Presence of phospholipase-D (dly) gene coding for damselysin production is not a pre-requisite for pathogenicity in Photobacterium damselae subsp. damselae.

The presence of the phospholipase-D (dly) gene as pre-requisite for virulence of Photobacterium damselae subsp. damselae for poikilotherm and homoiotherm animals was investigated in a total of 17 strains isolated from fish, shellfish, mammals and seawater. With this aim, we developed two PCR protocols. A simple PCR using primers flanking the almost complete dly gene, and a multiplex-PCR using two sets of primers directed to internal fragments of the dly and 16S rRNA genes. Only six of the 17 Ph. damselae subsp. damselae strains studied harboured the dly gene regardless of their haemolytic activity against sheep or rabbit erythrocytes as well as their virulence for mammals and marine fish. In fact, all strains but one were pathogenic for one or both animals, with LD(50)values ranging from 1x10(3)and 3x10(5)bacteria for turbot, and 2x10(6)and 8x10(7)cells for mice. The PCR results were corroborated in dot blot hybridization experiments employing a DNA probe directed to an internal region of the dly gene. From the data obtained in this work, we can conclude that the presence of the dly gene is not an indicative of the pathogenicity of Ph. damselae subsp. damselae and, therefore, the role of damselysin as the main virulence factor of this marine bacterium for poikilotherm and homoiotherm hosts should be re-evaluated.