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Several experiments were designed to elucidate the modes of transmission of the myxozoan parasite Enteromyxum scophthalmi to turbot Scophthalmus maximus. Direct transmission of the infections was achieved by cohabitation of infected and test fish, through waterborne contamination from the effluent of a tank containing infected fish, and via the oral route using parasite-infected intestines. The transmission of the turbot enteromyxosis was successful in all the fish exposed to the parasite by the 3 routes; accumulated mortality reached 100% at the end of most experiments. The progress of the infections was monitored by study of the histopathology. Influence of the mode of exposure was observed, with the oral route the fastest to initiate the parasite infections. The temperature also affected the course of the infections, which were established earlier at higher water temperature. Direct fish-to-fish transmission of the disease explains the rapid spreading of the turbot enteromyxosis in farms.

Bacteria inhibitory to fish larval pathogenic bacteria were isolated from two turbot larva rearing farms over a 1-year period. Samples were taken from the rearing site, e.g., tank walls, water, and feed for larvae, and bacteria with antagonistic activity against Vibrio anguillarum were isolated using a replica plating assay. Approximately 19,000 colonies were replica plated from marine agar plates, and 341 strains were isolated from colonies causing clearing zones in a layer of V. anguillarum. When tested in a well diffusion agar assay, 173 strains retained the antibacterial activity against V. anguillarum and Vibrio splendidus. Biochemical tests identified 132 strains as Roseobacter spp. and 31 as Vibrionaceae strains. Partial sequencing of the 16S rRNA gene of three strains confirmed the identification as Roseobacter gallaeciensis. Roseobacter spp. were especially isolated in the spring and early summer months. Subtyping of the 132 Roseobacter spp. strains by randomly amplified polymorphic DNA with two primers revealed that the strains formed a very homogeneous group. Hence, it appears that the same subtype was present at both fish farms and persisted during the 1-year survey. This indicates either a common, regular source of the subtype or the possibility that a particular subtype has established itself in some areas of the fish farm. Thirty-one antagonists were identified as Vibrio spp., and 18 of these were V. anguillarum but not serotype O1 or O2. Roseobacter spp. strains were, in particular, isolated from the larval tank walls, and it may be possible to establish an antagonistic, beneficial microflora in the rearing environment of turbot larvae and thereby limit survival of pathogenic bacteria.

The present report describes an intestinal disease which causes important losses in farmed turbot. Mortality rates were higher in summer and reached 100% in all tanks where the disease was confirmed. Affected fish showed external signs consisting of anorexia, sunken eyes and a typical prominent bony ridge on the skull. These signs can be considered the pathognomonic signs of the disease, together with the gut lesions seen in the histological study. Pallor of the internal organs, intestinal haemorrhages and the presence of liquid in the intestine were also observed, with ascites in heavily infected fish. Histopathological damage was evident in the gut, with severe enteritis, detachment of epithelium, haemorrhages and inflammation of the subepithelial connective tissue. The myxosporean aetiology was demonstrated in all the fish showing the characteristic signs of disease. Myxosporean stages, including scarce spores, were found in the affected epithelium or free in the intestinal lumen together with epithelial debris. The present study demonstrates the importance of this myxosporean disease which represents a serious threat for turbot culture. This is also the first record of a member of the Myxosporea in turbot.

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) occurring abundantly in eutrophic coastal and saprobic maricultural waters. Some of them can behave as opportunistic histophagous parasites causing severe infections in crustaceans and fish. Small (1967) proposed the new order Scuticociliatida including the suborders Pleuronematina, Pseudocohnilembina and Philasterina. Further revisions of the Ciliophora (Lynn and Small 2000) granted scuticociliates the level of subclass (Scuticociliatia), and the suborder Philasterina was ranked as order (Philasterida). Since the criteria used hitherto to classify the families, genera and species of scuticociliates have been variable, the taxonomy of these organisms is currently rather confusing.Recently, different scuticociliates have become an important threat for cultured marine fish such as tuna and different flatfish. Fatal encephalitis of bluefin tuna, attributed to Uronema nigricans, was reported by Munday et al. (1997). Among flatfish, Jee et al. (2001) described scuticociliatosis of Japanese flounder, Paralichthys olivaceus. In turbot, different episodes of scuticociliatosis have been reported in Spain (Dyková and Figueras 1994, Iglesias et al. 2001, authors' unpublished data) and Norway (Sterud et al. 2000). Only in one of these turbot epizootics the causative organism was identified to specific level as Philasterides dicentrarchi Dragesco, Dragesco, Coste, Gasc, Romestand, Raymond et Bouix, 1995by Iglesias et al. (2001. This species was originally described from an episode of Mediterranean seabass (Dice...

Aims: To characterize bacteria associated with turbot larvae feeding on Artemia and identify pathogens causing mortalities in larvae. Methods and Results: To identify bacteria associated with mortalities in larval turbot rearing, bacteria were isolated from homogenates of Artemia or from several batches of well-performing or poorly performing turbot larvae. Samples were plated onto marine agar and were characterized using biochemical tests and BIOLOG GN plates. Total culturable aerobic bacteria ranged from 1AE9 · 10 5 to 1AE8 · 10 6 CFU per larva and >96% of bacteria identified were vibrios. Almost all bacteria were haemolytic and clustered into two phenons represented by Vibrio alginolyticus and Vibrio splendidus. The bacterial flora of Artemia was almost entirely V. alginolyticus, whereas V. splendidus biotype 1 dominated the larval turbot gut flora (69/115 isolates in seven experiments) and formed four different groups based on BIOLOG GN reactions. Of 16 isolates tested for virulence towards turbot larvae, four of the 11 V. splendidus biotype 1 isolates were lethal and all belonged to the same group of V. splendidus biotype 1 isolates. Conclusions: In a commercial turbot hatchery, the microbial flora of the larval gut was dominated by V. splendidus biotype 1. Four of the 11 V. splendidus biotype 1 isolates caused mortalities in larval turbot and all belonged to one group of the biotype 1 strains identified. Significance and Impact of the Study: Identification of four isolates of V. splendidus that are pathogenic for turbot larvae from three separate batches of larval turbot will allow these to be compared with avirulent isolates to define how V. splendidus causes mortalities in larval turbot.