Summary of Awards to Date

Developing Cross-Assembly Phage as a Viral Indicator for Irrigation Waters


Jan. 1, 2017 - Dec. 31, 2018

Award Number


Funding Agency

Center for Produce Safety

Amount Awarded



Kyle Bibby, Ph.D.
University of Pittsburgh


Ensuring high-quality irrigation water is necessary to protect the public when consuming minimally processed produce. The highest risk from exposure to contaminated water is due to viruses; however, water quality is currently monitored using bacteria that are poor representatives of viruses. All previous viral indicators are limited by a low abundance (i.e. difficult to detect) in the environment. Recently, a bacteriophage (virus that infects bacteria) named ‘cross-assembly phage’ (crAssphage) was discovered that is more abundant than all other bacteriophages in the human gut combined. Investigations in the PI’s research group have shown crAssphage to be highly abundant in sewage. As crAssphage is a virus, it will be a better representative of viral contamination in the environment. In this investigation, I propose to sample irrigation water samples and measure crAssphage, viruses, and indicators in these samples to demonstrate the correlation of crAssphage and pathogens. I also proposed to determine how much sample volume is necessary to accurately measure crAssphage. The development of this viral monitoring tool, catalyzed by funding this project, will enable risk-managers to have an accurate and abundant indicator of viral contamination. This will ultimately provide greater protection of public health and improve consumer confidence in produce consumption.

Technical Abstract

Surface irrigation water represents a potential transmission source of viruses to produce - ensuring irrigation water quality is critical to limiting produce-associated disease transmission. The greatest infectious risk from exposure to contaminated water is due to viral pathogens. All existing indicators of viral pollution are challenged by low abundance in the environment. Irrigation waters are typically monitored for biological quality utilizing fecal indicator bacteria (FIB). FIB are inadequate representatives of viruses due to differential occurrence, persistence, source, and abundance in the environment. Recently, a highly abundant bacteriophage, named cross-assembly phage (abbreviated as crAssphage), was found in the human gut. Initial investigations in the PI’s group have found this viral indicator to be highly abundant in sewage and specific to sewage (i.e. not identified in animal feces). An end-point PCR assay for crAssphage has been developed and quantitative PCR development is underway and expected to be completed by project initiation. This assay is at least as rapid as existing bacterial measurement assays and exhibits a comparable limit of detection in sewage. To evaluate the suitability of crAssphage in irrigation waters, I propose to sample irrigation waters and measure crAssphage, and existing pathogens and indicators. This will enable the correlation of crAssphage with pathogens and existing indicators that are used for risk-based decision making. The limit of detection of crAssphage in irrigation waters will also be determined. A two-tiered sampling approach will be used to provide adequate depth and breadth in sampling. Surface water irrigation sources with a high potential for sewage contamination will be specifically targeted. First, samples in South-Central Pennsylvania will be collected from farms utilizing surface water irrigation sources monthly for two summers (>100 samples). Second, samples will be collected from surface water irrigation sources in Arizona and central California yearly for two growing seasons (20-30 samples). Impacted irrigation sources will be specifically targeted. CrAssphage, viruses, and FIB will be measured by PCR and culture and a correlation analysis will be conducted to demonstrate the suitability of crAssphage as an indicator of viral contamination. The crAssphage limit of detection in irrigation waters will also be determined to inform necessary sampling volume. Ultimately, support of this project will develop a tool that is not currently available to growers or decision makers – an abundant and sewage-specific indicator of viral contamination. This tool will enable a risk-based decision process that includes a more accurate measure of viral pathogens.

Research Objectives

  1. Collect irrigation water samples.

Irrigation waters will be collected and processed from western Pennsylvania (high sample coverage), as well as Arizona and California (once annual sampling). We will specifically target sampling impacted surface waters utilized for irrigation. Additional regions and sampling sites will be explored for sampling.

  1. Measure and determine correlation of crAssphage, viral pathogen, and FIB levels in irrigation water samples.

Previously developed methods for crAssphage detection, viral pathogens, and FIB will be used on collected irrigation water samples. A statistical analysis will then be employed to determine the correlation between crAssphage, pathogens, and existing indicators.

  1. Determine crAssphage limit of detection.

A critical question in the evaluation of crAssphage as a viral indicator for irrigation waters is the amount of sample volume necessary. To address this question, the limit of detection will be determined.