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The proposed study has 3 objectives that focus on crucial areas of preharvest food safety where data is lacking. The first is to determine the sensitivity of human pathogenic viruses to commonly used pesticides and fertilizers. The second is to determine the survival of human pathogenic viruses in bacterial biofilms that could occur in an agricultural environment. Lastly, the information gained from objectives 1 and 2 will be used to identify potential reservoirs, routes of transmission, and develop methods to reduce transmission of these agents to agricultural crops.
NON-TECHNICAL SUMMARY: Contamination of ground waters and food supplies by pathogenic microorganisms is common in many areas of the United States and public health concerns are increasingly focused on viruses. This project examines the survival of viruses in specific agricultural applications including pesticides and fertilizers and biofilms. <P>APPROACH: Viruses vary greatly in their surface properties and survival characteristics. The selection of viruses for this project was based on several criteria: (1) their potential to cause serious diseases in humans, (2) limited information available on their environmental fate, and (3) implication in food- and water-borne outbreaks. Norovirus and hepatitis A virus (HAV) are the two most widely reported food- and water-borne viruses. HAV and other enteric viruses are primarily transmitted through fecal contamination and common-source epidemics from contaminated foods and water are well-documented. Norovirus has a high prevalence as an environmental contaminant as shown during the hurricane Katrina aftermath in Louisiana and Houston, where a large number of evacuees contracted gastroenteritis associated with Norovirus. Aichi virus is an infectious virus associated with consumption of contaminated oysters in Asia, Europe, and South America. It may be possible that Aichi virus has caused disease in North America as well, but was not detected. Symptoms are similar to Norovirus, and young healthy people are most infected. Specifically the four viruses selected are representative of two virus families (2 from each family as family members do not necessarily act alike). Studies will include two picornaviruses (hepatitis A virus and Aichi virus) and two important caliciviruses as Norovirus surrogates (the most closely related murine Norovirus and the traditional surrogate feline calicivirus). Norovirus cannot be laboratory cultured and the comparison of the murine Norovirus and feline calicivirus is an important aspect of this work, as to date only one date has studied them simultaneously. Viruses will be propagated and analyzed in mammalian cell culture. At least 5 different broad spectrum pesticides will be evaluated. Fertilizers high in either nitrogen or phosphorous will also be evaluated. A point will be made to evaluate those chemicals which have previously been tested on pathogenic bacteria for which there was differential survival, and in order to have the complete spectrum of information. Virus survival will be monitored in liquid preparations and in land applications. In a related project, virus survival will be monitored in bacterial biofilms. Biofilms of Escherichia coli O157:H7 and Pseudomonas aeruginosa will be grown on stainless steel and plastic coupons and spinach leaves. Bacterial cultures will be grow for 24 hours at 4C in 50ml centrifuge tubes on coupons and plant materials as previously described. The experimental goal is to determine how and if viruses are incorporated into a growing bacterial biofilm. Viruses will be added at different timepoints (0, 24, 48, 96, 168 hr) and virus survival will be tested periodically as described above (every 3-6 days). Biofilms will be scraped from their support surface, bacterial cells pelleted, and viruses analyzed by PCR (for presence) and cell culture (for infectivity) as described above.