Frank J. Loge

Concentration of enteroviruses, adenoviruses, and noroviruses from drinking water by use of glass wool filters

ABSTRACT Available filtration methods to concentrate waterborne viruses are either too costly for studies requiring large numbers of samples, limited to small sample volumes, or not very portable for routine field applications. Sodocalcic glass wool filtration is a cost-effective and easy-to-use method to retain viruses, but its efficiency and reliability are not adequately understood. This study evaluated glass wool filter performance to concentrate the four viruses on the U.S. Environmental Protection Agency contaminant candidate list, i.e., coxsackievirus, echovirus, norovirus, and adenovirus, as well as poliovirus. Total virus numbers recovered were measured by quantitative reverse transcription-PCR (qRT-PCR); infectious polioviruses were quantified by integrated cell culture (ICC)-qRT-PCR. Recovery efficiencies averaged 70% for poliovirus, 14% for coxsackievirus B5, 19% for echovirus 18, 21% for adenovirus 41, and 29% for norovirus. Virus strain and water matrix affected recovery, with significant interaction between the two variables. Optimal recovery was obtained at pH 6.5. No evidence was found that water volume, filtration rate, and number of viruses seeded influenced recovery. The method was successful in detecting indigenous viruses in municipal wells in Wisconsin. Long-term continuous filtration retained viruses sufficiently for their detection for up to 16 days after seeding for qRT-PCR and up to 30 days for ICC-qRT-PCR. Glass wool filtration is suitable for large-volume samples (1,000 liters) collected at high filtration rates (4 liters min−1), and its low cost makes it advantageous for studies requiring large numbers of samples.

Simultaneous Discrimination between 15 Fish Pathogens by Using 16S Ribosomal DNA PCR and DNA Microarrays

ABSTRACT We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55°C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 × 106 genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.

Factors influencing ultraviolet disinfection performance Part II: association of coliform bacteria with wastewater particles

A technique is described for quantifying the number of particles with embedded coliform bacteria in wastewater samples. This technique was applied to eight different wastewater samples collected from eight significantly different treatment processes (five variations of the activated-sludge process, a trickling filter, an aerated lagoon, and a facultative lagoon). For all treatment process types that did not use chemical coagulants, total number of particles with embedded coliform bacteria correlated linearly with number of residual coliform bacteria following high doses of UV light. Lagoon treatment processes led to the formation of fewer bacteria-associated particles than either the activated-sludge or trickling-filter processes. Chemical coagulants showed a potential to significantly reduce the inactivation fraction of coliform bacteria associated with particles.

Viruses in Nondisinfected Drinking Water from Municipal Wells and Community Incidence of Acute Gastrointestinal Illness

Background: Groundwater supplies for drinking water are frequently contaminated with low levels of human enteric virus genomes, yet evidence for waterborne disease transmission is lacking. Objectives: We related quantitative polymerase chain reaction (qPCR)–measured enteric viruses in the tap water of 14 Wisconsin communities supplied by nondisinfected groundwater to acute gastrointestinal illness (AGI) incidence. Methods: AGI incidence was estimated from health diaries completed weekly by households within each study community during four 12-week periods. Water samples were collected monthly from five to eight households per community. Viruses were measured by qPCR, and infectivity assessed by cell culture. AGI incidence was related to virus measures using Poisson regression with random effects. Results: Communities and time periods with the highest virus measures had correspondingly high AGI incidence. This association was particularly strong for norovirus genogroup I (NoV-GI) and between adult AGI and enteroviruses when echovirus serotypes predominated. At mean concentrations of 1 and 0.8 genomic copies/L of NoV-GI and enteroviruses, respectively, the AGI incidence rate ratios (i.e., relative risk) increased by 30%. Adenoviruses were common, but tap-water concentrations were low and not positively associated with AGI. The estimated fraction of AGI attributable to tap-water–borne viruses was between 6% and 22%, depending on the virus exposure–AGI incidence model selected, and could have been as high as 63% among children < 5 years of age during the period when NoV-GI was abundant in drinking water. Conclusions: The majority of groundwater-source public water systems in the United States produce water without disinfection, and our findings suggest that populations served by such systems may be exposed to waterborne viruses and consequent health risks.

Comparative toxicity of hydrogen peroxide, hydroxyl radicals, and superoxide anion to Escherichia coli

The toxicity of hydrogen peroxide, hydroxyl radical, and superoxide radical anion to Escherichia coli was investigated as a basis for understanding the effects of hydrogen peroxide when it is injected into the subsurface for in situ bioremediation or in situ chemical oxidation. Hydrogen peroxide toxicity was evaluated by maintaining its steady state concentration at a series of concentrations ranging from 0.7 to 3.0 mM in the presence of E. coli. Hydroxyl radical toxicity was studied by conducting parallel reactions of equal steady state concentrations of hydrogen peroxide, but using an iron (III)–nitrilotriacetic acid complex to decompose hydrogen peroxide to hydroxyl radicals. Superoxide was also generated from equal steady state concentrations of hydrogen peroxide, but with the addition of pyrolusite (manganese oxide) to catalyze its decomposition to superoxide radical. Hydrogen peroxide was toxic to E. coli at all concentrations investigated. The generation of hydroxyl radicals in hydrogen peroxide solutions showed no increase in toxicity relative to hydrogen peroxide toxicity, indicating minimal additional toxicity of the hydroxyl radicals. Hydrogen peroxide solutions of equal concentrations in which superoxide was generated showed less toxicity relative to hydrogen peroxide systems. The results indicate that toxicity of hydrogen peroxide to microorganisms may be lower when it is injected to subsurface systems containing high manganese oxide contents. � 2002 Elsevier Science Ltd. All rights reserved.

Disease susceptibility of salmon exposed to polybrominated diphenyl ethers (PBDEs)

The health effects of the flame retardant polybrominated diphenyl ethers (PBDEs) in fish are not well understood. To determine the potential effects of this ubiquitous contaminant class on fish health, juvenile subyearling Chinook salmon (Oncorhynchus tshawytscha) were fed a diet that reflected the PBDE congeners found in the stomach contents of subyearling Chinook salmon collected from the highly urbanized and industrialized lower Willamette River in the Columbia River Basin of North America. The diet, consisting of five PBDE congeners (BDE-47, BDE-99, BDE-100, BDE-153 and BDE-154), was fed to the salmon at 2% of their body weight in food per day for 40 days. Two concentrations of the diet (1x and 10x PBDE) were fed to the salmon. The 1x PBDE diet reflected the concentration of PBDEs (190 ng PBDEs/g food) found in the stomach contents of juvenile subyearling Chinook salmon; the 10x diet was prepared at 10 times that concentration. The fish were then exposed to the marine bacterial pathogen Listonella anguillarum to assess susceptibility to infectious disease. Juvenile Chinook salmon fed the 1x PBDE diet were more susceptible to L. anguillarum than salmon fed the control diet. This suggests that juvenile salmonids in the lower Willamette River exposed to PBDEs may be at greater risk for disease than nonexposed juvenile salmonids. In contrast, salmon that consumed the 10x PBDE diet were not more susceptible to the pathogen than salmon fed the control diet. The mechanisms for the dichotomous results observed in disease susceptibility between salmon fed the 1x and 10x PBDE diets are currently not known but have also been observed in other species exposed to PBDEs with respect to immune function.

Post-anoxic denitrification driven by PHA and glycogen within enhanced biological phosphorus removal.

The objective of this research was to interrogate and develop a better understanding for a process to achieve post-anoxic denitrification without exogenous carbon augmentation within enhanced biological phosphorus removal (EBPR). Sequencing batch reactors fed real wastewater and seeded with mixed microbial consortia were operated under variable anaerobic-aerobic-anoxic and organic carbon loading conditions. The process consistently achieved phosphorus and nitrogen removal, while the observed specific denitrification rates were markedly higher than expected for post-anoxic systems operated without exogenous organic carbon addition. Investigations revealed that post-anoxic denitrification was predominantly driven by glycogen, an intracellular carbon storage polymer associated with EBPR; moreover, glycogen reserves can be significantly depleted post-anoxically without compromising EBPR. Success of the proposed process is predicated on providing sufficient organic acids in the influent wastewater, such that residual nitrate carried over from the post-anoxic period is reduced and polyhydroxyalkanoate (PHA) synthesis occurs.

Biomarker responses and disease susceptibility in juvenile rainbow trout Oncorhynchus mykiss fed a high molecular weight PAH mixture

Juvenile rainbow trout were fed a diet containing an environmentally relevant mixture of 10 high molecular weight polycyclic aromatic hydrocarbons (PAHs) at a dose of 0.66 or 7.82 µg PAH · g fish(-1) · d(-1). At 3, 7, 14, and 28 d, biomarkers of aryl hydrocarbon receptor activation (AHR), hepatic microsomal ethoxyresorufin-O-deethylase (EROD) activity, and cytochrome P4501A (CYP1A)-associated staining increased 14- to 26-fold and 6- to 14-fold, respectively, in fish fed 7.82 µg PAH · g fish (-1) · d(-1). Cytochrome P4501A-associated staining increased 2- to 9-fold on days 3, 7, and 28 in fish fed 0.66 µg PAH · g fish(-1) · d(-1). Bile fluorescent aromatic compounds served as a biomarker of exposure and confirmed that PAH exposure was consistent over 50 d. DNA damage in blood cells, protein oxidation, and lipid peroxidation in the kidney were biomarkers of oxidative stress and all increased in fish fed 7.82 µg PAH · g fish(-1) · d(-1). Fish fed 0.66 µg PAH · g fish(-1) · d(-1) had elevated DNA damage in blood cells but increased protein oxidation or lipid peroxidation in the kidney were not observed. Challenge with Aeromonas salmonicida, at lethal concentration (LC) 20, decreased survival in fish previously fed either 0.66 µg PAH · g fish(-1) · d(-1) or 7.82 µg PAH · g fish(-1) · d(-1) relative to fish fed the control diet. In general, biomarkers of both AHR activation and oxidative stress peaked at 3 to 14 d then declined at 28 to 50 d of PAH exposure and an increase in susceptibility to disease was observed at 50 d. These results link PAH exposure to biomarker responses that may be useful as early indicators of population level responses, such as mortality resulting from an increase in disease susceptibility.

Preliminary assessment of transport processes influencing the penetration of chlorine into wastewater particles and the subsequent inactivation of particle-associated organisms.

The diffusion of a chemical disinfectant into wastewater particles may be viewed as a serial two-step process involving transport through a macroporous network of pathways to micropores that lead into dense cellular regions. Previous research reveals that ultraviolet (UV) light penetration into wastewater particles is limited primarily to macropores, resulting in a residual concentration of targeted organisms in post-disinfected effluents that reflects the number of organisms embedded in the dense cellular regions of particles. Conversely, chlorine was demonstrated as part of this research to penetrate into both the macroporous and microporous network of pathways, implying that the application of chlorine may be designed feasibly to achieve a desired level of inactivation of particle-associated organisms. In the short term, a disinfection model previously developed for UV irradiation may be used to assess the inactivation of particle-associated organisms with chlorine. However, in the long-term, a more rigorous and complete understanding of the transport of chemical disinfectants into particles can be explored utilizing existing mathematical expressions commonly used to model mass transport into porous media. The parameters of interest in this modeling approach include the reaction rate of chlorine with particulate material, the diffusion rate of chlorine within a particle, the mass-transfer rate coefficient across the particles boundary, and the particle porosity.

Association of coliform bacteria with wastewater particles: impact of operational parameters of the activated sludge process.

The fraction of particles with associated coliform bacteria (PAC) in the activated sludge process was evaluated using a 16S rRNA oligonucleotide probe specific to the family Enterobacteriaceae. The PAC was found to decline exponentially with increasing mean cell residence times (MCRTs). The factors influencing the formation of PAC, identified with simplified mass balance relationships. are the concentration of particles, the concentration of dispersed (non-particle associated) coliform bacteria, and the MCRT. The concentration of dispersed coliform bacteria was found to decline with increasing MCRTs. The rate of decline was greater than the typical half-life attributed to endogenous decay, suggesting that other factors (e.g., predation by protozoa) influence the concentration of dispersed coliform bacteria, and subsequently the formation of PAC. Given that the association of targeted organisms with particles adversely impacts the performance of a disinfection system, studies targeted at the fate of organisms other than coliform bacteria in the activated sludge process are of paramount importance in assessing the health risks of post-disinfected effluents.