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Pseudomonas baetica: pathogenicity for marine fish and development of protocols for rapid diagnosis

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.