R. Iglesias

Piperazine N-substituted naphthyridines, pyridothienopyrimidines and pyridothienotriazines: new antiprotozoals active against Philasterides dicentrarchi

New antiprotozoals active against Philasterides dicentrarchi, the causative agent of scuticociliatosis in farmed turbot and Black Sea bass-bream, have been synthesised and tested. The most active compounds posses a piperazine ring, generally N-bonded to the heterocycle, and are the 1,8-naphthyridines, 2f and 5o, the pyridothienopyrimidine (7), and the pyridothienotriazines, 8, 9, 12d, 12f, 12h, 12m and 12k. Pyridothienotriazine (12k) presents the same activity (Lethal Dose, LD=0.8/1.5 mg L(-1)) as the well-known antiparasitics niclosamide and oxyclozanide.

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.

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.

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.

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.

Molecular analysis of antigens targeted by protective antibodies in rapid expulsion of Trichinella spiralis

Rapid expulsion is a protective immune mechanism which eliminates as much as 99% of a challenge infection of Trichinella spiralis muscle larvae from the gastrointestinal tract of suckling rats. Protective monoclonal antibodies (mAbs) generated against larval excretory-secretory antigens (ESA) specifically recognize a 45-kDa glycoprotein, gp45, in addition to a distinct profile of other cross-reactive antigens that are also recognized by non-protective mAbs. Recent data indicate that protective mAbs recognize carbohydrate epitopes. To complement biochemical studies on the target(s) of rapid expulsion, we describe here the cloning and characterization of the cDNA, TspE1, which belongs to a multigene family and encodes several larval proteins in the 40-50-kDa range. A second cDNA, TspM6 encodes a 45-kDa antigen and is homologous to the published sequence of gp45. Anti-TspE1 antibodies detected antigens within beta- and gamma-stichocytes while anti-TspM6 antibodies detected antigens within alpha-stichocytes of the secretory organs of muscle larvae. Sequence analysis has provided no functional information on the encoded gene products. Neither recombinant antigen is recognized by the mAbs but native parasite molecules with peptide homology to both the TspE1 and TspM6 recombinant antigens bear the glycan recognized by the protective mAbs. These molecules are candidate targets in rapid expulsion.

Abdominal macroparasites of commercially important flatfishes (Teleostei: Scophthalmidae, Pleuronectidae, Soleidae) in northwest Spain (ICES IXa)

Abstract This study investigated the helminth fauna of the abdominal viscera and cavity of 484 flatfishes caught in three areas off northwest Spain: the inner third of the Muros Estuary (MI), offshore from the Muros Estuary (ME) and offshore from the Arousa Estuary (AE). The following helminths were found: (a) nematodes— Cucullanus heterochrous Rudolphi, 1802, C. minutus Rudolphi, 1819, Anisakis simplex (Rudolphi, 1819), Hysterothylacium aduncum (Rudolphi, 1802), Contracaecum sp. and Capillaria gracilis Bellingham, 1840; (b) digeneans— Podocotyle atomon (Rudolphi, 1802), P. angulata (Dujardin, 1845), Macvicaria soleae (Dujardin, 1845), Derogenes varicus (Muller, 1784), Lecithochirium rufoviride (Rudolphi, 1819), L. musculus (Looss, 1907), Synaptobothrium caudiporum (Rudolphi, 1819), Zoogonus rubellus (Olsson, 1868), Zoogonoides viviparus (Olsson, 1868), Rudolphinus crucibulum (Rudolphi, 1819), Proctoeces maculatus (Looss, 1901), Homalometron galaicus (Sanmartin, Alvarez, Quinteiro et Paniagua, 1995) and Lomasoma stephanskii Dollfus, 1960; (c) cestodes—adult Bothriocephalus barbatus Renaud, Gabrion et Pasteur, 1983, B. gregarius Renaud, Gabrion et Pasteur, 1983, Bothriocephalus sp. and Bothrimonus nylandicus (Schneider, 1902), plerocercoids of Nybelinia lingualis (Cuvier, 1817), Grillotia sp. and unidentified tetraphyllidean plerocercoids; and (d) acanthocephalans— Acanthocephaloides propinquus (Dujardin, 1845). Most of these helminths were found in more than one host species and many at more than one capture site. Of the studied hosts, Microchirus variegatus and Platichthys flesus had the richest helminth communities with 13 species. Within each capture area, the helminth faunas of the various host species were similar except P. flesus (Linnaeus), which is infected by five helminth species not present in any other host ( Z. rubellus , R. crucibulum , C. minutus , Contracaecum sp. and C. gracilis ). There were marked differences between the three areas (particularly between the offshore areas and MI) in the identity of the helminth species present and their abundances, so some of the parasites found may thus be useful markers of different flatfish populations in this area.

Anatomical location of phosphorylcholine and other antigens on encystedTrichinella using immunohistochemistry followed by Wheatley's trichrome stain

This work investigated the location on the parasite ofTrichinella antigens recognized by the mouse immune system and the question as to which of them bear the epitope phosphorylcholine (PC). Wheatleys trichrome stain (initially developed for faecal smears) proved to be excellent for visualization ofTrichinella structures, enabling four types of stichocyte to be distinguished. By applying this stain on infected muscle sections after immunocytochemistry using (a) anti-PC BH8 monoclonal antibodies, (b) serum from mice that had been infected twice in the presence of 0.05% thiabendazole (to prevent reproduction by adult females) and then bled on day 7 post-reinfection, (c) serum from infected mice that were bled on day 14 postinfection, or (d) serum from infected mice that were bled on day 42 postinfection, we found (1) that PC is an abundant structural epitope on the hypodermis/muscle, genital primordium and intestinal tract but is absent from the cuticle and stichosome; (2) that the principle secretory cells of adult worms are delta- and beta-stichocytes, whereas those of migrating and encysted L1 larvae are alpha-stichocytes; and (3) thatTrichinella antigens recognized in the encysted phase of the parasites life cycle are present in parasitized myofibres in the sarcoplasmic matrix and in the nucleoplasm of hypertrophic nuclei. The significance of these findings is discussed.