Summary
New rules proposed under the Food Safety Modernization Act (FSMA) establish monitoring frequencies and Escherichia coli (E. coli) concentrations for characterizing agricultural water quality. In addition to monitoring for E. coli, other strategies for collecting and testing irrigation water can provide farm operators with a better understanding of the quality of water used in crop production. These strategies include collecting source water samples during times of greater potential risk for contamination (e.g., after rain events) and testing for pathogens and alternative water quality surrogates. In this project, ultrafiltration will be used to collect large-volume irrigation water samples from three farms in Georgia to investigate the benefits of collecting such samples for microbial water quality testing. Baseline and precipitation-impacted samples will be collected to enhance the comparison of large- versus small-volume collection procedures. Samples will be tested for traditional indicators of fecal contamination (E. coli and enterococci), alternative surrogates of fecal contamination (F+ coliphages), pathogens (Salmonella, Cryptosporidium and E. coli O157:H7), and analytes that can be used to identify sources of fecal contamination affecting agricultural water quality. This study will result in development of sampling and testing procedures for analysis of large-volume irrigation water samples for alternative microbial water quality parameters.
Technical Abstract
The new Produce Safety and Preventive Controls for Human Food Rules established under the Food Safety Modernization Act (FSMA) identify monitoring frequencies and Escherichia coli concentrations for characterizing agricultural water quality. In addition to monitoring for E. coli, risk-based collection and testing of agricultural source water for pathogens and alternative water quality surrogates can provide farm operators with a better understanding of the quality of water used in crop production. In this project, an emerging water sampling technique—ultrafiltration (UF)—will be used to collect large-volume irrigation water samples from three farms in Georgia to investigate the benefits of collecting large-volume samples for pathogens and alternative microbial water quality parameters. UF and grab samples will be collected at least every month for one year from the primary irrigation pond at each of the three farms. Precipitation-impacted samples will be collected for four rain events to enhance the comparison of large- versus small-volume collection procedures and evaluate this risk-based sampling approach. UF and grab samples will be analyzed for Salmonella, Cryptosporidium, E. coli O157:H7, pathogen surrogates, and indicators of fecal contamination [including “microbial source tracking” (MST) analytes], which can identify potential sources of fecal contamination contributing to elevated microbial levels. Controlled laboratory experiments will also be conducted to determine the effectiveness of UF for recovering Salmonella, Cryptosporidium and Cyclospora cayatenensis from agricultural water. This two-year project is a collaboration between the Centers for Disease Control and Prevention (CDC), University of Georgia (UGA), and Emory University (Emory). UGA staff will collect UF and grab samples from three farms in the area of Tifton, Georgia to enable statistical comparison of microbial detection rates using small-volume and large-volume sampling. UGA staff will provide the samples to CDC and Emory staff, who will process and analyze the samples at CDC in Atlanta, Georgia. Samples will be analyzed for E. coli, enterococci, F+ coliphages, and Salmonella using quantitative culture techniques. Salmonella will also be serotyped to identify potential host-specific serovars. Cryptosporidium will be analyzed by real-time PCR and sequencing to identify Cryptosporidium genotypes and subtypes for risk characterization (potential human infection risks) and fecal source tracking (identification of human and non-human animal hosts in the watershed of the farms). E. coli O157:H7 will be analyzed by real-time PCR and isothermal amplification, in conjunction with propidium monoazide (PMA) to detect the presence of viable cells. Molecular microbial source tracking analytes will include human-specific analytes [Bacteroides (HF183 human marker), human polyomaviruses BK and JC (HpYVs), and Methanobrevibacter smithii ], and mitochondrial markers for domestic animals (dogs, cats), wildlife (birds and deer), and livestock (cow, chicken, horse, pig). This project will result in large-volume water sample collection and testing protocols to enable sensitive detection of pathogens, pathogen surrogates, and fecal source tracking analytes. Produce industry operators and technical consultants can use these tools to better characterize the microbial quality of irrigation water. This project will also provide an evidence base for evaluating seasonal and precipitation-related sampling as risk-based approaches for agricultural water quality monitoring.
Research Objectives
1. Develop DEUF protocols and determine recovery efficiencies for pathogens (Salmonella, C. parvum, and C. cayatenensis) and alternative water quality parameters (e.g., Bacteroides) in 50-L surface water samples of varying quality (e.g., different turbidity levels). Establish protocols for minimizing the effect of inhibitors on the molecular methods used for qPCR and isothermal DNA amplification.
2. Utilize DEUF to determine if seasonality or weather-related events, such as rainfall, affect the occurrence or concentration of pathogens, surrogates and MST indicators in agricultural water.
3. Assess whether utilizing DEUF to collect large volume water samples improves detection and quantitation of target pathogens and pathogen surrogates versus small-volume (1-L) grab samples.
4. Determine if the presence and/or concentration of pathogen surrogates correlates with the occurrence and/or concentration of pathogens in agricultural water.
5. Demonstrate the utility of DEUF to facilitate detection of MST indicators in agricultural water.
Findings & Recommendations
• DEUF procedures were established for agricultural irrigation water applications, and recovery efficiencies for the DEUF method were determined for pathogenic bacteria, human parasites, and human-specific fecal bacteria and viruses from irrigation water.
• The DEUF method had higher detection rates of the low-level microbes, Salmonella and F+ coliphage, compared to grab samples, but minimal impact on concentration estimates. The DEUF method did not adversely affect detection of E. coli and enterococci compared to grab sampling. The DEUF method also increased detection rates of the molecular analytes Cryptosporidium and the Bacteroides Hf183 human-associated microbial source tracking marker.
• DEUF is recommended for advanced monitoring of irrigation water when target analytes are suspected to be present at low concentrations, as with pathogens or alternative indicators such as fecal source tracking markers. • The Bacteroides Hf183 human-associated marker and F+ coliphage were detected more frequently after rain events, suggesting a runoff-related human contamination source.
• E. coli concentration increased after a rain event and was more likely to exceed the geometric mean level, but this increase was not associated with an increase in Salmonella detection or concentration.
• Overall, Salmonella detections were not associated with E. coli concentrations above the geometric mean or statistical threshold value levels, indicating that these microbial water quality thresholds for E. coli were not predictive of Salmonella presence in the irrigation ponds studied during this project. However, increased E. coli and enterococci concentrations (by either grab or DEUF sampling) were associated with increased Salmonella detection and concentration (by DEUF only).
• Neither Cryptosporidium nor any of the MST markers were associated with Salmonella detection.
• Of the 131 subtyped Salmonella isolates collected from the irrigation ponds, approximately half matched PFGE patterns of clinical Salmonella isolates in the PulseNet database, and all isolates cultured from the ponds were serotypes potentially pathogenic to humans.