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United States Department of Agriculture

Agricultural Research Service

Research Project: ENVIRONMENTAL AND GENETIC FACTORS AFFECTING PATHOGEN PERSISTENCE IN ANIMAL WASTE AND TRANSFER TO CROPS
2006 Annual Report


1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
Americans suffer from over 75 million cases of food-borne illnesses each year, resulting in over 5,000 deaths. Financial losses associated with human and animal food-borne disease are estimated to exceed $20 billion annually. Many human and animal food-borne illnesses are caused by the consumption of foods that were contaminated by animal waste containing pathogenic bacteria including E. coli, Salmonella, Campylobacter, and Mycobacterium avium subsp. paratuberculosis (MAP).

Confined animal feeding operations (CAFOs), including dairies and beef feed lot operations, are often located near crops, where the animal manure is used as a low cost fertilizer. If disease-causing bacteria are present in the manure, it is possible that the crops will become contaminated, leading to food-borne illness. One way contamination may occur is from dust generated by waste handling, which may cause disease either directly by ingestion or indirectly through the contamination of crops. In addition to disease, animal waste generates large amounts of volatile organic chemicals, ammonia, methane, and hydrogen sulfide that cause nuisance odor and air quality problems in rural farming areas.

To prevent foodborne illnesses associated with the use of animal waste as a fertilizer we are developing new treatment methodologies for the treatment of waste before it is composted or stored in liquid wastewater holding lagoons. These methodologies include aerobic and anaerobic digestion technologies that not only have the potential to reduce pathogens but also decrease emissions of volatile chemicals into the atmosphere. We are also evaluating the potential of pathogen transmission via aerosols using state of the art air sampling techniques. Finally, using new DNA microarray technology, we are identifying the genes in MAP bacteria that allow them to persist in the environment and cause disease.

This research is administered under National Program 108 Food Safety.


2.List by year the currently approved milestones (indicators of research progress)
Year 1 (FY06) Characterize gene expression of MAP in vitro. Characterize bacterial communities in waste after aerobic and anaerobic digestion. Develop culture and immunomagnetic methods for detection of pathogens in aerosol/fog. Evaluate aerosol collection devices.

Year 2 (FY07) Characterize gene expression of MAP in vivo. Study effect of Monensin addition on microbial population and waste chemistry in aerobic and anaerobic manure digestion. Use cytometry to characterize protozoan hosts for MAP. Validate aerosol samplers with trapping media. Validate coupled immunomagnetic enrichment with quantitative PCR for detection of MAP in aerosols. Use polymerase chain reaction with differential gradient gel electrophoresis of 16S RNA genes for characterization of predominant members of the bacterial community. Compare microbial community of soil and corn plants grown with manure versus chemical fertilizers.

Year 3 (FY08) Generate green fluorescent protein-expressing MAP, infect protozoan hosts, and maintain in culture. Characterize gene expression of MAP in protozoan hosts. Compare microbial communities and profile waste chemistry in dairy manure from animals fed with low versus high protein diet. Determine susceptibility of crops to pathogen contamination and colonization by aerosol transmission of pathogens. Characterize time-of-day fluctuations in aerosol (fog) transmission of pathogens.

Year 4 (FY09) Determine whether protozoan hosts facilitate multiplication of MAP. Determine whether aerobic/anaerobic digestion reduce pathogen load in animal waste. Characterize seasonal fluctuations of bacteria in aerosols. Compare culturable and non-culturable bacterial species in aerosols Compare microbial community of soil and cantaloupe plants grown with manure versus chemical fertilizers.

Year 5 (FY10) Determine changes in MAP virulence caused by protozoan hosts. Track movement of bacteria from aerosols to crops. Characterize survival of bacteria on crops.


4a.List the single most significant research accomplishment during FY 2006.
Aerobic and anaerobic treatment of dairy waste: Dairy waste is a visible and contentious source of pathogens and volatile compound emissions. We compared the effect of aerobic and anaerobic digestion with subsequent storage in simulated wastewater lagoons on the bacterial population, chemical composition, and volatile compound emissions as compared to no treatment before storage. Our results show that both treatment methods substantially altered the bacterial population of waste, and reduced the levels of nutrients and volatile compound emissions. These methods may be adopted by CAFOs to reduce the spread of foodborne illness and decrease noxious odor. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statement 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships.


4b.List other significant research accomplishment(s), if any.
Testing for bacteria in dairy environments. It is unclear which disease-causing microbes are present in various places on a dairy. We characterized manure, drinking trough water and air samples from a Sonoma dairy for total aerobic bacteria, fungi, coliforms, and the pathogens E. coli O157:H7 and Salmonella. In aerosols we measured about 10,000 bacteria per liter of air, but only 1-10% of these were alive. And we were unable to detect pathogens in any samples. Thus if pathogen contamination occurs via aerosol, regrowth from extremely low levels is necessary for disease transmission. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statements: 1.1.1 Methodology; 1.1.2 Epidemiology; 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships; and 1.2.1 Detection and Validation.

MAP gene expression. Although known to be responsible for Johne’s disease, MAP remains poorly characterized. Using DNA microarray technology we identified genes that may be involved in the persistence of MAP in dairy wastewater environments. This information should prove useful in preventing spread of MAP and Johne’s disease. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statements: 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships; and 1.2.5 Omics.

Potential MAP virulence gene discovered. Because MAP is unusually slow-growing and difficult to study in the lab, little is known about how it causes disease. We have identified a gene in MAP that may be involved in virulence. We then constructed a mutant strain of MAP that has a deletion in this gene. To determine the role of this gene we will determine whether this mutant is capable of causing disease. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statements: 1.1.1 Methodology; 1.1.2 Epidemiology; 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships.


4c.List significant activities that support special target populations.
None.


4d.Progress report.
None.


5.Describe the major accomplishments to date and their predicted or actual impact.
Aerobic and anaerobic treatment of dairy waste. Dairy waste is a visible and contentious source of pathogens and volatile compound emissions. We compared the effect of aerobic and anaerobic digestion with subsequent storage in simulated wastewater lagoons on the bacterial population, chemical composition, and volatile compound emissions as compared to no treatment before storage. Our results show that both treatment methods substantially altered the bacterial population of waste, and reduced the levels of nutrients and volatile compound emissions. These methods may be adopted by CAFOs to reduce the spread of foodborne illness and decrease noxious odor. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statement 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships.


6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
None.


7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
J. McGarvey, 2006. “Why should you want purple photosynthetic bacteria in your wastewater lagoons?” Presented to: The California Diary Campaign members and other stakeholders, Modesto, CA.


Review Publications
Mcgarvey, J.A., Barak Cunningham, J.D., Miller, W.G. 2005. Surface sanitization of cantaloupes using thymol/cinnamaldehyde. California Melon Research Board Annual Meeting, November 20-23, 2005, Hermosilla, MX.

Ravva, S.V. 2006. Nutrients and wastewater components influence the survival and growth of pathogenic escherichia coli o157:h7 in dairy lagoons [Abstract]. 2nd Federation of European Microbioilogy Societies (FEMS) Congress of European Microbiologists. Poster #P.ENV.109.

Ravva, S.V., Sarreal, C.Z., Duffy, B., Stanker, L.H. 2006. Survival of escherichia coli o157:h7 and salmonella enterica in manure waste water from dairy lagoons. Journal of Applied Microbiology. 101(2006):891-902

Ravva, S.V., Stanker, L.H. 2005. Real-time quantitative pcr detection of mycobacterium avium subsp. paratuberculosis and differentiation from other mycobacteria using sybr green and taqman assays. Journal of Microbiological Methods. 63(2005) 305-317.

Last Modified: 12/22/2014
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