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      Preliminary Results on the Evaluation of the Occurrence of Tetrodotoxin Associated to Marine Vibrio spp. in Bivalves from the Galician Rias (Northwest of Spain)

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          Abstract

          Tetrodotoxins (TTX) are a potent group of natural neurotoxins putatively produced by symbiotic microorganisms and affecting the aquatic environment. These neurotoxins have been recently found in some species of bivalves and gastropods along the European Coasts (Greece, UK, and The Netherlands) linked to the presence of high concentrations of Vibrio, in particular Vibrio parahaemolyticus. This study is focused on the evaluation of the presence of Vibrio species and TTX in bivalves (mussels, oysters, cockles, clams, scallops, and razor clams) from Galician Rias (northwest of Spain). The detection and isolation of the major Vibrio spp. and other enterobacterial populations have been carried out with the aim of screening for the presence of the pathways genes, poliketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) possibly involved in the biosynthesis of these toxins. Samples containing Vibrio spp. were analyzed by biochemical (API20E-galery) and genetic tests (PCR-RT). These samples were then screened for TTX toxicity by a neuroblastoma cell-based assay (N2a) and the presence of TTX was further confirmed by LC-MS/MS. TTX was detected in two infaunal samples. This is the first confirmation of the presence of TTX in bivalve molluscs from the Galician Rias.

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          Vibrio parahaemolyticus: a concern of seafood safety.

          Vibrio parahaemolyticus is a human pathogen that is widely distributed in the marine environments. This organism is frequently isolated from a variety of raw seafoods, particularly shellfish. Consumption of raw or undercooked seafood contaminated with V. parahaemolyticus may lead to development of acute gastroenteritis characterized by diarrhea, headache, vomiting, nausea, and abdominal cramps. This pathogen is a common cause of foodborne illnesses in many Asian countries, including China, Japan and Taiwan, and is recognized as the leading cause of human gastroenteritis associated with seafood consumption in the United States. This review gives an overview of V. parahaemolyticus food poisoning and provides information on recent development in methods for detecting V. parahaemolyticus and strategies for reducing risk of V. parahaemolyticus infections associated with seafood consumption.
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            On the origins and biosynthesis of tetrodotoxin.

            The potent neurotoxin tetrodotoxin (TTX) has been identified from taxonomically diverse marine organisms. TTX possesses a unique cage-like structure, however, its biosynthesis has yet to be elucidated. Biosynthetic studies in the TTX-producing newt Taricha torosa, and in bacterial genera, including Vibrio, have proven inconclusive. Indeed, very few studies have been performed that address the cellular production of TTX. Here we review the sources of TTX described to date and provide evidence for the biosynthesis of TTX by symbiotic microorganisms in higher taxa. Chemical and genetic based biosynthesis studies of TTX undertaken thus far are discussed and we outline approaches which may be useful for expanding upon the current body of knowledge. The complex biosynthesis of structurally similar toxins, that reveal clues into the biosynthetic pathway of TTX, is also presented. Copyright © 2011 Elsevier B.V. All rights reserved.
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              Tetrazolium-based cell bioassay for neurotoxins active on voltage-sensitive sodium channels: semiautomated assay for saxitoxins, brevetoxins, and ciguatoxins.

              In the present study we have developed an assay for the detection of sodium channel-specific marine toxins based upon mitochondrial dehydrogenase activity in the presence of veratridine and ouabain. This cell bioassay allows detection of either sodium channel enhancers, such as the brevetoxins and the ciguatoxins, or sodium channel blocking agents, such as the saxitoxins. The assay responds in a dose dependent manner and differentiates the toxic activity as either sodium channel blocking or enhancing. In addition, the assay is highly sensitive, with present detection limits of 2 ng/ml for either saxitoxins or brevetoxins (PbTx-1 and PbTx-3). Assay response to a ciguatoxic extract and to brevetoxins is rapid, allowing dose dependent detection within 4 to 6 h. The method is simple, utilizes readily available reagents, uses substantially less sample than required for mouse bioassay, and is well within the scope of even modest tissue culture facilities. This cell-based protocol has the potential to serve as an alternate and complementary method to the standard mouse bioassay.
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                Author and article information

                Journal
                Mar Drugs
                Mar Drugs
                marinedrugs
                Marine Drugs
                MDPI
                1660-3397
                06 March 2018
                March 2018
                : 16
                : 3
                : 81
                Affiliations
                [1 ]Department Analytical and Food Chemistry, University of Vigo, 36310 Vigo, Spain; leao@ 123456uvigo.es (J.M.L.); amcbeluso21@ 123456gmail.com (A.L.-L.); jgiraldez@ 123456uvigo.es (J.G.); ovilarino@ 123456uvigo.es (Ó.V.)
                [2 ]EU Reference Laboratory for Marine Biotoxins, 36310 Vigo, Spain
                [3 ]Laboratory ASMECRUZ, 36939 Bueu, Pontevedra, Spain
                Author notes
                [* ]Correspondence: anagago@ 123456uvigo.es ; Tel.: +34-647-343-417
                Article
                marinedrugs-16-00081
                10.3390/md16030081
                5867625
                29509715
                a7ac20c7-4c1d-40ed-b7d2-d581b81cd14d
                © 2018 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 08 January 2018
                : 01 March 2018
                Categories
                Article

                Pharmacology & Pharmaceutical medicine
                tetrodotoxin,vibrio,nrps,pks,hilic-lc-ms/ms,bivalve molluscs
                Pharmacology & Pharmaceutical medicine
                tetrodotoxin, vibrio, nrps, pks, hilic-lc-ms/ms, bivalve molluscs

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