Progress 04/03/00 to 04/02/05
Outputs
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? What does it matter? The food and water supply of the United States is among the safest in the world. An increasing number of outbreaks of human disease are, however, occurring as a result of pathogen contamination of fruits, vegetables, and water resources. Liquid and solid animal wastes originating from Concentrated Animal Feeding Operations are believed to be a major source of pathogenic microorganisms. At present, our ability to detect and quantify pathogenic microorganisms, and predict their fate and transport in the environment is not understood. Hence, the two primary objectives of this research are to: (I) develop methods for the detection, quantification, and source identification of pathogenic microorganisms; and (II) quantify mechanisms and processes affecting the transport and survival of pathogenic
microorganisms. To this end, an interdisciplinary team of research scientists is conducting laboratory, growth chamber, field, and numerical experiments to develop novel detection techniques and to explore the influence of microbe, water, manure, and soil characteristics on pathogen fate and transport in the environment. Pathogens are considered by most consumers, food processors, food safety researchers and regulatory agencies to be the most important food safety problem today. Research that helps to detect, and minimize the transport and survival of pathogens from animal manure to the environment is important for maintaining confidence in the food and water supply. Insight gained from these studies should aid in the development of control strategies to prevent the transmission of pathogenic microorganisms to food-producing animals, agricultural crops, and the environment in general. 2. List the milestones (indicators of progress) from your Project Plan. This project expired after 5
years and replaced by the new CRIS 5310- 32000-002-00D, Detection, Source Identification, Environmental Transport, Fate, and Treatment of Pathogenic Microorganisms Derived from Animal Wastes. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. The effect of temperature, moisture and pH on the environmental survival of enteric pathogens and parasites will be determined. Milestone Fully Met 2. Mechanisms controlling pathogen transport and source identification to surface and ground waters will be determined. These prospective results should lead to the development and validation of predictive transport models. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2006- Experiments will examine the
influence of microorganism input duration and mass on retention mechanisms. Time dependent deposition and hydraulic properties will be areas of special research attention in these studies. Other experiments will examine mechanisms of microorganism transport and retention in unsaturated porous media, including: attachment/detachment, straining, interfacial sorption, film straining, and exclusion. These experiments will encompass both steady-state and transient water flow conditions. Source tracking by amplified fragment length polymorphism will continue and the use of Neural Network for multivariate statistics will be initiated to compared 16S sequencing and multi-antibiotic resistance patterning for differentiating E. coli and Enterococcus isolates of livestock, wildlife, or human sources in polluted water. The use of a PCR assay that targets specific genes of Enterococcus will be developed to test the specificity of the assay in identifying different entocococci at the species
levels. This assay will be critical in identifying the different species of Enterococcus and their sources. Second year study will continue at the watershed scale. FY 2007: Comparison of bacterial source tracking analysis will continue. Additional work will be done to determine the survival of E. coli in surface water, sand materials from the river, and aquifer materials to determine carbon sources responsible for the survival and growth the E. coli in different locations along the river. The overall out come of the research will be the development of management strategies for the management of the watershed for the improvement of water quality. Fate and transport of pathogens will continue and will include more locations throughout the watershed. Size exclusion and straining are anticipated to produce differences in transport velocities and hydrodynamic dispersion for solutes and colloids at greater transport distances. To quantify these factors, microbe transport experiments will
be conducted at different spatial scales. Other laboratory experiments will examine the influence of solution composition and ionic strength on microbe migration. Solution composition is expected to influence microbe deposition as a result of changes in electrostatic interactions between colloids and porous media, as well as due to differences in the transport induced aggregation (higher colloid-colloid collision frequencies). Milestone - A numerical code will be developed that will quantify the transport and survival of bacterial human pathogen in the natural environment. The model will consider the most relevant physical, chemical and biological processes affecting the transport of pathogens. 4a What was the single most significant accomplishment this past year? This project was expired after 5 years and replaced by the new CRIS 5310- 32000-002-00D, Detection, Source Identification, Environmental Transport, Fate, and Treatment of Pathogenic Microorganisms Derived from Animal
Wastes. 4b List other significant accomplishments, if any. This project was expired after 5 years and replaced by the new CRIS 5310- 32000-002-00D, Detection, Source Identification, Environmental Transport, Fate, and Treatment of Pathogenic Microorganisms Derived from Animal Wastes. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A quantitative real-time PCR methodology was developed to quantify E. coli O157:H7 in manure, soil, waste and irrigation water. This method enables the collection of a large data set with a quality, reliable, and high through-put technology. These assays were optimized to specifically quantify very low levels; >100 bacterial cells per gram of soil without enrichment or 1 to 10 bacterial cells per gram of soil with enrichment. Impact: This work represents a considerable advancement in pathogen quantification in different ecosystems. These methods will allow for routine monitoring of surface and
ground water soil and food for the occurrence/ prevalence of pathogenic E. coli. NP 206 Action Plan Component III, Goal 2. Detection of pathogens and adapt existing techniques for the detection of pathogenic bacteria and protozoa. Survival of pathogens: E. coli O157:H7 survived more than 45 days in rhizosphere soil, which was determined with a newly developed real time PCR method. Impact: A sensitive method to measure the actual fate of Escherichia coli 0157:H7 is available so that researchers and regulators no longer need to only use less reliable indicator organisms. NP 206 Action Plan Component III, Goal 3. Loading rates and survival. Constructed wetlands for removal of contaminants: The removal of the main pollutants from the dairy washwater has had a beneficial impact on the surface and groundwater in the Chino Basin, and, in turn, has improved the quality of water leading into the Santa Ana River and the Orange County groundwater basin. Impact: The wetlands provided a
cost-effective, low-maintenance process that can be independently built and managed. This project has already benefitted the water district by reducing cost of contaminant removal down stream and the contamination of ground water with nitrate. NP 206 Action Plan Component III, Goal 5. Treatment technologies. Recognition that pathogen transport is highly dependent on pathogen and porous media size, on colloid concentration, and the magnitude and distribution of manure particles in suspension. An improved understanding of pathogen transport and deposition behavior across soil textural interfaces and in heterogeneous aquifer systems. A mathematical model has been developed to describe pathogen attachment, detachment, straining, blocking and size exclusion. Quantification of physical and chemical factors that influence pathogen release and loading rates from animal waste to water, and the development of a conceptual model to describe and predict this process. Impact. This accurate
information on pathogen transport processes discussed above is needed to predict and numerical model pathogen transport in subsurface environments, to assess the vulnerability and risk of resource contamination, and to develop cost effective remediation technologies and best management practices to protect water supplies. NP 206 Action Plan Component III, Goals 3 and 4. This project was terminated after five years and replaced by the new CRIS, 5310-32000-002-00D. 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? Quantitative Real-time PCR methods for quantitative detection of E. coli O157:H7 in environmental samples has been transferred to other scientists and commercial water testing laboratories. The major constrain is that this is a PCR based assay
and may be subjected to PCR artifacts. Constructed wetland technology has been transfer to some local farmers in the Chino area. This may be a possible alternative to direct spreading of wash water on their pasture. Research findings on pathogen transport and release will continue to be transferred to other scientists via peer- reviewed publications and presentations at national and international meetings. The developed models of pathogen attachment, straining, and exclusion have been incorporated into the HYDRUS 1D computer program. These modifications have been disseminated to several user of this software in the scientific community. Pathogen Transport Processes and Models Laboratory and modeling studies were undertaken to better quantify mechanisms of Cryptosporidium oocyst transport and retention in saturated porous media, and colloid deposition at soil textural interfaces. Research findings were disseminated to other scientists by presentations at national (W188) and
international meetings (invited presentations at conferences in Germany and the Netherlands), and by publishing peer- reviewed journal articles. The Hydrus computer model, for variably saturated flow and transport processes, was also refined to account for relevant pathogen transport processes. Specifically, we developed and implemented conceptual models to account for filling and accessibility of pathogens and colloids to straining sites, ripening (pathogen attachment that increases with increasing time), and nonlinear pathogen attachment and detachment. These modifications have been disseminated to several user of this software (in the United States, the Netherlands, and New Zealand). 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). This project was expired after 5 years. The related publications can be found in the report for the replacement
project 5310-32000-002-00D.
Impacts
(N/A)
Publications
- Abriola, L.M., Bradford, S.A., Lang, J., Gaither, C.L. 2004. Volatilization of binary NAPL mixtures in unsaturated porous media. Vadose Zone Journal. 3:645-655.
- Bradford, S.A., Rathfelder, K.M., Lang, J., Abriola, L.M. 2003. Entrapment and dissolution of DNAPLs in heterogeneous porous media. Journal of Contaminant Hydrology. 67:133-157.
- Bradford, S.A., Bettahar, M., Simunek, J., Van Genuchten, M.T. 2004. Straining and attachment of colloids in physically heterogeneous porous media. Vadose Zone Journal. 3:384-394.
- DUNGAN, R.S., IBEKWE, A.M., YATES, S.R. EFFECT OF PROPARGYL BROMIDE AND 1, 3-DICHLOROPROPENE ON SOIL MICROBIAL COMMUNITIES. 2003. FEDERATION OF EUROPEAN MICROBIOLOGICAL SOCIETIES (FEMS) MICROBIOLOGY ECOLOGY. 43:75-87.
- Ibekwe, A.M. 2004. Effects of fumigants on non-target organisms in soils. In: Sparks, D.L., editor. Advances in Agronomy. Vol. 83. San Diego, CA:Academic Press. p. 1-35.
- Ibekwe, A.M., Grieve, C.M., Lyon, S.R. 2003. Characterization of microbial communities and composition in constructed dairy wetland wastewater effluent. Applied and Environmental Microbiology. 69(9):5060-5069.
- Ibekwe, A.M., Lyon, S.R. 2003. Constructed wetlands for the removal of contaminants. Proceedings of Watershed Management Conference, Total Maximum Daily Load (TMDL) Environmental Regulations-II:477-485.
- Ibekwe, A.M., Lyon, S. 2004. Impact of dairy production on microbial characteristics through drinking water aquifer materials. (CD-ROM). American Society for Microbiology Annual Conference, New Orleans, LA. May, 2004.
- Ibekwe, A.M., Watt, P.M., Papiernik, S.K. 2003. Impact of fumigants on soil bacteria and E. coli O157:H7. (CD-ROM) American Society of Agronomy Annual Meeting. Denver, CO, November, 2003.
- Ibekwe, A.M., Grieve, C.M. 2004. Changes in developing plant microbial community structure as affected by contaminated irrigation water. Journal of Microbiological Methods. 48:239-248.
- Phelan, T.J., Lemke, L.D., Bradford, S.A, O'Carroll, D.M., Abriola, L.M. 2004. Influence of textural and wettability variations on predictions of DNAPL persistence and plume development in saturated porous media. Advances in Water Resources. 27:411-427.
- Schijven, J.F., Bradford, S.A., Yang, S. 2004. Release of Cryptosporidium and Giardia from dairy cattle manure: Physical factors. Journal of Environmental Quality. 33(4):1499-1508.
- YANG, Y., DUNGAN, R.S., IBEKWE, A.M., VALENZUELA-SOLANO, C., CROHN, D.M., CROWLEY, D.E. 2003. EFFECT OF ORGANIC MULCHES ON SOIL MICROBIAL ACTIVITIES AND COMMUNITIES ONE YEAR AFTER APPLICATON. BIOLOGY AND FERTILITY OF SOILS. 38:273-281.
- Ibekwe, A.M., Jenkins, M. 2004. Identifying and quantifying sources of bacteria. In: Hargrove, W.L., editor. Pathogens in the Environment Workshop Proceedings, Feb. 23-25, 2004, Kansas City, MO. p. 18-21.
|
Progress 10/01/03 to 09/30/04
Outputs
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? What does it matter? The food and water supply of the United States is among the safest in the world. An increasing number of outbreaks of human disease are, however, occurring as a result of pathogen contamination of fruits, vegetables, and water resources. Liquid and solid animal wastes originating from Concentrated Animal Feeding Operations are believed to be a major source of pathogenic microorganisms. At present, our ability to detect and quantify pathogenic microorganisms, and predict their fate and transport in the environment is not understood. Hence, the two primary objectives of this research are to: (I) develop methods for the detection, quantification, and source identification of pathogenic microorganisms; and (II) quantify mechanisms and processes affecting the transport and survival of pathogenic
microorganisms. To this end, an interdisciplinary team of research scientists is conducting laboratory, growth chamber, field, and numerical experiments to develop novel detection techniques and to explore the influence of microbe, water, manure, and soil characteristics on pathogen fate and transport in the environment. Pathogens are considered by most consumers, food processors, food safety researchers and regulatory agencies to be the most important food safety problem today. Research that helps to detect, and minimize the transport and survival of pathogens from animal manure to the environment is important for maintaining confidence in the food and water supply. Insight gained from these studies should aid in the development of control strategies to prevent the transmission of pathogenic microorganisms to food-producing animals, agricultural crops, and the environment in general. 2. List the milestones (indicators of progress) from your Project Plan. These milestones are
unofficial and will be included on the next project plan. The previous OSQR review in FY 1999/2000 did not include milestones. To develop methods for the readily detection of specific pathogens in different types of soils and manures. The effect of temperature, moisture and pH on the environmental survival of enteric pathogens and parasites will be determined. Methods will be developed for quantitating enteric pathogens and parasites in soil, manure, and runoff water. Mechanisms controlling pathogen transport and source identifications to surface and ground waters will be determined. These prospective results should lead to the development and validation of predictive transport models. A numerical code will be developed that will quantify the transport and survival of bacterial human pathogen in the natural environment. The model will consider the most relevant physical, chemical and biological processes affecting the transport of pathogens. Predictive transport models will be used
as a tool for recommending and evaluating manure management strategies which minimize/prevent pathogen transport to surface or groundwater. Results from these investigations will lead to estimation of environmental pathogen loading from manures. 3. Milestones: Milestone: Methods will be developed for quantitating enteric pathogens and parasites in soil, manure, and runoff water. FY 2004: We have fully developed methods for accurate quantification of E. coli 157:H7 in environmental samples. These methods are used to study survival and transport of E. coli O157:H7 in the environment. The development of methods for bacterial source identification started last year and will continue at least for the next two years. The experimental sites were not ready for sampling and we have hired a new postdoc during FY 2004 who will work on this milestone. Milestone: Mechanisms controlling pathogen transport and source identifications to surface and ground waters will be determined. These
prospective results should lead to the development and validation of predictive transport models. FY 2004: Many fundamental questions concerning processes and mechanisms that control the environmental transport and fate of pathogens (biocolloid) remain unanswered and are topics of ongoing research. This past year we have focused on quantifying mechanisms of Cryptosporidium oocyst transport and retention in saturated porous media, and colloid deposition at soil textural interfaces. Milestone: A numerical code will be developed that will quantify the transport and survival of bacterial human pathogen in the natural environment. The model will consider the most relevant physical, chemical and biological processes affecting the transport of pathogens. FY 2004. The Hydrus computer model, for variably saturated flow and transport processes, was further refined to account for pathogen transport processes. Specifically, we developed and implemented conceptual models to account for
filling and accessibility of pathogens and colloids to straining sites, ripening (pathogen attachment that increases with increasing time), and nonlinear pathogen attachment and detachment. B. List the milestones (from the list in Question #2) that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The effect of temperature, moisture and pH on the environmental survival of enteric pathogens and parasites will be determined. Mechanisms controlling pathogen transport and source identification to surface and ground waters will be determined. These prospective results should lead to the development and validation of predictive transport models. A numerical code will be developed that will quantify the transport and survival of bacterial human pathogen in the natural environment. The model will consider the most relevant physical, chemical and biological processes affecting the
transport of pathogens. Predictive transport models will be used as a tool for recommending and evaluating manure management strategies which minimize/prevent pathogen transport to surface or groundwater. Results from these investigations will lead to estimation of environmental pathogen loading from manures Milestone: The effect of temperature, moisture and pH on the environmental survival of enteric pathogens and parasites will be determined. Research Plans FY 2005: Studies will be conducted in the growth chamber to assess pathogen dissemination from manure and irrigation water to forage crop, and determine the influence of manure salinity, pH, temperature, and moisture on pathogen survival and penetration into the internal organs of plants. Milestone: Mechanisms controlling pathogen transport and source identification to surface and ground waters will be determined. These prospective results should lead to the development and validation of predictive transport models. FY 2005:
Source tracking of E. coli and Enterococcus by amplified fragment length polymorphism will be initiated and compared with 16S sequencing and multi-antibiotic resistance patterning for differentiating E. coli and Enterococcus isolates of livestock, wildlife, or human sources in polluted water. Studies will continue on the concentrations of bacteria in the constructed wetlands and determine the different processes of contaminant removal. Bacteria identified in the wetlands will provide a major source for differentiating between host genetic characteristic and the genetic characteristic of the secondary source. We will start our work on fate and transport of pathogens at the watershed scale. We will use the Santa Ana River at Prado Dam that is managed by USGS and received urban runoff, agricultural waste, and waste water discharge from treatment plants. Additional laboratory studies will be initiated to further quantify the influence of manure suspensions on specific microorganism
(Giardia, coliphage, E. coli, and enterrococcus) transport and fate in the saturated zone, including the potential for manure-facilitated transport. The release behavior of variously sized microorganisms from animal manure to flowing water under a variety of environmental conditions will also be studied. FY 2006- Experiments will examine the influence of microorganism input duration and mass on retention mechanisms. Time dependent deposition and hydraulic properties will be areas of special research attention in these studies. Other experiments will examine mechanisms of microorganism transport and retention in unsaturated porous media, including: attachment/detachment, straining, interfacial sorption, film straining, and exclusion. These experiments will encompass both steady-state and transient water flow conditions. Source tracking by amplified fragment length polymorphism will continue and the use of Neural Network for multivariate statistics will be initiated to compared 16S
sequencing and multi-antibiotic resistance patterning for differentiating E. coli and Enterococcus isolates of livestock, wildlife, or human sources in polluted water. The use of a PCR assay that targets specific genes of Enterococcus will be developed to test the specificity of the assay in identifying different entocococci at the species levels. This assay will be critical in identifying the different species of Enterococcus and their sources. Second year study will continue at the watershed scale. FY 2007: Comparison of bacterial source tracking analysis will continue. Additional work will be done to determine the survival of E. coli in surface water, sand materials from the river, and aquifer materials to determine carbon sources responsible for the survival and growth the E. coli in different locations along the river. The overall out come of the research will be the development of management strategies for the management of the watershed for the improvement of water quality.
Fate and transport of pathogens will continue and will include more locations throughout the watershed. Size exclusion and straining are anticipated to produce differences in transport velocities and hydrodynamic dispersion for solutes and colloids at greater transport distances. To quantify these factors, microbe transport experiments will be conducted at different spatial scales. Other laboratory experiments will examine the influence of solution composition and ionic strength on microbe migration. Solution composition is expected to influence microbe deposition as a result of changes in electrostatic interactions between colloids and porous media, as well as due to differences in the transport induced aggregation (higher colloid-colloid collision frequencies). Milestone - A numerical code will be developed that will quantify the transport and survival of bacterial human pathogen in the natural environment. The model will consider the most relevant physical, chemical and
biological processes affecting the transport of pathogens. FY 2005- The Hydrus computer model will continue to be adapted to describe pathogen transport in the presence of manure suspensions. Potential modifications include improved descriptions for time dependent straining behavior and hydraulic properties, and manure-facilitated pathogen transport. Conceptual models for pathogen release will also be refined to include a dependence on pathogen size and other relevant environmental factors. FY 2006- The Hydrus computer model will be adapted to describe pathogen transport processes in the unsaturated zones. Potential modifications include the dependence of attachment, straining, and size exclusion on water content (accessible pore space concepts), interfacial sorption, and film straining. FY 2007- The Hydrus computer model will be further modified to describe pathogen transport for various solution chemistries. These adaptations may include consideration of temporal changes in the
colloid size distribution as a result of solution chemistry and transport induced aggregation, and their impact on straining (highly dependent on colloid size). Milestone - Predictive transport models will be used as a tool for recommending and evaluating manure management strategies which minimize/prevent pathogen transport to surface or groundwater. Results from these investigations will lead to estimation of environmental pathogen loading from manures. FY 2005- Numerical experiments will be conducted using the refined Hydrus model to predict pathogen transport in heterogeneous aquifer formations. Topics that will be examined include the degree and structure of aquifer heterogeneity, and the presence and absence of manure suspensions. FY 2006- Numerical experiments will be conducted to examine pathogen transport in heterogeneous unsaturated soil systems. Topics that will be examined include the degree and structure of the soil profile, precipitation rates and intensity, and the
presence and absence of manure suspensions. Other simulations will examine the potential for pathogen migration at sites where comprehensive nutrient management plans are being used to apply farm lagoon wastewater at agronomic rates to crops. FY 2007- Numerical experiments will be conducted to examine the influence of best management practices for manage handling and storage, on release rates of pathogens and manure to flowing water. Factors to be considered include pathogen type, manure distribution and type, mixing of cow and calf manure, solution salinity, precipitation rate and duration. 4. What were the most significant accomplishments this past year? A. During environmental contamination of fresh produce, the pathogenic strain will compete with other bacteria in the environment for nutrients and this will determine their rate of survival in that environment. A growth chamber study was conducted at the GEB Jr. Salinity Laboratory to determine the interaction of Escherichia coli
O157:H7 with total microbial community in different soils that may provide different nutrient levels for bacterial survival. PCR analysis was faster and more reliable in enumerating the pathogen than plate counts. This accomplishment clearly shows the importance of E. coli O157:H7 in the environment and the risk of recontamination. B. Relatively little research attention has focused on mechanisms that control pathogen (biocolloid) deposition in heterogeneous soil and aquifer systems. Laboratory and modeling studies were undertaken at the GEBJr. Salinity lab, in collaboration with researchers from the USDA (Drs. Martinus Th. van Genuchten and Scott Yates) and UCR (Drs. Jirka Simunek and Medhi Bettahar), to characterize colloid deposition behavior at soil textural interfaces. Results indicate that colloid straining occurred at soil textural interfaces when water flowed from coarser to finer textured soils (especially for larger colloids and greater textural contrasts), and that
blocking (filling) and accessibility of straining sites needs to be considered in models of pathogen transport. This information improved our understanding and ability to model pathogen transport and fate in heterogeneous subsurface environments. Quality of surface water in the Santa Ana watershed, a major source of drinking water, has been affected significantly by intensive dairy operations and the disposal of untreated wastewater into the Chino Basin. In a cooperative study with Stephen Lyons, Orange County Sanitation District, constructed wetlands were used as treatment option for the removal of high concentrations of contaminants in agricultural waste water prior to land application. In this study, the wetland effluent was suitable for on-site reuse and has reduced the amount of contaminants entering groundwater supplies as a result of percolation of the washwater stored in ponds and sprayed on disposal lands. The wetland project has served as an innovative model for waste
management for the dairy industry and other confined animal facilities by providing a cost-effective, low- maintenance process that can be independently built and managed. Many waterborne disease outbreaks have been attributed to Cryptosporidium parvum oocysts, and accurate knowledge of the processes that control oocyst movement and fate is therefore required to better protect food and water resources. Laboratory and mathematical modeling studies were undertaken at the GEBJr. Salinity Lab, in collaboration with Dr. Mehdi Bettahar (UCR), to better quantify mechanisms of oocyst transport and retention in groundwater systems. Results indicate that straining, attachment, and detachment are important transport processes that should be included in mathematical models to predict oocyst fate. This information increased our knowledge and improved models for oocyst transport and treatment by soil passage. C. None to report D. None to report. 5. Describe the major accomplishments over the life
of the project, including their predicted or actual impact. A quantitative real-time PCR methodology was developed to quantify E. coli O157:H7 in manure, soil, waste and irrigation water. This method enables the collection of a large data set with a quality, reliable, and high through-put technology. These assays were optimized to specifically quantify very low levels; >100 bacterial cells per gram of soil without enrichment or 1 to 10 bacterial cells per gram of soil with enrichment. Impact: This work represents a considerable advancement in pathogen quantification in different ecosystems. These methods will allow for routine monitoring of surface and ground water soil and food for the occurrence/prevalence of pathogenic E. coli. NP 206 Action Plan Component III, Goal 2. Survival of pathogens: E. coli O157:H7 survived more than 45 days in rhizosphere soil, which was determined with a newly developed real time PCR method. Impact: A sensitive method to measure the actual fate of
Escherichia coli 0157:H7 is available so that researchers and regulators no longer need to only use less reliable indicator organisms. NP 206 Action Plan Component III, Goal 3. Constructed wetlands for removal of contaminants: The removal of the main pollutants from the dairy washwater has had a beneficial impact on the surface and groundwater in the Chino Basin, and, in turn, has improved the quality of water leading into the Santa Ana River and the Orange County groundwater basin. Impact: The wetlands provided a cost-effective, low-maintenance process that can be independently built and managed. This project has already benefitted the water district by reducing cost of contaminant removal down stream and the contamination of ground water with nitrate. NP 206 Action Plan Component III, Goal 5. Recognition that pathogen transport is highly dependent on pathogen and porous media size, on colloid concentration, and the magnitude and distribution of manure particles in suspension. An
improved understanding of pathogen transport and deposition behavior across soil textural interfaces and in heterogeneous aquifer systems. A mathematical model has been developed to describe pathogen attachment, detachment, straining, blocking and size exclusion. Quantification of physical and chemical factors that influence pathogen release and loading rates from animal waste to water, and the development of a conceptual model to describe and predict this process. Impact. This accurate information on pathogen transport processes discussed above is needed to predict and numerical model pathogen transport in subsurface environments, to assess the vulnerability and risk of resource contamination, and to develop cost effective remediation technologies and best management practices to protect water supplies. NP 206 Action Plan Component III, Goals 3 and 4. 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? Quantitative Real-time PCR methods for quantitative detection of E. coli O157:H7 in environmental samples has been transferred to other scientists and commercial water testing laboratories. The major constrain is that this is a PCR based assay and may be subjected to PCR artifacts. Constructed wetland technology has been transfer to some local farmers in the Chino area. This may be a possible alternative to direct spreading of wash water on their pasture. Research findings on pathogen transport and release will continue to be transferred to other scientists via peer- reviewed publications and presentations at national and international meetings. The developed models of pathogen attachment, straining, and exclusion have been incorporated into the HYDRUS 1D computer program. These modifications have been disseminated to several
user of this software in the scientific community. Pathogen Transport Processes and Models Laboratory and modeling studies were undertaken to better quantify mechanisms of Cryptosporidium oocyst transport and retention in saturated porous media, and colloid deposition at soil textural interfaces. Research findings were disseminated to other scientists by presentations at national (W188) and international meetings (invited presentations at conferences in Germany and the Netherlands), and by publishing peer- reviewed journal articles. The Hydrus computer model, for variably saturated flow and transport processes, was also refined to account for relevant pathogen transport processes. Specifically, we developed and implemented conceptual models to account for filling and accessibility of pathogens and colloids to straining sites, ripening (pathogen attachment that increases with increasing time), and nonlinear pathogen attachment and detachment. These modifications have been disseminated
to several user of this software (in the United States, the Netherlands, and New Zealand).
Impacts
(N/A)
Publications
- Ibekwe, A.M., Grieve, C.M. 2004. Changes in developing plant microbial community structure as affected by contaminated irrigation water. Journal of Microbiological Methods. 48:239-248.
- Phelan, T.J., Lemke, L.D., Bradford, S.A, O'Carroll, D.M., Abriola, L.M. 2004. Influence of textural and wettability variations on predictions of DNAPL persistence and plume development in saturated porous media. Advances in Water Resources. 27:411-427.
- Schijven, J.F., Bradford, S.A., Yang, S. 2004. Release of Cryptosporidium and Giardia from dairy cattle manure: Physical factors. Journal of Environmental Quality. 33(4):1499-1508.
- YANG, Y., DUNGAN, R.S., IBEKWE, A.M., VALENZUELA-SOLANO, C., CROHN, D.M., CROWLEY, D.E. 2003. EFFECT OF ORGANIC MULCHES ON SOIL MICROBIAL ACTIVITIES AND COMMUNITIES ONE YEAR AFTER APPLICATON. BIOLOGY AND FERTILITY OF SOILS. 38:273-281.
- Ibekwe, A.M., Jenkins, M. 2004. Identifying and quantifying sources of bacteria. In: Hargrove, W.L., editor. Pathogens in the Environment Workshop Proceedings, Feb. 23-25, 2004, Kansas City, MO. p. 18-21.
- Abriola, L.M., Bradford, S.A., Lang, J., Gaither, C.L. 2004. Volatilization of binary NAPL mixtures in unsaturated porous media. Vadose Zone Journal. 3:645-655.
- Bradford, S.A., Rathfelder, K.M., Lang, J., Abriola, L.M. 2003. Entrapment and dissolution of DNAPLs in heterogeneous porous media. Journal of Contaminant Hydrology. 67:133-157.
- Bradford, S.A., Bettahar, M., Simunek, J., Van Genuchten, M.T. 2004. Straining and attachment of colloids in physically heterogeneous porous media. Vadose Zone Journal. 3:384-394.
- DUNGAN, R.S., IBEKWE, A.M., YATES, S.R. EFFECT OF PROPARGYL BROMIDE AND 1, 3-DICHLOROPROPENE ON SOIL MICROBIAL COMMUNITIES. 2003. FEDERATION OF EUROPEAN MICROBIOLOGICAL SOCIETIES (FEMS) MICROBIOLOGY ECOLOGY. 43:75-87.
- Ibekwe, A.M. 2004. Effects of fumigants on non-target organisms in soils. In: Sparks, D.L., editor. Advances in Agronomy. Vol. 83. San Diego, CA:Academic Press. p. 1-35.
- Ibekwe, A.M., Grieve, C.M., Lyon, S.R. 2003. Characterization of microbial communities and composition in constructed dairy wetland wastewater effluent. Applied and Environmental Microbiology. 69(9):5060-5069.
- Ibekwe, A.M., Lyon, S.R. 2003. Constructed wetlands for the removal of contaminants. Proceedings of Watershed Management Conference, Total Maximum Daily Load (TMDL) Environmental Regulations-II:477-485.
- Ibekwe, A.M., Lyon, S. 2004. Impact of dairy production on microbial characteristics through drinking water aquifer materials. (CD-ROM). American Society for Microbiology Annual Conference, New Orleans, LA. May, 2004.
- Ibekwe, A.M., Watt, P.M., Papiernik, S.K. 2003. Impact of fumigants on soil bacteria and E. coli O157:H7. (CD-ROM) American Society of Agronomy Annual Meeting. Denver, CO, November, 2003.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? The food and water supply of the United States is among the safest in the world. An increasing number of outbreaks of human disease are, however, occurring as a result of pathogen contamination of fruits, vegetables, and water resources. Liquid and solid animal wastes originating from Concentrated Animal Feeding Operations are believed to be a major source of pathogenic microorganisms. At present, our ability to detect and quantify pathogenic microorganisms, and predict their fate and transport in the environment is not understood. Hence, the two primary objectives of this research are to: (I) develop methods for the detection, quantification, and source identification of pathogenic microorganisms; and (II) quantify mechanisms and processes affecting the transport and survival of pathogenic microorganisms. To this end, an interdisciplinary team of research scientists is conducting
laboratory, growth chamber, field, and numerical experiments to develop novel detection techniques and to explore the influence of microbe, water, manure, and soil characteristics on pathogen fate and transport in the environment. Insight gained from these studies should aid in the development of control strategies to prevent the transmission of pathogenic microorganisms to food-producing animals, agricultural crops, and the environment in general. 2. How serious is the problem? Why does it matter? Animal manure represents a valuable source of fertilizer and soil conditioning material. On the other hand, pathogenic microorganisms contained in animal manure can cause product and environmental contamination that can place the public at risk of disease. Pathogens are considered by most consumers, food processors, food safety researchers and regulatory agencies to be the most important food safety problem today. It is estimated that there are 10 million to 100 million cases of
food-borne illness each year. In addition, there is concern that the virulence of some pathogens may be increasing (e.g.hemolytic uremic syndrome, Guillain-Barre Syndrome, reactive arthritis). The recent E. coli outbreak in Walkerton, Canada also suggests that drinking water supplies may be vulnerable to pathogen contamination from animal production operations. Hence, food- and water-borne pathogens originating from animal manure represent an important problem for U.S. agriculture. Research that helps to detect, and minimize the transport and survival of pathogens from animal manure to the environment is important for maintaining confidence in the food and water supply. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? Our project is addressing many of the goals of the National Program 206 including: Problem Area 1: Develop methods for sensitive detection and accurate quantification of pathogens in complex matrices such
as manure and soil. Problem Area 2: Determination of the environmental loading rate of bacterial pathogens from land application of manure and the survival of various types of fecal pathogens. Problem Area 3: Predict and control transport and dissemination of pathogens. 4. What were the most significant accomplishments this past year? A. Although large volumes of manure and pathogen contaminated water are generated from animal feeding operations, only limited research has investigated the influence of manure contamination on the transport and dissemination of pathogenic microorganisms. Soil column experiments and modeling studies were conducted at the Salinity Laboratory to examine the transport behavior of Escherichia coli O157:H7 through several aquifer sands in the presence and absence of a manure suspension. The transport of E. coli O157:H7 was greatly enhanced in the presence of manure presumably due to preferential filling of soil retention sites by manure particles,
although manure components also altered the surface charge characteristics of the bacteria. Results from this study indicate that the magnitude and distribution of manure particles in suspension can increase the transport potential of pathogenic microorganisms. B1. Subsurface pathogen transport and fate is commonly assumed to be controlled by chemical interactions between the soil and the pathogen, whereas only limited research has addressed the influence of straining (entrapment of pathogens in small pores) on pathogen migration. Laboratory and modeling studies were undertaken at the Salinity Laboratory to explore the influence of sand size on the transport and fate of a pathogenic bacterium (E. coli O157:H7), a protozoan parasite (Cryptosporidium), and bacteriophages (MS2 and PhiX174) in saturated porous media. Increasing the biocolloid size (bacteriophages<E. coli O157:H7<Cryptosporidium) and/or decreasing the porous medium size led to lower peak effluent concentrations,
increased mass removal of the microorganisms at the column inlet, and an inability of conventional pathogen transport theory (models) to adequately describe or predict the experimental data. Results from this study suggest that pathogen transport will be highly dependent on the size pathogen and the porous medium, and that mathematical models need to account for pore straining. B2. Surface and groundwater quality in the Chino-Santa Ana River Basin of California is a major source of drinking water supply for the Los Angeles metropolitan area, however, the quality of surface water in the watershed has been affected significantly by intensive dairy operations and the disposal of untreated wastewater into the Chino Basin. In cooperation with Dr. Steven Lyons, Orange County Water Control District, a study was initiated to determine the effectiveness of constructed wetlands located at Chino, CA for removal of high concentrations of contaminants in agricultural waste water prior to land
application. Data from our study showed that wetland effluent was suitable for on-site reuse and that different kinds of bacteria were involved in the treatment process in the wetlands, which ultimately influenced the final effluent water quality. The wetland project serves as an innovative model for waste management for the dairy industry and other confined animal facilities by providing a cost-effective, low-maintenance process than can be independently built and managed. B3. One of the most common vehicles by which E. coli O157:H7 may be introduced into crops is by flood irrigation with water contaminated with cattle feces or by contaminated surface water. Researchers at GEB Jr. Salinity Laboratory have used bacteria tagged with green fluorescent protein to quantify survival of pathogens in flooded irrigation water, soil, roots and leaf surfaces. After plating and PCR analysis, E. coli O157:H7 concentrations were higher on the rhizosphere than the non- rhizosphere soils and leaf
surfaces. PCR analysis provides a faster and more reliable procedure in enumerating the pathogen than plate counts. B4. There is an increasing concern about on-farm contamination of fresh produce by pathogens from contaminated water or manure used for fertilization. A growth chamber study was conducted at the GEB Jr. Salinity Laboratory to determine the interaction of Escherichia coli O157:H7 with total microbial community of fresh produce grown in different soils that may provide different nutrient levels for bacterial survival. As different bacterial groups increased with plant age, there was a corresponding decrease in the concentration of pathogens, and the microbial community became stable after 9 days. These results emphasized that irrigation water must be free of contaminants especially during the early stages of plant growth, as this may be the time for maximum pathogen contamination in plants. 5. Describe the major accomplishments over the life of the project, including their
predicted or actual impact. Recognition that pathogen transport is highly dependent on pathogen and porous media size, on colloid concentration, and the magnitude and distribution of manure particles in suspension. A mathematical model has been developed to describe pathogen attachment, detachment, straining, and size exclusion. Quantification of physical and chemical factors that influence pathogen release and loading rates from animal waste to water, and the development of a conceptual model to describe and predict this process. The accomplishment of the research proposed herein will provide Innovative methods for quantifying pathogens in manure and other environmental samples; i.e., detection of E. coli O157:H7 by multiplex real-time PCR in soil and manure samples at very low concentration. The use of constructed wetlands for the removal of contaminants in dairy wash water. 6. What do you expect to accomplish, year by year, over the next 3 years? FY 2004: Data from column studies
examining the transport of E. coli O157:H7, Cryptosporidium, and bacteriophages will be further analyzed and modeled. Additional studies will be conducted to characterize the influence of the colloid size distribution, including manure suspensions, on the transport of pathogenic microorganisms. The potential for colloid- facilitated transport of viruses will be examined. Source tracking of E. coli by amplified fragment length polymorphism will be initiated and compared with 16S sequencing and multi-antibiotic resistance patterning for differentiating E. coli isolates of livestock, wildlife, or human sources in polluted water. Studies will be continued on the concentrations of different pathogens in the constructed wetlands and determine the different processes of contaminant removal. In addition, studies will be conducted in the growth chamber to assess pathogen dissemination from manure and irrigation water to forage crop, and determine the influence of manure salinity on pathogen
penetration into the internal organs of plants. FY 2005. Experiments will examine the release behavior of various sized pathogens (phages, E. coli O157, and Giardia) from manure to flowing water. This information will help identify indicator organisms of pathogen release. Unsaturated soil column experiments will be initiated to explore the influence of pathogen and sand size on transport and fate. Mathematical models to describe pathogen transport and release will continue to be refined to account for experimental observations. Source tracking of E. coli by amplified fragment length polymorphism will continue and the use of Neural Network for multivariate statistics will be initiated to compared 16S sequencing and multi-antibiotic resistance patterning for differentiating E. coli isolates of livestock, wildlife, or human sources in polluted water. FY 2006. Additional laboratory and lysimeter scale experiments will be conducted to explore the influence of various physical,
chemical, and biological conditions on pathogen transport and fate in saturated and unsaturated systems. Numerical models will continue to be refined and validated using laboratory and lysimeter data and independently determined transport and release parameters. Comparison of bacterial source tracking analysis will continue. Additional work will be done to determine the survival of E. coli in surface water, sand materials from the river, and aquifer materials to determine carbon sources responsible for the survival and growth the E. coli in different locations along the river. 7. 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? Research findings on pathogen transport and release will continue to be transferred to other scientists via peer-reviewed
publications and presentations at national and international meetings. The developed models of pathogen attachment, straining, and exclusion have been incorporated into the HYDRUS 1D computer program. These modifications have been disseminated to several user of this software in the scientific community. Quantitative Real-time PCR methods for quantitative detection of E. coli O157:H7 in environmental samples have been transferred to other scientists and commercial water testing laboratories. The major constrain is that this is a PCR based assay and may be subjected to PCR artifacts. Constructed wetland technology has been transfer to some local farmers in the Chino area. This may be a possible alternative to direct spreading of wash water on their pasture.
Impacts
(N/A)
Publications
- Ibekwe., A. M. P. M. Watt, P. J. Shouse , C. M. Grieve. Quantification in Rhizosphere and Phyllosphere of E. coli O157:H7 by Plating and Real- Time (TaqMan) PCR. Agronomy Abstracts, American Society of Agronomy Meeting. CD-ROM. Madison, WI 2002.
- Ibekwe, A.M., Watt, P. M., Shouse, P.J., Grieve, C.M. Real-Time PCR-Based Quantification of Survival of Escherichia coli O157:H7 on Plants affected by Contaminated Irrigation Water. American Society of Microbiology Annual Meeting. CD-ROM. Washington DC . 2003.
- Bradford, S.A., Bettahar, M., Simunek, J., van Genuchten, M.Th. Transport and fate of colloids in physically heterogeneous porous media. Proceedings of the International Workshop on Colloids and Colloid-Facilitated Transport of Contaminants in Soils and Sediments. 2002. Danish Institute of Agricultural Sciences Report No. 80. p. 123-130.
- Bradford, S.A., Schijven, J. Release of Cryptosporidium and Giardia from dairy calf manure: Impact of solution salinity. Environmental Science and Technology. 2002. v. 36. p. 3916-3923.
- Bradford, S.A., Simunek, J., Bettahar, M., van Genuchten, M.Th., Yates, S. R. Modeling colloid attachment, straining, and exclusion in saturated porous media. Environmental Science and Technology. 2003. v. 37. p. 2242- 2250.
- Bradford, S.A., Simunek, J., Bettahar, M., van Genuchten, M.Th., Yates, S. R. Experimental and modeling studies of colloid attachment, straining, and exclusion in saturated porous media. Proceedings of the International Workshop on Colloids and Colloid-facilitated Transport of Contaminants in Soils and Sediments. 2002. Danish Institute of Agricultural Sciences Report No. 80. p. 289-296.
- Ibekwe., A. M. Influence of Microbial Community Composition and Function on Water Quality from Dairy Constructed Wetland Wastewater. Agronomy Abstracts, American Society of Agronomy Meeting. CD-ROM. Madison, WI 2002.
- Ibekwe, A.M., Grieve, C.M. Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR. Journal of Applied Microbiology. 2003. v. 94. p. 421-431.
- Ibekwe, A.M., Papiernik, S. K., Grieve, C.M. Impact of fumigants on the survival of Escherichia coli O157:H7 in animal manure. American Society of Microbiology Annual Meeting. CD-ROM. Washington DC. 2003.
- Ibekwe, A.M., Watt, P.M., Grieve, C.M., Sharma, V.K., Lyons, S.R. Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli O157:H7 in dairy wastewater wetlands. Applied and Environmental Microbiology. 2002. v. 69. p. 4853-4862.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? The food and water supply of the United States is among the safest in the world. An increasing number of outbreaks of human disease are, however, occurring as a result of pathogen contamination of fruits, vegetables, and water resources. Liquid and solid animal wastes originating from Concentrated Animal Feeding Operations are believed to be a major source of pathogenic microorganisms. At present, our ability to detect and quantify pathogenic microorganisms, and predict their fate and transport in the environment is not understood. Hence, the two primary objectives of this research are to: (I) develop methods for the detection, quantification, and source identification of pathogenic microorganisms; and (II) quantify mechanisms and processes affecting the transport and survival of pathogenic microorganisms. To this end, an interdisciplinary team of research scientists is conducting
laboratory, growth chamber, field, and numerical experiments to develop novel detection techniques and to explore the influence of microbe, water, manure, and soil characteristics on pathogen fate and transport in the environment. Insight gained from these studies should aid in the development of control strategies to prevent the transmission of pathogenic microorganisms to food-producing animals, agricultural crops, and the environment in general. 2. How serious is the problem? Why does it matter? Animal manure represents a valuable source of fertilizer and soil conditioning material. On the other hand, pathogenic microorganisms contained in animal manure can cause product and environmental contamination that can place the public at risk of disease. Pathogens are considered by most consumers, food processors, food safety researchers and regulatory agencies to be the most important food safety problem today. It is estimated that there are 10 million to 100 million cases of
food-borne illness each year. In addition, there is concern that the virulence of some pathogens may be increasing (e.g.hemolytic uremic syndrome, Guillain-Barre Syndrome, reactive arthritis). The E. coli outbreak in Walkerton, Canada (May, 2000) also suggests that drinking water supplies may be vulnerable to pathogen contamination from animal production operations. Hence, food- and water-borne pathogens originating from animal manure represent an important problem for U.S. agriculture. Research that helps to detect, and minimize the transport and survival of pathogens from animal manure to the environment is important for maintaining confidence in the food and water supply. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? Our project is addressing many of the goals of the National Program 206 including: Problem Area 1 ? Develop methods for sensitive detection and accurate quantification of pathogens in complex
matrices such as manure and soil. Problem Area 2 ? Determination of the environmental loading rate of bacterial pathogens from land application of manure and the survival of various types of fecal pathogens. Problem Area 3 ? Predict and control transport and dissemination of pathogens. 4. What was your most significant accomplishment this past year? A. Knowledge of the release behavior of pathogens from animal waste is needed to determine cumulative loading of pathogens to surface and groundwater. A conceptual model was developed at the George E. Brown Jr. Salinity Laboratory to describe and predict manure and protozoan parasite (oo)cyst (Giardia cysts and Cryptosporidium oocysts) release rates and cumulative loading; i.e., manure release into water was characterized as a diffusion controlled processes, and then the pathogen concentration was predicted from the dissolved manure concentration, the initial pathogen concentration in the manure, and the release efficiency of the
pathogens (relative to that of manure). The proposed model was successfully calibrated and applied to previously obtained release data collected over a range of physical (water flow rate, application method and duration, temperature, and manure type) and chemical (solution salinity) conditions. This release model will enhance the ability of researchers to characterize manure and pathogen release and loading to surface and groundwater, and therefore better evaluate the impact of hydrologic conditions and animal management practices on water quality. B. (1) Methods for the detection and quantification of enterohemorrhagic E. coli O157:H7 in manure, soil, waste, and surface waters are typically laborious, expensive, and insensitive. Researchers at the GEB Jr. Salinity Laboratory have applied a previously developed quantitative real-time PCR fluorogenic probe technique (Taqman PCR) and nucleic acid sequence-based amplification method to determine concentrations of E. coli O157:H7 in
dairy cow manure, wash water, and rhizosphere and phyllosphere samples. The Taqman PCR assay had an E. coli O157:H7 detection limit of 10 to 100 cfu in manure and wash water, and 2.6x100,000 cfu in rhizosphere and phyllosphere samples. The multiplexing of this novel TaqMan PCR protocol holds promise for the quantitative detection of E. coli O157:H7 and other pathogens in other difficult environmental matrices and for use in transport studies. (2) Although straining and size exclusion are typically neglected in pathogen transport models, recent experimental studies indicate that the subsurface transport and fate of pathogens is dependent on both of these processes as well as on attachment and detachment. A conceptual model was developed at the George E. Brown Jr. Salinity Laboratory that accounts for attachment and detachment using conventional first-order rate expressions, describes straining using a depth-dependent first-order rate expression, and models exclusions by employing
the pathogen accessible pore space in transport parameters. The calibrated model yielded a significantly improved description of both the effluent concentration curves and the spatial distribution of variously sized colloids (a surrogate for pathogenic microbes) in four soils than conventional pathogen transport models. The developed model provides an improved characterization of the processes that control pathogen transport, and will therefore aid researchers to assess the vulnerability and minimize the potential of pathogen contamination to water supplies. (3) The concentration of colloid particles in soil solution and groundwater can vary widely, typically ranging from 1,000,000,000 to 1, 000,000,000,000,000,000 particles per liter, and has been reported to influence the transport behavior of bacteria in porous media. A series of soil column experiments was undertaken at the George E. Brown Jr. Salinity Laboratory to further explore the influence of colloid input concentration
on the transport and fate of several colloid sizes in three soils. Results from these experiments could not be explained by conventional colloid blocking theory, because decreasing the colloid input concentration and porous medium grain size, and increasing the colloid size, all resulted in increased mass retention at the column inlet and lower relative (normalized to input concentration) concentrations in the effluent. This data suggests that theory and models for colloid transport need to be improved to account for the observed concentration dependent transport behavior, and that pathogen transport may be enhanced in porous media at higher colloid concentrations typical of manure release. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? Innovative methods for detecting pathogens in manure and other environmental samples; i.e., detection of E. coli O157:H7 by multiplex PCR in soil and manure samples at very low
concentration. Recognition that pathogen transport will be highly dependent on pathogen and porous media size, and on colloid concentration. A mathematical model has been developed to describe pathogen attachment, detachment, straining, and size exclusion. Quantification of physical and chemical factors that influence pathogen release and loading rates from animal waste to water, and the development of a conceptual model to describe and predict this process. 6. What do you expect to accomplish, year by year, over the next 3 years? FY 2003: Concentrations of different pathogens in an on-farm study will be monitored by real-time PCR in soil and manure samples at very low concentration. In addition, studies will be conducted in the growth chamber to assess pathogen dissemination from manure and irrigation water to pre-harvest fresh produce. Additional laboratory and modeling studies will be initiated to explore the role of input colloid concentration and duration (mass input) on
transport and fate. A complimentary study will examine the subsurface transport behavior of E.coli O157:H7 for various input concentrations of dissolved manure. Studies will also be initiated to characterize the electrophoretic mobility and aggregating behavior of E. coli O157:H7 as a function of solution salinity, pH, and pathogen growth stage. A plot on a local dairy farm will be instrumented to collect runoff water following precipitation events and to monitor surface water concentrations of manure and pathogens. Data from this study will be used to examine pathogen release issues and modeling at larger scales. The packed lysimeter will be instrumented and prepared for transport studies. FY 2004: Source tracking of E. coli by amplified fragment length polymorphism will be initiated and compared with 16S sequencing and multi- antibiotic resistance patterning for differentiating E. coli isolates of livestock, wildlife, or human sources in polluted water. Unsaturated soil column
experiments will be initiated to explore the influence of colloid size, soil size, and physical heterogeneity on the transport and fate of pathogens in the unsaturated systems. Plot scale pathogen release studies will be initiated at a local dairy farm, and pathogen transport studies will be conducted on the lysimeter. Mathematical models to describe pathogen transport and release will continue to be refined to account for experimental observations. FY 2005: Additional laboratory and lysimeter scale experiments will be conducted to explore the influence of various physical, chemical, and biological conditions on pathogen transport and fate in saturated and unsaturated systems. Plot scale experiments examining pathogen release will also be continued. Numerical models will continue to be refined and validated using laboratory, lysimeter, and plot scale data and independently determined transport and release parameters.
Impacts
(N/A)
Publications
- Ibekwe, A.M., Kennedy, A.C., Frohne, P.S., Papiernik, S.K., Yang, C.-H., Crowley, D.E. Microbial diversity along a transect of agronomic zones. FEMS Microbiology Ecology. 2002. v. 39. p. 183-191.
- Ibekwe, A.M., Papiernik, S.K., Gan, J., Yates, S.R., Crowley, D.E. Microcosm enrichment of 1,3-dichloropropene-degrading soil microbial communities in a compost-amended soil. Journal of Applied Microbiology. 2001. v. 91. p. 668-676.
- Bradford, S.L., Yates, S.R., Bettahar, M., Simunik, J., Van Genuchten, M.T. Physical factors affecting the transport and fate of colloids. ASA-CSSA- SSSA Annual Meeting. CD-ROM. Charlotte, NC: Agronomy Abstracts. 2001.
- Ibekwe, A.M., Watt, P.M., Lyon, S. Detection of pathogens by multiplex PCR in a constructed dairy wash water wetland. ASA-CSSA-SSSA Annual Meeting. CD-ROM. Charlotte, NC: Agronomy Abstracts. 2001.
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Progress 10/01/00 to 09/30/01
Outputs
1. What major problem or issue is being resolved and how are you resolving it?
The food and water supply of the United States is among the safest in the world. An increasing number of outbreaks of human disease are, however, occurring as a result of pathogen contamination of fruits, vegetables, and water resources. Liquid and solid animal wastes originating from Concentrated Animal Feeding Operations are believed to be a major source of pathogenic microorganisms. At present, our ability to detect and quantify pathogenic microorganisms, and predict their fate and transport in the environment is not understood. Hence, the two primary objectives of this research are to: (I) develop methods for the detection, quantification, and source identification of pathogenic microorganisms; and (II) quantify mechanisms and processes affecting the transport and survival of pathogenic microorganisms. To this end, an interdisciplinary team of research scientists is conducting laboratory, growth chamber, field, and numerical experiments to develop novel detection techniques
and to explore the influence of microbe, water, manure, and soil characteristics on pathogen fate and transport in the environment. Insight gained from these studies should aid in the development of control strategies to prevent the transmission of pathogenic microorganisms to food-producing animals, agricultural crops, and the environment in general.
2. How serious is the problem? Why does it matter?
Animal manure represents a valuable source of fertilizer and soil conditioning material. On the other hand, pathogenic microorganisms contained in animal manure can cause product and environmental contamination that can place the public at risk of disease. Pathogens are considered by most consumers, food processors, food safety researchers and regulatory agencies to be the most important food safety problem today. It is estimated that there are 10 million to 100 million cases of food-borne illness each year. In addition, there is concern that the virulence of some pathogens may be increasing (e.g.hemolytic uremic syndrome, Guillain- Barre Syndrome, reactive arthritis). The recent E. coli outbreak in Walkerton, Canada also suggests that drinking water supplies may be vulnerable to pathogen contamination from animal production operations. Hence, food- and water-borne pathogens originating from animal manure represent an important problem for U.S. agriculture. Research that helps to
detect, and minimize the transport and survival of pathogens from animal manure to the environment is important for maintaining confidence in the food and water supply.
3. How does it relate to the National Program(s) and National Component(s)?
Our project is addressing many of the goals of the National Program 206 including: Problem Area 1 Develop methods for sensitive detection and accurate quantification of pathogens in complex matrices such as manure and soil. Problem Area 2 Determination of the environmental loading rate of bacterial pathogens from land application of manure and the survival of various types of fecal pathogens. Problem Area 3 Predict and control transport and dissemination of pathogens.
4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment during FY 2001: Current methods for detecting and quantifying enterohemorrhagic E. coli O157 in manure, soil, waste and surface waters are laborious, expensive, relatively insensitive, and do not provide quantitative data which is necessary for developing predictive models. We are evaluating a new methodology for the quantitative detection of pathogens in environmental samples using immunomagnetic bead separation in combination with real-time PCR using fluorogenic probes (TaqMan PCR). A TaqMan PCR protocol has been developed and optimized for the quantitative fluorogenic multiplex detection of enterohemorrhagic E. coli O157 in environmental samples. This improved method permits detection of E. coli O157:H7 by multiplex PCR in soil and manure samples at very low concentrations. B. Other Significant Accomplishments during FY 2001: Subsurface pathogen transport and fate is typically believed to be controlled by sorption / desorption processes,
only limited research has addressed the influence of physical factors on pathogen migration. Laboratory and modeling studies were undertaken at USSL to explore the influence of colloid and soil size, and physical heterogeneity on the transport and fate of pathogens in saturated porous media. Increasing the colloid (a surrogate for pathogenic microbes) size and/or decreasing the porous medium size lead to increased pore straining, lower peak effluent concentrations, increased surface mass removal of colloids, and an inability of conventional colloid filtration theory (models) to adequately describe or predict the experimental data. Results from this study suggest that pathogen transport will be highly dependent on its size and porous medium characteristic, and that mathematical models need to be refined to account for pore straining and size exclusion of larger pathogens. Reliable data on the environmental factors which affect the release and loading rates of pathogenic parasites,
such as Giardia and Cryptosporidium (oo)cysts, from animal manure is needed to accurately assess and minimize contamination potential to surface and drinking water supplies. Studies were initiated at USSL to determine release rates of naturally occurring (oo)cysts from Holstein dairy calf manure under various physical (water application rate, duration, and method) and chemical (pH and ionic strength) conditions. Effluent (oo)cyst concentrations were initially several orders of magnitude below the starting (oo)cyst concentration in the manure, after continued application of water the (oo)cyst concentration gradually decreased and then exhibited persistent concentration tailing. Results indicate a high correlation between (oo)cyst concentrations and solution optical density, and that chemical factors had a relatively minor impact on (oo)cyst release rates compared to physical factors.
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
Innovative methods for detecting pathogens in manure and other environmental samples; i.e., detection of E. coli O157:H7 by multiplex PCR in soil and manure samples at very low concentration. Recognition that pathogen transport will be highly dependent on their size and mathematical models need to be refined to account for pore straining and size exclusion of larger pathogens. Development of a correlation between protozoan (oo)cyst concentrations and solution optical density, and finding that chemical factors had a relatively minor impact on (oo)cyst release rates compared to physical factors.
6. What do you expect to accomplish, year by year, over the next 3 years?
FY 2002 TaqMan PCR methods will be developed for the quantitative detection of enterohemorrhagic E. coli O157:H7 in feces/manure, soil, and waste water samples. A series of soil column experiments will be conducted to explore the role of influent colloid concentration and duration on transport and fate of colloids in saturated porous media. A numerical model will also be refined and utilized to describe and predict the influence of colloid straining and size exclusion in subsurface systems, and the release and transport of pathogenic protozoa parasites from animal manure. FY 2003 TaqMan PCR methods will also be developed for other pathogenic bacteria. Also, on-farm studies will be conducted to assess the reduction in pathogen transport from dairy waste water to surface waters through a constructed subsurface wetland. Unsaturated soil column experiments will be initiated to explore the influence of colloid size, soil size, and physical heterogeneity on the transport and fate of
pathogens in the vadose systems. Instrument and calibrate lysimeters for future experimental studies. Mathematical models to describe pathogen transport will continue to be refined to account for experimental observations. FY 2004 Transport studies will be continued and concentrations of different pathogens in the on-farm study will be monitored by real-time PCR. In addition, studies will be conducted in the growth chamber to assess pathogen dissemination from manure and irrigation water to preharvest fresh produce. Conduct additional experiments exploring the influence of various physical, chemical, and biological conditions on pathogen transport and fate in saturated and unsaturated systems. Initiate large-scale lysimeter experiments to investigate plot-scale pathogen transport scenarios under controlled conditions. Continue to refine and validate the numerical model using experimental two-dimensional and lysimeter data and independently determined transport (soil column)
parameters.
7. 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 & durability of the technology product?
Real-time PCR methods for quantitative detection of enterohemorrhagic E. coli O157:H7 in environmental samples will be transferred to other scientists and commercial water testing laboratories (via publications and presentations) in 2001 and 2002.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
This is a new project; none to report.
Impacts
(N/A)
Publications
- Ibekwe, A.M., Papiernik, S.K., Gan, J., Yates, S.R. Impact of fumigants on the structural diversity of ammonia-oxidizing bacteria. American Society of Agronomy. 2000. Abstract p. 256.
- Ibekwe, A.M. and Shannon, M.C. Detection of E. coli 0157:H7 in environmental samples. American Society of Agronomy. 2000. Abstract p. 398.
- Ibekwe, A.M. Detection of Eschericia coli 0157:H7 in Environmental Samples by Quantitative, Multiplex, Fluorogenic PCR Assay. American Society for Microbiology. 2001. Abstract p. 668.
- Ibekwe A.M., Papiernik, S.K., Gan, J., Crowley, D., Yang, C.-H., Yates, S.R. Microcosm enrichment of 1,3-Dichloropropene-degrading microbial communities. Journal of Applied Microbiology. 2001. v. 91. p. 625-634.
- Ibekwe A.M., Papiernik, S.K., Gan, J., Crowley, D., Yang, C.-H, Yates, S.R. Impact of fumigants on soil microbial community. Aplied and Environmental Microbiology. 2001. v. 67. p. 3245-3257.
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