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Aeromonas salmonicida subsp. masoucida, designated as laboratory strain MHJM250, was characterized from a naturally infected farmed golden mahseer, Tor putitora. The infected fish exhibited clinical signs of erosion at the caudal fin and hemorrhage onx the ventral body surface. Molecular identification through 16 S rDNA and phylogenetic analysis revealed 100% similarity with a known strain A. salmonicida subsp. masoucida (MT122821.1). MHJM250 exhibited positive reactions for oxidase, catalase, esculin, MR-VP, O/F and utilized arginine and lysine. It also demonstrated siderophore activity, thrived at various NaCl concentrations, hydrolyzed gelatinase, skimmed milk and casinase. In vitro studies exhibited its hemolytic nature, significant biofilm production in glucose-rich tryptone soya broth and beta-hemolysis. MHJM250 didn't produce slime and was non-precipitated upon boiling. It showed crystal violet binding characteristics and auto-agglutination with relatively weak hydrophobicity (25%). In the challenge assay, intraperitoneal administration of MHJM250 to T. pitutora fingerlings at 108 CFU mL-1 resulted in pathogenicity with 3% mortality and mild hemorrhagic symptoms. Histopathological analysis revealed degenerative changes in gill, kidney, liver, muscle, and intestine samples. The bacterium displayed resistance to several antibiotics (µg/disc); ampicillin (10 µg), ampicillin/ sulbactam (10/10 µg), clindamycin (2 µg), linezolid (30 µg), penicillin G (10 µg) and rifampicin (5 µg) and varied minimum inhibitory concentrations against oxytetracycline, erythromycin and florfenicol. Transmission electron microscopy showed its rod-shaped structure with single polar flagellum and lophotrichous flagella. An investigation on the molecular basis for virulence factors of A. salmonicida subsp. masoucida MHJM250 may offer crucial understandings to formulate disease prevention and control strategies in aquaculture.

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Aeromonas salmonicida is the typical pathogen causing furunculosis, reported widely in salmonids. Because of multiple serotypes, the control of A. salmonicida-caused disease has increasingly received much attention. Recently, A. salmonicida infection was reported in non-salmonid fish species. Here, a pathogenic A. salmonicida, named as As-s, was isolated from cultured snakehead (Channa argus) in a local fish farm in Shandong, China. As-s displayed clear hemolysis, amylase, and positive catalase activities, and grew at a wide range of temperatures (10-37 °C) and pH values (5.5-8.5). As-s was highly sensitive to cefuroxime sodium, ceftriaxone, ceftazidime, piperacillin, and cefoperazone and also apparently sensitive to chloramphenicol, erythromycin, and 25% cinnamaldehyde. The Virulence array protein gene cloning' results suggested that As-s has this gene compared with the other two vapA-containing strains, despite a close relationship of these strains via phylogenetic analysis. Severe ulcers on skin, muscle, and abnormal liver, and hemorrhage in pectoral/ventral fins and anal region were observed, and exophthalmos were also noticed in infected juvenile snakehead, as well as necrosis and infiltration of blood cells emerged in the internal organs using pathological section. In addition, As-s caused high mortality in snakehead, consistently with its immune gene response. This study reports the first isolation of vapA-absent A. salmonicida in snakehead.

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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.

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The Gram-negative bacterium A. salmonicida is the causal agent of furunculosis and used to be one of the most loss-causing bacterial infections in the salmonid aquaculture industry with a mortality rate of about 90% until the 1990s, when an inactivated vaccine with mineral oil as adjuvant was successfully implemented to control the disease. However, the use of this vaccine is associated with inflammatory side effects in the peritoneal cavity as well as autoimmune reactions in Atlantic salmon, and incomplete protection has been reported in rainbow trout. We here aimed at developing and testing a recombinant alternative vaccine based on virus-like particles (VLPs) decorated with VapA, the key structural surface protein in the outer A-layer of A. salmonicida. The VLP carrier was based on either the capsid protein of a fish nodavirus, namely red grouper nervous necrotic virus (RGNNV) or the capsid protein of Acinetobacter phage AP205. The VapA and capsid proteins were expressed individually in E. coli and VapA was fused to auto-assembled VLPs using the SpyTag/SpyCatcher technology. Rainbow trout were vaccinated/immunized with the VapA-VLP vaccines by intraperitoneal injection and were challenged with A. salmonicida 7 weeks later. The VLP vaccines provided protection comparable to that of a bacterin-based vaccine and antibody response analysis demonstrated that vaccinated fish mounted a strong VapA-specific antibody response. To our knowledge, this is the first demonstration of the potential use of antigen-decorated VLPs for vaccination against a bacterial disease in salmonids.

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This study aimed to identify the molecular mechanisms regulated by a combined vaccine against Aeromonas salmonicida and Vibrio anguillarum (O1 serotype). These bacteria cause furunculosis and vibriosis, respectively, and are associated with a high mortality in rainbow trout in Korea. The vaccine upregulated gene expression of TCRα, T-bet, sIgM, and mIgM, markers of an activated adaptive immune response. On days 1, 3, and 5, transcriptome analysis revealed 862 (430 up- and 432 downregulated), 492 (204 up- and 288 downregulated), and 741 (270 up- and 471 downregulated) differentially expressed genes (DEGs), respectively. Gene ontology (GO) enrichment analysis identified 377 (108 MF, 132 CC, 137 BP), 302 (60 MF, 180 CC, 62 BP), and 314 (115 MF, 129 CC, 70 BP) GOs at days 1, 3, and 5, respectively. Kyoto Encyclopedia of Genetic and Genomic enrichment analysis identified eight immune system-related pathways like cytokine-cytokine receptor interaction, NF-kappaB signaling pathway, TNF signaling pathway, NOD-like receptor signaling pathway, cytosolic DNA sensing pathway, cell adhesion molecule, complement and coagulation cascade, and antigen processing and presentation. In the analysis of the protein-protein interaction of immune-related DEGs, a total of 59, 21, and 21 interactional relationships were identified at days 1, 3, and 5, respectively, with TNF having the highest centrality at all three time points.

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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.

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In mammals, visceral adipose is increasingly seen as playing an important role in immune function with numerous pro-inflammatory, anti-inflammatory and immune-modulating proteins and peptides being identified in adipocytes. Adipose is also now known as a tissue that has an important role in the regulation of peritoneal immune responses. Despite this, only lately has consideration been given to visceral adipose as an important immune tissue in fish, especially in the context of intraperitoneal vaccination. The present study demonstrates that fish visceral adipose is capable of expressing a large range of immune molecules in response to stimulation with a live bacterium (A. salmonicida), a bacterial PAMP (Y. ruckeri flagellin), and the pro-inflammatory cytokines IL-1ß, TNF-α3 and IFN-γ. Following infection and stimulation with flagellin and IL-1ß a large upregulation of pro-inflammatory and antimicrobial molecules was seen, with a high degree of overlap. TNF-α treatment affected relatively few genes and the effects were more modest. IFN-γ had the smallest impact on adipose but IFN-γ inducible genes showed some of the largest effects. Overall, it is clear that adipose tissue should be considered an active immune site in fish, capable of participating in and influencing immune responses.

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Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen in aquaculture. Together with other pathogens, it is characterized by the presence of a type 3 secretion system (T3SS). The T3SS is the main virulence mechanism of A. salmonicida. It is used by the bacterium to secrete and translocate several toxins and effector proteins into the host cell. Some of these factors have a detrimental impact on the integrity of the cell cytoskeleton, likely contributing to impair phagocytosis. Furthermore, it has been suggested that effectors of the T3SS are able to modulate the host's immune response. Here we present the first partial characterization of the immune response in rainbow trout (Oncorhynchus mykiss) infected with distinct strains of A. salmonicida either carrying (i) a fully functional T3SS or (ii) a functionally impaired T3SS or (iii) devoid of T3SS ("cured" strain). Infection with an A. salmonicida strain either carrying a fully functional or a secretion-impaired T3SS was associated with a strong and persistent immune suppression. However, the infection appeared to be fatal only in the presence of a fully functional T3SS. In contrast, the absence of T3SS was neither associated with immune suppression nor fish death. These findings suggest that the T3SS and T3SS-delivered effector molecules and toxins of A. salmonicida do not only impair the host cells' cytoskeleton thus damaging cell physiology and phagocytosis, but also heavily affect the transcription of critical immune mediators including the shut-down of important warning signals to recognize infection and induce immune defense.

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The T-box transcription factor T-bet is expressed in a number of hematopoietic cell types in mammals and plays an essential role in the lineage determination of Th1 T-helper cells and is considered as an essential feature for both innate and adaptive immune responses in higher vertebrates. In the present study, we have identified and characterized the full-length Atlantic salmon T-bet cDNA (3502 bp). The putative primary structure of the polypeptide deduced from the cDNA sequence contained 612 aa, which possessed a T-box DNA binding domain. Phylogenetic study and gene synteny revealed it is as a homolog to mammalian T-bet. Quantitative PCR analysis of different tissues in healthy fish showed that salmon T-bet gene was highly expressed in spleen, followed by head kidney, and was expressed in intestine, skin, and liver at lower levels. Moreover, the time-dependent expression profile of T-bet, interferon gamma (IFNγ), interleukin-22 (IL-22), and natural killer enhancement factor in mucosal tissues during water-borne infection with live Aeromonas salmonicida, indicated the involvement of T-bet in mucosal immune response in Atlantic salmon.

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Furunculosis caused by Aeromonas salmonicida subsp. salmonicida is an epidemic disease among salmonids, including rainbow trout (Oncorhynchus mykiss). However, the immune mechanisms that are elicited in rainbow trout against the invasion of A. salmonicida are not yet fully understood. In this study, we examined the spleen to investigate the immune response of rainbow trout at 3days post-infection by A. salmonicida at the transcriptome and proteome levels by using Illumina-seq and iTRAQ methods, respectively. A total of 1036 genes and 133 proteins were found to undergo differential expression during the immune response of the spleen against A. salmonicida infection. Gene ontology and KEGG analysis were conducted among the differentially expressed genes and proteins, revealing that immune system process and response to stimulus were the top two biological processes, and immune system, signaling molecules and interaction, and immune diseases were the differential pathways activated. Correlation analysis of transcriptomic and proteomic results showed 17 proteins (11 upregulated and 6 downregulated) having consistent expression at RNA and protein levels. Moreover, protein-protein interaction analysis showed that diseases, proteasome, aminoacyl-tRNA biosynthesis, and nucleotide metabolism were the main interactions among the consistently expressed proteins. Consequently, these upregulated proteins, namely, ferritin, CD209, IL13Rα1, VDAC2, GIMAP7, PSMA1, and two ANXA11s could be considered as potential biomarkers for rainbow trout immune responses. BIOLOGICAL SIGNIFICANCE: This study provides the first identification of immune markers through an analysis of the differential expression of both genes and their corresponding protein products in the spleen of rainbow trout after infection by A. salmonicida, shedding light on the molecular mechanisms triggered in rainbow trout against A. salmonicida infection and providing new molecular targets for further immunological research in fish.

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OBJECTIVES: To characterize the chromosome-encoded metallo-ß-lactamase (MBL) from the psychrophilic, marine fish-pathogenic bacterium Aliivibrio salmonicida LFI1238 and check for the presence of the gene in other Aliivibrio isolates both connected to the fish-farming industry and from the environment. METHODS: The MBL gene was cloned and intracellularly expressed in Escherichia coli. Kinetic parameters, NaCl dependence, pH optimum and temperature optimum were determined using purified enzyme. The VIM-2 enzyme from a Pseudomonas aeruginosa hospital isolate was used as a counterpart in comparative analysis. PCRs with degenerate MBL primers were used to screen different A. salmonicida isolates for the presence of the gene. RESULTS: A. salmonicida MBL (ALI-1) is an Ambler class B ß-lactamase sharing 39% and 29% amino acid identity with IMP-1 and VIM-2, respectively. ALI-1 hydrolysed all ß-lactam antibiotics tested, except for the monobactam aztreonam and the penicillin piperacillin. A profound increase in activity was observed when adding NaCl to the assay mixture (60% active without addition of NaCl, increasing to 100% at 0.5 M NaCl). The increase was less noticeable for VIM-2 (100% active at 0.2 M NaCl). ALI-1 appears to be ubiquitous in nature as it is found in Aliivibrio isolates not affected by human activity. CONCLUSIONS: This work provides more data for the ever-expanding MBL group of enzymes. These periplasmic enzymes are activated by addition of NaCl, and the marine enzyme is highly salt tolerant and cold active. The observed enzyme properties very likely reflect the conditions that the enzymes face in situ.

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