Immunobiology of Eledone cirrhosa (Lamarck).

Reliable quantities of blood can be sampled repeatedly from the lesser octopus Eledone cirrhosa (Lam. ) and the haemocytes cultured for up to 72 h. Sampling causes an increase in the number of haemocytes/ml, in the percentage of haemocytes containing cytoplasmic granules and a change in the staining pattern of the haemocytes. Sampling also causes a decrease in the amount of copper (mg/ml) in the haemolymph and an initial decrease in the amount of protein (mg/ml), which returns to the original values over extended sampling periods. The haemocytes from E. cirrhosa will phagocytose bacteria (Vibrio anguillarum) in vitro in the absence of haemolymph (dependent on the temperature and duration of incubation) but enhanced phagocytosis will occur in the presence of haemolymph (10- 100% concentration). Opsonization is also dependent on the temperature and on the duration of exposure of the bacterium to the haemolymph. Haemocytes migrate towards low concentrations of blood preparations, to lipopolysaccharide (LPS) and to preparations which had contained live bacteria. Haemocytes also have a bacteriostatic effect on the growth of live bacteria with the effect being dependent on the temperature, duration of the assay and the bacterial species used. Haemocytes also produce intracellular reactive oxygen species, detected by nitroblue tetrazolium, after incubation with dead bacteria in particular, but also live bacteria and LPS. E. cirrhosa haemolymph agglutinates the bacteria V. anguillarum, Y. parahaemolyticus and Aeromonas salmonicida and exerts a bacteriostatic effect on these bacteria which is dependent on the temperature, the amount of haemolymph present and the bacterial species used. The haemolymph, haemocytes and certain tissues from E. cirrhosa exhibit lysozyme and antiprotease activity. The injection of live V. anguillarum causes an increase in lysozyme activity in the branchial heart (after 48 h) and a decrease in the haemocytes (after 24 h). Antiprotease activity increased in the haemocytes (4 h) after bacterial injection but decreased in the haemolymph. Live bacteria caused an increase in the number of circulating haemocytes. The bacteria were cleared from the circulation of E. cirrhosa in about 4h by both the haemocytes and tissues (branchial heart, branchial heart appendage and white body) where they were degraded. The large vacuole in branchial heart cells changed in appearance 4h after bacterial injection and the haemocyte nucleus became pleomorphic. Colloidal graphite was aggregrated in blood vessels only

Diseases, parasites, and contaminants

Cap. 3 - Biology and ecology of cephalopod species commercially exploited in Europe Cap. 3.4. - Diseases, parasites, and contaminant

Two-Step Concentration of Complex Water Samples for the Detection of Viruses

The accurate detection and quantification of pathogenic viruses in water is essential to understand and reduce the risk of human infection. This paper describes a two-step method suitable for concentrating viruses in water and wastewater samples. The method involves a tangential flow ultrafiltration step that reduces the sample volume of 1–10 L to approximately 50 mL, followed by secondary precipitation using polyethylene glycol 6000, which reduces the volume to 1–4 mL. For method validation, water samples were spiked with different concentrations of enteric viruses, and viral recovery in the concentrates exceeded 10% in all experiments. The method is suitable for water samples with high and low salinity and turbidity, allowing an accurate comparison of viral titers in a diverse range of water types. Furthermore, the method has the potential to concentrate other pathogens, e.g., bacteria or protozoa. Hence, the use of this method can improve the holistic assessment of risks associated with wastewater-contaminated environments

Evaluation of Molecular Methods for the Detection and Quantification of Pathogen-Derived Nucleic Acids in Sediment

The accurate detection of pathogens in environmental matrices, such as sediment, is critical in understanding pathogen fate and behavior in the environment. In this study, we assessed the usefulness of methods for the detection and quantification of Vibrio spp. and norovirus (NoV) nucleic acids in sediment. For bacteria, a commonly used direct method using hexadecyltrimethylammonium bromide (CTAB) and phenol-chloroform-isoamyl alcohol (PCI) extraction was optimized, whereas for NoV, direct and indirect (virus elution—concentration) methods were evaluated. For quantification, commercially available quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) kits were tested alongside a digital PCR (dPCR) approach. CTAB-based extraction combined with 16 h polyethylene glycol 6000 (PEG6000) precipitation was found to be suitable for the direct extraction of high abundance bacterial and viral nucleic acids. For the indirect extraction of viral RNA, beef extract-based elution followed by PEG6000 precipitation and extraction using the NucliSENS® MiniMag® Nucleic Acid Purification System and the PowerViral® Environmental RNA/DNA Isolation Kit and qRT-PCR resulted in 83–112 and 63–69% recoveries of NoV, respectively. dPCR resulted in lower viral recoveries (47 and 9%) and ~4 orders of magnitude lower Vibrio concentrations (3.6–4.6 log(10) gc/100 g sediment) than was observed using qPCR. The use of internal controls during viral quantification revealed that the RT step was more affected by inhibitors than the amplification. The methods described here are suitable for the enumeration of viral and/or bacterial pathogens in sediment, however the use of internal controls to assess efficiency is recommended

Viral dispersal in the coastal zone: A method to quantify water quality risk

Waterborne and shellfish-borne enteric viruses associated with wastewater-polluted coastal waters (e.g. Norovirus, Hepatitis A/E viruses, Adenovirus) represent a major threat to human health. Improved understanding of the locations and periods of heightened risks can help target mitigation measures and improve public health. We developed a river-estuary-coast model to simulate virus dispersal, driven by point source discharges and river flows in combination with tidal forcing. Viral inputs were based on measured wastewater adenovirus concentrations and the model was implemented with or without viral die-off. We applied the model to the Conwy river (North Wales, UK), through the estuary, to the Irish Sea coast where bathing waters and shellfisheries are known to be prone to viral contamination. Using a suite of scenarios, we showed that river flow was the primary control of viral export to the coast. Since the Conwy catchment is short and steep, and the estuary is small and river-dominated, short-duration high intensity ‘flash floods’ were shown to transport viruses through the estuary and out to sea, despite dilution or die-off effects. Duplicating flow events (i.e., storm clustering) did not double the virus export since the virus re-entered the estuary on the flood tide. The tidal magnitude and timing of high water relative to peak river flow were also important drivers regulating viral dispersal. A worst-case event simulation (i.e., combining high river flows with high viral loading and high spring tide) resulted in increased concentrations of virus at nearby coasts, although the spatial spread was similar to the previous scenarios. Our results suggest that impact models for predicting and mitigating episodes of poor microbiological water quality may require careful representation of the intensity and timings of river flow when evaluating pathogen exposure risk

Wastewater and public health: the potential of wastewater surveillance for monitoring COVID-19

Pathogenic viruses represent one of the greatest threats to human well-being. As evidenced by the COVID-19 global pandemic, however, halting the spread of highly contagious diseases is notoriously difficult. Successful control strategies therefore have to rely on effective surveillance. Here, we describe how monitoring wastewater from urban areas can be used to detect the arrival and subsequent decline of pathogens, such as SARS-CoV-2. As the amount of virus shed in faeces and urine varies largely from person to person, it is very difficult to quantitatively determine the number of people who are infected in the population. More research on the surveillance of viruses in wastewater using accurate and validated methods, as well as subsequent risk analysis and modelling is paramount in understanding the dynamics of viral outbreaks

Seasonal and spatial dynamics of enteric viruses in wastewater and in riverine and estuarine receiving waters

Enteric viruses represent a global public health threat and are implicated in numerous foodborne and waterborne disease outbreaks. Nonetheless, relatively little is known of their fate and stability in the environment. In this study we used carefully validated methods to monitor enteric viruses, namely adenovirus (AdV), JC polyomavirus (JCV), noroviruses (NoVs), sapovirus (SaV) and hepatitis A and E viruses (HAV and HEV) from wastewater source to beaches and shellfish beds. Wastewater influent and effluent, surface water, sediment and shellfish samples were collected in the Conwy catchment (North Wales, UK) once a month for one year. High concentrations of AdV and JCV were found in the majority of samples, and no seasonal patterns were observed. No HAV and HEV were detected and no related illnesses were reported in the area during the period of sampling. Noroviruses and SaV were also detected at high concentrations in wastewater and surface water, and their presence correlated with local gastroenteritis outbreaks during the spring and autumn seasons. Noroviruses were also found in estuarine sediment and in shellfish harvested for human consumption. As PCR-based methods were used for quantification, viral infectivity and degradation was estimated using a NoV capsid integrity assay. The assay revealed low-levels of viral decay in wastewater effluent compared to influent, and more significant decay in environmental waters and sediment. Results suggest that AdV and JCV may be suitable markers for the assessment of the spatial distribution of wastewater contamination in the environment; and pathogenic viruses can be directly monitored during and after reported outbreaks to prevent further environment-derived illnesses