Setting epidemiological cut-off values relevant to MIC and disc diffusion data for Aeromonas salmonicida generated by a standard method.

The Clinical and Laboratory Standards Institute has published epidemiological cut-off values for susceptibility data generated at 22°°C and read after 44-48 h for florfenicol, oxolinic acid and oxytetracycline against Aeromonas salmonicida. The cut-off values for the minimum inhibitory concentration (MIC) and disc diffusion were derived from data obtained by 1 laboratory and 2 laboratories respectively. The present work reports the generation of susceptibility data from additional laboratories and the calculation of provisional cut-off values from aggregations of these data with previously published data. With respect to MIC data, the provisional cut-off values, derived from aggregations of the data from 4 laboratories, were ≤4 µg ml-1 for florfenicol, ≤0.0625 µg ml-1 for oxolinic acid and ≤1 µg ml-1 for oxytetracycline. For disc diffusion data, the provisional cut-off values derived from aggregations of the data from 5 laboratories were ≥30 mm for florfenicol, ≥32 mm for oxolinic acid and ≥25 mm for oxytetracycline. In addition, a cut-off value of ≥29 mm for ampicillin was derived from the aggregation of data from 4 laboratories.

Characterization of the rainbow trout (Oncorhynchus mykiss) mucosal glycosphingolipid repertoire and Aeromonas salmonicida binding to neutral glycosphingolipids.

Infections pose a challenge for the fast growing aquaculture sector. Glycosphingolipids are cell membrane components that pathogens utilize for attachment to the host to initiate infection. Here, we characterized rainbow trout glycosphingolipids from five mucosal tissues using mass spectrometry and nuclear magnetic resonance and investigated binding of radiolabeled Aeromonas salmonicida to the glycosphingolipids on thin-layer chromatograms. 12 neutral and 14 acidic glycosphingolipids were identified. The glycosphingolipids isolated from the stomach and intestine were mainly neutral, whereas glycosphingolipids isolated from the skin, gills and pyloric caeca were largely acidic. Many of the acidic structures were poly-sialylated with shorter glycan structures in the skin compared to the other tissues. The sialic acids found were Neu5Ac and Neu5Gc. Most of the glycosphingolipids had isoglobo and ganglio core chains, or a combination of these. The epitopes on the rainbow trout glycosphingolipid glycans differed between epithelial sites leading to differences in pathogen binding. A major terminal epitope was fucose, that occurred attached to GalNAc in a α1-3 linkage but also in the form of HexNAc-(Fuc-)HexNAc-R. A. salmonicida were shown to bind to neutral glycosphingolipids from the gill and intestine. This study is the first to do a comprehensive investigation of the rainbow trout glycosphingolipids and analyze binding of A. salmonicida to glycosphingolipids. The structural information paves the way for identification of ways of interfering in pathogen colonization processes to protect against infections in aquaculture and contributes towards understanding A. salmonicida infection mechanisms.

Genome-Wide Identification and Interaction Analysis of Turbot Heat Shock Protein 40 and 70 Families Suggest the Mechanism of Chaperone Proteins Involved in Immune Response after Bacterial Infection.

Hsp40-Hsp70 typically function in concert as molecular chaperones, and their roles in post-infection immune responses are increasingly recognized. However, in the economically important fish species Scophthalmus maximus (turbot), there is still a lack in the systematic identification, interaction models, and binding site analysis of these proteins. Herein, 62 Hsp40 genes and 16 Hsp70 genes were identified in the turbot at a genome-wide level and were unevenly distributed on 22 chromosomes through chromosomal distribution analysis. Phylogenetic and syntenic analysis provided strong evidence in supporting the orthologies and paralogies of these HSPs. Protein-protein interaction and expression analysis was conducted to predict the expression profile after challenging with Aeromonas salmonicida. dnajb1b and hspa1a were found to have a co-expression trend under infection stresses. Molecular docking was performed using Auto-Dock Tool and PyMOL for this pair of chaperone proteins. It was discovered that in addition to the interaction sites in the J domain, the carboxyl-terminal domain of Hsp40 also plays a crucial role in its interaction with Hsp70. This is important for the mechanistic understanding of the Hsp40-Hsp70 chaperone system, providing a theoretical basis for turbot disease resistance breeding, and effective value for the prevention of certain diseases in turbot.

Comparative safety and efficacy of autogenous vaccine administrated by different routes against furunculosis caused by Aeromonas salmonicida sub. salmonicida in large Rainbow trout (Oncorhynchus mykiss).

The development and growth of fish farming are hindered by viral and bacterial infectious diseases, which necessitate effective disease control measures. Furunculosis, primarily caused by Aeromonas salmonicida, stands out as a significant bacterial disease affecting salmonid fish farms, particularly rainbow trout. Vaccination has emerged as a crucial tool in combating this disease. The objective of this experiment was to assess and compare the efficacy and duration of different vaccine protocols against furunculosis in large trout under controlled rearing conditions, utilizing single and booster administrations via intraperitoneal, oral, and immersion routes. Among the various vaccination protocols tested, only those involving intraperitoneal injection, administered at least once, proved truly effective in preventing the expression of clinical signs of furunculosis and reducing mortality rates. A single intraperitoneal administration provided protection for up to 2352°-days, equivalent to approximately 5 months in water at 16 °C. However, intraperitoneal vaccination may lead to reduced growth in the fish due to resultant intraperitoneal adhesions. Additionally, protocols incorporating booster doses via intraperitoneal injection demonstrated efficacy regardless of the administration route of the primary vaccination. Nevertheless, the use of booster vaccinations via the intraperitoneal route did not confer any significant advantage over a single intraperitoneal injection in terms of efficacy.

PACAP binds conserved receptors and modulates cytokine gene expression and protein secretion in trout cell lines.

Antimicrobial peptides (AMPs) are an alternative to antibiotics for treatment and prevention of infections with a lower risk of bacterial resistance. Pituitary adenylate cyclase activating polypeptide (PACAP) is an outstanding AMP with versatile effects including antimicrobial activity and modulation of immune responses. The objective of this research was to study PACAP immunomodulatory effect on rainbow trout cell lines infected with Aeromonas salmonicida. PACAP from Clarias gariepinus (PACAP1) and a modified PACAP (PACAP5) were tested. RT-qPCR results showed that il1b and il8 expression in RTgutGC was significantly downregulated while tgfb expression was upregulated after PACAP treatment. Importantly, the concentration of IL-1ß and IFN-γ increased in the conditioned media of RTS11 cells incubated with PACAP1 and exposed to A. salmonicida. There was a poor correlation between gene expression and protein concentration, suggesting a stimulation of the translation of IL-1ß protein from previously accumulated transcripts or the cleavage of accumulated IL-1ß precursor. In-silico studies of PACAP-receptor interactions showed a turn of the peptide characteristic of PACAP-PAC1 interaction, correlated with the higher number of interactions observed with this specific receptor, which is also in agreement with the higher PACAP specificity described for PAC1 compared to VPAC1 and VPACA2. Finally, the in silico analysis revealed nine amino acids related to the PACAP receptor-associated functionality.

A bacterial ghost vaccine against Aeromonas salmonicida infection in turbot (Scophthalmus maximus).

Aeromonas salmonicida is one of the most prevalent pathogens that causes huge economic losses to aquaculture. Effective vaccination is the first choice for preventing infection. Bacterial ghost (BG), an empty bacterial shell devoid of cytoplasm, is a promising vaccine antigen with distinct advantages. Herein, we established strategies for producing a substantial yield of A. salmonicida ghost (ASG) and investigated the immune-protective properties of it. As a result, 2.84 mg/ml NaOH was discovered to be capable of inducing considerable amounts of ASG. Furthermore, the ASG vaccine elicited adaptive immunity in turbots after rapid activation of innate immunity. Even though formalin-killed cells (FKC) produced a few more antibodies than ASG, ASG ultimately provided a much stronger immune protection effect because it strengthened cellular immunity, with a relative percentage survival (RPS) of 50.1 % compared to FKC. These findings demonstrated that ASG effectively activated cell-mediated immunity, which helped get rid of microorganisms inside cells. Therefore, this study presented novel perspectives for future research on furunculosis vaccine products based on ASG as an antigen.

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.

Role of cold shock proteins B and D in Aeromonas salmonicida subsp. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus).

Cold shock proteins (Csp) are pivotal nucleic acid binding proteins known for their crucial roles in the physiology and virulence of various bacterial pathogens affecting plant, insect, and mammalian hosts. However, their significance in bacterial pathogens of teleost fish remains unexplored. Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is a psychrotrophic pathogen and the causative agent of furunculosis in marine and freshwater fish. Four csp genes (cspB, cspD, cspA, and cspC) have been identified in the genome of A. salmonicida J223 (wild type). Here, we evaluated the role of DNA binding proteins, CspB and CspD, in A. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus). A. salmonicida ΔcspB, ΔcspD, and the double ΔcspBΔcspD mutants were constructed and characterized. A. salmonicida ΔcspB and ΔcspBΔcspD mutants showed a faster growth at 28°C, and reduced virulence in lumpfish. A. salmonicida ΔcspD showed a slower growth at 28°C, biofilm formation, lower survival in low temperatures and freezing conditions (-20°C, 0°C, and 4°C), deficient in lipopolysaccharide synthesis, and low virulence in lumpfish. Additionally, ΔcspBΔcspD mutants showed less survival in the presence of bile compared to the wild type. Transcriptome analysis revealed that 200, 37, and 921 genes were differentially expressed in ΔcspB, ΔcspD, and ΔcspBΔcspD, respectively. In ΔcspB and ΔcspBΔcspD virulence genes in the chromosome and virulence plasmid were downregulated. Our analysis indicates that CspB and CspD mostly act as a transcriptional activator, influencing cell division (e.g., treB), virulence factors (e.g., aexT), and ultimately virulence.

Sea water acclimation of rainbow trout (Oncorhynchus mykiss) modulates the mucosal transcript immune response induced by Vibrio anguillarum and Aeromonas salmonicida vaccine, and prevents further transcription of stress-immune genes in response to acute stress.

Mucosal tissues appear to be more important in fish than in mammals due to living in a microbial-rich aquatic milieu, yet the complex interaction between the immune and the neuroendocrine system in these tissues remains elusive. The aim of this work was to investigate the mucosal immune response in immunized rainbow trout vaccinated with Alpha ject vaccine (bivalent), kept in fresh water (FW) or transferred to seawater (SW), and to evaluate their response to acute stress (chasing). Acute stress resulted in higher levels of plasma cortisol (Sham + Stress and Vaccine + Stress). A similar response was observed in skin mucus, but it was lower in Vaccine + Stress compared with stressed fish. With a few exceptions, minimal alterations were detected in the transcriptomic profile of stress-immune gene in the skin of vaccinated and stressed fish in both FW and SW. In the gills, the stress elicited activation of key stress-immune components (gr1, mr, ß-ar, hsp70, c3, lysozyme, α-enolase, nadph oxidase, il1ß, il6, tnfα, il10 and tgfß1) in FW, but fewer immune changes were induced by the vaccine (nadph oxidase, il6, tnfα, il10 and igt) in both SW and FW. In the intestine, an array of immune genes was activated by the vaccine particularly those related with B cells (igm, igt) and T cells (cd8α) in FW with no stimulation observed in SW. Therefore, our survey on the transcriptomic mucosal response demonstrates that the immune protection conferred by the vaccine to the intestine is modulated in SW. Overall, our results showed: i) plasma and skin mucus cortisol showed no additional stress effect induced by prolonged SW acclimation, ii) the stress and immune response were different among mucosal tissues which indicates a tissue-specific response to specific antigens/stressor. Further, the results suggest that the systemic immune organs may be more implicated in infectious events in SW (as few changes were observed in the mucosal barriers of immunized fish in SW) than in FW.

Vegetable omega-3 and omega-6 fatty acids differentially modulate the antiviral and antibacterial immune responses of Atlantic salmon.

The immunomodulatory effects of omega-3 and omega-6 fatty acids are a crucial subject of investigation for sustainable fish aquaculture, as fish oil is increasingly replaced by terrestrial vegetable oils in aquafeeds. Unlike previous research focusing on fish oil replacement with vegetable alternatives, our study explored how the omega-6 to omega-3 polyunsaturated fatty acid (PUFA) ratio in low-fish oil aquafeeds influences Atlantic salmon's antiviral and antibacterial immune responses. Atlantic salmon were fed aquafeeds rich in soy oil (high in omega-6) or linseed oil (high in omega-3) for 12 weeks and then challenged with bacterial (formalin-killed Aeromonas salmonicida) or viral-like (polyriboinosinic polyribocytidylic acid) antigens. The head kidneys of salmon fed high dietary omega-3 levels exhibited a more anti-inflammatory fatty acid profile and a restrained induction of pro-inflammatory and neutrophil-related genes during the immune challenges. The high-omega-3 diet also promoted a higher expression of genes associated with the interferon-mediated signaling pathway, potentially enhancing antiviral immunity. This research highlights the capacity of vegetable oils with different omega-6 to omega-3 PUFA ratios to modulate specific components of fish immune responses, offering insights for future research on the intricate lipid nutrition-immunity interplay and the development of novel sustainable low-fish oil clinical aquaculture feeds.

Inactivated Aeromonas salmonicida impairs adaptive immunity in lumpfish (Cyclopterus lumpus).

Aeromonas salmonicida, a widely distributed aquatic pathogen causing furunculosis in fish, exhibits varied virulence, posing challenges in infectious disease and immunity studies, notably in vaccine efficacy assessment. Lumpfish (Cyclopterus lumpus) has become a valuable model for marine pathogenesis studies. This study evaluated several antigen preparations against A. salmonicida J223, a hypervirulent strain of teleost fish, including lumpfish. The potential immune protective effect of A. salmonicida bacterins in the presence and absence of the A-layer and extracellular products was tested in lumpfish. Also, we evaluated the impact of A. salmonicida outer membrane proteins (OMPs) and iron-regulated outer membrane proteins (IROMPs) on lumpfish immunity. The immunized lumpfish were intraperitoneally (i.p.) challenged with 104 A. salmonicida cells/dose at 8 weeks-post immunization (wpi). Immunized and non-immunized fish died within 2 weeks post-challenge. Our analyses showed that immunization with A. salmonicida J223 bacterins and antigen preparations did not increase IgM titres. In addition, adaptive immunity biomarker genes (e.g., igm, mhc-ii and cd4) were down-regulated. These findings suggest that A. salmonicida J223 antigen preparations hinder lumpfish immunity. Notably, many fish vaccines are bacterin-based, often lacking efficacy evaluation. This study offers crucial insights for finfish vaccine approval and regulations.

Climate change can impair bacterial pathogen defences in sablefish via hypoxia-mediated effects on adaptive immunity.

Low-oxygen levels (hypoxia) in aquatic habitats are becoming more common because of global warming and eutrophication. However, the effects on the health/disease status of fishes, the world's largest group of vertebrates, are unclear. Therefore, we assessed how long-term hypoxia affected the immune function of sablefish, an ecologically and economically important North Pacific species, including the response to a formalin-killed Aeromonas salmonicida bacterin. Sablefish were held at normoxia or hypoxia (100% or 40% air saturated seawater, respectively) for 6-16 weeks, while we measured a diverse array of immunological traits. Given that the sablefish is a non-model organism, this involved the development of a species-specific methodological toolbox comprised of qPCR primers for 16 key immune genes, assays for blood antibacterial defences, the assessment of blood immunoglobulin (IgM) levels with ELISA, and flow cytometry and confocal microscopy techniques. We show that innate immune parameters were typically elevated in response to the bacterial antigens, but were not substantially affected by hypoxia. In contrast, hypoxia completely prevented the ∼1.5-fold increase in blood IgM level that was observed under normoxic conditions following bacterin exposure, implying a serious impairment of adaptive immunity. Since the sablefish is naturally hypoxia tolerant, our results demonstrate that climate change-related deoxygenation may be a serious threat to the immune competency of fishes.

Revealing extracellular protein profile and excavating spoilage-related proteases of Aeromonas salmonicida based on multi-omics investigation.

Aeromonas is a ubiquitous aquatic bacteria, and it is a significant factor contributing to meat spoilage during processing and consumption. The abilities of Aeromonas salmonicida 29 and 57, which exhibit spoilage heterogeneity, to secrete protease, lipase, hemolysin, gelatinase, amylase, and lecithinase were confirmed by plate method. A total of 3948 proteins were identified by ITRAQ in extracellular secretions of A. salmonicida, and 16 proteases were found to be potentially related to spoilage ability. The complete genome sequence of A. salmonicida 57 consists of one circular chromosome and three plasmids, while A. salmonicida 29 consists of one circular chromosome, without a plasmid. Transcriptomic analysis revealed a significant number of DEGs were up-regulated in A. salmonicida 29, which were mainly enriched in metabolic pathways (e.g., amino acid metabolism, carbohydrate metabolism), indicating that A. salmonicida 29 had better potential to decompose and utilize nutrients in meat. Six protease genes (2 pepB, hap, pepA, ftsI, and pepD) were excavated by combined ITRAQ with transcriptome analysis, which potentially contribute to bacterial spoilage ability and exhibit universality among other dominant spoilage bacteria. This investigation provides new insights and evidence for elucidating metabolic and spoilage phenotypic differences and provides candidate genes and strategies for future prevention and control technology development.

Transcriptomic analysis of turbot (Scophthalmus maximus) treated with zymosan a reveals that lncRNAs and inflammation-related genes mediate the protection conferred against Aeromonas salmonicida.

Aeromonas salmonicida is one of the most harmful pathogens in finfish aquaculture worldwide. Immunostimulants such as ß-glucans are used to enhance the immunity of cultured fish. However, their effects on fish physiology are not completely understood. In the present work, we evaluated the effect of a single intraperitoneal (ip) injection of zymosan A on fish survival against A. salmonicida infection. A single administration of this compound protected fish against A. salmonicida challenge and reduce the bacterial load in the head kidney one week after its administration. Transcriptome analyses of head kidney samples revealed several molecular mechanisms involved in the protection conferred by zymosan A and their regulation by long noncoding RNAs. The transcriptome profile of turbot exposed only to zymosan A was practically unaltered one week after ip injection. However, the administration of this immunostimulant induced significant transcriptomic changes once the fish were in contact with the bacteria and increased the survival of the infected turbot. Our results suggest that the restraint of the infection-induced inflammatory response, the management of apoptotic cell death, cell plasticity and cellular processes involving cytoskeleton dynamics support the protective effects of zymosan A. All this information provides insights on the cellular and molecular mechanisms involved in the protective effects of this widely used immunostimulant.

Identification of the Csr global regulatory system mediated by small RNA decay in Aeromonas salmonicida.

We investigated the presence and functionality of the carbon storage regulator (Csr) system in Aeromonas salmonicida SWSY-1.411. CsrA, an RNA-binding protein, shared 89% amino acid sequence identity with Escherichia coli CsrA. CsrB/C sRNAs exhibited a typical stem-loop structure, with more GGA motifs, which bind CsrA, than E. coli. CsrD had limited sequence identity with E. coli CsrD; however, it contained the conserved GGDEF and EAL domains. Functional analysis in E. coli demonstrated that the Csr system of A. salmonicida influences glycogen biosynthesis, biofilm formation, motility, and stability of both CsrB and CsrC sRNAs. These findings suggest that in A. salmonicida, the Csr system affects phenotypes like its E. coli counterpart. In A. salmonicida, defects in csr homologs affected biofilm formation, motility, and chitinase production. However, glycogen accumulation and protease production were unaffected. The expression of flagellar-related genes and chitinase genes was suppressed in the csrA-deficient A. salmonicida. Northern blot analysis indicated the stabilization of CsrB and CsrC in the csrD-deficient A. salmonicida. Similar to that in E. coli, the Csr system in A. salmonicida comprises the RNA-binding protein CsrA, the sRNAs CsrB and CsrC, and the sRNA decay factor CsrD. This study underscores the conservation and functionality of the Csr system and raises questions about its regulatory targets and mechanisms in A. salmonicida.

Characterization of caspase gene family in Sebastes schlegelii and their expression profiles under Aeromonas salmonicida and Vibrio anguillarum infection.

The caspase, functioning as a proteinase, plays a crucial role in eukaryotic cell apoptosis, regulation of apoptosis, cellular growth, differentiation, and immunity. The identification of caspase gene family in Sebastes schlegelii is of great help to understand its antimicrobial research. In S. schlegelii, we totally identified nine caspase genes, including four apoptosis initiator caspases (caspase 2, caspase 8, caspase 9 and caspase 10), four apoptosis executioners (caspase 3a, caspase 3b, caspase 6, and caspase 7) and one inflammatory executioner (caspase 1). The duplication of caspase 3 genes on chr3 and chr8 may have been facilitated by whole genome duplication (WGD) events or other complex evolutionary processes. In general, the number of caspase genes relatively conserved in high vertebrates, while exhibiting variation in teleosts. Furthermore, syntenic analysis and phylogenetic relationships analysis supported the correct classification of these caspase gene family in S. schlegelii, especially for genes with duplicated copies. Additionally, the expression patterns of these caspase genes in different tissues of S. schlegelii under healthy conditions were assessed. The results revealed that the expression levels of most caspase genes were significantly elevated in the intestine, spleen, and liver. To further investigate the potential immune functions of these caspase genes in S. schlegelii, we challenged individuals with A. salmonicida and V. anguillarum, respectively. After infection with A. salmonicida, the expression levels of caspase 1 in the liver and spleen of S. schlegelii remained consistently elevated throughout the infection time points. The expression levels of most caspase family members in the intestine exhibited significant divergence following V. anguillarum infection. This study provides a comprehensive understanding of the caspase gene families in S. schlegelii, thereby establishing a solid foundation for further investigations into the functional roles of these caspase genes.

Aeromonas salmonicida, causative agent of salmonid furunculosis, isolated from the freshwater parasitic copepod, Salmincola californiensis.

Here, we provide evidence that the freshwater parasitic copepod, Salmincola californiensis, acts as a vector for Aeromonas salmonicida. While investigating the effects of S. californiensis on Chinoook salmon (Oncorhynchus tshawytscha), we tangentially observed that fish infected with the copepod developed furunculosis, caused by A. salmonicida. This occurred despite being reared in pathogen-free well water in a research facility with no prior history of spontaneous infection. We further investigated the possibility of S. californiensis to serve as a vector for the bacterium via detection of fluorescently labelled A. salmonicida inside the egg sacs from copepods in which the fish hosts were experimentally infected with GFP-A449 A. salmonicida. We then evaluated copepod egg sacs that were collected from adult Chinook salmon from a freshwater hatchery with A. salmonicida infections confirmed by either culture or PCR. The bacterium was cultured on tryptic soy agar plates from 75% of the egg sacs, and 61% were positive by PCR. These three separate experiments indicate an alternative tactic of transmission in addition to direct transmission of A. salmonicida in captivity. The copepod may play an important role in transmission of the bacterium when fish are more dispersed, such as in the wild.

Detection of Aeromonas salmonicida subsp. salmonicida infection in zebrafish by labelling bacteria with GFP and a fluorescent probe based on the siderophore amonabactin.

Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.

Transcriptome analysis of turbot (Scophthalmus maximus) kidney responses to inactivated bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda.

Turbot (Scophthalmus maximus) is a commercially important marine flatfish for global aquaculture. With intensive farming, turbot production is limited by several diseases, in which Aeromonas salmonicida and Edwardsiella tarda are two main causative agents. Vaccination is an effective and safe alternative to disease prevention compared to antibiotic treatment. In the previous study, we developed an inactivated bivalent vaccine against A. salmonicida and E. tarda with relative percent survival (RPS) of 77.1 %. To understand the protection mechanism in molecular basis of the inactivated bivalent vaccine against A. salmonicida and E. tarda, we use RNA-seq to analyze the transcriptomic profile of the kidney tissue after immunization. A total of 391,721,176 clean reads were generated in nine libraries by RNA-seq, and 96.35 % of the clean reads were mapped to the reference genome of S. maximus. 1458 (866 upregulated and 592 downregulated) and 2220 (1131 upregulated and 1089 downregulated) differentially expressed genes (DEGs) were obtained at 2 and 4 weeks post-vaccination, respectively. The DEGs were enriched in several important immune-related GO terms, including cytokine activity, immune response, and defense response. In addition, the analysis of several immune-related genes showed upregulation and downregulation, including pattern recognition receptors, complement system, cytokines, chemokines and immune cell surface markers. Eight DEGs (ccr10, calr, casr, mybpha, cd28, thr18, cd20a.3 and c5) were randomly selected for qRT-PCR analysis, which confirmed the validity of the RNA-seq. Our results provide valuable insight into the immune mechanism of inactivated bivalent vaccine against A. salmonicida and E. tarda in Scophthalmus maximus.

Efficacy of bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda infections in turbot.

In recent years, more than one pathogenic organism has usually been isolated from diseased turbot Scophthalmus maximus, creating a pressing need for the development of combination vaccines to prevent fish diseases brought on simultaneously by various infections. In this study, the inactivated bivalent vaccine of Aeromonas salmonicida and Edwardsiella tarda was prepared by the formalin inactivation method. After challenge with A. salmonicida and E. tarda at 4 weeks post-vaccination in turbot, the relative percentage survival (RPS) of the inactivated bivalent vaccine was 77.1%. In addition, we assessed the effects of the inactivated bivalent vaccine and evaluated the immunological processes after immunization in a turbot model. Serum antibody titer and lysozyme activity of the vaccinated group were both upregulated and higher than that in control group after vaccination. The expression levels of genes (TLR2, IL-1ß, CD4, MHCI, MHCⅡ) that related to antigen recognition, processing and presentation were also studied in the liver, spleen and kidney tissues of vaccinated turbot. All the detected genes in the vaccinated group had a significant upward trend, and most of them reached the maximum value at 3-4 weeks, which had significant differences from the control group, suggesting that antigen recognition, processing and presentation pathway was activated by the inactivated bivalent vaccine. Our study provides a basis for further application of the killed bivalent vaccine against A. salmonicida and E. tarda in turbot, making it good potential that can be applied in aquaculture.