Depuration of Mytilus galloprovincialis experimentally contaminated with hepatitis A virus

Mussels (Mytilus galloprovincialis) were contaminated with known amounts of laboratory strains of hepatitis A virus and Poliovirus 1 and the effectiveness of a self-cleansing mechanism was studied using a pilot depuration system. Both viruses were rapidly bioaccumulated by mussels and the maximal concentration of about 10(4) TCID50/ml was reached within 1.5 hours. Depuration was carried out up to 24 h; infectivity titer decreased to 10(2) TCID50/ml and 10(3.2) TCID50/ml within 6 h in hepatitis A virus and Poliovirus 1 contaminated mussels, respectively, but only a very slight further decrease was obtained after 24 h. E. coli was used as a control; within 24 h the concentration decreased from 40 to 2 bacteria/ml of mussel (MPN). The elimination of bacteria is not a reliable parameter to control the effectiveness of viral depuration.     

Canning process that diminishes paralytic shellfish poison in naturally contaminated mussels (Mytilus galloprovincialis).

Changes in toxin profile and total toxicity levels of paralytic shellfish poison (PSP)-containing mussels were monitored during the standard canning process of pickled mussels and mussels in brine using mouse bioassays and high-performance liquid chromatography. Detoxification percentages for canned mussel meat exceeded 50% of initial toxicity. Total toxicity reduction did not fully correspond to toxin destruction, which was due to the loss of PSP to cooking water and packing media of the canned product. Significant differences in detoxification percentages were due to changes in toxin profile during heat treatment in packing media. Toxin conversion phenomena should be determined to validate detoxification procedures in the canning industry.     

Detection of hepatitis A virus in shellfish by nested reverse transcription-PCR.

A method for the detection of HAV in shellfish, based on the use of guanidinium isothiocyanate-containing solution for RNA extraction and purification steps, followed by nested PCR, is hereby proposed. Tests were carried out on mollusc samples spiked with HAV strain FG. Results showed that in samples subjected only to one round of PCR it was possible to detect HAV at concentrations of 10(3)-10(4) TCID50/10 g of mollusc. The use of the nested PCR renders the system more sensitive and specific enabling the identification of HAV concentrations as low as 1 TCID50/10 g of mollusc. Furthermore thus method, in addition to allowing the avoidance of confirming tests, such as hybridization, proved to be inexpensive and simple to perform.     

Thermal inactivation of hepatitis A virus in suspension and in dried mussels (Mytilus edulis)

We examined the effects of three different temperatures (60, 85 and 100 °C) and durations of time (1 min at 100  ° C to 30 min at 60  ° C) on hepatitis A virus (HAV) in suspension and dried mussels (Mytilus edulis). In suspension, 3.61, 4.48, 5.06 and 5.66 log₁₀ tissue culture infectious dose (TCID)₅₀/mL were reduced by 60  ° C for 5 min, 60  ° C for 15 min, 60  ° C for 30 min and 85  ° C for 3 min, respectively. In dried mussels, 1.34, 1.94, 3.16, 2.36, 3.53 and 4.38 log₁₀TCID₅₀/mL were reduced by 60  ° C for 5 min, 60  ° C for 15 min, 60  ° C for 30 min, 85  ° C for 3 min, 85  ° C for 6 min and 85  ° C for 10 min, respectively. HAV inactivation from suspension and dried mussels was achieved by 85  ° C for 6 min and 15 min, respectively, and also by a 1 min at 100  ° C. At 60, 85 and 100  ° C, the 1‐log (D‐values) inactivation from both suspension and dried mussels was 6.33 and 7.93, 0.98 and 3.05, and 0.28 and 0.38 min, respectively. A higher temperature and/or a thermal treatment time shorter than 85  ° C for 6 min (100  ° C for 1 min) could be used for commercial target foods for complete HAV inactivation.     

Occurrence of giardia cysts in mussels (Mytilus galloprovincialis) destined for human consumption

Between January and June 2004, a total of 200 nondepurated mussel samples of the Galician coast (northwest Spain) were examined for Giardia cysts with a direct immunofluorescence antibody test. Giardia cysts were found in mussels from all of the estuaries studied, with an overall rate of contamination of 41.5%. There was relation between the presence of Giardia cysts, the microbiological contamination (expressed as most probable number of Escherichia coli) detected in the samples, and the harvesting area. This is the first work that describes the presence of Giardia cysts in mussels (Mytilus galloprovincialis) destined for human consumption.     

Heavy metals in mussels (Mytilus galloprovincialis) from the lonian Sea, Italy

Concentrations of six heavy metals (Hg, Pb, Cd, Cr, Zn, and Sn) were determined in mussels (Mytilus galloprovincialis) collected between June and September 1997 from 10 locations along a sound formed by two inlets (Mar Piccolo) near the Gulf of Taranto (Ionian Sea, Italy). The average concentrations of the heavy metals found in mussels samples were 0.15 mg/kg for Hg, 1.19 mg/kg for Pb, 0.64 mg/kg for Cd, 0.31 mg/kg for Cr, 5.15 mg/kg for Zn, and 0.54 mg/kg for Sn. The concentrations of heavy metals in mussels from the first inlet did not differ greatly from those observed in mussels from the second inlet. The concentrations of heavy metals in the mussels analyzed were below acceptable levels for human consumption.     

Detection of hepatitis A virus in oysters

A digoxigenin-labelled RNA probe with a sensitivity of 800 50% tissue culture infectious dose (TCID₅₀) was used to detect Hepatitis A Virus (HAV) in oysters. We studied the influence of extraction methodology on riboprobe detection. Oyster samples obtained by four methods of extraction and extraction-concentration were spiked with HAV (CF53 strain). There was no correlation between protein concentration and turbidity of samples, and anti-digoxigenin antibodies showed a non specific reaction. Background noise was independent of protein concentration and disappeared when HAV RNA isolation by phenol/chloroform extraction was introduced, but HAV RNA could not be detected by this technique. In the presence of Acid Guanidinium Thiocyanate (AGT), RNA from HAV suspension was detected following phenolic extraction with a detection threshold of 8.10⁴ TCID₅₀ of spotted virus. HAV detection in oyster extract by a digoxigenin-labelled riboprobe appeared useful in shellfish virology, at least for a primary screening of samples.     

Polycyclic aromatic hydrocarbons in mussels (Mytilus galloprovincialis) from the Ionian Sea, Italy

Concentrations of eight polycyclic aromatic hydrocarbons (PAHs) (phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, benzo[a]pyrene, dibenz[a,h]anthracene, benzo[ghi]perylene) were determined in mussels (Mytilus galloprovincialis) collected between June and September 1995 from 10 locations along a sound of sea formed by two inlets (Mar Piccolo) close to the Gulf of Taranto (Ionian Sea, Italy). In mussels the concentrations of total PAHs were between 14.8 and 645.3 microg/kg wet weight. Among the single identified compounds, the predominance of phenanthrene (29.5 microg/kg wet weight) and anthracene (64.7 microg/kg wet weight) was evident. Another relevant pollutant was pyrene (18.4 microg/kg wet weight) followed by fluoranthene (7.2 microg/kg wet weight), whereas the other compounds showed low levels. The mussels that showed the highest total concentrations of PAHs were collected from stations affected by stronger human activities (industrial fallout, urban wastewaters, and contaminants transported via riverine discharge). Our results were similar to those found in areas classified as moderately polluted. This observation suggests the need for an increased effort in controlling sources of pollution in this area recognized as one of the most productive mussel-farming areas in the Italy.     

Cooking mussels (Mytilus galloprovincialis) by steam does not destroy the infectivity of Cryptosporidium parvum

The consumption of shellfish has increased considerably worldwide, with an associated increase in foodborne illnesses. Among the bivalves, the mussels are usually cooked by steam, which constitutes a typical dish in several regions. In this article, we demonstrate that this preparation is not sufficient to destroy completely the infectivity of Cryptosporidium parvum. Oocysts recovered from experimentally contaminated mussels (Mytilus galloprovincialis) were infectious to neonatal mice after cooking. Although, to date, no official cases of cryptosporidiosis linked to shellfish consumption have been reported, we recommend that people with reduced immunity avoid this type of food because they are at high risk of being infected with Cryptosporidium spp. after eating raw or undercooked contaminated bivalves.     

Effects of copper nanoparticles exposure in the mussel Mytilus galloprovincialis

CuO NPs are widely used in various industrial and commercial applications. However, little is known about their potential toxicity or fate in the environment. In this study the effects of copper nanoparticles were investigated in the gills of mussels Mytilus galloprovincialis, comparative to Cu(2+). Mussels were exposed to 10 μg Cu·L(-1) of CuO NPs and Cu(2+) for 15 days, and biomarkers of oxidative stress, metal exposure and neurotoxicity evaluated. Results show that mussels accumulated copper in gills and responded differently to CuO NPs and Cu(2+), suggesting distinct modes of action. CuO NPs induced oxidative stress in mussels by overwhelming gills antioxidant defense system, while for Cu(2+) enzymatic activities remained unchanged or increased. CuO NPs and Cu(2+) originated lipid peroxidation in mussels despite different antioxidant efficiency. Moreover, an induction of MT was detected throughout the exposure in mussels exposed to nano and ionic Cu, more evident in CuO NPs exposure. Neurotoxic effects reflected as AChE inhibition were only detected at the end of the exposure period for both forms of copper. In overall, these findings show that filter-feeding organisms are significant targets for nanoparticle exposure and need to be included when evaluating the overall toxicological impact of nanoparticles in the aquatic environment.     

Evaluation of an extracting method for the detection of Hepatitis A virus in shellfish by SYBR-Green real-time RT-PCR

Consumption of virus-contaminated shellfish has caused numerous outbreaks of gastroenteritis and hepatitis worldwide. In the present study, we evaluated a rapid and simple extraction method to concentrate and purify enteric viruses from shellfish tissues for their detection by real-time RT-PCR. This procedure consists of an alkaline elution with a glycine buffer, solids removal by slow speed centrifugation, purification by chloroform extraction and virus concentration by ultracentrifugation. The efficiency of this method to recover Hepatitis A virus (HAV) from oysters seeded with this virus, was assessed by real-time RT-PCR and conventional RT-nested PCR after extracting viral RNA by a commercial isolation kit. Real-time RT-PCR yielded higher detection sensitivity than the obtained by conventional RT-nested PCR. Besides the improvements in detection sensitivity, the real-time RT-PCR, by quantifying HAV RNA, allowed to check the overall extraction procedure and the recovery efficiency after each processing step. After the last phase, i.e. virus concentration by ultracentrifugation, the RNA purity was high but the estimated HAV recovery efficiency was however low, probably due to virus losses and the presence of RT-PCR inhibitors in sample concentrates. In contrast, the HAV recovery percentage was higher after the virus elution step while the RNA purity was lower. Real-time RT-PCR detection could allow to eliminate some purification and concentration steps that are required for conventional RT-nested PCR detection. The overall procedure for detecting HAV could be then simplify avoiding virus losses during manipulation.     

Heavy metals at trace level in edible mussels (Mytilus galloprovincialis Lamarck) from the gulf of Trieste

The concentration of heavy metals (cobalt, nickel, copper, cadmium, mercury and lead), which are present at trace levels in the edible part of mussels (Mytilus galloprovincialis Lamarck) from hatcheries in the gulf of Trieste, is normally or log-normally distributed, according to a statistical test sensitive to asymmetry of the distribution. A log-normal distribution appears valid for describing nearly all trace metals, in particular toxic heavy metals like cadmium, mercury and lead. The frequency distribution of these metals has nearly the same asymmetry, although the one-way analysis of the variance shows that sample means come from different population means characterising the particular hatchery.     

Changes in quality characteristics during cold storage of shucked mussels (Mytilus galloprovincialis) and selected chemical decomposition indicators

The following chemical changes were observed during the cold storage of mussels for 6 days at 4 °C. Total volatile basic nitrogen (TVB‐N, mg N per 100 g) and trimethylamine values (TMA‐N, mg N per 100 g) were increased from 22.55 and 5.96 mg per 100 g at day 0 to 12.38 and 0.42 mg per 100 g, respectively, at the end of the storage period. The indole value and putrescine concentration were increased from 15.36 µg kg^?1 and 24.7mg kg^?1 to 34.46 µg kg^?1 and 63.86 mg kg^?1, respectively, on the fourth day of storage. The pH was slightly reduced and tyramine and cadaverine were not detected during storage. TVB‐N, TMA‐N and indole value could be selected as decomposition indicators for mussels during their cold storage. Acceptable limits of 15 mg per 100 g for TVB‐N, 3 mg per 100 g for TMA‐N, 35 µg kg^?1 for indole and 60 mg kg^?1 for putrescine are suggested. Sensory and chemical results indicated that the shelf‐life of mussels at 4 °C is limited to 4 days. Copyright © 2005 Society of Chemical Industry     

Vibrio parahaemolyticus, Vibrio vulnificus and microorganisms of fecal origin in mussels (Mytilus galloprovincialis) sold in the Puglia region (Italy)

Mytilus galloprovincialis is one of the most commonly consumed of all bivalve molluscs. The consumption of raw bivalve molluscs has caused outbreaks of food poisoning due to Vibrio parahaemolyticus and Vibrio vulnificus. This paper reports the results of a survey on the presence of V. parahaemolyticus, V. vulnificus fecal coliform bacteria, Escherichia coli and Salmonella spp. in 600 M. galloprovincialis samples collected from retail outlets in the Puglia region. V. parahaemolyticus and V. vulnificus were found in 47 (7.83%) and 17 (2.83%) of the samples, respectively. One sample (0.16%) was contaminated with Salmonella spp. but no relationship was observed between vibrios and fecal coliforms and E. coli. There were no significant differences among vibrios present in bivalve molluscs during the 3-year survey.     

Occurrence of Vibrio and other pathogenic bacteria in Mytilus galloprovincialis (mussels) harvested from Adriatic Sea, Italy.

Sixty-two samples of Mytilus galloprovincialis (mussels) harvested from approved shellfish waters in the Adriatic Sea were examined for the presence of Vibrio, Salmonella, Campylobacter, and verocytotoxin producing Escherichia coli. Vibrio spp. were isolated from 48.4% of samples; the species most frequently found were V. alginolyticus (32.2%) and V. vulnificus (17.7%), followed by V. cincinnatiensis (3.2%), V. parahaemolyticus (1.6%), V. fluvialis (1.6%) and V. cholerae non-O1 (1.6%). V. parahaemolyticus resulted negative to Kanagawa-phenomenon and to PCR amplification of tdh gene. V. cholerae resulted negative to PCR amplification of sto gene. No Salmonella, Campylobacter, or E. coli verocytotoxin-producing strains were isolated. The results of this study suggest the potential risk of ingesting raw or undercooked mussels due to the frequent presence of potentially pathogenic Vibrio species.     

Norovirus, hepatitis A virus and enterovirus presence in shellfish from high quality harvesting areas in Portugal

This is the first report on the screening of shellfish from Portugal for the presence of human enteropathogenic viruses. Approximately 2000 shellfish (Curbicula fluminea, Ruditapes decussatus, Tellina crassa, Spisula solida, Dosinia exoleta, Ensis spp., Mytilus spp., Ostrea edulis and Cerastoderma edule), organized in 49 batches, were collected between March 2008 and February 2009. They were tested for norovirus (NoV), hepatitis A virus (HAV) and enterovirus (EV) by RT-PCR followed by nucleotide sequencing. Bacterial contamination was also evaluated by Escherichia coli counts. Viral contamination was detected throughout the year in all shellfish species and in all collection areas, independently of their harvesting areas classification. Overall, 67% of all analyzed batches were contaminated by at least one of the studied viruses while the simultaneous presence of two and three viruses was detected in 22% and 6% batches, respectively. Of the three viruses, NoV was detected in 37% of the batches, followed by EV in 35%, and HAV in 33%. Nucleotide sequencing of the NoV and HAV RT-PCR products demonstrated that all strains belonged to NoV genotype GII.4 and HAV subgenotype 1B. The presence of NoV and HAV in shellfish from “A class” harvesting areas of Portugal can represent a potential health risk.     

Quantification of metallothionein-like proteins in the mussel Mytilus galloprovincialis using RP-HPLC fluorescence detection

A HPLC-fluorescence method, using the fluorophore SBD-F (ammonium-7-fluorobenz-2-oxa-1,3-diazole-4-sulfonate), was adapted for the quantification of metallothioneins and their isoforms from the Moroccan mussel Mytilus galloprovincialis. The method was first optimized using a rabbit liver metallothionein. The effects of EDTA, tris(2-carboxyethyl)phosphine, and SBD-F on the labeling efficiency were studied. The optimized method was then applied to evaluate the amount of metallothionein in the mussels either exposed to cadmium in the laboratory or collected from the Casablanca coast, Morocco. The concentrations of metallothioneins measured in the field samples describe the degree of contamination of the sites and are reflected by distinct isoform patterns.     

Survival of hepatitis A virus on modified atmosphere-packaged (MAP) lettuce

Experiments were designed to study the effect of various modified atmospheres (MA) on the survival rate of hepatitis A virus (HAV) on lettuce. Pieces of lettuce inoculated with HAV were incubated at room temperature (RT) and 4°C for 12 days in ambient air and under various modified atmospheres (CO₂:N₂at 30:70, 50:50, 70:30 and 100% CO₂) inside plastic bags of low O₂permeability. Samples were removed on days 1, 3, 6, 9 and 12 and the virus was recovered and plaque-assayed to determine residual titer. Incubation for 12 days at 4°C showed that the lowest HAV survival rate (47·5%) was on lettuce stored in a petri-dish (atmospheric air), whereas the greatest survival rates (83·6%) was on lettuce stored under 70% CO₂. Statistical analysis of virus survival at 4°C indicated that HAV titers decreased for all packages, but without a significant (P>0·05) difference between the package types. At RT, however, a significantly (P<0·05) lower HAV survival rate (0·01%) was evident on lettuce stored in a petri dish, whereas survival rates as high as 42·8% were observed on lettuce stored under 70% CO₂; much lower survival rates (≤8·6%) were obtained on lettuce stored under other MAP environments at RT. Statistical analysis of the RT data indicated that there was a highly significant (P<0·05) decrease in HAV titre with increasing storage time and between package types, except for lettuce stored under 70% CO₂. These data indicate that MAP does not influence HAV survival when present on the surface of produce incubated at 4°C. A slight improvement in virus survival on lettuce was seen in the presence of high CO₂levels at RT. This may have been attributed to the inhibition of spoilage-causing enzymatic activities in the lettuce, which may have reduced exposure of the virus to potential toxic by-products.