Enteric viruses as new indicators of human and cattle fecal contamination of irrigation waters

The standards used by the produce industry to detect fecal contamination (by indirectly testing for indicator organisms) in irrigation waters are based on tests developed for drinking waters (rather than surface waters) and include risk threshold levels established by the Environmental Protection Agency for recreational (bathing) waters. There is little scientific evidence that this threshold is meaningful for waters used to irrigate food crops. Thus, it may not be appropriate for determining if there is a risk of contamination of crops irrigated with waters tested in this manner. We will use “next generation” sequencing to determine the identities and relative concentrations of the entire prokaryotic (bacterial) and eukaryotic (e.g., protozoan) communities in water samples to identify microbial species which are present in the samples when the presence of foodborne pathogens has been confirmed. This work will evaluate the accuracy of this threshold and hopefully result in the identification of new species or shifts in the microbial communities which are more reliable indicators of the presence of contamination of fecal irrigation waters and the presence of foodborne pathogens. This will allow the produce industry to make risk-based assessments of water quality to determine when it is safe to irrigate fields.

The use of current recreational water standards of the safety of irrigation water is considered to be an outdated and inaccurate approach. The EPA criteria were not intended to apply to risks associated with irrigation management of edible crops. The development of novel approaches to evaluate the presence or absence of fecal contamination in irrigation waters is needed to provide relevant exposure data for risk assessments for fresh produce. Without accurate exposure data, risk based analysis cannot be conducted and future “risk relevant” standards will be difficult to implement to improve regulatory efforts for produce safety. Recent research has shown that viral targets may provide more conservative and accurate estimates of human and animal fecal contamination. Evaluations of viruses such as pepper mild mottle virus (PMMoV), Aichivirus, the enteroviruses, and human and bovine adenoviruses have shown these viruses to be highly prevalent and abundant in waste water and human and cow fecal samples. In addition, the development of low cost and low sample volume collection methods has yielded efficient and rapid approaches for detecting viruses from environmental water samples. The proposed study has the following specific objectives: 1) to assess the occurrence of bovine polyomavirus, bovine adenovirus , human adenovirus , Aichivirus, Pepper Mild Mottle Virus, and the enteroviruses in irrigation waters of varying quality in Arizona, California, Georgia, and Wisconsin; 2) to evaluate these enteric viruses as novel indicators of fecal contamination of water via side-by-side comparisons with the presence of Salmonella and E. coli (the currently used fecal indicator) in the same water to determine if the presence of these enteric viruses can be correlated with the resence of bacterial pathogens in irrigation waters; and 3) this information will then be used to perform a quantitative microbial risk assessment to determine what levels of fecal contamination from irrigation water pose a public health risk. With these new advances in environmental virus detection, we hope to provide a more accurate quantitative assessment of the presence of both human and cow fecal contamination associated with irrigation water for fresh or ready to eat produce. We will attempt to quantify the level of fecal contamination to attempt to identify the level of fecal exposure. This information will then be used in improved risk based models. This work will inform future efforts with a more scientifically accurate approach to evaluating the use of irrigation water for fresh produce to better inform industry standards and to mitigate risks.

1. Identify an ideal workflow and protocols for using the “negatively charged membrane method” that optimizes cost and time requirements for surveillance of novel viral indicator organisms. 

2. To assess the occurrence of bovine polyomavirus (BPyV), bovine adenovirus (BAdV), human adenovirus (AdV), Aichivirus (AiV), pepper mild mottle virus (PMMoV), and the enteroviruses (EV) in irrigation waters of varying quality in Arizona, Georgia, and Wisconsin using qPCR for application as novel viral indicator organisms in relation to traditional cultural methods among and between irrigation districts. 

3. Based on the presence/absence and relative abundance of novel virus indicators in irrigation waters, we will estimate the amount of fecal contamination present based on the known levels of viral shedding from cow and human fecal contamination sources. 

4. Provide improvements to exposure data regarding risk associated with fecal contamination on fresh produce from irrigation water.

The findings and recommendations resulting from this study include the following: 

• Physical/chemical and microbial parameters can vary significantly between different regions 

• It is important to concentrate samples in order to detect viruses 

• It is feasible to sample small volumes (~3 liters) of irrigation waters to detect indicator viruses that are found in high numbers in human and animal feces. These viruses could be used for the routine monitoring of irrigation water quality 

• The presence of fecal indicator viruses can point to changes in water quality and may correlate with the presence of pathogens 

• PMMoV is readily detected in irrigation water samples (between 36% and 63%) and can be used to estimate the amount of fecal contamination in the water 

• Cattle viruses do not appear to be good fecal indicators in irrigation waters in the regions included in the current study 

• Estimates of fecal contamination obtained with PMMoV can be used to perform a quantitative microbial risk assessment to determine the risks of foodborne illness caused by various pathogens as a result of consuming fresh produce irrigated with contaminated water 

• The QMRA performed in the current study resulted in a conservative estimate of risk. Future studies should try to develop data to fill the gaps between an irrigation event using contaminated water and the survival/persistence of specific pathogens between this event and subsequent harvest, processing, transport, etc.