Ani s 11-Like Protein Is a Pepsin- and Heat-Resistant Major Allergen of Anisakis spp. and a Valuable Tool for Anisakis Allergy Component-Resolved Diagnosis.

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.

Removal of Anisakis simplex allergens from infected fish during the washing step of surimi production.

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.

Antigenicity of Anisakis simplex s.s. L3 in parasitized fish after heating conditions used in the canning processing.

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.

Proteomic profiling and characterization of differential allergens in the nematodes Anisakis simplex sensu stricto and A. pegreffii.

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).

IgE sensitization to Thaumetopoea pityocampa : diagnostic utility of a setae extract, clinical picture and associated risk factors.

BACKGROUND: Setae from Thaumetopoea pityocampa larvae (the pine processionary moth or PPM) can induce hypersensitivity reactions, but their clinical role in IgE-mediated responses is still subject to discussion. The aim of this study was to evaluate a setae extract for in vivo and in vitro diagnosis in nonhospitalized patients with reactions to PPM. METHODS: Forty-eight adult patients presenting with PPM cutaneous reactions were studied by skin prick test (SPT) and specific IgE using setae and whole larval (WL) extracts. Biological standardized extracts were used for skin tests. RESULTS: A total of 47.9% patients had a positive SPT for PPM (70% to both extracts, 17% only to the WL extract and 13% only to the setae extract). IgE immunoblotting detected several reactive bands in 91% of the SPT-positive cases. In multivariate analysis, male sex, immediate latency (<1 h) and duration of skin symptoms (<24 h) were independent predictors of a positive SPT. CONCLUSIONS: IgE sensitization to PPM was found in 48% of the study patients, which was associated with immediate reactions and evanescent cutaneous lesions. Most of these patients reacted to both WL and setae extracts, but some reacted to only one of them. According to our data, skin and in vitro tests to PPM should be performed using both extracts.

Setae from the pine processionary moth (Thaumetopoea pityocampa) contain several relevant allergens.

BACKGROUND: Pine processionary larvae produce urticating hairs (setae) that serve for protection against predators. Setae induce cutaneous reactions in animals and humans. The presence of toxic or allergic mechanisms is a matter of debate. OBJECTIVES: To detect the presence of allergens in setae and to characterize them. MATERIALS AND METHODS: Setae extracts were characterized by gel staining and immunoblot, with sera from patients with immediate reactions and positive prick test reactions, as well as a rabbit antiserum raised against setae. Setae proteins were fractionated by high-performance liquid chromatography. The most relevant allergen was analysed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), and its sequence was deduced from an expressed sequence tag bank. Results. Setae contained at least seven different allergens. The most intense detection corresponded to a protein of MW ~ 14,000 that was similar to thaumetopoein, a previously described protein with mast cell-degranulating properties. MALDI-MS-based de novo sequencing provided a partial amino acid sequence different from that of the previously described allergen Tha p 1, and it was named Tha p 2. This allergen was detected in 61% of patients, and it is therefore a new major caterpillar allergen. CONCLUSIONS: Penetration of the setae from the pine processionary caterpillar delivers their allergenic content in addition to causing mechanical or toxic injury.

Antigenicity and viability of Anisakis larvae infesting hake heated at different time-temperature conditions.

Heat treatments (40 to 94 degrees Celsius, 30 s to 60 min) were applied to different batches of Anisakis simplex L3 larvae isolated from hake ovaries and viscera to study the effect of heat on the viability of the larvae measured as mobility, emission of fluorescence under UV light, and changes in color after staining with specific dyes, and on A. simplex antigenic proteins. The aim was to determine the lowest time-temperature conditions needed to kill the larvae to avoid anisakiasis in consumers, and to evaluate whether high temperature modifies the antigenicity of A. simplex extracts. Heating at 60 degrees Celsius for 10 min (recommended by some authors) was considered unsafe, as differences in viability between batches were found, with some larvae presenting spontaneous movements in one batch. At higher temperatures (> or = 70 degrees Celsius for > or = 1 min), no movement of the larvae was observed. Antigenic protein Ani s 4 and A. simplex crude antigens were detected in the larvae heated at 94 + or - 1 degrees Celsius for 3 min. This indicates that allergic symptoms could be provoked in previously sensitized consumers, even if the larvae were killed by heat treatment.

Quantification of Anisakis simplex allergens in fresh, long-term frozen, and cooked fish muscle.

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.

Identification and allergenic characterisation of a new isoform of the A. simplex allergen Ani s 4.

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.

Cloning and expression of Ani s 9, a new Anisakis simplex allergen.

The larvae of the nematode Anisakis simplex parasitize seafood. When people eat raw or undercooked parasitized fish, they can suffer anisakiasis, an important immune human response to parasitic infection of the gastrointestinal tract. Even more, allergic manifestations like angioedema, urticaria or anaphylaxis can occur in sensitized patients. The aim of this work was to clone Ani s 9-cDNA and overproduce this recombinant allergen in Escherichia coli. The finding of this allergen was an unexpected result of a PCR using degenerate primers designed to amplify Ani s 5. The complete cDNA for Ani s 9 was obtained by RACE-PCR, cloned and sequenced. Expression of recombinant allergen was performed in E. coli. Immunodetection and immunoblot inhibition assays tests were carried out with sera from Anisakis allergic patients. The recombinant Ani s 9 (rAni s 9) is a protein of 147 amino acids. By immunoblot inhibition assay, it was located as a 14 kDa band present in a crude extract of the parasite. This new allergen is heat stable and is present in excretory/secretory products. Ani s 9 belongs to the SXP/RAL-2 family and shares amino acid sequence identity of 60% with As-14, an Ascaris suum allergen. Five of thirty-six Anisakis allergic patients (13.8%) were positive to rAni s 9 and natural Ani s 9 by immunodetection. In conclusion, Ani s 9 is a new allergen in Anisakis allergy and it has been cloned and successfully expressed in E. coli.

Anisakis antigens detected in fish muscle infested with Anisakis simplex L3.

Anisakis simplex is a fish parasite that is a public health risk to those consuming raw or poorly cooked marine fish and cephalopods because of the possibility of becoming infested with live larvae. In humans, penetration of the larvae into the gastrointestinal track can cause acute and chronic symptoms and allergic anisakiasis. Excretion and secretion products released by the larvae are thought to play a role in migration through the tissues and induce an immunoglobulin E-mediated immune response. The aim of this preliminary study was to detect parasite antigens and allergens in fish tissues surrounding the migrating larvae. Hake and anchovy fillets were artificially parasitized with Anisakis larvae and stored in chilled conditions for 5 days. Larvae were evaluated for fluorescence, fish muscle tissue was examined with transmission electron microscopy, and immunohistochemical reactions of two rabbit polyclonal antisera against a parasite crude extract and the allergen Ani s 4 were recorded. Larvae immediately migrated into the fish muscle, and no emission of bluish fluorescence was observed. Fish muscle areas in contact with the parasite showed disruptions in the structure and inclusion of granules within sarcomeres. Both parasite antigens and the Ani s 4 allergen were located in areas close to the larvae and where sarcomere structure was preserved. These findings indicate that parasite antigens and allergens are dispersed into the muscle and might cause allergic symptoms such as dyspnea, vomiting, diarrhea, urticaria, angioedema, or anaphylaxis in some individuals sensitive to A. simplex.

Allergenic properties and cuticle microstructure of Anisakis simplex L3 after freezing and pepsin digestion.

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.

Cloning and characterisation of the Anisakis simplex allergen Ani s 4 as a cysteine-protease inhibitor.

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.

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

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.

Multicenter validation of CSF neurofilaments as diagnostic biomarkers for ALS.

OBJECTIVE: Neurofilaments are leading neurochemical biomarkers for amyotrophic lateral sclerosis (ALS). Here, we investigated the effect of preanalytical factors on neurofilament concentrations in cerebrospinal fluid (CSF) in a "reverse" round-robin with 15 centers across Europe/U.S. METHODS: Samples from ALS and control patients (5/5 each center, n = 150) were analyzed for phosphorylated neurofilament heavy chain (pNfH) and neurofilament light chain (NfL) at two laboratories. RESULTS: CSF pNfH was increased (p < 0.05) in ALS in 10 out of 15 centers and NfL in 5 out of 12 centers. The coefficient of variation (CV%) of pNfH measurements between laboratories was 18.7 ± 19.1%. We calculated a diagnostic cut-off of >568.5 pg/mL for pNfH (sensitivity 78.7%, specificity 93.3%) and >1,431pg/mL for NfL (sensitivity 79.0%, specificity 86.4%). CONCLUSION: Values in ALS patients are already comparable between most centers, supporting eventual implementation into clinical routine. However, continuous quality control programs will be necessary for inclusion in the diagnostic work-up.

Identification of autoclave-resistant Anisakis simplex allergens.

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.

Sensitization to the fish parasite Anisakis simplex: clinical and laboratory aspects.

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.

Isolation of a heat-resistant allergen from the fish parasite Anisakis simplex.

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.