Caracterización del riesgo de exposición al parásito de pescado "Anisakis" sp

Uno de los agentes más relevantes causante de zoonosis por parásitos de pescado son los nematodos de la familia Anisakidae.La especie que se ha descrito más frecuentemente en humanos es el A. simplex.Las enfermedades que puede causar son debidas a la infestación después de la ingestión de larvas viables o a reacciones alérgicas (hipersensibilidad) por exposición a material parasitario sin requerir la presencia de larva viva,por lo que la evaluación de la exposición tiene características claramente distintivas en las dos entidades. Los alérgenos de A. simplex son muy resistentes al calor y a la congelación,de hecho, se ha descrito persistencia de alérgenos y antígenos de Anisakis sp. tras realizar diversos tratamientos donde se someten las larvas al efecto del calor o de la congelacióne incluso en productos procesados de forma industrial; estos tratamientos aplicados a los productos de la pesca matan al parásito pero puedeque no eviten el riesgo de alergiaen los consumidores.Para evaluar la exposición a alérgenos del parásito hay que tener en cuenta también el consumo de productos de pesca que representan una fuente insospechada de alérgenos como son los productos muy elaborados, los productos envasados y los pescados de acuicultura. En el caso de los pescados de acuicultura, la ausencia de larva viva no excluye la posibilidad de que pueda detectarse en el músculo del pescado alérgenos procedentes de los piensos que hayan sido elaborados con harinas de pescado parasitados. El nematodo Anisakis sp.es el único organismo conocido que es a la vez un parásito helminto y un alérgeno presente en productos de la pesca, por lo que el sistema inmune responde al Anisakis sp. como un parásito invasor y como un alérgeno..

A Red-Berry mixture as a Nutraceutical: detailed composition and neuronal protective effect

Recommendations towards increased consumption of fresh fruit and vegetables are well supported by epidemiological and clinical trials. However, in some specific cases, it is difficult to follow these recommendations and the use of nutraceuticals or, in the present work, a freezedried fruits mixture can be recommended in order to afford the optimal consumption of dietary polyphenols naturally present in fruits and vegetables. In this work we have carefully characterized a red-berry mixture in terms of polyphenol composition, encountering mainly anthocyanins, which account for a total of 2.8 mg/g as cyanidin-3-glucoside equivalents. Additionally, we have assayed the red-berry blend in a cell model of neurological damage by differentiating the cells and measuring the effect of red-berry polyphenols on cell viability and redox state by flow cytometry. The berry-fruit extract showed an inhibitory effect on differentiated SH-SY5Y ROS formation at a concentration as low as 250 µg/mL (33% inhibition). The results show the potential of this berry-fruit blend for its nutraceutical use in the prevention of the neurodegeneration associated with age or environmental agents

Aqueous Extract of Cocoa Phenolic Compounds Protects Differentiated Neuroblastoma SH-SY5Y Cells from Oxidative Stress

Cocoa is a rich source of polyphenols, especially flavanols and procyanidin oligomers, with antioxidant properties, providing protection against oxidation and nitration. Cocoa phenolic compounds are usually extracted with methanol/ethanol solvents in order to obtain most of their bioactive compounds; however, aqueous extraction seems more representative of the physiological conditions. In this study, an aqueous extract of cocoa powder has been prepared and chemically characterized, and its potential protective effect against chemically-induced oxidative stress has been tested in differentiated human neuroblastoma SH-SY5Y cells. Neuronal-like cultured cells were pretreated with realistic concentrations of cocoa extract and its major monomeric flavanol component, epicatechin, and then submitted to oxidative stress induced by a potent pro-oxidant. After one hour, production of reactive oxygen species was evaluated by two different methods, flow cytometry and in situ fluorescence by a microplate reader. Simultaneously, reduced glutathione and antioxidant defense enzymes glutathione peroxidase and glutathione reductase were determined and the results used for a comparative analysis of both ROS (reactive oxygen species) methods and to test the chemo-protective effect of the bioactive products on neuronal-like cells. The results of this approach, never tested before, validate both analysis of ROS and indicate that concentrations of an aqueous extract of cocoa phenolics and epicatechin within a physiological range confer a significant protection against oxidative insult to neuronal-like cells in culture

Quantitative proteomics comparison of total expressed proteomes of anisakis simplex sensu stricto, A. pegreffii, and their hybrid genotype

The total proteomes of Anisakis simplex s.s., A. pegreffii and their hybrid genotype have been compared by quantitative proteomics (iTRAQ approach), which considers the level of expressed proteins. Comparison was made by means of two independent experiments considering four biological replicates of A. simplex and two each for A. pegreffii and hybrid between both species. A total of 1811 and 1976 proteins have been respectively identified in the experiments using public databases. One hundred ninety-six proteins were found significantly differentially expressed, and their relationships with the nematodes’ biological replicates were estimated by a multidimensional statistical approach. Results of pairwise Log2 ratio comparisons among them were statistically treated and supported in order to convert them into discrete character states. Principal component analysis (PCA) confirms the validity of the method. This comparison selected thirty seven proteins as discriminant taxonomic biomarkers among A. simplex, A. pegreffii and their hybrid genotype; 19 of these biomarkers, encoded by ten loci, are specific allergens of Anisakis (Ani s7, Ani s8, Ani s12, and Ani s14) and other (Ancylostoma secreted) is a common nematodes venom allergen. The rest of the markers comprise four unknown or non-characterized proteins; five different proteins (leucine) related to innate immunity, four proteolytic proteins (metalloendopeptidases), a lipase, a mitochondrial translocase protein, a neurotransmitter, a thyroxine transporter, and a structural collagen protein. The proposed methodology (proteomics and statistical) solidly characterize a set of proteins that are susceptible to take advantage of the new targeted proteomicsThis work was partially supported by Spanish National Project AGL2015-68248-C2-1-R and the National Natural Science Museum. The proteomic analysis was performed in the Proteomics Facility of the Spanish National Center for Biotechnology (CNB-CSIC) that belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019.Peer reviewe

Thermal patterns of heat treated Anisakis L3-infected fishery products allow separation into low, intermediate and high risk groups of potential use in risk management

Anisakis third-stage larvae (L3) is moderately tolerant to heat and, to mitigate the risk of live L3 intake in cooked seafood, it is important to define with precision at which point after heat treatment the parasite is no longer infective. We aimed to find thermal patterns that allowed to classify fish sandwiches spiked with Anisakis L3 into “low” (100% probability of mortality), “intermediate” and “high” risk groups. For that, experiments with varying set temperatures and heating times have been performed in conditions of different external heating tempera-tures. Decision points to classify the samples into terminal nodes associated to different risk groups have been obtained with decision tree analyses and then confirmed with linear discriminant analysis. Separation into two (i. e. low vs high +intermediate risk) or three (i.e. low, intermediate, and high risk) distinct thermal patterns (98% and 95.9% correct classifications by cross-validation respectively) was achieved. These results refine heating conditions reported in the EU Regulation, since reaching 60 ◦C for 1 min in the thermal centre is not sufficient to kill all L3. However, when factors such as relative temperature of heating or time to reach the set temperature are taken into account, other thermal conditions are found that are equally safe in terms of Anisakis L3 inactivation. This, together with the description of “intermediate” and “high” risk groups can help in the risk identification and management, as well as in providing clearer recommendations to consumersThis work was financed by the European Union's Seventh Framework Programme for Research, Technological Development and Demonstration under Grant Agreement 312068 (EU PARASITE) and Spanish (AGL2015-68248-C2-1-R) MINECO/FEDER (ANIRISK)

Respiratory analysis of intact Anisakis L3 as physiological tool to identify subtle changes in larvae subjected to thermal and/or chemical stress and still considered viable

Trabajo presentado al 48th West European FishTechnologists Association Meeting (WEFTA), celebrado en Lisboa (Portugal) del 15 al 18 de octubre de 2018.Human infection due to eating fish parasitized by live Anisakis larvae in the third stage (L3) is considered an important health problem and the application of treatments to ensure their mortality is crucial to prevent the risk of infection. Mobility is used as the method to assess viability. However, mobile larvae may not always be infective, and there is recognised a need to establish other methods to assess whether these larvae are capable of infecting humans. We suggest that mitochondria may become dysfunctional owing to various physical or chemical treatments applied to inactivate Anisakis L3, even if larvae survive these treatments and the oxygen consumption rate (OCR) could give valuable information about the actual mitochondrial function. We aimed to establish whether respiratory analysis of Anisakis L3 could identify differences between larvae considered viable, but that had been subjected to stress (thermal and/or chemical). The modulators FCCP and azide were used to obtain the basal, maximum, spare and residual respiration rates. The respiration analysis of larvae subjected to a certain temperature or environmental stress, (i.e. storage at 37 °C or in gastric juice), showed that mitochondria were affected compared to the untreated controls. The maximum respiratory capacity of larvae subjected to freezing could initially decrease immediately after thawing, but after some acclimatization they were able to recover their respiratory capacity fully. However, when treated larvae were stored at refrigeration temperatures their mitochondria became dysfunctional faster than those of untreated larvae. To conclude, Anisakis L3 responds to mitochondrial respiration modulators, so respiration analysis can be incorporated for in vivo assessment of mitochondrial function in this nematode. These measurements can be used as a tool to characterize L3 subjected to different stresses which, together with other indicators, help to give a broader picture of Anisakis L3 characteristics and potential infectivity.This work was supported by the Project ANIRISK (AGL2015-68248-C2), funded by the Spanish Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER).Peer Reviewe

Absorption of Anisakis spp. proteins using human Caco-2 cellular model

Trabajo presentado a la 49th West European Fish Technologists Association (WEFTA), celebrada en Tórshavn (Islas Feroe) del 15 al 17 de octubre de 2019.The risk of exposure Anisakis spp. allergens is mainly through the digestive tract, its pathogenic potential depends not only on its resistance to gastrointestinal digestion but also on its ability to cross the barrier of the intestinal epithelium. Previous studies have demonstrated the resistance to gastrointestinal digestion of allergens, mainly low molecular weight (unpublished data). The aim of this piece of work was to study the ability to cross the intestinal epithelial barrier of Anisakis spp. antigens/allergens from Anisakis crude extract (CE) using human Caco-2 cellular model. The integrity of the Caco-2 cells monolayer was characterised measuring the transepithelial electrical resistance (TEER) and paracellular permeability and the analysis of the antigens/allergens transported through the epithelium was performed by SDS-PAGE and immunoblotting. The analysis of TEER shows a significant decrease after two hours in samples with Anisakis CE added. Immunodetection assays (WB) show that the passage of the allergen Ani s 4 might combine paracellular and transcellular transport. Regarding the paracellular permeability evaluated by Lucifer Yellow, the results obtained after incubation of cells with CE, support that the Anisakis CE compromises the integrity of the Caco-2 monolayer. Viability assays show there is not any cytotoxic effect. Putative mechanisms underlying the decrease in TEER have been analysed (protease and phosphatase inhibition, reactive oxygen species determination). However, we have not currently identified the mechanism involved and further studies will be performed. Associated to the TEER changes, we have observed disorganization of occludin by confocal microscopy. Anisakis allergens are able to cross the intestinal barrier, which would justify the presence of symptoms in some patients sensitized to Anisakis ssp. after the consumption of seafood products contaminated Anisakis allergens.This work was financed by the Spanish Project ANIRISK (AGL201568248C1C2) MINECO/FEDER

Changes over Time in IgE Sensitization to Allergens of the Fish Parasite Anisakis spp

© 2016 Carballeda-Sangiao et al.[Background]: Sensitization to Anisakis spp. can produce allergic reactions after eating raw or undercooked parasitized fish. Specific IgE is detected long after the onset of symptoms, but the changes in specific IgE levels over a long follow-up period are unknown; furthermore, the influence of Anisakis spp. allergen exposure through consumption of fishery products is also unknown.[Objective]: To analyse the changes in IgE sensitization to Anisakis spp. allergens over several years of follow-up and the influence of the consumption of fishery products in IgE sensitization. [Methods]: Total IgE, Anisakis spp.-specific IgE, anti-Ani s 1 and anti-Ani s 4 IgE were repeatedly measured over a median follow-up duration of 49 months in 17 sensitized patients.[Results]: Anisakis spp.-specific IgE was detected in 16/17 patients throughout the follow-up period. The comparison between baseline and last visit measurements showed significant decreases in both total IgE and specific IgE. The specific IgE values had an exponential or polynomial decay trend in 13/17 patients. In 4/17 patients, an increase in specific IgE level with the introduction of fish to the diet was observed. Three patients reported symptoms after eating aquaculture or previously frozen fish, and in two of those patients, symptom presentation was coincident with an increase in specific IgE level.[Conclusions]: IgE sensitization to Anisakis spp. allergens lasts for many years since specific IgE was detectable in some patients after more than 8 years from the allergic episode. Specific IgE monitoring showed that specific IgE titres increase in some allergic patients and that allergen contamination of fishery products can account for the observed increase in Anisakis spp.-specific IgE level. Clinical Relevance: Following sensitization to Anisakis spp. allergens, the absence of additional exposure to those allergens does not result in the loss of IgE sensitization. Exposure to Anisakis spp. allergens in fishery products can increase the specific IgE level in some sensitized patients.The research leading to these results received funding from the European Union’s Seventh Framework Programme for Research, Technological Development and Demonstration under grant agreement n° 312068. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer Reviewe

Respiratory analysis as a tool to detect physiological changes in Anisakis larvae subjected to stress

Human infection due to eating fish parasitized by live Anisakis larvae in the third stage is considered an important health problem, and the application of treatments to ensure their mortality in the fish products is crucial to prevent the risk of infection. Mobility is used to assess viability, but mobile larvae may not always be infective and immobile larvae may be erroneously considered as non-viable. The objective was to establish whether the analysis of respiratory activity by means of the oxygen consumption rate (OCR) of Anisakis could be used to identify subtle differences between larvae that were still considered viable in terms of their mobility but had been subjected to thermal and/or chemical stress. The metabolic modulators FCCP [carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone] and sodium azide were used and the basal, maximum, spare and residual respiration rates calculated. Results showed that maximum respiratory capacity of larvae subjected to freezing significantly decreased immediately after thawing, but after some acclimatization, they recovered their capacity fully. However, when these larvae were stored at 4.6 °C, their mitochondria became dysfunctional faster than those of untreated larvae. OCR also showed that mitochondria of larvae were affected by incubation at 37 °C in NaCl or gastric juice. To conclude, OCR of Anisakis in the presence of metabolic modulators can help to identify subtle changes that occur in the larva. These measurements could be used to characterize larvae subjected to various stresses so that a broader picture of Anisakis pathogenic potential can be gained.This work was supported by the Project ANIRISK (AGL2015-68248-C2), under the Research, Development and Innovation Program oriented to Societal Challenges founded by the Spanish Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER)