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Systemic amoebiasis of Senegalese sole Solea senegalensis is caused by Endolimax piscium Constenla, Padrós & Palenzuela, 2014 a cryptic parasitic member of the Archamoebae whose epidemiology is yet unknown. To test whether the parasite can be transmitted horizontally, an experimental trial by cohabitation between non-infected and infected fish was designed. Transmission of the parasite from naturally infected to healthy fish was confirmed in the experiment, with the water as the most likely route of infection. Under the conditions of the study, the infection process was remarkably slow, as parasites could be detected by in situ hybridization within the intestinal mucosa of recipient fish only after 17 wk of cohabitation, and none of the new hosts displayed clinical signs of disease. Long prepatent period and the need for additional triggering factors for the development of the clinical condition are suggested. The intestinal mucosa is proposed as the tissue where the amoeba can survive as endocommensal, but also as an invasion route from which the parasite would disperse to other organs.

Three bacterial isolates were recovered from a disease outbreak with high mortality affecting brill Scophthalmus rhombus (Linnaeus, 1758). Moribund fish showed no external signs of disease, but plentiful haemorrhages were observed in liver. On the basis of phenotypic and genotypic characterization, the isolates were identified as Aliivibrio fischeri. The phenotypic profile of the isolates was basically similar to that of the type strain of this species, although some discrepancies were observed, mainly in the BIOLOG GN profile. The main cellular fatty acids of strain a591 were also consistent with this species. The highest 16S rDNA sequence similarities were recorded with the type strain of A. fischeri (99.07%); other Aliivibrio species showed similarity values below 96%. The highest sequence similarities with gyrB, rpoD and recA genes were also recorded with A. fischeri type strain (99.31, 98.99 and 95.29% similarity, respectively). DNA-DNA hybridization assays confirmed that these isolates belong to A. fischeri; levels of DNA relatedness were 73.5 to 86.2% with isolate a591 (reciprocal values of 86.9 to 99.04%). Finally, a virulence evaluation of the isolates using Senegalese sole fry was also performed; significant mortalities (100% mortality within 5 d) were recorded by intraperitoneal injection, but only with high doses of bacteria (2 × 106 cfu g-1 body weight).

Virulence factors for Aeromonas salmonicida subsp. salmonicida (ASS) strains isolated from cultured turbot Psetta maxima L. are unknown with regard to this host. The presence of virulence genes associated with different stages of ASS infection in salmonids (vapA, tapA, fla, ascV, ascC, aexT, satA and aspA) was analysed using a polymerase chain reaction (PCR) technique in ASS strains isolated from turbot. Other ASS strains isolated from salmonids and environmental A. salmonicida (AS) strains were included for comparison. The presence of the genes was evaluated with respect to ASS virulence in turbot based on intraperitoneal and bath challenges. The genetic profile, including all of the genes studied, that was linked to virulent behaviour after intraperitoneal challenge was significantly more frequent in strains isolated from turbot than in those from salmonids or the environment. The data prove that it is not possible to predict the virulence of ASS in turbot based only on the presence of all genes tested. Moreover, the combined PCR results of vapA, aexT, ascV and ascC were useful for separating most of the ASS from environmental A. salmonicida strains. An association between virulence or genetic profile and the geographical or facility origin of the strains was not found.

We report the first isolation of Vibrio harveyi from wedge sole Dicologoglossa cuneata. The pathogen was recovered from ulcers and internal organs of ailing cultured fish, from 7 different outbreaks between 2004 and 2006. The 15 isolates found were phenotypically characterized using biochemical tests and BIOLOG GN plates, which revealed high phenotypic diversity. Diagnosis was confirmed with PCR using V harveyi specific primers and partial 16S and 23S rRNA gene sequencing. A virulence evaluation of the isolates was also performed using fry and juvenile wedge sole. Significant mortalities were recorded by intraperitoneal injection; however, no mortalities were recorded by bath immersion.

A bacteria-free halibut larval rearing system was used to test 20 bacterial isolates, from British halibut hatcheries, for their toxicity towards halibut yolk-sac larvae under microbially controlled conditions. The isolates tested spanned a range of genera and species (Pseudoalteromonas, Halomonas marina, Vibrio salmonicida-like, Photobacterium phosphoreum and V. splendidus species). A pathogen of turbot, Scophthalmus maximus, V. anguillarum 91079, and 2 isolates from adult halibut were also included. Isolates were inoculated, at a concentration of 5 x 10(2) cfu ml(-1), into flasks containing 25 recently hatched axenic halibut larvae, using a minimum of 3 flasks for each treatment. Control survivals to 38 d post-hatch for the 3 experiments averaged 84, 51.5 and 49%, respectively. With the exception of V. anguillarum 91079, which was highly pathogenic towards halibut yolk-sac larvae, there was no statistically significant difference in survival between the controls and the different treatments. This suggests that most of the bacteria routinely isolated from halibut hatcheries are not harmful to yolk-sac larvae, even though most flasks contained in excess of 5 x 10(6) cfu m(-1) of the inoculated organism when the experiments were terminated. Three organisms previously shown to inhibit growth of bacteria in vitro were tested for their ability to protect halibut yolk-sac larvae against invasion by V. anguillarum. In 4 separate challenge experiments none of the test isolates, a Pseudoalteromonas strain and 2 Carnobacterium-like organisms, showed any protective effect. To investigate how particular bacteria influence their start-feed response, larvae were fed axenic and gnotobiotic Artemia colonized with a range of different Vibrio spp., and examined after 8 d. There were no statistically significant between-treatment differences in the proportion of Artemia-containing larvae, indicating that bacterial contamination of the live food does not appear to influence initiation of the feeding response.

The fate of Streptococcus parauberis in seawater and sediment microcosms at different temperatures (6 and 22 degrees C) was investigated by comparing the survival dynamics of 2 strains of this bacterial species, isolated respectively from diseased turbot and cattle. The turbot and the bovine isolate showed similar survival kinetics, remaining culturable for approximately 1 mo in water and 6 mo in sediment. A slight influence of temperature on the stability of the cells was observed, in that the number of culturable cells was about 1 log10 unit higher at 6 than at 22 degrees C. During the starvation period, the metabolic activity of the cells, after suffering a strong reduction during the first 12 d, stabilized at levels ranging from 20 to 40% of the initial values. However, in all the microcosms, the acridine orange (AO) and 4',6-diamidino-2-phenilindole (DAPI) counts remained at about 10(5) cells ml(-1) throughout the experimental period, even when cells became undetectable by standard plate count methods. The addition of fresh medium to microcosms containing nonculturable cells induced the return to culturability of S. parauberis strains. On the basis of these results, it seems that S. parauberis has the ability to enter into a viable but nonculturable (VBNC) state. Dormant cells of the turbot isolate maintained their infectivity and pathogenic potential for fish.