| 1. |
- Formiga-Cruz, M, et al.
(författare)
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Evaluation of potential indicators of viral contamination in shellfish and their applicability to diverse geographical areas.
- 2003
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 69:3, s. 1556-63
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Formiga-Cruz, M., et al.
(författare)
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Distribution of human virus contamination in shellfish from different growing areas in Greece, Spain, Sweden, and the United Kingdom
- 2002
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 68:12, s. 5990-5998
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Hernroth, Bodil, 1951, et al.
(författare)
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Environmental factors influencing human viral pathogens and their potential indicator organisms in the blue mussel, Mytilus edulis: the first Scandinavian report
- 2002
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 68:9, s. 4523-4533
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Hernroth, Bodil, 1951-
(författare)
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Factors influencing bactericidal activity of blue mussel (Mytilus edulis) haemocytes against Salmonella typhimurium
- 2003
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Ingår i: Fish and Shellfish Immunology. - 1050-4648 .- 1095-9947. ; 14:2, s. 93-104
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Hernroth, Bodil, 1951-, et al.
(författare)
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Influence on uptake, distribution and elimination of Salmonella typhimurium in the blue mussel, Mytilus edulis, by the cell surface properties of the bacteria
- 2000
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Ingår i: Journal of Shellfish Research. - 0730-8000 .- 1943-6319. ; 19:1, s. 167-174
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Tidskriftsartikel (refereegranskat)abstract
- This study was carried out to investigate whether the cell surface charge of Salmonella typhimurium could influence the kinetics of uptake, distribution, and elimination in the blue mussel, Mytilus edulis. The bacteria (1 mu m) were labeled with Tc-99(m) in the presence of stannous fluoride. Two different concentrations of stannous fluoride were used to produce differences in the cell surface charges of the bacteria. A set of mussels in the investigation were also given Sn-113-labeled microspheres (15 mu m) together with bacteria to compare the impact between particle size and cell surface properties on the distribution kinetics. The distribution of radiolabeled particles in the mussel was followed and analyzed with a computer-aided gamma camera that can detect two isotopes simultaneously. Finally. the mussels were dissected and the radioactivity in the fractions was measured with a well-shielded NaI(Tl) detector. The reduced cell surface charge of S. typhimurium enhanced the preingestive selection on the gills or labial palps as well as the postingestive selection in the digestive glands in such a way that it became similar to the microspheres, despite the size differences. The uptake of the bacteria labeled in the presence of less stannous fluoride was significantly lower. However, the subsequent absorption of these bacteria in the digestive gland was greater, because the recovery of radioactivity outside the digestive tract was higher than for the more manipulated bacteria and the microspheres. Likewise, the elimination of the more manipulated bacteria was similar to that of the microspheres and significantly higher than that of the less affected bacteria. It is concluded that the cell surface properties of bacteria, possibly the charge, influence the uptake, distribution, and elimination in M. edulis and that this factor could have the same influence as size on the uptake capacity.
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| 6. |
- Hernroth, Bodil, 1951, et al.
(författare)
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Manganese induced immune suppression of the lobster, Nephrops norvegicus
- 2004
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Ingår i: Aquatic Toxicology. - : Elsevier BV. - 0166-445X .- 1879-1514. ; 70:3, s. 223-231
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Tidskriftsartikel (refereegranskat)abstract
- Manganese (Mn) is one of the most abundant elements on earth, particularly in the soft bottom sediments of the oceans. As a micronutrient Mn is essential in the metabolic processes of organisms. However, at high concentrations the metal becomes a neurotoxin with well-documented effects. As a consequence of euthrophication, manganese is released from bottom sediments of coastal areas and the Norway lobsters, Nephrops norvegicus, can experience high levels of bioavailable Mn2+. Here, we present the first report showing that Mn also affects several fundamental processes in the mobilisation and activation of immunoactive haemocytes. When N. norvegicus was exposed to a realistic [Mn2+] of 20 mg l(-1) for 10 days 24.1 mug ml(-1) was recorded in the haemolymph. At this concentration the total haemocyte count was reduced by ca. 60%. By using BrdU as a tracer for cell division, it was shown that the proliferation rate in the haematopoietic tissue did not increase, despite the haemocytepenia. A gene coding for a Runt-domain protein, known to be involved in maturation of immune active haemocytes in a variety of organisms, was identified also in haemocytes of N. norvegicus. The expression of this gene was >40% lower in the Mn-exposed lobsters as judged by using a c DNA probe and the in situ hybridisation technique. In response to non-self molecules, like lipopolysaccharide, (LPS), the granular haemocytes of arthropods are known to degranulate and thereby release and activate the prophenoloxidase, system, necessary for their immune defence. A degranulation assay, tested on isolated granular haemocytes, showed about 75% lower activity in the Mn-exposed lobsters than that for the unexposed. Furthermore, using an enzymatic assay, the activation per se of prophenoloxidase by LPS was found blocked in the Mn-exposed lobsters. Taken together, these results show that Mn exposure suppressiA fundamental immune mechanisms of Norway lobsters. This identifies a potential harm that also exists for other organisms and should be considered when increasing the distribution of bioavailable Mn, as has been done through recently introduced applications of the metal.
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| 7. |
- Hernroth, Bodil, 1951-
(författare)
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The influence of temperature and dose on antibacterial peptide response against lipopolysaccharide in the blue mussel, Mytilus edulis.
- 2003
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Ingår i: Fish and Shellfish Immunology. - 1050-4648 .- 1095-9947. ; 14:1, s. 25-37
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Tidskriftsartikel (refereegranskat)abstract
- Blue mussels (Mytilus edulis) were inoculated with two different doses of lipopolysaccharides (LPS) or phosphate-saline (PS) buffer under different temperature conditions (6 and 20 degrees C). The activity of the antibacterial peptide fraction, purified through reverse phase chromatography from mussel haemolyph, was compared at different time intervals after the inoculation. The activity was determined as the minimal peptide concentration that inhibited growth of the Gram-negative bacteria Escherichia coli D21, by using radial diffusion assay. The antibacterial activity for mussels inoculated with LPS changed over time, both at 6 and 20 degrees C, but those inoculated with PS-buffer did not. The response was enhanced within a time course of 3h. The higher temperature did increase the inhibitory activity and made the mussel respond at an earlier stage, in comparison to that at 6 degrees C. At 20 degrees C, mussels inoculated with 10 microg of LPS responded faster than those inoculated with 0.1 microg of LPS. In addition, cytotoxic effects of LPS on mussel haemocytes were investigated in vitro, using a colorimetric assay. The survival index (SI%) for haemocytes decreased with 76% at 6 degrees C but increased with 100% at 20 degrees C, irrespective of the dose of LPS. This indicated that LPS did not influence the viability of the haemocytes but the high temperature increased their metabolic state. Likely, antibacterial response was provoked by LPS in a dose-dependent manner and favoured by higher metabolic state of the haemocytes, elicited at higher temperature. These results provide important considerations for variability in the internal defence of mussels and consequently, also the retention of viable human pathogens in mussels.
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| 8. |
- Hernroth, Bodil, 1951
(författare)
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Uptake and fate of pathogenic microbes in the blue mussel, Mytilus edulis
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bivalves, like the blue mussel Mytilus edulis, are filter-feeders and can therefore accumulate high numbers of microbes from the surrounding water. These shellfish are considered the main risk for bringing pathogens from domestic outlets in the sea, back to the community. Although Sweden has strict regulations for discharging waste-water, enteric human viruses were frequently found in mussels from the west coast. The viruses occurred even when the present indicator organism for faecal contamination, Escherichia coli, was found in low numbers. The European Commission has suggested bacteriophages (somatic coliphages, F-specific RNA phages, phages infecting Bacteroides fragilis) as more reliable indicators. However, environmental factors, such as water temperature and land run-off, influenced the indicators and human viruses differently and therefore they did not coexist. Furthermore, a series of laboratory experiments were performed in order to investigate if the content of viable bacteria in mussels can be affected by the cell surface properties of the bacteria and by the surrounding temperature. (i) The ingestion of Salmonella typhimurium was significantly increased when the negative cell surface charge of the bacteria was reduced through chemical modification. This treatment also altered the handling of the bacteria in the mussel stomach. Thus, the bacteria were recovered in different amounts and in different organs of the mussels. (ii) Survival of mutants of S. typhimurium when exposed to mussel haemocytes (in vitro) or inoculated into mussel haemolymph, was enhanced for mutants expressing more complete lipopolysaccharide (LPS). These mutants also managed to multiply in vivo and kill mussels when the temperature favoured bacterial growth. (iii) LPS extracted from S. typhimurium showed to be a trigger for antibacterial peptide activity when inoculated in the mussel haemolymph. The activity responded in a dose dependent manner and was enhanced by temperature.It can be concluded that the enrichment in the mussel can differ between microbes due to their cell surface properties. The content of viable microbes in the mussel tissue seemed to be more pronounced for the human bacterial pathogens. Such bacteria had a survival advantage particularly at higher temperatures. Therefore it does not seem adequate to use non-pathogenic E. coli as an indicator for human intestinal pathogens. These results as well as the fact that pathogens circulating in the community can be brought to the mussels via domestic drainage, should be taken into consideration when modelling risk assessment. Indicators should be evaluated with respect to the most risky pathogens present, and how the bivalves can handle these in relation to the particular environmental conditions in the area. Thus, we may have to accept that the choice of reliable indicators will differ between geographical areas and probably also on a seasonal basis.
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