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PubMed1993

Anisakis simplex: antigen recognition and antibody production in experimentally infected mice.

The kinetics of antibody response to intraperitoneal infection of mice with third stage larvae of Anisakis simplex was investigated by ELISA. Maximum antibody response to excretion-secretion (ES) antigens was reached before maximum response to somatic (SA) antigens. Total immunoglobulin (Ig) production (consisting mainly of IgM and IgG1 isotypes) was very similar in both cases. Immunoblotting was used to characterize the antigens recognized by the host in the presence or absence of the metabolic products released by the parasite in vivo. Sera from mice infected with live larvae (anti-L3 L serum) and immunized with dead larvae (anti-L3 D serum) recognized a similar pattern of bands in immunoblots of ES and SA antigen preparations. In the latter, however, three bands at 14, 17 and 18 kD were only recognized by the anti-L3 L serum. A possible explanation is that these low molecular weight antigens are ES products released only in vivo. Finally, the immune response in mouse was compared using ELISA and immunoblotting with the response of a human anisakiasis reference serum, and was found to display considerable similarities. This suggests that the mouse may be a useful model for studying the immunobiology of A. simplex in man.

Animals↗
PubMed1997

Recidivous acute urticaria caused by Anisakis simplex.

This study aimed to determine the cause of acute recidivous urticaria in patients who usually eat fish or other seafood. Twenty-five patients were studied. The skin prick test with larval Anisakis simplex extract was performed; total and specific IgE against A. simplex was measured with the CAP System; specific antibodies to A. simplex were determined by ELISA; and immunorecognition patterns of the sera were studied by Western blot. Nineteen patients showed specific IgE to A. simplex, but specific IgE to Ascaris was demonstrated in only two patients. No patients reacted to Toxocara canis or Echinoccocus granulosus antigens with the same test. The skin prick test was positive in 16 patients, in two of them persisting for 48 h. Five patients showed neither skin reaction nor specific IgE to A. simplex. Sera showed specific immunoglobulin levels against A. simplex larval crude extract, by both ELISA and Western blot. Likewise, specific immunoglobulin levels against excretory-secretory antigen were also measured by ELISA. Only one patient showed sensitization to fish. A. simplex was found to be the main cause of acute recidivous urticaria in patients who usually eat fish and are not sensitized to it.

Adolescent↗
PubMed1997

Periodate treatment of Anisakis simplex allergens.

Anaphylactic reactions after parasitized fish consumption are mediated by an IgE response. However, positive skin tests and specific IgE can also be found in many asymptomatic subjects who recognize a single medium-mol.-wt. antigen by IgE immunoblot. The study aimed to find out whether this unspecificity was due to the carbohydrate moieties of parasite antigens. Sixty-two patients with suspected parasite allergy, 51 blood donors, 18 bakers, and 38 atopic patients were studied by blotting. Parasite proteins were treated with periodate. Several selected sera were inhibited with a crude wheat extract and fungal amylase. Twelve patients (19%), eight donors (16%), six bakers (33%), and one atopic patient (3%) recognized a single medium-mol.-wt. band in blotting and should be considered false-positive. This band was periodate-sensitive, but specific IgE to this allergen could not be inhibited by a wheat extract nor by fungal amylase and was clinically irrelevant. Diagnosis of Anisakis simplex hypersensitivity by skin tests and/ or specific IgE values should always be confirmed by specific IgE immunoblotting in order to detect the presence of clinically unrelated antibodies directed to periodate-sensitive allergens. These allergens are probably not a carbohydrate moiety of a parasite glycoprotein.

Amylases↗
PubMed1997

Anisakis simplex, a relevant etiologic factor in acute urticaria.

Anisakis simplex, a parasite of fish and cephalopods, can induce IgE-mediated reactions. This study aimed to determine the etiologic role of A. simplex in patients affected by urticaria/angioedema (AE) or anaphylaxis. We studied 100 adult subjects suffering acute episodes of urticaria/AE, by anamnesis, prick tests with A. simplex and fish-mix extracts, and total and specific IgE to both A. simplex and cod. The following criteria of A. simplex allergy were considered: 1) urticaria/AE within 6 h after fish ingestion; 2) specific IgE to A. simplex; 3) positive prick test to A. simplex extract; 4) exclusion of other suspected causes. Double-blind, placebo-controlled food challenge was not carried out because ethical considerations forbid challenge with a parasite. Specific IgE to A. simplex (> 0.7 kU/l) was found in 22 subjects, but only eight were diagnosed as having A. simplex allergy. Other allergens were involved in 37 patients, and 55 cases were considered idiopathic. Specific IgE to fish (> 0.7 kU/l) was found in two patients, but only one was diagnosed as having fish allergy. We concluded that A. simplex is an important etiologic factor in acute urticaria. We suggest that it should be considered in cases of urticaria/AE or anaphylaxis, especially after fish ingestion.

Adolescent↗
PubMed2004

High prevalence of seropositivity to a major allergen of Anisakis simplex, Ani s 1, in dyspeptic patients.

Finding evidence of anisakidosis requires invasive methods. We have developed a serological assay based on the detection of an immunoglobulin E (IgE) specifically directed against Ani s 1 protein, a major parasite allergen of Anisakis simplex, which has shown a high level of accuracy in the diagnosis of anisakidosis. We used this tool to determine the prevalence of anti-Ani s 1 IgE in dyspeptic patients and to investigate if its seropositivity could be related to epidemiological factors other than raw fish consumption. A total of 174 dyspeptic patients who submitted to upper digestive tract endoscopy were studied. Specific IgE against Ani s 1 was determined by immunoblotting. Quantitative information on smoking, alcohol consumption, and fish consumption as well as a history of gastric surgery was recorded. Twenty-four (13.8%) patients were seropositive for Ani s 1 protein. The seroprevalence of anti-Ani s 1 IgE increased with age in patients who were less than 62 years old (P = 0.047). Seropositivity to Ani s 1 was associated with the consumption of fish in vinegar (P < 0.001), raw fish (P = 0.001), and smoked fish (P = 0.007). There was no relationship between anti-Ani s 1 IgE seropositivity and the number of cigarettes smoked (P = 0.098) or alcohol intake (P = 0.179). Five patients had undergone previous gastric surgery, and three of those patients were seropositive for Ani s 1 (P = 0.019). In multivariate analysis, the consumption of fish in vinegar (P = 0.006), raw fish (P = 0.017), and smoked fish (P = 0.002) and a history of gastric surgery (P = 0.005) were independent factors associated with anti-Ani s 1 IgE detection. In conclusion, at present, anisakidosis might frequently be underdiagnosed, and it might have a clinical role in patients with upper dyspeptic symptoms. Uncooked-fish ingestion and previous gastric surgery were associated with seropositivity for Ani s 1 protein.

Allergens↗
PubMedDate not supplied

Allergy and parasites reevaluated: wide-scale induction of chronic urticaria by the ubiquitous fish-nematode Anisakis simplex in an endemic region.

BACKGROUND: The ubiquitous fish-nematode Anisakis simplex produces acute urticaria or angioedema in the course of gastro-allergic anisakiasis. We studied the relationship between this nematode and chronic urticaria (CU), as well as the clinical usefulness of measuring specific IgG4 in A. simplex-sensitized patients with CU. METHODS: First, the prevalence of sensitization to A. simplex was estimated in 135 consecutive CU patients and the result was compared with known data about sensitization in a healthy population. Then, clinical response to a 2-month diet without fish was analyzed in 76 CU patients. The improvement rate in patients with and without sensitization to A. simplex was compared. Finally, the improvement rate, other clinical data and specific immunoglobulins in sensitized patients with and without detectable specific IgG4 were compared. RESULTS: a) The A. simplex sensitization rate in CU patients was 52.6 % compared with a known prevalence of between 16 and 20 % in our region. b) Of 65 sensitized patients, 52 experienced clinical improvement after the diet compared with only three of 11 patients without sensitization to A. simplex (p = 0.001). c) Of 43 patients with detectable specific IgG4, 38 showed clinical improvement compared with only 14 of 22 patients without detectable IgG4 (p = 0.02). Eight of nine patients with previous fish-associated cutaneous symptoms had detectable specific IgG4 compared with 15 of 32 patients who reported no previous fish-associated symptoms or acute urticaria (p = 0.03). CONCLUSIONS: Our results indicate that A. simplex is a possibly widespread etiologic agent able to induce CU. This parasite model constitutes the first report that associates an infectious agent with CU on a large scale. The detection of IgG4 antibodies reflects a previous acute parasitic infection and a temporary diet without fish improves symptoms in most patients with detectable specific IgG4.

Adult↗
PubMed2000

Molecular cloning of paramyosin, a new allergen of Anisakis simplex.

BACKGROUND: Anisakis simplex is a fish parasite that, when accidentally ingested by humans, may cause allergic reactions in sensitized individuals. The main objectives of our study were to: (1) construct a cDNA expression library of A. simplex; (2) identify clones producing specific IgE binding protein antigens, and (3) produce and purify the protein/s codified by the isolated clones produced in Escherichia coli. METHODS: An expression cDNA library from the third stage larvae (L3) of A. simplex was constructed. This library was first screened with a rabbit anti A. simplex hyperimmune serum. The positive clones, identified using the rabbit serum, were rescreened with a pool of human sera containing high titers of IgE antibodies against A. simplex. RESULTS: Two positive clones were isolated carrying the genes which codify for paramyosin. The paramyosin protein was produced in E. coli and purified. The partial sequence of a second paramyosin gene was also identified. The frequency of specific IgE binding to the recombinant and native forms of paramyosin using the sera of 26 A. simplex-sensitive individuals was 23 and 88%, respectively. Both paramyosins were able to inhibit 11% of the specific IgE binding to a total extract. CONCLUSIONS: We describe the primary structure of a paramyosin of A. simplex. It can be considered as an allergen based on its IgE binding capacity. We suggest that the recombinant protein does not maintain the complete allergenic properties of the native paramyosin, considering its lower IgE binding capacity of the recombinant protein. However, both proteins have the same specific IgE inhibition capacity. The recombinant protein can be produced in large quantities in E. coli. We propose the term Ani s 2 for this allergen.

Allergens↗
PubMed2001

Anaphylaxis due to hypersensitivity to Anisakis simplex.

Anaphylactic reactions are among the most frequent causes for requests for emergency medical care. It is possible that no clear precipitating factor can be found delaying the onset of adequate treatment and increasing the risk of new episodes of anaphylaxis in the future. Hypersensitivity to Anisakis simplex, an intestinal parasite found in fish, is an unusual cause of anaphylaxis but it should always be borne in mind in countries where a great deal of fish is consumed, especially if it is eaten raw or undercooked. 3 cases of anaphylaxis due to hypersensitivity to A. simplex are described.

Allergens↗
PubMed2005

Immunochemical and biological analysis of allergenicity with excretory-secretory products of anisakis simplex third stage larva.

BACKGROUND: Anisakis simplex third stage larvae (L3) are parasites that frequently give rise to allergic responses. The larvae molt into fourth stage larvae (L4), and at each stage they produce L3-excretory-secretory products (L3-ESP) and L4-ESP, respectively, which are different in their main protein constituents. Although the allergenicity of L4-ESP has been investigated by several research groups, research on the allergenicity of L3-ESP has not been carried out by any researcher. In this investigation, the allergenicity and antigenicity of L3-ESP were investigated in comparison with L4-ESP, using rat sera. METHODS: Rat sera were produced by L3 oral infection two times with a 9-week interval. Larvae ESP prepared by culture were concentrated and fractioned using lyophilizer and a centrifugal filter device, respectively. Immunochemical analysis was performed using both indirect ELISA and immunoblot. Biological allergenicity was analyzed by RBL-2H3 exocytosis. RESULTS: With the indirect ELISA, the optical density (OD) value of the nonfractioned (NF)-L3ESP was only one third of that of the NF-L4ESP in both specific IgM and IgG. On measuring specific IgE, the OD of NF-L3ESP was less than one tenth of that of NF-L4ESP. In addition, neither antigen nor allergen was shown in NF-L3ESP, but it was shown in NF-L4ESP with immunoblot. However, the biological allergenicity of NF-L3ESP was comparable to that of NF-L4ESP. To demonstrate the presence of any allergen, L3-ESP was fractioned and found to carry twelve visualized allergen bands from 10 to 186 kDa by immunoblot. CONCLUSIONS: These results indicate that L3-ESP may include the important allergens necessary to induce the allergy by L3 oral infection, as compared to L4-ESP.

Allergens↗
PubMedDate not supplied

Cross-reactivity between IgE-binding proteins from Anisakis simplex and Dermatophagoides pteronyssinus.

An association was found between Anisakis simplex (As) and Dermatophagoides pteronyssinus (Dp) sensitization. One recent study shows a cross-reactivity between As and Dp and tropomyosin (tr)is suspected as being one of the proteins responsible of this cross-reaction. The aim of our study was: 1) to confirm the cross-reactivity between Dp and As; 2) to determine the importance of tr in this cross reaction. SDS-PAGE analysis of Dp and As (metabolic and somatic) extracts was carried out. Then an IgE immunoblotting test using serum from a patient who had specific IgE only to Dp and As and immunoblotting inhibition experiments using Dp extract and tr as inhibitors were performed. We found that patients serum reacted: 1) against larval As antigens with a molecular weight (mw) of 25 kilodalton (kD) and a mw > than 100 kD, 2) against various metabolic As antigens with a mw > than 100 kD, a mw ranging approximately from 35 to 50 kD, and a mw around 20 kD, and 3) against Dp proteins with mw between 35 and 55 kD. Preincubation of patient's serum with Dp extract caused the disappearance of reactivity against antigens with a mw > than 100 kD in both larval and metabolic As extracts and against proteins with mw ranging approximately from 35 to 50 kD in the metabolic As extract. Preincubation of patients serum with As extract caused the disappearance of reactivity against antigens with mw between 35 and 55 kD in the Dp extract. Pre-incubation of patients serum with tr did not induce any change in the immunoblotting profile. The results show that 1) cross-reactive components between Dp and As are some proteins with a mw ranging approximately from 35 to 50 kD and with a mw > than 100 kD, and 2) tr is not involved in cross-reactivity between As and Dp.

Adult↗
PubMedDate not supplied

Acute intestinal anisakiasis in Spain: a fourth-stage Anisakis simplex larva.

A case of acute intestinal anisakiasis has been reported; a nematode larva being found in the submucosa of the ileum of a woman in Jaén (Spain). The source of infection was the ingestion of raw Engraulis encrasicholus. On the basis of its morphology, the worm has been identified as a fourth-stage larva of Anisakis simplex. In Spain, this is the ninth report of human anisakiasis and also probably the first case of anisakiasis caused by a fourth-stage larva of A. simplex.

Acute Disease↗
PubMed2001

Interleukin-4 production in BALB/c mice immunized with Anisakis simplex.

We investigated the interleukin (IL-4) levels in BALB/c mice immunized with Anisakis extract in single or multiple doses and in mice orally infected with a larva. From animals immunized maximum responses were obtained with the multiple doses with an only IL-4 peak. Conversely, in the mice inoculated with a larva per os, the IL-4 levels showed two peaks of different rates.

Animals↗
PubMed2002

Evaluation by ELISA of anisakis simplex larval antigen purified by affinity chromatography.

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of (1/4)00 and 1 microg/ml of antigenic concentration.

Animals↗
PubMed2000

Shared epitope for monoclonal IR162 between Anisakis simplex larvae and Clonorchis sinensis and cross-reactivity between antigens.

IR162 is a rat monoclonal IgE antibody (mAb). In the investigation of rat IgE production, the antigens recognized by IR162 (IR-Ags) were found to be expressed by a variety of helminthic parasites. By western blot analysis, IR162 detected bands in crude extracts of Anisakis simplex larvae, Clonorchis sinensis, Paragonimus westermani, plerocercoids of Spirometra mansoni, and Toxocara canis. Excretory-secretory material from A. simplex larvae also contained a protein recognized by IR162. IR162 mAb obtained from both Serotec and Zymed recognized identical bands of A. simplex larvae. Sandwich enzyme-linked immunosorbent assay analysis indicated that both IR-Ags of A. simplex larvae (IR-As) and C. sinensis (IR-Cs) were important antigens with respect to induction of specific IgG in human infections with these 2 organisms. Even though minimal cross-reaction between IR-As and IR-Cs was observed using sera from infected individuals, these results indicate that IR-Ags are found in the parasites examined, that IR-As and IR-Cs are the antigens that induce specific antibody in natural infection, and that the epitope that binds IR162 is shared by A. simplex larvae and C. sinensis.

Animals↗
PubMed2002

Effects of ivermectin and albendazole against Anisakis simplex in vitro and in guinea pigs.

The activity of ivermectin and albendazole against larval Anisakis simplex was tested in vitro and in experimentally infected guinea pigs. Before drug exposure the medium for half of the larvae was adjusted to pH 2.0 with 1 N HCl, whereas the other half was held at pH 7.0. To these solutions, ivermectin was added to full concentrations of 1, 2, 5, 10, 50, 100, or 200 microg/ml, and for albendazole, 300, 400, and 500 microg/ml. Animals from group I were given 0.1 ml of 1% (3.3 mg/kg) ivermectin, whereas guinea pigs from group II were each given 5-7 mg (16.6-23.3 mg/kg) of albendazole orally. The efficacy of both drugs against L, A. simplex was high in vitro and in vivo against the larvae in different organs of guinea pigs.

Albendazole↗
PubMed2004

Population structure of Anisakis simplex (Nematoda) in harbor porpoises Phocoena phocoena off Denmark.

The population structure and habitat selection of Anisakis simplex in 35 harbor porpoises off Denmark are described. The nematodes were collected from the stomach and duodenal ampulla and were categorized as third-stage larvae, fourth-stage larvae, subadults, and adults. The porpoises harbored 8,043 specimens of A. simplex. The proportion of adults and subadults increased with infrapopulation size. The number of development stages across infrapopulations covaried significantly (Kendall's test of concordance). Concordance was higher in hosts with the highest intensities than in those with low and medium intensities. All stages occurred mainly in the forestomach, but this trend was stronger for the adults. Adult and subadult sex ratios did not depart significantly from 1:1. Our data suggested that recruitment and duration of each stage were the main factors accounting for infrapopulation structure. The preference of A. simplex for the forestomach conformed with previous studies, but the narrower distribution of adults relative to other stages might indicate a strategy to enhance mating opportunities. Information on sex ratios of A. simplex is scarce and contradictory. We suggest that the discrepancies might partly reflect differences in categorization criteria and statistical methods.

Animals↗
PubMed2006

Radioresistance of Anisakis simplex third-stage larvae and the possible role of superoxide dismutase.

The radioresistance of Anisakis simplex third-stage larvae and the possible role of sublethal radiation on superoxide dismutase (SOD) were investigated. Larvae were isolated from the viscera of the sea eel Anago anago; irradiated with 10, 100, 200, 500, or 1,000 Gy; and then given orally to rats. Worms were recovered at 16 hr postinoculation. Most larvae were found to have invaded the gastric wall, omentum, and abdominal cavity, suggesting that their viability and infectivity were not controlled by irradiation with the doses used. To determine the relationship between SOD activities in parasites and their radiosensitivities, the larvae of A. simplex and the metacercariae of Neodiplostomum seoulense (a radiosensitive control) were irradiated with 0, 30, 100, or 500 Gy, and parasite SOD levels were measured. In nonirradiated A. simplex larvae, the average SOD level was 38.9 U/mg, and this increased to 51.3 U/mg at 500 Gy. However, at all radiation doses applied, SOD activities of N. seoulense metacercariae were significantly (P < 0.05) lower than those of A. simplex larvae. Our results demonstrate that A. simplex third-stage larvae are radioresistant, and suggest that SOD plays a role in this radioresistance.

Animals↗
PubMed2001

Fitness of the marine parasitic nematode Anisakis simplex s. str. in temperate waters of the NE Atlantic.

In temperate waters of the NE Atlantic, third-stage larvae of Anisakis simplex sensu stricto collected from 3 paratenic host species were identified by restriction fragment length polymorphisms. The condition of wild larval infrapopulations was assessed by examining morphometric and growth characteristics. The differentiation patterns and the excretory/secretory products of larvae grown in vitro were also examined. An extensive morphometric, growth and differentiation variability was found between parasite larvae collected from different paratenic host sources. Nematode infrapopulation larvae from the squid comprise those smaller individuals with the lowest values of survival rates and moult success. It may be then concluded that the fitness of A. simplex s. str. larvae is not the best possible in the squid, which impaired the competitiveness of the parasite and its chances of developing into an adult. This suggests that the microenvironments impaired by the paratenic host may provide larval infrapopulations with unique ecological factors probably influencing its recruitment to the final host populations.

Animals↗