Fish Parasite Anisakis Simplex Research Articles

Comparative studies of codon usage profile of Anisakis simplex (Nematoda) and Carassius gibelio (Prussian carp)

Aim: The aim of the present study was to understand the molecular relationship between nematode (parasite) and fish (host) through codon usage bias (CUB) analysis. Methodology: The Codon usage bias analysis has been performed in fish Carassius gibelio (Prussian carp) and nematode fish parasite Anisakis simplex. The complete coding sequences (CDS) of C. gibelio (Prussian carp) and A. simplex (Nematode) were retrieved from National Center for Biotechnology Information and followed to that we have performed bioinformatics analysis to understand the codon usage pattern between host and parasite. Results: Different CUB indices like Relative synonymous codon usage (RSCU), Effective number of codons (ENC), Codon adaptation index (CAI) and Codon bias index (CBI) revealed a similar pattern in the codon usage in C. gibelio and A. simplex. In addition, inclusive analysis using different plots (ENC, parity, neutrality) had shown the influence of both the evolutionary forces i.e mutational and translational selection on codon usage pattern. This describes the role of evolutionary forces in determining the conserved genome to establish species-specific function-level differences for efficient survival. Interpretation: The present study elucidated the association between Carassiusgibelio (host) and Anisakis simplex (parasite) based on the similar pattern of codon usage bias between both the species.

Fish feed as source of potentially allergenic peptides from the fish parasite Anisakis simplex (s.l.)

The carry-over of certain feed components into animal products can be of concern for human health. The safety assessment of chemical contaminants including natural toxins, agrochemicals, veterinary drugs, and environmental pollutants is a key element of the “farm-to-fork” (“One Health”) approach. The transmissibility of proteinaceous feed constituents such as enzymes, proteins from genetically engineered crops, and infectious prions in animal meal has also become of interest; but the transfer of proteins with allergic potential is little studied. In the present study, an exploratory zebrafish feeding trial using feed containing 20 percent of processed larvae of the marine fish parasite Anisakis simplex was performed as a proof-of-principle experiment. After a 2-week exposure period, anisakid peptides were detected in zebrafish tissue by high-resolution liquid-chromatography Orbitrap mass spectrometry and immunostaining using specific polyclonal antibodies or sera from patients with confirmed allergy to A. simplex. Since fishmeal produced from marine pelagic fish is an important feed component in the culture of Atlantic salmon and in the poultry industry, it should be considered as a source of potentially allergenic peptides in the final products. Furthermore, the substitution of fishmeal with plant proteins would not eliminate the potential health risk by allergen carry-over since crops of high nutritional value such as legumes also contain important food allergens. If our preliminary results from the present zebrafish feeding trial should be confirmed in necessary follow-up experiments, the question of labeling information on fish and animal food products raised on feed containing potentially allergenic ingredients could arise in order to minimize the exposure risk of allergic consumers.

Detection of Proteins from the Fish Parasite Anisakis simplex in Norwegian Farmed Salmon and Processed Fish Products

Wild-caught marine fish are potentially carrying parasites. Larvae of the nematode Anisakis simplex (herring or whale worm) occur in almost all commercially exploited fish stocks in temperate seas. The presence of A. simplex in fish and fish products is not only an economic concern but represents a significant consumer health risk. Anisakiasis, human infection with live larvae, can occur by consuming raw or undercooked fish while allergy symptoms can also be elicited by the presence of A. simplex proteins in processed seafood. The European Food Safety Authority (EFSA) has concluded in a scientific opinion that routine testing of seafood products for A. simplex is needed. In the present study, we have determined A. simplex proteins in farmed salmon intended for use in sushi and fish products from the Norwegian market by quantitative sandwich ELISA, immunostaining and mass spectrometry. Analytical methods detecting anisakid proteins at the single-digit milligram level appear to be sufficiently sensitive for the protection of allergic consumers. Only trace amounts (<10 mg/kg) were detected in a few samples showing that contamination with A. simplex is apparently not an immediate health problem given the results from this survey.

Characterisation of potential novel allergens in the fish parasite Anisakis simplex

The parasitic nematode Anisakis simplex occurs in fish stocks in temperate seas. A. simplex contamination of fish products is unsavoury and a health concern considering human infection with live larvae (anisakiasis) and allergic reactions to anisakid proteins in seafood. Protein extracts of A. simplex produce complex band patterns in gel electrophoresis and IgE-immunostaining. In the present study potential allergens have been characterised using sera from A. simplex-sensitised patients and proteome data obtained by mass spectrometry. A. simplex proteins were homologous to allergens in other nematodes, insects, and shellfish indicating cross-reactivity. Characteristic marker peptides for relevant A. simplex proteins were described.

An Extended Study of Seroprevalence of Anti‐Anisakis simplex IgE Antibodies in Norwegian Blood Donors

During the last decade, cases of the fish parasite Anisakis simplex infection and allergy in human have increased in countries with high fish consumption. Our aim was to perform an extended seroprevalence study of anti-IgE antibodies against this parasite in Norway, one of the high fish-consuming countries. At the Department of Immunology and Transfusion Medicine and the Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway, two main groups of anonymized serum samples were collected; the first (n = 993) from recently recruited blood donors (designated 'BDO') and the second (n = 414) from patient with total IgE levels ≥1000 kU/l (designated 'IGE+'). The sera were analysed by the ImmunoCAP(®) method for total IgE and IgE antibodies against A. simplex, house dust mite (HDM), shrimp, cod, crab, brine shrimp and shrimp tropomyosin. The A. simplex positive sera were further tested by an enzyme-linked immunosorbent assay (ELISA) method, which uses 2 recombinant (r) major allergens, rAni s 1 and rAni s 7 as target antigens. SDS-PAGE and Western immunoblotting analyses were also performed. Whereas the prevalences by ImmunoCAP(®) were 0.4% and 16.2% in the BDO and IGE+ groups, respectively, analyses with recombinant allergens showed only 0.0% and 0.2%. Cross-reactivity and immunoblotting analyses suggested that most of the ImmunoCAP(®) positive sera were probably false-positive due to cross-sensitization to shrimp and HDM. However, positivity due to other A. simplex antigens should also be considered. Compared with other high fish-consuming countries, we observed a very low seroprevalence of anti-Anisakis IgE antibodies in a Norwegian population.

Different Fish-Eating Habits and Cytokine Production in Chronic Urticaria with and without Sensitization against the Fish-Parasite Anisakis simplex

Anisakis simplex sensitization has been associated with acute, but also with chronic urticaria. The objective of this study is to characterize chronic urticaria with (CU+) and without sensitization (CU-) against the ubiquitous fish parasite A. simplex in a transversal and longitudinal evaluation. 16 CU+ and 22 CU- patients were included and assessed for Urticaria activity score (UAS), fish-eating habits by standardized questionnaire and cytokine production (assessed by flow cytometric bead-based array) of peripheral blood mononuclear cells after stimulation with A. simplex extract or Concanavalin A (Con A). Patients were randomly put on a fish-free diet for three months and UAS, as well as cytokine production were again assessed. A difference of ≥1 in UAS was defined as improvement. There was no difference in UAS in both groups. Anisakis induced IL-2, IL-4 and IFN-γ production was higher in CU+. Con A induced IL-6 and IL-10 production was higher in CU+. CU+ was associated with higher total fish intake, whereas CU- was associated with oily fish intake. The correlation of UAS was positive with oily fish, but negative with total fish intake. There was a better UAS-based prognosis in CU+ without diet. Improvement was associated with higher Con A induced IL-10/IFN-γ as well as IL-10/IL-6 ratios. Further, previous higher oily fish intake was associated with improvement. Our data confirm the different clinical and immunological phenotype of CU+. Our results show a complex relationship between fish-eating habits, cytokine production and prognosis, which could have important consequences in dietary advice in patients with CU. When encountering A. simplex sensitization, patients should not be automatically put on a diet without fish in order to reduce contact with A. simplex products.

IgE Sensitization to the Fish Parasite Anisakis simplex in a Norwegian Population: A Pilot Study

The reports on fish parasite Anisakis simplex allergy have increased in countries with high fish consumption in the last decade. In Norway, a high consumption country, the prevalence of immunoglobulin E (IgE) sensitization to A. simplex was still unknown. Thus, our objective was to investigate the sensitization prevalence in this country. At the Haukeland University Hospital, Bergen, Norway, two main groups of surplus serum samples were collected: one from newly recruited blood donors (BDO) and the other from the Allergy laboratory (ALL) after analysing IgE and IgE antibodies. The latter was divided into three series: one containing unsorted sera and two sorted by either Phadiatop(®) ≥0.35 kU(A)/l or total IgE ≥1000 kU/l. The sera were analysed for total IgE and IgE antibodies against A. simplex, shrimp, house dust mite (HDM), cod and cross-reactive carbohydrates (CCDs). The prevalence of IgE sensitization to A. simplex was 2.0%, 2.2% and 6.6% in BDO, the unsorted and Phadiatop(®) positive serum groups, respectively. A considerable degree of cross-sensitization to shrimp and HDM is further suggested. Unspecific binding because of high total IgE or by binding to CCDs seemed to play a minor role. The prevalence of IgE sensitization to A. simplex appears to be lower in a Norwegian population than in other high fish-consuming countries, but might still be overestimated owing to cross-sensitization.

IgE sensitisation to the fish parasite Anisakis simplex in a Norwegian population . A pilot study

The reports on fish parasite Anisakis simplex allergy have increased in countries with high fish consumption in the last decade. In Norway, a high consumption country, the prevalence of immunoglobulin E (IgE) sensitization to A. simplex was still unknown. Thus, our objective was to investigate the sensitization prevalence in this country. At the Haukeland University Hospital, Bergen, Norway, two main groups of surplus serum samples were collected: one from newly recruited blood donors (BDO) and the other from the Allergy laboratory (ALL) after analysing IgE and IgE antibodies. The latter was divided into three series: one containing unsorted sera and two sorted by either Phadiatop®≥0.35 kUA/l or total IgE ≥1000 kU/l. The sera were analysed for total IgE and IgE antibodies against A. simplex, shrimp, house dust mite (HDM), cod and cross-reactive carbohydrates (CCDs). The prevalence of IgE sensitization to A. simplex was 2.0%, 2.2% and 6.6% in BDO, the unsorted and Phadiatop® positive serum groups, respectively. A considerable degree of cross-sensitization to shrimp and HDM is further suggested. Unspecific binding because of high total IgE or by binding to CCDs seemed to play a minor role. The prevalence of IgE sensitization to A. simplex appears to be lower in a Norwegian population than in other high fish-consuming countries, but might still be overestimated owing to cross-sensitization.

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.

Der Fang des Tages: Scombrotoxin und Anisakis simplex als Ursache allergieähnlicher Krankheitsbilder

Introduction: By the changing attitude of eating habits, increasing import of exotic food into industrialized countries and growing worldwide tourism, also in temperate climate zones, patients with diseases elicited by marine parasites or toxins may be observed. From an epidemiological point of view the most common and important diseases are scombroid or histamine fish poisoning, anisakiasis and ciguatera. Whereas the former two may be also encountered in northern countries, ciguatera intoxications are only observed from tropical sea fish. In all three allergy-like symptoms may occur, they represent, therefore, an important differential diagnosis from IgE-mediated allergic reactions to fish and seafood allergens. Methods/patients: We present five typical cases of scombroid poisoning and a case of anisakidosis, and discuss the characteristics, elicitors, diagnostic criteria and differential diagnosis of these disorders. Results and discussion: Scombroid poisoning is an intoxication from biogene amines that are formed due to inadequate storing and insufficient freezing after the catch. Particularly the odor- and tasteless histamine, possibly together with other substances or cofactors, may induce acute allergy-like symptoms such as flush, pruritus, urticaria and gastrointestinal symptoms. The reaction is self-limiting, it lasts a few hours and it usually results in complete recovery. Anisakidosis or anisakiasis is caused by the fish parasite Anisakis simplex. The acute parasitic infestation of the accidental human host by live parasites results in acute severe abdominal pain, caused by an eosinophilic inflammation in the gastrointestinal wall, which subsides within a few hours after the death of the worm. In sensitized individuals exposure to undercooked fish infested by anisakis elicits allergic symptoms ranging from urticaria, angioedema to dyspnoe and life-threatening anaphylactic shock. Some authors have reported a combination of parasitic and allergic reaction called gastroallergic anisakiasis. Ciguatoxins are lipid-soluble toxins known as ciguatoxins accumulated in the muscles of carnivorous subtropical and tropical fish. Ciguatoxins arise from biotransformation in the fish from gambier-toxins produced by Gambierdiscus toxicus, a marine dinoflagellate that lives on macroalgae in corals. The toxins and their metabolites are concentrated in the food chain when carnivorous fish prey on smaller herbivorous fish. More than 400 species offish can be vectors of ciguatoxins. Symptoms include gastrointestinal, neurological and cardiovascular disturbances, in severe cases paralysis, coma and death may occur. Symptoms subside typically in few days but may persist for months or years, or recur periodically. In the differential diagnosis toxins from seafood such as saxatoxin and brevitoxin have to be considered. Conclusions: In patients with suspected allergic symptoms after the consumption of fish or seafood, the differential diagnosis should include disorders from marine toxins or parasites, which may elicit allergy-like symptoms and signs. This permits an exact diagnosis, therapy and adequate preventive measures.

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.

Isolation and characterization of a major allergen from the fish parasite Anisakis simplex

Background: Ingestion of raw or undercooked fish can lead to infection of human subjects by the fish parasite Anisakis simplex , a disease known as anisakiasis or anisakidosis. Patients sensitized to this fish parasite show high levels of total and specific IgE. Cross-reactions seem to explain the fact that specific IgE antibodies are also found in a high number of normal subjects, as reported in other parasitoses. Objective: We sought to purify and characterize a major IgE-binding protein from the parasite. Methods: A protein was purified from the crude parasite extract by means of ethanol precipitation and reversed-phase HPLC. Its clinical relevance was tested on 20 parasite-positive sera by using IgE and IgG4 immunoblotting. A monospecific human serum was used to study its localization in the parasite body. Results: A 24-kd protein was purified, to which only 45% of the sera had specific IgG4, but 85% of sera had specific IgE. The protein was present only in the excretory gland, as shown by immunohistochemistry. N-terminal amino acid sequence (17 residues) showed no homology to previously described proteins. Conclusion:A simplex contains a potent allergen in the excretory gland. This major parasite allergen, named Ani s 1, could have important clinical relevance, as shown by the high number of positive sera in the specific IgE immunoblotting. (J Allergy Clin Immunol 2000;106:177-82.)

The use of IgE immunoblotting as a diagnostic tool in Anisakis simplex allergy

Background: The fish parasite Anisakis simplex is the etiologic agent of anisakiasis and induces IgE-mediated reactions. Skin prick tests (SPTs) and the measurement of specific IgE to A. simplex were, in our experience, not valid tools with which to discriminate between allergic and nonallergic patients because many control subjects also had positive results. Objective: The study was carried out to assess the usefulness of IgE immunoblotting in the diagnosis of allergy to A. simplex. Methods: We have studied 61 patients with acute symptoms of urticaria, angioedema, or anaphylaxis and positive specific IgE to A. simplex. According to the anamnesis, time interval between ingestion of fish and clinical onset of symptoms, and exclusion of other causes of allergy, three different groups of patients were established: group A (allergic), group NA (nonallergic), and group D (doubtful). Fifty-one healthy donors were included as control subjects (group C). IgE immunoblotting with A. simplex whole-body extract was performed in all patients and control subjects. Results: Four patterns of immunoblotting were observed: type 1, with a group of several bands of medium molecular weight and others of low molecular weight; type 2, two or more bands of medium molecular weight; type 3, only one band of medium molecular weight; type 4, without any band. There was a significant predominance of blotting type 1 in group A and type 4 in group C. Conclusion: These data suggest that IgE immunoblotting is the most useful approach to A. simplex allergy diagnosis. (J Allergy Clin Immunol 1997;99:497-501.)