Detection of Ciguatoxins and Tetrodotoxins in Seafood with Biosensors and Other Smart Bioanalytical Systems

The emergence of marine toxins such as ciguatoxins (CTXs) and tetrodotoxins (TTXs) in non-endemic regions may pose a serious food safety threat and public health concern if proper control measures are not applied. This article provides an overview of the main biorecognition molecules used for the detection of CTXs and TTXs and the different assay configurations and transduction strategies explored in the development of biosensors and other biotechnological tools for these marine toxins. The advantages and limitations of the systems based on cells, receptors, antibodies, and aptamers are described, and new challenges in marine toxin detection are identified. The validation of these smart bioanalytical systems through analysis of samples and comparison with other techniques is also rationally discussed. These tools have already been demonstrated to be useful in the detection and quantification of CTXs and TTXs, and are, therefore, highly promising for their implementation in research activities and monitoring programs.info:eu-repo/semantics/publishedVersio

Estudio de la sensibilidad de las células de neuroblastoma Neuro-2a a estándares de toxinas marinas

[ESP] Los métodos de detección de toxinas marinas basados en el estudio del efecto tóxico en cultivos celulares aportan información relevante respecto a la actividad tóxica de las toxinas y eventualmente contribuyen a describir su mecanismo de acción. Al igual que todas las metodologías de detección de toxinas, los ensayos de citotoxicidad necesitan de validaciones para convertirse en métodos de uso convencional. En éste trabajo abordamos la respuesta de células Neuro-2a (ATCC, CCL131) frente a diferentes estándares de toxinas marinas utilizando un método dependiente de ouabaina y veratridina (O/V). Este modelo celular, rico en canales de sodio, es un modelo habitualmente empleado para estudios de toxinas que actúan sobre estos canales. Con el fin de estudiar su aplicabilidad a otras toxinas, hemos realizado un estudio multi-toxínico. En las condiciones utilizadas en presencia de O/V se obtuvieron EC50s de 8,6 nM para la saxitoxina (bloqueadora de canales de sodio), 8,2 nM para la brevetoxina-3 y 36,9 nM para la brevetoxina-2 (activadoras de canales de sodio). Entre las toxinas que no actúan sobre canales de sodio, se produjo mortalidad en las células Neuro-2a sin tratamiento previo con O/V de forma dosis- dependiente, obteniéndose las siguientes EC50s: palitoxina 0,1nM, pectenotoxina-2 28,3 nM, ácido okadaico 21,9 nM y dinophysistoxina-1 20,6 nM. El ácido domoico no produjo mortalidad en las células Neuro-2a con o sin O/V. El modelo Neuro-2a se configura como una estrategia potente para el estudio de un amplio espectro de toxinas marinas.Al equipo de R.W. Dickey en el Gulf Coast Seafood Laboratory del FDA (Alabama, U.S.A)

Detection of Ostreopsis cf. ovata in environmental samples using an electrochemical DNA-based biosensor

Ostreopsis cf. ovata is a benthic microalga distributed in tropical and temperate regions worldwide which produces palytoxins (PlTXs). Herein, an electrochemical biosensor for the detection of this toxic microalga is described. The detection strategy involves isothermal recombinase polymerase amplification (RPA) of the target using tailed primers and a sandwich hybridisation assay on maleimide-coated magnetic beads immobilised on electrode arrays. The biosensor attained a limit of detection of 9 pg/μL of O. cf. ovata DNA (which corresponds to ~640 cells/L), with no interferences from two non-target Ostreopsis species (O. cf. siamensis and O. fattorussoi). The biosensor was applied to the analysis of planktonic and benthic environmental samples. Electrochemical O. cf. ovata DNA quantifications demonstrated an excellent correlation with other molecular methods (qPCR and colorimetric assays) and allowed the construction of a predictive regression model to estimate O. cf. ovata cell abundances. This new technology offer great potential to improve research, monitoring and management of O. cf. ovata and harmful algal blooms.info:eu-repo/semantics/acceptedVersio

Detoxification of paralytic shellfish poisoning toxins in naturally contaminated mussels, clams and scallops by an industrial procedure

Paralytic shellfish poisoning (PSP) episodes cause important economic impacts due to closure of shellfish production areas in order to protect human health. These closures, if are frequent and persistent, can seriously affect shellfish producers and the seafood industry, among others. In this study, we have developed an alternative processing method for bivalves with PSP content above the legal limit, which allows reducing toxicity to acceptable levels. A modification of the PSP detoxifying procedure stablished by Decision 96/77/EC of the European Union in Acanthocardia tuberculatum, was developed and implemented for PSP elimination in other bivalves species. The procedure was applied to 6 batches of mussels, 2 batches of clams and 2 batches of scallops, achieving detoxification rates of around 85%. A viable industrial protocol which allows the transformation of a product at risk into a safe product was developed. Although a significant reduction was obtained, in a sample circa 9000 μg STX diHCl equiv/kg, the final toxin level in these highly toxic mussels did not fall below the European limit. The processing protocol described may be applied efficiently to mussels, clams and scallops and it may be a major solution to counteract the closure of shellfish harvesting areas, especially if persistent.info:eu-repo/semantics/acceptedVersio

A fast magnetic bead-based colorimetric immunoassay for the detection of tetrodotoxins in shellfish

Tetrodotoxin (TTX) is a potent neurotoxin responsible for many food poisoning incidents and some fatalities. Although mainly associated with the consumption of pufferfish, in recent years, TTX has been found in shellfish, particularly in Europe. In this work, a magnetic bead (MB)-based colorimetric immunoassay was applied to the detection of TTX in Pacific oysters (Crassostrea gigas), razor clams (Solen marginatus) and mussels (Mytilus galloprovincialis). Effective LODs (eLODs) for TTX of 1 μg/kg in oysters and razor clams and 3.3 μg/kg in mussels, significantly below the EFSA guidance threshold (44 μg/kg), were obtained. The strategy was applied to the analysis of naturally-contaminated Pacific oysters (Crassostrea gigas) and mussels (Mytilus edulis) from the Netherlands, and TTX was detected in all samples. The approach, which takes less than 1.5 h, proved to be useful as a rapid and simple method to detect TTX, support shellfish safety and protect consumers.info:eu-repo/semantics/acceptedVersio

Dual quantitative PCR assay for identification and enumeration of Karlodinium veneficum and Karlodinium armiger combined with a simple and rapid DNA extraction method

Karlodinium is a dinoflagellate genus responsible for massive fish mortality events worldwide. It is commonly found in Alfacs Bay (NW Mediterranean Sea), where the presence of two Karlodinium species (K. veneficum and K. armiger) with different toxicities has been reported. Microscopy analysis is not able to differentiate between these two species. Therefore, new and rapid methods that accurately and specifically detect and differentiate these two species are crucial to facilitate routine monitoring, to provide early warnings and to study population dynamics. In this work, a quantitative real-time PCR (qPCR) method to detect and enumerate K. veneficum and K. armiger is presented. The ITS1 region of the ribosomal DNA was used to design species-specific primers. The specificity of the primers together with the melting curve profile provided a reliable qualitative identification and discrimination between the two Karlodinium species. Additionally, a simple and rapid DNA extraction method was used. Standard curves were constructed from 10-fold dilutions of cultured microalgae cells. Finally, the applicability of the assay was tested with field samples collected from Alfacs Bay. Results showed a significant correlation between qPCR determinations and light microscopy counts (y = 2.838 x + 564; R2 = 0.936). Overall, the qPCR method developed herein is specific, rapid, accurate, and promising for the detection of these two Karlodinium species in environmental samples.info:eu-repo/semantics/acceptedVersio

Toxicity characterisation of Gambierdiscus species from the Canary Islands

In the last decade, several outbreaks of ciguatera fish poisoning (CFP) have been reported in the Canary Islands (central northeast Atlantic Ocean), confirming ciguatera as an emerging alimentary risk in this region. Five Gambierdiscus species, G. australes, G. excentricus, G. silvae, G. carolinianus and G. caribaeus, have been detected in macrophytes from this area and are known to produce the ciguatoxins (CTXs) that cause CFP. A characterization of the toxicity of these species is the first step in identifying locations in the Canary Islands at risk of CFP. Therefore, in this study the toxicity of 63 strains of these five Gambierdiscus species were analysed using the erythrocyte lysis assay to evaluate their maitotoxin (MTX) content. In addition, 20 of the strains were also analysed in a neuroblastoma Neuro-2a (N2a) cytotoxicity assay to determine their CTX-like toxicity. The results allowed the different species to be grouped according to their ratios of CTX-like and MTX-like toxicity. MTX-like toxicity was especially high in G. excentricus and G. australes but much lower in the other species and lowest in G. silvae. CTX-like toxicity was highest in G. excentricus, which produced the toxin in amounts ranging between 128.2 ± 25.68 and 510.6 ± 134.2 fg CTX1B equivalents (eq) cell−1 (mean ± SD). In the other species, CTX concentrations were as follows: G. carolinianus (100.84 ± 18.05 fg CTX1B eq cell−1), G. australes (31.1 ± 0.56 to 107.16 ± 21.88 fg CTX1B eq cell−1), G. silvae (12.19 ± 0.62 to 76.79 ± 4.97 fg CTX1B eq cell−1) and G. caribaeus (<LOD to 90.37 ± 15.89 fg CTX1B eq cell−1). Unlike the similar CTX-like toxicity of G. australes and G. silvae strains from different locations, G. excentricus and G. caribaeus differed considerably according to the origin of the strain. These differences emphasise the importance of species identification to assess the regional risk of CFP.info:eu-repo/semantics/publishedVersio

Marine biotoxins in the Catalan littoral: could biosensors be integrated into monitoring programmes?

Aquest article descriu els sensors enzimàtics i immunosensors electroquímics que s'han desenvolupat als nostres grups per a la detecció de la biotoxina marina àcid okadaic (OA), i discuteix la possibilitat d'integrar-los en programes de seguiment. Els sensors enzimàtics per a OA que es presenten es basen en la inhibició de la proteïna fosfatasa (PP2A) per aquesta toxina i la mesura electroquímica de l'activitat enzimàtica mitjançant l'ús de substrats enzimàtics apropiats, electroquímicament actius després de la seva desfosforació per l'enzim. Els immunosensors electroquímics descrits en aquest article es basen en un enzimoimmunoassaig sobre fase sòlida competitiu indirecte (ciELISA), amb fosfatasa alcalina (ALP) o peroxidasa (HRP) com a marcatges, i un sistema de reciclatge enzimàtic amb diaforasa (DI). Els biosensors presentats aquí s'han aplicat a l'anàlisi de dinoflagel·lats, musclos i ostres. Les validacions preliminars amb assaigs colorimètrics i LC-MS/MS han demostrat la possibilitat d'utilitzar les bioeines desenvolupades per al cribratge preliminar de biotoxines marines en mostres de camp o de cultiu, que ofereixen informació complementària a la cromatografia. En conclusió, tot i que encara cal optimitzar alguns paràmetres experimentals, la integració dels biosensors a programes de seguiment és viable i podria proporcionar avantatges respecte a altres tècniques analítiques pel que fa al temps d'anàlisi, la simplicitat, la selectivitat, la sensibilitat, el fet de poder ser d'un sol ús i l'efectivitat de cost.This article describes the electrochemical enzyme sensors and immunosensors that have been developed by our groups for the detection of marine biotoxin okadaic acid (OA), and discusses the possibility of integrating them into monitoring programmes. The enzyme sensors for OA reported herein are based on the inhibition of immobilised protein phosphatase 2A (PP2A) by this toxin and the electrochemical measurement of the enzyme activity through the use of appropriate enzyme substrates, which are electrochemically active after dephosphorylation by the enzyme. The electrochemical immunosensors described in this article are based on a competitive indirect Enzyme- Linked ImmunoSorbent Assay (ciELISA), using alkaline phosphatase (ALP) or horseradish peroxidase (HRP) as labels, and an enzymatic recycling system with diaphorase (DI). The biosensors presented herein have been applied to the analysis of dinoflagellates, mussels and oysters. Preliminary validations with colorimetric assays and LC-MS/MS have demonstrated the possibility of using the developed biotools for the preliminary screening of marine biotoxins in field or cultured samples, offering complementary information to chromatography. In conclusion, although optimisation of some experimental parameters is still required, the integration of biosensors into monitoring programmes is viable and may provide advantages over other analytical techniques in terms of analysis time, simplicity, selectivity, sensitivity, disposability of electrodes and cost effectiveness

Toxicidad de la dinoflagelada Gambierdiscus sp. aislada de las Islas Canarias

[ESP] En este trabajo presentamos la caracterización de la toxicidad de una cepa de Gambierdiscus sp. procedente de las Islas Canarias mediante la utilización de cultivos celulares de neuroblastomas Neuro-2a. Los resultados obtenidos dan una toxicidad muy elevada del extracto bruto. La utilización de un activador específico del canal de sodio (Veratridina) y de un inhibidor de la bomba de sodio potasio (Ouabaina) aplicados conjuntamente con el extracto, permite aumentar la sensibilidad de la respuesta tóxica de las células Neuro-2a ante la presencia de toxinas neurotóxicas que actúan sobre canales de sodio (como las ciguatoxinas). La diferencia de respuesta tóxica obtenida con el extracto de Gambierdiscus sp. procedente de las Islas Canarias sin presencia de Ouabaina/Veratridina (IC50= 3,84 células mL-1 RPMI) o con presencia de Ouabaina/Veratridina (IC50= 2,04 células mL-1 RPMI) no es estadísticamente significativa (p > 0,05) pero la respuesta no descarta la posible presencia de neurotoxinas de tipo activadoras del canal de sodio.A Javier Fernández, de la Universidad de la Laguna, y a J. Franco del CSIC por su colaboración. Este trabajo ha sido financiado parcialmente con una beca predoctoral INIA (Ministerio Español de Educación y Ciencia)

Assessment of cytotoxicity in ten strains of Gambierdiscus australes from Macaronesian Islands by neuro-2a cell-based assays

Within the dinoflagellate genus Gambierdiscus several species are well-known producers of ciguatoxins (CTXs) and maitotoxins (MTXs). These compounds are potent marine toxins that accumulate through the food chain, leading to a foodborne disease known as ciguatera when contaminated fish is consumed. Given the threat that the presence of these toxins in seafood may pose to human health and fisheries, there is an evident necessity to assess the potential toxicity of Gambierdiscus sp. in a particular area. Thus, the purpose of this work was to evaluate the production of CTX and MTX of 10 strains of Gambierdiscus australes isolated from the Selvagem Grande Island (Madeira, Portugal) and El Hierro Island (Canary Islands, Spain). The strains were first characterized by light microscopy and species were confirmed by molecular biology, being identified as G. australes. Following the species identification, CTX and MTX production of G. australes extracts was evaluated at the exponential growth phase using neuro-2a cell-based assays. Additionally, the production of MTX was also investigated in two of the G. australes strains collected at the stationary growth phase. Interestingly, 9 out of 10 strains were found to produce CTX-like compounds, ranging from 200 to 697 fg equiv. CTX1B cell−1. None of the G. australes strains showed MTX-like activity at the exponential phase, but MTX production was observed in two strains at the stationary growth phase (227 and 275 pg equiv. MTX cell−1). Therefore, the presence of G. australes strains potentially producing CTX and MTX in these Macaronesian Islands was confirmed.info:eu-repo/semantics/acceptedVersio