The study region in Sagres, SW Portugal, is subject to natural eutrophication of coastal waters by wind-driven upwelling, which stimulates high primary productivity facilitating the recent economic expansion of bivalve aquaculture in the region. However, this economic activity is threatened by harmful algal blooms (HAB) caused by the diatoms Pseudo-nitzschia spp., Dinophysis spp. and other HAB dinoflagellates, all of which can produce toxins, that can induce Amnesic Shellfish Poisoning (ASP), Diarrhetic Shellfish Poisoning (DSP) and Paralytic Shellfish Poisoning (PSP). This study couples traditional microscopy with 18S/28S rRNA microarray to improve the detection of HAB species and investigates the relation between HAB and the specific oceanographic conditions in the region. Good agreement was obtained between microscopy and microarray data for diatoms of genus Pseudo-nitzschia and dinoflagellates Dinophysis spp., Gymnodinium catenatum and raphidophyte Heterosigma akashiwo, with less effective results for Prorocentrum. Microarray provided detection of flagellates Prymnesium spp., Pseudochattonella spp., Chloromorum toxicum and the important HAB dinoflagellates of the genera Alexandrium and Azadinium, with the latter being one of the first records from the study region. Seasonality and upwelling induced by northerly winds were found to be the driving forces of HAB development, with Pseudo-nitzschia spp. causing the risk of ASP during spring and summer upwelling season, and dinoflagellates causing the risk of DSP and PSP during upwelling relaxation, mainly in summer and autumn. The findings were in agreement with the results from toxicity monitoring of shellfish by the Portuguese Institute for Sea and Atmosphere and confirm the suitability of the RNA microarray method for HABs detection and aquaculture management applications.
Monitoring drinking water quality is an important public health issue. Two objectives from the 4 years, six nations, EU Project μAqua were to develop hierarchically specific probes to detect and quantify pathogens in drinking water using a PCR-free microarray platform and to design a standardised water sampling program from different sources in Europe to obtain sufficient material for downstream analysis. Our phylochip contains barcodes (probes) that specifically identify freshwater pathogens that are human health risks in a taxonomic hierarchical fashion such that if species is present, the entire taxonomic hierarchy (genus, family, order, phylum, kingdom) leading to it must also be present, which avoids false positives. Molecular tools are more rapid, accurate and reliable than traditional methods, which means faster mitigation strategies with less harm to humans and the community. We present microarray results for the presence of freshwater pathogens from a Turkish lake used drinking water and inferred cyanobacterial cell equivalents from samples concentrated from 40 into 1 L in 45 min using hollow fibre filters. In two companion studies from the same samples, cyanobacterial toxins were analysed using chemical methods and those dates with highest toxin values also had highest cell equivalents as inferred from this microarray study.
Current knowledge about aquatic pathogens are scarce because bacteria, protozoans, algae and their toxins occur at low concentrations, making them difficult to measure directly or to filter sufficient volumes to facilitate detection. We developed and validated tools to detect pathogens in freshwater systems. To evaluate impacts on water quality, a phylogenetic microarray was developed in the EU project μAQUA to detect simultaneously numerous pathogens and was applied in MicroCoKit, to samples taken from four locations from two seasons for two years along the length of the Tiber River, Italy. The sites ranged from a pristine site near its source to ones contaminated by agricultural, industrial and anthropogenic waste moving downstream to near its mouth. Fifty litres were collected and concentrated using a hollow-fibre ultrafiltration, a rapid method with minimal cell loss to provide a concentrate for downstream analysis. The 60 Da cut-off ensures many organics, such as toxins, will be concentrated for analysis. Aliquots from the concentrate were preserved in TRI-Reagent and total RNA extracted, labelled and hybridised to the phylochip to detect pathogenic bacteria, protozoa and toxic cyanobacteria. The microarray results gave positive signals for all pathogens. Calibration curves enabled us to infer cell concentrations. Cross validation was performed using FISH probes for selected toxic cyanobacteria and hybridised to aliquots taken from the raw water prior to concentration by the hollow fibre filters.
Monitoring the quality of drinking water is an important issue for public health. Two of the main objectives of the European Project μAQUA were (i) the development of specific probes to detect and quantify pathogens in drinking water and (ii) the design of standardized sampling programs of water from different sources in Europe in order to obtain sufficient material for downstream analysis. Our phylochip contains barcodes that specifically identify freshwater pathogens for enabling the detection of organisms that can be risks for human health. Monitoring for organisms with molecular tools is rapid, more accurate and more reliable than traditional methods. Rapid detection means that mitigation strategies come into play faster with less harm to the community and to humans. Samples were collected from several waters in France, Germany, Ireland, Italy and Turkey over 2 years. We present microarray results for the presence of freshwater pathogens from brackish and freshwater sites in Northern Germany, and cyanobacterial cell numbers inferred from these sites. In a companion study from the same samples, cyanobacterial toxins were analyzed using two methods and those sites with highest toxin values also had highest cell numbers as inferred from this microarray study.
The transmission of water-borne pathogens typically occurs by a faecal-oral route, through inhalation of aerosols, or by direct or indirect contact with contaminated water. Previous molecular-based studies have identified viral particles of zoonotic and human nature in surface waters. Contaminated water can lead to human health issues, and the development of rapid methods for the detection of pathogenic microorganisms is a valuable tool for the prevention of their spread. The aims of this work were to determine the presence and identity of representative human pathogenic enteric viruses in water samples from six European countries by quantitative polymerase chain reaction (q-PCR) and to develop two quantitative PCR methods for Adenovirus 41 and Mammalian Orthoreoviruses. A 2-year survey showed that Norovirus, Mammalian Orthoreovirus and Adenoviruses were the most frequently identified enteric viruses in the sampled surface waters. Although it was not possible to establish viability and infectivity of the viruses considered, the detectable presence of pathogenic viruses may represent a potential risk for human health. The methodology developed may aid in rapid detection of these pathogens for monitoring quality of surface waters.
Monitoring the quality of freshwater is an important issue for public health. In the context of the European Project µAqua (FP7-KBBE-2010-4, 265409) a total of 150 samples were collected from several waters in France, Germany, Ireland, Italy and Turkey over two years. These samples were analyzed using two multi-toxin detection methods previously developed: (i) a microsphere-based method coupled to flow-cytometry and (ii) an Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) method. The presence of microcystins (MCs), nodularin (NOD), domoic acid (DA), cylindrospermopsin (CYN) and several analogues of anatoxin-a (ATX-a) was monitored. No traces of CYN or DA were found in any of the environmental samples. MC-LR and MC-RR were detected in two samples from Turkey and Germany. In the case of ATX-a derivatives, 75% of samples contained mainly H2-ATX-a and small amounts of H2-homo-ATX-a, whereas ATX-a and homo-ATX-a were found only in one sample. From these results the presence and wide distribution of dihydro derivatives of ATX-a toxins in European freshwaters is confirmed. This article is protected by copyright. All rights reserved
Distinct distribution patterns of members of the major bacterial clades SAR11, SAR86 and Actinobacteria were observed across a transect from the Marquesas islands through the ultra-oligotrophic South Pacific Gyre into the Chilean upwelling using 16S rRNA gene sequencing and RNA-DNA fingerprinting. Three different Actinobacteria sequence clusters belonging to "Candidatus Actinomarinidae" were localised in the western half of the transect, one was limited to the gyre deep chlorophyll maximum and sequences affiliated to the OCS155 clade were unique to the upwelling. The structure of the surface bacterial community was highly correlated with water mass and remained similar across the whole central gyre (1300 nautical miles). The surface hyperoligotrophic gyre was dominated (>70% of all sequences) by highly diverse SAR11 and SAR86 operational taxonomic units and these communities were significantly different from those in the deep chlorophyll maximum. Analysis of 16S rRNA fingerprints generated from RNA allowed insights into the potential activity of assigned bacterial groups. SAR11 and Prochlorococcus showed the highest potential activity in all water masses except for the upwelling, accounting together for 65% of the total bacterial 16S rRNA in the gyre surface waters in equal proportions whereas the contribution of SAR11 decreased significantly at the deep chlorophyll maximum.
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