Elisabetta Lambertini

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.

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.

Virus contamination from operation and maintenance events in small drinking water distribution systems

We tested the association of common events in drinking water distribution systems with contamination of household tap water with human enteric viruses. Viruses were enumerated by qPCR in the tap water of 14 municipal systems that use non-disinfected groundwater. Ultraviolet disinfection was installed at all active wellheads to reduce virus contributions from groundwater to the distribution systems. As no residual disinfectant was added to the water, any increase in virus levels measured downstream at household taps would be indicative of distribution system intrusions. Utility operators reported events through written questionnaires. Virus outcome measures were related to distribution system events using binomial and gamma regression. Virus concentrations were elevated in the wells, reduced or eliminated by ultraviolet disinfection, and elevated again in distribution systems, showing that viruses were, indeed, directly entering the systems. Pipe installation was significantly associated with higher virus levels, whereas hydrant flushing was significantly associated with lower virus levels. Weak positive associations were observed for water tower maintenance, valve exercising, and cutting open a water main. Coliform bacteria detections from routine monitoring were not associated with viruses. Understanding when distribution systems are most vulnerable to virus contamination, and taking precautionary measures, will ensure delivery of safe drinking water.

New mathematical approaches to quantify human infectious viruses from environmental media using integrated cell culture-qPCR

Quantifying infectious viruses by cell culture depends on visualizing cytopathic effect, or for integrated cell culture-PCR, attaining confidence a PCR-positive signal is the result of virus growth and not inoculum carryover. This study developed mathematical methods to calculate infectious virus numbers based on viral growth kinetics in cell culture. Poliovirus was inoculated into BGM cell monolayers at 10 concentrations from 0.001 to 1000 PFU/ml. Copy numbers of negative-strand RNA, a marker of infectivity for single-stranded positive RNA viruses, were measured over time by qRT-PCR. Growth data were analyzed by two approaches. First, data were fit with a continuous function to estimate directly the initial virus number, expressed as genomic copies. Such estimates correlated with actual inoculum numbers across all concentrations (R(2)=0.62, n=17). Second, the length of lag phase appeared to vary inversely with inoculum titers; hence, standard curves to predict inoculum virus numbers were derived based on three definitions of lag time: (1) time of first detection of (-)RNA, (2) second derivative maximum of the fitted continuous function, and (3) time when the fitted curve crossed a threshold (-)RNA concentration. All three proxies yielded standard curves with R(2)=0.69-0.90 (n=17). The primary advantage of these growth kinetics approaches is being able to quantify virions that are unambiguously infectious, a particular advantage for viruses that do not produce CPE.

Quantitative Microbial Risk Assessment for Escherichia coli O157:H7 in Fresh-Cut Lettuce

Leafy green vegetables, including lettuce, are recognized as potential vehicles for foodborne pathogens such as Escherichia coli O157:H7. Fresh-cut lettuce is potentially at high risk of causing foodborne illnesses, as it is generally consumed without cooking. Quantitative microbial risk assessments (QMRAs) are gaining more attention as an effective tool to assess and control potential risks associated with foodborne pathogens. This study developed a QMRA model for E. coli O157:H7 in fresh-cut lettuce and evaluated the effects of different potential intervention strategies on the reduction of public health risks. The fresh-cut lettuce production and supply chain was modeled from field production, with both irrigation water and soil as initial contamination sources, to consumption at home. The baseline model (with no interventions) predicted a mean probability of 1 illness per 10 million servings and a mean of 2,160 illness cases per year in the United States. All intervention strategies evaluated (chlorine, ultrasound and organic acid, irradiation, bacteriophage, and consumer washing) significantly reduced the estimated mean number of illness cases when compared with the baseline model prediction (from 11.4- to 17.9-fold reduction). Sensitivity analyses indicated that retail and home storage temperature were the most important factors affecting the predicted number of illness cases. The developed QMRA model provided a framework for estimating risk associated with consumption of E. coli O157:H7-contaminated fresh-cut lettuce and can guide the evaluation and development of intervention strategies aimed at reducing such risk.

Kinetics of conjugative gene transfer on surfaces in granular porous media.

The transfer of genetic material among bacteria in the environment can occur both in the planktonic and attached state. Given the propensity of organisms to exist in sessile microbial communities in oligotrophic subsurface conditions, and that such conditions typify the subsurface, this study focuses on exploratory modeling of horizontal gene transfer among surface-associated Escherichiacoli in the subsurface. The mathematics so far used to describe the kinetics of conjugation in biofilms are developed largely from experimental observations of planktonic gene transfer, and are absent of lags or plasmid stability that appear experimentally. We develop a model and experimental system to quantify bacterial filtration and gene transfer in the attached state, on granular porous media. We include attachment kinetics described in Nelson et al. (2007) using the filtration theory approach of Nelson and Ginn (2001, 2005) with motility of E. coli described according to Biondi et al. (1998).

Effects of Etiological Agent and Bather Shedding of Pathogens on Interpretation of Epidemiological Data Used to Establish Recreational Water Quality Standards

The overall goal of the study reported herein was to use techniques in the field of risk assessment (specifically a state-space population dynamic model of disease transmission within recreational waters) to explore the relative significance of (1) active shedding of microorganisms from bathers themselves, and (2) the type and concentration of etiological agent on the observed heterogeneity of the incidence of illness in epidemiological studies that have been used to develop ambient water quality criteria. The etiological agent and corresponding dose ingested during recreational contact was found to significantly impact the observed incidence of illness in an epidemiological study conducted in recreational water. In addition, the observed incidence of illness was found not to necessarily reflect background concentrations of indicator organisms, but rather microorganisms shed during recreational contact. Future revisions to ambient water quality criteria should address the etiological agent, dose, and the significance of microbial shedding relative to background concentrations of pathogens and indicator organisms in addition to the incidence of illness and concentration of indicator organisms. Without a quantitative assessment of these additional variables, study findings may potentially be site specific and not representative of the health risks associated with specific indicator concentrations in all recreational waters.

Transcriptional changes in innate immunity genes in head kidneys from Aeromonas salmonicida-challenged rainbow trout fed a mixture of polycyclic aromatic hydrocarbons

We previously observed that exposure to a complex mixture of high molecular weight polycyclic aromatic hydrocarbons (PAHs) increased sensitivity of rainbow trout (Oncorhynchus mykiss) to subsequent challenge with Aeromonas salmonicida, the causative agent of furunculosis. In this study, we evaluate potential mechanisms associated with disease susceptibility from combined environmental factors of dietary PAH exposure and pathogen challenge. Rainbow trout were fed a mixture of ten high molecular weight PAHs at an environmentally relevant concentration (7.82μg PAH mixture/g fish/day) or control diet for 50 days. After 50 days of PAH exposure, fish were challenged with either Aeromonas salmonicida at a lethal concentration 30 (LC30) or growth media without the pathogen (mock challenge). Head kidneys were collected 2, 4, 10 and 20 days after challenge and gene expression (q<0.05) was evaluated among treatments. In animals fed the PAH contaminated diet, we observed down-regulation of expression for innate immune system genes in pathways (p<0.05) for the terminal steps of the complement cascade (complement component C6) and other bacteriolytic processes (lysozyme type II) potentially underlying increased disease susceptibility after pathogen challenge. Increased expression of genes associated with hemorrhage/tissue remodeling/inflammation pathways (p<0.05) was likely related to more severe head kidney damage due to infection in PAH-fed compared to control-fed fish. This study is the first to evaluate transcriptional signatures associated with the impact of chronic exposure to an environmentally relevant mixture of PAHs in disease susceptibility and immunity.