Progress 09/01/13 to 08/31/16
Outputs
Target Audience:This seed project reached an audience of North Dakota and Minnesota produce growers from two farms via interactions during field research. Native American middle school students, high school students and tribal community college instructors at five tribal colleges were reached through a food safety workshop. Food safety researchers were reached via intramural presentations and extramural presentations at two conferences. Food safety and computer science researchers through publication of a journal article. The project reached three environmental scientists through a collaboration with the USGS. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?An entry level technician was hired as a result of this grant. A graduate student and the PD collaborated with those wetlands researchers and gained access and additional training in wetland ecosystems livestock farms, crop farms and in protected areas. Two graduate students and a technician were trained in survival experiments, microbial genetics, quantitative PCR, statistical analysis and predictive microbiology. One graduate student was trained in environmental (soil, sediment, and aquatic) microbiome analysis associated with this project. An undergraduate student, majoring in Computer Science and Microbiology, was trained in agent-based model coding and development. The graduate student presented his work at internal lab meetings and extramural meetings with collaborators. Three undergraduates were trained in molecular biology, field sampling, food safety and laboratory safety as part of this project. How have the results been disseminated to communities of interest?Experiments were recently completed, so have not been disseminated yet. We plan to publish the results in the coming year. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overall goal of this project is to advance techniques to predict and prevent foodborne illness outbreaks. While outbreaks of Listeria monocytogenes in fruits and vegetables remain rare, there have been several recent recalls, and one recent outbreak was linked to consumption of packaged salads in 2016. Two key challenges are being addressed in this seed project using controlled experiments. First, a reliable molecular method for detecting live L. monocytogenes is required. The efficacy of quantitative real-time PCR probes to detect L. monocytogenes was evaluated by inoculating known numbers of the foodborne pathogen into actual field-collected samples from fruit and vegetable farms in North Dakota and Minnesota. The overall data from this project may impact agricultural practices in three ways. First, it may not be possible to detect the L. monocytogenes pathogen in farm samples unless contamination is recent and the levels of contamination are high. Second, while proximity to surface water and wetlands may improve our chances to detect L. monocytogenes in on-farm audits (based on prior work), those areas do not necessarily have greater abundance or persistence of the pathogen (based on this project). Third, and most importantly, when the L. monocytogenes pathogen is present in soil, rainfall can increase its abundance and potentially increase the chances that produce commodities become contaminated in the farm field prior to or during harvest. Objective 1 - Evaluate the efficacy of quantitative real-time PCR (qPCR) to measure abundance of L. monocytogenes in inoculated environmental samples from produce fields. Only live L. monocytogenes cause disease, but most molecular (DNA-based) moethods to quantify pathogens canoot distinguish between DNA from live cells and DNA from dead cells. Our work in this areas focused on evaluating molecular methods for the detection of live L. monocytogenes in soil Changes in knowledge - We determined that strain J0161 from L. monocytogenes lineage II had a slower death rate in Fargo clay soil than strains from Lineage I or III (Year 1). We determined that the prs gene exhibited the best sensitivity and specificity for quantitative PCR (qPCR) detection of L. monocytogenes genomes in soil DNA extracts out of four candidate genes for which probes were evaluated (Year 1). We determined that propidium monoazide could be used to distinguish DNA from live vs. dead L. monocytogenes when a large halogen lamp was used to activate the dye in soil slurries (blue light was ineffective at activating the dye in soil slurries; Years 1 and 2). Using PMA-qPCR on samples with known quatities of live and dead L. monocytogenes and closely related negative controls (L. seeligeri), we showed that PMA qPCR can quantify live L. monocytogenes in soil, water, drag swab and plant leaf surfaces. Quantification from water and soil samples was most accurate (Year 2). So, sample type impacts our ability to detect L. monocytogenes that has been intentionally added to samples. However, the impacts were small and mainly the product of DNA extraction method, rather than anything intrinsic to the PMA-qPCR method. Based on our results, dead L. monocytogenes would not be expected to interfere with the PMA-qPCR based detection of live L. monocytogenes in these sample types. Objective 2 - Quantify the impact of precipitation events on the abundance and prevalence of L. monocytogenes under controlled conditions in soils from produce fields. Changes in knowledge (All in year 2) - Listeria monocytogenes has been reported to favor moist environments and prevalence of L. monocytogenes in farm fields is associated with higher available water storage in soil and closer proximity to surface water and wetlands. These findings beg the question, "Are L. monocytogenes better able to grow or persist in soils with elevated moisture?" and "How does rainfall affect the persistence and growth of L. monocytogenes?". We conducted two microcosm experiments to answer these questions about soil moisture and precipitation. To address the effect of rainfall on survival and growth, we inoculated L. monocytogenes J0161 expressing a greenfluorescent protein into soil microcosms at an initial abundance of 105 colony forming units (CFU) per g soil. These microcosms were subjected to one of three simulated rainfall treatments. Treatment 1 was a single precipitation event equivalent to 26 mm of rainfall at three days post-inoculation. Treatment 2 was repeated precipitation events equivalent to 10 mm rainfall at days 3, 7, 11 and 15 post inoculation. Treatment 3 received no simulated rainfall at all. Following the simulated rainfall events on day 3 for treatments 1 and 2, the abundance of L. monocytogenes J0161 increased approximately 100-fold. No increase in population size was observed in the no-precipitation controls. Rate of population decline was slower in treatments 1 and 2 than in the no-precipitation controls. The final cell density of L. monocytogenes in treatments 1 and 2 was approximately 100-fold greater than in the no-precipitation controls and was approximately 104 CFU per g soil. L. monocytogenes was detectable in treatment groups 1 and 2 out to 80 days post-inoculation but became undetectable by 60 days in no-precipitation controls. The results of this experiment suggest that rainfall increases the abundance of L. monocytogenes. This result is concerning because such large populations in recently wetted soils may be mobilized by subsequent rainfall events to reach leafy greens. It also provides initial data for adjusting geospatial models of Listeria monocytogenes abundance based on recent weather. Using this lab experiment as a proxy, the data suggest that abundance of L. monocytogenes, and our expections for its environmental prevalence, should be adjusted about 100-fold higher after recent rainfall events, though repeated rainfall had no obvious effect when compared to a single large rainfall event in this experiment. Published studies by Weller, Strawn, Wiedmann and Bergholz have also indicated that Listeria monocytogenes prevalence increases in proximity to surface water, though these studies have mainly focused on streams and rivers with limited attention to the impact of wetlands. We sought to test whether soils within and surrounding wetlands in North Dakota farms were more hospitable to Listeria monocytogenes. We expected that transition zone and toe-slope soils surrounding would promote survival of L. monocytogenes J0161, because these soils exhibit elevated moisture due to their hydrographic proximity to the wetland. Contrary to our expectations, transition zone sediments did not support enhanced survival of L. monocytogenes J0161, but toe-slope and shoulder-slope soils exhibited slower death rates after inoculation. While soil moisture was lower in toe- and shoulder-slope sites, salinity in those soils was lower than in transition and submerged sediments. The increase in salinity was substantial, wetland transition zone and submerged sediments were also overwhelmingly anaerobic with elevated levels of sulfide far greater than those detected in soil samples. Therefore, it seems likely that even if wetland margin and submerged sediments are more hospitable to L. monocytogenes due to their moisture, other toxic metabolic products from the native microbiota may be decreasing their abundance in sediments. Microbiome studies are in progress to assess positive and negative interactions between L. monocytogenes death rate and the community composition in these soils. It may be that only specific types of wetlands, or only non-stagnant surface water may contribute to elevated prevalence of L. monocytogenes. These results clearly should affect environmental models of L. monocytogenes prevalence, but only after some more experimental and field validation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Bergholz, P.W. and Wiedmann, M. Toward agent-based models for pre-harvest food safety. IBM J. Res. Devel. 60(5/6)
Paper 8.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Shiwakoti, S. and P.W. Bergholz (2014). Evaluation of quantitative PCR with propidium monoazide for the specific detection of live Listeria monocytogenes in soil. ND-EPSCoR/IDeA 2014 State Conference, Grand Forks, ND
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Schmidt, K.N., M.A. Mann, J.P. Schmidt, and P.W. Bergholz (2015) Evaluation of sample effects on detection of Listeria monocytogenes in small produce farms. USDA-NIFA, Institute of Food Safety and Nutrition, Division of Food Safety, Joint Project Directors Meeting. Portland, OR.
- Type:
Other
Status:
Published
Year Published:
2016
Citation:
Bergholz, P.W. "Probing the role of the soil microbiome in susceptibility of prairie pothole wetlands to invasion by a foodborne pathogen". An invited seminar for the Microbiome Seminar Series, Center for Protease Research, North Dakota State University, Fargo, ND.
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Progress 09/01/14 to 08/31/15
Outputs
Target Audience:During this reporting period, this seed project reached produce growers via interactions during field research and food safety researchers during project directors' meeting and the IAFP conference. Changes/Problems:A no-cost extension was requested and granted due to unforeseen delays in the execution of Objective 2.The reasons for delay are a combination of technical challenges in objective 2 and additional institutional oversight of BSL-2 research in our greenhouse facilities. Construction of recombinant Listeria monocytogenes, expressing green-fluorescent protein (GFP) from an inducible promoter, proved too challenging for our group; this led to a delay of approximately 4.5 months in the research on Obj. 2. We have since obtained constitutive GFP expressing Listeria monocytogenes strains from Cathy Webb at the University of Georgia. In addition to this, new institutional requirements for approval of our BSL-2 work in NDSU's research greenhouse facility were introduced during this year of the project. Work in the greenhouse was delayed while approval was obtained. This caused an additional 2 month delay in the project. While these challenges prevented us from completing the project along the original timeline, the work on Objective 2 is now underway and we anticipate completion before the project ends in August, 2016. What opportunities for training and professional development has the project provided?The PD connected with other environmental modeling researchers in food safety during and after the NIFA project directors meeting. Two undergraduate researchers and one graduate researcher received extensive training in experimental design and execution of foodborne pathogen detection protocols during this project period. How have the results been disseminated to communities of interest?The PD presented a poster at the annual NIFA Food Safety Project Directors meeting. What do you plan to do during the next reporting period to accomplish the goals?1) Finalize and publish the results from Objective 1 in a peer-reviewed food safety or applied microbiology journal. 2) Complete the research on Objective 2 by conducted side-by-side studies on changes in L. monocytogenes abundance using PMA-qPCR and viable plate countsin soil microcosms under different precipitation regimes. 3) Present the results of both research objectives at the annual meeting of the International Association for Food Protection.
Impacts
What was accomplished under these goals?
Two key challenges are being addressed by this seed project. First, a reliable molecular method for detecting live L. monocytogenes is required. Second, a controlled greenhouse experiment is being conducted to quantify how simulated rainfall changes the population size and mobility of L. monocytogenes residing in farm soil samples. Objective 1 - Evaluate the efficacy fo quantitative real-time PCR (qPCR) to measure abundance of L. monocytogenes in inoculated environmental samples from produce fields. Changes in knowledge resulting from work on Objective 1 - To successfully quantify the abundance of L. monocytogenes requires a molecular method that can distinguish DNA originating from live cells in mixtures containing both live and dead cells. To test the efficacy of qPCR to quantify the number of live L. monocytogenes in environmental samples from farms, we collected soil, drag swab, irrigation water and vegetable (parsley, cabbage, and green leaf lettuce) samples from three production blocks across two farms in North Dakota and Minnesota. We then inoculated the samples with known amounts (102 to 106) of live L. monocytogenes J0161 with and without 103 cells per sample of dead L. monocytogenes and/or 106cells of live L. seeligeri. This permited us to test our ability to quantify live L. monocytogenes in the presence of dead L. monocytogenes and/or a non-target Listeria species. After an incubation period, dead cell DNA was crosslinked to propidium monoazide to prevent its detection.In our first project period, we faced significant difficulties with the use of propidium monoazide to differentiate DNA from dead cells from that of living cells. We purchased a large, high powered halogen lamp and were able to use propidium monoazide (PMA) to successfully distinguish DNA from live cells against dead cells even in inoculated soil samples, provided that dead cell DNA was at less than 104 copies in the sample. Then, bulk DNA (our inoculum plus other DNA originating from these environmental samples) was extracted using methods accorded to each sample type. From these efforts, we showedthat L. monocytogenescould be quantified in all four sample types, but that there were quantifiable sample type effects. DNA from soil samples resulting, on average, with a 3-fold lower estimate than drag swabs. Drag swabs, in turn, produced approximately a 4-fold lower estimate than water samples. Produce leaf samples produced a 104 fold lower estimate than water samples. Neither L. seeligeri (non-target Listeria) nor dead L. monocytogenes interfered with the assay as utilized in this study. Our relative inability to detect L. monocytogenes on produce leaf samples was determined to be a product of suboptimal DNA extraction from these samples. Since conducting this initial study, we have since repeated the experiment using romaine lettuce in a greenhouse and determined that a new DNA extraction method leads to only about a 10-fold under estimate of L. monocytogenes. The bottom line is that sample type did impact our ability to detect L. monocytogenes that had been intentionally added to the samples. However, the impacts were generally small and mainly a product of DNA extraction efficiency. Dead L. monocytogenes would not, generally, be expected to interfere with the detection of live L. monocytogenes in these sample types. Objective 2 - Quantify the impact of precipitation events on the abundance and prevalence of L. monocytogenes under controlled conditions in soils from produce fields. Changes in knowledge resulting from work on Objective 2. During the first project period, we determined that qPCR with PMA may not be able to quantify live L. monocytogenes with great accuracy in the presence of dead L. monocytogenes. While our updated method may lead to successful use of qPCR with PMA in the objective 2 experiments, we have also optimized the use of recombinant L. monocytogenes J0161 (from Cathy Webbat UGA)expressing enhanced green fluorescent protein for use in these greenhouse experiments. Tests of both methods, qPCR and viable plate countsof recombinant L. monocytogenes,were promising for detectinglive L. monocytogenes in soils and in lettuce leaf surfaces under controlled precipitation experiments. Due to significant delays in starting these experiments (see Changes/Problems), we expect to complete this during our next reporting period.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Schmidt, K.N., M.A. Mann, J.P. Schmidt, and P.W. Bergholz. (2015). Evaluation of sample effects on detection of Listeria monocytogenes in small produce farms. USDA NIFA, Institute of Food Safety and Nutrition, Division of Food Safety, Joinr Project Directors Meeting. Portland, OR.
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Progress 09/01/13 to 08/31/14
Outputs
Target Audience: During this reporting period, this seed project reached produce growers via interactions during field research, Native American high school students during afood safety workshop,and food safety researchers via presentations and conferences. Changes/Problems: We changed the plan for Objective 2, because PMA-qPCR will be ineffective. The switch to recombinant L. monocytogenes for Objective 2 introduced delays due to the need to construct a recombinant eGFP-expressing strain of L. monocytogenes J0161. We continue to expect completion of Objective 2 during the project period (up to September, 2015). A new Institutional Biosafety Committee Protocol has been approved for this work - NDSU IBC protocol number B15011. What opportunities for training and professional development has the project provided? An entry level laboratory technician, Ms. Kaycie Schmidt,was hired and is devoting 0.5 FTE to this project. As part of this project, Ms. Schmidt, is developing her skills as in molecular biology, field sampling, genetics, food safety, laboratory safety,and management skills. A first year doctoral student in the Environmental and Conservation Sciences program,Mr. Suvash Shiwakoti,is focus on this project as part of his thesis. As part of his training, he has been trained in molecular biology, microbial genetics, food safety, and the conduct of science, including responsible conduct of research. Mr. Shiwakoti has presented a poster at the ND-EPSCoR state conference and has attended several local conferences as part of his professional development. Three undergraduates received training in molecular biology, field sampling, food safety, and laboratory safety as part of this project. Additionally, all participants have also gotten training in reviewing the food safety research as part of discussions focused on the published literature in lab meeting. How have the results been disseminated to communities of interest? The results were communicated to both environmental and agricultural scientists via a poster presentation at the ND-EPSCoR state conference in April, 2014. Some results were also shared with other Food Safety AFRI PDs via a poster presentation at the Project directors' meeting in 2014. What do you plan to do during the next reporting period to accomplish the goals? 1) Complete the research on Objective 1 by conducting PMA-qPCR to discern whether sample type and/or amount ofdead cell masshas impacts on the efficacy of PMA. 2) Complete the research on Objective 2 by conducting studies of the changes in L. monocytogenes abundance under different simulated rainfall regimes. 3) Present the resultsassociated withone or both research objectives at the annual meeting of the International Association for Food Protection. 4) Publish research results in peer-reviewed journals such as Journal of Food Protection or Applied and Environmental Microbiology. 5) Publish the "Food Safety on Farms" workshop design and plan in a peer-reviewed repository of learning activities.
Impacts
What was accomplished under these goals?
This seed project is focused on developinginitial dataon the impacts of detection method andfield weather conditions on thethedetection and abundance of Listeria monocytogenes, a foodborne pathogen, in pre-harvest environments of fruit and vegetable farms. Successful completion of this seed project will enable the deeper research and technology development leading to computer tools predicting the risk of fruit and vegetable contamination in the field. These computing tools will use information about the soil, soil amendments, weather and the lands surrounding a farm to produce maps depicting the risk of contamination in different parts of a farm landscape. Two key challenges are being address by this seed project. First, a reliable molecular method for detecting live L. monocytogenes is required.Second, a controlled greenhouse experiment to quantify howsimulated rainfallchanges the population size and mobility of L. monocytogenes residing in farm soil samples. Objective 1 - Evaluate the efficacy of quantitative real-time PCR to quantify L. monocytogenes abundance in inoculated environmental samples from produce fields. Only live L. monocytogenes cause disease, but most molecular (DNA-based) methods to quantify pathogens cannot distinguish between the DNA from live cells and the DNA from dead cells. Our initial work in this area has focused on evaluating molecular methods for the detection of L. monocytogenes. As a result, we now have key information about our ability to specifically detect live L. monocytogenes in soil. Changes in knowledge resulting from work on Objective 1. We have quantified the survival rates of three different strains of L. monocytogenes by viable plate count after incubation in North Dakota Fargo clay soil. L. monocytogenes J0161 (a representative of genetic lineage II) had a significantly slower death rate compared to L. monocytogenes H7858 (a representative of genetic lineage I). This result is sensible, as lineage II is more frequently found outside hosts than lineage I - which is predominantly composed of isolates from clinical listeriosis cases. We opted to use L. monocytogenes J0161 as our strain of focus for molecular quantification experiments. Quantitative PCR results require the use of oligonucleotide probes and primers. The choice of primer-probe combinations can greatly effect the sensitivity and specificity of a qPCR assay for pathogen detection. We tested the efficacy of three qPCR primer-probe sets for the detection of L. monocytogenes: one targeting the inlA gene, one targeting gene lm0398 and one targeting the prs gene. All probes were found to be specific for L. monocytogenes in computational analysis. The three probe sets also performed similarly in qPCR tests using known quantities of L. monocytogenes DNA. However, the lm0398 primer-probe set was less sensitive than the others, and the prs primer-probe set was somewhat more accurate in quantifying small amounts of L. monocytogenes genomes (102 genomes/ reaction). To successfully quantify the abundance of L. monocytogenes requires a molecular method that candistinguish DNA originating from live cells in mixtures containing both live and dead cells. Propidium monoazide (PMA)is a reactive DNA-binding dye that binds free floating DNA from dead cells in the presence of blue light. While propidium monoazide is thought to be effective at this activity in cell culture and in some food matrices, it is unclear whether it can successfully bind DNA in soil because: a) soil may interact with free-floating DNA and protect it from PMA exposure and b) soil quenches blue light very effectively, meaning that PMA may not react with free-floating DNA. We tested the efficacy of PMA to prevent the detection of DNA from dead cells in soil. PMA was unable to prevent the detection of dead cells that had been inoculated into soil microcosms in combination with live cells - though it remains possible that the level of dead cells used, which was unrealistically large at 109 CFU per g soil, was also too large to be effectively treated with PMA. To complete this objective, the efficacy of PMA to detect DNA from live cells must be assessed in all common sample types from pre-harvest environments. Since this is the PD's first project in North Dakota, relationships with regional produce farms had to be established in order to obtain samples. Sampling was conducted on two privately operated produce farms in North Dakota and Minnesota. Soil, surface drag swab, water, and vegetable samples were obtained. These samples were returned to the lab and inoculated with varying levels of live and dead L. monocytogenes J0161, incubated for 24 h at 17 degrees C, and processed for PMA treatment followed by DNA extraction. This work is ongoing. Objective 2 - Quantify the impact of precipitation events on the abundance and prevalence of L. monocytogenes under controlled conditions in soils from produce fields. Prior work by the PD determined that land management, soil characteristics and recentthermal and precipitationconditions all interacted to determine the presence of L. monocytogenes in samples collected from pre-harvest environments. However, weather conditions are highly dynamic, and a predictive model for L. monocytogenes abundance must be able to take information from recent weather events and calculate the impact of those events on the abundance of the pathogen.Our university sports a new biosafety level 2 greenhouse complex, including contained spray chambers capable of simulating rainfall. We will use these facilities to conduct controlled experiments on the impact of the timing and amount of rainfall on the abundance of L. monocytogenes in soil. However, we expect both live and dead cells to be present in soil microcosms in this experiments,but our experiments with PMA have demonstrated that qPCR cannot be used successfully in these experiments. To complete this objective, the graduate student has been working to construct recombinant Listeria monocytogenes J0161 expressing an inducible green fluorescent protein. We expect the recombineering work to be complete within the month at which point this strain will constitute not only a tool for our own work but for the food safety community at large.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Shiwakoti, S. and PW Bergholz. (2014) Evaluation of quantitative PCR with propidium monoazide for the specific detection of live Listeria monocytogenes in soil. ND EPSCoR/IDeA 2014 State Conference. Grand Forks, ND.
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