Presence of phospholipase-D (dly) gene coding for damselysin production isnot a pre-requisite for pathogenicity in Photobacterium damselae subsp damselae

The presence of the phospholipase-D (dly) gene as pre-requisite for virulen ce of Photobacterium damselae subsp. damselae for poikilotherm and homoioth erm animals was investigated in a total of 17 strains isolated from fish, s hellfish, mammals and seawater. With this aim, we developed two PCR protoco ls. A simple PCR using primers flanking the almost complete dry gene, and a multiplex-PCR using two sets of primers directed to internal fragments of the dry and 16S rRNA genes. Only six of the 17 Ph. damselae subsp. damselae strains studied harboured the dly gene regardless of their haemolytic acti vity against sheep or rabbit erythrocytes as well as their virulence for ma mmals and marine fish. in fact, all strains but one were pathogenic for one or both animals, with LD50 values ranging from 1 x 10(3) and 3 x 10(5) bac teria for turbot, and 2 x 10(6) and 8 x 10(7) cells for mice. The PCR resul ts were corroborated in dot blot hybridization experiments employing a DNA probe directed to an internal region of the dry gene. From the data obtaine d 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, there fore, the role of damselysin as the main virulence factor of this marine ba cterium for poikilotherm and homoiotherm hosts should be re-evaluated. (C) 2000 Academic Press.