Biotransformations of paralytic shellfish toxins by bacteria isolated from bivalve molluscs

Due to the possibility that bacteria could be involved in the clearance of paralytic shellfish toxins (PST) from bivalve molluscs, investigations into which, if any, bacteria were able to grow at the expense of PST focused on several common shellfish species. These species were blue mussels, oysters, razor fish, cockles, and queen and king scallops. Bacteria associated with these shellfish were isolated on marine agar 2216 and characterized by their carbon utilization profiles (BIOLOG). Selected isolates from groups demonstrating 90% similarity were screened for their ability to metabolize a range of PST (gonyautoxins 1 and 4 [GTX 1/4], GTX 2/3, GTX 5, saxitoxin, and neosaxitoxin) using a novel screening method and confirming its results by high-performance liquid chromatography. Results suggest that molluscan bacteria have different capacities to utilize and transform PST analogues. For example, isolates M12 and R65 were able to reductively transform GTX 1/4 with concomitant production of GTX 2/3, while isolate Q5 apparently degraded GTX 1/4 without the appearance of other GTXs. Other observed possible mechanisms of PST transformations include decarbamoylation by isolate M12 and sulfation of GTXs by isolates Q5, R65, M12, and C3. These findings raise questions as to the possible role of bacteria resident in the shellfish food transport system. Some researchers have suggested that the microflora play a role in supplying nutritional requirements of the host. This study demonstrates that bacteria may also be involved in PST transformation and elimination in molluscan species.     

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.     

Standardized extraction method for paralytic shellfish toxins in phytoplankton

The optimal conditions were established for extraction of paralytic shellfish toxins from a Danish clone of Alexandrium tamarense using extraction with acetic acid and HCl in the concentration range 0.01–1.0 N. Physical destruction of the cells was investigated microscopically to select the most efficient extraction procedure.The toxin content was quantitated by an automized isocratic reversed-phase high-performance liquid chromatography (HPLC) method. The best results as judged from the total amount of toxins and the toxin profile were obtained using 0.05–1.0 N acetic acid and 0.01–0.02 N HCl. Hydrochloric acid in the concentration range 0.03–1.0 N caused the amount of C1 and C2 toxins to decrease sharply and concomitant increase of gonyautoxins 2 and 3.The phytoplankton extracts with 0.1 to 0.5 N acetic acid or 0.01 N HCl were stable during 6 months at –20 °C, but the extracts with HCl 0.02 N underwent a change in toxin profile, although the total amount of toxins was constant.     

Three estuarine Australian dinoflagellates that can produce paralytic shellfish toxins

Three toxic dinoflagellates that can cause paralytic shellfishpoisoning (PSP) in humans are reported for the first time fromestuarine Australian waters. Blooms of the chain-forming, unarmouredGymnodinium catenatum Graham resulted in closures of shellfishfarms in summer-autumn 1986 and 1987 in southern Tasmanian estuaries.The chain-forming, armoured Alexandrium catenella (Whedon etKofoid) Balech occurred in April 1986 in Port Phillip Bay, Melbourne.Alexandrium minutum Halim produced red water in October 1986and 1987 in Port River, Adelaide. For the first time in thisspecies PSP toxin production was demonstrated by mouse bioassaysand HPLC analyses. Biogeographic aspects of these dinoflagellatesand the apparent global spreading of toxic plankton blooms arediscussed.     

Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp. (Dinophyta) in culture.

A substantial proportion of bacteria from five Alexandrium cultures originally isolated from various countries produced sodium channel blocking (SCB) toxins, as ascertained by mouse neuroblastoma assay. The quantities of SCB toxins produced by bacteria and dinoflagellates were noted, and the limitations in comparing the toxicities of these two organisms are discussed. The chemical nature of the SCB toxins in selected bacterial isolates was determined as paralytic shellfish toxins by pre- and postcolumn high-performance liquid chromatography, capillary electrophoresis-mass spectrometry, and enzyme immunoassay.     

Single-cell analysis of paralytic shellfish toxins in Alexandrium tamarense by HPLC with post-column fluorescent derivatization

We developed a methodology for analyzing the C-toxin (C2) content in single Alexandrium tamarense cells; this method was based on high performance liquid chromatography (HPLC). C2 is the main paralytic shellfish toxin (PST) detected in a clonal culture of A. tamarense, which is a common causative organism in cases of paralytic shellfish poisoning in Japan. This HPLC method employs post-column fluorescent derivatization (FL). Mobile phase, column size, flow rate, reagent concentrations, and lamp type for the fluorescent detector were all optimized for the detection of C2. With this improved methodology, we could measure 1 fmol of C2 with a signal to noise ratio (S/N) = 2. Clonal heterogeneity within the toxic strain, which was maintained for 13 years after re-isolation from the original clonal culture, ranged from <1 fmol to 700 fmol cell⁻¹. This report is the first to demonstrate definitively that PST content varies on a cell-by-cell basis in a clonal culture of a dinoflagellate that causes paralytic shellfish poisoning.     

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.     

Copepods induce paralytic shellfish toxin production in marine dinoflagellates

Among the thousands of unicellular phytoplankton species described in the sea, some frequently occurring and bloom-forming marine dinoflagellates are known to produce the potent neurotoxins causing paralytic shellfish poisoning. The natural function of these toxins is not clear, although they have been hypothesized to act as a chemical defence towards grazers. Here, we show that waterborne cues from the copepod Acartia tonsa induce paralytic shellfish toxin (PST) production in the harmful algal bloom-forming dinoflagellate Alexandrium minutum. Induced A. minutum contained up to 2.5 times more toxins than controls and was more resistant to further copepod grazing. Ingestion of non-toxic alternative prey was not affected by the presence of induced A. minutum. The ability of A. minutum to sense and respond to the presence of grazers by increased PST production and increased resistance to grazing may facilitate the formation of harmful algal blooms in the sea.     

A receptor binding assay for paralytic shellfish poisoning toxins: recent advances and applications

We recently described a high throughput receptor binding assay for paralytic shellfish poisoning (PSP) toxins, the use of the assay for detecting toxic activity in shellfish and algal extracts, and the validation of 11-[3H]-tetrodotoxin as an alternative radioligand to the [3H]-saxitoxin conventionally employed in the assay. Here, we report a dramatic increase in assay efficiency through application of microplate scintillation technology, resulting in an assay turn around time of 4 h. Efforts are now focused on demonstrating the range of applications for which this receptor assay can provide data comparable to the more time consuming, technically demanding HPLC analysis of PSP toxins, currently the method of choice for researchers. To date, we have compared the results of both methods for a variety of sample types, including different genera of PSP toxin producing dinoflagellates (e.g. Alexandrium lusitanicum, r2 = 0.9834, n = 12), size-fractioned field samples of Alexandrium spp. (20-64 microm; r2 = 0.9997, n = 10) as well as its associated zooplankton grazer community (200-500 microm: r2 = 0.6169, n = 10; >500 microm: r2 = 0.5063, n = 10), and contaminated human fluids (r2 = 0.9661, n = 7) from a PSP outbreak. Receptor-based STX equivalent values for all but the zooplankton samples were highly correlated and exhibited close quantitative agreement with those produced by HPLC. While the PSP receptor binding assay does not provide information on toxin composition obtainable by HPLC, it does represent a robust and reliable means of rapidly assessing PSP-like toxicity in laboratory and field samples. Moreover, this assay should be effective as a screening tool for use by public health officials in responding to suspected cases of PSP intoxication.     

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.     

Reevaluation of production of paralytic shellfish toxin by bacteria associated with dinoflagellates of the Portuguese coast

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by certain dinoflagellate and cyanobacterial species. The autonomous production of PSTs by bacteria remains controversial. In this study, PST production by two bacterial strains, isolated previously from toxic dinoflagellates, was evaluated using biological and analytical methods. Analyses were performed under conditions determined previously to be optimal for toxin production and detection. Our data are inconsistent with autonomous bacterial PST production under these conditions, thereby challenging previous findings for the same strains.     

Distribution and selective elimination of paralytic shellfish toxins in different tissues of Octopus vulgaris

Octopus (Octopus vulgaris, Cuvier) plays a central role in the marine food web, being an important consumer with high metabolic rates and at the same time an important food item for higher predators. After harmful algal blooms, octopus can accumulate high levels of marine toxins trough trophic interrelationships. The aim of this study is to characterize the distribution of paralytic shellfish toxins (PSTs) in selected tissues of the O. vulgaris, in order to assess the translocation of toxins among organs with different physiological functions. Different retention times and selective elimination of particular toxin analogues were also investigated. Twenty three specimens of O. vulgaris were captured in Peniche (NW coast of Portugal) after PSTs have been detected in molluscan bivalves. Tissue matrices were dissected from organs with digestive function (digestive gland, stomach and salivary glands) and excretory function (kidneys and branchial hearts) and analyzed for toxin determination. Toxin analysis was carried out by high performance liquid chromatography with fluorescence detection (LC-FLD). PSTs were found in all tissues analyzed. The highest toxin concentrations were found in the digestive gland, reaching a maximum of 2980 μg STX equiv. kg⁻¹. The toxin profile was constituted by dcSTX, B1, C1 + 2, dcGTX2 + 3, dcNEO, STX and GTX2 + 3. A lower number of toxins were identified in the remaining organs, with B1 and dcSTX compromising more than 90% in molar fraction. Decarbamoyl saxitoxin was the most abundant toxin detected in digestive gland, stomach and salivary glands, while B1 was dominant in organs with excretory function. A positive correlation of concentrations of B1 and dcSTX were found in the organs analyzed. Results indicate that B1 and dcSTX are assimilated into the digestive gland in a similar proportion. Selective elimination of toxins with higher elimination of B1 and retention of dcSTX is suggested. This study contributes to better understanding of the dynamics of PSTs in O. vulgaris and the fate of PSTs in the food web.     

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.     

In vitro transformation of paralytic shellfish toxins in the clams Mya arenaria and Protothaca staminea

Dissected tissues of two clam species, the Pacific littleneck, Protothaca staminea, and soft-shell, Mya arenaria, were evaluated for in vitro conversion of paralytic shellfish poisoning (PSP) toxins. Tissue homogenates were incubated with purified PSP toxins to determine the time-course of toxin conversion. The effects of boiling and addition of a natural reductant (glutathione) on toxin conversion were also assessed. For P. staminea, the digestive gland showed the greatest capacity for biotransformation, followed by gill, but mantle, adductor muscle, and siphon tissues exhibited very low conversion. In this species, the production of decarbamoyl derivatives was much greater from low potency N-sulfocarbamoyl toxins than from carbamate analogues. Decarbamolyation exhibited apparent specificity for α-epimers of all toxin substrates and this reaction was inhibited by boiling. Glutathione-mediated desulfation was tissue specific and had apparent specificity for β-epimers. These observations on P. staminea suggest that the above reactions are enzyme-mediated. In contrast, there was little toxin conversion in M. arenaria homogenates, but even this low activity was heat-labile and thus likely enzyme-mediated.     

Polyamine profile in the paralytic shellfish poison-producing alga Alexandrium minutum

The polyamine profiles of the toxic dinoflagellate Alexandriumminutum during different growth stages were measured by high-performanceliquid chromatography. Both free and conjugated polyamines werefound, including putrescine, cadaverine, spermine, spermidineand norspermidine. During the growth cycle of A. minutum T1,the levels of norspermidine and putrescine in the free polyamineswere the highest after 3 days culture. Putrescine and cadaverinewere the major components in the conjugated polyamines. Theamount of conjugated amines was higher than that of free aminesduring the exponential phase of A. minutum T1.     

Recent trends in paralytic shellfish toxins in Puget Sound,relationships to climate,and capacity for prediction of toxic events

Temporal and spatial trends in paralytic shellfish toxins (PSTs) in Puget Sound shellfish and their relationships with climate are investigated using long-term monitoring data since 1957. Data are selected for trend analyses based on the sensitivity of shellfish species to PSTs and their depuration rates, and the frequency of sample collection at individual sites. These criteria limit the analyses to the shellfish species Mytilus edulis at 20 sites from 1993 to 2007. Blue mussel toxicity is highly variable, but typically exceeds the regulatory limit for human consumption from July to November annually, with most closures occurring early in fall. Using blue mussel data only, we find no robust evidence to suggest that the frequency, magnitude, duration, or geographic scope of PST events in Puget Sound increased between 1993 and 2007. However, there is a significant basin-wide trend for closures to occur earlier in the year. There are no significant correlations between annual indices of mussel toxicity and aspects of the local and large-scale climate. Case studies of daily variations in local environmental factors leading up to exceptionally toxic events identify a combination of conditions that generally precedes most closures from 1993 to 2007. These results suggest that periods of warm air and water temperatures and low streamflow on sub-seasonal timescales may facilitate toxin accumulation in mussels. No relationships were found between water residence times in the surface layer and either streamflow or mussel toxicity. Recommendations are made for future monitoring to improve forecasting of PST risks in Puget Sound, an important region for recreational, commercial, and tribal subsistence shellfish harvesting.     

Winter accumulation of paralytic shellfish toxins in digestive glands of mussels from Arcachon and Toulon (France) without detectable toxic plankton species revealed by interference in the mouse bioassay for lipophilic toxins

Since January 1993, neurological symptoms and rapid deaths (5 to 10 min) were typically observed in the mouse bioassay of acetone extracts of digestive glands from Arcachon and Toulon (France) during the winter season. It was assumed initially that a new lipophilic toxin was present because tests using the AOAC mouse bioassay for paralytic shellfish toxins on acid extracts of whole shellfish meat were negative, no known lipophilic toxins were detected and no toxic phytoplankton species were observed in the area during the poisoning events. In this study, however, preparative isolation of the toxic factor from toxic mussel digestive glands has revealed the presence of paralytic shellfish toxins, the principal ones being gonyautoxins-2 and -3 at Arcachon and gonyautoxins-1, -4, -2 and -3 at Toulon. The toxin concentrations recorded were below levels harmful to consumers and therefore represent a false positive in the mouse bioassay for lipophilic toxins based upon acetone extraction. The origin of the toxins remains to be determined.     

淡水藻类产毒研究进展

淡水藻类的许多种属都可产生毒素,如微囊藻属(Microcystis)、节球藻属(Nodularia)、鱼腥藻属(Anabaena)等。藻类毒素可通过多种途径,直接或间接引起人类或动物的健康问题。严重者,甚至导致死亡。试就国内外近年来对藻类毒素的毒理、性质、结构和检测方法等方面的研究作一简要综述。     

The effect of iron on Chilean Alexandrium catenella growth and paralytic shellfish toxin production as related to algal blooms

BioMetals - The dinoflagellate Alexandrium catenella is a well-known paralytic shellfish toxin producer that forms harmful algal blooms (HABs) worldwide. Blooms of this species have repeatedly...