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.

     

Assessment of Viral Contamination of Five Brazilian Artisanal Cheese Produced from Raw Milk: a Randomized Survey

Food and Environmental Virology - Enteric viruses have been described as important contaminants in fresh and ready-to-eat foods such as sandwiches, deli meat and dairy products. This is a...     

Polymer composite material fabricated from recycled polyethylene terephthalate (PET) with polyurethane binder for potential noise control applications

Journal of Material Cycles and Waste Management - This paper shows a composite material fabricated from plastic waste of polyethylene terephthalate (PET) and polyurethane as binder, in a ratio of...     

The impact of COVID-19 pandemic in medical waste amounts: a case study from a high-populated city of Turkey

Journal of Material Cycles and Waste Management - In this study, the amount of medical waste generated in a high-populated city in Turkey between January 2018 and August 2021 was evaluated, and the...     

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,...     

Fine-scale chemical fingerprinting of an open coast crustacean for the assessment of population connectivity

Chemical fingerprinting techniques recently have been used to track larval dispersal of estuarine species that bear calcified structures, but the applicability of this important approach may be limited on the open coast where chemical signatures may be less distinctive and for the many species that do not retain calcified structures throughout development. Externally brooded embryos of the porcelain crab, Petrolisthes cinctipes, and inductively coupled plasma mass spectrometry were used to determine whether fine-scale variation in trace-elemental composition occurred along an open coast. Embryos were collected from 16 sites from 37.8° to 39.5° north latitude along the Pacific Coast of California, USA during late January and early February 2003. Discriminant function analysis revealed that collection sites, many separated by only a few kilometers along an open coast, could be differentiated with an overall accuracy of 73%, and combining the sites into three regions increased the accuracy to 88%. Thus, distinctive elemental signatures can be detected in open coast species even at a fine scale raising the possibility that larval tags can be developed for many more species than previously thought possible.     

Size–frequency dynamics of NE Pacific abyssal ophiuroids (Echinodermata: Ophiuroidea)

The 17-year time-series study at Station M in the NE Pacific has provided one of the longest datasets on deep-sea ophiuroids to date. Station M is an abyssal site characterized by low topographical relief and seasonal and interannual variation in surface-derived food inputs. From 1989 to 2005, over 31,000 ophiuroid specimens were collected. Size–frequency distributions of the four dominant species, Ophiura bathybia, Amphilepis patens, Amphiura carchara and Ophiacantha cosmica, were examined for recruitment and the role of surface-derived food supplies on body size distributions. Juveniles were collected in sediment traps and used to investigate settlement patterns and seasonality. Trawl samples showed no indication of seasonal changes in recruitment to larger size classes; however, there was evidence of seasonal settling of juveniles. Interannual differences in median disk diameters and size distributions of trawl-collected adults are greater than those at the seasonal scale. Three of the four species, O. bathybia, A. patens and O. cosmica, had co-varying monthly median disk diameters, suggesting they may have a similar factor(s) controlling their growth and abundance. Interannual differences in monthly size distributions were generally greater than those between seasons. Cross-correlations between the particulate organic carbon (POC) flux (food supply) and size distribution indices for O. bathybia, A. patens and O. cosmica all were significant indicating that increases in food supply were followed by increases in the proportion of smaller size classes after approximately 17–22 months. These findings suggest that food inputs are indeed an important factor influencing deep-sea ophiuroid populations on interannual time scales, more generally supporting the long-hypothesized connection between food availability and population size structure in the deep sea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nutrient transfer in a marine mutualism: patterns of ammonia excretion by anemonefish and uptake by giant sea anemones

Many symbioses involve multiple partners in complex, multi-level associations, yet little is known concerning patterns of nutrient transfer in multi-level marine mutualisms. We used the anemonefish symbiosis as a model system to create a balance sheet for nitrogen production and transfer within a three-way symbiotic system. We quantified diel patterns in excretion of ammonia by anemonefish and subsequent absorption by host sea anemones and zooxanthellae under laboratory conditions. Rates of ammonia excretion by the anemonefish Amphiprion bicinctus varied from a high of 1.84 μmole g⁻¹ h⁻¹ at 2 h after feeding, to a basal rate of 0.50 μmole g⁻¹ h⁻¹ at 24–36 h since the last meal. Conversely, host sea anemones Entacmaea quadricolor absorbed ammonia at a rate of 0.10 μmole g⁻¹ h⁻¹ during the daytime in ammonia-enriched seawater, but during the night reduced their absorption rate to near zero, indicating that ammonia uptake was driven by zooxanthella photosynthesis. When incubated together, net ammonia excretion was virturally zero, indicating that host anemones absorbed most of the ammonia produced by resident fish. Adult anemonefish weighed about 11 g under laboratory conditions, but on the coral reef may reach up to 64 g, resulting in a maximal potential ammonia load of >200 μmole h⁻¹ produced by two adult fish during daylight hours. In contrast, host sea anemones weighed about 47 g in the laboratory, but under field conditions, large individuals may reach 680 g, so their maximal ammonia clearance rates may reach about 70 μmole h⁻¹ during the daytime. As such, the ammonia load produced by adult anemonefish far exceeds the clearance rate of host anemones and zooxanthellae. Ammonia transfer likely occurs mainly during the daytime, when anemonefish consume zooplankton and excrete rapidly, and in turn the zooxanthellae are photosynthetically active and drive rapid ammonia uptake. We conclude that zooplanktivorous fishes that form mutualisms with coral reef cnidarians may serve as an important link between open water and benthic ecosystems, through the transfer of large quantities of nutrients to zooxanthellate hosts, thus enhancing coral reef productivity.     

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.     

Composition of plastics from waste electrical and electronic equipment (WEEE) by direct sampling

This paper describes a direct analysis study carried out in a recycling unit for waste electrical and electronic equipment (WEEE) in Portugal to characterize the plastic constituents of WEEE. Approximately 3400 items, including cooling appliances, small WEEE, printers, copying equipment, central processing units, cathode ray tube (CRT) monitors and CRT televisions were characterized, with the analysis finding around 6000 kg of plastics with several polymer types. The most common polymers are polystyrene, acrylonitrile-butadiene-styrene, polycarbonate blends, high-impact polystyrene and polypropylene. Additives to darken color are common contaminants in these plastics when used in CRT televisions and small WEEE. These additives can make plastic identification difficult, along with missing polymer identification and flame retardant identification marks. These drawbacks contribute to the inefficiency of manual dismantling of WEEE, which is the typical recycling process in Portugal. The information found here can be used to set a baseline for the plastics recycling industry and provide information for ecodesign in electrical and electronic equipment production.