Shellfish Flesh Research Articles

Impact of weather conditions on Escherichia coli accumulation in oysters of the Thau lagoon (the Mediterranean, France)

The Thau lagoon shellfish are regularly impacted by microbial pollution of faecal origin, which cause European health standards to be exceeded and closure of the shellfish harvest. The aims of this study were to investigate the impact of weather and hydrological conditions on Escherichia coli (E. coli) accumulation factor (AF) between water and oysters and to evaluate the relevance of the use of this ratio for the purpose of sanitary risk assessment. Water and oysters (Crassostrea gigas) were sampled simultaneously in situ during 18 months in periods of dry weather and after rainfall events. Shellfish sanitary thresholds were exceeded in both periods. The E. coli AFs measured after rainfall (median = 6) were lower than in dry weather (median = 32), suggesting different shellfish faecal contaminations were operating in this system process. The AFs we measured appeared to be relevant markers for generating sanitary risk assessments for Thau lagoon shellfish. The results of the study address the need to assess the relationship between water quality measures and shellfish flesh quality. This study will contribute to the elaboration of a management tool to guide local authorities in prioritizing the sources of pollution and in optimizing public investment in the watershed.

Determination of Brevetoxins in Shellfish by LC/MS/MS: Single-Laboratory Validation

A single-laboratory validation is reported for an LC/MS/MS quantification of six brevetoxins in four matrixes (Greenshell mussel, eastern oyster, hard clam, and Pacific oyster). Recovery and precision data were collected from seven analytical batches using shellfish flesh at 0.05 mg/kg. Method recoveries and within-laboratory reproducibility ranged from 73 to 112%, with an RSD between 14 and 18% for brevetoxin-3, brevetoxin B5, brevetoxin B2, and S-desoxy brevetoxin B2. The recovery and within-laboratory reproducibility for brevetoxin-2 was 61%, with an RSD of 27%. Brevetoxin B1 gave an RSD of 12%, but no reference material was available and this toxin was only recorded in a hard clam sample naturally contaminated with brevetoxins. One naturally contaminated sample of each shellfish matrix, with brevetoxin levels ranging from 0.012 to 9.9 mg/kg, was tested in multiple batches, and the RSDs were similar to those for fortified samples at 0.05 mg/kg. Comparisons with limited data for the neurotoxic shellfish poisoning mouse bioassay for four naturally contaminated shellfish samples showed that the regulatory action limit of 0.8 mg/kg is conservative with respect to the bioassay regulatory limit of 20 mouse units/100 g.

A sensitive assay for palytoxins, ovatoxins and ostreocins using LC-MS/MS analysis of cleavage fragments from micro-scale oxidation

Palytoxin is a highly toxic non-proteinaceous marine natural product that can pass through the food chain and result in human illnesses. A recent review by the European Food Safety Authority concluded that palytoxin requires regulation in seafood and a limit of 30 μg kg−1 for shellfish flesh was suggested. Current methods based on LC-MS detection of intact palytoxins do not have sufficient sensitivity to enforce this limit for palytoxin. To improve sensitivity for trace analysis, a novel screen approach has been developed that uses LC-MS/MS analysis of substructures generated by oxidative cleavage of vicinal diol groups present in the intact toxin. Oxidation of palytoxins, ovatoxins or ostreocins using periodic acid generates two nitrogen-containing aldehyde fragments; an amino aldehyde common to these toxins, and an amide aldehyde that may vary depending on toxin type. Conditions for micro-scale oxidation of palytoxin were optimised, which include a novel SPE cleanup and on-column oxidation step. Rapid analysis of cleavage fragments was established using LC-MS/MS. Linear calibrations were established for the amino aldehyde from a palytoxin reference standard, which is suitable for all known palytoxin-like compounds, and for the confirmatory amide aldehydes of palytoxin and ostreocin-D. Palytoxin recoveries (at 10 μg kg−1) from shellfish and fish tissues were 114–119% (as amine aldehyde) and 90–115% (as amide aldehyde) with RSDs for both of ≤18% (all tissues, n = 12). The method LOD was determined to be approximately 1 ng mL−1 and the LOQ 4 ng mL−1, which corresponds to 10 μg kg−1 in tissue (flesh of shellfish or fish). The method has potential for use in research and is sufficiently sensitive for regulatory testing, should it be required.

Comparative Evaluation of Enzyme-Linked Immunoassay and Reference Methods for the Detection of Shellfish Hydrophilic Toxins in Several Presentations of Seafood

A comparative study was conducted to determine the feasibility of enzyme-linked immunosorbent assays (ELISAs) for the detection of amnesic shellfish poisoning (ASP) and paralytic shellfish poisoning (PSP) toxins in nine naturally contaminated species in fresh, frozen, boiled and canned fish and shellfish. PSP and ASP were analyzed in 138 shellfish samples (mussels, clams, barnacles, razor shells, scallops and cockles) and anchovies by mouse bioassay (MBA) and high performance liquid chromatography with ultraviolet detection (HPLC-UV), respectively. Results were compared with toxin concentrations obtained using two commercial competitive ELISAs, saxitoxin and ASP kits. Immunoassays were able to quantify toxins in different matrices showing excellent Pearson's correlation coefficients (r = 0.974 for saxitoxin ELISA and r = 0.973 for ASP ELISA) and to detect PSP and ASP with a lower limit of detection (LOD), namely, 50 microg saxitoxin equivalent/kg shellfish meat for PSP and 60 microg/kg domoic acid in shellfish flesh for ASP, than the reference methods (350 microg saxitoxin equivalent/kg shellfish meat and 1.6 mg/kg domoic acid in shellfish flesh, respectively). These results suggest that the ELISA method could be used as screening systems in a variety of species without matrix interference.

Comparative risk of type 2 diabetes mellitus among vegetarians and non-vegetarians

Sir, The prevalence of type 2 diabetes in vegetarians was compared to that in non-vegetarians in 724 people in the Bijapur district of Karnataka in a hospital-based survey. The type of vegetarian diet was categorized based on a food-frequency questionnaire. A vegetarian diet, in the broad sense, is defined as the one that does not include meat, fish, or fowl. An ovolactovegetarian is a vegetarian who does not eat beef, lamb, pork, poultry, fish, shellfish, or animal flesh of any kind, but is willing to consume dairy and egg products. Pescetarianism, also called pescovegetarianism, is the practice of a diet that includes seafood and excludes mammals and birds. In addition to fish or shellfish, a pescetarian diet typically includes some or all of vegetables, fruit, nuts, grains, beans, eggs, and dairy. Vegetarian diets offer a number of nutritional benefits including lower levels of saturated fat, cholesterol, and animal protein as well as higher levels of carbohydrates, fiber, magnesium, potassium, folate, antioxidants such as vitamins C and E, and phytochemicals.(1)Vegans tend to be thinner, have lower serum cholesterol, and lower blood pressure, reducing their risk of heart diseases and diabetes mellitus. However, eliminating all animal products from the diet increases the risk of certain nutritional deficiencies. Micronutrients of special concern for the vegans include vitamins B12and D, calcium, and long-chain n-3 (omega-3) fatty acids.(2)On the other hand, the omission of meat and fish from the diet increases the risk of certain nutritional deficiencies.(3)There is convincing evidence that vegetarians have lower rates of coronary heart disease, largely explained by low LDL cholesterol, probable lower rates of hypertension and diabetes mellitus, and lower prevalence of obesity.(4)In our study, mean BMI was lowest in vegans (23.9 kg/m2) and incrementally higher in ovolactovegetarians (25.9 kg/m2), pescovegetarians (26.4 kg/m2), semivegetarians (27.36 kg/m2), and non-vegetarians (29.2 kg/m2). The prevalence of type 2 diabetes increased from 3.1% in vegans to 8.2% in non-vegetarians; the prevalence was intermediate in ovolactovegetarians (3.6%), pescovegetarians (4.9%), or semi-vegetarians (6.4%). After adjustment for age, sex, ethnicity, education, income, physical activity, television watching, sleep habits, alcohol, and BMI, vegans (OR 0.53 [95% CI 0.36–0.60]), ovolactovegetarians (0.55 [0.47–0.64]), pescovegetarians (0.68 [0.61–0.78]), and semi-vegetarians (0.79 [0.64–0.88]) had a lower risk of type 2 diabetes than non-vegetarians. The results show that increased conformity to vegetarian diets protected against risk of type 2 diabetes.

Evaluation of Fecal Pollution Indicators in an Oyster Quality Assurance Program: Application of Epidemiological Methods

Shellfish Quality Assurance programs rely on the use of bacteriological fecal pollution indicators as a routine monitoring tool for risk assessment and management in shellfish growing areas. Internationally, shellfish programs vary in detail but are typically based on either the United States model, as in Australia, which relies on the enumeration of indicator bacteria in water as a measure of fecal pollution, or the European Union model, which assesses the exposure of production sites to fecal pollution by determining indicator bacteria present in shellfish flesh. The equivalency of these approaches for delivering the same level of public health protection is not immediately apparent Both indicator tests are used in combination in a very conservative approach adopted in the Australian state of New South Wales (NSW) by the Shellfish Program for the oyster (predominately Sydney rock oyster) fishery, the country's oldest and the state's most productive fishery. This study reports on the analysis of a large data set of these two indicators taken at multiple sites within three NSW estuaries between 2000 and 2005. We report on performance measures used routinely in epidemiological studies to compare the two tests. These measures demonstrate poor agreement between the two tests. When the harvest area is deemed closed by the water test the odds of a negative test based on shellfish flesh is approximately one in four. Conversely, the odds of a positive test based on shellfish flesh occurring during extended periods of dry weather when the water test is clear exceed one in five and are seemingly random in occurrence. We are able to demonstrate a highly significant statistical relationship between the water test and environmental covariates of rainfall, salinity, and water temperature. We use k-fold cross validation methods to develop predictive models using these environmental covariates that can account for over 90% of the variation in water test readings. In contrast we are unable to demonstrate a valid predictive model for the oyster flesh test using these covariate. We discuss the results of these analyses and suggest that there are significant issues with the efficacy of the shellfish flesh test and, in particular, the unexplained systematic error that occurs at a high rate in the current program. The dual use of these tests in harvest management results in routine uncertainty that must create significant unpredictability and costs in trade. In turn, this uncertainty and the costs of testing result in levels of commercial risk that could prove unsustainable. The study highlights the need for the performance of tests to be rigorously appraised in shellfish quality assurance programs. In so doing it may be possible to maintain public health standards while minimizing unnecessary disruptions and costs in the trade of fresh oysters.

Influence of sampling depth and post-sampling analysis time on the occurrence of coliforms and vibrio in water and shellfish

The bacteriological quality was examined at the water surface, 3 m depth and in the shellfish flesh, and the results were compared to other potential pathogenic indicator organisms. The study was conducted at Long Harbour (mussel farm), St. John's and Outer Cove sites of Newfoundland, Canada. Bacteriological analysis was carried out for samples taken at water depth and at 1, 6, 12 and 24 hours post-sampling. It was observed that the total and faecal coliform bacteria were significantly higher in the 3 m water depth samples than in the surface water samples (ANOVA, F = 59.41, 26.751, 9.82 (T.C); 46.41, 26.81, 10.72 (F.C); P

Shellfish toxicity in UK waters: a threat to human health?

The potential for poisoning of humans through their consumption of shellfish which have themselves consumed biotoxin producing marine phytoplankton exists in the UK. Toxins are bio-accumulated within the shellfish flesh allowing them to reach harmful concentrations. This threat is in most part mitigated by monitoring programmes that assess both the presence of potentially harmful phytoplankton and shellfish flesh toxicity. However, the medical profession in the UK remains relatively ignorant of the potential for biotoxin derived shellfish toxicity, preventing quantification of magnitude, frequency, and severity of health effects in the community or the medical significance of more recently discovered toxins. While the current causative species and their toxins are relatively well characterised there remains a lack of understanding of the factors governing the temporal and spatial appearance of harmful phytoplankton. Expansion of shellfish aquaculture is likely both worldwide and in the UK. Better understanding of how harmful phytoplankton interact with their environment to promote the sporadic harmful blooms that we observe is required to underpin risk assessments.

Azaspiracid Shellfish Poisoning: A Review on the Chemistry, Ecology, and Toxicology with an Emphasis on Human Health Impacts

Azaspiracids (AZA) are polyether marine toxins that accumulate in various shellfish species and have been associated with severe gastrointestinal human intoxications since 1995. This toxin class has since been reported from several countries, including Morocco and much of western Europe. A regulatory limit of 160 μg AZA/kg whole shellfish flesh was established by the EU in order to protect human health; however, in some cases, AZA concentrations far exceed the action level. Herein we discuss recent advances on the chemistry of various AZA analogs, review the ecology of AZAs, including the putative progenitor algal species, collectively interpret the in vitro and in vivo data on the toxicology of AZAs relating to human health issues, and outline the European legislature associated with AZAs.

Azaspiracid Shellfish Poisoning: A Review on the Chemistry, Ecology, and Toxicology with an Emphasis on Human Health Impacts

Azaspiracids (AZA) are polyether marine toxins that accumulate in various shellfish species and have been associated with severe gastrointestinal human intoxications since 1995. This toxin class has since been reported from several countries, including Morocco and much of western Europe. A regulatory limit of 160 μg AZA/kg whole shellfish flesh was established by the EU in order to protect human health; however, in some cases, AZA concentrations far exceed the action level. Herein we discuss recent advances on the chemistry of various AZA analogs, review the ecology of AZAs, including the putative progenitor algal species, collectively interpret the in vitro and in vivo data on the toxicology of AZAs relating to human health issues, and outline the European legislature associated with AZAs.

Results of field investigations into the impact of intermittent sewage discharges on the microbiological quality of wild mussels ( Mytilus edulis) in a tidal estuary

Field surveys were designed to examine the effects of sewage contamination from storm overflow effluent on faecal coliform and Escherichia coli concentrations in the flesh of wild mussels ( Mytilus edulis). Bags containing 30 mussels each were fixed at known inter-tidal locations and retrieved at intervals following discharge from a nearby combined sewer overflow (CSO). Concentrations of faecal coliform bacteria and E. coli were measured in the shellfish flesh and in samples of overlying water prior to collection of the mussel samples. Faecal coliform and E. coli concentrations in shellfish increased rapidly after CSO discharge. E. coli concentrations exceeded the European shellfish hygiene class C limit of 46,000 100 g −1, and decayed during subsequent CSO discharge-free periods. The concentration and depuration response was independent of the magnitude of CSO spill volume. First-order exponential decay functions were fitted to the data. Decay rates were lower than those found in corresponding microcosm experiments. This relates to the repeated pattern of inundation and exposure associated with the tidal cycles in the estuary. Relationships between E. coli and faecal coliform concentrations in the shellfish and overlying water samples were relatively weak ( r<0.60), a pattern often seen with data from uncontrolled environmental experiments.

Determination of Domoic Acid Toxins in Shellfish by Biosense ASP ELISAA Direct Competitive Enzyme-Linked Immunosorbent Assay: Collaborative Study

A collaborative study was conducted on the Biosense amnesic shellfish poisoning (ASP) enzyme-linked immunosorbent assay (ELISA) for the determination of domoic acid (DA) toxins in shellfish in order to obtain interlaboratory validation data for the method. In addition, a method comparison study was performed to evaluate the ASP ELISA as an alternative to the current liquid chromatography (LC) reference method for DA determination. The study material comprised 16 shellfish samples, including blue mussels, Pacific oysters, and king scallops, spiked with contaminated mussel homogenates to contain 0.1-20 mg DA/kg shellfish flesh. The shellfish samples were extracted with 50% aqueous methanol, and the supernatants were directly analyzed. Sixteen participating laboratories in 10 countries reported data from the ASP ELISA, and 4 of these laboratories also reported data from instrumental LC analysis. The participating laboratories achieved interlaboratory precision estimates for the 8 Youden paired shellfish samples in the range of 10-20% for RSD(r) (mean 14.8 +/- 4%), and 13-29% for RSDR (mean 22.7 +/- 6%). The precision estimates for the ELISA data did not show a strong dependence on the DA concentration in the study samples, and the overall precision achieved was within the acceptable range of the Horwitz guideline with HorRat values ranging from 1.1 to 2.4 (mean HorRat 1.7 +/- 0.5). The analysis of shellfish samples spiked with certified reference material (CRM)-ASP-MUS-b gave recoveries in the range of 88-122%, with an average recovery of 104 +/- 10%. The estimate on method accuracy was supported by a correlation slope of 1.015 (R2 = 0.992) for the determined versus the expected DA values. Furthermore, the correlation of the ASP ELISA results with those for the instrumental LC analyses of the same sample extracts gave a correlation slope of 1.29 (R2 = 0.984). This indicates some overestimation of DA levels in shellfish by the ELISA, but it is also a result of apparent low recoveries for the LC methods. This interlaboratory study demonstrates that the ASP ELISA is suitable for the routine determination and monitoring of DA toxins in shellfish, and that it offers a rapid and cost-effective methodology with high sample throughput.

Development of an ultra-performance liquid chromatography–mass spectrometry method for the detection of lipophilic marine toxins

A rapid method for the detection of marine toxins was developed using an ultra-performance liquid chromatography (UPLC) system coupled to a latest generation mass spectrometry (MS) system. The analysis of 21 lipophilic marine toxins was achieved on an Acquity C18 column using a water–acetonitrile gradient with a cycle time of 6.6 min, reducing analysis time by more than a factor two compared to HPLC while maintaining peak resolution. Linear ranges, limits of detection and limits of quantification were established for okadaic acid (OA), pectenotoxin-2, azaspiracid-1 (AZA1), yessotoxin, gymnodimine and 13-desmethylspirolide C. The method was found to be accurate when using a triplicate methanolic extraction. Matrix effects were assessed by standard addition of OA and AZA1 in extracts of raw and heat-treated flesh of mussels and oysters. For the analysis of AZA1, the UPLC–MS method was always prone to signal suppression, while for OA analysis signal suppression was observed in extracts of raw shellfish flesh and signal enhancement in extracts of heat-treated flesh. Matrix effects occurring in the method presented are diminished compared to previous studies.

Assessment of fitness for purpose of an insect bioassay using the desert locust ( Schistocerca gregaria L.) for the detection of paralytic shellfish toxins in shellfish flesh

We have developed a bioassay using 5th instar desert locusts ( Schistocerca gregaria L.) for the detection of saxitoxin—the paralytic shellfish poison in shellfish flesh. The bioassay procedure is to inject 10 locusts with a shellfish extract, and assess their conditions at time points up to 2 h post injection, looking for an endpoint of paralysis. From the proportion responding, the equivalent dose of pure saxitoxin could be estimated. Performance characteristics of the bioassay were assessed using shellfish samples spiked with saxitoxin, and we found the bioassay could detect and quantify toxin levels in the range of regulatory relevance. Relative toxicities of selected saxitoxin analogues differed from those reported in mammalian systems. Variation for repeatability conditions was acceptable but variation was higher under reproducibility conditions. This was related to (a) batches of insects from different suppliers, (b) different operators, and (c) different observers assessing the endpoint. We also noted adverse reactions with some shellfish species. These problems may be resolved by further refinement of the method and operator training, before formal validation. Nevertheless, we suggest the method potentially offers a simple, ethically acceptable, broad-specificity functional bioassay, which is desirable in any toxin-monitoring programme.

Effect of storage on amnesic shellfish poisoning (ASP) toxins in king scallops (Pecten maximus)

Abstract Since 1998, king scallops ( Pecten maximus ) obtained from Scottish offshore sites have been monitored for domoic acid (DA) and epi -domoic acid ( epi -DA), the principal toxic compounds associated with amnesic shellfish poisoning (ASP). The presence of these toxins in king scallops harvested from Scottish waters at concentrations exceeding the current regulatory limit (20 μg g −1 shellfish flesh) is a recurrent event. However, little information was available to determine the effects that different storage conditions experienced during sample transportation to the monitoring laboratory may have on the toxin concentrations, which are subsequently detected. Furthermore, the stability of DA and epi -DA in the solvents (methanol:water (1:1, v/v) and citric acid buffer (0.5 M, pH 3.2)) routinely used for their extraction from shellfish has not previously been assessed. Results from this study demonstrate that when king scallop samples were stored for 2–3 days at 12 °C, a significantly higher toxin concentration was detected in the gonad than when samples were stored at 4 °C and analysed within 48 h. This implies that monitoring programmes must consider transport and storage conditions between harvest and analysis. Stability studies showed rapid decomposition of DA and epi -DA in aqueous methanol extracts while DA and epi -DA seem acceptably stable when stored refrigerated in citrate buffer.

Assessment of MIST Alert™, a Commercial Qualitative Assay for Detection of Paralytic Shellfish Poisoning Toxins in Bivalve Molluscs

A recently developed commercial rapid test kit (MIST Alert) was assessed for determination of the presence of paralytic shellfish poisoning (PSP) toxins in shellfish. Several commercially important shellfish species obtained from the UK shellfish toxin monitoring program, containing a range of total PSP toxicities as determined by the mouse bioassay (MBA), were tested. The kit detected toxin in all samples containing the European Community tolerance level of 80 microg saxitoxin (STX) equivalents/100 g shellfish flesh as determined by the MBA. With one exception, the kit detected toxin in all samples that contained >40 microg STX equivalents/100 g according to the MBA. Among samples in which the MBA did not detect toxin, the kit disagreed in 25% of the tests, although further analysis by liquid chromatography (LC) and MBA of some samples confirmed the presence of toxins. These results suggest that MIST Alert may be suitable as an initial screen for PSP toxins as part of routine monitoring programs, thereby greatly reducing the number of MBAs. Trials were also performed by nonscientific personnel to evaluate the ease of use and interpretation of results obtained by MIST Alert. The results indicated that the kits could be readily used and accurately interpreted by individuals with no technical or scientific background.

The NIST Natural Matrix Radionuclide Standard Reference Material Program for Ocean Studies

In 1977, the Low-level Working Group of the International Committee on Radionuclide Metrology met in Boston, MA (USA) to define the characteristics of a new set of environmental radioactivity reference materials. These reference materials were to provide the radiochemist with the same analytical challenges faced when assaying environmental samples. It was decided that radionuclide bearing natural materials should be collected from sites where there had been sufficient time for natural processes to redistribute the various chemically different species of the radionuclides. Over the succeeding years, the National Institute of Standards and Technology (NIST), in cooperation with other highly experienced laboratories, certified and issued a number of these as low-level radioactivity Standard Reference Materials (SRMs) for fission and activation product and actinide concentrations. The experience of certifying these SRMs has given NIST the opportunity to compare radioanalytical methods and learn of their limitations. NIST convened an international workshop in 1994 to define the natural-matrix radionuclide SRM needs for ocean studies. The highest priorities proposed at the workshop were for sediment, shellfish, seaweed, fish flesh and water matrix SRMs certified for mBq per sample concentrations of 90 Sr, 137 Cs and 239 Pu + 240 Pu. The most recent low-level environmental radionuclide SRM issued by NIST, Ocean Sediment (SRM 4357) has certified and uncertified values for the following 22 radionuclides: 40 K, 90 Sr, 129 I, 137 Cs, 155 Eu, 210 Pb, 210 Po, 212 Pb, 214 Bi, 226 Ra, 228 Ra, 228 Th, 230 Th, 232 Th, 234 U, 235 U, 237 Np, 238 U, 238 Pu, 239 Pu + 240 Pu, and 241 Am. The uncertainties for a number of the certified radionuclides are non-symmetrical and relatively large because of the non-normal distribution of reported values. NIST is continuing its efforts to provide the ocean studies community with additional natural matrix radionuclide SRMs. The freeze-dried shellfish flesh matrix has been prepared and recently sent to participating laboratories for analysis and we anticipate receiving radioanalytical results in 2000. The research and development work at NIST produce well characterized SRMs that provide the world's environment-studies community with an important foundation component for radionuclide metrology.

Evaluation of F-specific RNA bacteriophage as a candidate human enteric virus indicator for bivalve molluscan shellfish.

Escherichia coli is a widely utilized indicator of the sanitary quality of bivalve molluscan shellfish sold for human consumption. However, it is now well documented that shellfish that meet the E. coli standards for human consumption may contain human enteric viruses that cause gastroenteritis and hepatitis. In this study we investigated using F-specific RNA bacteriophage (FRNA bacteriophage) to indicate the likely presence of such viruses in shellfish sold for consumption. FRNA bacteriophage and E. coli levels were determined over a 2-year period for oysters (Crassostrea gigas) harvested from four commercial sites chosen to represent various degrees of sewage pollution. Three sites were classified as category B sites under the relevant European Community (EC) Directive (91/492), which required purification (depuration) of oysters from these sites before sale. One site was classified as a category A site, and oysters from this site could be sold directly without further processing. Samples were tested at the point of sale following commercial processing and packaging. All of the shellfish complied with the mandatory EC E. coli standard (less than 230 per 100 g of shellfish flesh), and the levels of contamination for more than 90% of the shellfish were at or below the level of sensitivity of the assay (20 E. coli MPN per 100 g), which indicated good quality based on this criterion. In contrast, FRNA bacteriophage were frequently detected at levels that exceeded 1,000 PFU per 100 g. High levels of FRNA bacteriophage contamination were strongly associated with harvest area fecal pollution and with shellfish-associated disease outbreaks. Interestingly, FRNA bacteriophage contamination exhibited a marked seasonal trend that was consistent with the trend of oyster-associated gastroenteritis in the United Kingdom. The correlation between FRNA bacteriophage contamination and health risk was investigated further by using a reverse transcription-PCR assay for Norwalk-like virus (NLV). NLV contamination of oysters was detected only at the most polluted site and also exhibited a seasonal trend that was consistent with the trend of FRNA bacteriophage contamination and with the incidence of disease. The results of this study suggest that FRNA bacteriophage could be used as viral indicators for market-ready oysters.

Polyclonal antibodies to domoic acid, and their use in immunoassays for domoic acid in sea water and shellfish

Ovine antibodies raised against conjugates linked through the secondary amino group of domoic acid (1) were used, together with activated-ester-derived conjugates of domoic acid (DA) as the plate coater, to develop a robust indirect competitive enzyme-linked immunosorbent assay (cELISA) for DA in shellfish and seawater. The ELISA was used to analyze shellfish samples for DA, and was compatible with several extraction procedures. The ELISA had a detection limit below 0.01 ng ml(-1), a limit of quantitation (LOQ) of 0.15 ng ml(-1) and a working range of 0.15-15 ng ml(-1) DA. The LOQ is equivalent to 38 ng g(-1) DA in shellfish flesh, assuming a 250-fold dilution during extraction. This is more than 500 times lower than the maximum permitted level (20 microg g(-1) flesh). The ELISA is designed for use alongside regulatory analyses, and, following formal validation, should be available for pre-screening of regulatory shellfish flesh samples. The ELISA was also shown to be appropriate for analysis of DA in algal cultures and in samples of seawater, and thus has the potential to provide early warning of developing algal blooms.

Pseudo-nitzschia in New Zealand and the role of DNA probes and immunoassays in refining marine biotoxin monitoring programmes

Domoic acid (DA) was first detected in shellfish in New Zealand after the implementation of a comprehensive biotoxin monitoring programme for amnesic, paralytic, diarrhetic and neurotoxic shellfish toxins, following a suspected neurotoxic shellfish poisoning (NSP) event in early 1993. Both phytoplankton monitoring and shellfish flesh testing programmes have led to an extensive database which has helped link species of Pseudo-nitzschia to specific DA outbreaks. In 1994, P. pungens and P. turgidula were associated with DA contamination of shellfish, and cultured isolates of these species proved to be toxin producers. During 1996 the use of species-specific ribosomal RNA (rRNA)-targeted oligonucleotide probes and DA immunoassays led to the discovery of toxin production by P. fraudulenta, and showed the nontoxic P. heimii to be a major bloom former. Pseudo-nitzschia delicatissima, P. pseudodelicatissima and P. multiseries, also identified using rRNA-targeted probes, have been linked to DA contamination of New Zealand shellfish; P. australis is the main cause of DA in scallops. The relative amnesic shellfish poisoning (ASP) risk associated with different species, largely determined by DA immunoassays of cultured isolates, is now used by some regulators to refine risk assessments. Species identification is therefore vital so that shellfish growers, and health and industry officials, can make safe and economically sound harvesting decisions. The development and field trialling of DNA probes is proving invaluable in this context.