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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
2008 Annual Report


1a.Objectives (from AD-416)
1: The identification of factors contributing to the persistence and horizontal transfer of MAP and other pathogens on dairy farms. We will examine the role alternate protozoan hosts play in MAP persistence and horizontal transfer. We will also perform functional genomic analysis of MAP in the environment to elucidate clues about the strategies employed by this organism for environmental survival. 2: The development of methodologies for the reduction of pathogens in waste. Two of the most common waste treatment methodologies are aerobic and anaerobic digestion, but little is known about the effect these processes have on the microbial population structure, pathogen levels, and volatile compound missions. The bacterial community structure, chemical composition and volatile compounds emitted from animal waste, aerobic and anaerobic digesters, and holding tanks receiving effluent will be characterized. We will also analyze the effect of diet and dietary supplements on the microbial population structure, pathogen levels, and volatile compound emissions from animal waste and animal waste treatment systems. 3: Pathogen transfer from manure and CAFOs (confined animal feeding operations) to crops. We will use high volume aerosol collection and agar impaction devices to collect bacteria from aerosols near CAFOs. Aerosol samples will be evaluated for pathogens using culture and non-culture based methods. We will examine the bacterial community structure and pathogen levels on crops fertilized with waste using culture and non-culture based methods and compare them to those of plants fertilized with only chemical fertilizers. We will study the colonization of crop plants by Salmonella enterica grown in soil amended with manure spiked with pathogens using standard culture and quantitative PCR methods.


1b.Approach (from AD-416)
The goal of this project is to reduce the incidence of foodborne illnesses by reducing the levels of pathogens in animal waste and in CAFO environments. In the next five years we will identify the locations in CAFOs that serve as reservoirs for pathogenic bacteria such as MAP, develop low cost pathogen abatement strategies for contaminated animal waste, examine the effect of diet on pathogen load and evaluate the risk of pathogen transfer from CAFOs to surrounding crop plants via aerosols and from fertilizing crop plants with contaminated manure. FORMERLY: 5325-32000-002-00D; 5325-42000-023-00D. 5325-42000-028-00D combined into this project (4/04). Replaces 5325-32000-005-00D (2/06).


3.Progress Report
We refined our microbiological methods for aerosol collection and processing via immunomagnetic bead capture. This let us conduct fairly large scale repetitive operations, studying aerosol transport of bacteria at various sites and seasons. Although we were unable to detect pathogens in naturally-occurring aerosol, we found bacteria that appeared characteristic for respective dairies. We continued to work on control of E. coli O157:H7 and other pathogens in animal waste by protozoan predation. We characterized common protozoans in manure lagoon water using rDNA sequence analysis. And using fluorescence activated cell sorting we directly observed protozoan predation of E coli bearing a fluorescent marker protein (green fluorescent protein). Our work on Mycobacterium avium subsp. paratuberculosis (MAP) includes looking for genes required by this pathogen for induction of intestinal disease. In FY08 we created a mutant MAP that survives poorly in a cell culture model of intestinal tissue. This project relates to pathogens, toxins and chemical containments prehavest NP108. Specifically addressess problem statement 1.1.1 Methodology; 1.1.3 Ecology, Host Pathogen and Chemical Containments relationships and 1.1.4 Intervention Strategies.


4.Accomplishments
1. Protozoa in wastewater consume E. coli O157:H7. E. coli O157:H7 is the causative agent for 48 produce-related and 144 beef-related outbreaks. ARS scientists in the Foodborne Contaminants Research Unit in Albany, CA observed a rapid consumption of E. coli O157:H7 by protozoa in wastewater from dairy lagoons. These protozoa were characterized using 18S rRNA sequencing. An understanding of the biological and environmental factors responsible for the survival of E. coli O157:H7 will help in developing on-farm pathogen control strategies. 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; 1.1.3 Ecology, Host Pathogen and Chemical Contaminants relationships; and 1.1.4 Intervention Strategies.

2. Transport of bacterial communities from dairies through aerosols. Concentrated animal feeding operations (CAFO) that generate large amount of animal waste that is treated on-site may aerosolize foodborne pathogens that can be transported via wind and fog. No known foodborne pathogens were detected from air samples collected from two dairies by ARS scientists in the Foodborne Contaminants Research Unit in Albany, CA with different manure management practices. However, the predominant bacteria found in aerosols from these dairies are from different classes (Betaproteobacteria vs Clostridia). Thus, bacteria specific to each dairy may be used as tracers for detecting the source of pathogens that are transported to crops grown in proximity. This accomplishment may aid in developing on-farm foodborne pathogen control. This research 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; 1.1.3 Ecology, Host Pathogen and Chemical Contaminants relationships; and 1.1.4 Intervention Strategies.

3. A Mycobacterium avium subsp. paratuberculosis mutant with reduced virulence. Mycobacterium avium subsp. paratuberculosis (MAP) causes disease in dairy cattle and is shed in milk. Drinking contaminated milk results in an infection, in which the bacteria grow inside macrophages (a type of white blood cell). In collaboration with scientists at California Dairy Campaign, ARS scientists in the Foodborne Contaminants Research Unit in Albany, CA observed that the MAP delta MAP0482-3 mutant survived poorly in laboratory-grown macrophages. We are currently collaborating with scientists at the University of Minnesota to examine the genes regulated by MAP0482-3 and are also beginning trials to use this strain as a live vaccine to prevent the infection of dairy cattle by this organism. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Pre-harvest. Specifically this research addresses Problem Statements: 1.1.1 Methodology; and 1.1.3 Ecology, Host Pathogen and Chemical Contaminants relationships; and 1.1.4 Intervention Strategies.

4. Flow-cytometric detection of protozoa that consume pathogenic bacteria. Protozoa in the environment are known to consume pathogenic bacteria and are believed to contribute to their survival and distribution. Understanding this relationship requires a method to locate and identify such protozoa. ARS scientists in the Foodborne Contaminants Research Unit in Albany, CA labeled a strain of disease-causing E. coli bacteria with a green fluorescent protein to identify and sort protozoa that ingest these bacteria. Application of this method to environmental samples should allow us to identify disease harboring protozoa and may aid in on-farm foodborne pathogen control. 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; 1.1.3 Ecology, Host Pathogen and Chemical Contaminants relationships; and 1.1.4 Intervention Strategies.


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of Non-Peer Reviewed Presentations and Proceedings1

Review Publications
Hernlem, B.J., Ravva, S.V., Sarreal, C.Z. 2007. Identification and Sorting of Bacterivorous Tetrahymena by Flow Cytometry. Journal of Environmental Monitoring. 9:1317-1322.

Romano, R.T., Zhang, R., Teter, S., Mcgarvey, J.A. 2009. The Effect of Enzyme Addition on Anaerobic Digestion of Jose Tall Wheat Grass. Bioresource Technology. 100(20):4564-4571.

Duffy, B., Ravva, S.V., Stanker, L.H. 2008. Cantaloupe Cultivar Differences as Opportunistic Hosts for Human Pathogenic Escherichia coli 0157:H7 and Salmonella. European Journal of Horticultural Science.73(2):73-75.

Last Modified: 7/24/2014
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