Exosomes are a class of extracellular vesicles released by bacteria and contain diverse biomolecules. In this study, we isolated exosomes from Vibrio harveyi and Vibrio anguillarum, which are both serious pathogens in mariculture, using a supercentrifugation method, and the proteins in the exosomes of these two vibrios were analyzed by LC-MS/MS proteomics. Exosome proteins released by V. harveyi and V. anguillarum were different; they not only contained virulence factors (such as lipase and phospholipase in V. harveyi, metalloprotease and hemolysin in V. anguillarum), but also participated in the important life activities of bacteria (such as fatty acid biosynthesis, biosynthesis of antibiotics, carbon metabolism). Subsequently, to verify whether the exosomes participated in bacterial toxicity, after Ruditapes philippinarum was challenged with V. harveyi and V. anguillarum, the corresponding genes of virulence factors from exosomes screened by proteomics were tested by quantitative real-time PCR. All the genes detected were upregulated which suggested that exosomes were involved in vibrio toxicity. The results could provide an effective proteome database for decoding the pathogenic mechanism of vibrios from the exosome perspective.

Accurate and rapid determination of bacterial disease agents of fish is an important step for sustainable and efficient aquaculture production. In general, biochemical and molecular methods are used for pathogen detection but they are usually time-consuming and required qualified personnel. Recently spectroscopic methods are preferred in clinical and food microbiology and declared as a promising alternative method for pathogens diagnosis with many advantages. In this study, the significant spectra of three important bacterial fish pathogens (Lactococcus garvieae, Vibrio anguillarum and Yersinia ruckeri) were determined by Raman spectroscopy. The first data of the pathogens were obtained and the distinctive differences in polysaccharides, nucleic acids, fatty acids and amino acids were identified. This preliminary study aimed to be pioneer for further studies in aquaculture and veterinary microbiology toward developing an alternative method for routine identification.

Vibrosis is one of the major threats in aquaculture farming, and due to the increasing antimicrobial resistance of different Vibrio species, there is an urgent need to replace conventional treatments with more sustainable solutions. Antimicrobial molecules such as organic acids (OA) and nature-identical compounds (NIC) are currently finding a central role in the infection management of terrestrial livestock, but little is known about their usage in aquaculture. The aim of this study was to perform a preliminary screening of the in vitro antimicrobial activity of a wide panel of OA and NIC against 2 Vibrio species characteristic of the Mediterranean area, Vibrio harveyi and Vibrio anguillarum, through minimal inhibitory/bactericidal concentration tests. The active principles that showed the best effective pathogen control were medium-chain fatty acids, sorbic and benzoic acid among OA and phenolic monoterpenoids (thymol, carvacrol and eugenol) and aromatic monoterpene aldehydes (vanillin and cinnamaldehyde) among NIC. These results showed how the usage of OA and NIC can open promising perspectives in terms of Vibrio spp. load control in aquaculture. Natural or nature-identical feed additives can make aquaculture production not only more efficient by reducing the need for medicated treatments, but also more sustainable.

The stress and immune-related effects of short-term (1, 6 and 24 h) air exposure stress (1 min), bath vaccination with Vibrio anguillarum bacterin, and both stressors combined were evaluated in liver and spleen of Sparus aurata, Danio rerio and Onchorhynchus mykiss. Expression profiles of immune (interleukin 1 beta: il1β; tumor necrosis factor alpha: tnfα; interleukin 10: il10; tumor growth factor beta: tgfβ1; immunoglobulin M: igm; lysozyme: lys; complement protein c3: c3) and stress-related genes (glucocorticoid receptor: gr; heat shock protein 70: hsp70; and enolase) were analysed by RT-qPCR. Cortisol level was assessed by radioimmunoassay. The gene expression patterns in liver and spleen were found to be differentially regulated in a time- and organ-dependent manner among species. In seabream, a higher il1β-driven inflammatory response was recorded. In zebrafish, air exposure stress but not bath vaccination alone modulated most of the changes in liver and spleen immune transcripts. Stressed and vaccinated trout showed an intermediate pattern of gene expression, with a lower upregulation of immune-related genes in liver and the absence of changes in the expression of hsp70 and enolase in spleen (as it was observed in seabream but not in zebrafish). Following air exposure, cortisol levels increased in plasma 1 h post-stress (hps) and then decreased at 6 hps in O. mykiss and D. rerio. By contrast, in S.aurata the cortisol level remained higher at 6 hps suggesting a greater degree of responsiveness to this stressor. When fish were exposed to combined air exposure plus bath vaccination cortisol levels were also augmented at 1 and 6 hps in O. mykiss and S.aurata and restored to basal level at 24 hps, whereas in D. rerio the response was higher in response to the combination of both stressors. In addition, V. anguillarum bacterin vaccination triggered cortisol secretion only in D. rerio, suggesting a greater responsiveness of D. rerio hypothalamic-pituitary-interrenal axis. Overall, comparing the tissue transcription responsiveness, liver was found to be more implicated in the response to handling stress compared to spleen. These results also indicate that a species-specific response accounts for the deviations of stress and immune onset in the liver and spleen in these fish species.

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Antimicrobial polypeptides (AMPPs) are humoral components of the vertebrates and invertebrates innate immune system. Their potent broad spectrum antimicrobial activities have drawn the attention of the scientific community to their potential use not only as an alternative to antibiotics but also as functional targets for immunostimulants in order to enhance the host immunity. Fish synthesize a great number of these peptides but in European sea bass, an important fish species in the Mediterranean aquaculture, only a few AMPPs have been studied and these surveys have highlighted their functional role as predictive markers of stressful conditions. Many aspects concerning AMPP mode of action in the host during bacterial infections are still unknown. In this work a 72 h time course experiment, performed on juvenile sea bass intraperitoneally (i.p.) injected with a sub-lethal dose of Vibrio anguillarum, was aimed to investigate the mRNA expression of four specific AMPP genes and interleukin-1β (IL-1β) in skin, gills, spleen, and head kidney. AMPP genes were: dicentracin (DIC), histone-like protein 1 (HLP-1), histone-like protein 2 (HLP-2) and hemoglobin-like protein (Hb-LP). The delta-delta C(T) method in real-time RT-PCR allowed to gain more knowledge about temporal dynamics, preferential sites of expression as well as immunological and physiological role of these molecular markers. DIC was significantly up-regulated mainly in head kidney at 1.5-3 h post-infection (p.i.). HLP-1 showed an extended-time overexpression in gills and a significant up-regulation in spleen. HLP-2 was interestingly overexpressed in gills at 24 h p.i., while Hb-LP showed a significant up-regulation in skin for all the 72 h trial as well as lower but always significant values either in gills or in spleen. Different was the response of IL-1β that showed a dramatic up-regulation in spleen and head kidney at 8 h p.i. whilst in gills it displayed a severe inhibition. During this survey the i.p. stimulus surely conditioned the AMPP expression in skin and gills, especially as regards the DIC that as piscidin-related gene has an important defensive role in the mucosal tissues. However, two unconventional AMPP genes such as HLP-2 and Hb-LP, strictly related to the physiological mechanisms of fish, were less affected in terms of expression by the route of infection, being more evident in peripheral loci. These findings might suggest them as potential markers to be analyzed within plans of health survey in fish farms.