Scientific Publications - Work Done by Microbiology Reader Bioscreen C

Ravva SV, Duffy B and Stanker LH, Control of Pathogens in Manure to Transfer to Plant Environments, Agricultural Research Service, National Program 108, Food Safety Progress Report 2001 (web version); Food Safety Research Information Office - FSRIO
 

SUMMARY

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 to all US citizens. Manure, irrigation water, and bioaerosols are recognized as potential sources of this contamination. More than eighty million tons of manure is produced each year in the United States. 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 and 2) development of sensitive real-time sequence detection methods for MAP. 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 produced
 

Summary FY-2001 Accomplishments:

Immunological and molecular detection methods for MAP: Because of the difficulty in culturing MAP (2-6 months to confirm presence), Mycobacterium avium paratuberculosis, the causal agent of Johnes disease, there are no studies on transport, survival and proliferation of this debilitating pathogen. Rapid, sensitive methods for detection of MAP by immunomagnetic separation and real-time detection of DNA insertion element (IS900) are in advanced stages of development. Monoclonal antibodies specific for various strains of MAP were produced. These antibodies do not cross react with organisms of other species of mycobacteria or closely related genera. The antibodies will be used for immunomagnetic separation of environmental isolates of MAP from complex manure and aerosol samples. Oligonucleotide primers specific for IS900 genetic element for use in real-time detection of MAP have been developed. We anticipate the detection of MAP in environmental samples in less than a day instead of the 2-6 month period by the traditional cultural methods.

Animal waste management on dairies: Liquid-solids separation. Improperly managed manure in confined animal feeding operations can harm the environment and become a serious food safety and public health issue. The life cycle of pathogen transmission from manure to crops and nutrient transport are being monitored on typical Central Valley California dairy farms with active manure management practices that include manure solid separators and aerators in lagoons. We continued collaborative research begun in late 2000 with California dairy producers on solid separation from dairy wastewater. The efficacy of a recently patented dual-separator system was evaluated for the first time. Over 90% of all large solids (>0.5 mm diameter) were removed from the wastewater at three commercial dairies with 800 to 6000 head. Total solids removed was approximately 40%, however, the remaining solids included clay and other non-manure components, and generally remain in suspension. On average, 139 tons per day of manure solids were removed from a dairy with 3500 milking cows. The separator system was found to effectively remove the portion of solids most responsible for filling storage lagoons, and for forming crusts that interfere with water recycling. Currently, we are developing research collaborations to study the utility of separated manures for composting material as animal bedding, and for on-site anaerobic digesters to generate energy. This project has received extraordinary attention from a variety of consumer groups. Those that have asked for consultation based on our results include: 1) county, state, and federal agencies dealing with issues that affect new dairy permits amid growing public concern over negative environmental impacts, 2) dairy producers interested in simple, relatively inexpensive technology for on-site waste management, and 3) agricultural structures companies that are interested in producing separators or integrating this system with their other products.

The manure solids removed were observed to contain higher populations of aerobic bacteria, fungi and coliforms than observed in manure flush/lagoon water and soils treated with lagoon water. Aerosol transport of microorganisms on a dairy farm: Transport through aerosols has long been suspected for foodborne pathogen contamination of agricultural crops produced in proximity to concentrated animal-raising operations. Bioaerosol samples were collected at different locations on a dairy farm by using an agar impaction device set at 100L of collection volume per minute. Aerobic bacteria and fungi were detected in all samples collected at several locations on the farm. Other cyclonic aerosol collection devices are currently being evaluated along with agar impaction devices for their efficiency of collection of pathogens from aerosols and fog.

Pathogen re-growth in compost extracts and transfer to crops: We began a project examining factors that influence re-growth of E. coli O157:H7 and Salmonella enterica Thompson strains in compost extracts, also called ‘teas’. Such teas are increasingly used for foliar disease suppression, foliar fertilization, and as a means of stretching compost supplies. Teas are also commonly used by organic producers and home gardeners on crops intended for fresh consumption. However, tea recipes are numerous and distributed primarily in trade journals with no peer review. Pathogen regrowth potential for different recipes has not been reported. First, we demonstrated that foliar treatment of strawberry plants with pathogen contaminated teas resulted in pathogen transfer. We then found that one of the most widely recommended amendments to teas, molasses, preferentially increases growth of pathogens in teas made from a variety of composts. Differences did exist in regrowth among the teas, but pathogens only regrew in teas amended with molasses. Regrowth was molasses concentration dependent with greatest regrowth at > 0.5%, which is below the 1-1.5% that is recommended in many tea recipes. We found that other common organic amendments differentially affected regrowth. We also found that certain plant compounds, such as clove and cinnamon oil, effectively prevented pathogen regrowth at 1% concentration. However, these compounds also reduced total numbers of aerobic bacteria, which may reduce the beneficial use of compost teas. Practical applications of this work are 1) providing information to extract users that will prevent potentially hazardous pathogen regrowth, 2) this system offers a simple model for evaluating factors that increase or decrease pathogen regrowth in compost and manure environments.

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 regrowth on dairies, 2) it provides evidence of some degree of host specificity for pathogens generally considered to be opportunistic invaders of agroecosystems, 3) it demonstrates 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 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:

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.

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

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 Environmental Committee of the California Almond Board. Results are regularly presented at scientific conferences
 

PROCEEDINGS/ABSTRACTS:

Ravva, S.V., Duffy, B.K., Stanker, L.H., and Mandrell, R.E. 2001. Foodborne Pathogens in Dairy Environments. 30th United States-Japan Cooperative Program in Natural Resources (UJNR) Protein Resources Panel Meeting, October 15-19, 2001, p 64-71, Tsukuba, Ibaraki, Japan.

Stanker, L.H., Sheffield, C., Beier, R.C., Andreotti, P., Venkateswaran, K., McBride, M.T., Ravva, S.V., Duffy, B.K., and Mandrell, R.E. 2001. Bacterial Detection Using Sensitive Immunological MethodsFoodborne Pathogens in Dairy Environments. 30th United States-Japan Cooperative Program in Natural Resources (UJNR) B Protein Resources Panel Meeting, October 15-19, 2001, p 64-71, Tsukuba, Ibaraki, Japan.

(Summary online)

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Last modified: May 25, 2005