to the same Vibrio isolates, similar to that previously described for clams (R. philippinarum) and oysters (C. gigas) (LeRoux et al 2002). Field studies have determined that bivalve mortality results from the interaction of environmental conditions, physiological status and the genetics of the host and the infectious agent, which could be an opportunist rather than a primary pathogen (Samain & McCombie 2008, Aranguren et al 2012). In our bath infection experiments, temperature changes and hypoxic stress were included to mimic environmental changes because they have been implicated in bivalve deaths (Soletchnik et al 1999, Lee et al 2001). In this disease model, deaths only occurred when animals were infected with a high concentration of bacteria in adverse environmental conditions. This result reflects the extreme resistance of mussels to the bacterial pathogens tested. Although the pathogenicity of the Vibrio strains isolated from wild mussel populations and natural reservoirs appeared to be low (high bacterial concentrations were needed to induce death in experimental infections), the fact that some strains induced more death than the reference strains highlights the potential risk of Vibrio spp. residing in plankton to bivalve populations. The differences in the mortality rates between different isolates of Vibrio splendidus were expected since, as occurs in other bacterial species, different strains could present different virulence degree. Moreover, Vibrio splendidus constitutes a clade containing numerous isolates of which only a few have been described (Thompson et al 2004). Otherwise the strains of V. splendidus isolated from the same animal after two different mortality events are never the same (Le Roux et al 2002, Waechter et al 2002). Moreover, several Vibrio species are generally isolated from the same individual and a complex of cooperative interactions emerged between them during the pathogenic process in the molluscan host (Gay et al 2004 b). In opposite, isolates of V. aestuarianus can be considered as only one species and only one strain could be isolated in the same animal.
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the Sor River. The bars represent the mean and SEM of the percentage of infected animals in one year. (*) significant differences regarding to results obtained in gills and gland (p≥0.05). Figure 5. A proportional representation of the Alcabre and Sor bacteria identified by 16S rDNA sequencing. The percentages indicate the representation of each group in the entire population of analysed bacteria from Alcabre and the Sor River (A). The distribution of bacteria within the different ecological compartments (water, sediment, plankton, mussel and oyster) in Alcabre (B) and the Sor River (C). Figure 6. (A) The cumulative mortality of mussels after a bath infection with V. splendidus and V. aestuarianus at 15 ºC (upper panel) and 25 ºC (lower panel). The effect of an 8 h emersion, which mimics hypoxia-induced stress, on the cumulative mortality was determined (left panels) (N=540 animals). (B) The cumulative mortality of mussels after an intramuscular injection of three different doses of V. splendidus and V. aestuarianus at 15 ºC (N=420 animals). Figure 7. The cumulative mortality from experimental infections by intramuscular injection of V. splendidus isolated from Alcabre (A) and the Sor River (B) and V. aestuarianus isolated from the Sor River (C). The results are presented as the mean and the SEM of two independent experimental infections (N=390 animals). Table 1. Detection of Vibrio splendidus (V. spl) and V. aestuarianus (V. aest) in environmental samples and macrofauna by qPCR. (+): positive detection; (-): negative detection; NA: not analysed.