M. F. Álvarez

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.

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.

Parasite community study of the undulate ray Raja undulata in the Ría of Muros (Galicia, northwest Spain)

Abstract The parasites present in 75 undulate rays (Raja undulata Lacepede, 1802) captured in the Ria of Muros, a coastal embayment (ria) in northwest Spain were studied. A total of five species of cestodes (Onchobothrium uncinatum, Phyllobothrium lactuca, Echeneibothrium beauchampi, Acanthobothrium benedeni and Grillotia sp.), three species of nematodes (Schulmanela [Piscicapillaria] sp., Pseudanisakis rotundata and Cystidicolidae gen. sp.) and one acanthocephalan (Acanthocephaloides propinquus) were detected. All species were elasmobranch-specific except Cystidicolidae sp. and Acan. propinquus, which can both be considered accidental in this host. Species diversity peaked over the period April–September (Shannon–Wiener diversity=1.9–2.2; Pielou evenness=0.8–0.9). The dominant species were cestodes, notably O. uncinatum (present in 73 of the 75 hosts, and accounting for 45% of all parasites detected), followed by A. benedeni, P. lactuca and Grillotia sp., all of which were present in more than 30% of the host individuals. All species detected were autogenous, and all individuals except those of Cystidicolidae gen. sp. were adults. The five species of cestodes were present in hosts of all sizes, though single-host species richness, single-host species diversity and overall intensity of infection increased with increasing host size. Nematodes appeared in larger individuals (body length >33 cm). Larval Cystidicolidae gen. sp. appeared only in the largest size class (>43 cm), probably because only rays of this size are capable of ingesting relatively large demersal fish, the usual intermediate hosts in this area. Analysis of the species compositions of single-host parasite communities, considering cestodes only, suggests that negative interspecific interactions do not occur, despite the typically large size and abundance of the parasites, and their spatial concentration in the spiral valve.