Loop-mediated isothermal amplification: Development, validation and application of simple and rapid assays for quantitative detection of species of Arcobacteraceae family- and species-specific Aliarcobacter faecis and Aliarcobacter lanthieri.

AIM: The family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis and Aliarcobacter lanthieri have been identified as emerging pathogens potentially cause health risks to humans and animals. This study was designed to develop/optimize, validate and apply Arcobacteraceae family- and two species-specific (A. faecis and A. lanthieri) loop-mediated isothermal amplification (LAMP) assays to rapidly detect and quantify total number of cells in various environmental niches. METHODS AND RESULTS: Three sets of LAMP primers were designed from conserved and variable regions of 16S rRNA (family-specific) and gyrB (species-specific) genes. Optimized Arcobacteraceae family-specific LAMP assay correctly amplified and detected 24 species, whereas species-specific LAMP assays detected A. faecis and A. lanthieri reference strains as well as 91 pure and mixed culture isolates recovered from aquatic and faecal sources. The specificity of LAMP amplification of A. faecis and A. lanthieri was further confirmed by restriction fragment length polymorphism analysis. Assay sensitivities were tested using variable DNA concentrations extracted from simulated target species cells in an autoclaved agricultural water sample by achieving a minimum detection limit of 10 cells mL-1 (10 fg). Direct DNA-based quantitative detection, from agricultural surface water, identified A. faecis (17%) and A. lanthieri (1%) at a low frequency compared to family-level (93%) with the concentration ranging from 2·1 × 101 to 2·2 × 105 cells 100 mL-1 . CONCLUSIONS: Overall, these three DNA-based rapid and cost-effective novel LAMP assays are sensitive and can be completed in less than 40 min. They have potential for on-site quantitative detection of species of family Arcobacteraceae, A. faecis and A. lanthieri in food, environmental and clinical matrices. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed LAMP assays are specific, sensitive, accurate with higher reproducibility that have potential to facilitate in a less equipped lab setting and can help in early quantitative detection and rate of prevalence in environmental niches. The assays can be adopted in the diagnostic labs and epidemiological studies.

Do reductions in agricultural field drainage during the growing season impact bacterial densities and loads in small tile-fed watersheds?

Predicting bacterial levels in watersheds in response to agricultural beneficial management practices (BMPs) requires understanding the germane processes at both the watershed and field scale. Controlling subsurface tile drainage (CTD) is a highly effective BMP at reducing nutrient losses from fields, and watersheds when employed en masse, but little work has been conducted on CTD effects on bacterial loads and densities in a watershed context. This study compared fecal indicator bacteria (FIB) [E. coli, Enterococcus, Fecal coliform, Total coliform, Clostridium perfringens] densities and unit area loads (UAL) from a pair of flat tile-drained watersheds (∼250-467 ha catchment areas) during the growing season over a 10-year monitoring period, using a before-after-control-impact (BACI) design (i.e., test CTD watershed vs. reference uncontrolled tile drainage (UCTD) watershed during a pre CTD intervention period and a CTD-intervention period where the test CTD watershed had CTD deployed on over 80% of the fields). With no tile drainage management, upstream tile drainage to ditches comprised ∼90% of total ditch discharge. We also examined FIB loads from a subset of tile drained fields to determine field load contributions to the watershed drainage ditches. Statistical evidence of a CTD effect on FIB UAL in the surface water systems was not strong; however, there was statistical evidence of increased FIB densities [pronounced when E. coli >200 most probable number (MPN) 100 mL-1] in the test CTD watershed during the CTD-intervention period. This was likely a result of reduced dilution/flushing in the test CTD watershed ditch due to CTD significantly decreasing the amount of tile drainage water entering the surface water system. Tile E. coli load contributions to the ditches were low; for example, during the 6-yr CTD-intervention period they amounted to on average only ∼3 and ∼9% of the ditch loads for the test CTD and reference UCTD watersheds, respectively. This suggests in-stream, or off-field FIB reservoirs and bacteria mobilization drivers, dominated ditch E. coli loads in the watersheds during the growing season. Overall, this study suggested that decision making regarding deployment of CTD en masse in tile-fed watersheds should consider drainage practice effects on bacterial densities and loads, as well as CTD's documented capacity to boost crop yields and reduce seasonal nutrient pollution.

Enhanced Single-tube Multiplex PCR Assay for Detection and Identification of Six Arcobacter Species.

AIM: A single-tube multiplex PCR (mPCR) assay was developed for rapid, sensitive and simultaneous detection and identification of six Arcobacter species including two new species, A. lanthieri and A. faecis, along with A. butzleri, A. cibarius, A. cryaerophilus and A. skirrowii on the basis of differences in the lengths of their PCR products. Previously designed monoplex, mPCR and RFLP assays do not detect or differentiate A. faecis and A. lanthieri from other closely related known Arcobacter spp. METHODS AND RESULTS: Primer pairs for each target species (except A. skirrowii) and mPCR protocol were newly designed and optimized using variable regions of housekeeping including cpn60, gyrA, gyrB and rpoB genes. The accuracy and specificity of the mPCR assay was assessed using DNA templates from six targets and 11 other Arcobacter spp. as well as 50 other bacterial reference species and strains. Tests on the DNA templates of target Arcobacter spp. were appropriately identified, whereas all 61 other DNA templates from other bacterial species and strains were not amplified. Sensitivity and specificity of the mPCR assay was 10 pg µl-1 of DNA concentration per target species. The optimized assay was further evaluated, validated and compared with other mPCR assays by testing Arcobacter cultures isolated from various faecal and water sources. CONCLUSIONS: Study results confirm that the newly developed mPCR assay is rapid, accurate, reliable, simple, and valuable for the simultaneous detection and routine diagnosis of six human- and animal-associated Arcobacter spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The new mPCR assay is useful not only for pure but also mixed cultures. Moreover, it has the ability to rapidly detect six species which enhances the value of this technology for aetiological and epidemiological studies.

Brominated flame retardants and perfluoroalkyl acids in groundwater, tile drainage, soil, and crop grain following a high application of municipal biosolids to a field.

Dewatered municipal biosolids (DMB) were applied at a rate of 22Mgdwha-1 to an agricultural field in fall 2008. Concentrations of polybrominated diphenyl ethers (PBDEs; BDE-47, -99, -100, -153, -154, -183, -197, -207, -209), other brominated flame retardants (BFRs; HBB, PBEB, DBDPE, BTBPE) and perfluoroalkyl acids (PFAAs; PFHxS, PFOS, PFDS, PFOSA, PFHpA, PFOA, PFNA, PFDA, PFUnA, PFDoA, PFTA) were monitored in tile drainage, groundwater (2m, 4m and 6m depth), soil cores (0-0.3m) pre- and post-application, DMB aggregates incorporated into the soil post-application, and in wheat (Triticum spp.) planted post-application. Several compounds were detected in soil and water pre-application and on a reference field plot. PBDEs, other BFRs and PFAAs were detected in tile drainage and 2m groundwater throughout the post-application study period; a few PBDEs were also detected sporadically at lower depths in groundwater. Some of these compounds had not been detected pre-application, while some exceeded reference field plot/pre-application levels (some significantly (p<0.05) in tile drainage); both cases indicating biosolid-based water contamination. In DMB aggregates, several PBDE congeners were found to have dissipated exponentially, with reductions >90% in many of them within 1year post-application. Exponential dissipation of other BFRs and PFAAs in DMB aggregates were not significant. No PBDEs, other BFRs, or PFAAs were detected in wheat grain.

Comparison of commercial DNA extraction kits and quantitative PCR systems for better sensitivity in detecting the causative agent of paratuberculosis in dairy cow fecal samples.

Mycobacterium avium ssp. paratuberculosis (MAP) causes ruminant paratuberculosis (Johne's disease) worldwide. Oral-fecal contamination is the most important mode of transmission of paratuberculosis, so eradicating MAP-shedding animals could prevent disease propagation. Fecal culture, a well-known method for MAP diagnosis, requires costly specialized media and a long incubation time that sometimes ends in disappointing bacterial contamination. To facilitate the efforts of control programs, we evaluated the performance of direct fecal quantitative PCR (qPCR) assays for their sensitivity and robustness for MAP detection. Commercial kits use different strategies for extracting DNA, combined with qPCR systems, to detect the presence of MAP in fecal samples. In this study, we compared the sensitivity of 3 commercially available DNA extraction kits (A, B, and C) combined with 2 qPCR systems (T and V) for the detection of MAP in infectious cows. A total of 49 dairy cows from 5 herds were sampled twice a year for 3 yr and diagnosed using fecal culture and ELISA. Eight replicates of their fecal samples from the first sampling were tested using each DNA extraction method and qPCR detection system. Although all 3 of the commercial DNA extraction kits have been previously described as very efficient for the diagnosis of paratuberculosis, kit B provided the highest sensitivity. Indeed, 89% of the cows declared positive for paratuberculosis by both fecal culture and ELISA were identified with kit B, whereas only 23 and 43% of the cows were identified with kits A and C, respectively. Interestingly, kit B was able to detect some low-MAP shedders. The qPCR detection system also played a critical role: system T yielded qPCR with the highest sensitivity. The results of this study suggest that DNA extraction kit B combined with detection system T provides the best amplification of MAP DNA from fecal samples with the highest sensitivity and specificity. Although 1 DNA extraction and qPCR analysis should be adequate to confirm that an animal with diarrhea or other signs of paratuberculosis is positive, detecting low shedders at the highest sensitivity should include repetitive testing. This study demonstrates the importance of repetitions using the most appropriate method for extracting DNA from fecal samples, combined with a compatible qPCR system for identifying MAP-shedding animals.

Agriculture and Agri-Food Canada's research program on antimicrobial resistance.

A key strategy for attenuating the development of antimicrobial resistance (AMR) is ensuring judicious use of antimicrobials in human and veterinary medicine and in agriculture. Research on AMR in agriculture includes risk assessment, risk management, and identifying the role of agricultural practices in development of AMR. Risk assessment includes an impact assessment of antimicrobial use in livestock and on the environment; for example, many antimicrobials are excreted unchanged and thus reach the environment through manure application. This creates the potential for AMR transmission through the food processing chain and into agro-ecosystems receiving the agricultural waste. Risk management includes the assessment of cost-effective methods to keep animals healthy without the need for antimicrobial use, such as the use of vaccines, nutritional supplements and pre-, pro- or synbiotics and of waste management strategies to avoid AMR transmission. Currently, there is an important gap in understanding the degree of human exposure to AMR that is generated through agriculture, the burden of illness of AMR pathogens in human populations and the relationship between exposure and burden of illness. It is important that research on the agricultural, environmental and human medicine dimensions of AMR not be undertaken in silos, which is why the United Nations and countries around the world are working together within the One Health Framework that considers the inter-relatedness of humans, animals and the environment.

Towards a more accurate quantitative assessment of seasonal Cryptosporidium infection risks in surface waters using species and genotype information.

Many Cryptosporidium species/genotypes are not considered infectious to humans, and more realistic estimations of seasonal infection risks could be made using human infectious species/genotype information to inform quantitative microbial risk assessments (QMRA). Cryptosporidium oocyst concentration and species/genotype data were collected from three surface water surveillance programs in two river basins [South Nation River, SN (2004-09) and Grand River, GR (2005-13)] in Ontario, Canada to evaluate seasonal infection risks. Main river stems, tributaries, agricultural drainage streams, water treatment plant intakes, and waste water treatment plant effluent impacted sites were sampled. The QMRA employed two sets of exposure data to compute risk: one assuming all observed oocysts were infectious to humans, and the other based on the fraction of oocysts that were C. hominis and/or C. parvum (dominant human infectious forms of the parasite). Viability was not considered and relative infection risk was evaluated using a single hypothetical recreational exposure. Many sample site groupings for both river systems, had significant seasonality in Cryptosporidium occurrence and concentrations (p ≤ 0.05); occurrence and concentrations were generally highest in autumn for SN, and autumn and summer for GR. Mean risk values (probability of infection per exposure) for all sites combined, for each river system, were roughly an order of magnitude lower (avg. of SN and GR 5.3 × 10-5) when considering just C. parvum and C. hominis oocysts, in relation to mean infection risk (per exposure) assuming all oocysts were infectious to humans (5.5 × 10-4). Seasonality in mean risk (targeted human infectious oocysts only) was most strongly evident in SN (e.g., 7.9 × 10-6 in spring and 8.1 × 10-5 in summer). Such differences are important if QMRA is used to quantify effects of water safety/quality management practices where inputs from a vast array of fecal pollution sources can readily occur. Cryptosporidium seasonality in water appears to match the seasonality of human infections from Cryptosporidium in the study regions. This study highlights the importance of Cryptosporidium species/genotype data to help determine surface water pollution sources and seasonality, as well as to help more accurately quantify human infection risks by the parasite.

Physical Characterization and Innate Immunogenicity of Aggregated Intravenous Immunoglobulin (IGIV) in an In Vitro Cell-Based Model.

PURPOSE: To investigate in vitro the innate immune response to accelerated stress-induced aggregates of intravenous immunoglobulin (IGIV) using a well-defined human cell-line model, and to correlate the innate response to physical properties of the aggregates. METHODS: IGIV aggregates were prepared by applying various accelerated stress methods, and particle size, count and structure were characterized. Immune cell activation as tracked by inflammatory cytokines released in response to aggregates was evaluated in vitro using peripheral blood mononuclear cells (PBMC), primary monocytes and immortalized human monocyte-like cell lines. RESULTS: IGIV aggregates produced by mechanical stress induced higher cytokine release by PBMC and primary monocytes than aggregates formed by other stresses. Results with the monocytic cell line THP-1 paralleled trends in PBMC and primary monocytes. Effects were dose-dependent, enhanced by complement opsonization, and partially inhibited by blocking toll-like receptors (TLR2 and TLR4) and to a lesser extent by blocking Fc gamma receptors (FcγRs). CONCLUSIONS: Stress-induced IGIV aggregates stimulate a dose-dependent cytokine response in human monocytes and THP-1 cells, mediated in part by TLRs, FcγRs and complement opsonization. THP-1 cells resemble primary monocytes in many respects with regard to tracking the innate response to IgG aggregates. Accordingly, the measurement of inflammatory cytokines released by THP-1 cells provides a readily accessible assay system to screen for the potential innate immunogenicity of IgG aggregates. The results also highlight the role of aggregate structure in interacting with the different receptors mediating innate immunity.

Long-Term Observations of Nitrogen and Phosphorus Export in Paired-Agricultural Watersheds under Controlled and Conventional Tile Drainage.

Controlled tile drainage (CTD) regulates water and nutrient export from tile drainage systems. Observations of the effects of CTD imposed en masse at watershed scales are needed to determine the effect on downstream receptors. A paired-watershed approach was used to evaluate the effect of field-to-field CTD at the watershed scale on fluxes and flow-weighted mean concentrations (FWMCs) of N and P during multiple growing seasons. One watershed (467-ha catchment area) was under CTD management (treatment [CTD] watershed); the other (250-ha catchment area) had freely draining or uncontrolled tile drainage (UCTD) (reference [UCTD] watershed). The paired agricultural watersheds are located in eastern Ontario, Canada. Analysis of covariance and paired tests were used to assess daily fluxes and FWMCs during a calibration period when CTD intervention on the treatment watershed was minimal (2005-2006, when only 4-10% of the tile-drained area was under CTD) and a treatment period when the treatment (CTD) watershed had prolific CTD intervention (2007-2011 when 82% of tile drained fields were controlled, occupying >70% of catchment area). Significant linear regression slope changes assessed using ANCOVA ( ≤ 0.1) for daily fluxes from upstream and downstream monitoring sites pooled by calibration and treatment period were -0.06 and -0.20 (stream water) (negative values represent flux declines in CTD watershed), -0.59 and -0.77 (NH-N), -0.14 and -0.15 (NO-N), -1.77 and -2.10 (dissolved reactive P), and -0.28 and 0.45 (total P). Total P results for one site comparison contrasted with other findings likely due to unknown in-stream processes affecting total P loading, not efficacy of CTD. The FWMC results were mixed and inconclusive but suggest physical abatement by CTD is the means by which nutrient fluxes are predominantly reduced at these scales. Overall, our study results indicate that CTD is an effective practice for reducing watershed scale fluxes of stream water, N, and P during the growing season.

Using AnnAGNPS to Predict the Effects of Tile Drainage Control on Nutrient and Sediment Loads for a River Basin.

Controlled tile drainage (CTD) can reduce pollutant loading. The Annualized Agricultural Nonpoint Source model (AnnAGNPS version 5.2) was used to examine changes in growing season discharge, sediment, nitrogen, and phosphorus loads due to CTD for a ∼3900-km agriculturally dominated river basin in Ontario, Canada. Two tile drain depth scenarios were examined in detail to mimic tile drainage control for flat cropland: 600 mm depth (CTD) and 200 mm (CTD) depth below surface. Summed for five growing seasons (CTD), direct runoff, total N, and dissolved N were reduced by 6.6, 3.5, and 13.7%, respectively. However, five seasons of summed total P, dissolved P, and total suspended solid loads increased as a result of CTD by 0.96, 1.6, and 0.23%. The AnnAGNPS results were compared with mass fluxes observed from paired experimental watersheds (250, 470 ha) in the river basin. The "test" experimental watershed was dominated by CTD and the "reference" watershed by free drainage. Notwithstanding environmental/land use differences between the watersheds and basin, comparisons of seasonal observed and predicted discharge reductions were comparable in 100% of respective cases. Nutrient load comparisons were more consistent for dissolved, relative to particulate water quality endpoints. For one season under corn crop production, AnnAGNPS predicted a 55% decrease (CTD) in dissolved N from the basin. AnnAGNPS v. 5.2 treats P transport from a surface pool perspective, which is appropriate for many systems. However, for assessment of tile drainage management practices for relatively flat tile-dominated systems, AnnAGNPS may benefit from consideration of P and particulate transport in the subsurface.

Resilience and recovery: the effect of triclosan exposure timing during development, on the structure and function of river biofilm communities.

Triclosan (TCS) is a ubiquitous antibacterial agent found in soaps, scrubs, and consumer products. There is limited information on hazardous effects of TCS in the environment. Here, rotating annular reactors were used to cultivate river biofilm communities exposed to 1.8 µg l(-1) TCS with the timing and duration of exposure and recovery during development varied. Two major treatment regimens were employed: (i) biofilm development for 2, 4 or 6 weeks prior to TCS exposure and (ii) exposure of biofilms to TCS for 2, 4 or 6 weeks followed by recovery. Biofilms not exposed to TCS were used as a reference condition. Communities cultivated without and then exposed to TCS all exhibited reductions in algal biomass and significant (p<0.05) reductions in cyanobacterial biomass. No significant effects were observed on bacterial biomass. CLSM imaging of biofilms at 8 weeks revealed unique endpoints in terms of community architecture. Community composition was altered by any exposure to TCS, as indicated by significant shifts in denaturing gradient gel electrophoresis fingerprints and exopolymer composition relative to the reference. Bacterial, algal and cyanobacterial components initially exposed to TCS were significantly different from those TCS-free at time zero. Pigment analyses suggested that significant changes in composition of algal and cyanobacterial populations occurred with TCS exposure. Bacterial thymidine incorporation rates were reduced by TCS exposure and carbon utilization spectra shifted in terms substrate metabolism. Direct counts of protozoans indicated that TCS was suppressive, whereas micrometazoan populations were, in some instances, stimulated. These results indicate that even a relatively brief exposure of a river biofilm community to relatively low levels of TCS alters both the trajectory and final community structure. Although some evidence of recovery was observed, removal of TCS did not result in a return to the unexposed reference condition.

The distribution of Salmonella enterica serovars and subtypes in surface water from five agricultural regions across Canada.

Serovar prevalence of the zoonotic pathogen, Salmonella enterica, was compared among 1624 surface water samples collected previously from five different Canadian agricultural watersheds over multiple years. Phagetyping, pulsed field gel electrophoresis (PFGE), and antimicrobial resistance subtyping assays were performed on serovars Enteritidis, Typhimurium, and Heidelberg. Serovars and subtypes from surface water were compared with those from animal feces, human sewage, and serovars reported to cause salmonellosis in Canadians. Sixty-five different serovars were identified in surface water; only 32% of these were isolated from multiple watersheds. Eleven of the 13 serovars most commonly reported to cause salmonellosis in Canadians were identified in surface water; isolates of these serovars constituted >40% of the total isolates. Common phagetypes and PFGE subtypes of serovars associated with illness in humans such as S. Enteritidis and S. Typhimurium were also isolated from surface water and animal feces. Antimicrobial resistance was generally low, but was highest among S. Typhimurium. Monitoring of these rivers helps to identify vulnerable areas of a watershed and, despite a relatively low prevalence of S. enterica overall, serovars observed in surface water are an indication of the levels of specific S. enterica serovars present in humans and animals.

Rainfall-induced runoff from exposed streambed sediments: an important source of water pollution.

When surface water levels decline, exposed streambed sediments can be mobilized and washed into the water course when subjected to erosive rainfall. In this study, rainfall simulations were conducted over exposed sediments along stream banks at four distinct locations in an agriculturally dominated river basin with the objective of quantifying the potential for contaminant loading from these often overlooked runoff source areas. At each location, simulations were performed at three different sites. Nitrogen, phosphorus, sediment, fecal indicator bacteria, pathogenic bacteria, and microbial source tracking (MST) markers were examined in both prerainfall sediments and rainfall-induced runoff water. Runoff generation and sediment mobilization occurred quickly (10-150 s) after rainfall initiation. Temporal trends in runoff concentrations were highly variable within and between locations. Total runoff event loads were considered large for many pollutants considered. For instance, the maximum observed total phosphorus runoff load was on the order of 1.5 kg ha. Results also demonstrate that runoff from exposed sediments can be a source of pathogenic bacteria. spp. and spp. were present in runoff from one and three locations, respectively. Ruminant MST markers were also present in runoff from two locations, one of which hosted pasturing cattle with stream access. Overall, this study demonstrated that rainfall-induced runoff from exposed streambed sediments can be an important source of surface water pollution.

Comprehensive nitrogen budgets for controlled tile drainage fields in eastern ontario, Canada.

Excessive N loading from subsurface tile drainage has been linked to water quality degradation. Controlled tile drainage (CTD) has the potential to reduce N losses via tile drainage and boost crop yields. While CTD can reduce N loss from tile drainage, it may increase losses through other pathways. A multiple-year field-scale accounting of major N inputs and outputs during the cropping season was conducted on freely drained and controlled tile drained agricultural fields under corn ( L.)-soybean [ (L.) Merr.] production systems in eastern Ontario, Canada. Greater predicted gaseous N emissions for corn and soybean and greater observed lateral seepage N losses were observed for corn and soybean fields under CTD relative to free-draining fields. However, observed N losses from tile were significantly lower for CTD fields, in relation to freely drained fields. Changes in residual soil N were essentially equivalent between drainage treatments, while mass balance residual terms were systematically negative (slightly more so for CTD). Increases in plant N uptake associated with CTD were observed, probably resulting in higher grain yields for corn and soybean. This study illustrates the benefits of CTD in decreasing subsurface tile drainage N losses and boosting crop yields, while demonstrating the potential for CTD to increase N losses via other pathways related to gaseous emissions and groundwater seepage.

Bacteria, viruses, and parasites in an intermittent stream protected from and exposed to pasturing cattle: prevalence, densities, and quantitative microbial risk assessment.

Over 3500 individual water samples, for 131 sampling times, targeting waterborne pathogens/fecal indicator bacteria were collected during a 7-year period from 4 sites along an intermittent stream running through a small livestock pasture system with and without cattle access-to-stream restriction measures. The study assessed the impact of cattle pasturing/riparian zone protection on: pathogen (bacterial, viral, parasite) occurrence, concentrations of fecal indicators, and quantitative microbial risk assessments (QMRA) of the risk of Cryptosporidium, Giardia and Escherichia coli O157:H7 infection in humans. Methodologies were developed to compute QMRA mean risks on the basis of water samples exhibiting potentially human infectious Cryptosporidium and E. coli based on genotyping Crytosporidium, and E. coli O157:H7 presence/absence information paired with enumerated E. coli. All Giardia spp. were considered infectious. No significant pasturing treatment effects were observed among pathogens, with the exception of Campylobacter spp. and E. coli O157:H7. Campylobacter spp. prevalence significantly decreased downstream through pasture treatments and E. coli O157:H7 was observed in a few instances in the middle of the unrestricted pasture. Densities of total coliform, fecal coliform, and E. coli reduced significantly downstream in the restricted pasture system, but not in the unrestricted system. Seasonal and flow conditions were associated with greater indicator bacteria densities, especially in the summer. Norovirus GII was detected at rates of 7-22% of samples for all monitoring sites, and rotavirus in 0-7% of samples for all monitoring sites; pasture treatment trends were not evident, however. Seasonal and stream flow variables (and their interactions) were relatively more important than pasture treatments for initially stratifying pathogen occurrence and higher fecal indicator bacteria densities. Significant positive associations among fecal indicator bacteria and Campylobacter spp. detection were observed. For QMRA, adjusting for the proportion of Cryptosporidium spp. detected that are infectious for humans reduces downstream risk estimates by roughly one order of magnitude. Using QMRA in this manner provides a more refined estimate of beneficial management practice effects on pathogen exposure risks to humans.

Measuring and modeling the effects of drainage water management on soil greenhouse gas fluxes from corn and soybean fields.

Controlled tile drainage can boost crop yields and improve water quality, but it also has the potential to increase GHG emissions. This study compared in-situ chamber-based measures of soil CH4, N2O, and CO2 fluxes for silt loam soil under corn and soybean cropping with conventional tile drainage (UTD) and controlled tile drainage (CTD). A semi-empirical model (NEMIS-NOE) was also used to predict soil N2O fluxes from soils using observed soil data. Observed N2O and CH4 fluxes between UTD and CTD fields during the farming season were not significantly different at 0.05 level. Soils were primarily a sink for CH4 but in some cases a source (sources were associated exclusively with CTD). The average N2O fluxes measured ranged between 0.003 and 0.028 kg N ha(-1) day(-1). There were some significantly higher (p ≤ 0.05) CO2 fluxes associated with CTD relative to UTD during some years of study. Correlation analyses indicated that the shallower the water table, the greater the CO2 fluxes. Higher corn plant C for CTD tended to offset estimated higher CTD CO2 C losses via soil respiration by ∼100-300 kg C ha(-1). There were good fits between observed and predicted (NEMIS-NOE) N2O fluxes for corn (R(2) = 0.70) and soybean (R(2) = 0.53). Predicted N2O fluxes were higher for CTD for approximately 70% of the paired-field study periods suggesting that soil physical factors, such as water-filled pore space, imposed by CTD have potentially strong impacts on net N fluxes. Model predictions of daily cumulative N2O fluxes for the agronomically-active study period for corn-CTD and corn-UTD, as a percentage of total N fertilizer applied, were 3.1% and 2.6%, respectively. For predicted N2O fluxes on basis of yield units, indices were 0.0005 and 0.0004 (kg N kg(-1) crop grain yield) for CTD and UTD corn fields, respectively, and 0.0011 and 0.0005 for CTD and UTD soybean fields, respectively.

Using Campylobacter spp. and Escherichia coli data and Bayesian microbial risk assessment to examine public health risks in agricultural watersheds under tile drainage management.

Human campylobacteriosis is the leading bacterial gastrointestinal illness in Canada; environmental transmission has been implicated in addition to transmission via consumption of contaminated food. Information about Campylobacter spp. occurrence at the watershed scale will enhance our understanding of the associated public health risks and the efficacy of source water protection strategies. The overriding purpose of this study is to provide a quantitative framework to assess and compare the relative public health significance of watershed microbial water quality associated with agricultural BMPs. A microbial monitoring program was expanded from fecal indicator analyses and Campylobacter spp. presence/absence tests to the development of a novel, 11-tube most probable number (MPN) method that targeted Campylobacter jejuni, Campylobacter coli, and Campylobacter lari. These three types of data were used to make inferences about theoretical risks in a watershed in which controlled tile drainage is widely practiced, an adjacent watershed with conventional (uncontrolled) tile drainage, and reference sites elsewhere in the same river basin. E. coli concentrations (MPN and plate count) in the controlled tile drainage watershed were statistically higher (2008-11), relative to the uncontrolled tile drainage watershed, but yearly variation was high as well. Escherichia coli loading for years 2008-11 combined were statistically higher in the controlled watershed, relative to the uncontrolled tile drainage watershed, but Campylobacter spp. loads for 2010-11 were generally higher for the uncontrolled tile drainage watershed (but not statistically significant). Using MPN data and a Bayesian modelling approach, higher mean Campylobacter spp. concentrations were found in the controlled tile drainage watershed relative to the uncontrolled tile drainage watershed (2010, 2011). A second-order quantitative microbial risk assessment (QMRA) was used, in a relative way, to identify differences in mean Campylobacter spp. infection risks among monitoring sites for a hypothetical exposure scenario. Greater relative mean risks were obtained for sites in the controlled tile drainage watershed than in the uncontrolled tile drainage watershed in each year of monitoring with pair-wise posterior probabilities exceeding 0.699, and the lowest relative mean risks were found at a downstream drinking water intake reference site. The second-order modelling approach was used to partition sources of uncertainty, which revealed that an adequate representation of the temporal variation in Campylobacter spp. concentrations for risk assessment was achieved with as few as 10 MPN data per site. This study demonstrates for the first time how QMRA can be implemented to evaluate, in a relative sense, the public health implications of controlled tile drainage on watershed-scale water quality.

Hormones, sterols, and fecal indicator bacteria in groundwater, soil, and subsurface drainage following a high single application of municipal biosolids to a field.

A land application of dewatered municipal biosolids (DMB) was conducted on an agricultural field in fall 2008 at a rate of 22Mg dry weight (dw) ha(-1). Pre- and post- application, hormone, sterol and fecal indicator bacteria concentrations were measured in tile drainage water, groundwater (2, 4, 6m depth), surface soil cores, and DMB aggregates incorporated in the soil (∼0.2m depth) for a period of roughly 1year post-application. Hormones and sterols were detected up to 1year post-application in soil and in DMB aggregates. Hormone (androsterone, desogestrel, estrone) contamination was detected briefly in tile water samples (22d and ∼2months post-app), at lowngL(-1) concentrations (2-34ngL(-1)). Hormones were not detected in groundwater. Sterols were detected in tile water throughout the study period post-application, and multiple fecal sterol ratios suggested biosolids as the source. Coprostanol concentrations in tile water peaked at >1000ngL(-1) (22d post-app) and were still >100ngL(-1) at 6months post-application. Fecal indicator bacteria were detected throughout the study period in tile water, groundwater (⩽2m depth), soil and DMB aggregate samples. These bacteria were strongly linearly related to coprostanol in tile water (R(2)>0.92, p<0.05). The limited transport of hormones and sterols to tile drainage networks may be attributed to a combination of the hydrophobicity of these compounds and limited macroporosity of the field soil. This transitory contamination from hormones and sterols is unlikely to result in any significant pulse exposure risk in subsurface drainage and groundwater.

Impact of riparian zone protection from cattle on nutrient, bacteria, F-coliphage, and loading of an intermittent stream.

This 5-yr study compared, via an upstream-downstream experimental design, nutrient and microbial water quality of an intermittent stream running through a small pasture (∼2.5 animals ha) where cattle are restricted from the riparian zone (restricted cattle access [RCA]) and where cattle have unrestricted access to the stream (unrestricted cattle access [URCA]). Fencing in the RCA excluded pasturing cattle to within ∼3 to 5 m of the stream. Approximately 88% (26/32) of all comparisons of mean contaminant load reduction for lower, higher, and all stream flow conditions during the 5-yr study indicated net contaminant load reductions in the RCA; for the URCA, this percentage was 38% (12/32). For all flow conditions, mean percent load reductions in the RCA for nutrients and bacteria plus F-coliphage were 24 and 23%, respectively. These respective percentages for the URCA were -9 and -57% (positive values are reductions; negative values are increases). However, potentially as a result of protected wildlife habitat in the RCA, the mean percent load reduction for for "all flow" was -321% for the RCA and 60% for the URCA; for , these respective percentages were -209% (RCA) and 73% (URCA). For "all flow" situations, mean load reductions for the RCA were significantly greater ( < 0.1) than those from the URCA for NH-N, dissolved reactive phosphorus (DRP), total coliform, , and . For "high flow" situations, mean load reductions were significantly greater for the RCA for DRP, total coliform, and . For "low flow" conditions, significantly greater mean load reductions were in favor of the RCA for DRP, total P, total coliforms, fecal coliforms, , and . In no case were mean pollutant loads in the URCA significantly higher than RCA pollutant loads. Restricting pasturing livestock to within 3 to 5 m of intermittent streams can improve water quality; however, water quality impairment can occur if livestock have unrestricted access to a stream.