Photobacterium damselae subsp. damselae, an Emerging Pathogen Affecting New Cultured Marine Fish Species in Southern Spain

Aquaculture is the fastest growing food-producing sector, accounting almost 50% of the world food fish demand. Considering the projected population growth over the next two decades, it is estimated that at least an additional 40 million tonnes of aquatic food will be required by 2030 to maintain the current per capita consumption (NACA/FAO, 2001). Marine aquaculture production was 30.2 million tonnes in 2004, representing 50.9% of the global aquaculture production (FAO, 2004). By major groupings, fish is the top group whether by quantity or by value at 47.4% and 53.9%, respectively. However, according to the World Aquaculture Society (WAS, 2006), the future of this sector must be based on the increase of scientific and technical developments, on sustainable practices, and, mainly, on the diversification of the cultured fish species. For this reason, the European Union has designed an innovative plan to increase the culture of new fish and shellfish species, mainly marine, maintaining the production of other consolidated species (UE, 2010). Marine fish farming is a very important activity of Spanish aquaculture industry. The main marine fish species intensively cultured are gilt-head seabream (Sparus aurata), European seabass (Dicentrarchus labrax), and turbot (Scophthalmus maximus), achieving production percentages of 47.91, 12.5 and 18.62%, respectively (MAPA, 2008). In last 7 years, several new marine fish species are being evaluated as potential candidates for aquaculture production. In Southern Spain, studies on the reproductive cycles, nutrition, growth, histology and immune system of species such as Senegelese sole (Solea senegalensis), redbanded seabream (Pagrus auriga), common seabream (Pagrus pagrus), white seabream (Diplodus sargus), and meagre (Argyrosomus regius) are ongoing (Cardenas & Calvo, 2003; Prieto et al., 2003; Ponce et al., 2004; Manchado et al., 2005; Fernandez-Trujillo et al., 2006; 2008; Martin-Antonio et al., 2007; Cardenas & Manchado, 2008). However, the intensive


Introduction
Aquaculture is the fastest growing food-producing sector, accounting almost 50% of the world food fish demand. Considering the projected population growth over the next two decades, it is estimated that at least an additional 40 million tonnes of aquatic food will be required by 2030 to maintain the current per capita consumption (NACA/FAO, 2001). Marine aquaculture production was 30.2 million tonnes in 2004, representing 50.9% of the global aquaculture production (FAO, 2004). By major groupings, fish is the top group whether by quantity or by value at 47.4% and 53.9%, respectively. However, according to the World Aquaculture Society (WAS, 2006), the future of this sector must be based on the increase of scientific and technical developments, on sustainable practices, and, mainly, on the diversification of the cultured fish species. For this reason, the European Union has designed an innovative plan to increase the culture of new fish and shellfish species, mainly marine, maintaining the production of other consolidated species (UE, 2010). Marine fish farming is a very important activity of Spanish aquaculture industry. The main marine fish species intensively cultured are gilt-head seabream (Sparus aurata), European seabass (Dicentrarchus labrax), and turbot (Scophthalmus maximus), achieving production percentages of 47.91, 12.5 and 18.62%, respectively (MAPA, 2008). In last 7 years, several new marine fish species are being evaluated as potential candidates for aquaculture production. In Southern Spain, studies on the reproductive cycles, nutrition, growth, histology and immune system of species such as Senegelese sole (Solea senegalensis), redbanded seabream (Pagrus auriga), common seabream (Pagrus pagrus), white seabream (Diplodus sargus), and meagre (Argyrosomus regius) are ongoing (Cardenas & Calvo, 2003;Prieto et al., 2003;Ponce et al., 2004;Manchado et al., 2005;Fernandez-Trujillo et al., 2006;Martin-Antonio et al., 2007;Cardenas & Manchado, 2008). However, the intensive culture of these new fish species has favoured the appearance of several outbreaks with varied mortality rates. The development of a fish disease is the result of the interaction among pathogen, host and environment. Therefore, only multidisciplinary studies involving the virulence factors of the pathogenic microorganisms, aspects of the biology and immunology of the fish, as well as a better understanding of the environmental conditions affecting fish cultures, will allow the application of adequate measures to control and prevent the microbial diseases limiting the production of marine fish. According to Toranzo et al. (2005), several aspects would be raised regarding the infectious d i s e a s e s c a u s e d b y b a c t e r i a i n m a r i n e f i s h : ( i ) o n l y a relatively small number of pathogenic bacteria are responsible of important and significant economic losses in cultured fish; (ii) several classical diseases considered as typical of fresh water aquaculture are today important problems in marine culture; (iii) clinical signs (external and internal) provoked by each pathogen depend on the host species, fish age and stage of the disease; (iv) there is no correlation between external and internal signs of the disease; and (v) the severity of the disease and the mortality are higher in cultured fish that in wild fish populations, because to the lack of the stressful conditions that usually occur in the culture facilities. In the present study, the description of the outbreaks and the characterization of the etiological agents involved are described in detail. From the results obtained, Photobacterium damselae subsp. damselae was the most frequently pathogenic bacteria implicated in these outbreaks. This microorganism has been recognized as a pathogen for a wide variety of aquatic animals, such as crustaceans, molluscs, fish and cetaceans. In addition, this bacterial pathogen has been reported to cause diseases in humans and, for this reason, it may be considered as an agent of zoonoses. We have revised the taxonomical position, and phenotypic and molecular characteristics of this microorganism. In addition, we describe the virulence properties and pathogenesis mechanisms of P. damselae subsp. damselae. Labella (2010), studying the microbial origin of diseases affecting new cultured marine fish species in Southern Spain, reported the occurrence of 9 epizootic outbreaks (from 2003 to 2006) affecting cultures of redbanded seabream (7 outbreaks), common seabream (3 outbreaks), white seabream (2 outbreaks), and meagre (1 outbreak). The mortality of these outbreaks varying between 5 and 94%, depending on season, affected fish species, and fish age (Table 1). Gross external and internal signs varied depending on the outbreak, being similar to those previously described for vibriosis in several fish species Balebona et al., 1998b). The main external signs were exophthalmia, dark skin pigmentation, and pale gills and eroded fins (Fig. 1), whilst in some specimens from outbreaks 3, 5 and 8 haemorrhagic areas and epidermic ulcers were observed. Internally, the predominant infection signs were the presence of a fatty liver, with or without petechiae, and abdominal swelling with ascistic liquid. Splecnomegaly and visceral fat accumulation were also recorded ( Fig. 1).

Macroscopic signs of disease in finding fish
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Organs used for bacterial culture and its isolation
Affected or moribund specimens were killed with an overdose of MS-222 in seawater, and immediately processed for bacteriological analyses. Samples collected from the skin, eyes, brain, liver, spleen and kidney were seeded onto several routine bacteriological culture media for isolation of bacterial fish pathogens. The bacterial isolates were subjected to phenotypic characterization by using the tests specified in Table 2, according to Bergey's Manual of Systematic Bacteriology (Thyssen & Ollevier, 2005). All isolated bacteria were Gram-negative short rods or cocobacilli, motile, oxidase and catalase positives, glucose fermenters, and sensitive to vibriostatic agent pteridine (O/129, 150 µg). Bacterial identification was confirmed by the analysis of 16S rRNA gene sequences, amplified and sequenced as previously described by Labella et al. (2006).

Antimicrobial resistance
The antimicrobial resistance pattern to 14 antimicrobials routinely used in aquaculture practice was determined for selected strains of each identified bacterial pathogen. All the bacterial strains tested showed sensitivity to chloramphenicol, enrofloxacine, flumequine and nalidixic acid (Table 3). On the other hand, all the bacterial strains presented resistance to streptomycin, and a variable resistance pattern to the other 9 antimicrobials tested (Table 3). Labella (2010) reported three different resistotype profiles for the bacterial pathogens involved in the epizootic outbreaks. The resistotype I consisted of all the Photobacterium damselae subsp. damselae strains, and it is characterized for the sensitivity to trimethoprimsulphametaxazole, oxolinic acid, and nitrofurantoine. The resitotype II grouped the isolates included in the Vibrio harveyi, V. splendidus, V. fischeri and V. alginolyticus species. This resistotype possessed sensitivity to oxolinic acid, erythromycin, tetracycline, oxytetracycline, nitrofurantoine and novobiocin. Finally, the resistotype III included all the strains of V. ichthyoenteri, and presented sensitivity to trimethoprim-sulphametaxazole, tetracycline, oxytetracycline, novobiocin and amoxycillin (Table 3). Table 3. Antimicrobial resistance patterns of the bacterial isolates involved in epizootic outbreaks.
On the basis of its high prevalence in the epizootic outbreaks recorded, P. damselae subsp. damselae was considered as the main bacterial pathogen affecting new cultured marine fish species in Southern Spain (Garcia-Rosado et al., 2007).

Taxonomical position
The taxonomic status of P. damselae subsp. damselae within the family Vibrionaceae has changed repeatedly. P. damselae subsp. damselae was initially isolated as Vibrio damselae from skin ulcers of temperate-water damselfish (Love et al., 1981), and was recognized as an opportunistic pathogen capable of causing disease in a variety of hosts, mainly fish and mammals. MacDonell & Colwell (1985) transferred this species to the new genus Listonella based on a review of phylogenetic relationships within the family by 5S rRNA sequence data. Later, Smith et al. (1991) transferred this species to the genus Photobacterium based on phenotypic data, which was further supported from the phylogenetic analysis carried out by Ruimy et al. (1994). Similarly, Gauthier et al. (1995) demonstrated that the fish pathogen Pasteurella piscicida was closely related to P. damselae on the basis of phylogenetic analysis of small-subunit rRNA sequences and DNA-DNA hybridization data. Accordingly, P. damselae was proposed to include two subspecies, P. damselae subsp. damselae and P. damselae subsp. piscicida. However, Thyssen et al. (1998) have showed that there is no phenotypic evidence that supports the inclusion of P. damselae subsp. piscicida as a subspecies of P. damselae. The distinctive diagnoses of both subspecies of P. damselae can be achieved by a multiplex PCR assay, which combines specific primers for 16S rRNA and urease C genes (Osorio et al., 2000b).
A new species, named P. histaminum, has been described by Okuzumi et al. (1994) as a halophylic potent histamine-producing bacterium. The new species has been distinguished from other members of the genus based on phenotypic characteristics, 16S rRNA gene sequence and DNA-DNA hybridization. A close physiological similarity between P. damselae subsp. damselae and P. histaminum has been reported (Dalgaard et al., 1997). Kimura et al. (2000) found that the type strain of P. damselae subsp. damselae has a histamine-producing ability as potent as P. histaminum. In addition, the levels of DNA relatedness between both species ranged from 80 to 88%. Regarding to the phenotypic differentiation of these organisms, the authors confirmed a biochemical profile identical for both species, except for the trehalose utilization. For these reasons, Kimura et al. (2000) proposed P. histaminum as a later subjective synonym of P. damselae subsp. damselae.

Phenotypic and molecular characteristics
According to Bergey's Manual of Systematic Bacteriology (Thyssen & Ollevier, 2005), P. damselae subsp. damselae belongs to the genus Photobacterium included in the family Vibrionaceae, displaying morphological characteristics typical of members of the family, appearing as coccobacilli. The flagellate organisms lack of a flagellar sheath, even P. damselae subsp. piscicida lacks flagella (Baumann & Baumann, 1981). Labella (2010) carried out a wide study on the phenotypic characteristics of P. damselae subsp. damselae, resulting positive the following traits: motility, catalase, cytochrome oxidase, growth at the range of 20-35ºC and in 1-6% NaCl, arginine dehydrolase, nitrate reduction, and fermentation of melibiose and maltose. P. damselae subsp. damselae uses the following compounds as unique carbon source: D-galactose, α-D-glucose, raffinose, turanose, D-ribose, N-acetylgalactosamine, glycogen, methyl α-D-glucoside, dextrin, Dglucose 6 phosphate, glycerol, sorbitol, succinate, D-L-lactic acid, glycil L-glutamic acid, tween 40, tween 80, L-glutamic acid, glycil L-aspartic acid, inosine, L-serine, L-aspartic acid, L-asparagine, L-alanine, uridine, L-alanylglycine and thymine. On the other hand, P. damselae subsp. damselae strains showed negative results for ornithine decarboxylase, production of H 2 S, indole production, alginase, and fermentation of D-mannitol, D-sorbitol, inositol, erytritol, D-adonitol, D-arabitol, dulcitol, raffinose, L-rhamnose and L-arabinose. This subspecies is unable to use D-fucose, α-D-lactose, L-arabinose, gentibiose, melibiose, Lrhamnose, D-mannitol, adonitol, myo-inositol, erytritol, xylitol, arabinitol, acetate, L-glutamate, formate, D-gluconic acid, propionic acid, L-leucine, D-alanine, L-proline, Lthreonine, L-ornithine, L-histidine, α-ceto glutaric acid, aconytic acid and ßhydroxyphenylacetic acid. The fatty acid profile of P. damselae subsp. damselae contains high concentrations of C 16:1 , C 16:0 , and C 18:1 fatty acids, and in lesser extent C 12:0 , C 14:0 , C1 4:1 , C 15:0 , C 17:0 , C 17:1 , and C 18:0 (Nogi et al., 1998). The electrophoretic analyses carried out revealed similar band pattern for P. damselae strains, sharing four major outer membrane proteins (OMP), with molecular masses of 20, 30, 42 and 53 kDa (Magariños et al., 1992). An OMP of 37 kDa (OMP-PD) forms a trimeric structure of approximately 110 kDa that conform an ion channel and acts as a porin in P. damselae (Gribun et al., 2004). These results have been confirmed by Western blot performed with anti-OMP polyclonal serum against the monomeric form of OMP-PD. As in the case of the OMP, all P. damselae strains showed the same silver-stained lipopolysaccharide (LPS) profile obtained by proteinase K digested whole cell lysates. This profile had a ladder like pattern, typical of smooth type LPS (S-LPS), with low amounts of 2keto-3-deoxyoctonate (KDO) (Kuwae et al., 1982). The G+C content of the genomic DNA of P. damselae subsp. damselae is 40.6-41.4 mol% (Thyssen & Ollevier, 2005). The DNA relatedness of P. damselae subsp. damselae and other classical Photobacterium species, demonstrated by DNA-DNA hybridization, varied between 12 and 37% with P. leiognathi, 21 and 30% with P. phosphoreum, 19.5% with P. profundum and 28% with P. augustum (Nogi et al., 1998;Kimura et al., 2000). Plasmids are present in most P. damselae subsp. damselae strains tested, with sizes ranging from 3.0 kb to higher than 190 kb (Pedersen et al., 1997). Several studies have demonstrated that strains of P. damselae subsp. damselae showed a high heterogeneity in biochemical and serological characteristics (Smith et al., 1991;Fouz et al., 1992;Pedersen et al., 1997;Labella et al., 2006). Botella et al. (2002) established that 11 biochemical features were variable among the 33 P. damselae subsp. damselae strains tested: acetoin production, luminescence, gas from glucose, lysine decarboxylase, growth at 4º and 40ºC, urease, and utilization of sucrose, D-mannose, D-cellobiose and D-gluconate. Labella et al. (2009) obtained that P. damselae subsp. damselae strains showed variability for the following tests: acetoin production, ß-galactosidase, lysine decarboxylase, growth at 4º and 40ºC, esculin hydrolysis, acid from mannitol, sorbitol and amygdalin, citrate utilization and assimilation of D-mannose, maltose, malate and N-acetylglucosamine. This variability led the authors to establish 8 different biotypes or phenotypic profiles among the 17 strains tested, isolated from cultured marine fish. However, the genetic variation of the strains of P. damselae subsp. damselae has received less attention. Botella et al. (2002), using the amplified fragment length polymorphism (AFLP) technique, demonstrated a high genetic variability among the P. damselae subsp. damselae strains isolated from gilthead seabream and European seabass cultured in the same geographical area and collected in a short time period (2 years). In fact, the 33 tested strains yielded 24 AFLP profiles, with almost every strain showing a different band pattern. Takahashi et al. (2008) established, using ribotyping, AFLP and pulsed-field gel electrophoresis (PFGE), that P. damselae subsp. damselae clinical isolates causing fatal cases in humans had similar genotypes, but they were not clearly distinguishable from environmental isolates (including isolates from fish). Nevertheless, the phenotypic profiles of the clinical isolates were clearly distinct from those showed by environmental isolates. The authors explained the inconsistency between the results obtained from genotypic and www.intechopen.com phenotypic analysis arguing that the divergence of clinical strains from environmental ones is a recent event, and these phenotypic differences are so small that they are not detected by whole genome typing techniques such as PFGE and AFLP. However, sequencing analysis of the gyrB, toxR and ompU genes showed larger differences between clinical and environmental isolates. Similar results were obtained by Labella (2010) comparing these clinical strains with fish isolates by repetitive extragenic palindromic (REP)-PCR. Labella et al. (2009) compared three PCR-based techniques [random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus (ERIC)-PCR, and REP-PCR] for the analysis of genetic variability within P. damselae subsp. damselae strains isolated from several fish species in outbreaks occurred in different geographical locations. All the PCRbased typing methods supported the high variability within P. damselae subsp. damselae, the strains being discriminated into 8-14 genetic groups, depending on the method employed. In addition, no concordance among the genetic assignation of the strains by the different PCR methods was obtained. These results suggest, as concluded also by Botella et al. (2002), that different clonal variants of P. damselae subsp. damselae potentially pathogenic for several fish species exist, and even can be involved in a single outbreak. On the other hand, and similarly to previous results (Botella et al., 2002), a relationship between the genetic profiles and the origin of isolation or the host fish species could not be established.

Pathogenicity and virulence factors
P. damselae subsp. damselae has been recognized as a bacterial pathogen in a wide variety of aquatic animals including fish, molluscs and crustaceans (Vera et al., 1991;Fouz et al., 1992;Company et al., 1999;Sung et al., 2001;Lozano-Leon et al., 2003;Labella et al., 2006;Wang & Cheng, 2006;Vaseeharan et al., 2007;Han et al. 2009;Kanchanopas-Barnette et al., 2009). This microorganism is an autochthonous inhabitant of aquatic ecosystems, which may survive in seawater and sediment for a long time, maintaining its infectivity and pathogenic properties (Ghinsberg et al., 1995;Fouz et al., 1998;2000). In addition, P. damselae subsp. damselae may be a primary pathogen for mammals, including humans (Morris et al., 1982;Clarridge & Zighelboim-Daum, 1985;Fujioka et al., 1988;Perez-Tirse et al., 1993;Yuen et al., 1993;Shin et al., 1996;Fraser et al., 1997;Tang & Wong, 1999;Goodell et al., 2004;Yamame et al., 2004). A comparatively small number of bacterial species belonging to the family Vibrionaceae causes diseases in both aquatic animals and humans. However, the fact that an organism provokes disease in an aquatic animal does not necessarily mean that this is the source for human infections (Austin, 2010). Indeed, the origin of some of these bacteria may be the waters in which the aquatic animals are found, and the transmission to humans may be via wound or may be food/water-borne. The extracellular products (ECPs) are produced by bacterial pathogens to facilitate the uptake of nutrients from the surrounding environment and/or for the successful penetration and survival of pathogens inside the host (Sakai, 1985;Bakopoulos et al., 2003). Main ECP components related to virulence include proteases, haemolysins, and siderophore-mediated iron sequestering systems (Norqvist et al., 1990;Balebona et al., 1998a;Rodkhum et al., 2005;Wang et al., 2007). These mechanisms can provoke host tissue destruction and haemorrhages, playing an important role in colonization, invasiveness and dissemination of the bacterial pathogen within the host (Finkelstein et al., 1992;Silva et al., 2003). However, only a few studies have been carried out on the role of ECPs in the pathogenesis of P. damselae subsp. damselae. Labella et al. (2010), studying the pathogenicity of P. damselae subsp. damselae strains isolated from cultured fish, demonstrated that the intraperitoneal inoculation of ECPs from virulent strains (mean LD 50 of about 1 x 10 5 CFU) was lethal for redbanded seabream at 2 to 4 h postinoculation, whilst ECP samples from a non-virulent strain (LD 50 > 10 8 CFU) did not produced toxic effects in fish after a 7d post-inoculation period. The inoculation of heated ECPs (100ºC, 10 min) to fish did not produce deaths, which suggests that the active toxic fraction present in the ECPs is secreted and thermolabile, and it is not associated with the thermorresistant bacterial lipopolysaccharide content. Similar results have been reported for several fish pathogens (Lamas et al., 1994), including P. damselae subsp. damselae (Fouz et al., 1995). Fish inoculated with heated ECPs (and also with ECPs from non-virulent strains) showed enlarged lymphohaematopoietic organs (Fig. 2), suggesting a stimulation of immune response with cellular accumulation, as also reported for Aeromonas hydrophila ECPs (Rey et al., 2009). P. damselae subsp. damselae ECPs displayed cytotoxic activity for different fish and mammalian cell lines (Wang et al., 1998;Labella et al., 2010). The cytotoxicity was limited to ECPs from virulent strains, and it was totally lost on heated ECP samples, which suggests the presence of thermolabile cytotoxic components in the raw ECPs . The main virulence factor characterized in P. damselae subsp. damselae is the damselysin, a thermolabile extracellular cytotoxin of 69 kDa, which is a phospholipase D active against the sphingomyelin of the sheep erythrocyte membrane (Kreger, 1984;Kothary & Kreger, 1985;Cutter & Kreger, 1990). The damselysin also presents haemolytic activity against several erythrocyte types, including fish (Kreger et al., 1987). Classically, damselysin production has been related to the pathogenicity of P. damselae subsp. damselae in diverse animal models (Kothary & Kreger, 1985;Fouz et al., 1993), although Osorio et al. (2000a) demonstrated that the presence of this toxin is not a requisite for the virulence of this bacterial pathogen. Labella et al. (2010) found that 75% of virulent P. damselae subsp. damselae strains showed phospholipase activity in their ECPs, but the specific 567 bp PCR amplicon corresponding to the phospholipase D (dly) gene was detected in only two www.intechopen.com strains (12.5%). Interestingly, the phospholipase activity in the dly + strains remained unaltered after thermal treatment, which differs from the behaviour described for phospholipase toxins in Vibrionaceae (Songer, 1987). Two types of bacterial phospholipases have been described, the extracellular phospholipases (A2, C or D), which are considered as virulence factors (Schmiel & Miller, 1999), and the phospholipases associated with the outer membrane (A type), whose role in pathogenesis had not been established (Dekker, 2000;Snijder & Dijkstra, 2000). Besides the phospholipase D activity associated to damselysin, other phospholipases (extracellular and/or A type) seem to be present in P. damselae subsp. damselae strains, although they are not directly related to the pathogenic properties of the strains . Several authors have pointed out that the pathogenicity of some bacterial fish pathogens was related to their ability to haemolyse the host erythrocytes (Borrego et al., 1991;Fouz et al., 1993;Grizzle & Kiryu, 1993;Pedersen et al., 2009). The haemolytic activity in Vibrionaceae can be related to extracellular enzymatic activities, such as phospholipases in V. parahaemolyticus, V. mimicus, V. harveyi and P. damselae subsp. piscicida (Shinoda et al., 1991;Lee et al., 2002;Zhong et al., 2006;Naka et al., 2007) and phospholipase D in P. damselae subsp. damselae (Kreger et al., 1987), or to the direct action of haemolysins that provoke the pore-structure formation on the erythrocyte membrane (Iida & Honda, 1997;Zhang & Austin, 2005). Labella et al. (2010) reported that all P. damselae subsp. damselae strains tested produced haemolysis of fish and/or sheep erythrocytes. This ability was exclusively associated with bacterial cultures in 81.25% of the strains. These results could suggest that the haemolysin is associated with the bacterial core or it is an extracellular haemolysin whose activity is inhibited by the enzymatic content of ECPs, as has been described for VTH haemolysin of V. tubiashii (Hasegawa & Hase, 2009). As in the case of phospholipases, a correlation between the haemolytic activity of P. damselae subsp. damselae strains and their virulence properties could not also be established . Several extracellular bacterial proteases, mainly metalloproteases and serine-proteases such as vibriolysins, are considered as virulence factors in numerous bacterial pathogens (Ishihara et al., 2002;Miyoshi et al., 2002;Farto et al., 2006). These proteases provoke tissue damages and degradation of host tissues, favouring the colonization and invasion of pathogens into the host (Miyoshi & Shinoda, 2000). In addition, proteases enable the evasion of the bacteria from several fish defence mechanisms (Vivas et al., 2004). A limited number of enzymatic activities has been detected in P. damselae subsp. damselae ECPs, including phosphatases, esterases, amylases and glycosidases, but their proteolytic activity was very low, lacking caseinase and gelatinase activities Fouz et al., 1993;Labella et al., 2010). Nevertheless, none of these enzymatic activities could be related with the degree of toxicity, both in vivo and in vitro, presented by the ECPs , in contrast to results reported for other fish pathogens such as Aeromonas (Esteve et al., 1995). In short, the presence of phospholipases (including damselysin), haemolysins or other enzymatic activities in the ECPs is not directly related to the pathogenicity of P. damselae subsp. damselae. Labella et al. (2010) hypothesized that another unknown type of toxin, different to the damselysin, could be involved in the toxicity of P. damselae subsp. damselae ECPs. A neurotoxin possessing an acetylcholine-esterase activity (ictiotoxin) has been described in strains of several species of Vibrionaceae, including P. damselae subsp. damselae (Balebona et al. 1998a;Perez et al. 1998), and may be responsible for several clinical signs observed by these authors.

Conclusions
In recent years, Photobacterium damselae subsp. damselae has been repeatedly isolated from epizootic outbreaks affecting several cultured fish species. In addition, this bacterial pathogen has been reported to cause diseases in humans, and for this reason, it may be considered as an agent of zoonoses. The unique virulence factor characterized in P. damselae subsp. damselae is the damselysin, a thermolabile extracellular cytotoxin, which is a phospholipase D and presents haemolytic activity against different erythrocytes types. However, recent results obtained by our research team demonstrate there is no correlation between the presence of the dly gene and the pathogenicity of P. damselae subsp. damselae, therefore, other virulence factors may be involved in the pathological damages that this microorganism caused in infected fish.