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PubMed2005

Use of rotavirus virus-like particles as surrogates to evaluate virus persistence in shellfish.

Rotavirus virus-like particles (VLPs) and MS2 bacteriophages were bioaccumulated in bivalve mollusks to evaluate viral persistence in shellfish during depuration and relaying under natural conditions. Using this nonpathogenic surrogate virus, we were able to demonstrate that about 1 log10 of VLPs was depurated after 1 week in warm seawater (22 degrees C). Phage MS2 was depurated more rapidly (about 2 log10 in 1 week) than were VLPs, as determined using a single-compartment model and linear regression analysis. After being relayed in the estuary under the influence of the tides, VLPs were detected in oysters for up to 82 days following seeding with high levels of VLPs (concentration range between 10(10) and 10(9) particles per g of pancreatic tissue) and for 37 days for lower contamination levels (10(5) particles per g of pancreatic tissue). These data suggest that viral particles may persist in shellfish tissues for several weeks.

Animals↗
PubMed2005

Accelerated sulfur cycle in coastal marine sediment beneath areas of intensive shellfish aquaculture.

Prokaryotes in marine sediments taken from two neighboring semi-enclosed bays (the Yamada and Kamaishi bays) at the Sanriku coast in Japan were investigated by the culture-independent molecular phylogenetic approach coupled with chemical and activity analyses. These two bays were chosen in terms of their similar hydrogeological and chemical characteristics but different usage modes; the Yamada bay has been used for intensive shellfish aquaculture, while the Kamaishi bay has a commercial port and is not used for aquaculture. Substantial differences were found in the phylogenetic composition of 16S rRNA gene clone libraries constructed for the Yamada and Kamaishi sediments. In the Yamada library, phylotypes affiliated with delta-Proteobacteria were the most abundant, and those affiliated with gamma-Proteobacteria were the second-most abundant. In contrast, the Kamaishi library was occupied by phylotypes affiliated with Planctomycetes, gamma-Proteobacteria, delta-Proteobacteria, and Crenarchaeota. In the gamma-Proteobacteria, many Yamada phylotypes were related to free-living and symbiotic sulfur oxidizers, whereas the Kamaishi phylotype was related to the genus Pseudomonas. These results allowed us to hypothesize that sulfate-reducing and sulfur-oxidizing bacteria have become abundant in the Yamada sediment. This hypothesis was supported by quantitative competitive PCR (qcPCR) with group-specific primers. The qcPCR also suggested that organisms closely related to Desulfotalea in the Desulfobulbaceae were the major sulfate-reducing bacteria in these sediments. In addition, potential sulfate reduction and sulfur oxidation rates in the sediment samples were determined, indicating that the sulfur cycle has become active in the Yamada sediment beneath the areas of intensive shellfish aquaculture.

Animals↗
PubMed2005

Artificial neural network prediction of viruses in shellfish.

A database was probed with artificial neural network (ANN) and multivariate logistic regression (MLR) models to investigate the efficacy of predicting PCR-identified human adenovirus (ADV), Norwalk-like virus (NLV), and enterovirus (EV) presence or absence in shellfish harvested from diverse countries in Europe (Spain, Sweden, Greece, and the United Kingdom). The relative importance of numerical and heuristic input variables to the ANN model for each country and for the combined data was analyzed with a newly defined relative strength effect, which illuminated the importance of bacteriophages as potential viral indicators. The results of this analysis showed that ANN models predicted all types of viral presence and absence in shellfish with better precision than MLR models for a multicountry database. For overall presence/absence classification accuracy, ANN modeling had a performance rate of 95.9%, 98.9%, and 95.7% versus 60.5%, 75.0%, and 64.6% for the MLR for ADV, NLV, and EV, respectively. The selectivity (prediction of viral negatives) was greater than the sensitivity (prediction of viral positives) for both models and with all virus types, with the ANN model performing with greater sensitivity than the MLR. ANN models were able to illuminate site-specific relationships between microbial indicators chosen as model inputs and human virus presence. A validation study on ADV demonstrated that the MLR and ANN models differed in sensitivity and selectivity, with the ANN model correctly identifying ADV presence with greater precision.

Adenoviruses, Human↗
PubMed1997

Evidence that mortality from Vibrio vulnificus infection results from single strains among heterogeneous populations in shellfish.

Vibrio vulnificus is the leading cause of food-related mortality reported in the state of Florida. It is normal microflora in marine environments, where seawater and molluscan shellfish are the primary vectors of V. vulnificus disease. Risk correlates with seasonally high numbers of V. vulnificus bacteria during the summer months. Currently, the infectious dose for humans, as well as whether the disease is caused by single or multiple strains found in molluscan shellfish, is unknown. In this work, we studied pulsed-field gel electrophoresis profiles of V. vulnificus strains isolated from blood and oysters associated with V. vulnificus disease. Results showed that ca. 10(3) V. vulnificus bacteria/gram of oyster and higher concentrations were associated with human infections and that a single V. vulnificus strain, evidenced by pulsed-field gel electrophoresis profiles, was isolated from human tissues.

Adult↗
PubMed2002

Molecular characterization of hepatitis a virus isolates from a transcontinental shellfish-borne outbreak.

One hundred eighty-four serologically confirmed cases of hepatitis A were reported in eastern Spain in 1999. A matched case-control study implicated imported coquina clams complying with European Union shellfish standards as the source of infection; this implication was confirmed by the detection by reverse transcription-PCR of hepatitis A virus (HAV) RNA in shellfish samples. In spite of the recognized low variability of HAV, genetic characterization of the complete capsid region of virus isolates from patient serum samples revealed the existence of both synonymous and nonsynonymous variants. Two antigenic variants were detected, one in a discontinuous epitope defined by monoclonal antibody K3-4C8 and a second in a linear VP1 epitope of the virus. In spite of these antigenic variants, all isolates were assigned to genotype IB, providing further evidence that the outbreak originated from a common source, although multiple strains were likely to be involved.

5' Untranslated Regions↗
PubMed1997

Immune bioactivity in shellfish toward serum-free cultured human cell lines.

The biologically functional effect of eight kinds of hot-water extracts of shellfish on cultured human cell lines was examined in a serum-free medium model. Meretrix lusoria and Sinonovacula constricta extracts enhanced IgM secretion of both hybridoma HB4C5 and SI102 cells when cultured with the respective extracts. The purified principle exhibited remarked activity in the adsorbed fraction in hydroxyapatite and Concanavalin A columns. The extracts of Corbicula fluminea, Crassostreas gigas, Meretrix lusoria, Anadara granosa, and Sinonovacula constricta enhanced in nitroblue tetrazolium (NBT)-reducing ability of macrophage U-M cells. Meretrix lusoria, Anadara granosa, and Sinonovacula constricta were specifically cytotoxic to both cultures of MCF-7 breast cancer cells and HuH-6KK hepatoblastoma. These findings imply that the extracts of shellfish that were examined exhibited a differential effect on immune cells and tumor cells.

Animals↗
PubMed1999

Effect of shellfish calcium on the apparent absorption of calcium and bone metabolism in ovariectomized rats.

Fossil shellfish powder (FS) and Ezo giant scallop shell powder (EG) were rendered soluble with lactate and citrate under decompression (FSEx and EGEx, respectively) and we examined the effects of lactate-citrate solubilization of FS and EG on mineral absorption, tissue mineral contents, serum biochemical indices and bone mineral density (BMD) in ovariectomized (OVX) rats. The apparent absorption ratios of minerals tended to be high in the rats fed with the solubilized mineral sources, those in the FSEx group being significantly higher than in the FS group. There was no significant difference in the tibia mineral content among the OVX groups. BMD at the distal femoral diaphysis was significantly increased by FSEx and EGEx feeding. It is suggested that solubilization with lactate and citrate under decompression increased the solubility and bioavailability of calcium from such natural sources of shellfish calcium as FS and EG.

Animals↗
PubMed1991

Creatine kinase MB elevation in paralytic shellfish poisoning.

An outbreak of paralytic shellfish poisoning occurred in southern Taiwan, affecting 116 persons who had consumed purple clams. Two victims died within four hours. Gonyautoxins were identified as causative toxins. During the outbreak, five patients with paralytic shellfish poisoning were seen in our hospital. All recovered following supportive treatments. Serum creatine kinase concentration was elevated in three of the five patients. The levels of the enzyme did not seem to correlate with the severity of poisoning. The most significant finding was the previously unreported observation of elevation of the creatine kinase MB level. In all four patients who had creatine kinase MB value determined, it was elevated.

Adult↗
PubMed2005

Paralytic shellfish poison (PSP) profiles and toxification of short-necked clams fed with the toxic dinoflagellate Alexandrium tamarense.

As a part of our studies on paralytic shellfish poison (PSP) accumulation kinetics in bivalves, short-necked clam Tapes japonia was experimentally contaminated with PSP by being fed with the toxic dinoflagellate Alexandrium tamarense for 2, 4, 6, 8 and 10 days, and the processes of PSP accumulation and bioconversion were investigated: the toxicity level was determined by mouse bioassay and toxin components were identified by high-performance liquid chromatography (HPLC). The strain of A. tamarense used in this study possessed a specific toxicity of 186.7 +/- 81 (mean +/- S.D., n = 5) x 10(-6) MU/cell. Total toxin concentration of this strain was 140.4 +/- 61 (mean S.D., n = 5) fmol/cell. The toxicity level of short-necked clams increased almost in parallel with the abundance of A. tamarense, reaching 1.8, 3.2, 3.8, 3.5 and 4.6 MU/g meat for 2, 4, 6, 8 and 10 days of feeding, respectively. The accumulation rates of PSP toxins, which are the ratio of the total amount of toxins accumulated in the bivalves to the estimated intake in each feeding experiment, were 7.5, 8.1, 5.7, 4.2 and 4.4% for 2, 4, 6, 8 and 10 days, respectively. At the end of each exposure period, many undigested algal cells were found in pseudofeces under microscopic observation. There was a remarkable difference in the relative proportions of the predominant toxin components between A. tamarense and short-necked clams. The most notable difference was the change in the relative amounts of C2 (carbamoyl-N-sulfo-11beta-hydroxysaxitoxin sulfate), GTX1 and GTX 4 during the first two days. In the toxic bivalves, the amount of C2, which is dominant in A. tamarense, decreased to below half a percent after being ingested. Subsequently, the amount of GTX1 in the shellfish meat reached 50.1 mol%, while that of GTX4 decreased to about half of that in A. tamarense. As for the configuration of 11-hydroxysulfate, PSP components in A. tamarense exist almost exclusively as beta-epimers (GTX3, GTX4, C2 and C4), accounting for 72.8 mol% of the total. This contrasts with the case of the short-necked clams, where the beta-epimers represented 25.8, 33.8, 30.8, 36.8 and 28.5 mol% of the total after 2, 4, 6, 8 and 10 days, respectively. PSP components seemed to be converted rapidly at an early stage of the feeding of A. tamarense.

Animals↗
PubMed1990

Lethal paralytic shellfish poisoning in Guatemala.

An outbreak of paralytic shellfish poisoning occurred in Champerico, on the Pacific coast of Guatemala, July-August 1987. Of 187 people affected with characteristic neurologic symptoms, 26 died. A case study implicated a species of clam, Amphichaena kindermani, harvested from local beaches as the vehicle of the neurotoxins (saxitoxins). Children less than 6 years old had a higher fatality rate (50%) than people greater than 18 years of age (7%). The minimum lethal dose for 1 child was estimated to have been 140 mouse units of toxin/kg body weight; thus children may be more sensitive to the saxitoxins than are adults. This is the first large outbreak of paralytic shellfish poisoning recognized in Guatemala.

Adolescent↗
PubMed2002

Development and application of an enzyme immunoassay based on a monoclonal antibody against gonyautoxin components of paralytic shellfish poisoning toxins.

With a gonyautoxin 2/3 (GTX2/3)-specific monoclonal antibody (designated GT-13A) and a saxitoxin-horseradish peroxidase conjugate (STX-HRP), a direct competitive enzyme immunoassay (GTX-EIA) was established and its sensitivity to various toxin components was investigated. The concentrations resulting in 50% inhibition of the binding of STX-HRP to the solid-phase GT-13A antibody for GTX2/3, decarbamoyl-GTX2/3 (dc-GTX2/3), N-sulfocarbamoyl-GTX2/3 (C1/2), GTX1/4, STX, and neosaxitoxin (neoSTX) in GTX-EIA were found to be 0.28, 0.41, 0.52, 3.46, 4.06, and 89.37 ng/ml, respectively. When the minimum detection limit was assumed to be at a toxin concentration causing 30% inhibition of the binding of STX-HRP to the solid-phase GT-13A antibody, the detection limits for GTX2/3, dc-GTX2/3, C1/2, GTX1/4, STX, and neoSTX were found to be 0.15, 0.18, 0.19, 1.09, 1.50, and 22.93 ng/ml, respectively. These results indicate that all of the GTX components examined and STX are detectable at concentrations lower than the regulatory limit of 80 microg/100 g of shellfish tissue, even when a minimum dilution factor of 100 is applied to tissue extracts with the extraction procedure of the Association of Official Analytical Chemists. Therefore, GTX-EIA is thought to be a useful qualitative screening method for GTX components and STX in the mass monitoring of toxin-contaminated shellfish.

Animals↗
PubMed2003

Identification of Salmonella serovars isolated from live molluscan shellfish and their significance in the marine environment.

A study on the presence of Salmonella spp. in live molluscs was performed, which included a description of the different serovars isolated and their relationship to the marine environment. A total of 2,980 samples of shellfish from Galicia (N.W. Spain) were tested for the presence of Salmonella spp. between September 1998 and August 2001. The overall incidence of Salmonella was 1.8% and showed a slight rise during the 3 years of the study. Mussels and oysters presented a higher incidence than clams and cockles, possibly because of their distinct growing habitat. A seasonal pattern was noted for the isolation of Salmonella spp.: 54% of the isolations were detected from September to November. That nearly 67% of the total Salmonella was isolated from shellfish with fecal coliform levels < 300/100 g (the maximum level criteria in the European Communities regulations) supported the view that low levels of fecal coliforms do not necessarily indicate the absence of Salmonella. A total of nine serovars were found in the 54 Salmonella isolated. Salmonella Senftenberg was the most frequent (50%), followed by Salmonella Typhimurium (18%) and Salmonella Agona (17%). Salmonella Senftenberg was detected frequently during the year, whereas the remaining serovars were detected only on occasional contamination events.

Animals↗
PubMedDate not supplied

Immunoassay methods for paralytic shellfish poisoning toxins.

The current status of immunochemical techniques for analysis of paralytic shellfish poisoning (PSP) toxins is summarized. Important aspects regarding production of the biological reagents necessary for immunochemical methods, the characteristics of polyclonal and monoclonal antibodies against saxitoxin and neosaxitoxin, and the importance of test sensitivity and specificity are discussed. Applications of immunochemical techniques for PSP toxins include microtiter plate enzyme immunoasays and enzyme-linked immunofiltration assays for toxin detection, and immunoaffinity chromatography (IAC) for sample extract cleanup. A major advantage of enzyme immunoassay (EIA) is simplicity and rapidity of the test procedure, and higher sensitivity than other methods. However, quantitative agreement between EIA and mouse bioassay is dependent on antibody specificity and the toxin profile in the shellfish; thus, both over- and underestimation of total toxicity may occur. For screening purposes, however, EIAs offer major advantages over the mouse bioassay, which is criticized in Europe because of animal welfare. A major application of antibodies against PSP toxins is their use for extract cleanup by IAC, which gives highly purified extracts, thereby enhancing determination of PSP toxins by conventional physicochemical methods such as liquid chromatography. IAC can also be used to isolate PSP toxins for preparation of analytical standard solutions.

Animals↗
PubMed2003

Kounis syndrome secondary to allergic reaction following shellfish ingestion.

Two cases of allergic angina and allergic myocardial infarction (Kounis syndrome) secondary to shellfish ingestion are described. The patients had pre-existing quiescent coronary artery disease (type II variant of the syndrome) and the allergic reaction following eating shellfish seemed to have triggered the development of an acute myocardial infarction. The clinical implications are also discussed.

Angina Pectoris↗
PubMedDate not supplied

Thermoluminescence detection of irradiated shellfish: international interlaboratory trial.

An international interlaboratory trial was conducted using thermoluminescence for the detection of irradiated shellfish, aimed at validating the method for routine use. Nephrops norvegicus, mussels, brown shrimps, black tiger prawns, and king scallops were presented as nonirradiated and irradiated to 0.5 and 2.5 kGy. The protocol called for the use of 3 preparation methods: extraction of silicates from whole shellfish by acid hydrolysis and physical separation, and of carbonates from powdered shells. Homogeneity was tested on each product and each treatment. Results verified that all methods were able to distinguish between nonirradiated and irradiated samples regardless of dose. Silicate methods produced better discrimination than powdered shell, and acid hydrolysis showed some evidence of better separation between the 2 doses than the physical method. Participants received each product in each treatment category for blind analysis. Six participants returned results for acid hydrolysis, 7 for physical separation, and 5 for the powdered shell method. Their results confirmed the homogeneity testing. Qualitative results gave 100% correct classification for both silicate methods and 85.3% for powdered shell. Silicate methods are therefore preferable unless only shell is available. Overall, the results confirmed the case for validation.

Carbonates↗
PubMed2004

Probing Norwalk-like virus presence in shellfish, using artificial neural networks.

A database was examined using artificial neural network (ANN) models to investigate the efficacy of predicting PCR-identified Norwalk-like virus presence and absence in shellfish. The relative importance of variables in the model and the predictive power obtained by application of ANN modelling methods were compared with previously developed logistic regression models. In addition, two country-specific datasets were analysed separately with ANN models to determine if the relative importance of the input variables was similar for geographically diverse regions. The results of this analysis found that ANN models predicted Norwalk-like virus presence and absence in shellfish with equivalent, and better, precision than logistic regression models. For overall classification performance, ANN modelling had a rate of 93%, vs 75% for the logistic regression. ANN models were able to illuminate the site-specific relationships between indicators and pathogens.

Databases, Factual↗
PubMedDate not supplied

Liquid chromatography with mass spectrometry--detection of lipophilic shellfish toxins.

A rapid multiple toxin method based on liquid chromatography with mass spectrometry (LC/MS) was developed for the detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1), DTX-2, yessotoxin (YTX), homoYTX, 45-hydroxy-YTX, 45-hydroxyhomo-YTX, pectenotoxin-1 (PTX-1), PTX-2, azaspiracid-1 (AZA-1), AZA-2, and AZA-3. Toxins were extracted from shellfish using methanol-water (80%, v/v) and were analyzed using a C8 reversed-phase column with a 5 mM ammonium acetate-acetonitrile mobile phase under gradient conditions. The method was validated for the quantitative detection of OA, YTX, PTX-2, and AZA-1 in 4 species (mussels, Mytilus edulis; cockles, Cerastoderma edule; oysters, Crassostrea gigas; king scallop, Pecten maximus) of shellfish obtained from United Kingdom (UK) waters. Matrix interferences in the determination of the toxins in these species were investigated. The validated linear range of the method was 13-250 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. Recovery and precision ranged between 72-120 and 1-22%, respectively, over a fortification range of 40-160 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. The limit of detection, reproducibility, and repeatability of analysis showed acceptable performance characteristics. A further LC/MS method using an alkaline hydrolysis step was assessed for the detection of OA, DTX-1, and DTX-2 in their esterified forms. In combination with the LC/MS multiple toxin method, this allows detection of all toxin groups described in Commission Decision 2002/225/EC.

Animals↗
PubMed2006

[Determination of 27 microelements in 11 shellfishes].

Shellfish samples were prepared by microwave, and 27 microelements including Ca, Mg, K, Na, P, S, Zn, Mn, Fe, Cu, Cr, Co, Se, Sn, Ni, Al, Ti, V, Mo, Ba, Ge, Si, Cd, I, Pb, As and Hg were determined by ICP-AES. It was showed that ICP-AES had an obvious advantage in the determination of microelements, and shellfishes contained upwards of 20 kinds of microelements. Calcium in fresh water was higher than that in seawater, and As and Pb were the major pollutants.

Arsenic↗