Photobacterium damselae subsp. damselae is considered to be an emerging pathogen of marine fish of importance in aquaculture, with a notable increase in its geographical distribution during the last several years. In this study, we carried out for the first time to our knowledge a genetic and pathobiological characterization of 14 strains isolated from sea bass (Dicentrarchus labrax) reared in the Southeastern Black Sea, where high mortalities were observed at two aquaculture farms during the summer and autumn of 2011. Heterogeneity was evidenced among strains in phenotypical traits, such as sucrose fermentation, motility, and hemolysis. Although 11 of 14 isolates were hemolytic, we found that all of the isolates lacked the pPHDD1 virulence plasmid that encodes the phospholipase-D damselysin (Dly) and the pore-forming toxin PhlyP, two hemolysins previously reported to constitute major virulence factors for turbot. Subsequent PCR and sequencing analyses demonstrated that the 11 hemolytic isolates harbored a complete hlyA ch gene, a chromosome I-borne gene that encodes HlyA ch hemolysin, whereas the three nonhemolytic isolates contained hlyA ch pseudogenes caused by insertion sequence elements. Virulence challenges with two representative strains revealed that, albeit less virulent than the pPHDD1-harboring strain RM-71, the plasmidless hlyA ch -positive and hlyA chnegative Black Sea isolates were pathogenic for sea bass. A phylogenetic analysis based on the toxR gene sequence uncovered a greater diversity in the isolates, indicating that the presence of this pathogen in the Black Sea was not caused by the introduction and spread of a single virulent clone but by the proliferation of different clones. IMPORTANCEThe geographical distribution of marine bacterial pathogens is undergoing a worldwide increase. In particular, bacteria of the group vibrios are increasingly being isolated as the causative agents of disease in novel species of cultivated fish in areas where they had not been previously reported. Here we characterize for the first time to our knowledge a collection of isolates of the fish and human pathogen Photobacterium damselae subsp. damselae from diseased sea bass reared in the Black Sea. We uncovered great genetic diversity in the Black Sea isolates of this pathogen, suggesting a multiclonal origin. We also demonstrate for the first time that these isolates bear pathogenic potential for sea bass cultures by virulence challenges. P hotobacterium damselae subsp. damselae is a marine bacterium of the family Vibrionaceae that is recognized as a pathogen for a wide variety of aquatic animals, including fish, molluscs, and crustaceans. In addition, it is a pathogen of concern for humans, as it is capable of causing fatal infections (1). Most of the reported infections in humans originated from wounds inflicted during the handling of fish and fishing tools or from exposure to marine animals or seawater. Notably, it is a primary pathogen of fish species of economical importance in aquaculture. During ...

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It was determined that there is a relationship between prevalence and mean densities of Trichodina spp. on Merlangius merlangus and organic pollution, measured as levels of nitrite, nitrate and phosphate in the surrounding environment. Monthly, two left anterior gill arcs of 60 M. merlangus (unless otherwise stated) captured with hook and line were fixed in 10% formalin. The number of Trichodina spp. was determined by counting all of the cells with a grid slide. The two right anterior gill arcs were used to prepare dry smears to picture the morphology of the trichodinids and to determine species composition. High levels of prevalence and densities of the protozoan were observed during the late fall, winter and early spring months. Then, prevalence decreased to lower levels but never below 60%, an indication of the important role of M. merlangus for Trichodina spp. in the studied region. A multivariate analysis revealed that the magnitude of prevalence was related to the level of all three parameters: nitrite, nitrate and phosphate (r2=0.59). However, a much stronger relationship between prevalence and nitrate, phosphate, oxygen and temperature (r2=0.89) was detected. Consequently, the seasonal parasite prevalence and density were affected by organic pollution. The timing for a high prevalence and mean densities of this parasite also suggests that primary production may be responsible for the observed seasonal variation in prevalence and mean densities. By using the model describing the relationship between the water quality parameters and the prevalence data of Trichodina spp. recalculated values and observed field data corresponded closely.

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