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Pseudomonas baetica is a pathogen isolated in 2012 from wedge sole (Dicologoglossa cuneata). The aims of this study were (i) to determine the influence of temperature on its virulence, and (ii) to develop specific protocols for rapid diagnosis. Virulence assays carried out by bath using Senegalese sole fry showed that virulence is strongly influenced by temperature: LD50 at 14°C was 8.5 × 105 cfu ml-1 while at 20°C no mortalities were recorded. On the other hand, the high mortality rates observed in virulence assays involving intraperitoneal injection of 2.5 × 105 cfu g-1 suggest that P. baetica may be pathogenic for the five fish species tested (wedge sole, Senegalese sole, sea bream, European sea bass and meagre). Two PCR protocols, using specific primers targeting the gyrB and rpoD genes, were developed for rapid diagnosis from pure cultures. An additional protocol, using both primer sets, was also optimized for detection from fish tissue samples. Specificity was tested using 81 strains from 66 bacterial species, taxonomically and/or ecologically related; only the P. baetica strains showed the expected DNA amplicons. A specific dot-blot assay using polyclonal antibodies was also developed for differentiation of P. baetica from related species. Altogether, the protocols described here will constitute useful tools for diagnosis and clarify the relevance of this pathogen.

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 × 10 6 cfu g −1 body weight).

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