lolB gene, a valid alternative for qPCR detection of Vibrio cholerae in food and environmental samples.

In recent years a new genetic target for Vibrio cholerae detection has been reported, but its application was limited to clinical samples. This target, lolB, has never been applied to either food or environmental samples. In the present study the development, as well as the evaluation and pre-validation, of a real-time PCR method based on lolB, is described. The method included a newly designed hydrolysis probe to enhance its specificity. After comparison against other molecular and traditional methods, similar results were obtained regarding relative sensitivity, relative specificity, relative accuracy, positive and negative predictive values and index kappa of concordance (all higher than 91%), as well as a very low limit of detection (2 cfu/25 g). Additionally, after the analysis of more than 160 different food and environmental samples, its applicability in the food industry was completely demonstrated.

Fast Real-Time PCR assay for detection of Tetramicra brevifilum in cultured turbot.

Global aquaculture production of turbot has rapidly increased worldwide in the last decade and it is expected to have even bigger growth in the next years due to new farms operating. The losses caused by pathogen infections have grown at the same time as the production of this species. Parasitological infections are among the main relevant pathologies associated with its culture and produce serious losses in aquaculture, reduce the growth rate in fish and may lead to unmarketable fish due to skeletal muscle abnormalities in cases with high intensity of infection. The microsporidian parasite Tetramicra brevifilum causes severe infections and generates major losses in farmed turbot. Infections are difficult to control due to spore longevity and its direct transmission. To facilitate the infection management, an effective tool for fast detection and identification of T. brevifilum is needed. This study provides a molecular methodology of fast Real-Time PCR for T. brevifilum detection to the aquaculture industry, useful for routine control of T. brevifilum at turbot farms. The method is characterized by its high specificity and sensitivity, and it can be applied to cultured turbot for parasite detection regardless of the life-cycle stage of the pathogen or the infection intensity.

Quantitative determination of the okadaic acid toxins group by a colorimetric phosphatase inhibition assay: interlaboratory study.

An interlaboratory collaborative study to validate a colorimetric phosphatase inhibition assay for quantitative determination of the okadaic acid (OA) toxins group in molluscs, OkaTest, was conducted. Eight test materials, including mussels, scallops, clams, and cockles, were analyzed as blind duplicates. Blank samples and materials containing different OA toxin levels ranging from 98 to 275 microg/kg OA equivalents were included. The study was carried out by a total of 16 laboratories from 11 different countries. Values obtained for repeatability relative standard deviations (RSDr) ranged from 5.4 to 11.2% (mean 7.5%). Reproducibility RSD (RSD(R)) values were between 7.6 and 13.2% (mean 9.9%). The Horwitz ratio (HorRat) values ranged between 0.4 and 0.6. A recovery assay was also carried out using a sample spiked with OA. A mean recovery of 98.0% and an RSD of 14.5% were obtained. The results obtained in this validation study indicate that the colorimetric phosphatase inhibition assay, OkaTest, is suitable for quantitative determination of the OA toxins group. OkaTest could be used as a test that is complementary to the reference method for monitoring the OA toxins group.

Cyclic imines: chemistry and mechanism of action: a review.

In recent years, there has been an increase in the production of shellfish and in global demand for seafood as nutritious and healthy food. Unfortunately, a significant number of incidences of shellfish poisoning occur worldwide, and microalgae that produce phycotoxins are responsible for most of these. Phycotoxins include several groups of small to medium sized natural products with molecular masses ranging from 300 to over 3000 Da. Cyclic imines (CIs) are a recently discovered group of marine biotoxins characterized by their fast acting toxicity, inducing a characteristic rapid death in the intraperitoneal mouse bioassay. These toxins are macrocyclic compounds with imine (carbon-nitrogen double bond) and spiro-linked ether moieties. They are grouped together due to the imino group functioning as their common pharmacore and due to the similarities in their intraperitoneal toxicity in mice. Spirolides (SPXs) are the largest group of CIs cyclic imines that together with gymnodimines (GYMs) are best characterized. Although the amount of cyclic imines in shellfish is not regulated and these substances have not been categorically linked to human intoxication, they trigger high intraperitoneal toxicity in rodents. In this review, the corresponding chemical structures of each member of the CIs and their derivatives are reviewed as well as all the data accumulated on their mechanism of action at cellular level.

Authentication of Atlantic salmon (Salmo salar) using real-time PCR.

A real-time PCR assay based on LNA TaqMan probe technology was developed for the detection and identification of Atlantic salmon (Salmo salar). Among the advantages it is worth highlighting simplicity, rapidity, highest potential for automation and minor risk of contamination of this technique. The TaqMan real-time PCR is the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabelling of this species. The method can be applied to all kind of products, fresh, frozen and processed products, including those undergoing intensive processes of transformation. The developed methodology using specific primers-probe set was validated and further applied to 20 commercial samples labelled as salmon or S. salar in order to determinate if the species used for their manufacturing corresponded to this species. The methodology herein developed is useful to check the fulfilment of labelling regulations for seafood products, verify the correct traceability in commercial trade and for fisheries control.

Whey protein-based coatings on frozen Atlantic salmon (Salmo salar): Influence of the plasticiser and the moment of coating on quality preservation.

The effects of different whey protein concentrate coating formulations (with or without glycerol or sorbitol in two proportions) on frozen Atlantic salmon quality parameters were evaluated. The influence of the moment of coating application (before or after freezing) was also studied. The coating application after freezing increased the thaw yield, decreased the drip loss, and modified colour parameters of frozen and thawed fillets, in comparison with application before freezing. The moment of coating also influenced the colour of cooked fish fillets. The type of plasticiser affects the colour of thawed and cooked samples, but not the colour of frozen samples. The protein coatings delayed lipid oxidation of salmon fillets, providing better protection against it than water glazing, and this effect was more pronounced when glycerol instead of sorbitol was used in the coating formulation. WPC+glycerol (1:1) coating was the best for frozen Atlantic salmon protection. The sensory properties of salmon fillets were not modified by the use of this coating.

Validation of a method for the detection of five species, serogroups, biotypes and virulence factors of Vibrio by multiplex PCR in fish and seafood.

In this work a sequential multiplex PCR system was designed and validated for the detection of most frequent foodborne pathogen Vibrio species in fish and seafood (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginoliticus and Vibrio mimicus). The method proposed functions in a hierarchical way, being composed of an end-point multiplex PCR to detect the presence of DNA belonging to the studied species, followed by multiplex PCR and fragment analysis allowing the viability assessment of the detected strains. The final multiplex PCR step of the method may be applied if identification of the serogroup, biotype and/or virulence factor level is necessary. Forty samples of commercial fish and seafood products were used at the method validation stage. Sixty three marine organism samples obtained from various estuarine areas of Spain including shrimps, crabs, bivalve mollusks and fishes were screened for presence of Vibrio species and 2 mussel samples were found positive for V. parahaemolyticus. On the whole, the proposed method is robust and readily adaptable in routine molecular diagnostic laboratories, allowing monitoring and simultaneous detection of all these bacterial pathogens in seafood samples, reducing the expenses and time consumed by other analytical methods.

Effects of environmental regimens on the toxin profile of Alexandrium ostenfeldii.

Environmental conditions are key factors in the development of marine toxic phytoplankton. Spirolides are marine toxins with a heptacyclic imine ring responsible for the toxicity in mice. Alexandrium ostenfeldii (A. ostenfeldii) is the main producer of these toxins, although this dinoflagellate often produces toxins belonging to the paralytic shellfish poisoning (PSP) group. The present study shows the first evidence that external environmental factors can influence the toxin profile produced by the dinoflagellate A. ostenfeldii. The species investigated is indigenous to the North Atlantic coast, and their cells grew under several environmental parameters. Toxin production was measured by means of liquid chromatography-mass spectrometry (LC-MS) and the chromatograms reflect the presence of two spirolides in all cultures; one in the region m/z 692.5, corresponding to 13-desmethyl spirolide C (13-desMeC) and the other in the region m/z 678.5, which corresponds to 13,19-didesmethyl spirolide C (13,19-didesMeC). The physical parameters studied were salinity, culture media, and photoperiod. The highest amount of toxin per cell was obtained when dinoflagellates grew in F/2 and Walne medium, 28 per thousand salinity, and 24 h of light. However, the highest proportion of 13,19-didesMeC with respect to 13-desMeC was achieved in L1 medium, 33 per thousand salinity, and 14:10 h light:dark. On the contrary, the highest proportion of 13-desMeC in cells was obtained when A. ostenfeldii was cultured in F/2 medium, 28 per thousand salinity, and the same photoperiod. Therefore, from these data the optimum conditions to culture A. ostenfeldii and to obtain the highest amount of spirolide per cell are shown. In addition, these environmental conditions can be considered a tool to predict and avoid A. ostenfeldii blooms.

Migration of BADGE (bisphenol A diglycidyl-ether) and BFDGE (bisphenol F diglycidyl-ether) in canned seafood.

Migration of potentially toxic materials used for the lining of commercial can goods remains an important issue, especially with respect to certain types of processed foods. Seafood is one type where more information is needed with respect to other ingredients used for adding value to fishery products. Most cans are internally coated with starters of resins such as bisphenol A diglycidyl-ether (BADGE) and bisphenol F diglycidyl-ether (BFDGE), both considered as toxic compounds. Several seafood products, sardines, tuna fish, mackerel, mussels, cod and mackerel eggs, were manufactured in different conditions changing covering sauce, time and temperature of storage and heat-treated for sterilization in cans. Migration kinetics of BADGE and BFDGE from varnish into canned products were evaluated by HPLC in 70 samples after 6, 12 or 18 months of storage. Results showed that there is no migration of BADGE in tuna fish, sardines, mussels or cod. However, migration of BFDGE occurs in all species, in a storage time-dependent way and content of fat, although migration of these compounds is not affected by sterilization conditions. All samples analyzed presented values lower than 9 mg BADGE/kg net product without exceeding European limits. However, concerning BFDGE migration, European legislation does not allow the use and/or the presence of BFDGE. Main migration takes place in mackerel reaching the highest values, 0.74 mg BFDGE/kg and 0.34 mg BADGE/kg net product, in red pepper sauce.

The cytoskeleton, a structure that is susceptible to the toxic mechanism activated by palytoxins in human excitable cells.

Palytoxin is a marine toxin responsible for a fatal type of poisoning in humans named clupeotoxism, with symptoms such as neurologic disturbances. It is believed that it binds to the Na(+)/K(+)-ATPase from the extracellular side and modifies cytosolic ions; nevertheless, its effects on internal cell structures, such as the cytoskeleton, which might be affected by these initial events, have not been fully elucidated. Likewise, ostreocin-D, an analog of palytoxin, has been only recently found, and its action on excitable cells is therefore unknown. Therefore, our aim was to investigate the modifications of ion fluxes associated with palytoxin and ostreocin-D activities, and their effects on an essential cytoskeletal component, the actin system. We used human neuroblastoma cells and fluorescent dyes to detect changes in membrane potential, intracellular Ca(2+) concentration, cell detachment, and actin filaments. Fluorescence values were obtained with spectrofluorymetry, laser-scanning cytometry, and confocal microscopy; the last of these was also used for recording images. Palytoxin and ostreocin-D modified membrane permeability as a first step, triggering depolarization and increasing Ca(2+) influx. The substantial loss of filamentous actin, and the morphologic alterations elicited by both toxins, are possibly secondary to their action on ion channels. The decrease in polymerized actin seemed to be Ca(2+)-independent; however, this ion could be related to actin cytoskeletal organization. Palytoxin and ostreocin-D alter the ion fluxes, targeting pathways that involve the cytoskeletal dynamics of human excitable cells.

Molecular detection of Xenostrobus securis and Mytillus galloprovincialis larvae in Galician Coast (Spain).

The mussel species Xenostrobus securis from New Zealand was detected in the Spanish coast recently, in the mouth of the Verdugo River into the Vigo Ria. In view of the great importance of the farm mussel sector in this region, the presence of this alien species greatly concerned producers and administration authorities, because of its potential medium- or long-term effects on the autochthonous species, Mytilus galloprovincialis, an important marine resource widely exploited in this location. The goal of this study was to develop a DNA-based technique to identify X. securis and M. galloprovincialis larvae in plankton samples, which would allow monitoring for the presence of X. securis in different points of the Vigo Ria. The techniques used were simplex and multiplex polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and fragment analysis. The application of this system to planktonic samples could be an effective means to assess the presence of the alien species, allowing monitoring if its dispersion is increasing, or on the contrary, if its distribution is restricted to the mouth of the Verdugo River, where X. securis was first detected. In addition, the application of this system at different times could be useful to assess the presence of larvae of these two species in the plankton.

Genetic identification of squids (families Ommastrephidae and Loliginidae) by PCR-RFLP and FINS methodologies.

Cephalopods are a taxonomic group that contains a great number of families, genera and species, with many of them very important at the commercial level. The existence of very similar species in this class added up to the transformation process applied to them makes it difficult or even impossible for species identification based on morphological characterization. Moreover, the global commerce makes it possible that one determined species can be marketed in its antipodes. These questions suggest the necessity of molecular techniques to solve this situation. In the present work, a genetic method was developed on the basis of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and forensically informative nucleotide sequencing (FINS) technique and makes possible the identification of more than 20 species belonging to the families Ommastrephidae and Loliginidae, as well as some octopus and sepia species. The PCR was employed to amplify 651 and 208 bp fragments of the mitochondrial cytochrome b gene. These molecular systems were applied to fresh, frozen, precooked, even canned cephalopods, allowing for the identification of the species included in these products. Therefore, these molecular tools could be applied in questions related to correct labeling, traceability, and importation controls of squids, sepias, and octopuses.

Detection of land animal remains in fish meals by the polymerase chain reaction-restriction fragment length polymorphism technique.

In the present study a technique was developed with the aim of guaranteeing the composition and security of fish meals, since it allows verification of whether these meals contain land animal remains. The method is based on polymerase chain reaction (PCR) and length polymorphism, followed by a restriction fragment length polymorphism (RFLP). Specific primers for every species were designed and calibrated, generating exclusively a PCR product with a specific size when DNA for each species was present in the sample. This technique allows the detection of land animal remains in fish meals, specifically cow, chicken, pig, horse, sheep, and goat. The identity of the PCR products can be confirmed by RFLP analysis using only one restriction enzyme. The selected restrictase generated one characteristic restriction profile for every species included in this study. The detection limit of this method was calculated by using mixtures of fish meals in different proportions and meal that exclusively contained remains of one of these land species studied. The analytical strategy herein proposed was applied to fish and meat meals, giving good results, both in the analyzed standards and in commercial samples.

Development of a method for the genetic identification of mussel species belonging to Mytilus, Perna, Aulacomya, and other genera.

Legislation regarding the labeling of processed products is an important issue in the protection of consumer rights. This labeling is especially important in products that cannot be identified on the basis of their morphological characters, because these are removed from the animal in the transformation process. The goal of this study was the identification of mussel species using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Forensically Informative Nucleotide Sequencing (FINS) methodologies. The molecular marker selected was 18S rDNA (nuclear small-subunit rDNA gene), which allows identification at the genus level and at the species level in some cases. The genera included in this study were Mytilus, Perna, Aulacomya, Semimytilus, Brachidontes, Choromytilus, and Perumytilus. Different markers were used for genetic identification at the species level. To identify the species included in the genus Perna and Choromytilus, a fragment of ITS 1 (Internal Transcribed Spacer 1) was amplified by multiplex PCR and digested with restrictases. The species of Mytilus were identified by length polymorphism and RFLP of the polyphenolic adhesive protein gene. This methodology was validated with products manufactured in the authors' pilot plant and applied to commercial samples. Therefore, this sequential method can be completely or partially used to determine the mussel genus or species present in any food product.

FAST Real Time PCR for control of intra-species recycling in aquaculture feed, focused to the most relevant fish species farmed in Europe.

Recent regulations in animal feed composition prohibit intra-species recycling, the recycling of one given animal species to the same species, in order to avoid potential safety risks to human and animal health. These regulations have generated the need of their control in aquaculture by effective and specific analytical techniques. To date, most studies of species identification and detection in feedstuffs are focused on land species, but few studies are focused on species composition in fish feed. The present work describes five methodologies based in Real Time PCR for detection of the most relevant fish species farmed in Europe: gilthead sea bream (Sparus aurata); sea bass (Dicentrarchus labrax); turbot (Scophthalmus maximus); rainbow trout (Onchorynchus mykiss); and salmon (Salmo salar), in order to guarantee the intra-species recycling regulation in aquaculture feedstuffs.

Effect of the industrial canning on the toxicity of mussels contaminated with diarrhetic shellfish poisoning (DSP) toxins.

The effect of canning in pickled sauce and autoclaving on weight, toxin content, toxin concentration and toxicity of steamed mussels was studied. Weight decreased by 25.5%. Okadaic acid (OA) and DTX2 content of mussel meat decreased by 24.1 and 42.5%, respectively. The estimated toxicity of the mussel remained nearly unchanged (increased by 2.9%). A part of the toxins lost by the mussels was leached to the sauce but the remaining part should have been thermally degraded. DTX2 underwent more degradation than OA and, in both toxins, free forms more than conjugated ones. This process, therefore, cannot be responsible for the large increments of toxicity of processed mussels -relative to the raw ones-sometimes detected by food processing companies. The final product could be monitored in several ways, but analysing the whole can content or the mussel meat once rehydrated seems to be the most equivalents to the raw mussel controls.

Azaspiracid-1, a potent, nonapoptotic new phycotoxin with several cell targets.

This paper reports on potential cellular targets of azaspiracid-1 (AZ-1), a new phycotoxin that causes diarrhoeic and neurotoxic symptoms and whose mechanism of action is unknown. In excitable neuroblastoma cells, the systems studied were membrane potential, F-actin levels and mitochondrial membrane potential. AZ-1 does not modify mitochondrial activity but decreases F-actin concentration. These results indicate that the toxin does not have an apoptotic effect but uses actin for some of its effects. Therefore, cytoskeleton seems to be an important cellular target for AZ-1 effect. AZ-1 does not induce any modification in membrane potential, which does not support for neurotoxic effects. In human lymphocytes, cAMP, cytosolic calcium and cytosolic pH (pHi) levels were also studied. AZ-1 increases cytosolic calcium and cAMP levels and does not affect pHi (alkalinization). Cytosolic calcium increase seems to be dependent on both the release of calcium from intracellular Ca(2+) pools and the influx from extracellular media through Ni(2+)-blockable channels. AZ-1-induced Ca(2+) increase is negatively modulated by protein kinase C (PKC) activation, protein phosphatases 1 and 2A (PP1 and PP2A) inhibition and cAMP increasing agents. The effect of AZ-1 in cAMP is not extracellularly Ca(2+) dependent and insensitive to okadaic acid (OA).

Collapse of mitochondrial membrane potential and caspases activation are early events in okadaic acid-treated Caco-2 cells.

Diarrhetic Shellfish Poisoning (DSP) results from the consumption of shellfish contaminated with okadaic acid (OA) or one of the dinophysistoxins (DTX). It has been reported that this toxin induces apoptosis in several cell models, but the molecular events involved in this process have not been clarified. In this report we studied intracellular signals induced by OA in Caco-2 cells: mitochondrial membrane potential, F-actin depolymerization, caspases activation, cell proliferation and cell membrane integrity. Results indicate that caspases-8 and -9 increased their activity after 30 min of OA treatment according to their role as initiator caspases. In contrast, activation of the downstream caspase-3 is a later event in the execution phase of apoptosis. Mitochondrial membrane potential changes are detected at 30 min of OA exposure indicating that this is an early response in the apoptotic cascade. F-actin depolymerization occurs after 24h of incubation with OA and this effect is significant at low doses of the toxin. LDH is released into the culture medium, although there is not PI uptake, indicative of a significant cell death in addition to apoptosis. Moreover, OA led to a dose- and time-dependent decrease in cellular proliferation.

ITS1-rDNA-based methodology to identify world-wide hake species of the Genus Merluccius.

Species-specific DNA-based tags are valuable tools for the management of both fisheries and commercial fish products. In this study, we have developed a two-step molecular tool to detect the presence of hake DNA (Merluccius spp.) and to identify the exact hake species present in an blind sample. The first test involves PCR amplification of an ITS1-rDNA fragment of 193 bp using nested primers that are interspecifically conserved in Merluccius spp. and Atlantic cod, Gadus morhua. The second test consists of the PCR amplification of a 602-659 bp DNA fragment spanning part of the ribosomal cluster 18S-ITS1-5.8S and digesting it with four restriction enzymes whose targets map at interspecifically nonconserved sites of the ITS1. Alternatively, the identification of hake species can be achieved by FINS or BLAST, using the nucleotide sequence of either the whole ITS1 sequence or its nested fragment of 193 bp. Because of their high reproducibility and ease of execution, these procedures allow for routine analysis and constitute high reliable tools for the rapid identification of 12 species of hake.

Detection of the causal agents of Keriorrhea (Lepidocybium flavobrunneum and Ruvettus pretiosus) by means of Real Time PCR.

Consumption of Lepidocybium flavobrunneum and Ruvettus pretiosus is related with the gastrointestinal disease called Keriorrhea. Sometimes, intentionally or not, these species are mislabelled as other harmless species, causing severe disruptions to consumers. The correct identification of these species helps to avoid food fraud and health problems. For this reason, a multiplex Real Time-PCR method based on TaqMan technology for the correct authentication of L. flavobrunneum and R. pretiosus has been developed. The method is based on a species-specific set of primers and TaqMan probe which amplifies a 276 bp fragment of the cytochrome oxidase I (COI) mitochondrial DNA region. This methodology allows your application to any type of product, regardless of the degree of processing it has undergone with high specificity, sensitivity and rapidity. Also, it might be a useful tool in monitoring and verifying labelling regulation and protect consumer rights.