Optimisation of a PMAxx™-RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels

Human noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate noroviruses in their digestive tissues, are a typical vector for human infection. RT-qPCR, the established method for human norovirus detection in food, does not allow discrimination between infectious and non-infectious viruses and can overestimate potentially infectious viral loads. To develop a more accurate method of infectious norovirus load estimation, we combined intercalating agent propidium monoazide (PMAxx™)-pre-treatment with RT-qPCR assay using in vitro-cultivable murine norovirus. Three primer sets targeting different genome regions and diverse amplicon sizes were used to compare one-step amplification of a short genome fragment to three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp and 2.3 kbp amplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated murine noroviruses in PBS suspension, PMAxx™ RT-qPCRs were implemented to detect murine noroviruses subsequent to their extraction from mussel digestive tissues; virus extraction via anionic polymer-coated magnetic beads was compared with the proteinase K-dependent ISO norm. The long-range RT-qPCR process detecting fragments of more than 2.3 kbp allowed accurate estimation of the infectivity of UV-damaged murine noroviruses. While proteinase K extraction limited later estimation of PMAxx™ pre-treatment effects and was found to be unsuited to the assay, magnetic bead-captured murine noroviruses retained their infectivity. Genome copies of heat-inactivated murine noroviruses differed by 2.3 log₁₀ between RT-qPCR and PMAxx™-RT-qPCR analysis in bivalve molluscs, the PMAxx™ pre-treatment allowing a closer approximation of infectious titres. The combination of bead-based virus extraction and PMAxx™ RT-qPCR thus provides a more accurate model for the estimation of noroviral bivalve mollusc contamination than the conjunction of proteinase K extraction and RT-qPCR and has the potential (once validated utilising infectious human norovirus) to provide an added measure of security to food safety authorities in the hazard assessment of potential bivalve mollusc contamination.

     

The combination of chemical,structural, and functional indicators to evaluate the anthropogenic impacts on agricultural stream ecosystems

Environmental Science and Pollution Research - Freshwater contamination by pesticides in agricultural landscapes is of increasing concern worldwide, with strong pesticide impacts on biodiversity,...     

Are sea snakes pertinent bio-indicators for coral reefs? a comparison between species and sites

Classical sampling methods often miss important components of coral reef biodiversity, notably organisms that remain sheltered within the coral matrix. Recent studies using sea kraits (sea snakes) as bio-indicators suggest that the guild of predators represented by anguilliform fish (Congridae, Muraenidae, Ophichthidae, henceforth “eels” for simplicity) were far more abundant and diverse than previously suspected. In the current study, eel diversity (similarity and species richness indices) estimated via sea snake sampling (SSS) was compared among six areas of one of the main oceanic biodiversity hotspot of the Pacific Ocean (southwest lagoon of New Caledonia). Based on the eel diversity in the snakes’ diet, the results obtained in six areas, in two snake species, and using different estimates (ANOSIM, Shannon index…) were consistent, suggesting that SSS provided robust information. Analyses also suggested subtle, albeit significant, differences in the eel assemblages among islets. Such spatial differences are discussed in light of local management practices. As SSS is easy to use, cost-effective, and provides the best picture of eel assemblages to date, it can be employed to monitor the eel assemblages in addition to the snakes themselves in many areas of the Indo-Pacific Ocean, thereby providing an index of the top predator biodiversity of many coral reefs.     

Aerobic Biodegradation of Polymers in Solid-State Conditions: A Review of Environmental and Physicochemical Parameter Settings in Laboratory Simulations

During the last few years, biodegradable polymers have been developed to replace petrochemical polymers. Until now, research devoted to these polymers essentially focused on their biodegradability. There is now a need to bear out their nontoxicity. To verify this, the biodegradation must be carried out in accelerated laboratory tests which allow the metabolites and residues to be recovered. To reproduce the natural conditions (compost, field) as closely as possible, degradation experiments must be run on solid-state substrates. We review studies of aerobic degradation in solid-state substrates. This article focuses in particular on the environmental, physical, and chemical parameters (such as substrate nature, moisture, temperature, C/N ratio, and pH) that influence biodegradation kinetics. This study also aims at finding the solid substrate most adapted to residues and metabolite recovery. The most significant parameters would appear to be the substrate type, moisture content, and temperature. Inert substrates such as vermiculite are well suited to residue extraction. This review also opens the field to new research aimed at optimizing conditions for aerobic solid-state biodegradation and at recovering the metabolites and residues of this degradation process.     

Prenatal exclusion of X-linked hydrocephalus-stenosis of the aqueduct of sylvius sequence using closely linked DNA markers

X-linked hydrocephalus-stenosis of the aqueduct of Sylvius sequence (H-SAS, MIM number 307 000) is a rare genetic disorder characterized by hydrocephalus, macrocephaly, adducted thumbs, spasticity, mental retardation, and cerebral malformations. This regularly lethal condition is usually diagnosed at birth or prenatally by ultrasound, but hydrocephalus may be moderate or even undetectable on fetal ultrasound examination. Moreover, since heterozygous women are asymptomatic, carrier detection is at present impossible before the birth of an affected son. Therefore, mapping the H-SAS locus to distal Xq (Xq28) was of primary importance for genetic counselling and prenatal diagnosis. Here, we report prenatal exclusion of H-SAS with a probability of 97.6 per cent in two male fetuses with a 50 per cent a priori risk of being affected using closely linked Xq28 DNA markers.     

A new C12 alcohol identified as a sex pheromone and a trail-following pheromone in termites: the diene (<Emphasis Type="Italic">Z</Emphasis>,<Emphasis Type="Italic">Z</Emphasis>)-dodeca-3,6-dien-1-ol

The diunsaturated C12 alcohol (Z,Z)-dodeca-3,6-dien-1-ol (dodecadienol) has been characterized by GC-MS and FTIR as a novel releaser pheromone in termites. This alcohol identified in Ancistrotermes pakistanicus (Termitidae, Macrotermitinae) possesses a double pheromonal function which again illustrates the chemical parsimony of termites compared with other social insects. In workers, dodecadienol elicits trail-following at a very low concentration (activity threshold at 0.1 pg/cm of trail); in male alates it induces trail-following at a low concentration (1–10 pg/cm) and sexual attraction at a higher concentration (about 1 ng). Traces of the monounsaturated C12 alcohol (Z)-dodec-3-en-1-ol (dodecenol), known as a trail pheromone of several Macrotermitinae, were also found in the sternal gland extracts of A. pakistanicus, although only dodecadienol was present at the surface of the sternal gland. Workers of A. pakistanicus are not sensitive to dodecenol, but they are as sensitive to dodecatrienol as to dodecadienol. However, in the study area (Vietnam), A. pakistanicus is living in sympatry only with those Macrotermitinae using dodecenol as a trail pheromone, the foraging populations therefore being well isolated through their respective trail pheromones. The presence of three types of unsaturated C12 alcohols as releaser pheromones in the only Macrotermitinae subfamily is discussed, and a possible biosynthetic pathway from linoleic acid is proposed for dodecadienol.     

Prospective maternal serum human chorionic gonadotropin screening for the risk of fetal chromosome anomalies and of subsequent fetal and neonatal deaths

A prospective study of maternal serum human chorionic gonadotrophin (hCG) measurement for the selection of pregnancies with an increased risk of fetal trisomy 21 was undertaken in 24 000 pregnancies from 1 January 1989 to 31 December 1990. Maternal serum was sampled at 15-18 weeks of gestation. hCG was measured in one laboratory, with one technique. This ‘hCG high level’ technique was developed for this screening. Amniocentesis was offered to each woman with a maternal serum hCG level above the cut-off. The follow-up of the pregnancies is known in 92 per cent of cases. The combination of hCG values and maternal age gave a detection efficiency of 63 per cent for trisomy 21 with rates of amniocentesis of 30 per cent for patients aged 37 years, 20 per cent for patients aged 35 or 36 years, and 5 per cent for patients under 35 years of age. Based on this prospective study, an individual risk was calculated combining the serum hCG value and maternal age. Seventy-four per cent of trisomy 13, trisomy 18, triploidy, and 5p- deletion were detected either in the same selected group of women or in combination with ultrasonography performed when hCG values were very low. The follow-up study showed that women who had high or low hCG values represented a group at high risk for fetal or perinatal death.     

Application of rough sets analysis to identify polluted aquatic sites based on a battery of biomarkers: a comparison with classical methods

The evaluation of toxicological effects at the cellular and molecular levels in organisms are often used to determine sites subjected to contamination problems that pose a threat to the long-term survival of organisms. However, the integration of multiple measurements on the health status of organisms into a model for site discrimination is challenging. This study compares two discrimination methods which are based on rule inference: rough sets (RS) analysis and classification trees (CT) with classical multivariate discriminant analysis (DA). Site classification was attempted with six biomarkers of effects: metallothionein levels, lipid peroxidation, DNA damage, levels of lipophosphoproteins (i.e., vitellins), phagocytosis activity and haemocyte cell viability on clam (Mya arenaria) populations from the Saguenay River fjord (Quebec, Canada). Rule based methods have the advantage of complete independence from data distribution constraints in contrast to the classification methods from multivariate analysis that are more commonly used in ecotoxicology. Results show that RS and CT gave better classifications than DA because they do not require strong distributional assumptions. Moreover, RS provided classification rules that could identify the most important biomarker(s) for site discrimination. RS and CT were shown to be simple and efficient methods for classifying multivariable ecotoxicological data. This methodology would be especially useful when freedom from distributional assumptions is required.     

Phytoremediation of petroleum hydrocarbons in tropical coastal soils II. microbial response to plant roots and contaminant

GOAL, SCOPE AND BACKGROUND: The goal of this study was to understand the interaction between plants and microorganisms during petroleum-hydrocarbon bioremediation in Pacific Islands coastal soils. Total bacteria and hydrocarbon-degrading microorganisms population dyanamics were examined in the rhizospheres of tropical trees and shrubs, which were evaluated for their phytoremediation potential in a greenhouse experiment. The respective and combined effects of plant roots and diesel contaminant on the microbial populations were determined in relation to diesel fuel depletion. An increase in the grading populations size of the hydrocarbon-degrading populations of microbes, elicited by rhizodeposition, is generally regarded as conducive to an enhanced degradation of petroleum hydrocarbon pollutants in vegetated soil. METHODS: The soil was a coastal sandy loam (pH 7.8) which was artificially contaminated with 10 g of No. 2 diesel fuel/kg soil or left uncontaminated. The pots were irrigated with fertilizer and 1% NaCl. The enumerations were carried out in the contaminated and uncontaminated rhizospheres of three trees, kiawe (Prosopis pallida), milo (Thespesia populnea), and kou (Cordia subcordata) and three shrubs, beach naupaka (Scaevola sericea), false sandalwood (Myoporum sandwicense), and oleander (Nerium oleander). Unplanted control soils were included in the experiment. Total bacteria and phenanthrene-degrading bacteria were enumerated on plates. Diesel- and pristane-degrading microorganisms were enumerated by the most-probable-number technique in tissue-culture plates. RESULTS AND DISCUSSION: All four types of microorganisms responded to the rhizosphere of the 6 plants in uncontaminated soil and to the diesel contaminant in unplanted soil. In contaminated rhizospheres, no effect of the plant on the hydrocarbon-degrader numbers was visible. Total bacteria responded more to the plant roots than to the contaminant. The phenanthrene-degrading bacteria and pristane-degrading microorganisms were more influenced by the contaminant than by the plants. The diesel-degrading microorganisms were equally stimulated by the plants and the contaminant. The numbers of hydrocarbon degraders were similar in the contaminated rhizospheres of the three effective plants (kiawe, kou, and milo) and in those of the three ineffective shrubs. CONCLUSION: The results suggest the quality of the rhizodeposition is plant-dependent and governs the type of diesel-degrader populations that will be enhanced by a given plant. RECOMMENDATIONS AND OUTLOOK: In the proposed phytoremediation-benefit model plant roots maintain high levels of hydrocaron degraders in uncontaminated soil. When the root enters a contaminated zone of soil, those hydrocarbon degraders that prefer the contaminant would switch to the contaminant as a carbon source, effectively removing the hydrocarbons. If the root exudates and the contaminant are equally attractive to the hydrocarbon degraders, the contaminant degradaton would be less effective.     

Conditions for effective removal of pyrene from an artificially contaminated soil using Pseudomonas aeruginosa 57SJ rhamnolipids

The efficacy of a new rhamnolipid biosurfactants mixture to enhance the removal of pyrene from a soil artificially contaminated was investigated. The molar solubilization ratio (MSR) and the partition coefficient between the micelles and water (log K(m)) were found to be 7.5 x 10(-3) and 5.7, respectively. From soil column studies, the pyrene removal increased linearly with the concentration of the injected biosurfactants solution above the effective critical micellar concentration (0.4 g L(-1)). Flushing with a 5.0 g L(-1) biosurfactants solution increased the pyrene concentration in the effluent by 178 times. At high biosurfactants' concentrations (2.5 and 5.0 g L(-1)), the cumulative pyrene recovery reached 70%. This pyrene remobilization takes place independently of the soil organic carbon solubilization. This study provides a combination of batch and column experiments in order to find the conditions for effective soil remediation using a new rhamnolipids mixture.