specificity when testing samples from various sources using a single molecular assay. In this study, a new sequencing-based method was designed targeting a specific fragment of C. cayetanensis cytochrome oxidase gene and developed as a complementary method to the TaqMan qPCR present in the U.S. FDA BAM Chapter 19b and Chapter 19c. The comparative results between the new PCR protocol and the qPCR for detection of C. cayetanensis in food and water samples provided similar results in both matrices with the same seeding level. The target region and primers in the protocol discussed in this study contain sufficient Cyclospora-specific sequence fidelity as observed by sequence comparison with other Eimeriidae species. The sequence of the PCR product appears to represent a robust target for identifying C. cayetanensis on samples from different sources. Such a sensitive method for detection of C. cayetanensis would add to the target repertoire of qPCR-based screening strategies for food and water samples.

Abstract: The impact of water application method on bacterial survival at or after the final irrigation was evaluated in bulb onions during commercially relevant field drying (curing). A three-strain rifampin-resistant cocktail of Escherichia coli was introduced to onions via a single overhead spray application in two separate trials (5.22 [trial 1] or 2.40 [trial 2] log CFU per onion) 2 to 3 days after the final irrigation. Onions were lifted from the soil 8 days after spray inoculation and, in some cases, foliage was removed (topping); onions remained in the field for an additional ca. 2 weeks (total ca. 3 weeks of curing). E. coli populations declined on the onions in the first 4 h after spray inoculation. E. coli was recovered from 38 (48%) or 28 (35%) of 80 whole-onion enrichments at the end of curing in trials 1 or 2, respectively. Topping did not significantly impact the percentage of E. coli-positive onions detected at the end of curing. From 8 h to 21 days, E. coli populations on positive onions ranged from 1 CFU per onion to 7 log CFU per onion in both trials, representing a potential risk of E. coli growth with overhead application of contaminated water at the end of onion production. In trial 2, additional rows of onions were inoculated via a 22-cm subsurface or surface drip irrigation line (1.94 log CFU/mL for 2.5 h). E. coli was detected in 0 (subsurface) and 4 (surface) of 50 whole-onion enrichments 3 h after the initiation of drip irrigation. Positive onions were detected at days 1 (4 of 50) and 7 (1 of 50) with subsurface drip inoculation, and at days 1 (7 of 50), 7 (2 of 50), and 14 (2 of 50) with surface drip inoculation. E. coli was not detected in whole-onion enrichments at the end of curing when inoculated by subsurface (0 of 50) or surface (0 of 50) drip irrigation. Application of contaminated water through drip irrigation, when coupled with field curing, results in low rates of contamination of bulb onions at the time of harvest., (Published 2022 by the International Association for Food Protection.)

The U.S. Food Safety Modernization Act (FSMA) Produce Safety Rule (PSR) requires that farmers generate a Microbial Water Quality Profile (MWQP) from 20 samples per agricultural water source, taken over 2-4 years and five annual samples thereafter. Farmers must use the MWQP to ascertain a geometric mean (GM) of ≤126 CFU/100 mL and statistical threshold value (STV) of ≤410 CFU/100 mL of generic Escherichia coli. Farmers are responsible for collecting samples and paying for testing, incurring a financial and time burden. To determine if testing frequency can be reduced without compromising accuracy, water samples (n = 279) were collected from twelve sites in the U.S. Mid-Atlantic region from 2016 to 2018 comprising tidal brackish river, non-tidal fresh river, pond, vegetable processing, and reclaimed water. The GM and STV were calculated for all sites and water types using all samples, and for multiple sub-samples of <20 from each site and water type. A Monte Carlo simulation was used to determine the proportion of sub-sample sizes that yielded the same determination as the entire sample size of PSR standard compliance. Four sites, two pond and two reclaimed water sites, complied with PSR GM and STV requirements when using the entire sample set. When a water source's calculated GM and STV using the entire sample set hovered close to the PSR thresholds, sub-sample sizes approached the recommended 20 samples to reach a congruent compliance determination. However, 99% agreement was obtained with a sub-sample of five when the absolute difference between the GM and STV from total samples and the PSR thresholds was ≥2.6 and 4.5 log CFU/100 mL E. coli, respectively. These findings suggest that under certain conditions the MWQP may be generated with well below 20 samples, reducing the economic burden on farmers while still maintaining a representative MWQP., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Public acceptance of treated sewage (TS) reuse in agriculture is a key element in successful implementation of TS reuse projects, but relevant research on the topic is limited. This study examined public willingness to accept (WTA) and willingness to pay (WTP) for agricultural products derived by irrigation with TS in Lorestan province, Iran, applying the contingent valuation (CV) method. The agricultural products determined for the study included corn, peaches, apricots, and plums, while TS was introduced in three qualities [Q1, complete TS treatment (i.e., physical treatment, aeration, and chemical treatment); Q2, partial TS treatment (i.e., physical treatment and aeration); and Q3, no TS treatment]. With reference to WTA, products irrigated with Q1 TS received 62.7% positive responses, while products irrigated with Q2 TS and Q3 TS received 21.3% and 17.0% positive responses, respectively. With reference to WTP, data showed that if the price of products derived by irrigation with TS was less than that of products irrigated with fresh water, some people would be willing to pay for these products (52.0% positive responses versus 48.0% negative responses). Awareness had a significantly positive impact on WTA1 and WTA2 (products irrigated with Q1 TS and Q2 TS, respectively) as well as on WTP1, WTP2, and WTP3 (products irrigated with Q1 TS, Q2 TS, and Q3 TS, respectively). Similarly, information sources had a significantly positive effect on WTP1, WTP2, and WTP3. Overall, individuals with high levels of health risk perception and high awareness showed high WTA, while non-processed corn and men were associated with low WTA. Concerning WTP, individuals with high awareness and those who had access to information sources showed high WTP, while men were associated with low WTP. Information about the importance of TS reuse, reduction of prices of products derived by irrigation with TS, and promotion of public confidence in TS are essential to increase public WTA and WTP., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

One solution to current water scarcity is the reuse of treated wastewater. Water reuse systems have to be examined as a whole, including the efficacy of water-reclamation treatments and the operation steps from the wastewater inlet into the WWTP to the irrigation endpoint, including the irrigated crop. In this study, the monitoring of human enteric viruses and coliphages were assessed in two water reused systems. The presence of hepatitis A virus (HAV) and human noroviruses genogroups I and II (GI and GII) were analyzed by real-time RT-PCR (RT-qPCR) in water (n = 475) and leafy green samples (n = 95). Total coliphages were analyzed by the double-layer agar plaque technique. The prevalence of HAV in water samples was very low (c.a. 2%), mostly linked to raw sewage, while for leafy green samples, none was positive for HAV. In leafy greens, prevalence of norovirus was low (less than 5-6%). The highest reductions for norovirus were observed in samples taken from the water reservoirs used by the growers near the growing field. The virus die-off during water storage due to solar radiation could be considered as an additional improvement. Reclamation treatments significantly reduced the prevalence and the counts of noroviruses GI and GII and coliphages in reclaimed water. However, the coliphage reductions (c.a. 5 log) do not comply with the specifications included in the new European regulation on reclaimed water (≥6.0 log). Correlations between noroviruses GI and GII and coliphages were found only in positive samples with high concentrations (>4.5 log PFU/100 mL). A high percentage of samples (20-25%) negative for total coliphages showed moderate norovirus counts (1-3 logs), indicating that coliphages are not the most suitable indicator for the possible presence of human enteric viruses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appealed to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Pond irrigation water comprises a major pathway of pathogenic bacteria to fresh produce. Current regulatory methods have been shown to be ineffective in assessing this risk when variability of bacterial concentrations is large. This paper proposes using mechanistic modeling of bacterial transport as a way to identify improved strategies for mitigating this risk pathway. If the mechanistic model is successfully tested against observed data, global sensitivity analysis (GSA) can identify important mechanisms to inform alternative, preventive bacterial control practices. Model development favored parsimony and prediction of peak bacterial concentration events. Data from two highly variable surface water irrigation ponds showed that the model performance was similar or superior to that of existing pathogen transport models, with a Nash-Sutcliffe efficiency of 0.48 and 0.18 for the two ponds. GSA quantified bacterial sourcing and hydrology as the most important processes driving pond bacterial contamination events. Model analysis has two main implications for improved regulatory methods: that peak concentration events are associated with runoff-producing rainfall events and that intercepting bacterial runoff transport may be the best option to prevent bacterial contamination of surface water irrigation ponds and thus fresh produce. This research suggests the need for temporal management strategies. IMPORTANCE Preventive management of agricultural waters requires understanding of the drivers of bacterial contamination events. We propose mechanistic modeling as a way forward to understand and predict such events and have developed and tested a parsimonious model for rain-driven surface runoff contributing to generic Escherichia coli contamination of irrigation ponds in Central Florida. While the model was able to predict the timing of peak events reasonably well, the highly variable magnitude of the peaks was less well predicted. This indicates the need to collect more data on the fecal contamination inputs of these ponds and the use of mechanistic modeling and global sensitivity analysis to identify the most important data needs.

Water-choice trial experiments revealed that Anguilla japonica glass eels collected in southern Japan possess strong preferences for fresh water and agricultural water. Their locomotor activity and preference for fresh water were higher and stronger, respectively, in this study when compared to previous studies conducted at lower temperatures. These results suggest that their locomotor activity and preference for fresh water is influenced by water temperature. The attraction to agricultural water indicates their upstream migration and habitat selection could be influenced by agricultural water., (© 2021 Fisheries Society of the British Isles.)

The safety of groundwater has been a great concern for irrigation and drinking purposes in recent decades due to the increasing impacts of anthropogenic activities. There are several standards to evaluate the groundwater quality for different utilization purposes. In this paper, 804 samples covering the entire Ordos Basin across five provinces were used to evaluate the irrigation suitability and human health risks. The results showed that the sequence of cationic concentration was Na +  > Ca 2+  > Mg 2+  > K +  > NH 4 + , and the anion concentration was HCO 3 -  > SO 4 2-  > Cl -  > NO 3 -  > F -  > NO 2 - . For drinking purposes, TDS, Na + , SO 4 2- , F - , TH and NO 3 - exceed seriously the regulated standard in the study area. For irrigating purposes, 80% of the water in the study area belongs to "good water" according to the evaluation of salinity and alkalinity. Saline water which is not suitable for irrigation accounts for about 9%. High health risks of fluoride ions are mainly observed to the samples representing the western part of the study area, while the health risks of nitrates spread throughout the study area. Health risk is not only related to location, but also varies with age, and it is found that children suffer more threats than adults.

Abstract: A comprehensive understanding of foodborne pathogen diversity in preharvest environments is necessary to effectively track pathogens on farms and identify sources of produce contamination. As such, this study aimed to characterize Listeria diversity in wildlife feces and agricultural water collected from a New York state produce farm over a growing season. Water samples were collected from a pond (n = 80) and a stream (n = 52). Fecal samples (n = 77) were opportunistically collected from areas <5 m from the water sources; all samples were collected from a <0.5-km2 area. Overall, 86 (41%) and 50 (24%) of 209 samples were positive for Listeria monocytogenes and Listeria spp. (excluding L. monocytogenes), respectively. For each positive sample, one L. monocytogenes or Listeria spp. isolate was speciated by sequencing the sigB gene, thereby allowing for additional characterization based on the sigB allelic type. The 86 L. monocytogenes and 50 Listeria spp. isolates represented 8 and 23 different allelic types, respectively. A subset of L. monocytogenes isolates (n = 44) from pond water and pond-adjacent feces (representing an ∼5,000-m2 area) were further characterized by pulsed-field gel electrophoresis (PFGE); these 44 isolates represented 22 PFGE types, which is indicative of considerable diversity at a small spatial scale. Ten PFGE types were isolated more than once, suggesting persistence or reintroduction of PFGE types in this area. Given the small spatial scale, the prevalence of L. monocytogenes and Listeria spp., as well as the considerable diversity among isolates, suggests traceback investigations may be challenging. For example, traceback of finished product or processing facility contamination with specific subtypes to preharvest sources may require collection of large sample sets and characterization of a considerable number of isolates. Our data also support the adage "absence of evidence does not equal evidence of absence" as applies to L. monocytogenes traceback efforts at the preharvest level., (Published 2021 by the International Association for Food Protection.)

Cyclospora cayetanensis is a protozoan parasite that causes foodborne and waterborne diarrheal illness outbreaks worldwide. Most of these outbreaks are associated with the consumption of fresh produce. Sensitive and specific methods to detect C. cayetanensis in agricultural water are needed to identify the parasite in agricultural water used to irrigate crops that have been implicated in outbreaks. In this study, a method to detect C. cayetanensis in water by combining dead-end ultrafiltration (DEUF) with sensitive and specific molecular detection was developed and evaluated. Triplicates of 10-liter agricultural water samples were seeded with 200, 100, 25, 12, and 6 C. cayetanensis oocysts. Surface water samples were also collected in the Mid-Atlantic region. All water samples were processed by DEUF and backflushed from the ultrafilters. DNA was extracted from concentrated samples and analyzed by quantitative PCR (qPCR) targeting the C. cayetanensis 18S rRNA gene. All water samples seeded with 12, 25, 100, and 200 oocysts were positive, and all unseeded samples were negative. Samples seeded with 6 oocysts had a detection rate of 66.6% (8/12). The method was also able to detect C. cayetanensis isolates in surface water samples from different locations of the Chesapeake and Ohio Canal (C&O Canal) in Maryland. This approach could consistently detect C. cayetanensis DNA in 10-liter agricultural water samples contaminated with low levels of oocysts, equivalent to the levels that may be found in naturally incurred environmental water sources. Our data demonstrate the robustness of the method as a useful tool to detect C. cayetanensis from environmental sources. IMPORTANCE is a protozoan parasite that causes foodborne and waterborne outbreaks of diarrheal illness worldwide. These foodborne outbreaks associated with the consumption of fresh produce and agricultural water could play a role in the contamination process. In this study, a method to detect Cyclospora cayetanensis in agricultural water by combining a robust filtration system with sensitive and specific molecular detection was developed and validated by the FDA. The results showed that this approach could consistently detect low levels of C. cayetanensis contamination in 10 liters of agricultural water, corresponding to the levels that may be found in naturally occurring environmental water sources. The method was also able to detect C. cayetanensis contamination in 10 liters of agricultural water, corresponding to the levels that may be found in naturally occurring environmental water sources. The method was also able to detect C. cayetanensis in agricultural water samples, which could be very useful to identify environmental sources of contamination.C. cayetanensis in agricultural water samples, which could be very useful to identify environmental sources of contamination.

While the Food Safety Modernization Act established standards for the use of surface water for produce production, water quality is known to vary over space and time. Targeted approaches for identifying hazards in water that account for this variation may improve growers' ability to address pre-harvest food safety risks. Models that utilize publicly-available data (e.g., land-use, real-time weather) may be useful for developing these approaches. The objective of this study was to use pre-existing datasets collected in 2017 ( N = 181 samples) and 2018 ( N = 191 samples) to train and test models that predict the likelihood of detecting Salmonella and pathogenic E. coli markers ( eaeA, stx ) in agricultural water. Four types of features were used to train the models: microbial, physicochemical, spatial and weather. "Full models" were built using all four features types, while "nested models" were built using between one and three types. Twenty learners were used to develop separate full models for each pathogen. Separately, to assess information gain associated with using different feature types, six learners were randomly selected and used to develop nine, nested models each. Performance measures for each model were then calculated and compared against baseline models where E. coli concentration was the sole covariate. In the methods, we outline the advantages and disadvantages of each learner. Overall, full models built using ensemble (e.g., Node Harvest) and "black-box" (e.g., SVMs) learners out-performed full models built using more interpretable learners (e.g., tree- and rule-based learners) for both outcomes. However, nested eaeA-stx models built using interpretable learners and microbial data performed almost as well as these full models. While none of the nested Salmonella models performed as well as the full models, nested models built using spatial data consistently out-performed models that excluded spatial data. These findings demonstrate that machine learning approaches can be used to predict when and where pathogens are likely to be present in agricultural water. This study serves as a proof-of-concept that can be built upon once larger datasets become available and provides guidance on the learner-data combinations that should be the foci of future efforts (e.g., tree-based microbial models for pathogenic E. coli )., Competing Interests: Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Surface water used for produce production is a potential source of pre-harvest contamination with foodborne pathogens. Decisions on how to mitigate food safety risks associated with pre-harvest water use currently rely on generic Escherichia coli -based water quality tests, although multiple studies have suggested that E. coli levels are not a suitable indicator of the food safety risks under all relevant environmental conditions. Hence, improved understanding of spatiotemporal variability in surface water microbiota composition is needed to facilitate identification of alternative or supplementary indicators that co-occur with pathogens. To this end, we aimed to characterize the composition of bacterial and fungal communities in the sediment and water fractions of 68 agricultural water samples collected from six New York streams. We investigated potential associations between the composition of microbial communities, environmental factors and Salmonella and/or Listeria monocytogenes isolation. We found significantly different composition of fungal and bacterial communities among sampled streams and among water fractions of collected samples. This indicates that geography and the amount of sediment in a collected water sample may affect its microbial composition, which was further supported by identified associations between the flow rate, turbidity, pH and conductivity, and microbial community composition. Lastly, we identified specific microbial families that were weakly associated with the presence of Salmonella or Listeria monocytogenes , however, further studies on samples from additional streams are needed to assess whether identified families may be used as indicators of pathogen presence., (Copyright © 2020 Chung, Weller and Kovac.)

Background: Free-living amoeba (FLA) are widely distributed in different environmental sources. The most genera of the amoeba are Acanthamoeba , Naegleria and Vermamoeba. The most common consequences of the infections in immune-deficient and immuno-competent persons are amoebic encephalitis and keratitis. The aim of this study was to investigate the presence of Acanthamoeba spp. and Naegleria spp., isolated from the main agricultural water canal in Qazvin., Methods: Totally, 120 water specimens were collected and later the specimens were cultured and cloned to identify positive samples. PCR amplification and sequencing were carried out to identify the isolated species as well as the genotypes of amoeba., Results: According to morphological surveys, 41.7% (50/120) of water specimens were positive for FLA. Molecular analysis revealed that 68.6% and 31.4% of Acanthamoeba specimens were identified as T3 and T4 genotypes, respectively. Also, two species of Naegleria named as N. lovaniensis (57.1%) and Naegleria sp. (42.8%) were identified. The results of pathogenicity assays demonstrated that 38.5% of T3 and 61.5% of T4 genotypes of Acanthamoeba were highly pathogenic parasites., Conclusion: The water flowing in the agricultural canal of the area is contaminated with potential pathogenic FLA, therefore, it is recommended that more attention to be paid towards proper treatment of water sources to prevent possible risk of the disease., Competing Interests: Conflict of interest The authors declare that there is no conflict of interest., (Copyright© Iranian Society of Parasitology & Tehran University of Medical Sciences.)

Agricultural water is an important source of foodborne pathogens on produce farms. Managing water-associated risks does not lend itself to one-size-fits-all approaches due to the heterogeneous nature of freshwater environments. To improve our ability to develop location-specific risk management practices, a study was conducted in two produce-growing regions to (i) characterize the relationship between Escherichia coli levels and pathogen presence in agricultural water, and (ii) identify environmental factors associated with pathogen detection. Three AZ and six NY waterways were sampled longitudinally using 10-L grab samples (GS) and 24-h Moore swabs (MS). Regression showed that the likelihood of Salmonella detection (Odds Ratio [OR] = 2.18), and eaeA-stx codetection (OR = 6.49) was significantly greater for MS compared to GS, while the likelihood of detecting L. monocytogenes was not. Regression also showed that eaeA-stx codetection in AZ (OR = 50.2) and NY (OR = 18.4), and Salmonella detection in AZ (OR = 4.4) were significantly associated with E. coli levels, while Salmonella detection in NY was not. Random forest analysis indicated that interactions between environmental factors (e.g., rainfall, temperature, turbidity) (i) were associated with likelihood of pathogen detection and (ii) mediated the relationship between E. coli levels and likelihood of pathogen detection. Our findings suggest that (i) environmental heterogeneity, including interactions between factors, affects microbial water quality, and (ii) E. coli levels alone may not be a suitable indicator of food safety risks. Instead, targeted methods that utilize environmental and microbial data (e.g., models that use turbidity and E. coli levels to predict when there is a high or low risk of surface water being contaminated by pathogens) are needed to assess and mitigate the food safety risks associated with preharvest water use. By identifying environmental factors associated with an increased likelihood of detecting pathogens in agricultural water, this study provides information that (i) can be used to assess when pathogen contamination of agricultural water is likely to occur, and (ii) facilitate development of targeted interventions for individual water sources, providing an alternative to existing one-size-fits-all approaches., (Copyright © 2020 Weller, Brassill, Rock, Ivanek, Mudrak, Roof, Ganda and Wiedmann.)

Agricultural water is considered as one of the main contamination source for produce prior to harvest. The purpose of study was to evaluate the fate of Shiga toxin-producing Escherichia coli (STEC), and generic E. coli in Central Florida agricultural surface water at different temperatures and the potential use of EPA Worst Case water as a standardized media. Cocktails of STEC (O145, O104, O111, O103, O157), and generic E. coli K-12 were inoculated into agricultural surface water samples (non-sterile and sterilized) and EPA Worst Case water, and enumerated for up to 168 days. E. coli was held at 15 and 25 ± 1 °C. Tested microorganisms decreased most rapidly in non-sterile surface water. At day 168, E. coli populations decreased to ≤2.5 log CFU/100 ml in non-sterile surface water and were 4.8 ≤ and ≤ 8.5 log CFU/100 ml in sterile surface water and EPA Worst Case water. Populations were significantly (P ≤ .05) higher in sterile surface water and EPA Worst Case water at all sampling points starting from Day 28. Rate of declines in non-sterile surface waters were between 32.8 and 50 days at both tested temperatures and microorganisms. Addition of cycloheximide to non-sterile surface waters resulted in no significant effect on behavior of E. coli populations. Monitoring generic E. coli (represented by K-12) population changes is a reasonable indicator of STEC survival in agricultural water. EPA Worst Case water is a suitable standard control for surface water microcosms., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

On February 27 to 28, 2018, the Produce Safety Alliance convened a national water summit in Covington, KY to discuss the requirements of the United States Food and Drug Administration's (FDA) Food Safety Modernization Act Standards for the Growing, Harvesting, Packing, and Holding of Produce for Human Consumption (Produce Safety Rule [PSR]). The goals of the meeting were to better understand the challenges growers face in implementing the requirements in Subpart E-Agricultural Water and work collaboratively to develop practical solutions to meet fruit and vegetable production needs while protecting public health. To meet these goals, the summit engaged a diverse group of stakeholders including growers, researchers, extension educators, produce industry members, and regulatory personnel. Key outcomes included defining implementation barriers due to diversity in water sources, distribution systems, commodity types, climates, farm size, and production activities. There was an articulated need for science-based solutions, such as the use of agricultural water system assessments and sharing of federal, state, and regional water quality data, to ensure qualitative and quantitative standards reduce microbial risks. These identified challenges and needs resulted in significant debate about whether reopening the PSR-Subpart E for modification or attempting to address concerns through guidance would provide the best mechanism for alleviating concerns. In addition, training, outreach, and technical assistance were identified as vital priorities once the concerns are formally addressed by FDA. The water summit highlighted the critical need for transparency of FDA's progress on reevaluating the Subpart E requirements to help guide growers' decisions regarding the use of agricultural water., (© 2019 Institute of Food Technologists®.)

Background: Irrigation water disinfection reduces the microbial load but it might lead to the formation and accumulation of disinfection by-products (DBPs) in the crop. If DBPs are present in the irrigation water, they can accumulate in the crop, particularly after the regrowth, and be affected by the postharvest handling such as washing and storage. To evaluate the potential accumulation of DBPs, baby lettuce was grown using irrigation water treated with electrolysed water (EW) in a commercial greenhouse over three consecutive harvests and regrowths. The impact of postharvest practices such as washing and storage on DBP content was also assessed., Results: Use of EW caused the accumulation of chlorates in irrigation water (0.02-0.14 mg L -1 ), and in the fresh produce (0.05-0.10 mg kg -1 ). On the other hand, the disinfection treatment had minor impact regarding the presence of trihalomethanes (THMs) in water (0.3-8.7 μg L -1 max), and in baby lettuce (0.3-2.9 μg kg -1 max)., Conclusions: Disinfection of irrigation water with EW caused the accumulation of chlorates in the crop reaching levels higher than the current maximum residual limit established in the EU legislation for leafy greens. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Salmonella ranks high among the pathogens causing foodborne disease outbreaks. According to the Centers for Disease Control and Prevention, Salmonella contributed to about 53.4% of all foodborne disease outbreaks from 2006 to 2017, and approximately 32.7% of these foodborne Salmonella outbreaks were associated with consumption of produce. Trace-back investigations have suggested that irrigation water may be a source of Salmonella contamination of produce and a vehicle for transmission. Presence and persistence of Salmonella have been reported in surface waters such as rivers, lakes, and ponds, while ground water in general offers better microbial quality for irrigation. To date, culture methods are still the gold standard for detection, isolation and identification of Salmonella in foods and water. In addition to culture, other methods for the detection of Salmonella in water include most probable number, immunoassay, and PCR. The U.S. Food and Drug Administration (FDA) issued the Produce Safety Rule (PSR) in January 2013 based on the Food Safety Modernization Act (FSMA), which calls for more efforts toward enhancing and improving approaches for the prevention of foodborne outbreaks. In the PSR, agricultural water is defined as water used for in a way that is intended to, or likely to, contact covered produce, such as spray, wash, or irrigation. In summary, Salmonella is frequently present in surface water, an important source of water for irrigation. An increasing evidence indicates irrigation water as a source (or a vehicle) for transmission of Salmonella . This pathogen can survive in aquatic environments by a number of mechanisms, including entry into the viable but nonculturable (VBNC) state and/or residing within free-living protozoa. As such, assurance of microbial quality of irrigation water is critical to curtail the produce-related foodborne outbreaks and thus enhance the food safety. In this review, we will discuss the presence and persistence of Salmonella in water and the mechanisms Salmonella uses to persist in the aquatic environment, particularly irrigation water, to better understand the impact on the microbial quality of water and food safety due to the presence of Salmonella in the water environment.

The Produce Safety Rule of the U.S. Food Safety Modernization Act includes restrictions on the use of agricultural water of poor microbiological quality. Mitigation options for poor water quality include the application of an irrigation-to-harvest interval of <4 days; however, dry bulb onion production includes an extended irrigation-to-harvest interval (<30 days). This study evaluated conventional curing practices for mitigating Escherichia coli contamination in a field setting. Well water inoculated with rifampin-resistant E. coli (1, 2, or 3 log CFU/mL) was applied to onion fields (randomized block design; n = 5) via drip tape on the final day of irrigation. Onions remained undisturbed for 7 days and were then lifted to the surface to cure for an additional 21 days before harvest. Water, onions, and soil were tested for presence of rifampin-resistant E. coli. One day after irrigation, 13.3% of onions (20 of 150) receiving the poorest quality water (3 log CFU/mL) tested positive for E. coli; this prevalence was reduced to 4% (6 of 150 onions) after 7 days. Regardless of inoculum level, E. coli was not detected on any onions beyond 15 days postirrigation. These results support conventional dry bulb onion curing practices as an effective strategy to mitigate microbiological concerns associated with poor quality irrigation water.

To establish the association between microbial indicators and the presence of foodborne pathogens in irrigation water, Escherichia coli was enumerated using two quantification methods (plate counts and PMA-qPCR) and presence/absence of pathogenic microorganisms, including five strains from the Shiga toxigenic E. coli (O157:H7, O26, O103, O111 and O145) and Salmonella spp. were evaluated. The results confirmed that surface water can be considered a microbial hazard when used for irrigation. The levels of viable E. coli were very similar to those of cultivable E. coli, except for irrigation water obtained from water reservoirs. Comparison between the E. coli counts in samples positive and negative for the presence of pathogenic bacteria for the evaluated water sources identified E. coli level of 2.35 log cfu/100 mL as a cut-off able to correctly predict positive and negative samples with 93% sensitivity and 66% specificity, respectively. Thus, for the samples with levels of E. coli under 2.35 log cfu/100 mL (e.g., 2.24 log cfu/100 mL) there was a 90% probability that the samples were not contaminated with pathogenic microorganism in locations with similar prevalence. E. coli levels in irrigation water were affected by the ambient temperature confirming that water source and climate conditions should be taken into account by growers when designing a sampling program and the frequency of the monitoring to make a better and more efficient use of their resources., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The U.S. Food and Drug Administration (FDA) has defined standards for the microbial quality of agricultural surface water used for irrigation. According to the FDA produce safety rule (PSR), a microbial water quality profile requires analysis of a minimum of 20 samples for Escherichia coli over 2 to 4 years. The geometric mean (GM) level of E. coli should not exceed 126 CFU/100 mL, and the statistical threshold value (STV) should not exceed 410 CFU/100 mL. The water quality profile should be updated by analysis of a minimum of five samples per year. We used an extensive set of data on levels of E. coli and other fecal indicator organisms, the presence or absence of Salmonella, and physicochemical parameters in six agricultural irrigation ponds in West Central Florida to evaluate the empirical and theoretical basis of this PSR. We found highly variable log-transformed E. coli levels, with standard deviations exceeding those assumed in the PSR by up to threefold. Lognormal distributions provided an acceptable fit to the data in most cases but may underestimate extreme levels. Replacing censored data with the detection limit of the microbial tests underestimated the true variability, leading to biased estimates of GM and STV. Maximum likelihood estimation using truncated lognormal distributions is recommended. Twenty samples are not sufficient to characterize the bacteriological quality of irrigation ponds, and a rolling data set of five samples per year used to update GM and STV values results in highly uncertain results and delays in detecting a shift in water quality. In these ponds, E. coli was an adequate predictor of the presence of Salmonella in 150-mL samples, and turbidity was a second significant variable. The variability in levels of E. coli in agricultural water was higher than that anticipated when the PSR was finalized, and more detailed information based on mechanistic modeling is necessary to develop targeted risk management strategies., (Copyright ©, International Association for Food Protection.)

Aim: The activity of chitosan microparticles (CM) was examined using a matrix of conditions in order to assess the efficacy of CM as a mitigation against various strains of Salmonella enterica in agricultural water., Methods and Results: Different concentrations of CM (0, 0·01, 0·1, 0·2, 0·3% w/v) were examined for antimicrobial activity against log vs stationary phase cells of Salmonella and at different conditions of temperature, salinity and pH. Results showed greatest activity with 0·3% CM at pH 7, 25-37°C without additional of salt. Significant reductions in Salmonella levels were also achieved in natural pond water, although decreases were reduced compared to sterile water. All serotypes were sensitive to CM, with minimal inhibitory concentrations ranging from 0·0031 to 0·0250% w/v. Phylogenic analysis of Javiana strains showed increased resistance appeared in multiple genetic lineages., Conclusion: Conditions demonstrating greatest CM activity were compatible with agricultural practices. Although sensitivity to CM varied among Salmonella strains, all strains were sensitive under conditions examined in this study., Significance and Impact of the Study: This research indicated that CM, a natural compound with minimal environmental impact, could be an effective alternative for mitigating Salmonella in agricultural water applications., (© 2017 The Society for Applied Microbiology.)

Chemical sanitizers may induce no injury (bacteria survive), sublethal injury (bacteria are injured), or lethal injury (bacteria die). The proportion of coliform bacteria that were injured sublethally by chlorine and fungicide mixed with agricultural water (pond water), which was used to dilute the pesticide solution, was evaluated using the thin agar layer (TAL) method. In pure cultures of Enterobacter cloacae , Escherichia coli , and E. coli O157:H7 (representing a human pathogen), the percentage of chlorine-injured cells was 69 to 77% for dilute electrolyzed water containing an available chlorine level of 2 ppm. When agricultural water was mixed with electrolyzed water, the percentage of injured coliforms in agricultural water was 75%. The isolation and identification of bacteria on TAL and selective media suggested that the chlorine stress caused injury to Enterobacter kobei . Of the four fungicide products tested, diluted to their recommended concentrations, Topsin-M, Sumilex, and Oxirane caused injury to coliform bacteria in pure cultures and in agricultural water following their mixture with each pesticide, whereas Streptomycin did not induce any injury to the bacteria. The percentage of injury was 45 to 97% for Topsin-M, 80 to 87% for Sumilex, and 50 to 97% for Oxirane. A comparison of the coliforms isolated from the pesticide solutions and then grown on either TAL or selective media indicated the possibility of fungicide-injured Rahnella aquatilis , Yersinia mollaretii , and E. coli . These results suggest the importance of selecting a suitable sanitizer and the necessity of adjusting the sanitizer concentration to a level that will kill the coliforms rather than cause sanitizer-induced cell injury that can result in the recovery of the coliforms.

Campylobacter species are one of the leading causes of bacterial gastroenteritis in humans worldwide. This twofold study was sought to: i) develop and optimize four single-tube multiplex PCR (mPCR) assays for the detection of six virulence (ciaB, dnaJ, flaA, flaB, pldA and racR), three toxin (cdtA, cdtB and cdtC) and one antibiotic resistance tet(O) genes in thermophilic Campylobacter spp. and ii) apply and evaluate the developed mPCR assays by testing 470 previously identified C. jejuni, C. coli and C. lari isolates from agricultural water. In each mPCR assay, a combination of two or three sets of primer pairs for virulence, antibiotic resistance and toxin (VAT) genes was used and optimized. Assay 1 was developed for the detection of dnaJ, racR and cdtC genes with expected amplification sizes of 720, 584 and 182bp. Assay 2 generated PCR amplicons for tet(O) and cdtA genes of 559 and 370bp. Assay 3 amplified cdtB ciaB, and pldA genes with PCR amplicon sizes of 620, 527 and 385bp. Assay 4 was optimized for flaA and flaB genes that generated PCR amplicons of 855 and 260bp. The primer pairs and optimized PCR protocols did not show interference and/or cross-amplification with each other and generated the expected size of amplification products for each target VAT gene for the C. jejuni ATCC 33291 reference strain. Overall, all ten target VAT genes were detected at a variable frequency in tested isolates of thermophilic Campylobacter spp. where cdtC, flaB, ciaB, cdtB, cdtA and pldA were commonly detected compared to the flaA, racR, dnaJ and tet(O) genes which were detected with less frequency. The developed mPCR assays are simple, rapid, reliable and sensitive tools for simultaneously assessing potential pathogenicity and antibiotic resistance profiling in thermophilic Campylobacter spp. The mPCR assays will be useful in diagnostic and analytical settings for routine screening of VAT characteristics of Campylobacter spp. as well as being applicable in epidemiological studies by providing information that could be related to the risk of human infection., (Copyright © 2016. Published by Elsevier B.V.)

The occurrence and diversity of thermophilic Campylobacter species (C. jejuni, coli, and lari) were studied in water samples from four river basins located across Canada. These basins located in Quebec (Bras d'Henri), Alberta (Oldman), Ontario (South Nation), and British Columbia (Sumas) represented some of the most intensive farming areas in Canada for hog, beef cattle, dairy cattle, and poultry, respectively. This study analyzed 769 water samples collected from 23 monitoring sites with agricultural influence, and four reference sites with limited or no agricultural influence. Water samples were collected bi-weekly over two years and analyzed for Campylobacter using a semi-quantitative minimum probable number (MPN) enrichment protocol. Putative isolates were confirmed by genus- and species-specific multiplex polymerase chain reaction (PCR) assays. A total of 377 (49%) water samples were positive for campylobacters with 355 samples having a cell density ranging from 4 to 4000 MPN L(-1). Campylobacters were more common at agricultural than reference sites in each river basin, although this difference was not significant in the Oldman and South Nation (p > 0.05). Campylobacter was significantly more common in the Bras d'Henri and Sumas (63%) compared to the South Nation (45%) and Oldman (33%) River basins (p < 0.05). C. jejuni, C. coli and C. lari were detected in each river basin, and these species occurred in 45% (n = 168), 34% (n = 128) and 19% (n = 73), of all Campylobacter positive samples, respectively. The remaining Campylobacter positive water samples without these three species (n = 67; 18%) were identified as other Campylobacter species. C. jejuni was the predominant species occurring in the Sumas, Oldman and South Nation River basins. However, in the Bras d'Henri River basin with intensive hog production, C. coli was the predominant species. This study found campylobacters to be common in some agricultural systems with intensive livestock farming activities, and different river basins could have strikingly different profiles of either C. jejuni or C. coli as the predominant waterborne thermophilic Campylobacter species., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)