(Ciliophora) Parasitizing Turbot Scophthalmus Maximus: Morphology, in Vitro Culture and Virulence
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FOLIA PARASITOLOGICA 51: 177–187, 2004 Histophagous scuticociliatids (Ciliophora) parasitizing turbot Scophthalmus maximus: morphology, in vitro culture and virulence Pilar Alvarez-Pellitero1(*), Oswaldo Palenzuela1(*), Francesc Padrós2, Ariadna Sitjà-Bobadilla1, Ana Riaza3, Raquel Silva3 and Javier Arán4 1Instituto de Acuicultura Torre de la Sal (CSIC), Ribera de Cabanes, 12595 Castellón, Spain; 2 Servicio de Diagnóstico Patológico en Peces, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain; 3Stolt Sea Farm, S.A., Lira, 15292 Carnota (La Coruña), Spain; 4Luso-Hispana de Acuicultura S.L., Muelle de S. Diego, 15006 La Coruña, Spain Key words: Scuticociliatia, Ciliophora, turbot, Scophthalmus, in vitro culture, virulence, aquaculture Abstract. Systemic ciliatosis caused by histophagous ciliates constitutes a serious disease of cultured turbot. Six ciliate isolates were obtained from parasitized turbot during six epizootics at four different farms located in Spain, France and Portugal. Axenic cultures of the six isolates were obtained by periodical subculturing in ATCC 1651MA or supplemented L-15 media. In basal media or seawater, the parasites could survive starving for long periods with no apparent proliferation. In adequate media, growth kinetics was found to be very similar for isolates A and B, with a clear influence of temperature. Morphological studies demonstrated that all isolates share common features that allows their assignment to either Philasterides Kahl, 1931 or Miamiensis Thompson et Moewus, 1964. However, statistically significant differences were evident in pairwise comparisons of the isolates from the four farm sites in 16 taxonomically relevant morphometric features. This could allow the discrimination of different species or strains. Virulence of isolates A and B for healthy turbot was tested in several experiments. Differences in the virulence were especially evident after long-term in vitro culturing, isolate A being clearly attenuated after 35–42 passages, whereas isolate B became more virulent after 20–42 passages. The need of further studies to confirm such virulence variability and its implications in pathogenesis and prevention of turbot scuticociliatoses is stressed. Scuticociliates are ciliate protozoans (Ciliophora) these turbot epizootics the causative organism was occurring abundantly in eutrophic coastal and saprobic identified to specific level as Philasterides dicentrarchi maricultural waters. Some of them can behave as oppor- Dragesco, Dragesco, Coste, Gasc, Romestand, Ray- tunistic histophagous parasites causing severe infections mond et Bouix, 1995 by Iglesias et al. (2001). This in crustaceans and fish. Small (1967) proposed the new species was originally described from an episode of order Scuticociliatida including the suborders Pleuro- Mediterranean seabass (Dicentrarchus labrax) mortal- nematina, Pseudocohnilembina and Philasterina. Further ity by Dragesco et al. (1995). Since the taxonomy of revisions of the Ciliophora (Lynn and Small 2000) scuticociliates is complicated, in several cases of scuti- granted scuticociliates the level of subclass (Scutico- cociliatosis the involved parasite has not been identified ciliatia), and the suborder Philasterina was ranked as to species level (Yoshinaga and Nakazoe 1993, Dyková order (Philasterida). Since the criteria used hitherto to and Figueras 1994, Umehara et al. 2003). classify the families, genera and species of scuticocili- The frequency and severity of scuticocilatoses have ates have been variable, the taxonomy of these organ- increased in recent years in turbot cultures in different isms is currently rather confusing. countries. Main clinical signs and general histopatho- Recently, different scuticociliates have become an logy are similar in the different outbreaks, but some important threat for cultured marine fish such as tuna differences have been pointed out (Iglesias et al. 2001). and different flatfish. Fatal encephalitis of bluefin tuna, Apart from the description of the disease, the most attributed to Uronema nigricans, was reported by Mun- complete information available deals with the parasite day et al. (1997). Among flatfish, Jee et al. (2001) de- reported as Philasterides dicentrarchi by Iglesias et al. scribed scuticociliatosis of Japanese flounder, Paral- (2001) and includes data on cultivation (Iglesias et al. ichthys olivaceus. In turbot, different episodes of scuti- 2003a), antigenicity (Iglesias et al. 2003b) and invasion cociliatosis have been reported in Spain (Dyková and routes of the ciliate (Paramá et al. 2003). Figueras 1994, Iglesias et al. 2001, authors’ unpublished This paper was presented at the 6th International Symposium on Fish data) and Norway (Sterud et al. 2000). Only in one of Parasites in Bloemfontein, South Africa, 22–26 September 2003. Address for correspondence: P. Alvarez-Pellitero, Instituto de Acuicultura Torre de la Sal (CSIC), Ribera de Cabanes, 12595 Castellón, Spain. Phone: ++34 964 319 500; Fax: ++34 964 319 509; E-mail. [email protected] (*)The first two authors, P. A.-P. and O. P., share the senior authorship for this work. 177 In recent years, we have studied different outbreaks did not support ciliate multiplication but did allow bacterial of scuticociliatosis in several turbot farms from four growth if cultures were contaminated. different geographic locations. In the present paper, Growth kinetics. Ciliates from an early stationary phase preliminary morphological studies suggesting the exis- culture of isolates A and B were seeded in 12-well plates tence of different species or strains are presented. In containing 1.5 ml supplemented L-15 medium per well and addition, data on in vitro cultivation, growth kinetics incubated at two different temperatures (15 and 20ºC). For and virulence of some isolates are included. both isolates and temperatures, wells were inoculated 375 or 750 ciliates per well. The cultures were monitored daily with an inverted microscope and the number of ciliates was counted MATERIALS AND METHODS in triplicate wells at 0, 6, 24, 48, 72, 96, 120, 144 and 168 h Outbreaks studied and obtainment of ciliates post-seeding at 20ºC and at 0, 24, 72, 96, 120, 144, 168, 216, Ciliates were isolated during outbreaks in four different 264 and 366 h at 15ºC. The presence of dividing or conjugat- turbot farms located in Spain, France and Portugal, as detailed ing ciliates was also assessed. in Table 1. All the farms had a flow-through water supply of Morphological studies seawater (pump-ashore sites) or well water. The morphological study of ciliates freshly obtained from During the outbreaks in the farms, samples of ascitic fluid diseased fish was performed using conventional and inverted and brain were taken from affected fish and sent refrigerated light microscopes. The observation under the light microscope to the Institute of Aquaculture Torre de la Sal (IATS) facilities was facilitated by adding 2% MgCl in basal artificial for further processing. Upon arrival to the laboratory, fresh 2 seawater (Repak 1992). Measurements of the observable smears of the ascitic fluid or squashes of brain were examined structures were taken. In addition, ciliates from the stationary for the presence of motile ciliates. This material was used for phase of axenic cultures of the different isolates were used for morphological studies and as inoculum for in vitro culture. morphological studies using two techniques: In vitro culture Silver impregnation. Ciliates were stained using the wet Ascitic fluid containing active ciliates was directly inocu- silver nitrate method described by Foissner (1992) with some lated in different media. In some cases, ciliates were previ- modifications. Briefly, ciliates were fixed in Champy’s fluid, ously washed and concentrated by dilution of the samples with washed, and stored in Da Fano’s fluid at 4ºC until further Hanks’ Balanced Salt Solution (HBSS) containing an antibio- processing. Between the following steps, ciliates were pelleted tic/antimycotic mixture (PSA) at 1× to 3× concentration (1× by centrifugation at 14,000 × g for 2 min. After removal of the PSA = 100 U·ml-1 penicillin, 100 µg·ml-1 streptomycin, and supernatant and washing with double-distilled water, ciliates 0.25 µg·ml-1 amphotericin B), followed by centrifugation at were silver-impregnated in suspension with a 1% silver nitrate 500 × g for 15 min. This process was repeated as needed to solution for 2 min, washed again and layered on Superfrost- obtain inocula with clean and viable parasites. plus slides (Menzel-Glaser, Germany), without additives. The Culture media. A sterile, artificial saltwater (20‰ salin- aqueous suspension of silver-impregnated ciliates was irradi- ity), prepared using Instant Ocean mixture (Aquarium Sys- ated under a coverslip with a 60 W incandescent lamp at 6 cm tems, Sarreborg, France), was used as a basal medium for the for 15–30 min. Slides were air-dried and dehydrated through a initial primary cultures and for starvation experiments. Other graded ethanol series, cleared with xylene and mounted in media tested include American Type Culture Collection DPX. (ATCC) 1651MA medium and modified Leibovitz’s L-15 Scanning electron microscopy (SEM). Ciliate suspen- medium (Gibco-Invitrogen, Paisley, UK) containing 1.28% ar- sions were fixed with an equal volume of fixative (2.5% tificial marine salts (for a final salinity equivalent to approxi- glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2) at mately 20‰).