Dispersal and Risk Factors for Airborne Escherichia coli in the Proximity to Beef Cattle Feedlots.

California Leafy Green Products Handler Marketing Agreement (LGMA) established food safety metrics with guidance recommendations of 366 m (1,200 feet) and 1,609 m (1 mile) distances between production fields of leafy greens and a concentrated animal feeding operation (CAFO) containing >1,000 and >80,000 head of cattle, respectively. This study evaluated the effect of these distance metrics and environmental factors on the occurrence of airborneEscherichia coliin proximity to seven commercial beef cattle feedlots located in Imperial Valley, California. A total of 168 air samples were collected from seven beef cattle feedlots during March and April 2020, which were the months implicated in the 2018 Yuma Arizona lettuce outbreak of E. coli O157:H7. The distance between air sampling sites and the edge of the feedlot ranged from ∼0 to ∼2,200 m (∼1.3 mile), with each sample comprised of 1,000 L of processed air taken at a 1.2 m elevation over a 10-minute duration. E. colicolonies were enumerated on CHROMagar ECC selective agar and confirmed with conventional PCR. Meteorological data (air temperature, wind speed, wind direction, relative humidity) were collectedin situ. The prevalence and mean concentration ofE. coliwere 6.55% (11/168) and 0.09 CFU per 1,000 L of air, with positive samples limited to within 37 m (120 ft) of the feedlot.Based on logistic regression, the odds of airborne E. coli detection were associated with little to no wind and close proximity to a feedlot. This pilot study found limited dispersal of airborne E. coli in proximity to commercial feedlots in Imperial Valley, with light-to-no wind and proximity within 37 m of a feedlot significant factor-associated airborne E. coli in this produce-growing region of California.

Low to Zero Concentrations of Airborne Bacterial Pathogens and Indicator E. coli in Proximity to Beef Cattle Feedlots in Imperial Valley, California.

This study characterized the effect of distance from beef cattle feedlots, environmental factors, and climate on the occurrence of airborne bacterial indicators and pathogens. Three hundred air samples were collected over 6 months from five feedlots, with each air sample comprising 6000 L of air. Air samples were processed onto TSB-enriched air filters, qPCR-screened, and then qPCR-confirmed for suspect positive colonies of E. coli O157, non-O157-Shiga-toxin-producing E. coli (STEC), Salmonella, and E. coli. Direct enumeration of E. coli was also collected. Although no bacterial pathogens were qPCR-confirmed for the 300 samples, E. coli was detected in 16.7% (50/300) of samples, with an overall mean concentration of 0.17 CFU/6000 L air. Logistic regression analyses revealed a higher odds of E. coli for samples in close proximity compared to >610 m (2000 ft) distance from feedlots, along with significant associations with meteorological factors, sampling hour of day, and the presence of a dust-generating activity such as plowing a field or nearby vehicular traffic. The lack of bacterial pathogen detection suggests airborne deposition from nearby feedlots may not be a significant mechanism of leafy green bacterial pathogen contamination; the result of our study provides data to inform future revisions of produce-safety guidance.

Wastewater reuse for irrigation of produce: A review of research, regulations, and risks.

The burden of disease caused by the contamination of ready-to-eat produce with common waterborne microbial pathogens suggests that irrigation supplies should be closely monitored and regulated. Simultaneously freshwater resources have become increasingly scarce worldwide while global demand continues to grow. Since the turn of the 20th century with the advent of modern wastewater treatment plants, the reuse of treated wastewater is considered a safe and viable water source for irrigation of ready-to-eat vegetables. However strict, and often costly, treatment regimens mean that only a fraction of the world's wastewater supplies are being put to reuse. The purpose of this review is to explore the available literature on the risks associated with reuse water for ready-to-eat produce production including different approaches to reducing those risks as the demand for reuse water increases. It is not the intent of the authors to determine which methods of treatment should be applied, which pathogens should be considered of greatest concern, or which regulations should be applied. Rather, it is meant to be a discussion of the evolving guidelines governing irrigation with reuse water, potential risks from known pathogens common to produce production and recommendations for improving the adoption of water reuse moving forward. To date, there is little evidence to suggest that adequately treated reuse water poses more risk for produce-related illness or outbreaks than other sources of irrigation water. However, multiple epidemiological and quantitative risk assessment models suggest that guidelines for the use of reuse water should be regionally specific and based on local growing practices, available technologies for wastewater treatment, and overall population health. Though research suggests water reuse is generally safe, the assumptions of risk are both personal and of public interest, they should be considered carefully before water reuse is either allowed or disallowed in produce production environments.

A comparison of amplification methods to detect Avian Influenza viruses in California wetlands targeted via remote sensing of waterfowl.

Migratory waterfowl, including geese and ducks, are indicated as the primary reservoir of avian influenza viruses (AIv) which can be subsequently spread to commercial poultry. The US Department of Agriculture's (USDA) surveillance efforts of waterfowl for AIv have been largely discontinued in the contiguous United States. Consequently, the use of technologies to identify areas of high waterfowl density and detect the presence of AIv in habitat such as wetlands has become imperative. Here we identified two high waterfowl density areas in California using processed NEXt generation RADar (NEXRAD) and collected water samples to test the efficacy of two tangential flow ultrafiltration methods and two nucleic acid based AIv detection assays. Whole-segment amplification and long-read sequencing yielded more positive samples than standard M-segment qPCR methods (57.6% versus 3.0%, p < .0001). We determined that this difference in positivity was due to mismatches in published primers to our samples and that these mismatches would result in failing to detect in the vast majority of currently sequenced AIv genomes in public databases. The whole segment sequences were subsequently used to provide subtype and potential host information of the AIv environmental reservoir. There was no statistically significant difference in sequencing reads recovered from the RexeedTM filtration compared to the unfiltered surface water. This overall approach combining remote sensing, filtration and sequencing provides a novel and potentially more effective, surveillance approach for AIv.

Microbiological safety of popular recreation swimming sites in Central California.

The objective of the study was to assess the microbiological safety of popular recreational swimming sites in Central California. Water samples were collected from eleven monitoring sites across the lower reaches of two watersheds for two consecutive swimming seasons (2012-2013), and levels of indicator and pathogenic microorganisms were determined. Data on ambient weather and water chemistry were collected for analyzing their associations with microorganisms in water. All water samples were positive for indicator E. coli with mean concentrations per site ranging from 3.07 to 216.11 MPN/100 ml in 2012 and 13.4 to 226.97 MPN/100 ml in 2013. Mean E. coli concentrations in 27% and 36% samplings sites exceeded the EPA 2012 Recreational Water Quality Criteria recommended mean concentration of ≤ 126 CFU/100 ml of E. coli, in 2012 and 2013, respectively. Cryptosporidium spp. oocysts were detected in all water samples from all sampling sites, with an overall prevalence of 50% and mean concentrations of 0.08 oocysts/l in 2012 and 0.19 oocysts/l in 2013. Giardia spp. cysts were detected at eight sites, with an overall prevalence of 28.8% and mean concentration of 0.2 cysts/l in both years. The majority of the detected Cryptosporidium spp. oocysts and Giardia spp. cysts appeared damaged under microscopy. E. coli O157:H7 was detected in 9% of water samples, with positive samples limited to three sites. Salmonella spp. were detected in all but one site across the two years, with mean concentrations of 0.94 MPN/l in 2012 and 1.85 MPN/l in 2013. Cryptosporidium spp. oocyst concentrations were negatively associated with 30-day mean wind speed and cumulative precipitation and dissolved oxygen in water. Giardia spp. cyst concentrations were positively associated with turbidity and pH of water and negatively associated with E. coli concentrations and 24-h mean air temperature. Salmonella spp. concentrations were positively associated with 30-day mean air temperature. The occurrence of E. coli O157:H7 was positively associated with previous 30-day cumulative precipitation.

Environmental inactivation and irrigation-mediated regrowth of Escherichia coli O157:H7 on romaine lettuce when inoculated in a fecal slurry matrix.

Field trials were conducted in July-August and October 2012 to quantify the inactivation rate of Escherichia coli O157:H7 when mixed with fecal slurry and applied to romaine lettuce leaves. Lettuce was grown under commercial conditions in Salinas Valley, California. One-half milliliter of rabbit, chicken, or pig fecal slurry, containing an average of 4.05 × 107 CFU E. coli O157:H7 (C0), was inoculated onto the upper (adaxial) surface of a lower leaf on 288 heads of lettuce per trial immediately following a 2.5 h irrigation event. To estimate the bacterial inactivation rate as a function of time, fecal matrix, irrigation and seasonal climate effects, sets of lettuce heads (n = 28) were sampled each day over 10 days and the concentration of E. coli O157:H7 (Ct) determined. E. coli O157:H7 was detected on 100% of heads during the 10-day duration, with concentrations ranging from ≤340 MPN/head (∼5-log reduction) to >3.45 × 1012 MPN/head (∼5-log growth). Relative to C0, on day 10 (Ct = 12) we observed an overall 2.6-log and 3.2-log mean reduction of E. coli O157:H7 in July and October, respectively. However, we observed relative maximum concentrations due to bacterial growth on day 6 (maximum Ct = 8) apparently stimulated by foliar irrigation on day 5. From this maximum there was a mean 5.3-log and 5.1-log reduction by day 10 (Ct = 12) for the July and October trials, respectively. This study provides insight into the inactivation and growth kinetics of E. coli O157:H7 on romaine lettuce leaves under natural field conditions. This study provides evidence that harvesting within 24 h post irrigation has the potential to increase the concentration of E. coli O157:H7 contamination, if present on heads of romaine lettuce; foliar irrigation can temporarily stimulate substantial regrowth of E. coli O157:H7.

Spatiotemporal Variability in Microbial Quality of Western US Agricultural Water Supplies: A Multistate Study.

In 2011, the US Congress passed the Food Safety Modernization Act, which tasks the US Food and Drug Administration to establish microbiological standards for agricultural water. However, little data are available for the microbiological quality of surface water irrigation supplies. During the 2015 irrigation season, we conducted a baseline study on the microbial water quality of large irrigation districts in California ( = 2) and Washington ( 4). Monthly samples ( 517) were analyzed for bacterial indicators (fecal coliforms, enterococci, and ) and pathogens ( spp., O157, and non-O157 Shiga toxin-producing [STEC]). Although there was a high degree of variability (µ ± SD = 59.13 ± 106.0), only 11% of samples (56/517) exceeded 126 colony-forming units (CFU) 100 mL, and only six samples exceeded 410 CFU 100 mL. Two volumes of water were collected for pathogen analysis (1 L and 10 L); prevalence of in 10-L samples (68149) was nearly double of that found in 1-L samples (132/517). We found STEC during ∼9% of sampling events (58/517); serotypes O26 and O45 were the most common at 31 and 26%, respectively. Pathogens were not associated with exceedance of the regulatory threshold, yet the odds of detecting increased approximately threefold (odds ration [O.R.] = 3.14, 0.0001) for every log increase in turbidity. Microbiological outcomes were highly district-specific, suggesting drivers of water quality vary across spatiotemporal scales. The true risk of contamination of produce from irrigation water supplies remains unknown, along with the optimal monitoring strategy to improve food safety.

Spatial and temporal variability of bacterial indicators and pathogens in six California reservoirs during extreme drought.

California has one of the largest systems of surface water reservoirs in the world, providing irrigation water to California's agriculturally productive Central Valley. Irrigation water is recognized as a vehicle for the microbial contamination of raw produce and must be monitored according to new federal regulation. The purpose of this study was to further understanding of the variability of fecal indicator bacteria (Escherichia coli and fecal coliforms) and pathogens (E. coli O157:H7 (O157), non-O157 Shiga toxin-producing E. coli (STEC) and Salmonella) along both horizontal and vertical profiles within California reservoirs. Monthly sampling was conducted in six reservoirs located in the foothills of the Western Sierra Nevada during the summer irrigation season and extreme drought conditions of 2014 (n = 257). Concentrations of fecal indicator bacteria were highly variable between reservoirs (p < 0.05) and along the horizontal profile (p < 0.001) from upstream to downstream, with higher concentrations typically found outside of the reservoirs than within. Though many of the reservoirs were thermally stratified, bacterial concentrations were not associated with water temperature (p > 0.05) or any one particular depth strata (p < 0.05). However, prevalence of Salmonella and STEC (16/70 and 9/70 respectively) was higher in the deep strata than in mid or surface layers. We found no statistical association between samples collected downstream of reservoirs and those from the reservoirs themselves. Continued monitoring and modeling of both bacterial indicators and enteric pathogens are critical to our ability to estimate the risk of surface irrigation water supplies and make appropriate management decisions.

Inactivation of Escherichia coli O157:H7 on Romaine Lettuce When Inoculated in a Fecal Slurry Matrix.

A field trial was conducted in July 2011 to quantify the inactivation rate of Escherichia coli O157:H7 when mixed with fecal slurry and applied to romaine lettuce leaves. Lettuce was grown under commercial conditions in Salinas Valley, CA. One-half milliliter of rabbit fecal slurry, containing 6.3 × 107 CFU of E. coli O157:H7, was inoculated onto the upper (adaxial) surface of a lower leaf on 240 heads of lettuce within 30 min after a 2.5-h irrigation event. Forty-eight romaine lettuce heads were collected per event at 2.5 h (day 0.1), 19.75 h (day 0.8), 43.25 h (day 1.8), 67.25 h (day 2.8), and 91.75 h (day 3.8) postinoculation and were analyzed for the concentration of E. coli O157:H7 (Ct). E. coli O157:H7 was detected on 100% of collected heads in concentrations ranging from 340 to 3.40 × 1010 most probable number (MPN) per head. Enumeration data indicate substantial growth of E. coli O157:H7 postinoculation (2.5 h), leading to elevated concentrations, 1 to 3 log above the starting inoculum concentration (Co). By the end of the 92-h trial, we observed a net 0.8-log mean reduction of E. coli O157:H7 compared with Co; however, after accounting for the substantial bacterial growth, there was an overall 2.3-log reduction by the final sampling event (92 h). On the basis of two different regression models that used either the raw data for Ct or log-transformed values of Ct/Co during the period 2.5 to 91.75 h postinoculation, there was an estimated 76 to 80% reduction per day in bacterial counts; however, more accurate predictions of MPN per head of lettuce were generated by using non-log-transformed values of Ct. This study provides insight into the survival of E. coli O157:H7 transferred via splash from a contaminated fecal source onto produce during irrigation. Moreover, these findings can help generate inactivation times following a potential contamination incident.

Monitoring bacterial indicators of water quality in a tidally influenced delta: A Sisyphean pursuit.

The Sacramento-San Joaquin Delta Estuary (Delta) is the confluence of two major watersheds draining the Western Sierra Nevada mountains into the Central Valley of California, ultimately terminating into San Francisco Bay. We sampled 88 sites once a month for two years (2006-2008) over 87 separate sampling events for a total of 1740 samples. Water samples were analyzed for fecal indicator bacteria (Escherichia coli, enterococci and fecal coliforms), and 53 other physiochemical, land use, and environmental characteristics. The purpose of the study was to create a baseline of microbial water quality in the Delta and to identify various factors (climatic, land use, tidal, etc.) that were associated with elevated concentrations of indicator bacteria. Fecal indicator bacteria generally had weak to modest relationships to environmental conditions; the strength and direction of which varied for each microbial indicator, drainage region, and across seasons. Measured and unmeasured, site-specific effects accounted for large portions of variance in model predictions (ρ=0.086 to 0.255), indicating that spatial autocorrelation was a major component of water quality outcomes. The effects of tidal cycling and lack of connectivity between waterways and surrounding landscapes likely contributed to the lack of association between local land uses and microbial outcomes, though weak associations may also be indicative of mismatched spatiotemporal scales. The complex nature of this system necessitates continued monitoring and regular updates to statistical models designed to predict microbial water quality.

Multistate Evaluation of Microbial Water and Sediment Quality from Agricultural Recovery Basins.

Agricultural recovery basins are an important conservation practice designed to provide temporary storage of sediment and water on farms before low-volume discharge. However, food safety concerns have been raised regarding redistribution of captured sediment and water to fields used for human food production. The purpose of this study was to examine the potential microbiological risk that recovery basins may contribute to nearby produce fields and to evaluate characteristics that may influence or mitigate those risks. Water and sediment samples were collected from participating farms in three states and evaluated for bacterial indicators and pathogens over several months. Overall, 45% ( = 48) of water samples and less than 15% ( = 13) of sediment samples were positive for spp. In water samples, the occurrence of was positively associated with the use of surface water as a source of irrigation compared with groundwater as well as log-scale increases in concentration. In sediment samples, was associated with basin location (region) and basin fill levels. Sediment exposed to drying during dewatering had lower concentrations of indicator and a lower proportion of positives than submerged sediment from the same pond. Surrounding landscape characteristics, including vegetative coverage, proximity to livestock operations, and evidence of wildlife, were not correlated with pathogen occurrence in either sediment or water samples, suggesting that although habitat surrounding ponds may be an attractant to wildlife, those features may not contribute to increased pathogen occurrence in agricultural recovery basins.

Fecal Indicator and Pathogenic Bacteria and Their Antibiotic Resistance in Alluvial Groundwater of an Irrigated Agricultural Region with Dairies.

Surveys of microbiological groundwater quality were conducted in a region with intensive animal agriculture in California, USA. The survey included monitoring and domestic wells in eight concentrated animal feeding operations (CAFOs) and 200 small (domestic and community supply district) supply wells across the region. was not detected in groundwater, whereas O157:H7 and were each detected in 2 of 190 CAFO monitoring well samples. Nonpathogenic generic and spp. were detected in 24.2% (46/190) and 97.4% (185/190) groundwater samples from CAFO monitoring wells and in 4.2% (1/24) and 87.5% (21/24) of CAFO domestic wells, respectively. Concentrations of both generic and spp. were significantly associated with well depth, season, and the type of adjacent land use in the CAFO. No pathogenic bacteria were detected in groundwater from 200 small supply wells in the extended survey. However, 4.5 to 10.3% groundwater samples were positive for generic and . Concentrations of generic were not significantly associated with any factors, but concentrations of were significantly associated with proximity to CAFOs, seasons, and concentrations of potassium in water. Among a subset of and isolates from both surveys, the majority of (63.6%) and (86.1%) isolates exhibited resistance to multiple (≥3) antibiotics. Findings confirm significant microbial and antibiotic resistance loading to CAFO groundwater. Results also demonstrate significant attenuative capacity of the unconfined alluvial aquifer system with respect to microbial transport.

Transfer of Escherichia coli O157:H7 from simulated wildlife scat onto romaine lettuce during foliar irrigation.

A field trial in Salinas Valley, California, was conducted during July 2011 to quantify the microbial load that transfers from wildlife feces onto nearby lettuce during foliar irrigation. Romaine lettuce was grown using standard commercial practices and irrigated using an impact sprinkler design. Five grams of rabbit feces was spiked with 1.29 × 10(8) CFU of Escherichia coli O157:H7 and placed - 3, - 2, and - 1 days and immediately before a 2-h irrigation event. Immediately after irrigation, 168 heads of lettuce ranging from ca. 23 to 69 cm (from 9 to 27 in.) from the fecal deposits were collected, and the concentration of E. coli O157:H7 was determined. Thirty-eight percent of the collected lettuce heads had detectable E. coli O157:H7, ranging from 1 MPN to 2.30 × 10(5) MPN per head and a mean concentration of 7.37 × 10(3) MPN per head. Based on this weighted arithmetic mean concentration of 7.37 × 10(3) MPN of bacteria per positive head, only 0.00573% of the original 5 g of scat with its mean load of 1.29 × 10(8) CFU was transferred to the positive heads of lettuce. Bacterial contamination was limited to the outer leaves of lettuce. In addition, factors associated with the transfer of E. coli O157:H7 from scat to lettuce were distance between the scat and lettuce, age of scat before irrigation, and mean distance between scat and the irrigation sprinkler heads. This study quantified the transfer coefficient between scat and adjacent heads of lettuce as a function of irrigation. The data can be used to populate a quantitative produce risk assessment model for E. coli O157:H7 in romaine lettuce to inform risk management and food safety policies.

Quantifying the sensitivity of scent detection dogs to identify fecal contamination on raw produce.

Consumption of raw produce commodities has been associated with foodborne outbreaks in the United States. In a recent Centers for Disease Control and Prevention report outlining the incidence of food-related outbreaks from 1998 to 2008, produce of all kinds were implicated in 46% of illnesses and 23% of deaths. Methods that quickly identify fecal contamination of foods, including produce, will allow prioritization of samples for testing during investigations and perhaps decrease the time required to identify specific brands or lots. We conducted a series of trials to characterize the sensitivity and specificity of scent detection dogs to accurately identify fecal contamination on raw agricultural commodities (romaine lettuce, spinach, cilantro, and roma tomatoes). Both indirect and direct methods of detection were evaluated. For the indirect detection method, two dogs were trained to detect contamination on gauze pads previously exposed to produce contaminated with feces. For the direct detection method, two dogs were trained to identify fecal contamination on fresh produce. The indirect method did not result in acceptable levels of sensitivity except for the highest levels of fecal contamination (25 g of feces). Each dog had more difficulty detecting fecal contamination on cilantro and spinach than on roma tomatoes. For the direct detection method, the dogs exhibited >75% sensitivity for detecting ≥0.25 g of feces on leafy greens (cilantro, romaine lettuce, and spinach) and roma tomatoes, with sensitivity declining as the amount of feces dropped below 0.025 g. We determined that use of a scent detection dog to screen samples for testing can increase the probability of detecting ≥0.025 g of fecal contamination by 500 to 3,000% when samples with fecal contamination are rare (≤1%).