Scientific Publications - Work Done by Microbiology Reader Bioscreen C

Ravva SV, Stanker LH, Control of Pathogens in Manure and Transfer to Plant Environments, Agricultural Research Service, National Program 108, Food Safety Progress Report 2002 (web version), Section 9: Animal Manure and Wastewater; Food Safety Research Information Office - FSRIO
 

ABSTRACT

Summary Project Aims:

The increasing number of outbreaks of human disease as a result of microbial contamination of meat, poultry, fruits and vegetables is a major concern. Manure, irrigation water, and aerosols are recognized as potential sources of this contamination. More than eighty million tons of manure is produced each year in the U.S. Due to the confinement of many food-producing animals, the manure is concentrated in relatively small areas and is difficult to control. Improperly managed manure can harm the environment and become a serious food safety and public health issue.

The aims of this project incorporate both basic and applied research to help understand the biology, ecology and control of food-borne pathogens in manure, water and air. Project areas include: development of methods for tracking pathogens in manure, compost, soils, irrigation water and aerosols; identifying mechanisms crucial to pathogen survival in manure; and development of new strategies for minimizing pathogens in manure and environments influenced by manure.

Summary Accomplishments During Entire Project:

Our group has developed a number of research directions related to understanding the biology of pathogens in food and environments crucial to food production. These include production of reagents for detection of pathogens, pathogen reporter strains for studies of gene regulation, assays for measuring and tracking pathogens in food, identification of non-toxic anti-microbials, methods for fast and sensitive identification of pathogens, and the role of biofilms in pathogen survival. Specific accomplishments of the Manure Pathogen Team are: 1) development of monoclonal antibodies that are specific for Mycobacterium avium paratuberculosis (MAP) for use in immunomagnetic concentration of this pathogen from environmental samples: 2) development of sensitive real-time sequence detection methods for MAP; 3) determined the influence of aeration and nutrient levels in manure lagoons on the survival of pathogenic E. coli and Salmonella; 4) removal of manure solids from manure lagoons reduce the microbial load that is being re-circulated on dairy farms; 5) pathogen regrowth potential of manure teas depend up on the amendments used; 6) pathogen transfer to plants occur during foliar application of manure teas; and 7) pathogen regrowth in maize rhizosphere differ with cultivars. It is anticipated that other innovative methods for detecting pathogens in manure and other environments will be developed, and new methods for minimizing pathogens in the environment will be developed.

Summary 2002 Accomplishments:

Survival of introduced pathogens in aerated and nonaerated manure water microcosms: The survival of human pathogenic E. coli O157:H7 marked with rifampin resistance is being monitored in aerated and non-aerated manure lagoon water from a California dairy farm with active manure management practices that include separation of manure solids followed by aeration in lagoons. Laboratory scale manure water microcosms aerated with miniaturized circulating aerators provided by our MOU partners were used to monitor the survival and proliferation of pathogens. The populations of E. coli O157:H7 declined rapidly from 105 cells/mL to undetectable levels in less than 1 week. Strain variations were noted for the extent of survival in manure lagoon microcosms. A strain of O157:H7 recently isolated from a dairy survived longer than the isolates from the Albany culture collection. The studies on pathogen survival in manure lagoons are critical in determining the potential for pathogen transmission through crops grown on-site by fertilization and irrigation with manure water. The findings were well received and appreciated by dairymen of Central Valley of California and the manufacturer of circulating aerators. This type of data on pathogen survival in aerated manure lagoons was not available until now.

Detection of Mycobacterium avium paratuberculosis (MAP): Real-time quantitative PCR methods to detect MAP at low numbers, the causal agent of Johne's disease were developed. Oligonucleotide primers and probes encoding the amplification of 50 to 65 base pair fragments of the DNA insertion sequence, IS900, unique for MAP were designed and optimized. Methods were developed to detect the presence of amplicons in both isolated DNA and whole cells of MAP. We anticipate the detection of MAP in environmental samples in less than half-a-day instead of the 2-6 month period by the traditional culture methods.

Pathogen survival under continuous fecal shedding to manure lagoons: The possibility of pathogen re-growth to outbreak proportions by continuous fecal shedding of pathogens in manure water lagoons was evaluated in laboratory microcosms. The survival of E. coli O157:H7 was monitored in manure lagoon waters repeatedly inoculated at weekly intervals for one month to the same population levels of 105 cells/mL. The pathogen failed to establish during the five weekly re-inoculation periods and the numbers declined to undetectable levels between re-inoculation intervals. The practical implication of absence of pathogen re-growth to outbreak levels requires evaluation on dairy farms during active fecal shedding of pathogenic E. coli.

Pathogen transfer to feed crops. We began a project examining factors that influence transfer of E. coli O157:H7 and Salmonella enterica Thompson to crops typically grown by California dairy producers for use as animal feed. These crops are irrigated and/or fertilized with animal waste, and if contaminated waste application may be a means of increasing pathogen numbers on crops. We screened five cultivars each of maize and wheat for relative ability to support growth of pathogens in the rhizosphere (root zone) and phyllosphere (foliage). There were significant differences in pathogen growth on the roots of different cultivars of each species, and there was relatively little transfer of pathogens to leaves. When we evaluated the effect of plant root exudates on bacterial growth in a Microbiology Workstation Bioscreen C system, we found that growth in exudates from different cultivars corresponded to growth in the rhizosphere. Generally, exudates from cultivars that supported high bacterial growth in the rhizosphere also stimulated growth in vitro, and vice versa. The practical implications of this work are 1) cultivars may be selected to reduce the potential risk of pathogen re-growth on dairies, 2) provides evidence of some degree of host specificity for pathogens generally considered to be opportunistic invaders of agroecosystems, and 3) indicates the need to consider host genotype when interpreting work on pathogen: plant interactions.

OSQR/Impact:

Both the planned objectives of determining the factors influencing the survival and re-growth of human pathogens in manure and related environments and designing integrated pathogen control strategies have been addressed during the past year. These studies resulted in developing sensitive methods for environmental detection of Mycobacterium avium paratuberculosis. We and efforts are in progress for evaluating on-site manure management practices for nutrient recycling. Laboratory microcosm based pathogen control strategies are being evaluated.

Projected Research Accomplishments During Next 3 Years:

In year 1, we will develop methods for separation and detection of bacterial pathogens in complex environments related to manure, water and compost. In the first year, we will give priority to evaluating the impact of manure solids removal on pathogen populations in a typical California dairy. This will involve quantification and typing of key pathogens (E. coli, Salmonella, Campylobacter), and monitoring population numbers in manure when it is deposited in free stall lanes, in lane flush water mixed with manure, in manure that is separated, and in wastewater holding lagoons. We will also focus in developing methods for recovering and sampling pathogens in aerosols and fog. We will develop immunomagnetic and molecular methods for sensitive detection of Mycobacterium avium paratuberculosis from manure.

In year 2, we will determine the survival and proliferation of multiple human pathogens in manure and crop environments. Develop methods for recovering and sampling pathogens in aerosols and fog. Screen for novel anti-pathogen compounds suitable for use in manure management. In the second year, we will determine the survival and proliferation of multiple human pathogens in manure and crop environments. Survival of pathogens in aerosols and their resultant deposition on plants will be studied. We will use the compost extract model to evaluate interactions between human pathogens and plant beneficial microorganisms that will likely be added to compost and compost extracts. This includes microorganisms that suppress plant diseases or pests, and microorganisms that improve plant nutrient availability. This model will also be used to isolate and evaluate microorganisms that may have direct antagonistic potential as biocontrol agents for human pathogens.

In year 3, we will develop a prototype strategy for minimizing pathogens in manure and crops treated with manure and compost. In the third year, we will further examine the life cycle of human pathogen transport from manure to crops. Develop a prototype strategy for minimizing pathogens in manure and crops treated with manure and compost. We will further examine the specific interactions between pathogens and feed crops. Specifically, having identified differences among crop cultivars for ability to support pathogen regrowth, we will now examine if all pathogen clones are competent colonizers of feed crops. This will have the practical impact of finding particularly competitive strains that can best serve in models for screening potential control strategies.

Technology Transfer:

We have continued working with a small agricultural structures company in an MOU agreement for evaluating the role of circulation systems in pathogen life cycles in dairy wastewater holding ponds. We have been regularly invited by small companies and California dairy producers to present results on dairy waste management strategies. Our research findings were presented to the Johne's Committee of the US Animal Health Association and members of the California Almond Board. Results are regularly presented at scientific conferences and ARS sponsored meetings.

PROCEEDINGS/ABSTRACTS:

Ravva, S.V., 2001. Environmental fate and transport of Mycobacterium avium partuberculosis. 105th Annual Meeting of the United States Animal Health Association. USAHA Johne's Disease Committee. November 1-8, 2001, Hershey, PA.

Duffy, B., Sarreal, C., Stevenson, R., Ravva, S. and Stanker. L., 2002. Regrowth of pathogenic bacteria in compost teas and risk of transmission to strawberry plants, p. 1142-1149. In F.C. Michel, Jr., R.F. Rynk and H.A.J. Hoitink (ed.), Proceedings of 2002 International Symposium: Composting and Compost Utilization, The JG Press, Inc., Emmaus, PA. (http://www.composting2002.org )

Stanker, L.H. and Ravva, S.V., 2002. Survival of E.coli O157:H7 in aerated dairy manure lagoons, 31st United States-Japan Cooperative Program in Natural Resources (UJNR) Protein Resources Panel Meeting, December 1-7, 2002, Monterey, California.

Stanker, L.H. 2002. Overview of research: Foodborne Contaminants Research Unit, Western Regional Research Center, Agricultural Research Service. 37th Joint Panel Meeting of the United States-Japan Cooperative Program on Development & Utilization of Natural Resources - Joint Panel on Toxic Microorganisms, November 10-16, 2002, Albany, California. "Survival of human pathogens in manure" was the focus of this presentation.

(Summary online)

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Last modified: May 25, 2005