J. Leiro

Antigenic cross-reactivity in mice between third-stage larvae of Anisakis simplex and other nematodes

Abstract We used ELISA and immunoblotting to investigate antigenic cross-reactivity in mice between third-stage larvae of Anisakis simplex and five other nematodes: the ascaridoids Ascaris suum, Toxocara canis and Hysterothylacium aduncum, and the nonascaridoids Trichinella spiralis and Trichuris muris. Two sera were raised against each species (including A. simplex, but excluding A. suum), by infection or by immunization with somatic antigens. Serum against A. suum was raised by immunization only. The reactivities of each serum with A. simplex somatic antigens (SA), excretion-secretion antigens (ES), pseudocoelomic fluid antigens (PF) and cuticular antigens (CA) were investigated. The results of ELISA indicated high antigenic cross-reactivity between A. simplex and the remaining ascaridoid nematodes, confirming that there is extensive antigenic similarity within this group of nematode parasites. Immunoblotting again confirmed the high degree of cross-reactivity between the SA of A. simplex and SAs of the other ascaridoids, although several A. simplex SA components in the 11–18 kDA range were only recognized by sera from mice infected with A. simplex. In addition, two A. simplex PF components of 22 and 27 kDA, were recognized only by sera from mice infected with, or immunized with the SA of, A. simplex. Finally, the anti-phosphorylcholine monoclonal antibody BH8 recognized only a small number of A. simplex antigens, indicating that phosphorylcholine epitopes are not significant contributors to the observed cross-reactivity with the other nematodes.

Philasterides dicentrarchi (Ciliophora: Scuticociliatida) expresses surface immobilization antigens that probably induce protective immune responses in turbot.

Philasterides dicentrarchi is a histophagous ciliate causing systemic scuticociliatosis in cultured turbot. This study demonstrates that turbot which survive this disease have serum antibodies that recognize ciliary antigens of this ciliate in ELISA and immobilize/agglutinate the ciliate in vitro. Mouse sera raised against ciliary antigens and integral membrane proteins are likewise capable of immobilizing/agglutinating the ciliates, indicating that P. dicentrarchi, like other ciliates, expresses surface immobilization antigens. Furthermore, the antigen agglutinating reaction induces the parasite to shed its surface antigens rapidly, replacing them with others with different specific serology. This antigen shedding and variation response is similar to that detected in other protozoan parasites. Immunization of turbot with ciliate lysate plus adjuvant or with formalin-fixed ciliates induced synthesis of agglutinating antibodies and conferred a degree of protection against challenge infection, suggesting that the response to surface antigens may play an important role in defence against this pathogen, SDS-PAGE and immunoblotting studies indicated the existence of a predominant polypeptide of about 38 kDa in the ciliary antigen and membrane protein fractions, and this may be the principal surface antigen of P. dicentrarchi.

Philasterides dicentrarchi (Ciliophora, Scuticociliatida): experimental infection and possible routes of entry in farmed turbot (Scophthalmus maximus)

Philasterides dicentrarchi is a histophagous ciliate infecting turbot (Scophthalmus maximus) in aquaculture, where it causes significant economic losses. However, the route of entry of these parasites in turbot is not known. In the present study, we attempted experimental infection by seven routes: nasal, oral, rectal, branchial/dermal (i.e. by immersion with or without previous abrasion of the gills and opercula), intraperitoneal, periorbital and intramuscular. Our results indicate that the natural route of infection is probably through lesions in the gills and/or skin, thus, assays which aim to mimic natural infection conditions should probably use immersion following branchial/dermal abrasion. However, in chemotherapeutic assays, where it is necessary to obtain severely infected fish in a fast and reliable manner, the most appropriate route for experimental infection is probably the intraperitoneal route.

Monoclonal antibodies to turbot (Scophthalmus maximus) immunoglobulins: characterization and applicability in immunoassays

Five monoclonal antibodies (mAbs) to immunoglobulins (Igs) of the turbot Scophthalmus maximus were produced and characterized. All the mAbs (denominated UR1, UR3, UR4, UR6 and UR7) are of isotype IgG1/kappa and show good anti-turbot Ig reactivity in enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results of competitive ELISA and immunoblotting analysis indicate that these five mAbs react with at least three different epitopes on the turbot Ig H chain. Except in the case of UR1, reactivity with periodate-treated purified turbot Ig was much lower than with the untreated Ig, suggesting that carbohydrate residues are involved in epitope recognition. All the mAbs showed reactivity with sera from the closely related species Scophthalmus rhombus but not with sera from species of other flatfish genera. One of these mAbs (UR3) has been successfully applied for the detection of antibodies against Vibrio anguillarum in ELISA.

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.

In vitro growth requirements for the fish pathogen Philasterides dicentrarchi (Ciliophora, Scuticociliatida)

Philasterides dicentrarchi is a scuticociliate causing fatal disease in farmed turbot and sea bass. In view of its high virulence and endoparasitic location, this parasite cannot be effectively controlled by formalin baths, and no systemic chemotherapeutic treatments have yet proved effective; immunoprophylaxis may thus be an attractive alternative approach. Since vaccine development is greatly facilitated by axenic culture of the pathogen, we have developed a simple axenic culture system based on commercially available Leibovitz L-15 medium, supplemented with fetal bovine serum, lipids (lecithin and Tween 80), nucleosides and glucose. After 1 weeks culture under optimal conditions (salinity 10 per thousand, pH 7.2, temperature between 18 and 23 degrees C), yields of 1-2 x 10(5)cells/ml were obtained. Even cultures with seeding densities as low as 20 cells/ml were found to produce a good yield of ciliates (about 6 x 10(4)cells/ml) after 11 days of incubation. The ciliates thus obtained were free of contamination by other microorganisms, enabling preparation of pure P. dicentrarchi antigens for vaccine development studies.

Cysteine proteinase activities in the fish pathogen Philasterides dicentrarchi (Ciliophora: Scuticociliatida).

This study investigated protease activities in a crude extract and in vitro excretion/secretion (E/S) products of Philasterides dicentrarchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS-PAGE analysis (pH 4, reducing conditions) detected 7 bands with gelatinolytic activity (approximate molecular weights 30-63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptide p-nitroanilide substrates, the crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-I collagen and haemoglobin in vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology of Philasterides dicentrarchi.

O-glycans as a source of cross-reactivity in determinations of human serum antibodies to Anisakis simplex antigens

Anisakis simplex is a seafood‐borne parasite that may both infect humans and cause allergy. Serodiagnosis of anisakiasis and allergy caused by this nematode is difficult since most Anisakis antigens show cross‐reactivity problems.

Helminth fauna of Falconiform and Strigiform birds of prey in Galicia, Northwest Spain

This is a survey of the helminth fauna of 285 individuals of 14 species of birds of prey (Falconiformes and Strigiformes) from Galicia (northwest Spain), namely Buteo buteo, Accipiter nisus, A. gentilis, Milvus migrans, M. milvus, Pernis apivorus, Circus pygargus, Falco tinnunculus, F. peregrinus, F. subbuteo, Tyto alba, Strix aluco, Asio otus and Athene noctua. A total of 15 helminth species were detected, namely 8 nematodes (Eucoleus dispar, Capillaria tenuissima, Synhimantus laticeps, Microtetrameres sp., Physaloptera alata, Procyrnea leptoptera, Hovorkonema variegatum and Porrocaecum angusticolle), 4 cestodes (Cladotaenia globifera, Paruterina candelabraria and Mesocestoides sp.), 2 trematodes (Neodiplostomum attenuatum and Strigea falconis), and 1 acanthocephalan (Centrorhynchus globocaudatus). The helminth communities observed were basically similar, although there were marked differences in species richness, which was higher in falconiforms (except for A. gentilis) than in strigiforms. More specifically, species richness was highest in B. buteo (13 species), followed by A. nisus (11 species). In the falconiforms, the helminth species present generally exhibited a clear relationship with host diet. In the strigiforms, by contrast, species richness was lower than expected given the host’s diet, suggesting that a different explanation is needed.

Monoclonal antibodies against diagnostic Anisakis simplex antigens

Abstract Five monoclonal antibodies (UA2, UA3, UA5, UA6, and UA8) specific for Anisakis simplex are described. All are IgG1/κ monoclonal antibodies, except for UA2, which is an antibody IgM/κ. The molecular weights of the major components recognized in immunoblotting are 48 and 67 kDa (UA2); 139 kDa (UA3 and UA5; same epitope); 35, 38, and 139 kDa (UA6); and 205 kDa (UA8). UA2 was the only monoclonal antibody to recognize both components of an excretion-secretion antigen preparation and antigens in the excretory cell and esophageal glands of third-stage A.␣simplex larvae; antigens in the excretory cell were also recognized by UA3 and UA6. Cross-reactivity studies using a hyperimmune polyclonal rabbit serum reacting with various ascaridoid nematodes indicated that the antigens captured by our monoclonal antibodies were specific for A. simplex. Finally, comparative studies of our monoclonal antibodies and An2 (the only monoclonal antibody currently available for serodiagnosis of human anisakiasis), based on the calculation of multiples of normal activity for human anisakiasis sera, indicated that our monoclonal antibodies (and particularly UA3) recognized antigens that are good candidates for serodiagnostic purposes.