Exposure to human pathogenic viruses in recreational waters has been shown to cause disease outbreaks. In the context of Article 14 of the revised European Bathing Waters Directive 2006/7/EC (rBWD, ...CEU, 2006) a Europe-wide surveillance study was carried out to determine the frequency of occurrence of two human enteric viruses in recreational waters. Adenoviruses were selected based on their near-universal shedding and environmental survival, and noroviruses (NoV) selected as being the most prevalent gastroenteritis agent worldwide. Concentration of marine and freshwater samples was done by adsorption/elution followed by molecular detection by (RT)-PCR. Out of 1410 samples, 553 (39.2%) were positive for one or more of the target viruses. Adenoviruses, detected in 36.4% of samples, were more prevalent than noroviruses (9.4%), with 3.5% GI and 6.2% GII, some samples being positive for both GI and GII. Of 513 human adenovirus-positive samples, 63 (12.3%) were also norovirus-positive, whereas 69 (7.7%) norovirus-positive samples were adenovirus-negative. More freshwater samples than marine water samples were virus-positive. Out of a small selection of samples tested for adenovirus infectivity, approximately one-quarter were positive. Sixty percent of 132 nested-PCR adenovirus-positive samples analysed by quantitative PCR gave a mean value of over 3000 genome copies per L of water. The simultaneous detection of infectious adenovirus and of adenovirus and NoV by (RT)PCR suggests that the presence of infectious viruses in recreational waters may constitute a public health risk upon exposure. These studies support the case for considering adenoviruses as an indicator of bathing water quality.
Human adenoviruses (HAdV) may be implicated in some disease outbreaks associated with recreational water exposures, typically in swimming pools. Modern molecular methods can be used to detect HAdV in ...environmental water samples. During the EU FP6 Project VIROBATHE a database of over 290 HAdV analyses with corresponding faecal indicator organism (FIO) determinations was gathered and used to explore statistical associations between HAdV and FIO results. The FIOs measured were Escherichia coli, intestinal enterococci and somatic coliphage. Statistically significant trends of increasing proportions of HAdV-positive results in categories of increasing FIO concentration were found in freshwater but not seawater samples. The analysis of these trends in freshwater samples was refined, the trends remaining statistically significant when using categories of 0.5 log10 intervals of FIO concentration. Logistic regression models were then developed to predict the probability of a HAdV-positive outcome from FIO concentration. Potential applications of these models to predict the probability of HAdV-positive outcomes from routine FIO determinations used to describe recreational water quality exposures and to classify recreational water quality are discussed.
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► Over 290 samples were collected from diverse European freshwater and marine sites. ► Samples were analyzed for the presence of human adenovirus using PCR. ► Concentrations of traditional faecal indicator organisms (FIOs) were also measured. ► Adenovirus presence was relatively high at low FIO concentrations. ► Adenovirus presence increased with FIO concentration in freshwater samples.
A novel and simple procedure for concentrating adenoviruses from seawater samples is described. The technique entails the adsorption of viruses to pre-flocculated skimmed milk proteins, allowing the ...flocs to sediment by gravity, and dissolving the separated sediment in phosphate buffer. Concentrated virus may be detected by PCR techniques following nucleic acid extraction. The method requires no specialized equipment other than that usually available in routine public health laboratories, and due to its straightforwardness it allows the processing of a larger number of water samples simultaneously. The usefulness of the method was demonstrated in concentration of virus in multiple seawater samples during a survey of adenoviruses in coastal waters.
Potatoes, tomatoes, and aubergines are all species of the Solanum genus and contain a vast array of secondary metabolites including calystegine alkaloids, phenolic compounds, lectins, and ...glycoalkaloids. Glycoalkaloids have been the subject of many literature papers, occur widely in the human diet, and are known to induce toxicity. Therefore, from a food safety perspective further information is required regarding their analysis, toxicity, and bioavailability. This is especially important in crop cultivars derived from wild species to prevent glycoalkaloid-induced toxicity. A comprehensive review of the bioactivity of glycoalkaloids and their aglycones of the Solanum species, particularly focused on comparison of their bioactivities including their anticancer, anticholesterol, antimicrobial, anti-inflammatory, antinociceptive, and antipyretic effects, toxicity, and synergism of action of the principal Solanum glycoalkaloids, correlated to differences of their individual molecular structures is presented.
Fascioliasis caused by the trematodes Fasciola hepatica and F. gigantica, is a global neglected zoonotic disease estimated to cost the livestock industry over €2.5 billion annually. Farm management ...measures and sustainable use of anthelmintics can, in principle, effectively control trematode infection in livestock and reduce the rate of developing anthelmintic resistance. Previously, we designed an environmental DNA (eDNA) assay to identify a common trematode intermediate host, the freshwater snail Galba truncatula, in water sources to measure specific trematode infection risk areas on pasture-land. To improve this procedure, we now report a loop-mediated isothermal amplification (LAMP) assay to identify G. truncatula eDNA.
A LAMP assay was designed and optimised (e.g. temperature, time duration and primer concentration) to identify G. truncatula DNA. The ability of the LAMP assay to target G. truncatula DNA was identified, and LAMP assay limit of detection was investigated in comparison to conventional PCR. In the field, 48 water samples were collected from stream, ditch and water pool habitats in four locations at two Aberystwyth University farms over a seven week period to investigate the applicability of the LAMP assay for use on eDNA samples, in comparison to conventional PCR.
The LAMP assay delivered detectable results in 30 min at 63 °C. The assay discriminated between G. truncatula DNA and non-target DNA, presenting a level of DNA detection comparable to conventional PCR. No significant difference was found between the ability of the LAMP and PCR assay to identify G. truncatula eDNA in water samples. Kappa coefficient analysis revealed a moderate level of agreement between LAMP and PCR assays.
This study demonstrated that the LAMP assay can detect G. truncatula eDNA in a simple and rapid manner. The LAMP assay may become a valuable tool to determine optimum pasture management for trematode parasite control.
Integrated river basin management planning to mitigate the impacts of economic, demographic and climate change is an important issue for the future protection of water resources. Identifying sources ...of microbial contamination via the emerging science of Microbial Source Tracking (MST) plays a key role in risk assessment and the design of remediation strategies. Following an 18-month surveillance program within the EU-FP7-funded VIROCLIME project, specific MST tools were used to assess human markers such as adenoviruses (HAdV) and JC polyomaviruses (JCPyV) and porcine and bovine markers such as porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) via quantification with real-time PCR to analyze surface water collected from five sites within different climatic zones: the Negro River (Brazil), Glafkos River (Greece), Tisza River (Hungary), Llobregat River (Spain) and Umeälven River (Sweden). The utility of the viral MST tools and the prevalence and abundance of specific human and animal viruses in the five river catchments and adjacent seawater, which is impacted by riverine contributions from the upstream catchments, were examined. In areas where no sanitation systems have been implemented, sewage can directly enter surface waters, and river water exhibited high viral loads; HAdV and JCPyV could be detected at mean concentrations of 105 and 104 Genome Copies/Liter (GC/L), respectively. In general, river water samples upstream of urban discharges presented lower human viral loads than downstream sampling sites, and those differences appeared to increase with urban populations but decrease in response to high river flow, as the elevated river water volume dilutes microbial loads. During dry seasons, river water flow decreases dramatically, and secondary effluents can represent the bulk of the riverine discharge. We also observed that ice cover that formed over the river during the winter in the studied areas in North Europe could preserve viral stability due to the low temperatures and/or the lack of solar inactivation. Porcine and bovine markers were detected where intensive livestock and agricultural activities were present; mean concentration values of 103 GC/L indicated that farms were sometimes unexpected and important sources of fecal contamination in water. During spring and summer, when livestock is outdoors and river flows are low, animal pollution increases due to diffuse contamination and direct voiding of feces onto the catchment surface. The field studies described here demonstrate the dynamics of fecal contamination in all catchments studied, and the data obtained is currently being used to develop dissemination models of fecal contamination in water with respect to future climate change scenarios. The results concerning human and animal targets presented in this study demonstrate the specificity and applicability of the viral quantitative parameters developed to widely divergent geographical areas and their high interest as new indicators of human and animal fecal contamination in water and as MST tools.
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•Bovine, porcine and human fecal pollution dynamics were characterized in five rivers.•The viral MST tools used are applicable in all geographical areas.•The skimmed milk concentration method is robust and effective for viral trackability.
Environmental DNA (eDNA) is a powerful tool for identifying the spatial and temporal presence and density of species in a range of aquatic habitats. The analysis of eDNA has a wide range of ...application, one of which may be to inform of Fasciola hepatica infection risk on pastures based on the detection of its eDNA as well as that of its intermediate snail host, Galba truncatula eDNA. Here, droplet digital PCR (ddPCR) and quantitative real‐time PCR (qPCR) assays were developed to detect the eDNA of F. hepatica, and its intermediate snail host, G. truncatula in water samples collected from pastures grazed by cattle and/or sheep. Environmental factors associated with species presence, as detected via an eDNA survey, were identified using zero‐inflated linear mixed models. Sixty‐four habitats were sampled across six farms in Ceredigion, Wales, UK, with ddPCR and qPCR identifying 42 and 33 habitats to be positive for G. truncatula eDNA, respectively. G. truncatula eDNA was significantly less likely to be detected in habitats fully shaded by trees, those that contained black or dark brown soils and habitats that contained deep water pools (p < 0.05). Significantly higher G. truncatula eDNA concentrations were observed in habitats that tend to dry up during Summer (i.e., temporary habitats) (p < 0.05). ddPCR also identified five habitats to be positive for F. hepatica eDNA; however, questions remain regarding the utility of F. hepatica eDNA detection due to a lack of specificity toward infective F. hepatica larval stages. The results of this study inform of factors which influences G. truncatula distribution and ecology on pastures and also provided practical information for farmers to aid F. hepatica control in their flocks and herds.
ddPCR‐ and qPCR‐based eDNA assays were developed and applied to survey Galba truncatula on pastureland with the aim of identifying Fasciola hepatica infection risk areas. The likelihood of G. truncatula eDNA presence was lower in habitats that were fully shaded by trees, had black or dark brown soils, and contained deep water pools. The tools developed and results can support future fasciolosis control strategies on cattle and sheep farms.
We report the synthesis of organometal halide perovskites by milling CH3NH3I and PbI2 directly with an Al2O3 scaffold to create hybrid Al2O3-CH3NH3PbI3 perovskites, without the use of organic capping ...ligands that otherwise limit the growth of the material in the three dimensions. Not only does this improve the ambient stability of perovskites in air (100 min versus 5 min for dimethylformamide (DMF)-processed material), the method also uses much fewer toxic solvents (terpineol versus dimethylformamide). This has been achieved by solid-state reaction of the perovskite precursors to produce larger perovskite nanoparticles. The resulting hybrid perovskite–alumina particles effectively improve the hydrophobicity of the perovskite phase whilst the increased thermal mass of the Al2O3 increases the thermal stability of the organic cation. Raman data show the incorporation of Al2O3 shifts the perovskite spectrum, suggesting the formation of a hybrid 3D mesoporous stack. Laser-induced current mapping (LBIC) and superoxide generation measurements, coupled to thermogravimetric analysis, show that these hybrid perovskites demonstrate slightly improved oxygen and thermal stability, whilst ultra-fast X-ray diffraction studies using synchrotron radiation show substantial (20×) increase in humidity stability. Overall, these data show considerably improved ambient stability of the hybrid perovskites compared to the solution-processed material.
The therapeutic application of siRNA shows promise as an alternative approach to small-molecule inhibitors for the treatment of human disease. However, the major obstacle to its use has been the ...difficulty in delivering these large anionic molecules in vivo. In this study, we have investigated whether siRNA-mediated knockdown of p38 MAP kinase mRNA in mouse lung is influenced by conjugation to the nonviral delivery vector cholesterol and the cell penetrating peptides (CPP) TAT(48−60) and penetratin. Initial studies in the mouse fibroblast L929 cell line showed that siRNA conjugated to cholesterol, TAT(48−60), and penetratin, but not siRNA alone, achieved a limited reduction of p38 MAP kinase mRNA expression. Intratracheal administration of siRNA resulted in localization within macrophages and scattered epithelial cells and produced a 30−45% knockdown of p38 MAP kinase mRNA at 6 h. As with increasing doses of siRNA, conjugation to cholesterol improved upon the duration but not the magnitude of mRNA knockdown, while penetratin and TAT(48−60) had no effect. Importantly, administration of the penetratin or TAT(48−60) peptides alone caused significant reduction in p38 MAP kinase mRNA expression, while the penetratin−siRNA conjugate activated the innate immune response. Overall, these studies suggest that conjugation to cholesterol may extend but not increase siRNA-mediated p38 MAP kinase mRNA knockdown in the lung. Furthermore, the use of CPP may be limited due to as yet uncharacterized effects upon gene expression and a potential for immune activation.
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