Immunomodulatory effects of single and combined exposure to ZnO and TiO2 nanoparticles on rainbow trout challenged with Aeromonas salmonicida achromogenes.

The increasing release of engineered nanoparticles (ENPs) in aquatic ecosystems stresses the need for stringent investigations of nanoparticle mixture toxicity towards aquatic organisms. Here, the individual and combined immunotoxicity of two of the most consumed ENPs, the ZnO and the TiO2 ones, was investigated on rainbow trout juveniles (Oncorhynchus mykiss). Fish were exposed to environmentally realistic concentrations (21 and 210 µg L-1 for the ZnO and 210 µg L-1 for the TiO2) for 28 days, and then challenged with the pathogenic bacterium, Aeromonas salmonicida achromogenes. Antioxidant and innate immune markers were assessed before and after the bacterial infection. None of the experimental conditions affected the basal activity of the studied innate immune markers and the redox balance. However, following the bacterial infection, the expression of genes coding for pro and anti-inflammatory cytokines (il1ß and il10), as well as innate immune compounds (mpo) were significantly reduced in fish exposed to the mixture. Conversely, exposure to ZnO NPs alone seemed to stimulate the immune response by enhancing the expression of the IgM and c3 genes for instance. Overall, our results suggest that even though the tested ENPs at their environmental concentration do not strongly affect basal immune functions, their mixture may alter the development of the immune response when the organism is exposed to a pathogen by interfering with the inflammatory response.

Mobile Colistin Resistance Enzyme MCR-3 Facilitates Bacterial Evasion of Host Phagocytosis.

Mobile colistin resistance enzyme MCR-3 is a phosphoethanolamine transferase modifying lipid A in Gram-negative bacteria. MCR-3 generally mediates low-level (≤8 mg L-1 ) colistin resistance among Enterobacteriaceae, but occasionally confers high-level (>128 mg L-1 ) resistance in aeromonads. Herein, it is determined that MCR-3, together with another lipid A modification mediated by the arnBCADTEF operon, may be responsible for high-level colistin resistance in aeromonads. Lipid A is the critical site of pathogens for Toll-like receptor 4 recognizing. However, it is unknown whether or how MCR-3-mediated lipid A modification affects the host immune response. Compared with the wild-type strains, increased mortality is observed in mice intraperitoneally-infected with mcr-3-positive Aeromonas salmonicida and Escherichia coli strains, along with sepsis symptoms. Further, mcr-3-positive strains show decreased clearance rates than wild-type strains, leading to bacterial accumulation in organs. The increased mortality is tightly associated with the increased tissue hypoxia, injury, and post-inflammation. MCR-3 expression also impairs phagocytosis efficiency both in vivo and in vitro, contributing to the increased persistence of mcr-3-positive bacteria in tissues compared with parental strains. This study, for the first time, reveals a dual function of MCR-3 in bacterial resistance and pathogenicity, which calls for caution in treating the infections caused by mcr-positive pathogens.

Immunodetection of rainbow trout IL-8 cleaved-peptide: Tissue bioavailability and potential antibacterial activity in a bacterial infection context.

Chemokines such as IL-8 are part of an important group of proinflammatory response molecules, as well as cell recruitment. However, it has been described in both higher vertebrates and fish that IL-8 has an additional functional role by acting as an antimicrobial effector, either directly or by cleavage of a peptide derived from its C-terminal end. Nevertheless, it is still unknown whether this fragment is released in the context of infection by bacterial pathogens and if it could be immunodetected in tissues of infected salmonids. Therefore, the objective of this research was to demonstrate that the C-terminal end of IL-8 from Oncorhynchus mykiss is cleaved, retaining its antibacterial properties, and that is detectable in tissues of infected rainbow trout. SDS-PAGE and mass spectrometry demonstrated the cleavage of a fragment of about 2 kDa when the recombinant IL-8 was subjected to acidic conditions. By chemical synthesis, it was possible to synthesize this fragment called omIL-8α80-97 peptide, which has antibacterial activity against Gram-negative and Gram-positive bacteria at concentrations over 10 µM. Besides, by fluorescence microscopy, it was possible to locate the omIL-8α80-97 peptide both on the cell surface and in the cytoplasm of the bacteria, as well as inside the monocyte/macrophage-like cell. Finally, by indirect ELISA, Western blot, and mass spectrometry, the presence of the fragment derived from the C-terminal end of IL-8 was detected in the spleen of trout infected with Piscirickettsia salmonis. The results reported in this work present the first evidence about the immunodetection of an antibacterial, and probably cell-penetrating peptide cleaved from the C-terminal end of IL-8 in monocyte/macrophage-like cell and tissue of infected rainbow trout.

Agreement between the categorization of isolates of Aeromonas salmonicida and Yersinia ruckeri by disc diffusion and MIC tests performed at 22℃.

Standard disc diffusion and MIC test procedure were used to investigate the susceptibility of two hundred and fifty-one isolates collected from infected fish in France to florfenicol, oxolinic acid and tetracycline. The tests were performed at 22 ± 2℃ and for the 177 Yersinia ruckeri they were read after 24-28 hr incubation and for the 74 Aeromonas salmonicida isolates they were read after 44-48 hr. Applying epidemiological cut-off values to the susceptibility data generated in these tests, the isolates were categorized as wild-type or non-wild-type. The agent-specific categories into each isolate were placed on the basis of the data generated by the two methods were in agreement in 98% of the determinations made. It is argued that, with respect to categorising isolates, disc diffusion and MIC methods can be considered as equally valid at this temperature and after both periods of incubation.

Antibiotics modulate biofilm formation in fish pathogenic isolates of atypical Aeromonas salmonicida.

Atypical Aeromonas salmonicida causes furunculosis infections of non-salmonid fish, which requires antibiotic therapy. However, antibiotics may induce biofilm in some bacteria, which protects them against hostile conditions while allowing them to persist on surfaces, thus forming a reservoir for infection. The aim of this study was to determine whether atypical isolates of A. salmonicida increased biofilm in the presence of two antibiotics, florfenicol and oxytetracycline. A microtitre plate assay was used to quantify biofilm in the presence and absence of each antibiotic. Fifteen of 28 isolates formed biofilms under control conditions, while 23 of 28 isolates increased biofilm formation in the presence of at least one concentration of at least one antibiotic. For oxytetracycline, the most effective concentration causing biofilm to increase was one-quarter of that preventing visible bacterial growth, whereas for florfenicol it was one-half of this value. This is the first study to demonstrate that a bacterial pathogen of fish increases biofilm in response to antibiotics. Biofilm formation may increase the risk of re-infection in culture systems and this lifestyle favours the transmission of genetic material, which has implications for the dissemination of antibiotic-resistance genes and demonstrates the need for enhanced disease prevention measures against atypical A. salmonicida.

The determination of antimicrobial susceptibility by MIC and epidemiological cut-off values and the detection of resistance genes in Aeromonas species isolated from cultured fish.

The present study was aimed at determining antimicrobial susceptibility by a CLSI standard microdilution testing protocol and detecting the resistance genes of motile Aeromonas species isolated from cultured fish. The importance of the minimum inhibitory concentrations was assessed based on statistically determined epidemiological cut-off values calculated by normalized resistance analysis. Unfortunately, CLSI epidemiological cut-off values are available only for Aeromonas salmonicida, and there is no further detailed data on Aeromonas isolated from aquatic animals. The antimicrobial susceptibilities of pre-identified motile Aeromonas species to florfenicol, tetracycline and sulfamethoxazole were determined by calculating epidemiological cut-off values with fully automated and freely available Excel spreadsheets, applying the normalized resistance interpretation (NRI) method. Furthermore, the presence of the antimicrobial resistance genes floR, tetA, tetB, tetC, tetD, tetE, tetH, sulI, sulII and sulIII was detected by PCR analysis and confirmed by sequence analysis. The presence of up to six different genes (multiple antimicrobial resistance) was determined in the Aeromonas isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: In this study, we investigated phenotypic and genotypic antimicrobial resistance characteristics by a novel method based on epidemiological cut-off values. This is the second comprehensive study on the antimicrobial susceptibility characteristics of Aeromonas species using NRI and epidemiological cut-off values. The present research is related to our previous researches focussed on the identification of motile Aeromonads, their prevalence in relation to different fish lengths, seasons and regions, and covered the investigation of Lactococcus garvieae, Yersinia ruckeri, Flavobacterium spp., Enterobacter spp. and Citrobacter spp.

Characterization of Aeromonas salmonicida and A. sobria isolated from cultured salmonid fish in Korea and development of a vaccine against furunculosis.

Previously, Aeromonas sobria and A. salmonicida were identified to be the most prevalent species in salmonid farms in Korea. In this study, we evaluated the biochemical characteristics, antibiotic susceptibility and pathogenicity of A. salmonicida (3 isolates) and A. sobria (8 isolates) isolated from salmonids, and further investigated efficacy of A. salmonicida vaccine. In antibiotic susceptibility test, all of A. sobria isolates were resistant to amoxicillin and ampicillin. Six A. sobria and two A. salmonicida isolates were resistant to oxytetracycline. In challenge test, A. sobria isolates exhibited low pathogenicity in rainbow trout (Oncorhynchus mykiss) while one A. salmonicida isolate showed high pathogenicity with LD50 of 6.4 × 103  CFU/fish in rainbow trout and coho salmon (Oncorhynchus kisutch). Among virulence factors, secretion apparatus (ascV and ascC) and transcription regulatory protein (exsA) of type 3 secretion system and A-layer protein genes were differentially detected in DNA or cDNA of A. salmonicida isolates, indicating their contribution to the pathogenicity. A formalin-killed vaccine of highly pathogenic A. salmonicida isolate exhibited a protective effect with relative survival rate of 81.8% and 82.9% at 8 weeks and 16 weeks post-vaccination, respectively, in challenge test.

In vitro antimicrobial effect of various commercial essential oils and their chemical constituents on Aeromonas salmonicida subsp. salmonicida.

AIMS: This study aimed to evaluate in vitro efficacy of essential oils (EOs) and their compounds (EOCs) alone or in combination against Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in salmonid fish. METHODS AND RESULTS: Antimicrobial activity of 13 EOs and 16 EOCs was investigated for four A. salmonicida subsp. salmonicida strains using broth microdilution. The checkerboard assay was used to evaluate a putative synergy between the most efficient EOs and EOCs against the tested strains. Cinnamon bark, oregano, clove, and thyme oils and their major compounds cinnamaldehyde, eugenol, carvacrol and thymol showed the lower minimum inhibitory concentration and minimum bactericidal concentration values. The association of cinnamaldehyde and eugenol (V/V: 30%/70%) showed a synergistic activity against three tested strains. The combinations of cinnamon with oregano, clove or thyme EOs showed a neutral or additive activity against all the tested strains. CONCLUSIONS: Cinnamon, oregano, clove and thyme oils and their major phytochemical compounds showed strong activities against A. salmonicida subsp. salmonicida strains. SIGNIFICANCE AND IMPACT OF THE STUDY: To reduce the use of antibiotics in aquaculture, phytochemicals such as cinnamaldehyde and eugenol can be tested alone or in combination in in vivo studies as functional feed alternatives.

Kuma Bamboo Grass (Sasa veitchii) Extracts Exhibit Protective Effects Against Atypical Aeromonas salmonicida Infection in Goldfish (Carassius auratus).

Atypical Aeromonas salmonicida ( i.e. subsp. achromogenes and subsp. masoucida) are one of the major opportunistic pathogens that cause ulcer diseases in a variety of fishes, in which this pathogen has become a worldwide economic threat in sectors that handle of particular high-priced ornamental fishes like varicolored carp and goldfish due to appearance damages. Here we reported that the kuma bamboo grass (Sasa veitchii) extracts (KBGE) that contained a variety of fatty acids, exhibited antibacterial activity against nine Aeromonas strains including 5 atypical A. salmonicida strains. Experimental challenges with four atypical A. salmonicida strains revealed that supplementation with 375 to 750 µg/ml of the KBGE restored the survival of goldfish in coincidence of inhibition of both bacterial replication and superoxide dismutase (SOD) activity upon infection, compared with those of untreated control. Together, our data demonstrating the antibacterial effects of the plant extracts proposes its possible implication for prevention of Aeromonas infection in the ornamental fish.

The Outer Membrane Protein FstC of Aeromonas salmonicida subsp. salmonicida Acts as Receptor for Amonabactin Siderophores and Displays a Wide Ligand Plasticity. Structure-Activity Relationships of Synthetic Amonabactin Analogues.

Amonabactins are a group of four related catecholate siderophores produced by several species of the genus Aeromonas, including A. hydrophila and the fish pathogen A. salmonicida subsp. salmonicida. Although the gene cluster encoding amonabactin biosynthesis also contains a gene that could encode the ferri-siderophore receptor (fstC), to date there is no experimental evidence to explain its role. In this work, we report the identification of the amonabactins' outer membrane receptor and the determination of the minimal structural parts of these siderophores involved in the molecular recognition by their cognate receptor. The four natural amonabactin forms (P750, T789, P693, and T732) and some mono and biscatecholate amonabactin analogues were chemically synthesized, and their siderophore activity on A. salmonicida FstC(+) and FstC(-) strains was evaluated. The results showed that each amonabactin form has quite different growth promotion activity, with P750 and T789 the most active. The outer membrane receptor FstC recognizes more efficiently biscatecholate siderophores in which the length of the linker between the two iron-binding catecholamide units is 15 atoms (P750 and T789) instead of 12 atoms (P693 and T732). Analysis of the siderophore activity of synthetic analogues indicated that the presence of Phe or Trp residues is not required for siderophore recognition. The results together point toward evidence that the amonabactin receptor FstC admits a high degree of ligand plasticity. We also showed that FstC is present in most Aeromonas species, including relevant human and animal pathogens as A. hydrophila. From the results obtained, we concluded that the ferri-amonabactin uptake pathway involving the outer membrane transporter FstC possesses a considerable functional plasticity that could be exploited for delivery of antimicrobial compounds into the cell. This would allow the use of the siderophore-based iron uptake mechanisms to combat infections caused by species of the genus Aeromonas.

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm.

Our ability to predict evolutionary trajectories of pathogens in response to antibiotic pressure is one of the promising leverage to fight against the present antibiotic resistance worldwide crisis. Yet, few studies tackled this question in situ at the outbreak level, due to the difficulty to link a given pathogenic clone evolution with its precise antibiotic exposure over time. In this study, we monitored the real-time evolution of an Aeromonas salmonicida clone in response to successive antibiotic and vaccine therapies in a commercial fish farm. The clone was responsible for a four-year outbreak of furunculosis within a Recirculating Aquaculture System Salmo salar farm in China, and we reconstructed the precise tempo of mobile genetic elements (MGEs) acquisition events during this period. The resistance profile provided by the acquired MGEs closely mirrored the antibiotics used to treat the outbreak, and we evidenced that two subclonal groups developed similar resistances although unrelated MGE acquisitions. Finally, we also demonstrated the efficiency of vaccination in outbreak management and its positive effect on antibiotic resistance prevalence. Our study provides unprecedented knowledge critical to understand evolutionary trajectories of resistant pathogens outside the laboratory.

Effects of Vitamin D2 (Ergocalciferol) and D3 (Cholecalciferol) on Atlantic Salmon (Salmo salar) Primary Macrophage Immune Response to Aeromonas salmonicida subsp. salmonicida Infection.

Vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) are fat-soluble secosteroid hormones obtained from plant and animal sources, respectively. Fish incorporates vitamin D2 and D3 through the diet. In mammals, vitamin D forms are involved in mineral metabolism, cell growth, tissue differentiation, and antibacterial immune response. Vitamin D is an essential nutrient in aquafeeds for finfish. However, the influence of vitamin D on fish cell immunity has not yet been explored. Here, we examined the effects of vitamin D2 and vitamin D3 on Salmo salar primary macrophage immune response to A. salmonicida subspecies salmonicida infection under in vitro conditions. We determined that high concentrations of vitamin D2 (100,000 ng/ml) and D3 (10,000 ng/ml) affect the growth of A. salmonicida and decrease the viability of S. salar primary macrophages. In addition, we determined that primary macrophages pre-treated with a biologically relevant concentration of vitamin D3 for 24 h showed a decrease of A. salmonicida infection. In contrast, vitamin D2 did not influence the antibacterial activity of the S. salar macrophages infected with A. salmonicida. Vitamin D2 and D3 did not influence the expression of canonical genes related to innate immune response. On the other hand, we found that A. salmonicida up-regulated the expression of several canonical genes and suppressed the expression of leukocyte-derived chemotaxin 2 (lect-2) gene, involved in neutrophil recruitment. Primary macrophages pre-treated for 24 h with vitamin D3 counteracted this immune suppression and up-regulated the transcription of lect-2. Our results suggest that vitamin D3 affects A. salmonicida attachment to the S. salar primary macrophages, and as a consequence, the A. salmonicida invasion decreased. Moreover, our study shows that the positive effects of vitamin D3 on fish cell immunity seem to be related to the lect-2 innate immunity mechanisms. We did not identify positive effects of vitamin D2 on fish cell immunity. In conclusion, we determined that the inactive form of vitamin D3, cholecalciferol, induced anti-bacterial innate immunity pathways in Atlantic salmon primary macrophages, suggesting that its utilization as a component of a healthy aquafeed diet in Atlantic salmon could enhance the immune response against A. salmonicida.

Silver nanoparticles: Their role as antibacterial agent against Aeromonas salmonicida subsp. salmonicida in rainbow trout (Oncorhynchus mykiss).

The rise of bacterial resistance to antibiotics is one of the great challenges of our age. One of the strategies to limit the development of antibiotics resistance is the investigation of alternative antimicrobials. As silver nanoparticles demonstrated a potent bactericidal activity in vitro, the aim of this study was to evaluate the in vivo antibacterial activity of silver nanoparticles against Aeromonas salmonicida subsp. salmonicida. Rainbow trout (n = 120) were divided into four groups of 30 fish each. First group was challenged with A. salmonicida (Positive control), the second group was challenged with A. salmonicida and exposed to silver nanoparticles by immersion for three hours (100 µg/L), the third group was challenged with A. salmonicida and intraperitoneally injected with silver nanoparticles (17 µg/mL) and the fourth group was sham-treated and served as a negative control group. At the 7th day post challenge, histopathology of the positive control group revealed the presence of bacterial aggregates in tissues with degenerative and necrotic changes, while at the 35th day post challenge, only liver necrosis persisted. Silver nanoparticles-treated and negative control groups did not show any clinical signs, mortalities or histopathological alterations and they were tested negative for A. salmonicida. The immersion in silver nanoparticles did not result in detectable residues of silver in the muscles 35 days after treatment. These findings demonstrate the antibacterial properties of silver nanoparticles against A. salmonicida infection. Therefore, they could be used for development of antibacterial agents in aquaculture.

Dissection of the antimicrobial and hemolytic activity of Cap18: Generation of Cap18 derivatives with enhanced specificity.

Due to the rapid emergence of resistance to classical antibiotics, novel antimicrobial compounds are needed. It is desirable to selectively kill pathogenic bacteria without targeting other beneficial bacteria in order to prevent the negative clinical consequences caused by many broad-spectrum antibiotics as well as reducing the development of antibiotic resistance. Antimicrobial peptides (AMPs) represent an alternative to classical antibiotics and it has been previously demonstrated that Cap18 has high antimicrobial activity against a broad range of bacterial species. In this study we report the design of a positional scanning library consisting of 696 Cap18 derivatives and the subsequent screening for antimicrobial activity against Y. ruckeri, A. salmonicida, S. Typhimurium and L. lactis as well as for hemolytic activity measuring the hemoglobin release of horse erythrocytes. We show that the hydrophobic face of Cap18, in particular I13, L17 and I24, is essential for its antimicrobial activity against S. Typhimurium, Y. ruckeri, A. salmonicida, E. coli, P. aeruginosa, L. lactis, L. monocytogenes and E. faecalis. In particular, Cap18 derivatives harboring a I13D, L17D, L17P, I24D or I24N substitution lost their antimicrobial activity against any of the tested bacterial strains. In addition, we were able to generate species-specific Cap18 derivatives by particular amino acid substitutions either in the hydrophobic face at positions L6, L17, I20, and I27, or in the hydrophilic face at positions K16 and K18. Finally, our data showed the proline residue at position 29 to be essential for the inherent low hemolytic activity of Cap18 and that substitution of the residues K16, K23, or G21 by any hydrophobic residues enhances the hemolytic activity. This study demonstrates the potential of generating species-specific AMPs for the selective elimination of bacterial pathogens.

Identification of a moronecidin-like antimicrobial peptide in the venomous fish Pterois volitans: Functional and structural study of pteroicidin-α.

The present study characterizes for the first time an antimicrobial peptide in lionfish (Pterois volitans), a venomous fish. Using a peptidomic approach, we identified a mature piscidin in lionfish and called it pteroicidin-α. We detected an amidated form (pteroicidin-α- CONH2) and a non-amidated form (pteroicidin-α-COOH), and then performed their functional and structural study. Interestingly, the two peptides displayed different antibacterial and hemolytic activity levels. Pteroicidin-α-CONH2 was bactericidal on human pathogens like Staphylococcus aureus or Escherichia coli, as well as on the fish pathogen Aeromonas salmonicida, while pteroicidin-α-COOH only inhibited their growth. Furthermore, the two peptides induced hemolysis of red blood cells from different vertebrates, namely humans, sea bass and lesser-spotted dogfish. Hemolysis occurred with low concentrations of pteroicidin-α-CONH2, indicating greater toxicity of the amidated form. Circular dichroism analysis showed that both peptides adopted a helical conformation, yet with a greater α-helix content in pteroicidin-α-CONH2. Overall, these results suggest that amidation strongly influences pteroicidin-α by modifying its structure and its physico-chemical characteristics and by increasing its hemolytic activity.

Alternatives to mineral oil adjuvants in vaccines against Aeromonas salmonicida subsp. salmonicida in rainbow trout offer reductions in adverse effects.

In an effort to reduce the frequency and severity of adverse reactions seen from the use of mineral oil adjuvants in salmonid fish, the effects of two alternative adjuvants were assessed, focusing on the induction of adverse effects as well as protection. Using rainbow trout (Oncorhynchus mykiss) as recipients, injection vaccines based on formalin-inactivated Aeromonas salmonicida subspecies salmonicida were formulated with CpG oligodeoxynucleotides, the liposomal cationic adjuvant formulation 01 (CAF01) or with Freund's incomplete adjuvant and administered intraperitoneally. Control groups of unvaccinated, Tris-buffered saline-injected or bacterin-injected individuals were included, and each group included in the study held a total number of 240 individuals. Subsequently, individuals from each group were examined for differences in Fulton's condition factor, macro- and microscopic pathological changes, as well as protection against experimental infection with A. salmonicida. While adverse effects were not eliminated, reductions in microscopic and macroscopic adverse effects, in particular, were seen for both the nucleotide- and liposome-based vaccine formulations. Furthermore, the induced protection appears similar to that of the benchmark formulation, thus introducing viable, potential alternative types of adjuvants for use in future fish vaccines.

The protective effect of humic-rich substances on atypical Aeromonas salmonicida subsp. salmonicida infection in common carp (Cyprinus carpio L.).

When challenged with atypical Aeromonas salmonicida subsp. salmonicida, exposure of the common carp (Cyprinus carpio L.) to different humic-rich compounds resulted in a significant reduction in infection rates. Specifically, in fish exposed to (i) humic-rich water and sludge from a recirculating system, (ii) a synthetic humic acid, and (iii) a Leonardite-derived humic-rich extract, infection rates were reduced to 14.9%, 17.0% and 18.8%, respectively, as compared to a 46.8% infection rate in the control treatment. An additional set of experiments was performed to examine the effect of humic-rich components on the growth of the bacterial pathogen. Liquid culture medium supplemented with either humic-rich water from the recirculating system, the synthetic humic acid or the Leonardite humic-rich extract resulted in a growth reduction of 41.1%, 45.2% and 61.6%, respectively, as compared to the growth of the Aeromonas strain in medium devoid of humic substances. Finally, in a third set of experiments it was found that while the innate immune system of the carps was not affected by their exposure to humic-rich substances, their acquired immune system was affected. Fish, immunized against bovine serum albumin, displayed elevated antibody titres as compared to immunized carps which were not exposed to the various sources of humic substances.

In vitro effects of Italian Lavandula multifida L. leaf extracts on gilthead seabream (Sparus aurata) leucocytes and SAF-1 cells.

Lavandula multifida is very appreciated by pharmaceutical and cosmetic industries. In Italy is only found in Calabria and Sicily and, at present, urge its valorization due to its high extinction and genetic erosion risks. Possible applications of L. multifida extracts as immunostimulant in fish aquaculture were assayed by using gilthead seabream (Sparus aurata) as a marine fish model, due to its importance in fish aquaculture. The in vitro effects of both aqueous and ethanolic leaf extracts obtained from two Italian populations of L. multifida on head kidney leucocyte activities (viability, phagocytosis, respiratory burst and peroxidase content) were assessed. Furthermore, the possible cytotoxic effects of the extracts on SAF-1 cells and their bactericidal effects on three fish pathogenic bacteria (Vibrio harveyi, Vibrio anguillarum, Aeromonas salmonicida) were also evaluated. All the assays were performed in comparison with leaf extracts obtained from a widely-distributed species as L. angustifolia. Results showed that water and ethanolic leaf extracts obtained from L. multifida enhanced innate immune activities of S. aurata HK leucocytes. Furthermore, SAF-1 cell viability was not affected significantly after being incubated with the extracts. These extracts did not exert any bactericidal activity on the pathogenic bacterial strains tested in the present study. Results obtained in the present work suggested the possibility of use such extracts in in vivo studies in order to corroborate the possibility of their use in aquaculture. Their use could prevent to improve fish defense against pathogenic infections through enhancement of the fish immune status.

Characterization and mechanism of anti-Aeromonas salmonicida activity of a marine probiotic strain, Bacillus velezensis V4.

The bacterium Aeromonas salmonicida is the causative agent of furunculosis, a systemic, ubiquitous disease of fish in the salmon family, characterized by high mortality and morbidity. Probiotics are a promising approach for prevention of furunculosis in aquaculture. A bacterial strain with anti-A. salmonicida properties, Bacillus velezensis V4, was isolated and the mechanisms underlying these properties were investigated. Anti-A. salmonicida compounds present in cell-free supernatant of V4 were purified and structurally identified as members of the iturin, macrolactin, and difficidin groups. The compounds contributed jointly to inhibition of A. salmonicida, and the diversity of the compounds was related to the versatility of their mode of action. Addition of the compounds to A. salmonicida cell suspensions reduced cell density. Analyses by confocal microscopy and scanning electron microscopy revealed cell membrane disruption, deletion of cellular content, and cell lysis of A. salmonicida. The V4 genome was sequenced, and gene clusters involved in synthesis of anti-Aeromonas compounds were detected and identified. A possible probiotic effect on growth performance of Oncorhynchus mykiss (rainbow trout) was investigated by addition of 0, 1, and 3 % (v/w) V4. Relative to control, mortality was reduced 27.25 % in the 1 % addition group and 81.86 % in the 3 % addition group. Feed coefficient ratio was reduced 19.49 % and weight gain ratio was increased 71.22 % in the 1 % addition group. Our findings demonstrate that V4 is an effective probiotic strain in O. mykiss and has clear potential for both control of furunculosis and growth promotion of aquaculture animals.