Development of a molecular methodology for fast detection of Photobacterium damselae subspecies in water samples

Photobacterium damselae subspecies damselae and piscicida are known fish pathogens responsible for disease outbreaks in several cultured fish species. Fast detection of these pathogens is important for management and control of disease outbreaks. In this study, we developed a molecular approach for quantification of P. damselae species and detection of its subspecies (piscicida and damselae) based on real time PCR (RT-PCR) and PCRdenaturing gradient gel electrophoresis (PCR-DGGE) of toxR gene fragments. In addition, the efficacy of this molecular methodology was tested against water samples from a natural environment (Ria de Aveiro estuary, Aveiro, Portugal) and from an intensive recirculating aquaculture system (RAS). In the first phase of this study we developed a RT-PCR targeting the toxR gene suitable for detection and quantification of P. damselae species. After the first RT-PCR trial, a nested PCR-DGGE was developed to determine the presence of P. damselae subspecies in the positive samples. Confirmation of the identity of different subspecies was obtained by Sanger DNA sequencing and phylogenetic analysis of toxR gene fragments obtained from excised DGGE bands. Our RT-PCR analysis detected between 1797.85± 376.15 (ca 0.71 fg) and 2976.68± 1253.63 (ca 1.76 fg) gene copy numbers of P. damselae toxR genes in the aquaculture samples. The DGGE analysis of these samples detected two equally abundant bands near the DGGE reference position for P. damselae subsp. piscicida. The sequence and phylogeny analyses of excised bands revealed the presence of two populationswith distinct toxR gene sequences suggesting a close phylogenetic relationship with P. damselae subsp. piscicida. Contrary to aquaculture samples, no RT-PCR signal was obtained for DNA extracted from estuarine water samples. Here we provide evidences that themolecularmethodology developed in this study can be used for overall quantification of P. damselae and subsequent detection of its subspecies in natural ecosystems and aquaculture systems