Daniel R Shelton

Agricultural Research Service, Kerrville, Texas, United States

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Publications (94)215.06 Total impact

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    ABSTRACT: Biofilm-forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been shown to promote the incorporation of Escherichia coli O157:H7 into dual-species biofilms. In this study, interactions between E. coli O157:H7 and R. insidiosa were examined under different incubating conditions. Under static culture conditions, the incorporation of E. coli O157:H7 into biofilms with R. insidiosa was not significantly affected by either low incubating temperature (10°C) or by limited nutrient availability. Greater enhancement of E. coli O157:H7 incorporation in dual-species biofilms was observed by using a continuous culture system with limited nutrient availability. Under the continuous culture conditions used in this study, E coli O157:H7 cells showed a strong tendency of colocalizing with R. insidiosa on a glass surface at the early stage of biofilm formation. As the biofilms matured, E coli O157:H7 cells were mostly found at the bottom layer of the dual-species biofilms, suggesting an effective protection by R. insidiosa in the mature biofilms.
    Journal of food protection 01/2015; 78(1):121-7. DOI:10.4315/0362-028X.JFP-14-302 · 1.80 Impact Factor
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    ABSTRACT: Infiltration is important to modeling the overland transport of microorganisms in environmental waters. In watershed- and hillslope scale-models, infiltration is commonly described by simple equations relating infiltration rate to soil saturated conductivity and by empirical parameters defining changes in infiltration rate with soil water content. For the microbial transport model KINEROS2/STWIR used in this study, infiltration in unsaturated soil is accounted for by a net capillary drive parameter, G, in the Parlange equation. Scarce experimental data and multiple approaches for estimating parameter G introduce uncertainty, reducing reliability of overland water flow and microbial transport models. Our objectives were to evaluate reliability and robustness of three methods to estimate parameter G and associated accuracy and uncertainty in predicting runoff and fecal coliform (FC) transport. These methods include (i) KINEROS2 fitting to the experimental cumulative runoff data; (ii) estimating solely on soil texture; and (iii) estimating by individual pedotransfer functions (PTFs) and an ensemble of PTFs from basic soil properties. Results show that the most accurate prediction was obtained when the G parameter was fitted to the cumulative runoff. The KINEROS2-recommended parameter slightly overestimated the calibrated value of parameter G and yielded less accurate predictions of runoff, FC concentrations and total FC. The PTFs-estimated parameters systematically deviated from calibrated G values that caused high uncertainty in the KINEROS2/STWIR predictions. Averaging PTF estimates considerably improved model accuracy, reducing the uncertainty of runoff and FC concentration predictions. Overall, ensemble-based PTF estimation of the capillary drive can be efficient for simulations of runoff and bacteria overland transport when a single effective value is used across the study area.
    Journal of Hydrology 11/2014; 519:644-655. DOI:10.1016/j.jhydrol.2014.08.005 · 2.69 Impact Factor
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    M D Stocker, Y A Pachepsky, D R Shelton
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    ABSTRACT: Modeling inactivation of indicator microorganisms is a necessary component of microbial water quality forecast and management recommendations. The linear semi-logarithmic (LSL) model is commonly used to simulate the dependencies of bacterial concentrations in waters on time. There were indications that assumption of the semi-logarithmic linearity may not be accurate enough in waters. The objective of this work was to compare performance of the LSL and the two-parametric Weibull inactivation models with data on survival of indicator organism in various types of water from a representative database of 167 laboratory experiments. The Weibull model was preferred in >99% of all cases when the root mean squared errors and Nash-Sutcliffe statistics were compared. Comparison of corrected Akaike statistic values gave the preference to the Weibull model in only 35% of cases. This was caused by (i) a small number of experimental points on some inactivation curves, (ii) closeness of the shape parameter of the Weibull equation to one, and (iii) piecewise log-linear inactivation dynamic that could be well described by neither of the two models compared. Based on the Akaike test, the Weibull model was favored in agricultural, lake, and pristine waters, whereas the LSL model was preferred for groundwater, wastewater, rivers, and marine waters. The decimal reduction time parameter of both the LSL and Weibull models exhibited an Arrhenius-type dependence on temperature. Overall, the existing inactivation data indicate that the application of the Weibull model can improve the predictive capabilities of microbial water quality modeling. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.
    Journal of Environmental Quality 09/2014; 43(5):1559-65. DOI:10.2134/jeq2014.01.0023 · 2.35 Impact Factor
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    ABSTRACT: As sediments increasingly become recognized as reservoirs of indicator and pathogen microorganisms, an understanding of the persistence of indicator organisms becomes important for assessment and predictions of microbial water quality. The objective of this work was to observe the response of water column and sediment coliform populations to the change in nutrient concentrations in the water column. Survival experiments were conducted in flow-through chambers containing sandy sediments. Bovine feces were collected fresh and introduced into sediment. Sixteen days later, the same fecal material was autoclaved and diluted to provide three levels - 1×, 0.5×, and 0.1× of nutrient concentrations - spike in water column. Total coliforms, Escherichia coli, and total aerobic heterotrophic bacterial concentrations were monitored in water and sediment. Bacteria responded to the nutrient spike with initial growth both in the water column and in sediment. The response of bacterial concentrations in water column was nonlinear, with no significant changes at 0.1 and .5× spikes, but a substantial change at 1× spike. Bacteria in sediment responded to the spikes at all added nutrient levels. Coliform inactivation rates both in sediment and in water after the initial growth occurred, were not significantly different from the inactivation rates before spike. These results indicate that introduction of nutrients into the water column results in nonlinear response of E. coli concentrations both in water and in sediments, followed by the inactivation with the same rate as before introduction of nutrients.
    Water Research 04/2014; 59C:316-324. DOI:10.1016/j.watres.2014.04.019 · 5.32 Impact Factor
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    ABSTRACT: The objective of this study was to compare dependency of survival rates on temperature for indicator organisms E. coli and Enterococcus and the pathogen Salmonella in surface waters. A database of 86 survival datasets from peer-reviewed papers on inactivation of E. coli, Salmonella, and Enterococcus in marine waters and of E. coli and Salmonella in lake waters was assembled. The Q10 model was used to express temperature effect on survival rates obtained from linear sections of semi-logarithmic survival graphs. Available data were insufficient to establish differences in survival rates and temperature dependencies for marine waters where values of Q10=3 and a survival rate of 0.7 day−1 could be applied. The Q10 values in lake waters were substantially lower in marine waters, and Salmonella inactivation in lake water was, on average, twice as fast as E. coli; data on E. coli substantially outnumber data on Enterococcus and Salmonella. The relative increase in inactivation with increase in temperature is higher in marine waters than lake water, and differences in inactivation between Salmonella and E. coli at a given temperature were significant in lake water but not in marine waters.This article is protected by copyright. All rights reserved.
    Letters in Applied Microbiology 04/2014; 59(3). DOI:10.1111/lam.12272 · 1.75 Impact Factor
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    ABSTRACT: Spinach plants were irrigated biweekly with water containing 2.1 log CFU Salmonella/100 ml water (the maximum Escherichia coli MPN recommended by the Leafy Greens Marketing Agreement; LGMA), or 4.1 CFU Salmonella/100 ml water to determine Salmonella persistence on spinach leaves. Green Fluorescent protein expressing Salmonella were undetectable by most-probable number (MPN) at 24 h and 7 days following each irrigation event. This study indicates that Salmonella are unlikely to persist on spinach leaves when irrigation water is contaminated at a level below the LGMA standards. In a parallel study, persistence of Salmonella isolated from poultry or produce was compared following biweekly irrigation of spinach plants with water containing 6 log CFU Salmonella/100 ml. Produce Salmonella isolates formed greater biofilms on polystyrene, polycarbonate and stainless steel surfaces and persisted at significantly higher numbers on spinach leaves than those Salmonella from poultry origin during 35 days study. Poultry Salmonella isolates were undetectable (<1 log CFU/g) on spinach plants 7 days following each irrigation event when assayed by direct plating. This study indicates that Salmonella persistence on spinach leaves is affected by the source of contamination and the biofilm forming ability of the strain.
    Food Microbiology 12/2013; 36(2):388-94. DOI:10.1016/j.fm.2013.06.019 · 3.37 Impact Factor
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    ABSTRACT: Biofilm formation is a mechanism adapted by many microorganisms that enhances the survival in stressful environments. In food processing facilities, foodborne bacterial pathogens, which many are poor biofilm formers, could potentially take advantage of this protective mechanism by interacting with other strong biofilm producers. The objective of this study was to determine the influence of bacteria native to fresh produce processing environments on the incorporation of Escherichia coli O157:H7 in biofilms. Bacteria strains representing 13 Gram-negative species isolated from two fresh produce processing facilities in a previous study were tested for forming dual-species biofilms with E. coli O157:H7. Strong biofilm producing strains of Burkholderia caryophylli and Ralstonia insidiosa exhibited 180% and 63% increase in biofilm biomass, and significant thickening of the biofilms (B. caryophylli not tested), when co-cultured with E. coli O157:H7. E. coli O157:H7 populations increased by approximately 1 log in dual-species biofilms formed with B. caryophylli or R. insidiosa. While only a subset of environmental isolates with strong biofilm formation abilities increased the presence of E. coli O157:H7 in biofilms, all tested E. coli O157:H7 exhibited higher incorporation in dual-species biofilms with R. insidiosa. These observations support the notion that E. coli O157:H7 and specific strong biofilm producing bacteria interact synergistically in biofilm formation, and suggest a route for increased survival potential of E. coli O157:H7 in fresh produce processing environments.
    International journal of food microbiology 11/2013; 171C:15-20. DOI:10.1016/j.ijfoodmicro.2013.11.007 · 3.16 Impact Factor
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    ABSTRACT: Microbial quality of irrigation water attracts substantial attention due to the increased incidence of gastrointestinal illness caused by contaminated produce. Little is known about the changes in microbial quality of water during its delivery to crops. Studies were conducted to compare the biofilm formation and changes in microbial water quality in aluminum and PVC irrigation pipes. Irrigation events were conducted weekly and sections of irrigation pipe (coupons) analyzed for total protein, for total and fecal coliform bacteria, and for Escherichia coli. Coliform and E. coil concentrations along with nitrate, orthophosphate, and total organic carbon were monitored in the intake surface water, output irrigation water, and measured in residual standing water in pipes just prior to each irrigation event. Proteins accumulated to a greater extent in aluminum-associated biofilms than in plastic-associated biofilms. Numbers of total coliforms associated with aluminum coupons increased with time while numbers of total coliforms associated with plastic coupons fluctuated. Nitrates disappeared in standing water after one week in aluminum pipes and remained present in plastic. No detectable E. coil remained in standing water in both types of pipe. There was a high probability that coliform concentrations in output irrigation water were different from the intake concentrations in plastic pipes but not in aluminum pipes. Further research is required to evaluate how pipe material may affect the potential of biofilms in irrigation distribution systems to serve as reservoirs of pathogens that can be disseminated to crops during irrigation. Published by Elsevier B.V.
    Agricultural Water Management 11/2013; 129:145-151. DOI:10.1016/j.agwat.2013.07.021 · 2.33 Impact Factor
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    ABSTRACT: Release of manure components is an important element of modeling applications in environmental water quality. The scarcity of experimental data and the multiplicity of the approaches for modeling release kinetics of the manure components introduce uncertainty and reduce reliability of overland flow and contaminant transport models. The goal of this study was to estimate release parameters for different manure components and provide input for KINEROS2/STWIR model developed for pathogen risk assessment associated with livestock operations. The objectives of this work were to evaluate reliability and robustness of the manure release parameters estimated based on individual and grouped release kinetics of soluble, particulate and combination of particulate and soluble materials from surface applied manure. The parameters of Bradford–Schijven model were evaluated from the experimental data on release of chloride, water-extractable phosphate-P, total bioactive P, organic carbon, enterococci and E. coli from surface applied manure measured in the runoff-box and runoff-plot experiments. The results showed that release of different manure components from surface applied manure can be reliably predicted with just a single set of parameters characterizing the kinetics of manure mass release. We demonstrated that the manure release parameters could be estimated more reliably when the model fit was performed using data for different manure components pooled together, while the model fit to a single release curve produced correlated parameters. The model parameters appeared to be robust and transferable from the calibration to validation datasets without any or with only minor losses of the model accuracy.
    Ecological Modelling 08/2013; 263:126–138. DOI:10.1016/j.ecolmodel.2013.05.008 · 2.07 Impact Factor
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    ABSTRACT: Representative food contact and nonfood contact surfaces in two mid-sized, fresh-cut processing facilities were sampled for microbiological analyses after routine daily sanitization. Mesophilic and psychrotrophic bacteria on the sampled surfaces were isolated by plating on nonselective bacterial media. Alternatively, bacteria were isolated after an incubation period that allowed the formation of heterogeneous biofilms on stainless steel beads. Of over 1,000 tested isolates, most were capable of forming biofilms, with approximately 30 % being strong or moderate biofilm formers. Selected isolates (117) were subjected to species identification by using the Biolog Gen III microbial identification system. They distributed among 23 genera, which included soil bacteria, plant-related bacteria, coliforms, and opportunistic plant- or human-pathogenic bacteria. The most commonly identified bacteria species were Pseudomonas fluorescens, Rahnella aquatilis, and Ralstonia insidiosa. The high prevalence of R. insidiosa, a strong biofilm former, and P. fluorescens, a moderate biofilm former, suggests that they were established residents in the sampled plants. These results suggest that native microflora capable of forming biofilms are widely distributed in fresh-produce processing environments.
    Journal of food protection 05/2013; 76(5):827-32. DOI:10.4315/0362-028X.JFP-12-433 · 1.80 Impact Factor
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    ABSTRACT: Escherichia coli is the leading indicator of microbial contamination of natural waters, and so its in-stream fate and transport needs to be understood to eventually minimize surface water contamination by microorganisms. To better understand mechanisms of E. coli release and transport from soil sediment in a creek the artificial high-water flow events were created by releasing 60-80 m(3) of city water on a tarp-covered stream bank in four equal allotments in July 2008, 2009 and 2010. A conservative tracer difluorobenzoic acid (DFBA) was added to the released water in 2009 and 2010. Water flow rate, E. coli and DFBA concentrations as well as water turbidity were monitored with automated samplers at three in-stream weirs. A one-dimensional model was applied to simulate water flow, and E. coli and DFBA transport during these experiments. The Saint-Venant equations were used to calculate water depth and discharge while a stream solute transport model accounted for release of bacteria by shear stress from bottom sediments, advection-dispersion, and exchange with transient storage (TS). Reach-specific model parameters were estimated by evaluating observed time series of flow rates and concentrations of DFBA and E. coli at all three weir stations. Observed DFBA and E. coli breakthrough curves (BTC) exhibited long tails after the water pulse and tracer peaks had passed indicating that transient storage (TS) might be an important element of the in-stream transport process. Comparison of simulated and measured E. coli concentrations indicated that significant release of E. coli continued when water flow returned to the base level after the water pulse passed and bottom shear stress was small. The mechanism of bacteria continuing release from sediment could be the erosive boundary layer exchange enhanced by changes in biofilm properties by erosion and sloughing detachment.
    Water Research 02/2013; 47(8). DOI:10.1016/j.watres.2013.02.011 · 5.32 Impact Factor
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    ABSTRACT: Microbiological quality of surface waters can be affected by microbial load in runoff from grazing lands. This effect, with other factors, depends on the survival of microorganisms in animal waste deposited on pastures. Since temperature is a leading environmental parameter affecting survival, it indirectly impacts water microbial quality. The Q(10) model is widely used to predict the effect of temperature on rates of biological processes, including survival. Objectives of this work were to (i) evaluate the applicability of the Q(10) model to Escherichia coli inactivation in bovine manure deposited on grazing land (i.e., cowpats) and (ii) identify explanatory variables for the previously reported E. coli survival dynamics in cowpats. Data utilized in this study include published results on E. coli concentrations in natural and repacked cowpats from research conducted the U.S. (Virginia and Maryland), New Zealand, and the United Kingdom. Inspection of the datasets led to conceptualizing E. coli survival (in cowpats) as a two-stage process, in which the initial stage was due to growth, inactivation or stationary state of the population and the second stage was the approximately first-order inactivation. Applying the Q(10) model to these datasets showed a remarkable similarity in inactivation rates, using the thermal time. The reference inactivation rate constant of 0.042 (thermal days)(-1) at 20°C gave a good approximation (R(2)=0.88) of all inactivation stage data with Q(10)=1.48. The reference inactivation rate constants in individual studies were no different from the one obtained by pooling all data (P<0.05). The rate of logarithm of the E. coli concentration change during the first stage depended on temperature. Duration of the first stage, prior to the first-order inactivation stage and the initial concentration of E. coli in cowpats, could not be predicted from available data. Diet and age are probable factors affecting these two parameters however, until their environmental and management predictors are known, microbial water quality modeling must treat them as a stochastic source of uncertainty in simulation results.
    Environment international 01/2013; 54C:1-10. DOI:10.1016/j.envint.2012.12.013 · 5.66 Impact Factor
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    ABSTRACT: Chlorine is widely used as a sanitizer in commercial fresh‐cut produce wash operations. However, chlorine reacts rapidly with organic materials, leading to potential pathogen survival when chlorine concentration nears depletion. This study evaluated a new process aid, T128, for its capacity to enhance chlorine efficacy against Escherichia coli O157:H7 in solutions with high organic loads. Chlorine solutions were prepared with either T128 or citric acid (control) to adjust pH. Survival of E. coli O157:H7 was assayed during washing of increasing amounts of shredded lettuce, or addition of lettuce juice as organic load. The application of T128 significantly reduced survival of E. coli O157:H7 when free chlorine in solution decreased to levels approaching depletion. In the presence of T128, no pathogen survival was observed with the free chlorine concentration as low as 0.05 mg/L, while 0.4 mg/L free chlorine was required to kill pathogens in the absence of T128 in the wash solution. Practical ApplicationsThe foodborne illness outbreaks that plague the fresh‐cut produce industry are a testament to the need for a more effective sanitizer to prevent pathogen survival and cross contamination in commercial produce wash systems with large influxes of organic matter. This paper reports the investigation of the effect of a new process aid, T128, on reducing the survival of E. coli O157:H7 in wash solutions in which chlorine is near depletion due to high organic load, and thus the potential to improve the safety margin of fresh‐cut process control.
    Journal of Food Processing and Preservation 12/2012; 36(6). DOI:10.1111/jfpp.12000 · 0.94 Impact Factor
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    ABSTRACT: Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q(10) model. This suggestion was made 34 years ago based on 20 survival curves taken from published literature, but has not been revisited since then. The objective of this study was to re-evaluate the accuracy of the Q(10) equation, utilizing data accumulated since 1978. We assembled a database of 450 E. coli survival datasets from 70 peer-reviewed papers. We then focused on the 170 curves taken from experiments that were performed in the laboratory under dark conditions to exclude the effects of sunlight and other field factors that could cause additional variability in results. All datasets were tabulated dependencies "log concentration vs. time." There were three major patterns of inactivation: about half of the datasets had a section of fast log-linear inactivation followed by a section of slow log-linear inactivation; about a quarter of the datasets had a lag period followed by log-linear inactivation; and the remaining quarter were approximately linear throughout. First-order inactivation rate constants were calculated from the linear sections of all survival curves and the data grouped by water sources, including waters of agricultural origin, pristine water sources, groundwater and wells, lakes and reservoirs, rivers and streams, estuaries and seawater, and wastewater. Dependency of E. coli inactivation rates on temperature varied among the water sources. There was a significant difference in inactivation rate values at the reference temperature between rivers and agricultural waters, wastewaters and agricultural waters, rivers and lakes, and wastewater and lakes. At specific sites, the Q(10) equation was more accurate in rivers and coastal waters than in lakes making the value of the Q(10) coefficient appear to be site-specific. Results of this work indicate possible sources of uncertainty to be accounted for in watershed-scale microbial water quality modeling.
    Water Research 11/2012; 47(2). DOI:10.1016/j.watres.2012.10.027 · 5.32 Impact Factor
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    ABSTRACT: Irrigation waters have come under increasing scrutiny as a potential source of pathogenic microorganisms contaminating fresh produce. It is generally assumed that the microbial concentrations entering and leaving irrigation pipe networks are identical. However, this may not be true if biofilms form on the inner surfaces of irrigation pipes. The retention and release of pathogens in biofilms are well documented in drinking water distribution systems, but very little data are available for irrigation systems. We examined the attachment and/or incorporation of total coliform, fecal coliform, and Escherichia coli bacteria into biofilms in new and used aluminum irrigation pipe. Water from a local creek in Maryland was used to conduct weekly irrigation events. Prior to each event, removable sections of pipe (coupons) were scraped to determine the extent of bacterial attachment; in addition, bacterial concentrations in residual water were determined. Substantial populations of coliform bacteria were found on the pipe surfaces. Old pipes had fewer attached or biofilm-incorporated coliforms and lower coliform concentrations in the residual water. High probabilities were found for average fecal coliform and total coliform concentrations being different between creek water and sprinkler water. These results have implications for monitoring and control of microbial quality of irrigation waters.
    Irrigation Science 09/2012; 31(5). DOI:10.1007/s00271-012-0373-x · 2.84 Impact Factor
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    ABSTRACT: Aims: The focus of this work was to compare the survival of Escherichia coli introduced into streambed sediments from goose, deer and bovine faeces vs indigenous E. coli. Methods and Results: The survival experiments were conducted in flow-through chambers for 32 days using two sediments (mineral and organic) obtained from a first-order creek in Maryland. Bovine, goose and deer faeces were collected fresh and diluted or enriched so that added E. coli and indigenous populations were equivalent. Escherichia coli and total coliforms were enumerated using the Colilert-18 Quanti-Tray system. Patterns of E. coli survival and inactivation rates were virtually identical for indigenous strains in both mineral and organic sediments. The addition of E. coli strains from bovine, goose or deer faeces had relatively little impact on final E. coli concentrations, with the exception of deer-borne E. coli populations in the organic sediment. Conclusion: These results indicate that indigenous sediment-borne E. coli strains are generally, or more, persistent than those deposited into sediments, including wildlife. Significance and Impact of the Study: This is the first study on the survival of E. coli originating from wildlife faeces, in sediments, as opposed to bovine faeces or laboratory-cultured strains. As wildlife are likely to be the primary source of E. coli in most non agricultural watersheds, an understanding of the persistence of these strains is important to understanding microbial water quality.
    Letters in Applied Microbiology 08/2012; 55(5). DOI:10.1111/j.1472-765X.2012.03296.x · 1.75 Impact Factor
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    ABSTRACT: Row-crop and poultry production have been implicated as sources of water pollution along the Choptank River, an estuary and tributary of the Chesapeake Bay. This study examined the effects of land use, subwatershed characteristics, and climatic conditions on the water quality parameters of a subwatershed in the Choptank River watershed. The catchments within the subwatershed were defined using advanced remotely-sensed data and current geographic information system processing techniques. Water and sediment samples were collected in May-October 2009 and April-June 2010 under mostly baseflow conditions and analyzed for select bacteria, nitrate-N, ammonium-N, total arsenic, total phosphorus (TP), orthophosphate (ortho-P), and particle-phase phosphorus (PP); n=96 for all analytes except for arsenic, n=136, and for bacteria, n=89 (aqueous) and 62 (sediment). Detections of Enterococci and Escherichia coli concentrations were ubiquitous in this subwatershed and showed no correlation to location or land use, however larger bacterial counts were observed shortly after precipitation. Nitrate-N concentrations were not correlated with agricultural lands, which may reflect the small change in percent agriculture and/or the similarity of agronomic practices and crops produced between catchments. Concentration data suggested that ammonia emission and possible deposition to surface waters occurred and that these processes may be influenced by local agronomic practices and climatic conditions. The negative correlation of PP and arsenic concentrations with percent forest was explained by the stronger signal of the head waters and overland flow of particulate phase analytes versus dissolved phase inputs from groundwater. Service roadways at some poultry production facilities were found to redirect runoff from the facilities to neighboring catchment areas, which affected water quality parameters. Results suggest that in this subwatershed, catchments with poultry production facilities are possible sources for arsenic and PP as compared to catchment areas where these facilities were not present.
    Science of The Total Environment 05/2012; 430:270-9. DOI:10.1016/j.scitotenv.2012.03.056 · 3.16 Impact Factor
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    ABSTRACT: Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10(5) cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10-10(4) cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.
    Biosensors & Bioelectronics 12/2011; 30(1):337-41. DOI:10.1016/j.bios.2011.09.029 · 6.45 Impact Factor
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    ABSTRACT: The focus of this work was to investigate the contribution of native Escherichia coli to the microbial quality of irrigation water and to determine the potential for contamination by E. coli associated with heterotrophic biofilms in pipe-based irrigation water delivery systems. The aluminium pipes in the sprinkler irrigation system were outfitted with coupons that were extracted before each of the 2-h long irrigations carried out with weekly intervals. Water from the creek water and sprinklers, residual water from the previous irrigation and biofilms on the coupons were analysed for E. coli. High E. coli concentrations in water remaining in irrigation pipes between irrigation events were indicative of E. coli growth. In two of the four irrigations, the probability of the sample source, (creek vs sprinkler), being a noninfluential factor, was only 0.14, that is, source was an important factor. The population of bacteria associated with the biofilm on pipe walls was estimated to be larger than that in water in pipes in the first three irrigation events and comparable to one in the fourth event. Biofilm-associated E. coli can affect microbial quality of irrigation water and, therefore, should not be neglected when estimating bacterial mass balances for irrigation systems. This work is the first peer-reviewed report on the impact of biofilms on microbial quality of irrigation waters. Flushing of the irrigation system may be a useful management practice to decrease the risk of microbial contamination of produce. Because microbial water quality can be substantially modified while water is transported in an irrigation system, it becomes imperative to monitor water quality at fields, rather than just at the intake.
    Letters in Applied Microbiology 12/2011; 54(3):217-24. DOI:10.1111/j.1472-765X.2011.03192.x · 1.75 Impact Factor

Publication Stats

1k Citations
215.06 Total Impact Points

Institutions

  • 2001–2013
    • Agricultural Research Service
      Kerrville, Texas, United States
    • Maryland Department Of Agriculture
      Annapolis, Maryland, United States
  • 2009
    • Creatv MicroTech, Inc
      Maryland, United States
  • 1996–2002
    • Natural Resources Research Institute
      Silver Spring, Maryland, United States