Michael B. Jenkins

Performance assessment PCR-based assays targeting bacteroidales genetic markers of bovine fecal pollution.

ABSTRACT There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.

Preferential Flow and Transport of Cryptosporidium parvum Oocysts through the Vadose Zone: Experiments and Modeling

in the form of 4- to 6-m-long ovoid-shaped oocysts, with a double wall that is resistant to most oxidation As a result of Cryptosporidium parvum in drinking water, several processes such as ozonation and chlorination (Current, outbreaks of cryptosporidiosis have occurred in the last 10 yr. Al1986; Atwill et al., 1997). though it is generally believed that movement of pathogens through the soil is minimal, recent research has shown that appreciable num- During the past two decades, the presence of C. parbers of C. parvum oocysts may be transported via preferential or vum in surface- and groundwaters in the United States fingered flow to groundwater. The objective of the present research and Great Britain (Galbraith et al., 1987; Rose et al., was to further investigate and model the transport of oocysts through 1991; Craun et al., 1998) has been associated with several preferential flow paths in the vadose zone under a “worst-case” sce- major outbreaks of cryptosporidiosis (Hayes et al., 1989; nario. This was studied by adding calves feces containing C. parvum MacKenzie et al., 1994). Among the different pathways oocysts with a Cl tracer to undisturbed silt loam columns and disfor the transport of oocysts to drinking water sources, turbed sand columns during a simulated steady-state rain. The sand columns exhibited preferential flow in the form of fingers whereas downward percolation is usually considered to be insigmacropore flow occurred in the undisturbed cores. In the columns nificant, because soils are generally assumed to be an with fingered flow, oocysts and Cl were transported rapidly with the effective filter for a wide range of pathogens. Studies same velocity through the columns. Although only 14 to 86% of the of packed columns with saturated flow by Brush et al. amount applied, the number of oocysts transported across the columns (1999) and Harter et al. (2000) and undisturbed columns was several orders of magnitude above an infective dose. The macwith unsaturated flow (Mawdsley et al., 1996), however, ropore columns had only a very limited breakthrough of oocysts, showed that C. parvum oocysts could be transported which appeared several pore volumes after the Cl broke through initially. A simulation model for the transport of oocysts via preferen- rapidly downward through the soil. Although transport tial flow was developed on the basis of an existing preferential flow of C. parvum oocysts in saturated flow has been studied model for nonadsorbing solutes, with addition of a first-order sink experimentally and described mathematically (Brush et term for adsorbance of the C. parvum to the air–water–solid (AWS) al., 1999; Harter et al., 2000), detailed observations of interfaces, and with velocity and dispersivity parameters derived from the transport and persistence of C. parvum oocysts in Cl transport. The breakthrough of C. parvum oocysts could be de- unsaturated soils with preferential flow are still lacking, scribed realistically for the sand columns. However, the model could particularly in the presence of preferential flow pronot describe oocyst transport in the columns with macropores. cesses.

Preferential transport of Cryptosporidium parvum oocysts in variably saturated subsurface environments

When oocysts of the protozoan Cryptosporidium parvum contaminate drinking water supplies, they can cause outbreaks of Cryptosporidiosis, a common waterborne disease. Of the different pathways by which oocysts can wind up in drinking water, one has received little attention to date; that is, because soils are often considered to be perfect filters, the transport of oocysts through the subsoil to groundwater is generally ignored. To evaluate the significance of this pathway, a series of laboratory experiments investigated subsurface transport of oocysts. Experiment 1 was carried out in a vertical 18-cm-long column filled either with glass beads or silica sand, under conditions known to foster fingered flow. Experiment 2 involved undisturbed, macroporous soil columns subjected to macropore flow. Experiment 3 aimed to study the lateral flow on an undisturbed soil block. The columns and soil samples were subjected to artificial rainfall and were allowed to reach steady state. At that point, feces of contaminated calves were applied at the surface along with a known amount of potassium chloride to serve as a tracer, and rainfall was continued at the same rate. The breakthrough of oocysts and chloride, monitored in the effluent, demonstrate the importance of preferential flow on the transport of oocysts. Compared with chloride, peak oocyst concentrations were not appreciably delayed and, in some cases, occurred even before the chloride peak. Recovery rates for oocysts were low, ranging from 0.1 to 10.4% of the oocysts originally applied on the columns. However, the numbers of oocysts present in the effluents were still orders of magnitude higher than 10 oocysts, the infectious dose considered by the U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, to be sufficient to cause Cryptosporidiosis in healthy adults. These results suggest that the transport of oocysts in the subsurface via preferential flow may create a significant risk of groundwater contamination in some situations.

Rainfall and tillage effects on transport of fecal bacteria and sex hormones 17β-estradiol and testosterone from broiler litter applications to a Georgia Piedmont Ultisol

Poultry litter provides nutrients for crop and pasture production; however, it also contains fecal bacteria, sex hormones (17beta-estradiol and testosterone) and antibiotic residues that may contaminate surface waters. Our objective was to quantify transport of fecal bacteria, estradiol, testosterone and antibiotic residues from a Cecil sandy loam managed since 1991 under no-till (NT) and conventional tillage (CT) to which either poultry litter (PL) or conventional fertilizer (CF) was applied based on the nitrogen needs of corn (Zea mays L) in the Southern Piedmont of NE Georgia. Simulated rainfall was applied for 60 min to 2 by 3-m field plots at a constant rate in 2004 and variable rate in 2005. Runoff was continuously measured and subsamples taken for determining flow-weighted concentrations of fecal bacteria, hormones, and antibiotic residues. Neither Salmonella, nor Campylobacter, nor antimicrobial residues were detected in litter, soil, or runoff. Differences in soil concentrations of fecal bacteria before and after rainfall simulations were observed only for Escherichia coli in the constant rainfall intensity experiment. Differences in flow-weighted concentrations were observed only for testosterone in both constant and variable intensity rainfall experiments, and were greatest for treatments that received poultry litter. Total loads of E. coli and fecal enterococci, were largest for both tillage treatments receiving poultry litter for the variable rainfall intensity. Load of testosterone was greatest for no-till plots receiving poultry litter under variable rainfall intensity. Poultry litter application rates commensurate for corn appeared to enhance only soil concentrations of E. coli, and runoff concentrations of testosterone above background levels.

Comparative die-off of Escherichia coli O157:H7 and fecal indicator bacteria in pond water.

In situ and in vitro experiments were performed to assess the effects of solar radiation and predation by indigenous microflora on the relative die-off rates of a toxigenic strain of Escherichia coli O157:H7, commensal E. coli, and fecal enterococci in surface waters from ponds in agricultural watersheds. The objective of these experiments was to discern a mechanism of persistence of E. coli O157:H7 in surface waters compared to fecal indicator bacteria. Results of these experiments indicated that E. coli and fecal enterococci were affected by both insolation and apparent predation; whereas E. coli O157:H7 appeared to be resistant to both of these environmental stressors. The number of days to reach 99% die-off (T(99)-values) for E. coli O157:H7 was significantly greater than that for the indicator bacteria. The capacity to prolong die-off may be connected to the apparent persistence of E. coli O157:H7 in surface waters.

EFFECTS OF FREEZE–THAW EVENTS ON THE VIABILITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS IN SOIL

The effects of freeze–thaw events on the inactivation of Cryptosporidium parvum oocysts in soil were examined. Oocysts were inoculated into distilled water in microcentrifuge tubes or into chambers containing soil the water content of which was maintained at 3%, 43%, or 78% of the container capacity. The chambers and tubes were then embedded in 3 soil samples from different aspects of a hillside landscape (Experiments 1 and 2) and in 3 distinct soil types (Experiment 3) and frozen at −10 C. Containers were thawed every 3 days for a period of 24 hr in 1–9 freeze–thaw cycles over 27 days (Experiments 1 and 2) and 2–5 freeze–thaw cycles over 15 days (Experiment 3). Oocyst viability was measured using the fluorescent dyes 4′6-diaminidino-2-phenylindole and propidium iodide. Inactivation rates were greater in soils than in water and greater in dry soil than in moist and wet soils. Soil type showed no effect on inactivation. Oocysts subjected to freeze–thaw cycles had inactivation rates not significantly different from those of oocysts subjected to −10 C under static conditions. The results indicated that 99% of oocysts exposed to soils that are frozen at −10 C will become inactivated within 50 days whether or not freeze–thaw cycles occur.

17β-Estradiol and testosterone in drainage and runoff from poultry litter applications to tilled and no-till crop land under irrigation.

Thirteen million [corrected] metric tons of poultry litter are produced annually by poultry producers in the U.S. Poultry litter contains the sex hormones estradiol and testosterone, endocrine disruptors that have been detected in surface waters. The objective of this study was to evaluate the potential impact of poultry litter applications on estradiol and testosterone concentrations in subsurface drainage and surface runoff in irrigated crop land under no-till and conventional-till management. We conducted an irrigation study in fall of 2001 and spring of 2002. Four treatments, no-till plus poultry litter, conventional-till plus poultry litter, no-till plus conventional fertilizer, and conventional-till plus conventional fertilizer, were evaluated. Flow-weighted concentration and load ha(-1) of the two hormones were measured in drainage and runoff. Soil concentrations of estradiol and testosterone were measured. Based on comparisons to the conventional fertilizer (and control) treatments, poultry litter did not add to the flow-weighted concentration or load ha(-1) of either estradiol or testosterone in subsurface drainage or surface runoff. Significant differences were, however, observed between tillage treatments: flow-weighted concentrations of estradiol were greater for no-till than conventional-till plots of the June irrigation; and runoff loads of both estradiol and testosterone were less from no-till than conventional-till plots for the November irrigation. Although the differences between no-till and conventional-tillage appeared to affect the hydrologic transport of both hormones, the differences appeared to have inconsequential environmental impact.

Assessing short-term responses of prokaryotic communities in bulk and rhizosphere soils to tall fescue endophyte infection

In contrast to endophyte-free (E−) tall fescue, endophyte-infected (E+) tall fescue pastures appear to enhance soil carbon sequestration. A hypothetical mechanism that may account for the enhanced carbon sequestration is that the E+ tall fescue affects the soil microbial community or components of it that are involved in organic carbon turnover. A 60-week mesocosm study with a factorial arrangement of soil type, loamy sand (LS) and clay loam (CL), and E+ and E− tall fescue was conducted to determine if the soil microbial communities were affected by the presence of the endophyte. Bulk and rhizosphere soil samples were fixed in paraformaldehyde, and prepared for total direct microbial counts, and with a combination of one of a domain or subdivision fluorescent oligonucleotide probe for enumerating metabolically active Eubacteria, bacterial subdivisions, and Archaea. E+ tall fescue suppressed the archaeal and high G+C gram-positive bacterial communities of the bulk CL, the delta-proteobacterial community in the rhizosphere CL, and the Planctomycetes community of the rhizosphere LS. In the long-term, suppression of these microbial communities may be a factor in enhanced soil carbon sequestration associated with E+ tall fescue.

Significance of Wall Structure, Macromolecular Composition, and Surface Polymers to the Survival and Transport of Cryptosporidium parvum Oocysts

ABSTRACT The structure and composition of the oocyst wall are primary factors determining the survival and hydrologic transport of Cryptosporidium parvum oocysts outside the host. Microscopic and biochemical analyses of whole oocysts and purified oocyst walls were undertaken to better understand the inactivation kinetics and hydrologic transport of oocysts in terrestrial and aquatic environments. Results of microscopy showed an outer electron-dense layer, a translucent middle layer, two inner electron-dense layers, and a suture structure embedded in the inner electron-dense layers. Freeze-substitution showed an expanded glycocalyx layer external to the outer bilayer, and Alcian Blue staining confirmed its presence on some but not all oocysts. Biochemical analyses of purified oocyst walls revealed carbohydrate components, medium- and long-chain fatty acids, and aliphatic hydrocarbons. Purified walls contained 7.5% total protein (by the Lowry assay), with five major bands in SDS-PAGE gels. Staining of purified oocyst walls with magnesium anilinonaphthalene-8-sulfonic acid indicated the presence of hydrophobic proteins. These structural and biochemical analyses support a model of the oocyst wall that is variably impermeable and resistant to many environmental pressures. The strength and flexibility of oocyst walls appear to depend on an inner layer of glycoprotein. The temperature-dependent permeability of oocyst walls may be associated with waxy hydrocarbons in the electron-translucent layer. The complex chemistry of these layers may explain the known acid-fast staining properties of oocysts, as well as some of the survival characteristics of oocysts in terrestrial and aquatic environments. The outer glycocalyx surface layer provides immunogenicity and attachment possibilities, and its ephemeral nature may explain the variable surface properties noted in oocyst hydrologic transport studies.

Most probable number methodology for quantifying dilute concentrations and fluxes of Salmonella in surface waters.

Aims:  To better understand and manage the fate and transport of Salmonella in agricultural watersheds, we developed a culture‐based, five tube–four dilution most probable number (MPN) method for enumerating dilute densities of Salmonella in environmental waters.