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PubMed2005

Human intoxication with paralytic shellfish toxins: clinical parameters and toxin analysis in plasma and urine.

This study reports the data recorded from four patients intoxicated with shellfish during the summer 2002, after consuming ribbed mussels (Aulacomya ater) with paralytic shellfish toxin contents of 8,066 +/- 61.37 microg/100 gr of tissue. Data associated with clinical variables and paralytic shellfish toxins analysis in plasma and urine of the intoxicated patients are shown. For this purpose, the evolution of respiratory frequency, arterial blood pressure and heart rate of the poisoned patients were followed and recorded. The clinical treatment to reach a clinically stable condition and return to normal physiological parameters was a combination of hydration with saline solution supplemented with Dobutamine (vasoactive drug), Furosemide (diuretic) and Ranitidine (inhibitor of acid secretion). The physiological condition of patients began to improve after four hours of clinical treatment, and a stable condition was reached between 12 to 24 hours. The HPLC-FLD analysis showed only the GTX3/GTX2 epimers in the blood and urine samples. Also, these epimers were the only paralytic shellfish toxins found in the shellfish extract sample.

Aged↗
PubMed2000

Survey of paralytic toxins in shellfish in southern Taiwan between 1995 and 1997.

To establish the safety data of shellfish in southern Taiwan, a total of 3,074 specimens of 30 shellfish species were seasonally collected from August 1995 to March 1997. These samples were assayed for the presence of tetrodotoxin (TTX) and paralytic shellfish poisons (PSP) by the bioassay methods. It was found that some major shellfish including oyster, clam, ear shell, and purple clam were nontoxic, but four species, Babylonia formosae, Niotha clathrata, Natica lineata, and Natica vitellus, were toxic. The toxic percentages of these four species was 1% in B. formosae, 56% in N. clathrata, 37% in N. lineata, and 23% in N. vitellus. The toxic composition was TTX in B. formosae. In the other three shellfish types the toxic composition was TTX and PSP.

Animals↗
PubMed2006

Consumption of viscera as the most important risk factor in the largest outbreak of shellfish poisoning in Hong Kong, 2005.

Poisoning from shellfish toxins is associated with significant morbidity worldwide. During 10-15 March 2005, 36 clusters of shellfish poisoning related to consumption of fresh scallops (Atrina vexillum) were reported to the Center for Health Protection, Department of Health Hong Kong. We conducted a case-control study to identify the risk factors associated with shellfish poisoning. Detailed demographic, clinical, shellfish consumption data of these subjects were collected using standardized questionnaires. Fifty-eight cases and 44 controls were identified. The mean age for the cases was 37.5 years (range 10-81 years); 45% (n=26) were male. Ninety-five percent had onset of symptoms within 12 hours of consumption of scallops (range: 10 minutes to 30 hours, median: 45 minutes). Dizziness (87.9%) and blurred vision (53.4%) were the predominant symptoms. The mean number of pieces of scallop meat and viscera taken by the cases were 3.7 and 3.6, respectively, significantly higher than that for the controls, which were 1.6 (p<0.001) and 0.5 (p<0.001), respectively. Forty-two percent (n=22) and 19% (n=7) of cases and controls, respectively, took soup/sauce from the same dish that was cooked with the scallops (p=0.02). Consumption of scallop viscera was identified as the only significant risk factor (Adjusted OR=9.93, p=0.001) after adjusting for other risk factors. The result show that consumption of scallop viscera is an important risk factor for shellfish poisoning. The public should be warned specifically in health education messages to avoid eating viscera of scallops.

Adolescent↗
PubMedDate not supplied

Evaluation of laboratory performance of the AOAC method for PSP toxin in shellfish.

Laboratory performance of the official AOAC method for paralytic shellfish poison (PSP) toxin in shellfish was evaluated. Two series of naturally toxic shellfish split samples were distributed (15 in 1979 and 19 in 1982) to state shellfish-monitoring laboratories which participate in the National Shellfish Sanitation Program. The laboratories performed bioassays on duplicate 100 g portions of each 220 g split sample. Bioassays were consistent among the laboratories and compared favorably with those of previous studies.

Animals↗
PubMed1981

Absorption and transport of shellfish sterols in human subjects.

While individually cholesterol is the most abundant sterol in clams, oysters, and scallops, five different major noncholesterol sterols (a C-26 sterol, 22-dehydrocholesterol, brassicasterol, 24-methylene cholesterol, and a C-29 sterol) comprise some 60% of the total sterols, a situation unique among foods of animal origin that humans consume. In order to measure the intestinal absorption and transport of these noncholesterol shellfish sterols by humans, clams, oysters, and scallops were fed daily for 3-4 wk to 9 subjects and in a single meal to 8 subjects. The major shellfish sterols in the diet and in the plasma, individual plasma lipoproteins, red blood cells, and feces were analyzed by digitonin precipitation, thin-layer and gas-liquid chromatography-mass spectrometry. The absorption of these shellfish sterols was ascertained both by their appearance in the blood of the subjects fed shellfish and by their intake and output in the feces. After daily feeding for 3--4 wk, shellfish sterols were identified for the first time in the plasma lipids, in lipoproteins (chiefly in low-density lipoprotein), and in red blood cells of all subjects. The absorption of the dietary C-26 sterol, 22-dehydrocholesterol, brassicasterol, 24-methylene cholesterol was 54%, 35%, 4% and 11%, respectively. Our data indicated that the intestinal mucosa of humans absorbs these unusual sterols, and then they enter into the tissues and overall sterol metabolism of the body.

Adult↗
PubMed1980

Commentary on AOAC method for paralytic shellfish poisoning.

Paralytic shellfish poisoning (PSP) is caused by ingesting bivalve molluscan shellfish which have fed on the toxigenic marine dinoflagellates Gonyaulax catanella or G. tamarensis. The toxins from these organisms are neurotoxic alkaloids which interfere with nerve conduction and block muscle-action potential. The U.S. Food and Drug Administration cooperates with state shellfish control officials in the National Shellfish Sanitation Program to prevent marketing of toxin-contaminated shellfish. The toxins are quantitated by the standard mouse bioassay method, as found in Official Methods of Analysis of the AOAC. This paper discusses the procedure followed in the standard bioassay method in an attempt to clarify for the PSP analyst the rather complex official methodology, and, thus, promote more uniform results among laboratories.

Animals↗
PubMedDate not supplied

Shellfish hypersensitivity: clinical and immunological characteristics.

Shellfish is one of the most frequent causes of food allergy. We studied 48 patients (25 male and 23 female) with a mean age of 24.2 +/- 1.8 with shellfish hypersensitivity. A clinical questionnaire was carried out and prick tests were performed using a series of aeroallergens and a battery of extracts of squid, shrimp, lobster, crab, mussel and clam. Prick tests were also performed using raw and boiled extracts from fresh squid, octopus and limpet. Total and specific IgE to these allergens were determined. The most frequent causes of symptoms were shrimp (33 cases) and squid (24 cases). The most frequently found symptoms were Urticaria/angioedema (39 patients), asthma (18 patients) and rhinitis (14 patients). Clinical association was found between Cephalopoedae and Lamelibranquiae (p < 0.05 for clam and p < 0.01 for mussel), but not among both groups and Crustaceans. Association between history and Prick was statistically significant for Crustaceae and Cephalopoedae (p < 0.01) but not for Lamalibranquiae. Association between history and CAP was not found for shellfish. Significant differences among prick-tests with raw and boiled extracts were not found. These results suggest that prick test yields better results than CAP does it, in shellfish hypersensitivity, that clinical association among shellfish hypersensitivity can occurs within the same and different Phylum reflecting common epitopes and that squid, octopus and limpet extracts contain a large amount of heat-stable allergens.

Adolescent↗
PubMed1996

New molecular methods for the detection of hepatitis A and Norwalk viruses in shellfish.

Outbreaks of viral enteric diseases after consumption of shellfish are a major health risk. Methodological problems (such as toxicity for cell cultures and low viral concentrations) and the unculturability of some strains (i.e. hepatitis A virus, Norwalk virus) have made it difficult to study those viruses in the environmental samples. Currently, the analysis of the hygienic quality of marketable shellfish is determined by the use of fecal indicator bacteria, but their reliability in determining viral pollution of shellfish is very low. Recent biotechnology developments are providing available rapid, sensitive, and specific tools for detecting food-borne viruses in shellfish and in shellfish-growing waters. In this paper, a review of these new molecular methods is carried out, discussing their advantages and possible applications.

DNA Probes↗
PubMed1999

New approach to the direct detection of known and new diarrhoeic shellfish toxins in mussels and phytoplankton by liquid chromatography-mass spectrometry.

A new approach using combined liquid chromatography-mass spectrometry (LC-MS) with ionspray ionization is proposed for the direct detection of known and new toxins in mussels and phytoplankton. A first stage reversed-phase, negative ion mode, selected ion monitoring (SIM) LC-MS analysis was performed in order to detect DSP toxins in the same chromatographic run with a total run time of 20 min. The toxins analysed included yessotoxin (YTX), okadaic acid (OA) and four of its analogues, dinophysistoxins (i.e. DTX-1, DTX-2, DTX-2B, DTX-2C), and pectenotoxins (PTXs), involving PTX-2, two PTX-2 secoacids (PTX-2SAs), PTX-2SA, 7-epi-PTX-2SA, and AC1, the three isomeric toxins structurally related to PTX-2 recently identified in Irish phytoplankton. Positive samples can, therefore, be analyzed through reversed-phase, positive ion mode SIM LC-MS, in order to perform complete chromatographic separations of the structurally related toxins within the OA and PTX groups. Detailed toxin profiles of a number of toxic phytoplankton and shellfish, from different marine areas, were easily obtained through the new approach. PTX-2SAs and AC1 were found in phytoplankton and shellfish from Ireland as well as in Italian shellfish. Moreover, for the first time there was evidence of the presence of PTX-2 in Irish phytoplankton. YTX was present in Italian shellfish. Four isomeric OA toxins were detected in samples from Ireland with OA, DTX-2 and DTX-2B present in shellfish, and OA, DTX-2 and DTX-2C in phytoplankton. In contrast, OA was the only toxin from this group to be detected in Italian mussels.

Animals↗
PubMedDate not supplied

Limited selection of sodium channel blocking toxin-producing bacteria from paralytic shellfish toxin-contaminated mussels (Aulacomya ater).

Paralytic shellfish toxins (PSTs) are sodium channel blocking (SCB) toxins, produced by cyanobacteria, as well as by marine dinoflagellates and their associated bacteria, and cause serious health and economic concern worldwide. In a previous study, approximately 70% of the bacteria enriched from PST-contaminated shellfish tissue and isolated on marine agar medium were observed to produce SCB toxins. In the study reported here, the high percentage of cultivable toxigenic bacteria is demonstrated to be obtained through a marked selection on marine agar medium. The cultivable as well as the total bacterial diversity associated with PST-contaminated shellfish collected from the Magallanes region in the south of Chile has been analysed. Approximately 80% of bacterial isolates, analysed by restriction analysis of PCR amplified ribosomal DNA (i.e., ARDRA fingerprinting), were limited to only two genotypic OTUs (operational taxonomic unit). Sequence determination and analysis of the 16S rDNA from representative isolates of both OTUs established them to be closely related to species of the Psychrobacter genus of the gamma-subclass of the Proteobacteria. The total bacterial diversity in the shellfish was further analysed, using a cultivation-independent strategy of extraction of total DNA from contaminated tissue, PCR-amplification of bacterial 16S rRNA genes, cloning of the PCR products and analysis of the cloned 16S rDNA sequence types by fingerprinting and sequencing. Only 2% of the cloned sequence types corresponded to species of the Psychrobacter genus. The 16S rDNA sequence types detected clustered with species of the y-Proteobacteria subclass, the Cytophaga-Flexibacter-Bacteroides (CFB), the Fusobacteria and the Firmicutes phyla. The level of diversity observed within the libraries of cloned 16S rDNA was markedly greater than that observed among isolates obtained through marine agar enrichment cultures from the same shellfish tissue. Additionally the predominant cloned 16S rDNA sequence types detected from samples of the surrounding seawater demonstrated no correlation with those observed in the PST-contaminated mussels.

Animals↗
PubMedDate not supplied

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.

Animals↗
PubMed2005

Use of two-dimensional gel electrophoresis to differentiate morphospecies of Alexandrium minutum, a paralytic shellfish poisoning toxin-producing dinoflagellate of harmful algal blooms.

Contamination of shellfish with paralytic shellfish poisoning toxins (PST) produced by toxic harmful algal blooms (HABs) have been negatively affecting the shellfish and aquaculture industries worldwide. Therefore, accurate and early identification of toxic phytoplankton species is crucial in HABs surveillance programs that allow fish-farmers to take appropriate preventive measures in shellfish harvesting and other aquaculture activities to overcome the negative impacts of HABs on human health. The identification of toxic dinoflagellates present in the water is currently a time-consuming operation since it requires skillful taxonomists and toxicologists equipped with optical and scanning electron microscopes as well as sophisticated equipment, for example, high-performance liquid chromotography-fluorescence detection. In this paper, a two-dimensional gel electrophoresis (2-DE)-based proteomic approach was applied to discriminate between toxic and nontoxic strains of Alexandrium minutum. Variation in morphological features between toxic and nontoxic strains was minimal and not significant. Also, variation in 2-DE protein patterns within either toxic or nontoxic strains was low, but pronounced differences were detected between toxic and nontoxic strains. The most notable differences between these strains were several abundant proteins with pIs ranging from 4.8 to 5.3 and apparent molecular masses between 17.5 and 21.5 kDa. Groups of proteins, namely NT1, NT2, NT3, and NT4, were consistently found in all nontoxic strains, while T1 and T2 were prominent in the toxic strains. These specific protein spots characteristic for toxic and nontoxic strains remained clearly distinguishable irrespective of the various growth conditions tested. Therefore, they have the potential to serve as "taxonomic markers" to distinguish toxic and nontoxic strains within A. minutum. Initial studies revealed that the expression pattern of T1 was tightly correlated to toxin biosynthesis in the examined alga and may be used to serve as a potential toxin indicator.

Amino Acid Sequence↗
PubMed2003

The mouse bioassay for diarrhetic shellfish poisoning: a gross misuse of laboratory animals and of scientific methodology.

The UK shellfish industry has recently been affected by the statutory closure of several cockle beds, following the detection of samples causing rapid and severe reactions in the regulatory approved test for diarrhetic shellfish poisoning (DSP) toxins, the mouse bioassay (MBA). It is contended that these so-called atypical results are due to procedural artefacts of the MBA; so far, several studies have failed to identify their cause. This paper critically assesses the development, regulatory use and methodological deficiencies of the MBA. It also discusses how testing for DSP toxins could and should have been improved and made more humane by applying the Three Rs concept of Reduction, Refinement and Replacement, and by the proper validation of the test method used. It is concluded that the MBA should not have been developed for the routine screening of shellfish samples, as it has a substantially severe endpoint and is not used as part of a tiered-testing strategy with non-animal methods. Moreover, during the UK monitoring programme for DSP toxins, the assay has been used without an optimised and universal protocol, and apparently without due regard to the principles of basic scientific methodology. In view of this, the atypical results obtained for cockle samples cannot be relied on as evidence of a human health hazard. It is recommended that the use of the MBA should be discontinued as soon as possible, in favour of other methods, especially those involving non-animal techniques. In the short-term, these methods should be based on analytical chemical detection systems and the essential availability of the relevant pure toxin standards. The lack of any known toxins in samples should be taken as evidence of lack of contamination. The suitability of the existing non-animal methods needs to be assessed as a matter of urgency. It is crucial that all new methods should be properly validated, and that their acceptability for their stated purposes should be endorsed by recognised criteria and validation centres, before being recommended to, or required by, regulatory agencies. In this way, the possibility that scientifically unsuitable methods will once again be used for monitoring for the contamination of shellfish with toxins can be avoided. This gross misuse of laboratory animals and ill-judged application of science should never be allowed to occur again.

Animal Testing Alternatives↗
PubMed2005

[Analysis of paralytic shellfish poison of bivalves in seafood market in Guangzhou].

OBJECTIVE: The investigations of the paralytic shellfish poisoning (PSP) from Huangsha seafood market of Guangzhou was performed to assess the risk of PSP in bivalves. METHODS: The concentration and profiles of PSP toxins in bivalves were determined by mouse bioassay of AOAC and high-performance liquid chromatography (HPLC). The risk assessment of PSP in bivalves was conducted with FAO and Chinese Administration Organization of Fish Culture and Seaport. RESULTS: The content of PSP detected was lower than the safe standard (4 MU/g meat) in all of the 84 samples, one of which had the highest toxicity with 1.84 MU/g muscle. These results suggested that the bivalves in seafood market was safe to feed. It was 9 samples' gland in 2 species that be detected to have PSP in the bivalves being researched, the muscles had few PSP. The concentration of PSP in one sample's gland exceeded the threshold of FAO (4 MU/g) with 14.52 MU/g meat, and the profiles of PSP in the gland were B1, GTX2/3, GTX1/4 and C according to HPLC. CONCLUSION: These results suggested that both of the concentration and detection rate of PSP of bivalves in seafood market in Guangzhou were low as a whole, but the content and discovery rate of PSP were far higher in glands than in the muscles, and the PSP content in one gland exceeded the threshold of Standard. The levels of PSP contamination in shellfish was characteristic of season. The toxins level in shellfish were the maximum in spring, but the frequency of toxins detected in shellfish was higher in summer and autumn, so the detection and risk assessment of PSP in bivalves from seafood market was essential in the future.

Animals↗
PubMed2005

Hydrophilic interaction liquid chromatography/mass spectrometry for determination of domoic acid in Adriatic shellfish.

This paper describes a new method for sensitive, specific and direct determination of domoic acid (DA), the causative toxin of amnesic shellfish poisoning (ASP) syndrome, in shellfish. It is based on combination of hydrophilic interaction liquid chromatography with mass spectrometry (HILIC/MS). The high percentage of organic modifier in the mobile phase and the omission of ion-pairing reagents, both favoured in HILIC, result in enhanced detection limits with MS detection. The new method was set up either on an ionspray ion trap MS instrument operating in MS and MS/MS scanning acquisition modes, or on a turboionspray triple-quadrupole MS system operating in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) acquisition modes. Positive and negative ion experiments were performed. MRM experiments are recommended for screening contaminated shellfish tissue and for quantitative analyses due to highest sensitivity and selectivity. The minimum detection levels for the toxin in tissue were found to be 63 and 190 ng/g in positive and negative MRM experiments, respectively, which are well below the regulatory limit for DA in tissue (20 microg/g). Application to shellfish samples collected in the Adriatic Sea (Italy) in the period 2000-2004 demonstrated for the first time in Italy the presence of DA as a new toxin that has entered the Adriatic Mytilus galloprovincialis toxin profile.

Animals↗
PubMed2006

Heavy metal monitoring using bivalved shellfish from Zhejiang coastal waters, East China Sea.

The interest of culturing bivalved shellfish (including mussels, clams, oysters etc.), has increased in recent years, but the consumption has been hampered both by high levels of heavy metals and toxic algae. The levels of heavy metals (Hg, Cd, Pb, Zn, Cu and As) were determined in soft tissues of different shellfish from Zhejiang coastal waters, East China Sea. The average concentrations of the heavy metals analyzed exhibited the following decreasing order: Zn > Cu > As > Cd > Pb > Hg. Comparison of heavy metals concentrations among species indicated that Bivalves clearly have significant potential as useful bioindicators; however, the results indicated that no one species is universally suitable, as is true of any organism used in these studies. The distribution of the metals was also investigated along the Zhejiang coast, which the results showed the mean concentration of Hg and Cd detected did not vary greatly and presented a similar spatial pattern at different sampling stations, the concentrations of Zn and Pb were highly variable at different sampling sites. The amounts of heavy metals in soft tissue of shellfish were also compared with those from the related species from other seas in China and world marine waters. The results suggested that the coastal area might be considered relatively unpolluted with heavy metals and the concentrations of metals in shellfish also below the seafood safety limits for human consumption.

China↗
PubMed2006

Can general anaesthesia be used for the Paralytic Shellfish Poison bioassay?

The current method for Paralytic Shellfish Poisoning (PSP) testing in shellfish is based on the mouse bioassay (MBA), which involves injecting shellfish extract into a conscious mouse, and then converting its time to death into PSP toxicity using Sommer's table. To improve animal welfare, the present study investigated the use of anaesthesia. A saxitoxin (STX) calibration study was conducted where known amounts of STX were injected into both unanaesthetised and anaesthetised mice. Death time was approximately doubled when mice were anaesthetised. Both unanaesthetised and anaesthetised animals showed a linear relationship between the inverse death time and log(STX). Based on these data, new calibration curves were developed. This study revealed that the current method employing Sommer's table underestimates toxicity by up to 50% for higher toxin levels. Subsequently, shellfish samples were tested on both unanaesthetised and anaesthetised mice. Using the new calibration curves, the numbers of samples exceeding the field closure limit were similar for unanaesthetised and anaesthetised mice, and were nearly two-fold higher than those obtained with the current method. The studies showed that the bioassay gives variable results for both unanaesthetised and anaesthetised animals. Anaesthesia forms a viable and more ethical alternative to the current bioassay, at least in the short term. A practical summary on how to conduct this method is given.

Anesthetics, General↗
PubMed2001

A functional assay for paralytic shellfish toxins that uses recombinant sodium channels.

Saxitoxin (STX) and its derivatives are highly toxic natural compounds produced by dinoflagellates commonly present in marine phytoplankton. During algal blooms ("red tides"), shellfish accumulate saxitoxins leading to paralytic shellfish poisoning (PSP) in human consumers. PSP is a consequence of the high-affinity block of voltage-dependent Na channels in neuronal and muscle cells. PSP poses a significant public health threat and an enormous economic challenge to the shellfish industry worldwide. The standard screening method for marine toxins is the mouse mortality bioassay that is ethically problematic, costly and time-consuming. We report here an alternative, functional assay based on electrical recordings in cultured cells stably expressing a PSP target molecule, the STX-sensitive skeletal muscle Na channel. STX-equivalent concentration in the extracts was calibrated by comparison with purified STX, yielding a highly significant correlation (R=0.95; N=30) between electrophysiological determinations and the values obtained by conventional methods. This simple, economical, and reproducible assay obviates the need to sacrifice millions of animals in mandatory paralytic shellfish toxin screening programs.

Animals↗