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PubMed2006

Anisakis simplex: CO(2)-fixing enzymes and development throughout the in vitro cultivation from third larval stage to adult.

We studied the effect of CO(2) on the in vitro cultivation of Anisakis simplex, an aquatic parasitic nematode of cetaceans (final hosts) and fish, squid, crustaceans and other invertebrates (intermediate/paratenic hosts), and, occasionally, of man (accidental host). The results showed that a high pCO(2), at a suitable temperature, is vital for the optimum development of these nematodes, at least from the third larval stage (L3) to adult. After 30 days cultivation in air, molting to L4 (fourth larval stage) was reduced to 1/3, while survival was about 1/3 of that when cultivated in air + 5% CO(2). The activity of the CO(2)-fixing enzymes, PEPCK and PEPC, was also studied. Throughout the development of the worms studied, PEPCK activity was much higher than that of PEPC (e.g., 305 vs. 6.8 nmol/min.mg protein, respectively, in L3 collected from the host fish). The activity of these enzymes in the worms cultivated in air + 5% CO(2) was highest during M3, and was also generally higher than that of those cultivated in air only, especially during molting from L3 to L4 (e.g., in recently molted L4, PEPCK activity was 3.7 times greater than that of PEPC 2.9 times greater than when cultivated in air).

Animals↗
PubMed2006

Anisakis simplex: the activity of larval products on the complement system.

The effects of the larval products (crude extract and excretory-secretory) of Anisakis simplex on the classical and alternative pathways of human complement system were investigated. This could constitute a mechanism to evade host defences, similarly than in other parasitic diseases. The larval products showed a stronger effect on the classical pathway than on the alternative pathway. The most pronounced modulating effects were found for the excretory-secretory products. Chelation of bivalent cations (Ca(2+) or Mg(2+)) by these larval products may be responsible for their mode of action on the alternative pathway, whereas the chelation is not likely to be particularly involved in the anticomplementary activity found on the classical pathway. Detailed studies revealed that the larval products of A. simplex act at the level of the C3 and other complement components. Heating the crude parasite extract led to a notable loss of haemolysis inhibition activity, and the addition of PMSF (a serine protease inhibitor) also cause variation in the activity of the crude extract.

Animals↗
PubMed2005

The complete mitochondrial genome of Anisakis simplex (Ascaridida: Nematoda) and phylogenetic implications.

We determined the nucleotide sequence of the complete mitochondrial genome of the nematode species Anisakis simplex. The genome is circular, 13,916 bp in size and conforms to the general characteristics of nematode mitochondrial DNAs. The gene arrangement of A. simplex is the same as that of Ascaris suum and almost identical to those of rhabditid species with a minor exception concerning the relative position of the AT-rich and non-coding regions and radically different from those of spirurid species. Along with comparisons of gene arrangement, phylogenetic analyses (maximum parsimony, neighbour joining and maximum likelihood methods) based on concatenated amino acid sequences of 12 protein-coding genes from 13 nematode species provided strong support for the sister-group relationship between Ascaridida and Rhabditida. The Shimodaira-Hasegawa and Templeton's tests both rejected the alternative hypothesis of a closer relationship between Ascaridida and Spirurida. These results contradicted the traditional view of nematode classification and a recent molecular phylogenetic study of 18S rDNA data that assigned Ascaridida and Spirurida as being a sister-group. Mapping of gene arrangement across the phylogenetic tree lead to the assumption that the conserved gene arrangement found in Ascaridida-Rhabditida members might have been acquired after the most recent common ancestor of ascaridid/rhabditid members branched off from the basal stock of the rhabditid lineage.

Amino Acid Sequence↗
PubMed2004

Purification and molecular cloning of a major allergen from Anisakis simplex.

A heat-stable allergen with a molecular weight of 21 k was purified from larvae of the nematode Anisakis simplex by gel filtration, anion-exchange FPLC and reverse-phase HPLC. When analyzed by immunoblotting and ELISA, seven of eight patient sera reacted to the 21 k allergen, demonstrating that this protein is a major allergen of A. simplex. A full-length cDNA encoding the 21 k allergen was cloned by a combination of 3'RACE and screening of an expression library with DIG-labeled DNA probes. The precursor of the 21 k allergen was judged to be composed of a signal peptide (23 residues) and a mature protein (171 residues). As compared to the N-terminal amino acid sequence (up to the 17th residue) of Ani s 1 previously identified as the major allergen, the 21 k allergen has only one replacement, suggesting that the 21 k allergen belongs to the same protein family of Ani s 1. Although the 21 k allergen was found to have 30-40% sequence identity with Kunitz-type trypsin inhibitor domain containing hypothetical proteins of Caenorhabditis elegans, it lacked inhibitory activity against trypsin. The 21 k allergen was successfully expressed in Escherichia coli as a GST-fusion protein showing reactivity with IgE in patient sera.

Allergens↗
PubMed2005

In vivo larvicidal activity of monoterpenic derivatives from aromatic plants against L3 larvae of Anisakis simplex s.l.

In view of the lack of an effective pharmacological treatment against human anisakiosis, a disease produced by L(3) larvae of the genus Anisakis present in raw fish, we studied the in vivo larvicidal effect of certain monoterpenic derivatives against L(3) of A. simplex s.l. The aldehydic monoterpene citral and the alcoholic citronellol, when they are administered together to the larvae of the nematode at the concentration of 46.90 mg/0.5 ml in olive oil, achieve 85.90% and 67.53% dead L(3), respectively, and also stop rats suffering gastrointestinal hemorrhages produced by the larvae.

Acyclic Monoterpenes↗
PubMed2003

Cross-reactivity between Anisakis simplex sensitization and visceral larva migrans by Toxocara canis.

The aim of this work was to study cross-reactivity in the diagnosis of two related ascaridosis. Nineteen patients diagnosed with recidivous acute urticaria (RAU) caused by Anisakis simplex and 26 patients diagnosed with visceral larva migrans (VLM) caused by Toxocara canis were studied employing commercial diagnostic kits and "in house" assay kits. Cross-reactivity observed was greater when using "in house" assay kits, suggesting that T. canis excretory-secretory antigens were not only recognized by antibodies from patients with RAU but with greater intensity compared to the A. simplex excretory-secretory antigens.

Animals↗
PubMed1996

Laboratory determinations in Anisakis simplex allergy.

BACKGROUND: Anaphylactic reactions caused by the fish nematode, Anisakis simplex, after ingestion of parasitized fish, have been described. OBJECTIVE: This study was undertaken to confirm, by histamine release tests, that A. simplex is able to trigger IgE-mediated reactions and to describe the serologic profiles in this sensitization. METHODS: Twelve patients who had anaphylactic symptoms after ingestion of cooked fish and positive prick test results and determinations of IgE to A. simplex were studied by indirect IgG ELISA and IgG and IgE immunoblotting. Sera from subjects parasitized with other nematodes, patients with fish allergy, and healthy donors were included as controls. A histamine release test was performed in a representative case. RESULTS: IgE immunoblotting was a specific test to detect A. simplex allergy. IgE-reacting bands were found in serum samples from 11 of our patients. Specific IgG antibodies were found by ELISA and immunoblotting, but this response was less specific. Histamine release was positive with A. simplex extract and negative with fish. CONCLUSION: A specific and intense immune response to an A. simplex extract was found in our patients. A. simplex is able to elicit anaphylactic reactions, and A. simplex allergy should be suspected in patients with allergic symptoms after ingestion of fish. A positive prick test response to A. simplex and a negative response to fish is a good indication for a diagnosis of A. simplex allergy.

Adult↗
PubMed1997

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 (non-allergic), 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.

Acute Disease↗
PubMed2000

Molecular cloning and characterization of an IgE-reactive protein from Anisakis simplex: Ani s 1.

Ingestion of the parasitic nematode Anisakis simplex in undercooked fish can cause severe allergic reactions in some individuals. Using pooled human sera from sensitized patients we have probed an expression library for A. simplex antigens. One positive clone was found to encode a full length 21 kDa protein with strong homology to nematode troponins. The recombinant protein was expressed as a GST-fusion protein and found by immunoblot analysis to react with sera from 20% of allergic patients. The presence of functional EF-hand Ca(2+) binding motifs was demonstrated by gel-shift analysis.

Allergens↗
PubMedDate not supplied

Dietary recommendations for patients allergic to Anisakis simplex.

BACKGROUND: Anisakis simplex, a fish parasite, causes allergic urticaria, angioedema and anaphylactic shock through an IgE-mediated hypersensitivity mechanism. Consensus on the dietary recommendations that should be given to allergic patients is lacking. Our objective was to evaluate the usefulness of different types of diets in preventing further reactions in patients allergic to A. simplex. METHODS: Twenty-eight adult patients, who had suffered an allergic episode caused by A. simplex were asked to follow one of the following three diets for a mean period of 13.16 months: a fish-free diet (diet 1; n = 19), a diet including fish frozen for more than 48 hours (diet 2; n = 9) and a diet with fresh fish (diet 3; n = 12). In all patients raw fish was excluded. Relapse rates and changes in total serum IgE and specific IgE to A. simplex were studied during the follow up. RESULTS: During the 13-month follow-up none of the patients developed anaphylaxis. Urticaria symptoms were present in 5.8 %, 11.1 % (n.s) and 33.3 % (p = 0.016) of patients following diets 1, 2 and 3, respectively. Total IgE decreased by 64 % (p < 0.05), 48 % (p < 0.05) and 39.4 % (p < 0.05), respectively. Specific IgE to A. simplex decreased by 50.7 % (p < 0.05), 54.1 % (p < 0.05) and 23.6 % (p < 0.05) after diets 1, 2 and 3, respectively. No statistically significant differences were found among the groups in variations in total and specific IgE. CONCLUSIONS: Patients allergic to A. simplex can eat fish that has been frozen at -20 C for 48 hours without risk of a severe allergic reaction. Long term decreases in specific and total IgE may not be good markers of eventual contact with A. simplex.

Adult↗
PubMedDate not supplied

[Anisakis and anisakiosis].

The nematode Anisakis simplex (AS) is a world wide distributed parasite that infects consumers of raw or undercoocked parasitized fish. The clinical manifestation of Anisakiosis depend on the site in the digestive tract in which larva lodges. The symptoms develops as a result of the inflammation when the larvae penetrates the gastric mucous. Most of asymptomatic subjects show high levels of specific IgE to AS. Diagnosis of AS allergy is not simple, due to cross-reactivity with other allergens. In childhood is more difficult to make a right diagnosis than in adult population. Most of positive prick test to AS correspond to children with positive prick tests to other allergens. Cross-reactivity between this parasite and other parasites with a higher prevalence in childhood, is the cause of a false diagnosis. The secretor-excretory antigen shows a better specificity, recognizing the true parasitized patients. This antigen could be used as indicator of parasitization. To follow prevention rules AS, avoid consumption of raw fish unless frozen for 48 hours or ingestion of fresh fish always cooked for more than 20 minutes at least at 60 C.

Animals↗
PubMed2003

Gingivostomatitis after eating fish parasitized by Anisakis simplex: a case report.

Anisakis simplex (AS) is a nematode that may be encountered as a parasite in various kinds of seafood. Human beings may accidentally acquire AS larvae by eating raw or undercooked seafood. In addition to human parasitization (anisakiasis), this nematode can induce allergic reactions. AS-related diseases are frequent, especially in those countries with a high level of fish consumption and with traditions of eating raw or undercooked seafood. To our knowledge, this is the first report of gingivostomatitis secondary to the ingestion of fish with AS parasites.

Animals↗
PubMed2001

Anisakis simplex-sensitized patients: should fish be excluded from their diet?

BACKGROUND: Anisakis simplex (A.s.) allergy is an emerging disease. The third-stage larvae of this nematode are a source of hidden allergens in fish. There are no clear guidelines concerning dietary restrictions for patients with serum-specific IgE to this parasite. OBJECTIVE: To follow up the clinical data and immunological parameters of patients sensitized to A.s. during 6 to 23 months. METHODS: The clinical symptoms and serologic status of 17 patients with specific IgE and positive skin prick test results to A.s. were studied prospectively. Six of these had anaphylaxis (ANA) attributed to A.s. and 11 patients experienced concomitant chronic urticaria (CU). All patients were advised not to eat fish for 6 months. RESULTS: Four patients from the ANA group excluded fish, and ANA did not recur. Two other patients with ANA refused to exclude fish; one remained free of symptoms and the other experienced several urticarial episodes. During this 6-month period total IgE levels decreased in all six ANA patients; specific IgE for A.s. decreased in four patients and increased in two. Two patients from the CU group did not exclude fish, and symptoms persisted in these two patients. Clinical improvement was observed in 78% of the patients with CU who excluded fish. Total and specific IgE levels decreased in all the patients with CU. CONCLUSIONS: Because ANA symptoms are very severe, patients should always be advised to exclude fish until specific food allergens are identified. However, in patients with CU and specific IgE to A.s., only the clinical response to fish ingestion will determine the need for strict fish avoidance.

Adult↗
PubMed2001

Element concentration variability in the whaleworm Anisakis simplex s.l.

Concentrations of the elements C, N, Fe, Na, K, Ca and Mg were analysed by chromatography and atomic absorption spectrometry in the whaleworm Anisakis simplex sensu lato. Nearly all mean inorganic element concentrations measured were higher in adult worms than in larval forms. Similarly, adults contained significantly higher nitrogen (i.e. lower C/N ratio values) than either eggs or larvae. Evidence for the significance of the parasitic life cycle stage and the systematic position of their hosts on the element concentration variability is discussed.

Animals↗
PubMed2002

Anisakis simplex: dangerous--dead and alive?

The risk of infection with Anisakis simplex and related parasites of fish has been recognized for some time, but it is now emerging that ingestion of material from dead parasites in food is also potentially dangerous. The resulting allergic reactions range from rapid onset and potentially lethal anaphylactic reactions to chronic, debilitating conditions. This review discusses the problems and clinical implications associated with A. simplex, other related conditions, and the way in which disease manifestations vary from person to person.

Adolescent↗
PubMed1997

Genomic identification of Anisakis simplex isolates.

RAPD technique was used to differentiate individuals of Anisakis simplex obtained from Merluccius merluccius, Phycis blennoides, Conger conger and Lepidorhombus boscii, from the North Atlantic Ocean. The amplification patterns of the host DNA controls were markedly different from those obtained for the parasitic material. No variation within the same host was detected. The amplification patterns for larvae obtained from fish of the same genus were somewhat different. The amplification patterns of A. simplex isolates from M. merluccius, P. blennoides, C. conger and L. boscii, were different. These results suggest the possible existence of two populations with a considerable high genetic variability and a different adaptation to different host species.

Animals↗
PubMed1992

Molecular cloning of the cDNA encoding a 42 kDa antigenic polypeptide of Anisakis simplex larvae.

The gene encoding an antigenic polypeptide of Anisakis simplex larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from A. simplex larvae was ligated into phage vector lambda gtll DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gtll expression library was constructed. A cDNA clone encoding a 42 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing cDNA for a 42 kDa protein was isolated. The gene encoding this 42 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 42 kDa polypeptide. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.

Amino Acid Sequence↗
PubMed1995

Anisakis simplex: stage-specific antigens recognized by mice.

To investigate antigenic differences between the developmental stages of Anisakis simplex, somatic, excretion-secretion and detergent-solubilized surface antigen preparations of third- and fourth-stage A. simplex larvae, and a somatic antigen preparation of adults, were characterized by SDS-PAGE and immunoblotting using mouse immune serum containing antibodies against the two larval stages. Excretion-secretion and surface antigen preparations from third-stage larvae behaved very differently from those of fourth-stage larvae, in both SDS-PAGE and immunoblotting; this suggests that certain excretion-secretion and surface antigens are highly stage-specific. By contrast, somatic components of third- and fourth-stage larvae were found to have very similar banding patterns, suggesting conservation of these components during the development of the parasite in mice. The SDS-PAGE and immunoblotting results for adult somatic components seem to support this hypothesis.

Animals↗