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PubMedDate not supplied

Integrated enzyme-linked immunosorbent assay screening system for amnesic, neurotoxic, diarrhetic, and paralytic shellfish poisoning toxins found in New Zealand.

Enzyme-linked immunosorbent assays (ELISAs) were developed for amnesic, neurotoxic, and diarrhetic shellfish poisoning (ASP, NSP, and DSP) toxins and for yessotoxin. These assays, along with a commercially available paralytic shellfish poisoning (PSP) ELISA, were used to test the feasibility of an ELISA-based screening system. It was concluded that such a system to identify suspect shellfish samples, for subsequent analysis by methods approved by international regulatory authorities, is feasible. The assays had sufficient sensitivity and can be used on simple shellfish extracts. Alcohol extraction gave good recovery of all toxin groups. The ease of ELISAs permits the ready expansion of the system to screen for other toxins, as new ELISAs become available.

Amnesia↗
PubMed2002

[Capillary electrophoretic analysis of amnesic shellfish toxin--domoic acid].

Domoic acid, a main component of amnesic shellfish toxin (one of the four sorts of marine red tide bio-toxins) was determined by capillary electrophoresis with UV detection. The shellfish samples were prepared by solvent extraction, cleaned-up with a strong anion-exchange cartridge (SAX: Part No. 1210-2044, Lot No. 182639, Varian) and a cation-exchange cartridge (SCX: Part No. 1211-3039, Lot No. 171069, Varian). The quantitative analysis was performed with external standard under the optimum conditions of capillary electrophoretic analysis. The calibration curve of domoic acid showed good linearity in the range of 0.2 mg/L-50 mg/L with r = 0.9990, and the detection limit was 0.063 mg/L(S/N > 3). Spiked with standard at three levels (5.00 micrograms/g, 10.00 micrograms/g and 20 micrograms/g), the samples had the recoveries of 97.24%, 96.92% and 97.55%, and the RSDs of 2.74%, 2.59% and 1.95%, respectively. Five kinds of shellfish samples normally consumed in Dalian sea area were collected and quantified. The method has the advantages of being simple, convenient, sensitive and low cost. Therefore it can be used as one of the routine monitoring methods for amnesic shellfish toxin.

Animals↗
PubMed2003

Bacterial source tracking and shellfish contamination in a coastal catchment.

Introduced pathogens from faecal material can make their way into the aquatic environment from a number of catchment sources. These sources typically include sewage outfalls, seepage from septic tanks, and urban and agricultural runoff. Shellfish as filter feeders are particularly susceptible to contamination in faecally contaminated waters and a range of microbiological indicators have been developed to assess the levels of contamination and likely risks to public health (Hackney and Pierson, 1994). This paper outlines the application of bacterial source tracking (BST) in a shellfish growing area in part of the Port Stephens estuary along the NSW north coast. The approach is based on the premise that bacterial isolates from different faecal sources will have significantly different resistance patterns to the battery of antibiotics and concentrations tested. Faecal streptococci (FS) were isolated from several possible faecal sources: beef and dairy cattle, chickens and humans. The resistance patterns of these isolates to four different concentrations of four antibiotics were compared to those of FS isolates obtained from samples collected upstream and in the vicinity of the oyster leases. Discriminant analysis was performed using the patterns from the known source isolates and the rate of correct classification was determined for each source. The predictive function of discriminant analysis was then used to determine the most probable source of each of the unknown isolates from Tilligerry Creek, the drainage channels to the estuary, and the shellfish leases. Preliminary results are presented here and suggest that there is no single significant source of faecal contamination, rather there are contributions from a range of sources. The findings may have implications for the ways in which land use activities and catchments are managed in similar estuarine locations with a shellfish industry.

Agriculture↗
PubMed2004

Sewage impact on shellfish microbial contamination.

Coastal areas are frequently contaminated by microorganisms of human origin, due to high population density and low seawater renewal. To evaluate the impact of wastewater input on shellfish quality, a study was conducted in Brittany (France) over a period of 20 months. A hydrodynamic model was used to simulate wastewater impact on microbial water quality. To validate the model, wastewater from the three main sewage treatment plants and shellfish from three sites were sampled monthly. Bacterial indicators (E. coli), F-RNA phages were searched for by culture and noroviruses by RT-PCR and hybridisation. These microorganisms were detected in the three effluents and clams, with no marked seasonal variation. The microbial concentrations in the two oyster beds, distant from the effluent outfall, were low, and only three of the samples were positive for norovirus. For simulation, the winter wastewater inputs of E. coli and phages were calculated and an estimation for norovirus flux was made from the epidemic situation in the population. The microbial behaviour was included in the model by a decay-rate factor. Results from the model calculations were found to be very similar to E. coli and phage concentrations observed in shellfish. For noroviruses, the model indicated that shellfish distant from the wastewater input were under the detection limit of the RT-PCR method. This study demonstrated the use of modelisation to interpret norovirus contamination in various areas.

Animals↗
PubMed2005

Current scientific understanding of the environmental biosafety of transgenic fish and shellfish.

A fluorescent zebrafish was the first genetically engineered animal to be marketed, and biotechnologists are developing many transgenic fish and shellfish. Biosafety science is not sufficiently advanced to be able to draw scientifically reliable and broadly trusted conclusions about the environmental effects of these animals. The science is best developed for identifying hazards posed by environmental spread of a transgenic fish or shellfish and least developed for assessing potential ecological harms of spread. Environmental spread of certain transgenic fish or shellfish could be an indirect route of entry into the human food supply. The management of predicted environmental risks is in its infancy and has thus far focused on the first step of the risk management process, i.e. risk reduction, via a few confinement methods. There is a critical need to improve scientific methods of environmental safety assessment and management and to gather empirical data needed to substantiate biosafety conclusions and to effectively manage transgenic fish and shellfish. Scientists and potentially affected parties should participate in prioritising the knowledge gaps to be addressed.

Animals↗
PubMed2006

Rapid detection of noroviruses in fecal samples and shellfish by nucleic acid sequence-based amplification.

The purpose of this study was to determine the efficacy of a nucleic acid sequence-based amplification (NASBA) method of detecting noroviruses in artificially and naturally contaminated shellfish. We used 58 fecal samples that tested positive for noroviruses with electron microscopy (EM) to develop an NASBA assay for these viruses. Oligonucleotide primers targeting the polymerase coding region were used to amplify the viral RNA in an isothermal process that resulted in the accumulation of RNA amplicons. These amplicons were detected by hybridization with digoxigenin-labeled oligonucleotide probes that were highly specific for genogroup I (GI) and genogroup II (GII) of noroviruses. The expected band of 327 bp appeared in denaturing agarose gel without any nonspecific band. The specific signal for each amplicon was obtained through Northern blotting in many repeats. All fecal samples of which 46 (79.3%) belonged to GII and 12 (20.6%) belonged to GI were positive for noroviruses by EM and by NASBA. Target RNA concentrations as low as 5 pg/ml were detected in fecal specimens using NASBA. When the assay was applied to artificially contaminated shellfish, the sensitivity to nucleic acid was 100 pg/1.5 g shellfish tissue. The potential use of this assay was also confirmed in naturally contaminated shellfish collected from different ponds in Guangzhou city of China, of which 24 (18.76%) out of 128 samples were positive for noroviruses; of these, 19 (79.6%) belonged to GII and 5 (20.4%) belonged to GI. The NASBA assay provided a more rapid and efficient way of detecting noroviruses in fecal samples and demonstrated its potential for detecting noroviruses in food and environmental samples with high specificity and sensitivity.

Animals↗
PubMed1979

Shellfish quality standards.

Total coliforms, faecal coliforms, enterococci and heterotrophic bacteria were determined in shellfish samples collected both in the marine environment (at Bacoli and Coroglio in the Bay of Naples) and from the local market. The same bacteriological analyses were carried out on water samples obtained from the shellfish growing areas. The results are considered in relation to standards applied in Italy and elsewhere to shellfish and shellfish cultures.

Animals↗
PubMedDate not supplied

Recommended modification of dilution procedure used for bacteriological examination of shellfish.

The dilution procedure recommended by the American Public Health Association for use in bacteriological analysis of shellfish samples may cause an underestimation of the number of bacteria in shellfish. Modifications of that procedure to include preparation of the first dilution of the shellfish homogenate on a weight rather than a volume basis, and the inoculation of 1 g portions of the shellfish from a dilution of the homogenate, have been shown to significantly increase the recovery of fecal coliform bacteria from oyster samples. It is recommended that these modifications be adopted by the American Public Health Association.

Animals↗
PubMed1993

Quantification of diarrhetic shellfish toxins and identification of novel protein phosphatase inhibitors in marine phytoplankton and mussels.

Liquid chromatography (LC)-linked protein phosphatase 1/2A (PP-1/PP2A) bioassay was used to quantitatively identify diarrhetic shellfish toxins in marine phytoplankton (cultured and natural assemblages) and commercially available mussels. Using this approach, multiple protein phosphatase inhibitor profiles of varying composition were found in diarrhetic mussels from Holland and Canada. Based on LC elution positions and relative activity versus PP-1 and PP-2A, at least six inhibitors distinct from known diarrhetic shellfish toxins were identified and termed mussel phosphatase inhibitor (MPI) 19,22,23,25,33 and 42. The levels of these inhibitors, in okadaic acid equivalent units, varied from 100 pg to 3350 ng per g shellfish tissue. The combined levels of PP-1/2A inhibitors in all instances superseded that of okadaic acid/dinophysistoxin-1 and may contribute to the diarrhetic shellfish toxin profile of the contaminated mussels. The efficacy of LC-protein phosphatase bioassay was established for cultured phytoplankton where picogram levels of okadaic acid could be detected from microgram extracts of Prorocentrum lima. Analyses of plankton net tows from estuarine mussel culture sites in Eastern Canada revealed a heterogeneous population of protein phosphatase inhibitors, with dinophysistoxin-1 being most prevalent. This toxin was predominant for at least 2 months in mussel populations in the immediate vicinity of plankton sampling sites. The results are consistent with a hypothetical model in which marine bacteria, cyanobacteria and dinoflagellates combine to produce a variety of protein phosphatase inhibitors effective against signal transduction pathways in higher eukaryotes.

Animals↗
PubMed2003

Bacterial contamination of Mediterranean coastal seawater as affected by riverine inputs: simulation approach applied to a shellfish breeding area (Thau lagoon, France).

Consequences of short-term changes in thermotolerant coliform loads on their spatio-temporal distribution in a Mediterranean lagoon with large-scale mollusk farming (Thau lagoon, France) were explored using a simulation approach. Simulations were based on bacterial transport and survival coupled models forced by the input of bacterial loads from the two main rivers (Vène and Pallas) that flow into the lagoon. Different flow types (reference, sudden and constant), bringing the same bacterial load, were considered and subsequent spatial and temporal bacterial contamination of lagoon surface water and shellfish was estimated. Simulation results showed that as long as loads were high, hydrodynamical processes governed the distribution of bacterial abundance in receiving areas. As soon as loads decreased or when time supply increased, biological die-off processes became dominant. Bacterial contamination of shellfish induced by the different flow types appeared to depend on the receiving area. In the case of Pallas River area, a sudden input of bacteria led to a high bacterial contamination of shellfish but only during a short period ( approximately 1 day). A constant input of the same amount of bacteria induced a lower but significant contamination during all the simulation period (10 days). On the contrary, bacterial inputs from the Vène River led to shellfish contamination only when bacteria were delivered through a flood event. Exposure time of bacteria to adverse environmental conditions appeared to be the main explanation to the above-mentioned differences. Consequences of our results in terms of environmental management strategy were discussed.

Animals↗
PubMed2002

Utilization of shrimp shellfish waste as a substrate for solid-state cultivation of Aspergillus sp. S1-13: evaluation of a culture based on chitinase formation which is necessary for chitin-assimilation.

The utilization of shrimp shellfish waste as a substrate for solid-state cultivation of a filamentous fungus, Aspergillus sp. S1-13, was investigated. The organism was selected from among 220 isolates based on the productivity of its chitinolytic enzyme (chitinase), which might reflect microbial growth. The enzyme was produced only when the organism was grown on medium containing the shellfish waste. The addition of 58-65% water (w/w) to the medium was effective in enhancing production, and a certain amount of enzyme was observed in media of higher water content (up to about 75%). The initial pH and nitrogen source (ammonium sulfate) of the solid-state medium also affected the amount of enzyme. The amount of enzyme increased 2-fold in an optimum solid-state medium: 5 g of shrimp shellfish waste and 3 ml of basal medium (pH 5) containing 0.1% (NH4)2SO4 was inoculated with 4 ml of spore suspension; static cultivation at room temperature. The amount increased further (1.5-fold) when the cultivation was carried out at 37 degrees C, with 1.85 units of the enzyme formed from 1 g of shrimp shellfish waste. An analysis by ion-exchange column chromatography suggested the presence of at least two colloidal chitin-hydrolyzing enzymes and one p-nitrophenyl beta-D-N-acetylglucosaminide-hydrolyzing enzyme in an extract of the solid-state culture. The elution profile was similar to that obtained with a liquid culture filtrate.

Journal Article↗
PubMed1975

The quantitative bacteriology of some commercial bivalve shellfish entering British markets.

Incidents of non-specific illness associated with the consumption of oysters have highlighted the lack of published information on the bacteriology of shellfish suitable for consumption. Investigations showed that the majority of molluscan shellfish entering English markets conform to the accepted standard of less than 5 Escherichia coli/ml. tissue. The numbers of E. coli were related to the sanitary quality of the growing area but no relation could be established between numbers of E. coli and coliforms, faecal streptococci or Clostridium welchii. The numbers of non-specific bacteria varied considerably but shellfish from sources associated with non-specific illness yielded relatively high counts at 37 degrees C. The results showed that there was no justification for a standard based on total plate counts, which often exceeded 10(6)/g. Such a standard would have to be coupled with spoilage or the incidence of non-specific illness. The relation between the numbers of non-specific bacteria growing at 20 and 37 degrees C. appears to be a useful measure for assessing the likelihood that raw shellfish are a public health risk.

Animals↗
PubMed2003

Geographical, temporal, and species variation of the polyether toxins, azaspiracids, in shellfish.

Azaspiracid Poisoning (AZP) is a new toxic syndrome that has caused human intoxications throughout Europe following the consumption of mussels (Mytilus edulis), harvested in Ireland. Shellfish intoxication is a consequence of toxin-bearing microalgae in the shellfish food chain, and these studies demonstrated a wide geographic distribution of toxic mussels along the entire western coastal region of Ireland. The first identification of azaspiracids in other bivalve mollusks including oysters (Crassostrea gigas), scallops (Pecten maximus), clams (Tapes phillipinarium), and cockles (Cardium edule) is reported. Importantly, oysters were the only shellfish that accumulated azaspiracids at levels that were comparable with mussels. The highest levels of total azaspiracids (microg/g) recorded to-date were mussels (4.2), oysters (2.45), scallops (0.40), cockles (0.20), and clams (0.61). An examination of the temporal variation of azaspiracid contamination of mussels in a major shellfish production area revealed that, although maximum toxin levels were recorded during the late summer period, significant intoxications were observed at periods when marine dinoflagellate populations were low. Although human intoxications have so far only been associated with mussel consumption, the discovery of significant azaspiracid accumulation in other bivalve mollusks could pose a threat to human health.

Animals↗
PubMedDate not supplied

Paralytic shellfish poison reference materials: an intercomparison of methods for the determination of saxitoxin.

Within the framework of the European Commission's Measurements and Testing Programme (BCR) a project has been undertaken to develop shellfish reference materials for Paralytic Shellfish Poisons (PSP). In a preliminary phase of the project, an intercomparison study of methods was undertaken. In this exercise 18 laboratories were asked to analyse solutions of saxitoxin and PSP-containing shellfish extracts with a method of their choice. The study revealed that: all the methods considered (four HPLC methods, one ELISA method) were in principle adequate for the quantification of saxitoxin in solution in the absence of interfering substances (Coefficient of variation (CV) 33% at a concentration of 0.5 microgram/ml); three of the HPLC methods used were able to quantify saxitoxin in PSP-positive mussel extract, the fourth method gave significant overestimation; the CV of all HPLC results was 53% at a mean saxitoxin mass fraction of 2.06 mg/kg mussel meat, the recoveries varied from 59-173%; and the ELISA method grossly overestimated the saxitoxin content in mussel extract, probably due to cross reactions of the antibodies with other PSP. The feasibility of preparing a homogeneous batch of ampouled mussel extracts (CV 3.5% at a saxitoxin concentration of approximately 1.5 mg/kg shellfish), sufficiently stable for at least 4 months storage both at 4 degrees C and approximately 20 degrees C, was demonstrated. The performance of the different methods for the analysis of PSP other than saxitoxin has not yet been evaluated, due to the current lack of PSP standards. Some of the problems observed in the intercomparison study were partly due to the nature of the chromatographic columns used, the composition of the HPLC mobile phase (pH, ion strength), non-optimal conditions for derivatization and matrix interference. Following the outcome of this study, a three year multistage project involving 15-20 European laboratories has been initiated, aimed at improving the accuracy and comparability of PSP measurements as well as preparing reference materials for PSP.

Animals↗
PubMed2005

Persistent organochlorine pollutants in surface sediments and economic shellfish of Minjiang Estuary-Mazu and Xiamen-Jinmen sea areas.

Samples of surface sediment and shellfish were collected from Minjiang Estuary-Mazu and Xiamen-Jinmen sea areas and were analyzed for hexachlorocyclohexanes (HCHs), dichlorodiphenyl-trichlorophenyl (DDT) and its degraded derivatives dichloro-diphenyl-dichloroethane (DDT) and dichloro-dichlorophenyl-ethylene (DDE), and polychlorinated biphenyls (PCBs) by gas chromatography. Concentration ranges of HCHs, DDTs, and PCBs in the sediments were 0.03-0.48, 1.10-14.3, and ND to 0.76 ng/g dw, respectively; those for shellfish samples were ND to 1.5, 21.5-892, dw and ND-3.7 ng/g dw, respectively. The results showed that the concentrations of such oganochlorine compounds (OCs) in the sediments were usually low as compared with other sea areas and estuaries, and the organochlorines were accumulated in marine bivalve mollusks, particularly in oyster. With respect to the concentrations of HCHs, DDTs, and PCBs in the samples of either sediment or shellfish, DDTs were higher than HCHs and PCBs. The concentrations of OCs in shellfish samples varied with the sampling locations and organism species but were generally higher than those in the sediments. The concentration distributions, compositions, and residue levels of these OCs are extensively discussed and evaluated.

Animals↗
PubMed1986

A tentative national reference procedure for isolation and enumeration of Escherichia coli from bivalve molluscan shellfish by most probable number method.

In the UK several quantitative methods exist for the examination of bivalve molluscan shellfish for sewage contamination. These methods include roll tubes, pour plates and most probable number (MPN) techniques, but there is no national standard method. A comparative study was made of the most commonly used methods for detection of Escherichia coli in bivalve shellfish. Schemes employing solid media, such as the roll tube and pour plate methods, underestimated faecal contamination in shellfish tissue compared with a liquid MPN multiple test-tube method using minerals-modified-glutamate broth (MMGB) as primary enrichment medium. The composition of MMGB apparently permits repair of sublethally injured cells of E. coli. Incorporation of resuscitation stages into the pour plate technique did not yield higher counts. A standardized MPN technique for examination of bivalve molluscan shellfish for E. coli content is proposed as a possible national reference procedure pending further collaborative assessment.

Animals↗
PubMed1983

Accumulation of sediment-associated viruses in shellfish.

The present study focused on the importance of contaminated sediments in shellfish accumulation of human viruses. Epifaunal (Crassostrea virginica) and infaunal (Mercenaria mercenaria) shellfish, placed on or in cores, were exposed to either resuspended or undisturbed sediments containing bound poliovirus type 1 (LSc 2ab). Consistent bioaccumulation by oysters (four of five trials) was only noted when sediment-bound viruses occurred in the water column. Virus accumulation was observed in a single instance where sediments remained in an undisturbed state. While the incidence of bioaccumulation was higher with resuspended rather than undisturbed contaminated sediment, the actual concentration of accumulated viruses was not significantly different. The accumulation of viruses from oysters residing on uninoculated sediments. When clams were exposed to undisturbed, virus-contaminated sediments, two of five shellfish pools yielded viral isolates. Bioaccumulation of undisturbed sediments by these bivalves was considered marginal when related to the concentration of virus in contaminated sediments; they would only represent a significant threat when suspended in the water column. Arguments were advanced for water-column sampling in the region of the water-sediment interface to provide an accurate determination of the virological quality of shellfish harvesting waters.

Animals↗
PubMed1997

Evidence for paralytic shellfish poisons in the freshwater cyanobacterium Lyngbya wollei (Farlow ex Gomont) comb. nov.

Lyngbya wollei (Farlow ex Gomont) comb. nov., a perennial mat-forming filamentous cyanobacterium prevalent in lakes and reservoirs of the southeastern United States, was found to produce a potent, acutely lethal neurotoxin when tested in the mouse bioassay. Signs of poisoning were similar to those of paralytic shellfish poisoning. As part of the Tennessee Valley Authority master plan for Guntersville Reservoir, the mat-forming filamentous cyanobacterium L. wollei, a species that had recently invaded from other areas of the southern United States, was studied to determine if it could produce any of the known cyanotoxins. Of the 91 field samples collected at 10 locations at Guntersville Reservoir, Ala., on the Tennessee River, over a 3-year period, 72.5% were toxic. The minimum 100% lethal doses of the toxic samples ranged from 150 to 1,500 mg kg of lyophilized L. wollei cells-1, with the majority of samples being toxic at 500 mg kg-1. Samples bioassayed for paralytic shellfish toxins by the Association of Official Analytical Chemists method exhibited saxitoxin equivalents ranging from 0 to 58 micrograms g (dry weight)-1. Characteristics of the neurotoxic compound(s), such as the lack of adsorption by C18 solid-phase extraction columns, the short retention times on C18 high-performance liquid chromatography (HPLC) columns, the interaction of the neurotoxins with saxiphilin (a soluble saxitoxin-binding protein), and external blockage of voltage-sensitive sodium channels, led to our discovery that this neurotoxin(s) is related to the saxitoxins, the compounds responsible for paralytic shellfish poisonings. The major saxitoxin compounds thus far identified by comparison of HPLC fluorescence retention times are decarbamoyl gonyautoxins 2 and 3. There was no evidence of paralytic shellfish poison C toxins being produced by L. wollei. Fifty field samples were placed in unialgal culture and grown under defined culture conditions. Toxicity and signs of poisoning for these laboratory-grown strains of L. wollei were similar to those of the field collection samples.

Amphibian Proteins↗