A. Paramá

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.

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.

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.

Vaccination of turbot, Psetta maxima (L.), against the protozoan parasite Philasterides dicentrarchi: effects on antibody production and protection

The efficacy of a vaccine against the fish pathogen Philasterides dicentrarchi was evaluated in turbot by measuring the production of specific antibodies and duration of protection. Four groups of turbot were vaccinated twice, on days 0 and 30, with phosphate-buffered saline, mineral oil adjuvant, antigen or antigen plus adjuvant. Specific serum antibodies were determined on day 0 and 1 month after the first and the second vaccinations. Protection was evaluated 1 month after the first vaccination and 1 and 5 months after the second vaccination. Serum antibody titres, measured by enzyme-linked immunosorbent assay, and protection, assessed by challenges, increased significantly 1 month after the second vaccination in the group injected with antigen plus adjuvant and the protection lasted for at least a further 5 months in this group. The relative protection was 77% and 66% 1 and 5 months after the second vaccination, respectively. Administration of antigen or adjuvant separately had no effect on antibody response or protection. The results indicate that emulsion containing antigen plus adjuvant induced durable protection against P. dicentrarchi after the administration of the two vaccinations, and that this preparation can be used as a vaccine against the pathogen.

Ultrastructure and phylogeny of Philasterides dicentrarchi (Ciliophora, Scuticociliatia) from farmed turbot in NW Spain

Several species of opportunistic histophagous scuticociliates have been implicated in systemic infections of farmed fish. In turbot, scuticociliatosis is an emerging disease, and the identification of the parasite species involved is controversial. We have previously isolated Philasterides dicentrarchi from farmed turbot scuticociliatosis outbreaks in northwest Spain. In the present study, we report detailed ultrastructural studies of this parasite, and investigate phylogenetic relations with other members of the order Philasterida on the basis of sequence comparison of the small-subunit rRNA (SSUrRNA) gene. Ultrastructural study indicates the presence of dikinetids in the anterior two-thirds of the body; micronucleus closely associated with the macronucleus, though not physically connected; numerous mitochondria located below the cell cortex, parallel to the surface; numerous spherical and fusiform extrusomes located close to the plasma membrane. We consider that these characteristics are useful for diagnosis of infections by this parasite. A nested 350-bp nucleotide sequence of the SSUrRNA gene of the turbot P. dicentrachi isolate showed high identity with previously reported SSUrRNA gene sequences from 2 scuticociliates isolated from olive flounder Paralichthys olivaceus in Korea, namely P. dicentrarchi (98%) and Miamiensis avidus (99%); conversely, our P. dicentrarchi sequence showed low identity (86%) with that of Uronema marinum, a scuticociliate that has also been implicated in scuticociliatosis outbreaks in turbot in Europe and olive flounder in Asia. Phylogenetic tree construction on the basis of the SSUrRNA gene sequences, using the neighbour-joining method, confirm that the different P. dicentrarchi isolates and M. avidus are closely related and a possible synonymy between both ciliates species should be considered.

PCR detection of Tetramicra brevifilum (Microspora) infection in turbot (Scophthalmus maximus L.) musculature

This study investigated the spatial distribution of Tetramicra brevifilum spores in the musculature of infected turbot Scophthalmus maximus, with the aim of identifying the most appropriate body locations for diagnostic assays. A PCR protocol optimized for the detection of T. brevifilum spores in turbot muscle is also described. In fish showing low- and moderate-intensity infection, the spatial distribution of spores was best fitted by a negative binomial distribution, indicating a clumped spatial pattern; the negative binomial coefficient k was lower for fish with low-intensity infection, indicating a more markedly clumped pattern in these fish. In fish with high-intensity infection, the spatial distribution of spores was best fitted by the Poisson distribution, indicating a random pattern. In both low- and moderate-intensity infection, spores were present at highest density in the musculature adjoining the dorsal fins. Samples for PCR were therefore obtained from this location. PCR amplification was of the small subunit ribosomal DNA (SSUrDNA), using a pair of species-specific primers that amplify the 1250 bp product. The PCR protocol developed showed better sensitivity than microscopical techniques (detection rate by microscopy 25%, versus 42% by PCR), suggesting that it may be useful for routine screening for Tetramicra brevifilum infection in cultured turbot.

RFLP analysis of PCR-amplified small subunit ribosomal DNA of three fish microsporidian species.

The phylogenetic relationships of the microsporidian species Microgemma caulleryi, Pleistophora finisterrensis and Tetramicra brevifilum were investigated on the basis of restriction fragment length polymorphism (RFLP) analysis of PCR-amplified small-subunit rDNA (SSUrDNA). Using PCR primers specific for microsporidian SSUrDNA, a single product was obtained from each species, and heteroduplex analysis indicated a high degree of sequence homology among the 3 products. In RFLP analysis of the PCR-amplified SSUrDNA, the enzymes AluI and DdeI gave restriction patterns that differed among all 3 species. Phylogenetic analysis using restriction patterns as differential characters indicated that Microgemma caulleryi and Tetramicra brevifilum are more closely related to each other than to Pleistophora finisterrensis.

DNA probes for detection of the fish microsporidians Microgemma caulleryi and Tetramicra brevifilum

DNA probes were developed for the detection and identification of 2 microsporidian parasites of marine fishes, Microgemma caulleryi (infecting the liver of the greater sand-eel, Hyperoplus lanceolatus) and Tetramicra brevifilum (infecting muscle, intestine and liver of the turbot, Scophthalmus maximus, a commercially important species). The probe-development procedure used is fast and straightforward, and readily applicable to the development of probes for other microsporidian species. First, genomic DNA of microsporidian spores was isolated and digested with the restriction enzyme Hind III. The fragments obtained were ligated into the vector pBluescript SK(+) and cloned in Escherichia coli. Appropriate inserts were identified and then amplified by PCR, using primers specific for regions adjacent to the Hind III restriction site in the vector sequence (and thus avoiding the need to develop primers specific for the inserts themselves). The copies were labelled with digoxigenin, for subsequent use as probes, during PCR itself. The specificity of candidate probes was tested in dot-blot hybridization assays, with the target DNA being (a) genomic DNA of the microsporidian from which the probe had been obtained, or of another species, (b) the corresponding genomic DNA in the phagemid, or (c) DNA from the corresponding host tissue. These assays identified a ca 1180 bp probe for M. caulleryi, denominated C38, and a ca 1363 bp probe for T. brevifilum, denominated F9. Similar assays designed to assess sensitivity indicated that F9 showed detectable binding to as little as 500 ng of T. brevifilum genomic DNA, and C38 to as little as 125 ng of M. caulleryi DNA; these results were obtained with detection of DIG by enzyme immunoassay (i.e. using a phosphatase-coupled anti-DIG antibody), and could no doubt be improved if a radioactive labelling and detection system were used. The probes developed in this study will greatly facilitate detection and identification of M. caulleryi and T. brevifilum in fish tissues, and may prove useful for identifying possible intermediate hosts used by these species.