16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis

The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae sub sp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromo somal DNA-DNA hybridization data; thus, this organism belongs to the same s pecies as Photobacterium damselae subsp. damselae (formerly Vibrio damselae ). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic po sition of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR pr otocol for detection of P. damselae based on 16S rRNA was developed. This P CR protocol was validated by testing 35 target and 24 nontarget pure cultur es, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR pro tocol was applied to fish tissues spiked with bacteria. The PCR approach de scribed in this paper allows detection of the pathogen in mixed plate cultu res obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosi s.