Progress 09/15/07 to 08/31/12
Outputs
OUTPUTS: Movement of disease-causing bacteria from animal production into the human food chain and water bodies, typically as a result of land application of animal manures, has become a matter of heightened public, scientific and government concern. To limit the adverse impacts of food- and water-borne illness on society, it is vitally important to have a solid grounding in the behavior of these pathogens in the farm environment as a means to develop ways of suppressing and controlling them. In this project, we conducted a series of studies to investigate how two important zoonotic pathogens, Escherichia coli O157:H7 (E. coli O157:H7) and Salmonella enterica serovar Newport (S. Newport), survive and travel in the post-manure shedding environment under various manure handling and treatment conditions. Field-based experiments included survival studies of the two target pathogens in various locations on a working dairy operation, and an on-farm monitoring study examining presence of five pathogens on local dairy farms. Laboratory investigations were performed to determine the potential of modifying pH or temperature to suppress pathogens in manure up-front, before field application, and using temperature as a proxy for seasonal conditions. A second laboratory study focused on pathogen transport and retention in soil using leaching columns and simulated rainfall. Results from the project have been disseminated via various means: (i) in peer-reviewed publications, including the Journal of Dairy Science and the Open Waste Management Journal. A manuscript on pathogen leaching and soil retention behavior is currently under review at FEMS Microbiology and Ecology. A paper concerning findings from the on-farm monitoring study is in preparation. (ii) through reports and scientific exchanges with fellow researchers, extension and education personnel at regional, national, and international meetings such as the Land and Sea Grant National Water Conferences, the American Society of Agronomy Annual Meetings, the Joint Workshop in the Agricultural and Environmental Sciences, and the Third International Workshop on Nutrient Management: Technology and Policy, held in Beijing, China. (iii) as feedback to the producers who were involved in the project and participated in the on-farm monitoring. PARTICIPANTS: To fulfill the objectives of the on-farm pathogen monitoring phase of the project, thirteen dairy operations in southeastern and southcentral Pennsylvania were selected from participants in a separate dairy nutrition and profitability study. After arriving on the farms, operators were given a description of our objectives and sampling methods. Upon completion of the laboratory analyses, farmers were sent written summaries of pathogen occurrence and location on their farms. When requested, individual interpretations of results were provided. TARGET AUDIENCES: Target audiences included fellow researchers, extension and education personnel, and the farmers collaborating on the pathogen monitoring study. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
To increase the knowledge base and provide a comprehensive understanding of zoonotic bacteria survival and transport in the farm environment, we conducted a series of laboratory and field studies. 1) Pathogen persistence in the dairy farm environment. Environmental sentinel chambers containing manure or soil-manure mixtures were inoculated with S. Newport or E. coli O157:H7 and buried in a farm field, immersed in the effluent lagoon or under a static compost pile. The pathogens survived for up to 276 days in soil, and 137 days in the lagoon, whereas high temperatures generated in the compost eliminated them within 18 hours. Deactivation of the pathogens followed a log-linear decline model for 60-90 days, then trailed at concentrations of up to 100 CFU/g for extended periods of time. 2) Assess constraining growth of S. Newport and E. coli O157:H7 in dairy manure by modifying pH or temperature. By adjusting pH in the manure to either <4.2 or >11.0, the pathogens were eliminated within 6 days. The pathogens were deactivated within 2 weeks at 37 C, and 10 weeks at 22 C, but at 4 C, S. Newport was still present after 11 months. 3) Pathogen transport and retention in soil columns with simulated rainfall. Dairy manure inoculated with S. Newport or E. coli O157 was placed at the top of intact or tilled soil columns; simulated rainfall was applied, and leachate collected. Cumulative pathogen leaching loss ranged from 5.8 to 8.1 log CFU. Greater losses occurred in intact soil than tilled. Delay of the first simulated rainfall reduced pathogen losses by up to 12-fold. Pathogens in the leachate were as high as 4.6 log/mL even after several rainfall events. At the end of the 15-day trials, pathogens retained in soil were comparable to cumulative leaching loss, but the manure layer maintained greater pathogen amounts than initially inoculated. 4) Monitor presence of zoonotic pathogens on local dairy farms. Occurrence of Salmonella, E. coli O157:H7, Campylobacter, Mycobacterium paratuberculosis and the parasite Cryptosporidium was determined on 13 dairy farms, in feces, animal bedding, manure storages, milk filters, field soil and stream water. At least one pathogen species was present on 12 of the 13 farms. Most commonly found were Mycobacterium and E. coli O157:H7, in 19 and 20 percent of samples, followed by the other species in 3 to 7 percent of samples. Pathogens occurred in 50 percent of fecal samples, and in 73 percent of samples from manure storage. In summary, S. Newport and E. coli O157:H7 are variably present regionally on dairy operations. The pathogens are able to survive for months in soil and manure storages, and move readily through soil in leachate water, providing ample time and opportunity for subsequent dissemination into the agroecosystem. pH alteration to certain threshold values and composting give the best treatment alternatives for pathogen suppression in manure. Results from this project enhance our understanding of manure-borne pathogen fate in the farm environment and provide options for development of science-based and practically-useful interventions.
Publications
- (Refereed journals) Toth, J.D., H.W. Aceto, S.C. Rankin, and Z. Dou. 2011. Survival characteristics of Salmonella enterica serovar Newport in the dairy farm environment. J. Dairy Sci. 94:5238-5246.
- Toth, J.D, H.W. Aceto, S.C. Rankin, C. DebRoy, and Z. Dou. 2012. Accelerating the deactivation of Salmonella enterica serovar Newport and Escherichia coli O157:H7 in dairy manure by modifying pH or temperature. Open Waste Manage. J. 5:11-18.
- Toth, J.D., H.W. Aceto, S.C. Rankin, J.D. Vanderhoef, and Z. Dou. 2012. Leaching characteristics and soil retention of Salmonella enterica serovar Newport and Escherichia coli O157:H7 affected by rainfall pattern and tillage. FEMS Microbiol. Ecol. (in review).
- (Presentations) Toth, J.D., Z. Dou, S.C. Rankin, H.W. Aceto, C.F. Ramberg, Jr., and C. Debroy. Environmental survival of zoonotic pathogenic bacteria in manure-amended soil. Abstract and poster presentation at ASA-CSSA-SSSA Annual Meeting, Houston, TX, Oct., 2008.
- Dou, Z. J.D. Ferguson, J.D. Toth, S.C. Rankin, H.W. Aceto, C.F. Ramberg, Jr., and C. Debroy. Survival characterisitcs of Salmonella and E. coli in manure lagoon, compost, and field soil using environmental sentinel chambers. Oral presentation at NE 1024 regional project Annual Meeting, Tillamook, OR, Oct., 2008.
- Toth, J.D., Z. Dou, S.C. Rankin, H.W. Aceto, and C. Debroy. Environmental survival of zoonotic pathogenic bacteria in manure-amended soil. Abstract and poster presentation at USDA National Water Conference, St. Louis, MO, Feb., 2009.
- Toth, J.D., Z. Dou, S.C. Rankin, and H.W. Aceto. Deactivation of Salmonella Newport in dairy manure through modification of pH and temperature. Abstract and oral presentation at ASA-CSSA-SSSA Annual Meeting, Pittsburgh, PA, Nov., 2009.
- Dou, Z. and J.D. Toth. Survival and transport of food-borne pathogenic bacteria in the dairy farm environment. Joint Workshop in the Agricultural and Environmental Sciences, Newark, DE, Aug., 2010.
- Toth, J.D. and Z. Dou. Tracking pathogens from animal manure to the human food production chain. Abstract and oral presentation at the 3rd International Workshop on Nutrient Management: Technology and Policy. Beijing, China, Sept., 2010.
- Dou, Z., J.D. Toth, S.C. Rankin, H.W. Aceto, and C. DebRoy. Persistence and dynamics of manure-borne pathogens under natural conditions in the farm environment. Oral presentation at USDA National Water Conference, Washington DC, Feb., 2011.
- Toth, J.D., Z. Dou, S.C. Rankin, H.W. Aceto, J.D. Vanderhoef, and C. Debroy. Deactivation of pathogenic Escherichia coli O157:H7 in dairy manure through modification of pH and temperature. ASA-CSSA-SSSA Annual Meeting, San Antonio, TX, Oct., 2011.
- Dou, Z., S.C. Rankin, H.W. Aceto, J.D. Toth, and C. DebRoy. Survival and transport of E. coli O157 and Salmonella Newport in the farm environment. USDA National Water Conference, Portland, OR, May, 2012.
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Progress 09/15/08 to 09/14/09
Outputs
OUTPUTS: Movement of foodborne disease-causing pathogenic bacteria from farm animals into the human food production chain, typically as a result of land application of animal manures, is a matter of increasing public, scientific and government concern. To limit the adverse impacts of foodborne illness on society, it is of paramount importance to understand the behavior of these pathogens in the farm environment as a means to develop ways of suppressing and controlling them. As part of our ongoing comprehensive research project, we have concentrated in the past year on determining the effect of modifying pH on deactivating and killing the pathogens Salmonella Newport and E. coli O157:H7 in dairy manure. Two commercial chemicals commonly used in dairy production, alum and hydrated lime, were employed for this purpose. In addition, we investigated the effect of temperature on pathogen survival. Furthermore, through a complementary on-farm sampling program, we also determined the prevalence of pathogenic organisms including Salmonella, E. coli O157:H7, Campylobacter, Mycobacterium paratuberculosis and the water-borne parasite Cryptosporidium on thirteen dairy farms in Southeastern and Southcentral Pennsylvania. The on-farm study included samples of animal bedding, lactating cow and calf fecal material, manure from storage, milk filters, field soil and water from streams on the farms, which were collected and submitted to microbiology labs for determination of pathogen occurrence. Results from the previous phases of the research project have been disseminated in presentations to the Land and Sea Grant National Water Conferences and the American Society of Agronomy Annual Meetings. A paper on the survival and presence of Salmonella Newport in field soil, effluent lagoon and compost pile on a dairy operation has been accepted for publication in Journal of Dairy Science, and will appear later in 2011. Preliminary results from the on-farm monitoring have been summarized and reported to the relevant producers. Complete results will be given them once the Mycobacterium culturing is completed. PARTICIPANTS: Research was planned by Drs. Dou, Aceto and Rankin with input from Research Specialist John Toth, all University of Pennsylvania. Pure cultures of the pathogens were provided by Dr. Rankin, Department of Pathobiology and by Dr. DebRoy, of the partner organization Penn State University's E. coli Reference Center. Research was conducted by Mr. Toth, with the assistance of technical staff. Presentations of research findings and authorship of a submissions to peer-reviewed journals were done by Dr. Dou and Mr. Toth. Collaboration was provided by the thirteen private farmers who allowed us to collect sample materials on their dairy operations as part of the on-farm pathogen monitoring segment of the research program. Professionals in nutrient management and microbiological sciences encountered at national meetings also provided useful discussion of the topics covered in the research program. TARGET AUDIENCES: Target audiences include the research community involved in control of foodborne illness and food animal production management, and more importantly the dairy producers themselves who are on the front line of preventing potential pathogens moving off-farm. Findings from the project have been disseminated through presentations to research meetings and a peer-reviewed journal submission. Preliminary on-farm pathogen monitoring results have been presented to the farmer-collaborators. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
New information obtained and new knowledge generated in the past year include an estimation if pH or temperature modification of manure could suppress or kill pathogens, and across-farm and within-farm incidences of five pathogens on local dairy operations. Modifying pH was effective in killing Salmonella and E. coli O157:H7 in dairy manure when the pH was adjusted to certain threshold values. Salmonella was completely eliminated at pH <4.2 or pH >11. At intermediate pH (4.5-6, and 9-11), Salmonella was suppressed for 6 to 10 days then rapidly rebounded. E. coli O157:H7 was more sensitive to acidification, with lethal pH of 4.5 or lower. With pH raised in the manure, E. coli was killed by pH >11. At pH 10-11, E. coli O157:H7 concentration was reduced by 10,000-fold but not completely deactivated. In practical terms, achieving the lethal high pH would take 6 kilograms hydrated lime per metric ton of manure in order to eliminate the pathogens. Using alum, 7 to 8 times as much would be needed to achieve the same effect. From the temperature incubation trials, log reduction times (time for a ten-fold decrease in pathogen numbers) averaged 2, 12 and 34 days at 37, 22, and 4 degrees C, respectively. Assuming 22 and 4 degrees C temperatures represent summer and winter conditions, 99% of the pathogens would deactivate within 3 1/2 weeks in summer, but the pathogens could persist at low concentrations for much longer (over 300 days) in colder, non-growing season environments. Manure spreading in winter is currently discouraged because of the potential for nutrient losses through runoff. Long-term survival of pathogens in the colder months is another reason for limiting manure applications to the growing season if possible. Our on-farm monitoring studies at local dairies showed that the pathogenic organisms have frequent presence but are not ubiquitous. Nine of 13 farms tested was positive for at least one of the five pathogens. E. coli O157:H7 was encountered most frequently; found on 6 of the 13 farms, and in 23 of 120 (19%) of all samples collected. E. coli O157:H7 is of special concern in human health. While the number of cases per year in the U.S. is relatively low, the rate of hospitalization is high, and colitis can have serious side effects including kidney or intestinal damage. Campylobacter was found in 6% of the samples, and the Cryptosporidium parasite in 3 of the 39 fecal and manure samples. Interestingly, Salmonella Newport, which is reported as having an epidemic spread on dairy farms in the Northeastern states in the past decade, was not detected. On the other hand, Salmonella serovars Cerro and Montevideo were detected on 2 of the 13 farms. Pathogens were more likely to be found in lactating cow or calf feces (42% of samples positive for at least one of the pathogens) than in bedding, stored manure, milk filters, soil or water (15 to 29% of samples). E. coli O157:H7 was present in all types of samples collected. This on-farm monitoring allowed us to estimate how common pathogens are in regional dairy operations, and which locations on the farm are more likely to have pathogen contamination.
Publications
- Dou, Z., S.C. Rankin, H.W. Aceto, J.D. Toth, J. Vanderhoef, C.F. Ramberg, Jr., and C. DebRoy. 2011. Survival and transport of E. coli O157 and Salmonella in manure and manured soils. Land Grant and Sea Grant National Water Conference, Washington, D.C., 31 January-1 February 2011.
- Toth, J.D., Z. Dou, H.W. Aceto, S.R. Rankin, J. Vanderhoef, and C. DebRoy. 2011. Control of Salmonella Newport and E. coli O157:H7 in dairy manure through modification of pH and temperature. ASA Annual Meetings, San Antonio, TX, 16-19 October, 2011.
- Toth, J.D., H.W. Aceto, S.C. Rankin, and Z. Dou. 2011. Survival characteristics of Salmonella enterica serovar Newport in the dairy farm environment. J. Dairy Science (in press). doi:10.3168/jds.2011-4493.
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Progress 09/15/07 to 09/14/08
Outputs
OUTPUTS: Background Escherichia coli O157:H7 (E. coli O157) and Salmonella enterica serovar Newport (Salmonella Newport) are zoonotic pathogens imposing serious public health concerns. E. coli O157 is associated with 73,000 infections and >60 deaths in the US annually, whereas Salmonella Newport currently ranks third by CDC in causing human intestinal infections (CDC, 2006). Both pathogens have established reservoirs in dairy cattle. As infected animals shed the organisms in manure and manure is subsequently spread on agricultural land, the pathogens may be transmitted to plants, other animals or humans, or transported into waters. The purpose of this project is to enhance the understanding of the survival and environmental fate of these pathogens in the agroecosystem. Events and Activities Sentinel chambers were used to monitor the survival of the pathogens under field conditions. Pure cultures of Salmonella Newport or E. coli O157 were mixed with manure or soils. Three replicated treated sentinels and one without bacteria inoculation were included. Diluted extracts were plated on selective media (XLD agar for Salmonella Newport and sorbitol-MacConkey agar for E. coli O157). Two trials were conducted. In Trial 1 (Nov '07 - Feb '08 over-winter trial), Salmonella Newport or E. coli O157 were inoculated into soil-manure mixtures, sentinel chambers buried under grass sod. This was designed to "work the bugs out". S. Newport log reduction time 16 days, consistent with other Salmonella trials. Enumerations of E. coli were a problem, difficult to separate introduced, inoculated E. coli stx- from the native coliforms. The problem was resolved in subsequent trials, especially choice of plating medium. In Trial 2, Salmonella Newport or E. coli were inoculated into (i) soil-manure mixtures with sentinel chambers buried under grass sod, (ii) dairy manure with sentinel chambers immersed in dairy lagoon or buried in compost pile. Samples were collected periodically and bacterial populations were monitored. Products/Dissemination Results and findings from the above trials were communicated to: (i) students at the School of Veterinary Medicine, University of Pennsylvania as well as Cornell University via classroom teaching, (ii) researchers, educators, and extension personnel at USDA CSREES National Water Conference, (iii) Agronomists, Soil Scientists, and Environmental researchers at the annual meeting of ASA-CSSA-SSSA. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
New knowledge we learned from the trials are summarized below. - S. Newport and E. coli log reduction time in the lagoon system (7-9 days) approximately half that of the field soil samples. Competition or antagonistic interactions between the introduced pathogens and native manure microorganisms probably a contributing factor. - In the summer field soil sentinel trials, Salmonella and E. coli had different survival patterns, Salmonella rate of deactivation lower than that of E. coli. A hypothesis is that E. coli may be less tolerant of low soil moisture status, typical of growing season conditions. A possible way to test is to do a lab incubation of inoculated soil-manure mixtures, adjusting soil moisture to, say, 50, 70 and 90% of field capacity and follow survival. - S. Newport and E. coli in compost pile were killed within 18 hours of installation, presumably by high temperatures generated in the compost, 64 C. Native compost microorganisms killed off as well. We are discussing whether re-run the compost trial using aged compost, after the temperature has equilibrated. In July, the compost temperature dropped to 40 C after about a month. - Salmonella in the field trials showed a steady linear deactivation when log CFU was plotted against time, at least to the detection limit of 2 log10 g-1. After that, MPN analysis showed a long tail, a steady decline but over 5-6 months, making it a bimodal plot. We are discussing what models to use; of course a model assumes a mechanism behind it - what is the balance between goodness-of-fit and a likely mechanism - A.M. Ibekwe (USDA Riverside) gave a talk at the recent Agronomy Society meetings, E. coli O157 survival in competition with native microorganisms in the soil system. He suggested that the long tail in the deactivation plots showed two populations of the pathogen, the group more sensitive to environmental conditions and/or competition dying out rapidly and the more robust group surviving at low levels for longer periods.
Publications
- Dou, Z., J. Toth, S. Rankin, H. Aceto, and C. DeBroy. (2008). Survival of Salmonella Newport and Eschericia coli O157 in manure. Abstract and post presentation at USDA-CSREES National Water Conference, Sparks, NV, 2008.
- Toth, J., Z. Dou, S. Rankin, H. Aceto, C.F. Ramberg and C. Debroy. (2008). Environmental survival of zoonotic pathogenic bacteria in manure-amended soil. Abstract and post presentation at ASA-CSSA-SSSA annual meeting, Houston, TX, Oct. 2008.
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