Photobacterium damselae subsp. damselae is a marine halophilic bacterium, which may cause fatal infections in a wide variety of hosts, including fish, molluscs, crustaceans, humans and other mammals. It is an emerging pathogen of economically important aquaculture species with increasing geographical distribution worldwide. The major virulence factors of Pdd are plasmid (pPHDD1)-encoded phospholipase-D damselysin and pore-forming toxin phobalysin P. However, strains of Pdd without plasmid have also demonstrated pathogenicity due to the presence of chromosome I-encoded virulence factors. The present study was conducted to characterize the intraspecific variability in 16 isolates of P. damselae subsp. damselae collected from cultured yellowtail kingfish (YTK) in Australia by means of biochemical and molecular typing and to get the insights of pathogenicity of Pdd infections in YTK.
The intraspecific variability was conducted by means of biochemical and phenotyping characteristics while sequence analysis was done based on toxR gene to investigate genetic heterogenicity. An infection trial was conducted with healthy, unvaccinated cultured YTK, weighing an average 152.0±17.7 g. Three isolates of Pdd (one plasmid-positive, AS-16-0963#3; and two plasmid-negative, AS-15-3942#7 and AS-16-0963#1) were selected for experimental infection. There were six experimental treatments, with intraperitoneal injection, one of the three test isolates of Pdd at two different concentrations, 104 CFU/fish and 107 CFU/fish, and three replicate tanks per treatment. Control fish in two replicate tanks were sham-injected. Fish mortality was recorded daily for 10 days post-challenge. Blood samples from infected fish after 4 days of infection were collected to measure hematology indices.
There are considerable differences in phenotypic traits among isolates and five biotypes were established accordingly. toxR sequence analysis demonstrated substantial genetic diversity among isolates, suggesting the presence of several different clonal lineages of Australian Pdd isolates. Mortality rates for the plasmid-positive isolate, AS-16-0963#3, were 47% and 100% at 104 CFU/fish and 107 CFU/fish respectively, compared to13-20% and 3-13% respectively for the two plasmid-negative isolates. No mortality was observed in the control fish. There was no significant difference in hematology parameters among isolates. These results suggest that, while presence of the pPHDD1 plasmid is not essential for pathogenicity, the plasmid does increase the virulence of Photobacterium damselae subsp. damselae to Seriola lalandi in Australia.