Paralytic shellfish poisoning (PSP) in Margarita Island, Venezuela

A severe outbreak of Paralytic Shellfish Poisoning (PSP) occurred in Manzanillo and Guayacán, northwestern coast of Margarita Island, Venezuela, between August and October 1991. A bloom of dinoflagellates including Prorocentrum gracile, Gymnodinium catenatum and Alexandrium tamarense seemed to be responsible for this outbreak. Levels of PSP toxins in mussels (Perna perna) exceeded the international safety limit of saxitoxin, 80 microg STX/100 microg meat. PSP toxin values varied between 2548 and 115 microg STX/100 g meat in Manzanillo, and between 1422 and 86 microg STX/100 g meat in Guayacán. At both locations, the highest levels were detected in August, when 24 patients exhibited typical symptoms of PSP toxicity after consuming cooked mussels (16 required hospitalization). A high pressure liquid chromatographic (HPLC) procedure was recently used on the 1991 samples. The major toxin detected in samples of both locations was decarbamoyl saxitoxin (dcSTX), but low concentrations of saxitoxin were also found in Manzanillo samples. Gonyautoxins GTX1, GTX2 and GTX3 were detected only at Guayacán, while in both locations, decarbamoylgonyatouxin (dcGTX2,3) toxins were detected. These findings represent the first time that causative toxins of PSP in Venezuela have been chemically identified, and confirm the presence of dcSTX and dcGTX in mussels from the Caribbean Sea. The presence of dcSTX and dcGTX in shellfish is indicative that Gymnodinium catenatum was a causative organism for outbreak of PSP.     

Accumulation of PSP toxins in Atlantic mackerel: seasonal and ontogenetic variations

Atlantic mackerel Scomber scombrus are known to be lethal vectors of paralytic shellfish poisoning (PSP) toxins to predators. To elucidate dynamics of PSP toxin accumulation in this species, mackerel were sampled in the Gulf of St Lawrence from May to October 1993. Mackerel appear to retain toxins (saxitoxin, gonyautoxins 2 and 3) year-round. The toxin content of the liver, as determined by high performance liquid chromatography, increased significantly with fish age ( r² =0.40) and length ( r² =0.52), suggesting that mackerel progressively accumulate PSP toxins throughout their life. The toxin content of the liver also increased significantly during the summer feeding sojourn in the Gulf of St Lawrence. Comparison of profiles of saxitoxin derivatives indicated that zooplankton were the likely source of PSP toxins found in mackerel. The mean ± S.D toxin content was 17.4 ± 10.6 nmol liver⁻¹ and the mean ± S.D. PSP toxicity was 112.4 ± 67.0 μg saxitoxin equivalents 100 g⁻¹ liver wet weight ( n =247).     

A fluorimetric method based on changes in membrane potential for screening paralytic shellfish toxins in mussels

To prevent the consumption of bivalves contaminated with paralytic shellfish poisoning (PSP), toxin levels in seafood products are estimated by using the official mouse bioassay. Because of the limitations of this bioassay other methods of monitoring toxins are clearly needed. We have developed a test to screen for PSP toxins based on its functional activity; the toxins bind to the voltage-gated Na+ channels and block their activity. The method is a fluorimetric assay that allows quantitation of the toxins by detecting changes in the membrane potential of human excitable cells. This assay gives an estimate of toxicity, since each toxin present in the sample binds to sodium channels with an affinity which is proportional to its intrinsic toxic potency. The detection limits for paralytic shellfish toxins were found to be 1 ng saxitoxin equivalents/ml compared to the regulatory limit threshold of 400 ng/ml (equivalent to 80 microg/100 g) used in most countries. Our results indicate that this fluorescent assay is a specific, very sensitive, rapid, and reliable method of monitoring PSP toxin levels in samples from seafood products and toxic algae.     

Characterization of nontoxic and toxin-producing strains of Alexandrium minutum (Dinophyceae) in Irish coastal waters

A comparative analysis of the morphology, toxin composition, and ribosomal DNA (rDNA) sequences was performed on a suite of clonal cultures of the potentially toxic dinoflagellate Alexandrium minutum Halim. These were established from resting cysts or vegetative cells isolated from sediment and water samples taken from the south and west coasts of Ireland. Results revealed that strains were indistinguishable, both morphologically and through the sequencing of the D1-D2 domain of the large subunit and the ITS1-5.8S-ITS2 regions of the rDNA. High-performance liquid chromatography fluorescence detection analysis, however, showed that only strains derived from retentive inlets on the southern Irish coast synthesized paralytic shellfish poisoning (PSP) toxins (GTX2 and GTX3), whereas all strains of A. minutum isolated from the west coast were nontoxic. Toxin analysis of net hauls, taken when A. minutum vegetative cells were in the water column, revealed no PSP toxins in samples from Killary Harbor (western coast), whereas GTX2 and GTX3 were detected in samples from Cork Harbor (southern coast). These results confirm the identity of A. minutum as the most probable causative organism for historical occurrences of contamination of shellfish with PSP toxins in Cork Harbor. Finally, random amplification of polymorphic DNA was carried out to determine the degree of polymorphism among strains. The analysis showed that all toxic strains from Cork Harbor clustered together and that a separate cluster grouped all nontoxic strains from the western coast.     

The effect of mussel size, temperature, seston volume, food quality and volume-specific toxin concentration on the uptake rate of PSP toxins by mussels (Mytilus galloprovincialis Lmk)

The accumulation of paralytic shellfish poisoning (PSP) toxins by bivalves is a serious threat to public health all over the world. However, very little is known about the uptake kinetics of these toxins and the environmental factors that may modify this process. We have studied the effect of mussel size, temperature, seston volume, food quality, and volume-specific toxin concentration (VOSTOC), on the uptake rate of paralytic shellfish poisoning (PSP) toxins by mussels (Mytilus galloprovincialis), by means of a second order factorial experiment. Over a 3-day period, the mussels were fed artificial diets containing Alexandrium minutum AL1V (a PSP toxin producer), Tetraselmis suecica, Ensiculifera sp1 and silt, to the levels required by each treatment. Mussel size, seston volume and VOSTOC were found to be statistically significant when the total toxin accumulated per weight of wet tissue was considered. Mussel size affected the uptake negatively and latter two positively. The interactions, mussel size-VOSTOC and mussel size-food quality were also significant. The response was not linear as shown by the significance of the quadratic term of mussel size. Notwithstanding, when the PSP toxins accumulation per mussel was analysed, only one factor, the VOSTOC and the interactions, food quality-mussel size and food quality-seston volume, were found to be significant. VOSTOC was the most important factor in the accumulation of toxins, in our opinion, probably due to toxin assimilation being mainly regulated by the probability of contact between the toxins and the cellular walls of the digestive system. The size of the bivalve is also especially important because toxin concentration is usually calculated per weight of bivalve tissue and because the weight-specific ingestion increases with mussel size. The food quality, which was directly related to the assimilation of organic matter, had an inverse effect on toxin assimilation. In our opinion, this is probably due to the effect of inorganic particles in enhancing the disruption of Alexandrium cells. Temperature had no effect on the uptake rate except for the accumulation of the gonyautoxin GTX1.     

Erratum to Bacterial diversity in toxic<Emphasis Type="Italic"> Alexandrium tamarense</Emphasis> blooms off the Orkney Isles and the Firth of Forth

The genetic diversity of the bacterial community associated with Alexandrium tamarense blooms was studied in blooms of the toxic dinoflagellates in the waters around the Orkney Isles and the Firth of Forth (Scotland). For toxin and molecular analysis of the bacterial communities associated with the toxic bloom, water samples were taken in 1998 and 1999 from A. tamarense blooms. The bacterial community structure, as determined by DGGE (denaturing gradient gel electrophoresis) showed clear differences between all three investigated size fractions (dinoflagellate-associated bacteria, attached bacteria and free-living bacteria), with high diversity within each sample. DNA sequence analysis of the dominant and most frequent DGGE bands revealed the dominance of α Proteobacteria, mainly of the Roseobacter clade, with similarities of 91–99%. Moreover, DGGE bands occurring at the same position in the gel throughout in most samples corroborate the presence of several specific α Proteobacteria of the Roseobacter clade. Overall, 500 bacteria were isolated from the bloom and partly phylogenetically analysed. They were members of two prokaryotic phyla, the Proteobacteria and the Bacteroidetes, related to Proteobacteria of the α and γ subdivisions (Alteromonas, Pseudoalteromonas and Colwellia). All bacteria were tested for the production of sodium channel blocking (SCB) toxins using mouse neuroblastoma assay. No production of SCB toxins was found and high performance liquid chromatography (HPLC) analysis confirmed these results. The content of total paralytic shellfish poisoning (PSP) toxin in the water samples, as measured within the toxic dinoflagellate blooms using HPLC, ranged from 53 to 2191 ng PSP l^?1 in 1998 and from 0 to 478 ng PSP l^?1 in 1999. Changes in PSP toxin content were not accompanied by changes of DGGE band patterns. We therefore presume that the bacterial groups identified in this study were not exclusively associated with toxic A. tamarense, but were generally associated with the phytoplankton.     

Phycotoxin accumulation in zooplankton feeding on Alexandrium fundyense--vector or sink?

Zooplankton can consume toxic Alexandrium spp. dinoflagellatesin the Gulf of Maine and retain paralytic shellfish poisoning(PSP) toxins, potentially acting as toxin vectors. We performedexperiments to determine toxin budgets for common species ofcopepods (Acartia hudsonica, Eurytemora herdmani, Centropageshamatus) feeding on toxic Alexandrium fundyense, offered asmonocultures or in mixtures of algal prey, by comparing calculatedtoxin ingestion rates and toxin content of copepod body tissueand fecal pellets. When fed monocultures, both copepod tissueand fecal pellet fractions accounted for 5% each of the calculatedingested toxin, and thus by difference 90% was lost as a dissolvedfraction into the seawater medium. The presence of alternativefood did not significantly alter the efficiency of toxin retention.Sloppy feeding or regurgitation are probable mechanisms forrelease of toxin to sea water. Experiments using varying concentrationsof A. fundyense and alternativenon-toxic species did not showsignificant effects of cell concentration on toxin retentionefficiency. Total toxin retained and efficiency of retentionvaried among copepod species. Toxin profiles (% molar composition)of dinoflagellates, copepod tissues and fecal pellets differedslightly, suggesting some metabolic transformation. Becauseof their low retention efficiency, copepod grazers can effectivelydisperse PSP toxins produced by Alexandrium spp. into the environment,where they are much less likely to be harmful—zooplanktonact as a sink for PSP toxins. Nevertheless, sufficient toxinbody burdens are attained to contribute to propagation of PSPtoxins to other trophic levels.     

Toxin profile of Alexandrium catenella from the Chilean coast as determined by liquid chromatography with fluorescence detection and liquid chromatography coupled with tandem mass spectrometry

The profile of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP) was determined from a Chilean strain of the marine dinoflagellate Alexandrium catenella. The toxin composition was compared with that of toxic shellfish, presumably contaminated by natural blooms of A. catenella from the same region in southern Chile. Ion pair-liquid chromatography with post-column derivatization and fluorescence detection (LC-FD) was employed for relative quantitative analysis of the toxin components, whereas unambiguous identification of the toxins was confirmed by tandem mass spectrometry (LC–MS/MS). In the dinoflagellate strain from Chile, the N-sulfocarbamoyl derivatives (C1/C2, B1) and the carbamoyl gonyautoxins GTX1/GTX4 comprise >90% of the total PSP toxin content on a molar basis. This toxin composition is consistent with that determined for A. catenella populations from the Pacific coast in the northern hemisphere. The characteristic toxin profile is also reflected in the shellfish, but with evidence of epimerization and metabolic transformations of C1 and C2 to GTX2 and GTX3, respectively. This work represents the first unequivocal identification and confirmation of such PSP toxin components from the Chilean coast.     

Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR

The toxic dinoflagellate Alexandrium fundyense is widespread in the northeastern part of North America, including the Gulf of Maine, and is responsible for seasonal harmful algal blooms in these regions. Even at low cell densities, A. fundyense toxins can accumulate in shellfish and result in paralytic shellfish poisoning (PSP). PSP can be debilitating or lethal to humans and other shellfish consumers and is a public health concern. As a result, accurate measurements of A. fundyense distributions, particularly at low cell density, are critical to continued PSP monitoring and mitigation efforts. Towards this end we have developed a real-time quantitative PCR (qPCR) method to monitor A. fundyense. Laboratory validation indicates that the qPCR assay is sensitive enough to detect 10 cells per sample, and that it does not detect co-occurring dinoflagellates such as Alexandrium ostenfeldii. The qPCR methodology was used to quantify A. fundyense cell densities in samples collected during a spring 2003 transect in the Gulf of Maine, and the data were compared to those obtained in parallel from light microscope and DNA hybridization-based methods. Results show that A. fundyense cell density was low during this period relative to typical cell densities required for PSP contamination of local shellfish, and that qPCR values were comparable to numbers determined by independent methods.     

Characterization of Nontoxic and Toxin-Producing Strains of Alexandrium minutum (Dinophyceae) in Irish Coastal Waters

A comparative analysis of the morphology, toxin composition, and ribosomal DNA (rDNA) sequences was performed on a suite of clonal cultures of the potentially toxic dinoflagellate Alexandrium minutum Halim. These were established from resting cysts or vegetative cells isolated from sediment and water samples taken from the south and west coasts of Ireland. Results revealed that strains were indistinguishable, both morphologically and through the sequencing of the D1-D2 domain of the large subunit and the ITS1-5.8S-ITS2 regions of the rDNA. High-performance liquid chromatography fluorescence detection analysis, however, showed that only strains derived from retentive inlets on the southern Irish coast synthesized paralytic shellfish poisoning (PSP) toxins (GTX2 and GTX3), whereas all strains of A. minutum isolated from the west coast were nontoxic. Toxin analysis of net hauls, taken when A. minutum vegetative cells were in the water column, revealed no PSP toxins in samples from Killary Harbor (western coast), whereas GTX2 and GTX3 were detected in samples from Cork Harbor (southern coast). These results confirm the identity of A. minutum as the most probable causative organism for historical occurrences of contamination of shellfish with PSP toxins in Cork Harbor. Finally, random amplification of polymorphic DNA was carried out to determine the degree of polymorphism among strains. The analysis showed that all toxic strains from Cork Harbor clustered together and that a separate cluster grouped all nontoxic strains from the western coast.     

Alexandrium (Dinophyceae) species in Malaysian waters

A study was carried out to determine the presence of paralytic shellfish poisoning (PSP) toxin-producing dinoflagellates in the coastal waters of Peninsula Malaysia. This followed first ever occurrences of PSP in the Straits of Malacca and the northeast coast of the peninsula. The toxic tropical dinoflagellate Pyrodinium bahamense var. compressum was never encountered in any of the plankton samples. On the other hand, five species of Alexandrium were found. They were Alexandrium affine, Alexandrium leei, Alexandrium minutum, Alexandrium tamarense and Alexandrium tamiyavanichii. Not all species were present at all sites. A. tamiyavanichii was present only in the central to southern parts of the Straits of Malacca. A. tamarense was found in the northern part of the straits, while A. minutum was only found in samples from the northeast coast of the peninsula. A. leei and A. affine were found in both the north and south of the straits. Cultured isolates of A. minutum and A. tamiyavanichii were proven toxic by the receptor binding assay for PSP toxins but A. tamarense clones were not toxic. Mean toxin content for the A. tamiyavanichii and A. minutum clones were 26 and 15 fmol per cell STX equivalent, respectively. This study has provided evidence on the presence of PSP toxin-producing Alexandrium species in Malaysian waters which suggests that PSP could increase in importance in the future.     

Paralytic shellfish poisoning (PSP) toxin profiles and short-term detoxification kinetics in mussels Mytilus galloprovincialis fed with the toxic dinoflagellate Alexandrium tamarense

Mussels (Mytilus galloprovincialis) were experimentally contaminated with paralytic shellfish poisoning (PSP) toxins by being fed with the toxic dinoflagellate Alexandrium tamarense, and changes in toxin content and specific composition during the decontamination period were analyzed by high-performance liquid chromatography (HPLC). Toxins excreted by the mussels into the seawater were also recovered using an activated charcoal column and analyzed by HPLC. The predominant toxins in A. tamarense, mussels, and seawater were the N-sulfocarbamoyl-11-hydrosulfate toxins (C1,2) and carbamate gonyautoxins-1,4 (GTX1,4). There were no remarkable differences in the relative proportions of the predominant toxins within A. tamarense, mussels and seawater. Because the relative proportion of the various toxin analogues excreted by the mussels was similar to that within their tissues during detoxification, it appeared that the selective release of particular toxins by the mussels was unlikely. The total amount of toxin lost from mussels was nearly equal to that which was found dissolved in the seawater, suggesting that, at least the early stages of mussel detoxification, most losses can be accounted for by excretion.     

Determination of paralytic shellfish poisoning (PSP) toxins in UK shellfish

A study was conducted to aid the interpretation of data generated by parallel testing of the qualitative Jellett Rapid Test (JRT) and the mouse bioassay (MBA) for detection of paralytic shellfish poisoning (PSP) toxins within the UK statutory shellfish biotoxin monitoring programme. A selection of stored sample extracts subjected to testing by MBA and/or JRT were further analysed by liquid chromatography with fluorescence detection (LC–FLD) to provide additional information on the concentrations of PSP toxins and toxin profiles.Results, from this study, demonstrate the potential of the JRT to effectively screen out PSP toxin negative shellfish samples and samples containing low concentrations of toxins from UK monitoring programmes. Additionally, data generated using LC–FLD highlights the potential of introducing alternative analytical techniques to completely replace the requirement for the MBA.     

IDENTIFICATION AND TOXICITY OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN SCOTTISH WATERS1

Contamination of shellfish with paralytic shellfish poisoning (PSP) toxins produced by Alexandrium species poses a potential threat to the sustainability of the Scottish aquaculture industry. Routine LM analysis of water samples from around the Scottish coast has previously identified Alexandrium (Dinophyceae) as a regular part of the spring and summer phytoplankton communities in Scottish coastal waters. In this study, Alexandrium tamarense (M. Lebour) Balech isolated from sediment and water samples was established in laboratory culture. Species identification of these isolates was confirmed using thecal plate dissections and by molecular characterization based on their LSU and, in some cases, ITS rDNA sequence. Molecular characterization and phylogenetic analysis showed the presence of two ribotypes of A. tamarense: Group I (North American ribotype) and Group III (Western European ribotype). Assessment of PSP toxin production using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) showed that A. tamarense Group I produced a complex array of toxins (～2,000 fg STX equivalents · cell^?1) with the major toxins being C2, neosaxitoxin (NEO), saxitoxin (STX), gonyautoxin‐4 (GTX‐4), and GTX‐3, while A. tamarense Group III did not produce toxins. Historically, it was considered that all Alexandrium species occurring in Scottish waters produce potent PSP toxins. This study has highlighted the presence of both PSP toxin‐producing and benign species of A. tamarense and questions the ecological significance of this finding.     

In vitro transformation of PSP toxins by different shellfish tissues

Many in vivo shellfish feeding experiments have been carried out in order to investigate the fate of PSP toxins in the marine food chain. A focal point of these studies concerns the species- and tissue-specific differences in toxin metabolism. However, tissue specific effects are often overlapped by selective toxin retention as well as transfer between individual compartiments. In in vitro experiments presented here digestive tissue and adductor homogenates of 10 shellfish species (bivalvia: Mytilus edulis, Crassostrea gigas, Cardium edule, Arctica islandica, Ensis ensis, Modiolus modiolus, Mactra stultorum, Pecten maximus as well as two snails: Littorina littorea and Buccinum undatum) were incubated with an extract of the toxic strain Alexandrium fundyense CCMP 1719. After incubation, changes in the toxin pattern could be observed in all samples with significant differences occuring between both the species and tissues. The greatest metabolic activity was found in digestive tissue samples. Among the organisms, the species with a non-filtering lifestyle, L. littorea and B. undatum, showed the highest conversion rates. Interestingly, the high metabolic transformation rate of the PSP toxins was accompanied with a fast reduction (up to 73%) of toxicity in the homogenates.     

Detection of bacteria originally isolated from Alexandrium spp. in the midgut diverticula of Mytilus edulis after water-borne exposure

Bacteria associated with toxic dinoflagellates have been implicated in the production of paralytic shellfish poisoning (PSP) toxins, but it has not been substantiated that bacteria are truly capable of autonomous PSP toxin synthesis or what role bacteria may play in shellfish toxification. In this study, different putatively PSP toxin producing bacteria originally isolated from toxic Alexandrium spp. were exposed to the blue mussel Mytilus edulis. To document that these bacteria accumulated in the digestive tract of the mussels, hybridization techniques that use rRNA targeted oligonuceotides for in situ identification of these bacteria were applied. The mussel hepatopancreas was dissected and paraffin and frozen sections were made. The dissected glands were hybridized with digoxigenin-labelled 16S rRNA oligonucleotide probes. Results demonstrate that mussels will readily uptake and accumulate these bacteria in the hepatopancreas. However, the mussels were not rendered toxic by the ingestion of the bacteria as determined by HPLC with UV detection for PSP toxins and determination of sodium channel blocking activity using the mouse neuroblastoma assay. Thus, although the role that bacteria play in mussel toxification remains unclear, methods are now available which will aid in further investigation of this relatively unexplored area.     

Comparison of ELISA and SPR biosensor technology for the detection of paralytic shellfish poisoning toxins

An enzyme labeled immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor assay for the detection of paralytic shellfish poisoning (PSP) toxins were developed and a comparative evaluation was performed. A polyclonal antibody (BC67) used in both assay formats was raised to saxitoxin–jeffamine–BSA in New Zealand white rabbits. Each assay format was designed as an inhibition assay. Shellfish samples (n = 54) were evaluated by each method using two simple rapid extraction procedures and compared to the AOAC high performance liquid chromatography (HPLC) and the mouse bioassay (MBA). The results of each assay format were comparable with the HPLC and MBA methods and demonstrate that an antibody with high sensitivity and broad specificity to PSP toxins can be applied to different immunological techniques. The method of choice will depend on the end-users needs. The reduced manual labor and simplicity of operation of the SPR biosensor compared to ELISA, ease of sample extraction and superior real time semi-quantitative analysis are key features that could make this technology applicable in a high-throughput monitoring unit.     

Erratum to: Bacterial diversity in toxic<Emphasis Type="Italic"> Alexandrium tamarense</Emphasis> blooms off the Orkney Isles and the Firth of Forth

The genetic diversity of the bacterial community associated with Alexandrium tamarense blooms was studied in blooms of the toxic dinoflagellates in the waters around the Orkney Isles and the Firth of Forth (Scotland). For toxin and molecular analysis of the bacterial communities associated with the toxic bloom, water samples were taken in 1998 and 1999 from A. tamarense blooms. The bacterial community structure, as determined by DGGE (denaturing gradient gel electrophoresis) showed clear differences between all three investigated size fractions (dinoflagellate-associated bacteria, attached bacteria and free-living bacteria), with high diversity within each sample. DNA sequence analysis of the dominant and most frequent DGGE bands revealed the dominance of Proteobacteria, mainly of the Roseobacter clade, with similarities of 91–99%. Moreover, DGGE bands occurring at the same position in the gel throughout in most samples corroborate the presence of several specific Proteobacteria of the Roseobacter clade. Overall, 500 bacteria were isolated from the bloom and partly phylogenetically analysed. They were members of two prokaryotic phyla, the Proteobacteria and the Bacteroidetes, related to Proteobacteria of the and subdivisions (Alteromonas, Pseudoalteromonas and Colwellia). All bacteria were tested for the production of sodium channel blocking (SCB) toxins using mouse neuroblastoma assay. No production of SCB toxins was found and high performance liquid chromatography (HPLC) analysis confirmed these results. The content of total paralytic shellfish poisoning (PSP) toxin in the water samples, as measured within the toxic dinoflagellate blooms using HPLC, ranged from 53 to 2191 ng PSP l^–1 in 1998 and from 0 to 478 ng PSP l^–1 in 1999. Changes in PSP toxin content were not accompanied by changes of DGGE band patterns. We therefore presume that the bacterial groups identified in this study were not exclusively associated with toxic A. tamarense, but were generally associated with the phytoplankton.An erratum to this article can be found at Communicated by H.-D. Franke