Cod (Gadus morhua), an important fish species in the Baltic Sea, is the paratenic host for many parasite species, including the zoonotic nematodes, Anisakis sp. and Contracaecum osculatum. We aimed to identify which invertebrate species (found in situ in the fish stomach) are responsible for infection of cod with zoonotic nematodes. We found that Crangon crangon and Gammarus sp., both invertebrate prey species of cod, were infected with Anisakis simplex and C. osculatum, respectively. These host–parasite systems are reported here for the first time, implicating C. crangon and Gammarus sp. as sources of infection of Baltic cod with zoonotic nematodes.

Third-stage larvae of Pseudoterranova decipiens commonly infect whitefish such as cod, and the parasite can be transferred to humans through lightly prepared (sushi) meals. Because little is known about the nematode's cold tolerance capacity, we examined the nematode's ability to supercool, and whether or not cold acclimation could induce physiological changes that might increase its ability to tolerate freezing conditions. Even if third-stage Pseudoterranova decipiens larvae have some supercooling ability, they show no potential for freezing avoidance because they are not able to withstand inoculative freezing. Still, they have the ability to survive freezing at high subzero temperatures, something which suggests that these nematodes have a moderate freeze tolerance. We also show that acclimation to high temperatures triggers trehalose accumulation to an even greater extent than cold acclimation. Trehalose is a potential cryoprotectant which has been shown to play a vital role in the freeze tolerance of nematodes. We suggest that the trehalose accumulation observed for the cold acclimation is a general response to thermal stress, and that the nematode's moderate freeze tolerance may be acquired through adaptation to heat rather than coldness.

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 λ gtll DNA and packaged in vitro. The phages were propagated on Escherichia coli and a λ 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 β-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.

The relationships between larvae and adults of Anisakis from the Mediterranean Sea and North-East Atlantic Ocean were analysed by multilocus electrophoresis. The correspondence of type I larvae with the A. simplex complex, including the sibling species A. simplex A and B, and of type II larvae with A. physeteris is confirmed. 19 of the 22 loci studied discriminated between the two larval types. Biochemical keys are given for the electrophoretic identification of A. simplex A, A. simplex B and A. physeteris, at both the larval and adult stages.

To increase our understanding of the immune response to Anisakis infection, antigen specific IgG, IgA and IgE responses were identified using an immunoblot technique after polyacrylamide gel electrophoresis of excretory-secretory products from the larval stage of Anisakis simplex. Nine sera were drawn from proven cases of gastric anisakiasis within 3 days after symptoms had developed. The molecular weight of the major antigenic bands were distributed between 50 kDa and 120 kDa of the antigens. In nine cases of gastric anisakiasis, three of them were positive for IgG response, five for IgE, and six for IgA, respectively. None of control sera recognized the antigenic bands in IgA and IgE responses. In contrast, two controls had IgG antibodies against 1–2 proteins in the 65–95 kDa region. The antigenicity of the excretory–secretory products was lost following treatment by 0·2% trypsin, but not by 0·2 M periodic acid. Based on the results of reactivity to lectins, antigenic bands of the ES products possessed mucin type glycoconjugate residues in their protein portion. This indicates that the humoral responses of IgA and IgE antibodies to the larval ES antigens are a more reliable index of infection than that of the IgG response.

Anisakis simplex larvae were cultured in vitro in medium containing 35-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 immunocomplexes. 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.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1·8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

This paper describes the in vitro cultivation of the 3rd-larval stage (L3) of Anisakis simplex to adulthood in a much simpler and easier to prepare medium than those described to date. The adult males obtained are between 3.8 and 6.5 cm long and the females between 4.5 and 8.0 cm. Some individually cultivated females laid eggs which had an average size of 44.4×50.5 μm. The culture conditions were as follows: medium RPMI-1640 supplemented with 20% heat-inactivated fetal bovine serum and 1% commercial pepsin, at pH 4.0 and a temperature of 37 °C, and in air atmosphere with 5% CO2. The pepsin was found to be the key to the success of the culture. The average survival of the worms in the culture increased from 50 to 88 days, due to the fact that the survival of the adults practically doubled (increasing by 1.9 times). Furthermore, the number of worms that completed the 4th moulting (M4) increased by 4.2 times, from 22.9 to 95.6%. This culture medium may facilitate, due to its simplicity, the study of anisakids, or at least of A. simplex, constituting another step towards achieving a complete in vitro life-cycle for these parasites.