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PubMed1992

Radiolabelling of the excretory-secretory and somatic antigens of Anisakis simplex larvae.

Anisakis simplex larvae were cultured in vitro in medium containing 35S-methionine for ten days. The medium and the larval tissues were analysed for biosynthetically labelled polypeptide by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Immunoprecipitates with positive and negative human antisera were similarly analysed, using Staphylococcus aureus to absorb immuno-complexes. ES products of Anisakis larvae contained many polypeptides with molecular weights of less than 200 K. 180 KDa and 40 KDa polypeptides in ES products reacted with IgG in Anisakis-infected human sera. Somatic extracts also contained many polypeptides with molecular weights of less than 200 K. One of these polypeptides with a molecular weight of 130 K reacted with IgG in Anisakis-infected human sera. These polypeptides did not react with other nematode-infected human sera.

Animals↗
PubMed2005

Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography.

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.

Animals↗
PubMedDate not supplied

Eosinophilic gastritis due to Anisakis: a case report.

BACKGROUND: the parasite Anisakis simplex is a helminth included in the nematode class. When man eats raw or rare fish and cephalopods infested by Anisakis larvae, he can acquire the parasitic disease (anisakidosis). The parasite can also originate manifestations of immediate IgE mediated hypersensitivity in patients with sensitisation to it. METHODS AND RESULTS: we present the case of a 14 year old boy diagnosed of eosinophilic gastritis after endoscopic examination and biopsy associated to recurrent abdominal pain. After allergologic study, a type I hypersensitivity mechanism against Anisakis simplex is confirmed by means of prick test, antigen specific IgE determination and antigen specific histamine release test. Sensitisation against fish proteins is ruled out as well as parasitic infestation. CONCLUSIONS: in this case report we demonstrate a type I hypersensitivity mechanism against Anisakis simplex in a patient diagnosed of eosinophilic gastritis. This can be suspected in cases of gastritis or non filiated enteritis with a torpid evolution following the conventional treatment and especially if the onset of the symptoms is related with the intake of fish. The therapeutic success was reached when fish and shellfish were taken out of the diet. After two years without seafood ingestion our patient is asymptomatic and the allergologic study has been normalised.

Abdominal Pain↗
PubMed2000

Anisakis and fish induced urticaria.

Skin tests using prepared Anisakis antigen and commercially available fish antigen were done. Also, specific IgE detection for Anisakis was done by RAST method. Two patients out of 20 showed positive skin test and positive RAST to larval Anisakis antigen, and negative skin test to fish antigen. These 2 patients were considered hypersensitive to Anisakis.

Animals↗
PubMed1997

Genetic and ecological data on the Anisakis simplex complex, with evidence for a new species (Nematoda, Ascaridoidea, Anisakidae).

Isozyme analysis at 24 loci was carried out on anisakid nematodes of the Anisakis simplex complex, recovered from various intermediate/paratenic (squid, fish) and definitive (marine mammals) hosts from various parts of the world. A number of samples were found to belong to A. simplex sensu stricto and Anisakis pegreffii, widely extending the geographic ranges and the number of hosts of these 2 species. In addition, a new distinct gene pool was detected, showing different alleles with respect to A. simplex s. str and A. pegreffii at 5 diagnostic loci (99% level). Samples with this gene pool were assigned to a new species, provisionally labeled A. simplex C. Reproductive isolation between A. simplex C and the other 2 Anisakis species was directly assessed by the lack of hybrid and recombinant genotypes in mixed samples from sympatric areas, i.e., Pacific Canada for A. simplex C+A. simplex s. str., South Africa and New Zealand for A. simplex C+A. pegreffii, even when such samples were recovered from the same individual host. Similar levels of genetic divergence were observed among the three species (DNei from 0.36 to 0.45). At the intraspecific level, Canadian Pacific and Austral populations of A. simplex C were found to be genetically rather differentiated from one another (average DNei = 0.08), contrasting with the remarkable genetic homogeneity detected within both A. simplex s. str. and A. pegreffii (average DNei about 0.01). Accordingly, a lower amount of gene flow was estimated within A. simplex C (Nm = 1.6) than within the other 2 species (Nm = 5.4 and 17.7, respectively). Anisakis simplex C showed the highest average values of genetic variability with respect to both A. simplex s. str. and A. pegreffii, e.g., expected mean heterozygosity. Hr = 0.23, 0.16, and 0.11, respectively, in the 3 species. Data on geographic distribution and hosts of the 3 members so far detected in the A. simplex complex are given. Their ecological niche is markedly differentiated, with a low proportion of hosts shared. Intermediate and definitive hosts of A. simplex s. str. and A. pegreffii appear to belong to distinct food webs, benthodemersal, and pelagic, respectively; this would lead to different transmission pathways for the parasites.

Animals↗
PubMed2003

[Allergy to Anisakis simplex].

Anisakis simplex is a parasite, belonging to the Anisakidae family. The life cycle of the parasite can include one or more intermediary hosts, their final hosts being marine mammals or large fish, in which the larvae develop until the adult stage is reached. Man is an accidental host who acquires the larvae by eating raw or undercooked fish. Since the mid-50s, when the first case studies were published in Holland and Japan, new cases have been emerging in different countries including Spain. Parasitization of man by the living larva is known as anisakiasis, principally giving rise to digestive symptomology, with other rare cases described of invasion of other organs such as the lung, the liver, the spleen, the pancreas, etc. Clinical pictures of allergy to IgE mediated anisakis simplex have also been described: reactions by thermostable antigens of the parasite that develop in spite of the fish being cooked or frozen, and an acute digestive parasitization with allergic symptoms called gastro-allergic anisakiasis. In the diagnosis of anisakiasis and/or allergy to Anisakis, the antecedent of the prior ingestion of fish as well as the clinical accompaniment can form basic data of considerable orientational value, and endoscopy can reveal the presence of the larvae and make possible their extraction. Besides, in cases of allergy the detection test for specific IgE facing Anisakis simplex, and cutaneous tests with fish should be carried out. The best treatment for avoiding this parasitization is prophylactic, avoiding the consumption of raw or undercooked fish, while a fish free diet is necessary in cases of true allergy to the thermostable proteins of the parasite.

Anisakiasis↗
PubMed1990

Identification of the secreted neutral proteases from Anisakis simplex.

Ingestion of larval nematodes (family: Anisakidae) can cause the human disease known as anisakiasis. After ingestion, Anisakis larvae can be invasive, penetrating host stomach or intestinal wall. Observation of larvae penetrating the tissue layers of human stomach in vitro by SEM showed tunnels and burrows were formed in the mucosa and submucosa. Based on these observations, we hypothesized that secreted proteases may be involved in the degradation of host tissue macromolecules to allow tunnel formation. Using a model of connective tissue extracellular matrix (ECM), we found that as few as 5 Anisakis simplex larvae could degrade approximately 25% of the ECM in a 16-mm culture well in 24 hr. Further characterization of the secreted proteases using synthetic peptide substrates and inhibitors revealed that there were 2 classes of proteases present: a metallo aminopeptidase and a trypsinlike serine protease. Extracts of Anisakis larvae contained a 25-kDa protease that was recognized by rabbit anti-rat trypsin antibody on western blots. This suggests that there is structural as well as functional similarity between the Anisakis trypsin and vertebrate trypsins.

Animals↗
PubMed1998

Occupational asthma by Anisakis simplex.

BACKGROUND: No cases of occupational asthma caused by the inhalation of antigens from Anisakis simplex have been published. OBJECTIVE: The purpose of this study was to evaluate the possibility that A simplex can play a role in the asthma experienced by 2 workers when handling fish and fish flour. METHODS: Skin prick and bronchial challenge tests with A simplex were performed. We also carried out measurements of specific IgE to A simplex and immunoblotting. RESULTS: Both patients had strong positive skin test responses, challenge test responses, and specific IgE to A simplex. Immunoblotting showed that both patients also had IgE against several bands in the fish flour extract, suggesting contamination by Anisakis allergens. CONCLUSION: These 2 patients provide evidence for occupational asthma caused by A simplex, based on in vivo and in vitro tests for Anisakis-specific IgE.

Allergens↗
PubMed1999

Expression and characterization of elastase inhibitors from the ascarid nematodes Anisakis simplex and Ascaris suum.

Two elastase inhibitors, ASPI-1 and ASPI-2, from the parasitic nematode Anisakis simplex, have been isolated and characterized. Because these inhibitors are similar in size (60 amino acids in length) and primary sequence (52 and 47% identical) to the Ascaris suum chymotrypsin/elastase inhibitor-1 (AsC/E-1), we suggest that these Anisakis elastase inhibitors belong to the same unique class of canonical inhibitors formed by the family of Ascaris inhibitors (Huang K, Strynadka NCJ, Bernard VD, Peanasky RJ, James MG. Structure 1994;2:679-689). To compare ASPI-1 with AsC/E-1, we expressed both inhibitors in Pichia pastoris and found that: (1) the association constant of rASPI-1 with porcine pancreatic elastase (PPE) is similar to native inhibitor (Ka = 4.5 x 10(9) and 6.4 x 10(9) M(-1), respectively); (2) rASPI-1 is a potent inhibitor of PPE and human leukocyte elastase (Ka = 1.6 x 10(9) M(-1)); and (3) it is only a very weak inhibitor of chymotrypsin (CHYM) (Ka = 1.2 x 10(6) M(-1)). In contrast to the Anisakis inhibitor, however, rAsC/E inhibitor-1 is a very strong inhibitor of both PPE (Ka = 3.5 x 10(10) M(-1)) and CHYM (Ka = 3.6 x 10(12) M(-1)). We also found that the determined reactive sites (P1-P'1) of rASPI-1 and rAsC/E-1, as recognized by PPE, are Ala 28-Met 29 and Leu 31-Met 32, respectively. These P1-P'1 residues of AsC/E-1 constitute the same reactive site as that also recognized by CHYM (Peanasky RJ, Bentz Y, Homandberg GA, Minor ST, Babin DR. Arch Biochem Biophys 1994;232:135-142). The difference in specificities of ASPI-1 and AsC/E-1 toward their cognate serine proteases may be attributed to the P1 and P'3 residues in the inhibitors. Elastase, which recognizes both alanine and leucine, canaccommodate both ascarid inhibitors, whereas chymotrypsin, which prefers bulky, hydrophobic residues, only recognizes the Ascaris C/E inhibitor-1.

Amino Acid Sequence↗
PubMed2000

Association of hypersensitivity to the nematode Anisakis simplex with HLA class II DRB1*1502-DQB1*0601 haplotype.

Anisakiasis as well as allergic and anaphylactoid reactions to Anisakis simplex antigens are recently identified clinical entities. They are relatively frequent in countries with habitual raw food consumption, often in the form of large amounts of fish and sea food products. In this communication the relationship between HLA class II alleles and the IgE-specific immune response to A. simplex allergen was studied in a defined population in Northern Spain. Individuals with immediate-type Anisakis hypersensitivity and healthy controls were examined for HLA-DRB1, DQB1 and DQA1 alleles by sequence-specific oligonucleotide probe typing. Analysis of the HLA data among patients revealed increased phenotypic frequencies for DRB1*1502 and DRB1*0404 compared to healthy controls (p < 1 x 10(-7) and < 0.01, respectively). Analysis of haplotypic frequencies showed that the DRB1*1502-DQB1*0601 haplotype is significantly higher in patients with Anisakis hypersensitivity in comparison with the control population from the same region (p < 4 x 10(-8)). The data suggest that this haplotype can be considered to be a susceptibility factor for hypersensitivity to A. simplex antigens.

Animals↗
PubMed1994

Detection of antibodies to Anisakis simplex larvae by enzyme-linked immunosorbent assay and immunoelectrophoresis using crude or purified antigens.

The enzyme-linked immunosorbent assay (ELISA) and immunoelectrophoresis (IEP) were used for the serodiagnosis of larval Anisakis simplex infections in man and immunized rabbits. Sephacryl S-300 gel filtration was used for separating crude antigen. Four fractions were obtained. Sera from patients with other helminth infections sometimes cross reacted with Anisakis larval antigens. With IEP, crude antigen is more sensitive than purified antigens. With ELISA, the third fraction is the most sensitive for detecting antibodies to Anisakis larvae in the sera of humans and immunized rabbits.

Animals↗
PubMed2004

Cross-reactivity between antigens of Anisakis simplex s.l. and other ascarid nematodes.

A study of the cross-reactivity among somatic and excretory-secretory antigens of the third stage larvae of Anisakis simplex s.l. and somatic antigens of other ascarid nematodes (Ascaris lumbricoides, A. suum, Toxocara canis, Anisakis physeteris, Hysterothylacium aduncum and H. fabri) was carried out by immunoblotting. It was revealed a high degree of cross-reactivity among ascarids in the 30 and > 212 kDa range by using sera against somatic and excretory-secretory antigens of A. simplex s.l. It has been revealed also specific components of the Anisakis genus (< 7.2, 9, 19 and 25 kDa) that will be interesting in diagnosis.

Animals↗
PubMed1998

Eosinophilic gastroenteritis and Anisakis.

BACKGROUND: The differential diagnosis of eosinophilic gastroenteritis (EG) includes, among other diseases, parasitic infections such as anisakiasis, which has acquired worldwide importance. METHODS: We reviewed all patients referred to our allergy service who had been diagnosed as having primary EG to determine the possible role of Anisakis simplex in the etiopathology of the disease. All patients (n = 10) were studied and diagnosed as having primary EG between 1989 and 1996, inclusive. Two different groups of subjects were used as controls: group A (149 subjects without digestive disorder) and group B (10 subjects with digestive disorder different from EG). Cutaneous prick tests were performed with the main foods, aeroallergens, and commercial extract of A. simplex. Total and specific serum IgE was measured in all patients. Gastric or gut histologic specimens were re-examined in five cases. RESULTS: Peripheral eosinophilia was detected in 40% of the patients with EG, and sensitization to A. simplex was detected in 80% of these. In both control groups, the rate of sensitization to A. simplex was 10%. Sensitization to A. simplex in EG patients with respect to control groups A and B showed odds ratios (OR) of 36 and 40, respectively. In one case, serialization of the histologic section allowed us to observe a whole Anisakis larva. CONCLUSIONS: Immunologic methods to detect specific antibodies against Anisakis should be used routinely before diagnosing EG as primary disorder. Preventive measures are of capital importance.

Animals↗
PubMed1999

Survival of Anisakis simplex in microwave-processed arrowtooth flounder (Atheresthes stomias).

The purpose of this study was to define the relationship between survival and temperature of nematodes of the species Anisakis simplex in microwave-processed arrowtooth flounder (Atheresthes stomias). Ten fillets (each 126 to 467 g, 0.5 to 1.75 cm thick), with an average of five larvae of Anisakis simplex per fillet, were processed to target temperatures on high (100%) power using a commercial 700-W microwave oven. Fillets were neither covered nor rotated and had a temperature probe inserted to two-thirds depth into the thickest portion. After the fillet was digested using a 1% pepsin solution, the viability of nematodes was determined by viewing them under a dissecting microscope. Survival rates were 31% at 140 degrees F (60 degrees C), 11% at 150 degrees F (65 degrees C), 2% at 160 degrees F (71 degrees C), 3% at 165 degrees F (74 degrees C), and 0% at 170 degrees F (77 degrees C). Microwave processing of standardized fillet "sandwiches," 14 cm long, 4.5 cm wide, and approximately 1.75 cm high, each of which was preinoculated with 10 live nematodes, resulted in no survival at either 160 degrees F or 170 degrees F. Using ultraviolet light to detect both viable and nonviable nematodes in fillet sandwiches as an alternative method to pepsin digestion resulted in survival rates of 1% at 140 degrees F (60 degrees C), 3% at 145 degrees F (63 degrees C), and 0% at 150 degrees F (65 degrees C). Smaller fillet sandwiches, which most likely had fewer cold spots during microwave processing, required 150 degrees F (65 degrees C), whereas larger whole fillets required 170 degrees F (77 degrees C) to kill larvae of Anisakis simplex. The parasites were most likely inactivated by a thermal mechanism of microwave treatment. Damage to the nematodes was often evident from ruptured cuticles that were no longer resistant to digestive enzymes. The high hydrostatic pressure and low chloride content of the pseudocoelomic fluid probably contributed greatly to the damage incurred by the larvae.

Animals↗
PubMed2002

Anisakis simplex larva killed by high-hydrostatic-pressure processing.

Anisakis simplex is a common nematode parasite present in many marine fish, including finfish and squid. It can pose a public health problem if it is not destroyed during food processing. Anisakis larvae were isolated from fish tissue, and their survival of high-pressure treatments in distilled water and physiological isotonic solution was assayed. Treatment at a pressure of 200 MPa for 10 min at a temperature between 0 and 15 degrees C kills all Anisakis larvae, with a lack of motility being used as an indicator of larval death. Lower pressures can be successfully employed down to 140 MPa, but with lower pressures, the treatment time must be increased by up to I h to kill all larvae. Meanwhile, most larvae treated for >10 min at pressures of >120 MPa were dead, with the autofluorescence method being used to determine death. Cycles of compression and decompression increase the destruction of larvae compared with a single pressure treatment for a similar treatment time. Our results indicate that high-pressure treatment is an alternative nonthermal method for killing this nematode. The possible mechanism of death and damage by pressure is discussed, and uses for this treatment in food processing are suggested.

Animals↗
PubMed2005

A new procedure for marinating fresh anchovies and ensuring the rapid destruction of Anisakis larvae.

The consumption of marinated anchovies is the main route of transmission of anisakiasis in Spain. Because this country is one of the world's major tourist destinations, this traditional food also poses a potential health risk to millions of foreign visitors. Anisakis larvae are not destroyed by the traditional marinating procedure, and alternative methods, such as long-term storage in brine, freezing, or hydrostatic pressure treatment, all present major difficulties. In this study, we used high food-grade acetic acid concentrations (10, 20, 30, and 40% [vol/vol] in line with the quantum satis rule) to destroy these larvae rapidly, and we report data on the survival of Anisakis larvae exposed directly to different marinades and when the larvae are placed under the fish musculature. The percentage of salt and acetic acid in the fish tissue water phase was also determined. A marinating procedure is proposed that ensures the rapid death of Anisakis through the use of strong acetic acid concentrations. Posttreatment washes with water reduce these to levels acceptable to consumers. The sensory characteristics of the product were shown to be satisfactory. The actual selection of an acetic acid concentration for marinating depends on costs and the processing time available. The physiological stress of the larvae exposed to the different marinades was determined by measuring the levels of their stress proteins. The latter are good indicators of injury and might reflect the infectivity of larvae. In addition, we also used a rat model to determine the infectivity of larvae considered microscopically dead.

Acetic Acid↗
PubMed2006

Scanning electron microscopy of Anisakis larvae following different treatments.

Ingestion of fish parasitized with Anisakis larvae can produce infestation and/or allergy in consumers. Technological and food processing treatments have been applied to parasitized fish in order to kill the larvae and avoid the infestation; however, their influence on allergenicity has not been studied. Four lots of hake (Merluccius merluccius) steaks artificially parasitized with Anisakis larvae were subjected to two storage chilling (5 degrees C +/- 1 degrees C) and freezing (-20 degrees C +/- 1 degrees C) treatments and two food processing treatments of heat (final temperature 86.3 degrees C) and microwave (final temperature 66.9 degrees C) and studied by scanning electron microscopy, environmental scanning electron microscopy (ESEM) (acid [pH = 2] and water preparations), and emission of fluorescence. Anisakis larvae were resistant to acid conditions, remaining alive after treatment. Larvae in the heat- and microwave-treated lots presented coagulated and disrupted zones in the cuticle with release of fluids. The cylindrical shape changed to a dehydrated appearance mainly observed by ESEM. Fluorescence was only noticeable in the frozen larvae. Larvae without apparent changes, together with dehydrated ones, were observed by ESEM in the frozen lot; nevertheless, no disruptions in the cuticle were perceptible. Further studies are needed in order to elucidate if the changes observed in the cuticle reduce the resistance of the parasites to the action of gastric enzymes in the gastrointestinal tract and to determine the release of allergens to the flesh by the live larvae during chilled storage of the fish.

Animals↗
PubMedDate not supplied

[Peter-the-Great Gulf fishes are a source of human invasion of the nematode Anisakis simplex (the family Anisakidae)].

The paper presents the results of the rate of infection with the nematode Anisakis simplex in Peter-the-Great gulf fishes that are a source of human invasion. Five of 19 fish types were found to be nematode-free, and the extensiveness and intensity of invasion were observed in Siberian salmons (Oncorhynchus keta) (61% and 2-700 worms, respectively), Alaska pollacks (Theragra chalcogramma) (75.7% and 1-31 worms), and brown rock trouts (Hexagrammos octogrammus) (100% and 1-60 worms). Autopsy showed that the distribution of larvae was ambiguous in different fish species. Data on the survival of Anisakis were also obtained under experimental conditions. The nematodes were ascertained to die at -8 degrees C at the moment of complete fish freezing. Anisakis may be alive in the unfrozen fish for more than 40 days. The death of worms in the salted fish occurs within 3-8 days only when 6-10/1000 concentrations of NaCl salts are achieved at the sites of worms.

Animals↗