Multiple factors affecting Ixodes ricinus ticks and associated pathogens in European temperate ecosystems (northeastern France).

In Europe, the main vector of tick-borne zoonoses is Ixodes ricinus, which has three life stages. During their development cycle, ticks take three separate blood meals from a wide variety of vertebrate hosts, during which they can acquire and transmit human pathogens such as Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis. In this study conducted in Northeastern France, we studied the importance of soil type, land use, forest stand type, and temporal dynamics on the abundance of ticks and their associated pathogens. Negative binomial regression modeling of the results indicated that limestone-based soils were more favorable to ticks than sandstone-based soils. The highest tick abundance was observed in forests, particularly among coniferous and mixed stands. We identified an effect of habitat time dynamics in forests and in wetlands: recent forests and current wetlands supported more ticks than stable forests and former wetlands, respectively. We observed a close association between tick abundance and the abundance of Cervidae, Leporidae, and birds. The tick-borne pathogens responsible for Lyme borreliosis, anaplasmosis, and hard tick relapsing fever showed specific habitat preferences and associations with specific animal families. Machine learning algorithms identified soil related variables as the best predictors of tick and pathogen abundance.

Toxicity of lanthanides on various fish cell lines.

The growing use of Lanthanides in new technologies has increased their anthropogenic releases into the aquatic environment over the last decades. However, knowledge on their ecotoxicological impacts is still incomplete, especially with regard to biological effects of Lanthanides mixtures and the possible regular variation in toxicity along the Lanthanides series. The present study evaluated the individual toxicity of all Lanthanides and the toxicity of mixtures of three of them, namely Neodymium (Nd3+), Gadolinium (Gd3+), and Ytterbium (Yb3+) on Danio rerio fibroblast-like cells (ZF4). Individual and mixtures toxicity of Neodymium (Nd3+) and Ytterbium (Yb3+) were also assessed on Danio rerio hepatic cells (ZFL) and Oncorhynchus mykiss epithelial cells (RTgill-W1). The measured Lanthanide concentrations were close to the nominal ones in the culture media of ZF4, ZFL, and RTgill-W1 cells (85-99%). A toxic impact was observed on the three fish cell lines exposed to all Lanthanides tested individually. RTgill-W1 appeared as the less sensitive cells, compared to the two others. Four Lanthanides, Erbium (Er3+), Thulium (Tm3+), Ytterbium (Yb3+) and Lutetium (Lu3+) showed a higher toxicity than the others on ZF4 cells but no correlation could be established between the toxicity of Lanthanides and the order of the elements within the Lanthanides series. Exposures to binary mixtures highlighted the presence of synergistic effects on cell viability for all cell lines.

Effects and bioaccumulation of Cr(III), Cr(VI) and their mixture in the freshwater mussel Corbicula fluminea.

Chromium has two main oxidation states, Cr(III) and Cr(VI), that can occur simultaneously in natural waters. Current consensus holds that Cr(VI) is of high ecotoxicological concern, but regards Cr(III) as poorly bioavailable and relatively non-toxic. In this work, the effects and bioaccumulation of Cr(III), Cr(VI) and their mixture were studied using the freshwater clam Corbicula fluminea as a model organism. Mixture exposures were carried out using solutions isotopically enriched in 50Cr(III) or 53Cr(VI), allowing to quantify the contribution of each redox form to total Cr accumulation in the clams. Following exposure to individual redox forms, Cr(III) accumulated preferentially in the digestive glands and Cr(VI) in the gills of C. fluminea. In mixture exposures, both redox forms accumulated mainly in the gills; the concentration of Cr(III) in the digestive glands being much lowered compared with individual exposures. Both oxidation states affected the expression of biomarkers related to energy reserves, cellular damage and mitochondrial functioning, as well as the expression of mRNA for detoxification genes. The observed effects differed between gills and digestive glands. The present study suggests that Cr(III) is a bioavailable and biologically active elemental species deserving more consideration by the ecotoxicological community.

Variability in molecular characteristics of Hepatitis E virus quasispecies could modify viral surface properties and transmission.

Hepatitis E virus (HEV) usually causes self-limited liver diseases but can also result in severe cases. Genotypes 1 (G1) and 2 circulate in developing countries are human-restricted and waterborne, while zoonotic G3 and G4 circulating in industrialized countries preferentially infect human through consumption of contaminated meat. Our aims were to identify amino acid patterns in HEV variants that could be involved in pathogenicity or in transmission modes, related to their impact on antigenicity and viral surface hydrophobicity. HEV sequences from human (n = 37) and environmental origins (wild boar [n = 3], pig slaughterhouse effluent [n = 6] and urban wastewater [n = 2]) were collected for the characterization of quasispecies using ultra-deep sequencing (ORF2/ORF3 overlap). Predictive and functional assays were carried out to investigate viral particle antigenicity and hydrophobicity. Most quasispecies showed a major variant while a mixture was observed in urban wastewater and in one chronically infected patient. Amino acid signatures were identified, as a rabbit-linked HEV pattern in two infected patients, or the S68L (ORF2) / H81C (ORF3) residue mostly identified in wild boars. By comparison with environmental strains, molecular patterns less likely represented in humans were identified. Patterns impacting viral hydrophobicity and/or antigenicity were also observed, and the higher hydrophobicity of HEV naked particles compared with the enveloped forms was demonstrated. HEV variants isolated from human and environment present molecular patterns that could impact their surface properties as well as their transmission. These molecular patterns may concern only one minor variant of a quasispecies and could emerge under selective pressure.

F-Specific RNA Bacteriophages Model the Behavior of Human Noroviruses during Purification of Oysters: the Main Mechanism Is Probably Inactivation Rather than Release.

Noroviruses (NoV) are responsible for many shellfish outbreaks. Purification processes may be applied to oysters before marketing to decrease potential fecal pollution. This step is rapidly highly effective in reducing Escherichia coli; nevertheless, the elimination of virus genomes has been described to be much slower. It is therefore important to identify (i) the purification conditions that optimize virus removal and (ii) the mechanism involved. To this end, the effects of oyster stress, nutrients, and the presence of a potential competitor to NoV adhesion during purification were investigated using naturally contaminated oysters. Concentrations of NoV (genomes) and of the viral indicator F-specific RNA bacteriophage (FRNAPH; genomes and infectious particles) were regularly monitored. No significant differences were observed under the test conditions. The decrease kinetics of both virus genomes were similar, again showing the potential of FRNAPH as an indicator of NoV behavior during purification. The T90 (time to reduce 90% of the initial titer) values were 47.8 days for the genogroup I NoV genome, 26.7 days for the genogroup II NoV genome, and 43.9 days for the FRNAPH-II genome. Conversely, monitoring of the viral genomes could not be used to determine the behavior of infectious viruses because the T90 values were more than two times lower for infectious FRNAPH (20.6 days) compared to their genomes (43.9 days). Finally, this study highlighted that viruses are primarily inactivated in oysters rather than released in the water during purification processes.IMPORTANCE This study provides new data about the behavior of viruses in oysters under purification processes and about their elimination mechanism. First, a high correlation has been observed between F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and norovirus (NoV) in oysters impacted by fecal contamination when both are detected using molecular approaches. Second, when using reverse transcription-quantitative PCR and culture to detect FRNAPH-II genomes and infectious FRNAPH in oysters, respectively, it appears that genome detection provides limited information about the presence of infectious particles. The comparison of both genomes and infectious particles highlights that the main mechanism of virus elimination in oysters is inactivation. Finally, this study shows that none of the conditions tested modify virus removal.

Nano-exploration of organic conditioning film formed on polymeric surfaces exposed to drinking water.

Adsorption of organic macromolecules onto surfaces in contact with waters forms a so-called conditioning film and induces modifications of the surface properties. Here, we characterized conditioning films formed onto two hydrophobic materials (used as pipe liner) and immersed for 24 h in tap water. Using combination of atomic force microscopy (AFM), and chemical force microscopy (CFM), we detected some changes in roughness and hydrophilic/hydrophobic balance of the surface of the tested coupons, and also the deposition of numerous organic polymers (few millions/cm2) randomly distributed on the surface. The maximum molecular extension of these organic polymers was in the range of 250-1250 nm according to the tested materials. Systematic analysis of the force curves with the theoretical models (WLC and FJC) allowed determining the proportion of rupture events related to the unfolding of both polysaccharide and polypeptide segments, which represented 75-80% and 20-25% of the analyzed curves, respectively. The number of autochthonous drinking water bacteria, which attached to the material within the same period of time was 10000-folds lower than the detected number of polymers attached to the surface. Even in drinking water systems with relatively low organic matter (dissolved organic carbon < 1.1 mg/L), the potential of formation of a conditioning biofilm is important.

Heterogeneity: a major factor influencing microbial exposure and risk assessment.

Microbial risk assessment is dependent on several biological and environmental factors that affect both the exposure characteristics to the biological agents and the mechanisms of pathogenicity involved in the pathogen-host relationship. Many exposure assessment studies still focus on the location parameters of the probability distribution representing the concentration of the pathogens and/or toxin. However, the mean or median by themselves are insufficient to evaluate the adverse effects that are associated with a given level of exposure. Therefore, the effects on the risk of disease of a number of factors, including the shape parameters characterizing the distribution patterns of the pathogen in their environment, were investigated. The statistical models, which were developed to provide a better understanding of the factors influencing the risk, highlight the role of heterogeneity and its consequences on the commonly used risk assessment paradigm. Indeed, the heterogeneity characterizing the spatial and temporal distribution of the pathogen and/or the toxin contained in the water or food consumed is shown to be a major factor that may influence the magnitude of the risk dramatically. In general, the risk diminishes with higher levels of heterogeneity. This scheme is totally inverted in the presence of a threshold in the dose-response relationship, since heterogeneity will then have a tremendous impact, namely, by magnifying the risk when the mean concentration of pathogens is below the threshold. Moreover, the approach of this article may be useful for risk ranking analysis, regarding different exposure conditions, and may also lead to improved water and food quality guidelines.

New concepts in the evaluation of biodegradation/persistence of chemical substances using a microbial inoculum.

The European REACH Regulation (Registration, Evaluation, Authorization of CHemical substances) implies, among other things, the evaluation of the biodegradability of chemical substances produced by industry. A large set of test methods is available including detailed information on the appropriate conditions for testing. However, the inoculum used for these tests constitutes a "black box." If biodegradation is achievable from the growth of a small group of specific microbial species with the substance as the only carbon source, the result of the test depends largely on the cell density of this group at "time zero." If these species are relatively rare in an inoculum that is normally used, the likelihood of inoculating a test with sufficient specific cells becomes a matter of probability. Normally this probability increases with total cell density and with the diversity of species in the inoculum. Furthermore the history of the inoculum, e.g., a possible pre-exposure to the test substance or similar substances will have a significant influence on the probability. A high probability can be expected for substances that are widely used and regularly released into the environment, whereas a low probability can be expected for new xenobiotic substances that have not yet been released into the environment. Be that as it may, once the inoculum sample contains sufficient specific degraders, the performance of the biodegradation will follow a typical S shaped growth curve which depends on the specific growth rate under laboratory conditions, the so called F/M ratio (ratio between food and biomass) and the more or less toxic recalcitrant, but possible, metabolites. Normally regulators require the evaluation of the growth curve using a simple approach such as half-time. Unfortunately probability and biodegradation half-time are very often confused. As the half-time values reflect laboratory conditions which are quite different from environmental conditions (after a substance is released), these values should not be used to quantify and predict environmental behavior. The probability value could be of much greater benefit for predictions under realistic conditions. The main issue in the evaluation of probability is that the result is not based on a single inoculum from an environmental sample, but on a variety of samples. These samples can be representative of regional or local areas, climate regions, water types, and history, e.g., pristine or polluted. The above concept has provided us with a new approach, namely "Probabio." With this approach, persistence is not only regarded as a simple intrinsic property of a substance, but also as the capability of various environmental samples to degrade a substance under realistic exposure conditions and F/M ratio.

Occurrence, survival, and persistence of human adenoviruses and F-specific RNA phages in raw groundwater.

Detection of specific genetic markers can rapidly identify the presence of enteric viruses in groundwater. However, comparison of stability characteristics between genetic and infectivity markers is necessary to better interpret molecular data. Human adenovirus serotype 2 (HAdV2), in conjunction with MS2 phages or GA phages, was spiked into raw groundwater microcosms. Viral stability was periodically assessed by both infectivity and real-time PCR methods. The results of this yearlong study suggest that adenoviruses have the most stable persistence profile and an ability to survive for a long time in groundwater. According to a linear regression model, infectivity reductions of HAdV2 ranged from 0.0076 log(10)/day (4°C) to 0.0279 log(10)/day (20°C) and were significantly lower than those observed for phages. No adenoviral genome degradation was observed at 4°C, and the reduction was estimated at 0.0036 log(10)/day at 20°C. Occurrence study showed that DNA of human adenoviruses could be observed in groundwater from a confined aquifer (7 of the 60 samples were positive by real-time PCR), while no fecal indicators were detected. In agreement with the persistence of genetic markers, the presence of adenoviral DNA in groundwater may be misleading in term of health risk, especially in the absence of information on the infective status.

Occurrence and persistence of enteroviruses, noroviruses and F-specific RNA phages in natural wastewater biofilms.

Enteroviruses and noroviruses are pathogenic viruses excreted by infected individuals. Discharged in wastewaters, some of these viruses can be captured by biofilms. In the present study, we assessed the occurrence and persistence of these viruses in wastewaters and in corresponding biofilms. Natural wastewaters and biofilms were analyzed monthly from January to July using real-time RT-PCR. Enterovirus RNA was detected in wastewater in June while norovirus RNA was detected from January to March. In contrast, biofilm analysis revealed the presence of both enterovirus and norovirus genomes throughout the study period. For instance, enterovirus and norovirus genogroups (GG) I and II were detected in 50, 46 and 37% of the biofilm samples, respectively (n=24). In a laboratory experiment, persistence of norovirus GGI RNA (quantified using molecular techniques) and F-specific bacteriophages (quantified using both culture and molecular techniques) was assessed in wastewater and corresponding naturally-contaminated biofilms at both 4 and 20 degrees C. The concentrations of viral genomes (norovirus GGI and F-specific RNA phage) were very stable in biofilms. Indeed, no significant decrease was observed during the persistence experiment that lasted 49 days. Furthermore, regardless of our experimental conditions, viral genome and infectious F-specific bacteriophages persisted longer in biofilm than in wastewater. According to our results, wastewater biofilms may contribute to the persistence and dispersal of pathogenic viruses outside of epidemic periods.

Relationship between F-specific RNA phage genogroups, faecal pollution indicators and human adenoviruses in river water.

Recent studies have shown the increasing interest of F-specific RNA phage genotyping to identify major sources of faecal contamination in waters. This study, conducted in a river located in an urbanized watershed with recognized anthropogenic influences, was aimed at evaluating the relevance of direct phage genotyping by real-time RT-PCR. One hundred percent of positive results were obtained with a 5 mL aliquot of river water (n=31). Phage distribution was modified after cultivation, since the ratio of the two most abundant genogroups (II and I) reached 1.51 log(10) by direct RT-PCR-based method versus 0.30 log(10) after cultivation (n=8). For the first time, positive correlations between the concentrations of genogroup II, bacterial indicators and human adenoviruses were observed, which may indicate a human faecal pollution. No correlation between genogroups II and I has been revealed. The concentration of genogroup I was only correlated with water turbidity, suggesting an animal pollution coming from upstream after rainfall events. Among the microbiological parameters studied, only genogroup II/genogroup I ratio shows variations occurring in the major sources of faecal pollution.

Predicting uncertainty in the ecotoxicological assessment of solid waste leachates.

Environmental managers need suitable technological methods to use in optimization studies to improve management of hazardous waste. One of the challenges to achieving a reliable hazardous waste classification is the improvement of procedures used forthe ecotoxicological characterization of solid waste leachates. Indeed, this step requires data that meet levels of acceptable quality if scientifically based decisions are to be made. In this study, we illustrate how the variability associated with the successive steps of a procedure used to assess ecotoxicological hazard of solid waste (i.e., primary sampling, laboratory sampling, toxicity measurements) can contribute to the overall variability of the ecotoxicity results. To this end, a municipal solid waste incinerator bottom ash and a slag from a second smelting of lead were studied using a nested experimental design. The results showed that the waste sampling design is of major importance for limiting the final variability of toxicity test parameters. At the opposite, increasing the number of replicates at the toxicity test level has negligible impact on this variability. Our approach could be of great practical interest in ecotoxicological studies not only for ensuring a safe classification for these materials, but also for improving sampling protocols and facilitating less time-consuming and less expensive ecotoxicological evaluations.

Drugs and chronic alcohol abuse in drivers.

Blood specimens from 210 drivers (179 male and 31 female) apprehended in Luxembourg from autumn 2001 to spring 2002 and requested for the determination of their blood alcohol concentration (BAC) were tested for medicinal drugs, illicit drugs, and chronic alcohol abuse (by quantification of the carbohydrate-deficient transferrin: CDT). These additional analyses were performed anonymously and with permission of state prosecutor. The 22.8% had consumed medicinal drugs, with benzodiazepines and antidepressants (10.9 and 7.6%, respectively) as main psychoactive classes. Cannabis was the most detected illicit drug (9.5%) but only one in three had THC detectable in their blood. Association of two or more psychoactive substances (poly-drug use) was observed in 27.6% of drivers (90.6% of drug consumers). On the basis of CDT values, 29.5% of drivers investigated were assumed to be chronic alcohol abusers. Statistical analysis revealed that chronic alcohol abuse and medicinal psychoactive drugs were associated with significantly higher BAC. Medicinal psychoactive drugs were clearly associated with poly-drug use, and were furthermore detected at supra-therapeutic levels in 34.9%.

Relationship between blood alcohol concentration and carbohydrate-deficient transferrin among drivers.

Carbohydrate-deficient transferrin (CDT) was quantified in 408 blood specimens, randomly selected from 1260 drivers apprehended and submitted to blood alcohol concentration (BAC) determination. The first step of the study was to observe whether a BAC based pre-evaluation was relevant for deciding to test drivers for chronic alcohol abuse. For this purpose, the diagnosis of chronic alcohol abuse was verified by CDT quantification, with a voluntary high positive cut-off fixed at 3% for high specificity. The results display a significant increase in the part of chronic alcohol abusers with respect to increasing BAC: a few alcohol abusers were present in the BAC category below 0.5 g/L, and their frequency increased to 47 and 67% when BAC was between 3 and 3.5 g/L and above 3.5 g/L, respectively. Secondly, the usefulness of the biomarker CDT in the traffic safety context was investigated by observing whether drivers with abnormally increased CDT value had also higher BAC. The average BAC was 1.32 g/L in drivers with CDT below 1%, and increased to 2.28 g/L in drivers with CDT above 3%. Statistical analysis showed evidence of a monotone increasing link between BAC and CDT (P<0.0001). We confirmed here the relevance of BAC-based pre-evaluation before testing chronic alcohol abuse among drivers, and demonstrated that CDT is a biomarker suitable for traffic safety context, as drivers with increased CDT had significantly higher BAC.

Standard use conditions of terrestrial gastropods in active biomonitoring of soil contamination.

The rehabilitation of contaminated sites is becoming a rising preoccupation which requires the knowledge of their past before realization of a suitable remediation. Physicochemical analysis must be realized jointly with the use of bioindicator organisms, which, owing to their bioaccumulation capacities, will reveal the bioavailability of metals in soils. Among terrestrial invertebrates, gastropods like Helix aspersa aspersa possess an important organotropism for metals in their digestive gland and they can be used in active biomonitoring. During in situ monitoring, two parameters are tested: growth and accumulation of Fe, Mn, Zn, Cu, Cd, Ni, Pb, and Cr in viscera of snails. Environmental conditions, like humidity or autochthonous vegetation, are able to modify growth or bioaccumulation. In order to remove the variation of these factors, microcosms previously used must be improved: they were equipped with porous candles, which continually humidify soil. Concerning vegetation, an experimental plan was realized to determine the combination of food with the best compromise between growth and bioaccumulation: the combination clover-snail feed was chosen. Thus, in situ, the experimental environment will be repeated in all investigated sites and used to allow follow up of their contamination levels and intercomparison between different sites.