Tracing of norovirus outbreak strains in mussels collected near sewage effluents

Noroviruses from mussels collected near sewage effluents were compared with local patient outbreak strains. Sequence analyses of RNA polymerase-capsid-poly(A)-3' (3.1-kilobase) regions confirmed the 99.9% similarity between genotype I.1 strains from mussels and patient strains from recreational-bathing outbreaks, indicating the potential usefulness of sentinel norovirus mussel studies in tracing human norovirus contamination of coastal waters.     

Factors influencing bactericidal activity of blue mussel (Mytilus edulis) haemocytes against Salmonella typhimurium

This study showed that in vitro survival of Salmonella typhimurium, after exposure to haemocytes of Mytilus edulis, was significantly affected by the lipopolysaccharides (LPS) structures expressed on the cell surface of the bacteria. Survival seemed to be affected by the surrounding temperature as well. Mussel haemocytes were in vitro exposed to mutants of S. typhimurium, expressing differences in O -antigen polysaccharide chains and core sugars of LPS on their cell surface. Surviving cells of the mutants were determined after incubation with the haemocytes at different temperatures, using a colorimetric assay. In addition, a complementary study on clearance of these mutants, inoculated into the adductor muscle of mussels, was performed at 6 and 20 degrees C. It was concluded that the survival index (SI%) measured in vitro for the mutant with complete LPS was significantly lower at 6 degrees C (c.15%) compared to that at 14 and 20 degrees C (c.70%). SI% for the other mutants was c.35-45% and was not affected by temperature. The in vivo study at 20 degrees C showed that during the first 24h, the clearance rate for the mutants with complete LPS was significantly higher than for the others. Thereafter all mutants, with exception for the most deficient, started to increase in numbers and caused death to the mussels. At 6 degrees C the mutants were slowly reduced and after 17 days, viable cells of the mutant with complete LPS were still detectable in the haemolymph. The study indicated that the mussel haemocytes responded in relation to the LPS of the mutants. However, more intact LPS also seemed to protect the bacteria from being killed. The higher temperatures favoured the growth of the mutants that managed to resist the haemocyte defence. Cell surface properties and temperature seem to affect the survival of bacteria in mussels, which consequently can affect risk assessments in regard to public health.     

Factors influencing survival of enterotoxigenic Escherichia coli, Salmonella enterica (serovar Typhimurium) and Vibrio parahaemolyticus in marine environments

The presence and persistence of enterotoxigenic Escherichia coli (ETEC) is poorly investigated in marine habitats. Here we compared ETEC with the more studied fecal contaminant, Salmonella enterica serotype Typhimurium ( S. enterica ) and the marine bacteria Vibrio parahaemolyticus . All three species of bacteria were culturable on agar plates during 8 weeks of incubation in seawater. However, the culturability of ETEC was positively affected by low temperature whereas V. parahaemolyticus was negatively affected. High-nutrient conditions favored the growth of ETEC but not the other bacteria. When the bacteria were fed to blue mussels, V. parahaemolyticus inhibited the filtration activity and the ingestion was lower than that of the enterobacteria. On the other hand, the mussels were less efficient in eliminating V. parahaemolyticus and an in vitro study showed that the hemocytes of three different species of bivalves were not able to kill this strain of V. parahaemolyticus . The bactericidal capacity of bivalves was seemingly an efficient elimination pathway for S. enterica and ETEC. This study showed that ETEC in endemic areas should, to the same degree as S. enterica and V. parahaemolyticus , be taken in consideration when assessing the role of marine environments as a source of enteric infection.     

Sampling and filtration efficiency of two commonly used plankton nets. A comparative study of the Nansen net and the Unesco WP 2 net

The paper presents the results from a study in which two commonlyused plankton nets, the Nansen net and the WP 2 net, were compared.The study was performed at six stations in the Baltic Sea andthe samples were collected during seasons of both high and lowparticle abundance. The comparison included both qualitativeand quantitative filtration capacity of the nets under differentenvironmental conditions. Different mesh sizes were also tested.The results show that the Nansen net has an efficiency of 50–70%compared to the WP 2 net under favourable conditions. When conditionsare unfavourable, i.e. during periods of high particle abundanceor during long hauls, the efficiency may be as low as 25–30%.The mesh size of the nets proved to be of decisive importancefor the qualitative composition of the samples. Most of thesmaller size fractions such as rotifers, copepod nauplii andyoung copepodites passed through the 160 and 200 µm meshes.The 90 µm nets sampled these fractions well but with thedrawback of retaining vast amounts of phytoplankton at certaintimes of the year.     

Coelomocyte numbers and expression of HSP70 in wounded sea stars during hypoxia

Hypoxia, mainly caused by eutrophication, is a common and growing problem on marine soft bottoms. Echinoderms are known for their ability to regenerate tissue after wounding but hypoxia has a negative influence on regeneration and also on reproduction in echinoderms. We have investigated the cellular and molecular responses to wounding stress and hypoxia in the sea star Asterias rubens by using the total coelomocyte count (TCC) and the expression of heat shock proteins (HSPs). As early as 1 h after wounding, sea stars under hypoxic conditions show significantly increased TCC and, after 6 h, cell numbers increase approximately two-fold. After a 3-h hypoxia exposure of wounded animals, Western blot analysis reveals highly elevated coelomocyte cytoplasmic HSP70 expression. Non-wounded sea stars exposed to hypoxia and wounded animals kept in normoxia show enhanced HSP70 expression only after 24 h. Immunocytochemical analysis has not demonstrated any translocation of HSP70 from the cytoplasm to the nucleus. We conclude that both wounding and hypoxia elicit a stress response in sea stars and that the combined stress produces synergistic effects that may inhibit the initial processes of wound healing and regeneration.     

Distribution of human virus contamination in shellfish from different growing areas in Greece,Spain, Sweden,and the United Kingdom

Viral pollution in shellfish has been analyzed simultaneously across a wide range of geographical regions, with emphasis on the concomitant variations in physicochemical characteristics and social features. The methods for sample treatment and for the detection of human enteric viruses were optimized by the participating laboratories. The second part of this study involves the selection of a protocol for virus detection, which was validated by analyzing the distribution and concentration of human viral pathogens under diverse conditions during an 18-month period in four European countries. Shellfish-growing areas from diverse countries in the north and south of Europe were defined and studied, and the microbiological quality of the shellfish was analyzed. Human adenovirus, Norwalk-like virus, and enterovirus were identified as contaminants of shellfish in all the participating countries. Hepatitis A virus was also isolated in all areas except Sweden. The seasonal distribution of viral contamination was also described. Norwalk-like virus appeared to be the only group of viruses that demonstrated seasonal variation, with lower concentrations occurring during warm months. The depuration treatments currently applied were shown to be adequate for reducing Escherichia coli levels but ineffective for the elimination of viral particles. The human adenoviruses detected by PCR correlate with the presence of other human viruses and could be useful as a molecular index of viral contamination in shellfish.     

Environmental Factors Influencing Human Viral Pathogens and Their Potential Indicator Organisms in the Blue Mussel, Mytilus edulis: the First Scandinavian Report

This study was carried out in order to investigate human enteric virus contaminants in mussels from three sites on the west coast of Sweden, representing a gradient of anthropogenic influence. Mussels were sampled monthly during the period from February 2000 to July 2001 and analyzed for adeno-, entero-, Norwalk-like, and hepatitis A viruses as well as the potential viral indicator organisms somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, and Escherichia coli. The influence of environmental factors such as water temperature, salinity, and land runoff on the occurrence of these microbes was also included in this study. Enteric viruses were found in 50 to 60% of the mussel samples, and there were no pronounced differences between the samples from the three sites. E. coli counts exceeded the limit for category A for shellfish sanitary safety in 40% of the samples from the sites situated in fjords. However, at the site in the outer archipelago, this limit was exceeded only once, in March 2001, when extremely high levels of atypical indole-negative strains of E. coli were registered at all three sites. The environmental factors influenced the occurrence of viruses and phages differently, and therefore, it was hard to find a coexistence between them. This study shows that, for risk assessment, separate modeling should be done for every specific area, with special emphasis on environmental factors such as temperature and land runoff. The present standard for human fecal contamination, E. coli, seems to be an acceptable indicator of only local sanitary contamination; it is not a reliable indicator of viral contaminants in mussels. To protect consumers and get verification of “clean” mussels, it seems necessary to analyze for viruses as well. The use of a molecular index of the human contamination of Swedish shellfish underscores the need for reference laboratories with high-technology facilities.     

Marine pelagic rotifers and tintinnids - important trophic links in the spring plankton community of the Gullmar Fjord, Sweden

During the winter and spring of 1981, the dynamics of a marinepelagic rotifer, Synchaeta vorax Rousselet and tintinnids ofthe genus Tintinnopsis, were studied in the Gullmar Fjord onthe Swedish west coast. Although present in February and March,it was not until early April when the surface water temperatureexceeded 6?C that the rotifer population started to grow rapidly.Thereafter, the population increased exponentially for 6 weeks.Estimates of consumption of phytoplankton by rotifers and tintinnidsin their most dynamic phase approximated to that of the phyto-planktonproduction. Lack of data on the amount of detritus availableand the number of young rotifers passing the 90 µm netare however obvious limitations in such calculations. Nevertheless,the results indicate that the tintinnids and the rotifers arehighly important components in the area's spring plankton community.Through high turnover rates, they are able to utilize effectivelythe spring phytoplankton bloom and thus serve as a link betweenthe primary and the larger secondary producers.     

North Atlantic Oscillation primary productivity and toxic phytoplankton in the Gullmar Fjord,Sweden (1985—1996)

The evaluation of the time-series data set of primary production, chlorophyll a (1985-1996) and toxic phytoplankton species abundance measurements (1986–1996) in the Gullmar Fjord as part of the Swedish Monitoring Program, revealed the importance of considering climatic and environmental forces among the factors that may be responsible for the observed fluctuations. The results suggested that the occurrence of toxic phytoplankton blooms in the Skagerrak may be related to changes in the phase of the North Atlantic Oscillation (NAO). The Dinophysis species were related to the NAO, temperature and salinity and the fluctuations in the phytoplankton biomass expressed as chlorophyll a were also shown to be related to the NAO. The results pointed to the need to understand further the role played by climatic and environmental forces in relation to the formation and duration of harmful algal blooms and the need for continuous quality monitoring programmes and novel statistical methods for detecting trends and cycles in ecological time-series related to environmental forces.     

Evaluation of Potential Indicators of Viral Contamination in Shellfish and Their Applicability to Diverse Geographical Areas

The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of Escherichia coli, F-specific RNA bacteriophages, and phages infecting Bacteroides fragilis strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where E. coli would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of E. coli or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked.