Demonstrating the Benefits of Predictive Bayesian Dose-Response Relationships Using Six Exposure Studies of Cryptosporidium parvum.

A conventional dose-response function can be refitted as additional data become available. A predictive dose-response function in contrast does not require a curve-fitting step, only additional data and presents the unconditional probabilities of illness, reflecting the level of information it contains. In contrast, the predictive Bayesian dose-response function becomes progressively less conservative as more information is included. This investigation evaluated the potential for using predictive Bayesian methods to develop a dose-response for human infection that improves on existing models, to show how predictive Bayesian statistical methods can utilize additional data, and expand the Bayesian methods for a broad audience including those concerned about an oversimplification of dose-response curve use in quantitative microbial risk assessment (QMRA). This study used a dose-response relationship incorporating six separate data sets for Cryptosporidium parvum. A Pareto II distribution with known priors was applied to one of the six data sets to calibrate the model, while the others were used for subsequent updating. While epidemiological principles indicate that local variations, host susceptibility, and organism strain virulence may vary, the six data sets all appear to be well characterized using the Bayesian approach. The adaptable model was applied to an existing data set for Campylobacter jejuni for model validation purposes, which yielded results that demonstrate the ability to analyze a dose-response function with limited data using and update those relationships with new data. An analysis of the goodness of fit compared to the beta-Poisson methods also demonstrated correlation between the predictive Bayesian model and the data.

Centrifuge separation effect on bacterial indicator reduction in dairy manure.

Centrifugation is a commonly applied separation method for manure processing on large farms to separate solids and nutrients. Pathogen reduction is also an important consideration for managing manure. Appropriate treatment reduces risks from pathogen exposure when manure is used as soil amendments or the processed liquid stream is recycled to flush the barn. This study investigated the effects of centrifugation and polymer addition on bacterial indicator removal from the liquid fraction of manure slurries. Farm samples were taken from a manure centrifuge processing system. There were negligible changes of quantified pathogen indicator concentrations in the low-solids centrate compared to the influent slurry. To study if possible improvements could be made to the system, lab scale experiments were performed investigating a range of g-forces and flocculating polymer addition. The results demonstrated that polymer addition had a negligible effect on the indicator bacteria levels when centrifuged at high g forces. However, the higher g force centrifugation was capable of reducing bacterial indicator levels up to two-log10 in the liquid stream of the manure, although at speeds higher than typical centrifuge operations currently used for manure processing applications. This study suggests manure centrifuge equipment could be redesigned to provide pathogen reduction to meet emerging issues, such as zoonotic pathogen control.

Subsurface Transport of in Soils of Wisconsin's Carbonate Aquifer Region.

is a waterborne pathogen known to have a significant reservoir in bovine manure. Land-dependent manure disposal may not result in significant or reliable pathogen attenuation and, therefore, presents a risk for transport of pathogenic spp. to groundwater. One factor missing in the existing literature is the role soil characteristics play in affecting oocyst transport. Of specific concern in regions with carbonate geology are macropores and other soil structures that contribute to preferential flow. Therefore, research is needed to understand soil type effects and important transport pathways for pathogens such as oocysts to drinking water wells. This study investigated transport potential in several soils overlying Wisconsin's vulnerable carbonate aquifer and related the soil transport to soil series and textural class. Experimental work involved monitoring the transport of oocysts through intact soil columns of different soil series under simulated rain conditions. Results demonstrate that a significant portion of oocysts will sorb or be physically entrapped in the soil, especially in soil with high clay content. However, silt loam soils with comparatively lower clay content demonstrated an ability to transport oocysts through the soil profile primarily via the first flush of water infiltrating through soil macropores. The rate of oocyst migration in silt loam soils paralleled the bromide tracer front, thus bypassing the soils' ability to strain or adsorb oocysts out of infiltrating water. Nevertheless, proper manure treatment and management are necessary to minimize public health risks.

Bench-scale Analysis of Surrogates for Anaerobic Digestion Processes.

Frequent monitoring of anaerobic digestion processes for pathogen destruction is both cost and time prohibitive. The use of surrogates to supplement regulatory monitoring may be one solution. To evaluate surrogates, a semi-batch bench-scale anaerobic digester design was tested. Bench-scale reactors were operated under mesophilic (36 °C) and thermophilic (53-55 °C) conditions, with a 15 day solids retention time. Biosolids from different facilities and during different seasons were examined. USEPA regulated pathogens and surrogate organisms were enumerated at different times throughout each experiment. The surrogate organisms included fecal coliforms, E. coli, enterococci, male-specific and somatic coliphages, Clostridium perfringens, and bacterial spores. Male-specific coliphages tested well as a potential surrogate organism for virus inactivation. None of the tested surrogate organisms correlated well with helminth inactivation under the conditions studied. There were statistically significant differences in the inactivation rates between the facilities in this study, but not between seasons.

Use of cationic polymers to reduce pathogen levels during dairy manure separation.

Various separation technologies are used to deal with the enormous amounts of animal waste that large livestock operations generate. When the recycled waste stream is land applied, it is essential to lower the pathogen load to safeguard the health of livestock and humans. We investigated whether cationic polymers, used as a flocculent in the solid/liquid separation process, could reduce the pathogen indicator load in the animal waste stream. The effects of low charge density cationic polyacrylamide (CPAM) and high charge density cationic polydicyandiamide (PDCD) were investigated. Results demonstrated that CPAM was more effective than PDCD for manure coagulation and flocculation, while PDCD was more effective than CPAM in reducing the pathogen indicator loads. However, their combined use, CPAM followed by PDCD, resulted in both improved solids separation and pathogen indicator reduction.

The use of multiple tracers to evaluate the impact of sewered and non-sewered development on coastal water quality in a rural area of Florida.

When onsite wastewater treatment and disposal systems (OSTDS) are not sited appropriately or installed properly, wastewater constituents can be a source of adverse environmental impacts to soil and groundwater, which can lead to potential public health risks. A paired monitoring design developed to compare water quality in sewered and non-sewered areas is presented here. It is suggested as a possible monitoring scheme for assessing the impact of sewer installation projects. As such, two sets of single-family, rural residential Florida neighborhoods were evaluated over a two-year period to gain insight into the effects of small-community use of OSTDS on coastal water quality. One set of two neighborhoods were connected to the sanitary sewer network and the other set of two were served exclusively by OSTDS. Water quality sampling was conducted at the paired sites during seasonal high water table (SHWT) and seasonal low water table (SLWT) events. Measured surface water quality during the SHWT showed indications of environmental impacts from OSTDS in terms of nutrients, microbial pathogen indicators, and other water quality measures, such as turbidity and conductivity. However, during the SLWT events, no obvious impacts attributable to OSTDS were detected. The water quality results indicate that OSTDS impacts may be measureable in rural areas. Other factors, such as microbial indicator survival and regrowth potential, may confound the understanding of water quality impacts of sewer projects. For example, the microbial indicators Escherichia coli and enterococci were found to persist over time and therefore did not always represent true comparisons of OSTDS and sewered areas between seasons. The timeframe for evaluating the effects of sewer projects may be longer than anticipated because of this survival and regrowth phenomenon.

A comparison of rapid bacterial methods to U.S. EPA-approved methods for biosolids.

The U.S. Environmental Protection Agency (U.S. EPA) specifies methods for testing microorganisms in biosolids, including U.S. EPA Methods 1680 and 1682 for fecal coliforms and Salmonella sp., respectively. However, these methods require 3 to 5 days to complete. For research or nonregulatory process monitoring purposes, it would be desirable to have a more rapid, less labor-intensive method of enumerating these organisms. The purpose of this study was to compare a modified Colilert with Quanti-Tray/2000 method to U.S. EPA Method 1680 for enumeration of fecal coliforms and E. coli, and to compare spread plating on xylose-lysine deoxycholate (XLD) supplemented with 10 pg/L novobiocin to U.S. EPA Method 1682 for Salmonella sp. in biosolids. These alternative enumeration methods require only 1 day to complete, using fewer costly materials. This limited study indicated that the more rapid methods provide results that are statistically comparable to U.S. EPA methods under most conditions and would be acceptable for rapid, nonregulatory data collection.

Foaming phenomenon in bench-scale anaerobic digesters.

The Madison Metropolitan Sewerage District (The District) in Madison, Wisconsin has been experiencing seasonal foaming in their anaerobic biosolids digesters, which has occurred from mid-November to late June for the past few years. The exact cause(s) of foaming is unknown. Previous research findings are unclear as to whether applications of advanced anaerobic digestion processes reduce the foaming potential of digesters. The object of this study was to investigate how configurations of thermophilic and acid phase-thermophilic anaerobic digestion would affect foaming at the bench-scale level compared to single stage mesophilic digestion for The District. Bench-scale anaerobic digesters were fed with a 4 to 4.5% by dry weight of solids content blend of waste activated sludge (WAS) and primary sludge from The District. Foaming potential was monitored using Alka-Seltzer and aeration foaming tests. The bench-scale acid phase-thermophilic digester had a higher foaming potential than the bench-scale mesophilic digester. These results indicate that higher temperatures increase the foaming potential of the bench-scale anaerobic digesters. The bench-scale acid phase-thermophilic digesters had a greater percent (approximately 5 to 10%) volatile solids destruction and a greater percent (approximately 5 to 10%) total solids destruction when compared to the bench-scale mesophilic digester. Overall, for the full-scale foaming experienced by The District, it appears that adding an acid phase or switching to thermophilic digestion would not alleviate The District's foaming issues.

Assessment of the efficacy of the first water system for emergency hospital use.

OBJECTIVE: The First Water Responder B package water treatment device was evaluated for its ability to reduce the levels of spiked indicators and pathogens (Escherichia coli, MS2 coliphage, murine adenovirus, and Cryptosporidium oocysts) in a surface water to partially evaluate its appropriateness to be used to provide safe drinking water to hospitals during emergency situations. METHODS: Lake water was collected in 50-L carboys and spiked with selected indicators and pathogens (E coli, MS2 coliphage, murine adenovirus, and Cryptosporidium oocysts) at 2 different spike levels (low and high). This water was treated using the First Water Responder B, and the microorganisms were enumerated before and after treatment using US Environmental Protection Agency and Standard Methods. Microbial removal efficiencies were compared with Environmental Protection Agency guidelines. RESULTS: E coli spikes ranged from 2.9 to 1059 colony-forming units (CFU)/100 mL with removals to below detection limits (1 CFU/100 mL) to 2.8 CFU/100 mL or 0.98 to 3.5 log(10) reductions. MS2 coliphage spikes ranged from 3 plaque-forming units (PFU) to 837 PFU/100 mL with removals to below detection limits (1 PFU/100 mL) to 11.7 PFU/100 mL or 0.65 to 1.9 log(10) reductions. Murine adenovirus spikes ranged from 203 to 8410 most probable number (MPN) of infectious units/100 mL with removals to below detection limits (23 MPN infectious units/100 mL) to 1370 MPN infectious units/100 mL or 0.79 to >1.2 log(10) reductions. Cryptosporidium parvum oocyst spikes ranged from 52 to 853 oocysts per liter with removals to below detection limits (<1 oocyst per liter) to 0.3 oocysts per liter or >2.2 to 3.4 log(10) reductions. CONCLUSIONS: Although the First Water system could remove a significant portion of the spiked organisms, it is recommended that this point-of-use system be coupled with chemical disinfection in a multiple-barrier approach to provide water of the highest reasonably achievable quality for hospital use in emergency situations.

Torque teno virus: an improved indicator for viral pathogens in drinking waters.

BACKGROUND: Currently applied indicator organism systems, such as coliforms, are not fully protective of public health from enteric viruses in water sources. Waterborne disease outbreaks have occurred in systems that tested negative for coliforms, and positive coliform results do not necessarily correlate with viral risk. It is widely recognized that bacterial indicators do not co-occur exclusively with infectious viruses, nor do they respond in the same manner to environmental or engineered stressors. Thus, a more appropriate indicator of health risks from infectious enteric viruses is needed. PRESENTATION OF THE HYPOTHESIS: Torque teno virus is a small, non-enveloped DNA virus that likely exhibits similar transport characteristics to pathogenic enteric viruses. Torque teno virus is unique among enteric viral pathogens in that it appears to be ubiquitous in humans, elicits seemingly innocuous infections, and does not exhibit seasonal fluctuations or epidemic spikes. Torque teno virus is transmitted primarily via the fecal-oral route and can be assayed using rapid molecular techniques. We hypothesize that Torque teno virus is a more appropriate indicator of viral pathogens in drinking waters than currently used indicator systems based solely on bacteria. TESTING THE HYPOTHESIS: To test the hypothesis, a multi-phased research approach is needed. First, a reliable Torque teno virus assay must be developed. A rapid, sensitive, and specific PCR method using established nested primer sets would be most appropriate for routine monitoring of waters. Because PCR detects both infectious and inactivated virus, an in vitro method to assess infectivity also is needed. The density and occurrence of Torque teno virus in feces, wastewater, and source waters must be established to define spatial and temporal stability of this potential indicator. Finally, Torque teno virus behavior through drinking water treatment plants must be determined with co-assessment of traditional indicators and enteric viral pathogens to assess whether correlations exist. IMPLICATIONS OF THE HYPOTHESIS: If substantiated, Torque teno virus could provide a completely new, reliable, and efficient indicator system for viral pathogen risk. This indicator would have broad application to drinking water utilities, watershed managers, and protection agencies and would provide a better means to assess viral risk and protect public health.

Monitoring source water for microbial contamination: evaluation of water quality measures.

Watershed management programs often rely on monitoring for a large number of water quality parameters to define contaminant issues. While coliforms have traditionally been used to identify microbial contamination, these indicators cannot discriminate among potential contaminant sources. Microbial source tracking (MST) can provide the missing link that implicates the sources of contamination. The objective of this study was to use a weight-of-evidence approach (land use analysis using GIS, sanitary surveys, traditional water quality monitoring, and MST targets) to identify sources of pollution within a watershed that contains a raw drinking water source. For the study watersheds, statistical analyses demonstrated that one measure each of particulate matter (turbidity, particle counts), organic matter (total organic carbon, dissolved organic carbon, UV(254) absorbance), and indicator organisms (fecal coliforms, enterococci) were adequate for characterizing water quality. While these traditional parameters were useful for assessing overall water quality, they were not intended to differentiate between microbial sources at different locations. In contrast, the MST targets utilized (Rhodococcus coprophilus, sorbitol-fermenting Bifidobacteria, and male-specific coliphages) pinpointed specific sources of microbial pollution. However, these targets could not be used for routine monitoring due to a high percentage of non-detects.

Monod kinetics for aerobic biodegradation of petroleum hydrocarbons in unsaturated soil microcosms.

We use Monod kinetics to calibrate previously published data that documentthe aerobic biodegradation of hydrocarbon vapors in soil microcosms from a weathered petroleum spill site. Monod kinetics offer insight into biodegradation mechanics because they address biomass growth as well as substrate depletion. A blend of five aromatics and five alkanes dose the microcosm sets at four strengths, and a finite difference model describes the response superimposed across the constituent substrates. An observed initial biomass X0 of 125 g biomass/m(3) soil moisture and an endogendous decay rate b of 0.102 day(-1) calibrate all four dosages and agree with heterotrophic plate counts. Common maximum specific growth rates microMJ and half saturation constants KSJ calibrate each constituent across the four dosages. The biodegradable alkanes exhibit microMJ values ranging from 0.0190 to 0.0996 day(-1), while the aromatic rates vary from 0.0946 to 0.322 day(-1). One of the alkanes (2,2,4-trimethylpentane) is recalcitrant. The half saturation constants range from 0.000083 to 0.000355 g substrate/m(3) soil moisture for the biodegradable alkanes, which imply zero-order kinetics. The aromatic KSJ values vary from 5.02 to 14.3 g substrate/m(3) soil moisture, and suggest first-order kinetics. The yield YJ increases with dosage concentration for all the biodegradable constituents, varying from 0.0533 to 1.58 g biomass/g substrate.

Watershed issues associated with Clostridium botulinum: a literature review.

Botulism the disease, the related organism (Clostridium botulinum) and toxin have gained renewed attention in these times of heightened homeland security and bioterrorism preparedness. Since C. botulinum is ubiquitous in nature, botulism outbreaks resulting from environmental exposure can be of concern to watershed managers and drinking water utilities. This paper reviews aspects of naturally occurring C. botulinum in light of concerns for source water watersheds. Information regarding sources and occurrence of botulism, C. botulinum and botulism toxins are discussed. Ecology and physiology of environmental C. botulinum and cycles of disease are reviewed. Finally, the effectiveness of water treatment and disinfection measures is discussed.

An optimized enumeration method for sorbitol-fermenting Bifidobacteria in water samples.

With increased focus on watershed protection under the Surface Water Treatment Rule, indicators that discriminate among sources of microbial inputs (microbial source tracking) are needed to supplement the quantitative information provided by total and fecal coliform measurements for drinking water monitoring. Bifidobacteria are found in the digestive tract and feces of humans and other animals, and also in sewage. Sorbitol is a food additive used exclusively in food intended for human consumption. Therefore, the presence of sorbitol-fermenting Bifidobacteria in environmental waters can be indicative of sources of human fecal contamination. A series of media were evaluated using ATCC cultures of B. breve and B. adolescentis, feces from different animals, and domestic wastewater samples. The media evaluated were Human Bifid Sorbitol agar (HBSA), modified Human Bifid Sorbitol agar, Beerens Medium, modified Beerens Medium, Reinforced Clostridial agar, BIM-25 Medium, and modified BIM-25 Medium. Variables such as sample preservation, incubation time, different pH indicators, plating technique, and discontinuous exposure to sorbitol were also evaluated. A series of biochemical tests were used to confirm positive colonies enumerated on the various media. Membrane filtration and enumeration of sodium sulfite preserved samples on HBSA containing bromocresol purple using loose lidded plates for 48 h provided the best recoveries for presumptive positive colonies. A number of sorbitol-fermenters that were not Bifidobacteria were able to grow on all media tested, resulting in false-positives. Therefore, plating on HBSA should be followed by a confirmation step when monitoring for sorbitol-fermenting Bifidobacteria in environmental waters. A year-long sampling survey of a managed reservoir in Massachusetts provided field validation of the proposed methodology for sorbitol-fermenting Bifidobacteria as a human-related source tracking indicator tool.

Molecular detection and genotyping of male-specific coliphages by reverse transcription-PCR and reverse line blot hybridization.

In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Q beta, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies.

A comparison of the survival of F+RNA and F+DNA coliphages in lake water microcosms.

The survival of seven F+RNA phages (MS2 Group I ATCC type strain, two Group I environmental isolates, a Group II environmental isolate, a Group III environmental isolate, and two Group IV environmental isolates) and six F+DNA phages (M13, fd, f1, and ZJ/2 ATCC type strains, and two environmental isolates) were examined in microcosms using a surface drinking water source. Phages were spiked into replicate aliquots of a source water at about 20,000 pfu/ml. Replicate spikes were incubated at 4 and 20 degrees C and monitored for 110 days. At 4 degrees C, Groups I and II F+ RNA phages were detectable through 110 days, with reductions of about 1 and 3 log10, respectively. The Group III F+RNA phage demonstrated 5 log10 reduction after 3 weeks, and the Group IV F+RNA phages were reduced to detection limits (5 log10 reduction) within 10 days. Of the F+DNA phages, all four type strains were detectable with about 2.5 log10 reduction after 110 days at 4 degrees C. The F+DNA environmental isolates were detectable with about a 4 log10 reduction after 110 days at 4 degrees C. All phages demonstrated faster decay at 20 degrees C. These results suggest that differences in F+ phage survival may influence their prevalence in environmental waters and the ability to attribute their prevalence to specific human and animal sources of faecal contamination.

Evaluation of F+ RNA and DNA coliphages as source-specific indicators of fecal contamination in surface waters.

Male-specific (F+) coliphages have been investigated as viral indicators of fecal contamination that may provide source-specific information for impacted environmental waters. This study examined the presence and proportions of the different subgroups of F+ coliphages in a variety of fecal wastes and surface waters with well-defined potential waste impacts. Municipal wastewater samples had high proportions of F+ DNA and group II and III F+ RNA coliphages. Bovine wastewaters also contained a high proportion of F+ DNA coliphages, but group I and IV F+ RNA coliphages predominated. Swine wastewaters contained approximately equal proportions of F+ DNA and RNA coliphages, and group I and III F+ RNA coliphages were most common. Waterfowl (gull and goose) feces contained almost exclusively F+ RNA coliphages of groups I and IV. No F+ coliphages were isolated from the feces of the other species examined. F+ coliphage recovery from surface waters was influenced by precipitation events and animal or human land use. There were no significant differences in coliphage density among land use categories. Significant seasonal variation was observed in the proportions of F+ DNA and RNA coliphages. Group I F+ RNA coliphages were the vast majority (90%) of those recovered from surface waters. The percentage of group I F+ RNA coliphages detected was greatest at background sites, and the percentage of group II F+ RNA coliphages was highest at human-impacted sites. Monitoring of F+ coliphage groups can indicate the presence and major sources of microbial inputs to surface waters, but environmental effects on the relative occurrence of different groups need to be considered.