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Background: Freezing is considered the most suitable technological treatment to avoid Anisakis infection from eating raw or undercooked fish but modifications of their cuticles upon freezing may reduce their resistance to gastric fluids, provoking a greater release of allergens. This work aimed to study the relationship between freezing-induced modifications of Anisakis simplex s.l., antigen recognition, and resistance to oral and gastric digestion in spiked fish mince., Results: (i) Differences between non-treated larvae and larvae that survived freezing / thawing were studied in terms of respiratory capacity, survival in simulated gastric fluid (SGF), recognition of antigens and allergens. (ii) Untreated (i.e. chilled) mince containing live larvae, mince frozen at two freezing rates, with a negative (uninfected) mince and a positive mince (infected with broken larvae) as controls, were subjected to the oral and gastric phases of a simulated digestion process. Anisakis able to survive freezing showed lower resistance to gastric fluid (i.e. faster mortality as compared to controls). Untreated larvae released significantly more antigens than freeze-surviving larvae but only after 96 h in SGF. In treatments rendering complete larvae mortality, the highest loss of larvae integrity was found upon fast freezing. There was a positive correlation between antigen release and the number of ruptures of larvae after the oral digestion phase, whereas a more complex trend was observed after oral plus gastric digestion phases., Conclusion: These results suggest a new factor to consider for sensitized patients and suggest that the numbers of L3 should be reduced before industrial freezing to minimize risk. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

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 Log 2 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 proteomics.

Background: Anisakis spp. are nematode parasites found in a wide range of marine organisms. Human beings may accidentally become infected, showing the symptoms of anisakiasis and allergic responses. There has been evidence of increased intestinal permeability in A. simplex-sensitized subjects and that specific IgE titres increase in some allergic patients when fishery products are re-introduced into their diet. The aims of this work were to study the effect of A. simplex crude extract on the intestinal integrity and permeability by using Caco-2 cell monolayer. To analyse the capacity of Ani s 4 allergen to cross the epithelial barrier., Methodology/principal Findings: Cellular bioenergetics, transepithelial electrical resistance, viability, permeability, reactive oxygen species generation and immunofluorescent staining of tight junction proteins were analysed. A. simplex crude extract compromises the Caco-2 cell monolayer integrity in a dose-dependent manner. This effect is detected at 1 hour of culture and integrity is recovered after 24 hours of culture. The epithelial barrier disruption is accompanied by an increase in paracellular permeability and reactive oxygen species production and by a delocalization of occludin and zonula occludens-1. Finally, Ani s 4, a thermostable and resistant to digestion allergen with cystatin activity, is able to cross the epithelial barrier in Caco-2 monolayer and reach a cumulative mean percentage of 22.7% of total concentration in the basolateral side after 24 hours of culture., Conclusions/significance: Our results demonstrate that A. simplex induces an early and reversible alteration of integrity and permeability of Caco-2 cell monolayer and that an underlying mechanism of this effect would involve the oxidative stress and disruption of epithelial tight junctions. Additionally, it has been shown that Ani s 4 allergen is able to cross the epithelial barrier. These findings could explain the increased intestinal permeability observed in Anisakis-sensitized patients, the changes over time in IgE sensitization to A. simplex allergens, and the specific IgE persistence in Anisakis allergy., Competing Interests: The authors have declared that no competing interests exist

In plant and animal nematode parasites, proteins derived from esophageal gland cells have been shown to be important in the host-nematodes relationship but little is known about the allergenic potential of these proteins in the genus Anisakis . Taking into account the increase of anisakiasis and allergies related to these nematodes, immunoreactive properties of gland cell proteins were investigated. Two hundred ventricles were manually dissected from L3 stage larvae of Aniskakis simplex s.s . to allow direct protein analysis. Denaturing gel electrophoresis followed by monochromatic silver staining which revealed the presence of differential (enriched) proteins when compared to total nematode extracts. Such comparison was performed by means of 1D and 2D electrophoresis. Pooled antisera from Anisakis spp.-allergic patients were used in western blots revealing the presence of 13 immunoreactive bands in the ventricular extracts in 1D, with 82 spots revealed in 2D. The corresponding protein bands and spots were excised from the silver-stained gel and protein assignation was made by MALDI-TOF/TOF. A total of 13 (including proteoforms) were unambiguously identified. The majority of these proteins are known to be secreted by nematodes into the external environment, of which three are described as being major allergens in other organisms with different phylogenetic origin and one is an Anisakis simplex allergen.

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.

Purpose of Review: To compare the prevalence of sensitization in different countries based on specific IgE values and to evaluate the use of isolated native or recombinant allergens for diagnosis., Recent Findings: Isolated allergens help in the diagnosis of truly sensitized patients avoiding false positives due to cross-reactions. Their use is therefore highly recommended, especially when used as a combination of several relevant allergens. The use of purified allergens allows an accurate diagnosis and this has led to three important findings: (1) in addition to the digestive route of sensitization, occupational and non-digestive exposure seems to be clinically relevant. (2) The parasite appears as an important agent for chronic urticaria. And (3) in endemic countries, the amount of highly sensitized subjects in the general population could be as high as 7%. Adequate information to asymptomatic patients on fish consumption habits would avoid new contacts with parasite allergens and decrease their specific IgE levels and consequently the appearance of acute or chronic episodes induced by the parasite.

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.

Background: Anisakis simplex is a fish parasite responsible for gastrointestinal and allergic symptoms in humans. The Ani s 11-like protein has been proposed as an Anisakis allergen because its primary structure is similar to that of Ani s 11. The aims of this work were to analyse the frequency of detection of the Ani s 11-like protein and assess its diagnostic value., Methods: rAni s 11-like protein, rAni s 5 and rAni s 4 were expressed in Escherichia coli and rAni s 1 was produced in Pichia pastoris. Recombinant allergen detection patterns in 37 Anisakis-sensitised patients were determined. The stability to pepsin digestion and heat treatment of rAni s 11-like protein was also analysed by IgE immunoblotting., Results: Ani s 11-like protein is a major allergen detected by 78% of Anisakis-allergic patients, and 13.5% of patients detect only the rAni s 11-like allergen. This allergen is heat stable because it retains its capability of binding IgE after boiling for 30 min and it is resistant to pepsin digestion for 120 min., Conclusions: These data indicate that the Ani s 11-like protein is a pepsin- and heat-resistant major allergen (Ani s 11.0201) of Anisakis spp. and a valuable tool for Anisakis allergy component-resolved diagnosis., (© 2016 S. Karger AG, Basel.)

Backgound: The washing operation of fish muscle is one of the key steps in the production of surimi. The aim of this study was to assess in parasitised minced fish the effect of the washing steps on the allergen removal of Anisakis simplex and on protein yield during surimi processing. Experimentally infected hake (Merluccius merluccius) (50 Anisakis simplex s.s L3 larvae per 100 g of muscle) underwent three successive washing steps with water, phosphate buffer (20 mmol L(-1) ), sodium bicarbonate (60 mmol L(-1) ), or sodium hypochlorite (0.27 mmol L(-1) ) in the surimi processing (4 kg muscle, 1:4 w/v for each solution). Total protein concentration and A. simplex antigens and allergens were evaluated in each waste fraction., Results: The highest removal of Ani s 4 and A. simplex antigens was achieved by using phosphate buffer, together with a good protein yield in the raw surimi. Decrease of the concentration of allergens and antigens as a function of the washing steps rendered a linear trend (R(2) = 0.95 and 0.98 for Ani s 4 and A. simplex antigens, respectively)., Conclusion: The conditions for an optimal removal of Anisakis allergens can be established and calculated as a function of the washing steps. This approach opens a line to utilise parasitised fish in a safer way. © 2014 Society of Chemical Industry., (© 2014 Society of Chemical Industry.)

Background: Some technological and food processing treatments applied to parasitized fish kill the Anisakis larvae and prevent infection and sensitization of consumers. However, residual allergenic activity of parasite allergens has been shown. The aim here was to study the effect of different heat treatments used in the fish canning processing industry on the antigen recognition of Anisakis L3. Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) were experimentally infected with live L3 Anisakis. After 48 h at 5 ± 1 °C, brine was added to the muscle, which was then canned raw (live larvae) or heated (90 °C, 30 min) (dead larvae) and treated at 113 °C for 60 min or at 115 °C for 90 min. Anisakis antigens and Ani s 4 were detected with anti-crude extract and anti-Ani s 4 antisera respectively., Results: Ani s 4 decreased in all lots, but the muscle retained part of the allergenicity irrespective of the canning method, as observed by immunohistochemistry. Dot blot analysis showed a high loss of Ani s 4 recognition after canning, but residual antigenicity was present., Conclusion: The results indicate that heat treatment for sterilization under the conditions studied produces a decrease in Ani s 4 and suggest a potential exposure risk for Anisakis-sensitized patients., (© 2014 Society of Chemical Industry.)

The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In this study, the relationship of this Anisakis species complex and their allergenic capacities is assessed by studying the differences between the two most frequent species (A. simplex s.s., A. pegreffii) and their hybrid haplotype by studying active L3 larvae parasiting Merluccius merluccius. They were compared by 2D gel electrophoresis and parallel Western blot (2DE gels were hybridized with pools of sera from Anisakis allergenic patients). Unambiguous spot differences were detected and protein assignation was made by MALDI-TOF/TOF analysis or de novo sequencing. Seventy-five gel spots were detected and the corresponding proteins were identified. Differentially expressed proteins for A. simplex s.s., A. pegreffii, and their hybrid are described and results are statistically supported. Twenty-eight different allergenic proteins are classified according to different families belonging to different biological functions. These proteins are described for the first time as antigenic and potentially new allergens in Anisakis. Comparative proteomic analyses of allergenic capacities are useful for diagnosis, epidemiological surveys, and clinical research. All MS data have been deposited in the ProteomeXchange with identifier PXD000662 (http://proteomecentral.proteomexchange.org/dataset/PXD000662)., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Anisakis simplex is a fish parasite able to induce allergic reactions in humans infected when eating raw or undercooked fish parasitized with viable third-stage larvae. Some authors claim that exposure to nonviable Anisakis material can result in allergic symptoms in previously sensitized patients, indicating that parasite allergens are resistant to the thermal treatments of usual cooking procedures. Furthermore, some patients report symptoms after eating canned fish. The aim of this work was the analysis of parasite allergen stability in heating to 121 °C in an autoclave to simulate the thermal process applied to canned fish. Third-stage larvae were subjected to autoclaving for 20, 40, and 80 min, and parasite crude extracts were analyzed by electrophoresis, immunoblotting, and a flow-cytometric basophil activation test. Allergens resistant to autoclaving were separated by reversed-phase high-performance liquid chromatography and identified by ion trap mass spectrometry. Protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that autoclaving considerably reduced the number and intensity of identifiable protein bands in a time-dependent manner. Several allergens were detected by immunoblotting with a pool of A. simplex allergic patients' sera after autoclaving. Allergens of 9 and 14 kDa resistant to autoclaving were identified as Ani s 4 and Ani s 1 allergens, respectively. Functional analysis showed that allergens retain their capacity to activate basophils even after autoclaving for 80 min. In conclusion, some relevant A. simplex allergens retain their capacity to bind immunoglobulin E and activate basophils after being subjected to autoclaving, which is a method equivalent to that used in industrial canning processes.

Fish-borne parasitic zoonoses such as Anisakiasis were once limited to people living in countries where raw or undercooked fish is traditionally consumed. Nowadays, several factors, such as the growing international markets, the improved transportation systems, the population movements, and the expansion of ethnic ways of cooking in developed countries, have increased the population exposed to these parasites. Improved diagnosis technology and a better knowledge of the symptoms by clinicians have increased the Anisakiasis cases worldwide. Dietary recommendations to Anisakis-sensitized patients include the consumption of frozen or well-cooked fish, but these probably do not defend sensitized patients from allergen exposure. The aim of our work was to develop a sensitive and specific method to detect and quantify Anisakis simplex allergens in fish muscle and its derivatives. Protein extraction was made in saline buffer followed by preparation under acid conditions. A. simplex antigens were detected by IgG immunoblot and quantified by dot blot. The allergenic properties of the extracts were assessed by IgE immunoblotting and basophil activation test. We were able to detect less than 1 ppm of A. simplex antigens, among them the allergen Ani s 4, in fish muscle with no cross-reactions and with a recovery rate of 82.5%. A. simplex antigens were detected in hakes and anchovies but not in sardines, red mullets, or shellfish. We detected A. simplex allergens in cooked hakes and also in hake stock. We proved that A. simplex allergens are preserved in long-term frozen storage (-20 degrees C +/- 2 degrees C for 11 months) of parasitized hakes. Basophil activation tests have proven the capability of the A. simplex-positive fish extracts to induce allergic symptoms.

This article examines the viability of and the alterations to the larval cuticle and the pattern of the antigens released when live or frozen Anisakis simplex larvae were treated with acid and pepsin. The results showed that freezing did not greatly alter the larva body. If ruptures were observed, the antigen release to the incubation media was not enhanced, and most of the antigenic content was retained inside the bodies of the larvae. The immunoblotting assay demonstrated that most of the antigens released, including the allergen Ani s 4, were resistant to pepsin. Freezing killed the larvae, but their survival was not compromised by acid treatment or pepsin digestion when kept chilled. All these findings support recommendations about freezing fish for consumption raw or undercooked to prevent human infection by A. simplex larvae. However, our data show that the antigenicity of the larvae is preserved after freezing and may explain why some sensitized patients develop symptoms after ingestion of infested frozen fish.

Anisakis simplex hypersensitivity is a growing disease in developed countries. A positive diagnosis usually leads to the dietary recommendation of avoiding fish and seafood consumption. The protein Ani s 4 is a clinically relevant allergen due to its heat and pepsin resistant properties and its importance in the anaphylaxis process. The attempt of cloning Ani s 4 has led to the identification and characterisation of a new isoform that differs only in one amino acid with the previously published. This isoform was produced as an His tagged recombinant protein and its allergenic properties were tested by IgE immunoblot and by a flow cytometry basophil activation test. The results were compared to the allergenic properties of the isoform previously described. Both isoforms of Ani s 4 showed different capacities to bind IgE from sensitised patients and different potencies in the basophil activation test.

Hypersensitivity to Anisakis simplex is a worldwide medical problem. The parasite larvae die after freezing or cooking, but the tolerance of sensitized subjects to eating frozen fish remains a matter of controversy with contradictory findings. The aim of this study was to test if intolerance to properly cooked/frozen fish was due to the recognition of a particular allergen. Sixty-four patients with Anisakis simplex sensitization were studied by an IgE multiblot using simultaneously five different antigenic extracts. The antigens tested were a crude extract, excretory/secretory allergens, a heated extract, and two gradient ethanol precipitates of the crude extract. Intolerance was reported by 20% of the patients and was not related to the detection of any special allergen, nor to total or specific IgE levels. Intolerant patients only reported a higher frequency of digestive symptoms than the patients who tolerated fish ingestion. The most sensitive immunoblot antigen source was the 50-66% ethanol fraction of a crude extract (10x concentrated) that was found to be positive in 100% of the samples. Interestingly, 95% sensitivity in the IgE-immunoblot assay could be achieved using only two allergens, Ani s 1 and Ani s 4. Allergens from the dead larvae remain a problem for 20% of the sensitized subjects. The use of a fractionated and concentrated crude extract improved the sensitivity of the immunoblot assay.

Anisakis simplex is a nematode that can parasitise humans who eat raw or undercooked fish containing live L3s. Larvae invading the gastrointestinal mucosa excrete/secrete proteins implicated in the pathogenesis of anisakiasis that can induce IgE mediated symptoms. Misdiagnosis of anisakiasis, due to cross-reactivity, makes it necessary to develop new diagnostic tools. Recombinant allergens have proved to be useful for diagnosis of other parasitoses. Among the Anisakis allergens, Ani s 4 was considered to be a good potential diagnostic protein because of its heat resistance and its importance in the clinical history of sensitised patients. Therefore, the objective of this study was to clone and characterise the cDNA encoding this allergen. The Ani s 4 mRNA sequence was obtained using a PCR-based strategy. The Ani s 4 amino acid sequence contained the characteristic domains of cystatins. Mature recombinant Ani s 4 was expressed in a bacterial system as a His-tagged soluble protein. The recombinant Ani s 4 inhibited the cleavage of a peptide substrate by papain with a Ki value of 20.6 nM. Immunobloting, ELISA, a commercial fluorescence-enzyme-immunoassay and a basophil activation test were used to study the allergenic properties of rAni s 4, demonstrating that the recombinant allergen contained the same IgE epitopes as the native Ani s 4, and that it was a biologically active allergen since it activated basophils from patients with allergy to A. simplex in a specific concentration-dependent manner. Ani s 4 was localised by immunohistochemical methods, using a polyclonal anti-Ani s 4 anti-serum, in both the secretory gland and the basal layer of the cuticle of A. simplex L3. In conclusion, we believe that Ani s 4 is the first nematode cystatin that is a human allergen. The resulting rAni s 4 retains all allergenic properties of the natural allergen, and can therefore be used in immunodiagnosis of human anisakiasis.

Background: Laboratory diagnosis of anisakidosis is based on specific serum IgE detection. Recently, detection of allergen-induced basophil activation by flow cytometry has been proposed as a valuable tool for allergy diagnosis., Objective: To evaluate if detection of Anisakis-induced basophil activation by flow cytometry is a useful tool in the diagnosis of Anisakis allergy., Methods: Patients with Anisakis allergy (A.s.+, n = 37), patients reporting chronic urticaria or abdominal pain unrelated to fish ingestion (A.s.-, n = 51), and healthy controls (n = 12) were studied. Specific IgE to Anisakis simplex (A. simplex) was quantified with CAP-FEIA method, and basophil activation test was performed with three different concentrations of an Anisakis crude extract. Basophil gating was performed with CD123 and HLA-DR, and cellular activation was measured with CD63., Results: A.s.+ patients showed significantly higher age and total IgE levels than did the A.s.- patients. Specific IgE to A. simplex correlated with the activated basophil percentages obtained with 15 microg/mL (r = 0.80; P < 0.001), 1.5 microg/mL (r = 0.84; P < 0.001), and 0.15 microg/mL (r = 0.82; P < 0.001) of A. simplex crude extract. Nine individuals (3 in the A.s.+ group and 6 in the A.s.- group) were nonresponders to basophil stimulation with anti-IgE. Five A.s.- patients showed positive IgE values to A. simplex while the basophil activation test was negative. According to the receiver operating characteristics curves performed between A.s.+ vs. A.s.- and A.s.+ vs. healthy controls, the cutoff for a positive basophil activation test was >or=21% (specificity = 96%, sensitivity = 100%), and 16% (sensitivity and specificity of 100%) respectively. When nonresponders were included in the A.s.+ vs. A.s.- analysis, sensitivity decreased to 95%. Multivariate logistic analysis showed that the specific basophil activation was a factor independently associated with clinical symptoms of A. simplex allergy., Conclusions: Detection of A. simplex-induced basophil activation by flow cytometry is a useful laboratory technique for the diagnosis of anisakidosis, supplementing specific IgE determinations., ((c) 2005 Wiley-Liss, Inc.)

The thermal stability of allergenic peptides from the fish parasite Anisakis simplex has not been fully elucidated. This is of special relevance for physicians who should clearly indicate if sensitized patients should avoid ingestion of raw fish only or whether well-cooked fish should also be avoided, if allergenic peptides derived from the parasite remain immunologically detectable. An allergen was purified after heating a crude parasite extract for 30 min. The allergen was further purified by an ethanol fractionation procedure followed by a reversed-phase HPLC. The N-terminal amino acid sequence was obtained. This allergen was detected by 27% of sensitized subjects. The N-terminal amino acid sequence of the 9 kDa allergen showed no similarities to other known proteins. A minor low molecular weight allergen from A. simplex is highly resistant to heating and it could therefore have significant clinical relevance.

Abstract Serodiagnosis of human anisakidosis is presently hampered by the current lack of standardised serological assays that allow sensitive and specific detection of Anisakidae-specific antibodies in human patients. In the present study, we comparatively evaluated the diagnostic value (by IgG-ELISA) of excretory-secretory antigens (ESAgs) of Anisakis simplex , Pseudoterranova decipiens and Contracaecum osculatum , representing the most frequently found genera responsible for human infection. In addition, we tested also a mix of the three ES preparations (Mix-ESAgs) as well as two recombinant allergens of A. simplex , rAni s 1 and rAni s 7. ES antigen from C. osculatum yielded the best diagnostic performance in IgG-ELISA-based serodiagnosis of the Spanish anisakidosis patients investigated in this study (relative serodiagnostic sensitivity 100%; specificity 89%) as compared to A. simplex ES-antigen (93% versus 57%) and P. decipiens (67% versus 93%) or a mix of the three ES antigens (100% versus 44%), respectively. Cross-reactions of C. osculatum ES antigen with serum-antibodies from patients suffering from other helminth infections were rare and were exclusively found with few sera from toxocariasis, ascariasis, and filariasis patients. The two recombinant allergens rAni s 1 and rAni s 7 did not prove sufficiently sensitive and specific in order to justify a further evaluation of these antigens regarding their suitability in IgG-ELISA-based serodiagnosis of human anisakidosis. In conclusion, the C. osculatum -ESAg-ELISA remains as key candidate to be further assessed for the serodiagnosis of symptomatic anisakidosis in different endemic regions. Graphical abstract Image 1 Highlights • We assessed the utility of Anisakidae antigens for serodiagnosis of anisakidosis. • Excretory/secretory antigens (ESAg) demonstrated the best diagnostic performance. • Crude and recombinant antigens yielded low specificity and sensitivity. • Contracaecum osculatum ESAg exhibited the best performance in IgG-ELISA. • Anisakis simplex ESAg elicited lower specificity than C. osculatum ESAg. [ABSTRACT FROM AUTHOR]

Background: Anisakis simplex is the only fishery-product associated parasite causing clinical allergic responses in humans so far. However, other anisakids, due to the presence of shared or own allergens, could also lead to allergic reactions after sensitization. The aim of this study was to determine if Pseudoterranova decipiens belonging to the family Anisakidae has allergenic activity and is able to induce sensitization after oral administration in a murine (BALB/c mice) model. Results: The ingestion of A. pegreffii proteins by BALB/c mice, which had been previously sensitized by intraperitoneal inoculation with the corresponding live L3 larvae, triggers signs of allergy within 60 min, whereas P. decipiens did to a lesser extent. Beside symptoms, allergic reactions were furtherly supported by the presence of histamine in sera of sensitized mice. Specific IgG1 and IgE responses were detected in sera of all sensitized mice from week four. Specific IgG2a response was detected in sera from mice sensitized to P. decipiens. After polyclonal or specific activation with anti-CD3/anti-CD28 or antigens, respectively, splenocytes from mice infected i.p. with A. pegreffii or P. decipiens larvae showed significantly higher production of IL-10 than naïve mice. After stimulation with specific antigens, significantly higher IL-5 and IL-13 amounts were produced by specific antigen stimulated splenocytes than by the naïve cells; only P. decipiens proteins induced IFN-γ. Conclusions: The overall results suggest that infection with P. decipiens can sensitize mice to react to subsequent oral challenge with anisakid proteins, as described for A. simplex (sensu stricto) and A. pegreffii infections. The results show that anisakid proteins induce a dominant Th2 response, although P. decipiens could also induce a mixed type 1/type 2 pattern. [ABSTRACT FROM AUTHOR]

Anisakis L3 were subjected to freezing in domestic freezers and the risk posed by those larvae that might possibly survive freezing was analysed by determining their agar penetration ability, survival in gastric juice, allergenic potential and oxygen consumption rate. Anisakis L3 gradually became non-viable as the temperature fell from −1 °C to −28 °C. The faster the freezing rate, the greater the decrease in survival, but viability was not found to be affected by the proportions of Anisakis species per batch ( A. simplex s.s. 83–100%, A. pegreffii 0–17%, and their heterozygote genotypes at the ITS region of rDNA 0–10%). Surviving larvae after freezing presented a drastically reduced ability to penetrate into a layer of solid agar, as compared to controls, but still 5% of them penetrated into this medium. About 80% of surviving larvae died within 24 h under conditions simulating those of gastric fluid, whereas up to 85% of the controls were able to resist for 24 h. Allergen release during freezing and after freezing was higher than in controls, and this release was compatible with a contribution of both passive (by rupture of the cuticle) and active liberation of excretion/secretion allergens during and after freezing, suggesting that the pathogenic potential of these frozen and thawed larvae cannot be discarded. These results could be of use for more precise risk assessment and providing guidelines for consumers. [ABSTRACT FROM AUTHOR]

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 temperatures. 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 consumers. • Thermal conditions to inactivate Anisakis L3 have been studied. • Three thermal patterns from low to high risk were found. • Results refine and expand reported heating recommendations needed to kill L3. • These results can help in risk identification and management. [ABSTRACT FROM AUTHOR]

The effect of freezing rate, temperature and storage time of Anisakis L3 infecting fish mince in controlled conditions were studied in terms of larval viability and parasite antigen release. In order to determine the potential infectivity of larvae that might accidentally survive a freezing process, their agar penetration ability, survival in artificial gastric juice, and respiratory control were analysed in infected hake muscle in conditions that produced various survival rates. For that a series of experiments was conducted with 10–30 L3 per 75 g of mince, frozen and stored to provide a variety of freezing kinetic conditions and storage times. The results highlight the importance of a number of kinetic parameters in the survival of Anisakis larvae, not only final temperatures or storage times. High freezing rates, preferred in order to maintain optimum fish eating quality, are able to kill Anisakis larvae faster, but may cause a higher release of antigens to the surrounding medium. The characteristics of the surviving L3 were impaired as compared to the controls. No relationship between the latter and the percentage of viability due to different freezing conditions was found, which suggest that any larva which shows any sign of movement after freezing should be treated as equally infective. • Other kinetic parameters besides T or storage time affect Anisakis L3 survival. • Fast freezing rates are able to kill Anisakis larvae even at −10 °C. • Fast freezing rates may cause a higher release of antigens. • Biological or physiological parameters are impaired in freeze-surviving L3. [ABSTRACT FROM AUTHOR]

Abstract Background Nematodes of the genus Anisakis parasitize many commercial fish species and are responsible for a fish-borne zoonosis (anisakiasis) and allergic reactions. Anisakis can also cause consumer distrust in fishery products and economic losses to the fish industry. Scope and approach We review current socioeconomic, legislative, risk management and human health problems caused by the occurrence of Anisakis in fishery products and discuss possible strategies to mitigate them. Key findings and conclusions Visual inspection (and candling) of fishery products as required by EU legislation is not efficient for parasite detection. Consequently, visible (and non-visible) Anisakis reach the market and may be detected (and eaten) by consumers. Marine fish appears to be the only industrial food product that is at high risk of containing parasites when placed on the market. Anisakiasis and allergy to Anisakis are hidden, underestimated emerging zoonoses worldwide. There is a need to better understand the impact of these zoonoses on individual health and particularly exposed human populations, and to assess the risk posed by Anisakis allergens in fishery products. Quantitative risk assessment (QRA) is identified as an appropriate methodology as it estimates the risk from fishing ground to human disease. Improvements in parasite control legislation and procedures (e.g. establishment of research-based and standardized parasite detection methodologies, appropriate sampling strategies, development of non-destructive methods for detection and removal of nematodes from fish products), suitable for use by seafood businesses, are recommended to improve protection of consumers and to protect the industry by minimizing Anisakis -associated economic losses. QRA may help to provide the scientific basis for improved food safety legislation and strategies to reduce the risk of anisakiasis/allergy in humans. Highlights • Anisakid control measures implemented by seafood businesses are not efficient. • Visible Anisakis reach the fish market and may be detected and eaten by consumers. • The health risk posed by Anisakis allergens in fish products needs to be assessed. • We need to better understand the impacts of anisakiasis and Anisakis allergy. • Improved food safety legislation and procedures are needed; QRA methods may help. [ABSTRACT FROM AUTHOR]