Vibrio Anguillarum Research Articles

Structural analysis of the siderophore-interacting protein from Vibrio anguillarum and its implications in classification of Vibrio homologs

Bacteria secrete siderophores to sequester the scarce iron in the environments, then the iron is transported into the cell in a siderophore-complexed form, which can be released by siderophore-interacting protein (SIP). Vibrio species comprise an array of serious pathogens, whose iron releasing process by SIP remains poorly understood. Herein, we report the high-resolution (1.2 Å) structure of Vibrio anguillarum SIP (VaSIP) in complex with FAD, representing the first structure of Vibrio SIP. VaSIP consists of a FAD-bound β-barrel domain and a Rossmann-fold domain connected by a linker, like other subgroup I SIPs. FAD is bound to the inter-domain cavity by aromatic stacking and hydrogen bonding interactions. Structural comparison indicated a modified NAD(P)H-binding motif (DxTA–EVL–GE) for subgroup I SIPs. The putative siderophore-binding pocket of VaSIP contains three lysines to form the basic triad to bind siderophore. Phylogenetic analysis shows Vibrio SIPs are mainly divided into two clades, represented by VaSIP and Vibrio cholerae ViuB, respectively. Interestingly, the two clades adopt distinct siderophore-binding basic triads, suggesting functional divergence among Vibrio SIPs. Our results shed light on the structural and phylogenetic characteristics of Vibrio SIPs, providing molecular basis for understanding Vibrio iron metabolism and designing anti-Vibrio drugs.

Paradendryphiella arenariae an endophytic fungus of Centella asiatica inhibits the bacterial pathogens of fish and shellfish.

Aquaculture has been considered a major food-producing sector in the world during the last few decades. The foremost constraint in the development of aquaculture is bacterial disease control and management. Since various fish pathogens are resistant to conservative treatments, it is essential to screen new and effective alternative antibacterial agents. Endophytic fungi are microorganisms that live in the plant's internal tissues without harming its host. Endophytic fungi have proven themselves as reliable sources of novel bioactive compounds that can be used as antibacterial agents. In the present study, fifteen morphologically different endophytic fungi were isolated from the fresh and healthy stem section of Centella asiatica. The active endophytic fungal crude extracts were tested for agar well diffusion assay, Minimum Inhibitory Concentration, Minimum Bactericidal Concentration assays, Time-kill kinetic analysis, Brine shrimp lethality assay. Agar plug diffusion and agar well diffusion assays revealed that endophytic fungus CAS1 exhibits maximum antagonistic activity against bacterial fish pathogens. The ethyl acetate crude extract of CAS1 exhibited the maximum zone of inhibitions against Aeromonas hydrophila (21 ± 0.11 mm), Aeromonas caviae (18 ± 0.1 mm), Edwardsiella tarda (23 ± 0.11 mm), Vibrio anguillarum (19 ± 0.05 mm) and Vibrio harveyi (20 ± 0.27 mm). The MIC and MBC values extract varied reliant on the trial pathogens ranging between 12.5-100 μg/mL and 25-100μg/mL correspondingly. The morphological and molecular characterization of potential isolate CAS1 was confirmed as Paradendryphiella arenariae by 18S rRNA ITS gene sequencing with 99.18% identity. This is the foremost findings to study the antagonistic effect of Paradendryphiella arenariae isolated from the stem of Centella asiatica against bacterial fish pathogens which can be used as natural effective antibacterial agents in aquaculture.

Development and optimization of immunomagnetic bead separation-convective PCR (IMBS-CPCR) for on-site detection of four common Vibrio species in aquaculture

Foodborne infectious diseases, particularly those caused by pathogenic Vibrio species in aquaculture, are a significant concern in the field of seafood safety. They not only hinder the economic development of the industry but also pose a major threat to public health. Traditional detection methods are often too slow, insensitive, or impractical for portable use, highlighting the urgent need for a sensitive, on-site detection method for Vibrio that prioritizes convenience and speed. This study developed a visual IMBS-CPCR method for the detection of four common pathogenic Vibrio species: Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, and Vibrio harveyi. The method achieved high capture rates for target bacteria (82.6%–83.3%) and low capture rates for non-target bacteria (below 11%), demonstrating good sensitivity by detecting bacteria at concentrations as low as 10 CFU/mL. Optimal amplification conditions were determined for each species, with specificity tests confirming that only target bacteria produced amplification bands. Sensitivity tests revealed detection limits of 101 CFU/mL for V. alginolyticus, V. anguillarum, and V. fluvialis, and 102 CFU/mL for V. harveyi. The combined techniques improved detection sensitivity in simulated fish samples by 10–100 fold compared to direct CPCR or PCR methods.

Characterization of the ferric uptake regulator VaFur regulon and its role in Vibrio anguillarum pathogenesis.

The Gram-negative marine bacterium Vibrio anguillarum is able to cause vibriosis with hemorrhagic septicemia in many fish species, and iron acquisition is a critical step for virulence. Despite the fact that genes specific to certain processes of iron transport have been studied, the iron-regulated circuits of the V. anguillarum strains remain poorly understood. In this study, we showed that in V. anguillarum strain 775, iron could affect the expression of a number of critical metabolic pathways and virulence factors. The global iron uptake regulator VaFur is the major actor to control these processes for the bacterium to respond to different iron conditions. A VaFur binding motif was identified to distinguish directly and indirectly regulated targets. The absence of VaFur resulted in the aberrant expression of most iron acquisition determinants under rich-iron conditions. A similar regulation pattern was also observed in the transcription of genes coding for the type VI secretion system. The expression of peroxidase genes is positively controlled by VaFur to prevent iron toxicity, and the deletion of Vafur caused impaired growth in the presence of iron and H2O2. VaFur also upregulates some virulence factors under limited-iron conditions, including metalloprotease EmpA and motility, which are likely critical for the high virulence of V. anguillarum 775. The deletion of VaFur led to reduced swimming motility and decreased extracellular protease activity under limited-iron conditions, thereby leading to attenuated pathogenicity. Our study provides more evidence to better understand the VaFur regulon and its role in the pathogenesis of V. anguillarum.IMPORTANCEVibriosis, the most common disease caused by marine bacteria belonging to the genus Vibrio, leads to massive mortality of economical aquatic organisms in Asia. Iron is one of the most important trace elements, and its acquisition is a critical battle occurring between the host and the pathogen. However, excess iron is harmful to cells, so iron utilization needs to be strictly controlled to adapt to different conditions. This process is mediated by the global iron uptake regulator Fur, which acts as a repressor when iron is replete. On the other hand, free iron in the host is limited, so the reduced virulence of the Δfur mutant should not be directly caused by abnormally regulated iron uptake. The significance of this work lies in uncovering the mechanism by which the deletion of Fur causes reduced virulence in Vibrio anguillarum and identifying the critical virulence factors that function under limited-iron conditions.

Integrated microRNA study and pathological analysis provides new insights into the immune response of Ruditapes philippinarum under Vibrio anguillarum challenge

Manila clam (Ruditapes philippenarum) is an important shellfish aquaculture product. The large-scale breeding of clams is often affected by V. anguillarum and causes large-scale death. However, the pathogenesis, immune response and metabolic pathway of V. anguillarum are still unclear. In this study, we found that the bacterial load in the hepatopancreas of R. philippinarum peaked at 48 h after V. anguillarum infection, and then gradually decreased, while the activity of lysozyme reached the peak at 12 h. Tissue section observation reveals that the infected hepatopancreas cells lost normal structure or necrosis. Additionally, six small RNA libraries were constructed using hepatopancreas of clams. A total of 15 differentially expressed (DE) microRNA (miRNA) were identified at 48 h after V. anguillarum infection, including 8 upregulated and 7 downregulated miRNAs. GO and KEGG enrichment results indicated the prediction of 48 known miRNAs and 127 new miRNAs, with functional annotation suggests that endocytosis pathway and bacterial recognition proteins may play key roles in immune response. The sequencing results were basically consistent with the qRT-PCR validation, indicating the accuracy of the data. This study provides a new idea to explore the immune regulation mechanism of shellfish after V. anguillarum infection, which brings important reference significance for modern immunological research.

Characterization of NK-lysin A, a potent antimicrobial peptide from the zebrafish Danio rerio

Antimicrobial peptides (AMPs) are important players of the innate immune system with a major role in the defense against invading pathogens. AMPs belonging to the family of saposin-like proteins (SAPLIPs) include the porcine NK-lysin and the human granulysin. In the zebrafish Danio rerio, transcript analyses of NK-lysin encoding genes have been reported, but biochemical characterizations at the protein level are missing so far. Here, we present the recombinant expression, purification, and characterization of one of these homologs, namely of NK-lysin A (DaNKlA). To remove the affinity tag from DaNKlA, we made use of a self-splicing intein. Recombinant DaNKlA depolarized liposomes over a broad pH range and showed a preference for negatively charged lipids. DaNKlA inhibited the growth of and killed different Gram-positive and Gram-negative bacteria, including the fish pathogenic bacterium Vibrio anguillarum, by membrane permeabilization but displayed substantially lower activity against yeast cells. Structural modelling and bioinformatic comparison of DaNKlA with characterized SAPLIPs suggest membrane destabilization accompanied by strong electrostatic interactions as the mode of action.

Effect of Dietary Short-Chain Fatty Acids on the Immune Status and Disease Resistance of European Seabass Juveniles

(1) Background: This study aimed to evaluate the potential of short-chain fatty acids as functional ingredients to improve the immune status and disease resistance of European seabass (Dicentrarchus labrax) juveniles. (2) Methods: For that purpose, triplicate groups of fish with an initial body weight of 15.2 ± 0.03 g were fed isoproteic (43% crude protein) and isolipidic (18% crude lipids) diets supplemented with sodium acetate (SA), sodium propionate (SP), and sodium butyrate (SB) at two inclusion levels: 0.25% and 0.50%. An unsupplemented diet was used as a control. After 56 days of feeding with the experimental diets, fish were intraperitoneally (i.p.) injected with 100 µL of Vibrio anguillarum (1.2 × 107 Colony Forming Units (CFU)/mL) and mortality was recorded for 3 weeks. At the end of the trial, there were no differences in survival between the treatment groups and the control, but survival was higher in fish fed the diet supplemented with SB 0.50 than SP 0.25 (93.3 vs. 66.7%). Compared to the pre-challenge values, and regardless of diet composition, all hematological parameters (hemoglobin, hematocrit, red blood cells, white blood cells) measured decreased after 4 h of bacterial challenge, except for neutrophils which were increased. Independently of diet composition, lysozyme and nitric oxide decreased at 4 and 24 h post infection. Compared to the control, diets supplemented with SA and SP promoted an up-regulation of both pro- and anti-inflammatory cytokines at 4 h after the challenge, while the diets supplemented with SB promoted an up-regulation of pro- and anti-inflammatory cytokines at 24 h after the challenge. (3) Conclusions: Overall, present results suggest that SA and SP provide a fast response to a bacterial challenge in European sea bass juveniles, while SB provides increased survival.

Differential immune gene expression in rainbow trout, Oncorhynchus mykiss (walbaum), exposed to five pathogens: Aeromonas salmonicida, Flavobacterium psychrophilum, Vibrio anguillarum, Yersinia ruckeri and Ichthyophthirius multifiliis

Rainbow trout, Oncorhynchus mykiss, originating from the same outbred population and reared under the same conditions, were bath exposed to five different pathogens (Aeromonas salmonicida, Flavobacterium psychrophilum, Vibrio anguillarum, Y. ruckeri, Ichthyophthirius multifiliis). The exposures were conducted as independent experiments with non-exposed fish as time-point controls. At peak of infections, fish showing significant clinical signs, fish without clinical signs, and time-point control fish were euthanized, and gills, spleen, and liver were sampled for gene expression analysis. At the end of the infection course, i.e. a period without any disease signs, surviving fish were sampled as well. Genes encoding 14 cytokines, 5 immune cell markers, 5 immune effector molecules and 3 reference/housekeeping genes were analyzed. The host fish exhibited different reaction patterns dependent on the pathogen. Especially trout infected with I. multifiliis showed a characteristic Th2 expression signature in gills, whereas only innate responses occurred in internal organs. Rainbow trout exposed to F. psychrophilum displayed a relatively lower expression of immune genes contrasting the host responses to A. salmonicida, V. anguillarum and Y. ruckeri. The bacterial pathogens induced a relatively similar expression profile in internal organs of trout with a few exceptions. Thus, F. psychrophilum induced upregulation of genes earlier in gills than internal organs, and A. salmonicida infected fish showed a notable downregulation of immune genes in gills. The differential expression patterns may reflect the different invasion mechanisms and target tissues associated with the five pathogens.

Effect of Amorphous Halomonas-PHB on Growth, Body Composition, Immune-Related Gene Expression and Vibrio anguillarum Resistance of Hybrid Grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatu ♂) Juveniles.

Poly-β-hydroxybutyrate (PHB) is a bacterial metabolite produced by bacteria such as Halomonas sp. that serves as a carbon and energy storage compound for bacteria under nutrient-limited conditions. Two experiments were conducted to investigate the effects of dietary supplementation with Halomonas-PHB on hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatu ♂). In experiment I, juvenile groupers were fed basal diets supplemented with 3% Halomonas-PHB (3% HM-PHB) containing 1.4% PHB and 3% Halomonas (3% HM) without PHB, as well as a control diet, for seven weeks. The results showed no significant difference in survival rate, weight gain, and crude fat content between the 3% HM-PHB group and the control group; however, the crude protein of the 3% HM-PHB group was significantly lower than that of the control group. Furthermore, supplementation with 3% HM-PHB increased the fatty acids content in fish muscles, including long-chain unsaturated fatty acids C18:1n9, EPA, and DHA. In experiment II, groupers were fed a basal diet supplemented with 6.5% Halomonas-PHB (6.5% HM-PHB) containing 3% PHB and 6.5% Halomonas (6.5% HM) containing no PHB, as well as a basal diet (Control). After seven weeks of rearing, the fish were challenged with Vibrio anguillarum for 48 h. Although no significant difference in survival rate and growth was observed among different groups, the dietary supplement of 6.5% Halomonas-PHB improved the survival rate of V. anguillarum challenged grouper and significantly increased the gene expressions of catalase (CAT) and superoxide dismutase (SOD) in blood, interleukin 1 (IL1) and interleukin 10 (IL10) in the liver, spleen, head kidney, and blood (p < 0.05). In conclusion, dietary supplementation of Halomonas-PHB had no significantly positive effect on fish growth performance but increased the content of fatty acids, including long-chain unsaturated fatty acids C18:1n9, EPA, and DHA in fish muscle; it also improved the V. anguillarum resistance, possibly through increasing immune-related gene expression in different tissues and organs. Our findings offer compelling evidence that Halomonas-PHB can be utilized as a feed additive in intensive grouper farming to enhance the groupers' resistance to Vibrio.

Investigation of vibriosis caused by Vibrio anguillarum in rainbow trout (Oncorhynchus mykiss).

Identifying pathogenic microorganisms causing disease is important for epidemiological research, antimicrobial therapy, and control. The current study was carried out to use different methods for the identification of Vibrio anguillarum from diseased rainbow trout (Oncorhynchus mykiss) obtained from Türkiye (Muğla-Fethiye), the damage caused by the pathogenic microorganism in the tissues and organs, and the determination of the antibiotic effective against the pathogen. Hemorrhagic and ulcerative skin lesions and diffuse petechial hemorrhage in the internal organs were clinically detected in diseased fish obtained from the rainbow trout farm. Bacteria isolated from diseased fish were subjected to analysis using conventional bacteriological methods, a commercial bacterial identification test kit (API), an automated bacteria identification system known as Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), BD Phoenix™, and 16S rRNA sequence analysis. All isolated bacteria were identified as V. anguillarum by API 20E and conventional bacteriological method. These results have been confirmed with 16S rRNA sequence analysis. However, the isolated bacteria were identified as Grimontia hollisae (syn. Vibrio holisae) with the BD Phoenix system. Histologically, tissue damage such as melano-macrophage centers and necrosis in the kidney and spleen, hyperemia and mononuclear cell infiltration in the liver, as well as mononuclear cell infiltration on muscles, talengectiasis in the gill tissue was observed. In addition, it has been determined that the most effective antibiotic against the pathogen was enrofloxacin. When comparing all identification methods used for this pathogen causing tissue damage, it was demonstrated that the MALDI-TOF MS provides better results than other methods in terms of cost and identification time, and it could be used as an alternative to the conventional method to the identification of V. anguillarum.

Silver Nanoparticles Synthesized from Water Extracts of Artemisia annua and Their Antibacterial Activity Against Aquatic Pathogens

AbstractSilver nanoparticles (AgNPs) by biological synthesis have many advantages. Among them, herbs attract wide attention due to their unique medicinal effects. In this paper, the water/alcohol/ether extracts of dried or fresh Artemisia annua were used as reducing agents and stabilizers to synthesize AgNPs. Water is a highly polar solvent that can effectively extract polar compounds such as phenolic profile, which possesses strong reducing properties and enables the reduction of Ag+ to AgNPs. The prepared AgNPs were characterized using ultraviolet–visible (UV–vis) absorption spectra, X‐ray diffraction (XRD), Fourier transform infrared (FT‐IR) spectroscopy, transmission electron microscopy (TEM), and zeta potential analysis. The AgNPs prepared from the water extracts of dried Artemisia annua have obvious bacteriostatic effects on six common aquatic pathogens and have a wide application prospect in aquaculture. The inhibition zone against Vibrio anguillarum is up to 16.88 mm. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Vibrio anguillarum are 1.09 µg/mL and 2.17 µg/mL, respectively, which is lower than most published reports.

Epibiotic Bacteria Isolated from the Non-Indigenous Species Codium fragile ssp. fragile: Identification, Characterization, and Biotechnological Potential.

Due to their richness in organic substances and nutrients, seaweed (macroalgae) harbor a large number of epiphytic bacteria on their surfaces. These bacteria interact with their host in multiple complex ways, in particular, by producing chemical compounds. The released metabolites may have biological properties beneficial for applications in both industry and medicine. In this study, we assess the diversity of culturable bacterial community of the invasive alga Codium fragile ssp. fragile with the aim to identify key groups within this epiphytic community. Seaweed samples were collected from the Northern Tunisian coast. A total of fifty bacteria were isolated in pure culture. These bacterial strains were identified by amplification of the ribosomal intergenic transcribed spacer between the 16S and the 23S rRNA genes (ITS-PCR) and by 16S rRNA sequencing. Antimicrobial activity, biochemical, and antibiotic resistance profile characterization were determined for the isolates. Isolated strains were tested for their antimicrobial potential against human and fish bacterial pathogens and the yeast Candida albicans, using the in vitro drop method. About 37% of isolated strains possess antibacterial activity with a variable antimicrobial spectrum. Ba1 (closely related to Pseudoalteromonas spiralis), Ba12 (closely related to Enterococcus faecium), and Bw4 (closely related to Pseudoalteromonas sp.) exhibited strong antimicrobial activity against E. coli. The isolated strain Ba4, closely related to Serratia marcescens, demonstrated the most potent activity against pathogens. The susceptibility of these strains to 12 commonly used antibiotics was investigated. Majority of the isolates were resistant to oxacillin, cefoxitin, tobramycin, and nitrofurantoin. Ba7 and Ba10, closely related to the Vibrio anguillarum strains, had the highest multidrug resistance profiles. The enzymes most commonly produced by the isolated strains were amylase, lecithinase, and agarase. Moreover, nine isolates produced disintegration zones around their colonies on agar plates with agarolitic index, ranging from 0.60 to 2.38. This investigation highlighted that Codium fragile ssp. fragile possesses an important diversity of epiphytic bacteria on its surface that could be cultivated in high biomass and may be considered for biotechnological application and as sources of antimicrobial drugs.

LncRNA (BCO1-AS) regulate inflammatory responses in bacterial infection through caspase-1 in turbot (Scophthalmus maximus)

LncRNA plays key role in several biological processes, including transcriptional regulation, post transcriptional control and epigenetic regulation. However, research on the functional roles of lncRNAs in teleost species remains limited. Here, we discovered a lncRNA (BCO1-AS) with a critical role in antibacterial responses. Briefly, the full length of BCO1-AS was 2005 bp. Subsequently, BCO1-AS was distributed throughout the nucleus, where it may either trans- or cis-regulate the nearby genes. In addition, BCO1-AS was widely expressed in all the examined tissues with the highest expression level in intestine, while the lowest expression level was detected in muscle. Moreover, following Vibrio anguillarum challenge, BCO1-AS was significantly down-regulated in intestine, and up-regulated in gill and skin. In CHIRP experiment, BCO1-AS could effectively enrich RNA and might interact with several immune-related genes. Furthermore, we found that LPS could induce the expression of BCO1-AS. Finally, BCO1-AS could positively regulate caspase-1 at the mRNA and protein level. The BCO1-AS was speculated to inhibit the synthesis of inflammatory components. In summary, these results showed the roles of BCO1-AS in the regulation of inflammatory in turbot, which provided valuable information for further understanding the immune regulation network of lncRNA in teleost fish.

Characteristics and functional analysis of a novel mannose receptor in Penaeus vannamei

The mannose receptor (MR) plays a key role in the innate immune system as a pattern recognition receptor. Here, a novel type of mannose receptor, named PvMR2, was identified from Penaeus vannamei (P. vannamei). The PvMR2 coding sequence (CDS) obtained was 988 base pairs in length, encoding a protein consisting of 328 amino acids. This protein includes a signal peptide and two classical C-type lectin domains (CTLD). Quantitative real-time PCR showed that PvMR2 was distributed in all detected tissues, with the highest levels in the intestines and stomach. Following a bacterial challenge with Vibrio anguillarum (V. anguillarum), PvMR2 showed significant up-regulation in both the intestines and stomach of shrimp. To validate the function of PvMR2, recombinant proteins were extracted and purified using a His-tag. The resulting rPvMR2 demonstrated binding capability with lipopolysaccharides (LPS) and peptidoglycan (PGN) in a dose-dependent manner, affirming its binding affinity. The purified rPvMR2 demonstrated calcium-independent binding activity towards both Gram-positive bacteria (V. anguilliarum and Vibrio parahaemolyticus) and Gram-negative bacteria (Escherichia coli and Aeromonas Veronii). Antibacterial assays confirmed that rPvMR2 inhibits bacterial growth. Intestinal adhesion and adhesion inhibition experiments confirmed that the rPvMR2 can be used to reduce the adhesion capacity of harmful bacteria in the gut. Phagocytosis experiments have shown that rPvMR2 promotes phagocytosis in hemocytes and protects the host from external infection. Treatment with recombinant PvMR2 significantly bolstered bacterial clearance within the hemolymph and markedly augmented shrimp survival post-infection with V. anguillarum. These results suggest that PvMR2 has agglutination, growth inhibition, adhesion inhibition, clearance promotion, and phagocytosis effects on harmful bacteria, and plays a crucial role in the antimicrobial immune response of P. vannamei.

Ferredoxin: A novel antimicrobial peptide derived from the black scraper (Thamnaconus modestus)

Ferredoxin (FDX) is a highly conserved iron-sulfur protein that participates in redox reactions and plays an important role as an electron transport protein in biological processes. However, its function in marine fish remains unclear. We identified two ferrodoxin proteins, FDX1 and FDX2, from black scraper (Thamnaconus modestus) to confirm their genetic structures and expression profiles and to investigate their antimicrobial activity properties by fabricating them with antimicrobial peptides based on sequences. The two TmFDXs mRNAs were most abundant in peripheral blood leukocytes of healthy T. modestus. After artificial infection with Vibrio anguillarum, a major pathogen of T. modestus, TmFDX1 mRNA was significantly upregulated in the gills, heart, intestines, kidneys, liver, and spleen, but was consistently downregulated in the brain. The expression levels of TmFDX2 mRNA were significantly upregulated in the heart, intestines, kidneys, liver, and spleen; however, no significant changes in expression were observed in the brain or gills. Based on the 2Fe-2S ferredoxin-type iron-sulfur-binding domain sequence, two peptides (pFDX1 and pFDX2) were synthesized. The bactericidal effect, biofilm formation inhibition, and gDNA-binding activity of these peptides were investigated. These findings highlight the potential as a natural peptide candidate for TmFDXs.

Bioactive compound from marine seagrass Streptomyces argenteolus TMA13: combatting fish pathogens with time-kill kinetics and live-dead cell imaging.

Actinobacteria, pervasive in aquatic and terrestrial environments, exhibit a filamentous morphology, possess DNA with a specific G + C content and production of numerous secondary metabolites. This study, focused on actinobacteria isolated from marine seagrass, investigating their antibacterial activity against fish pathogens. Among 28 isolates, Streptomyces argenteolus TMA13 displayed the maximum zone of inhibition against fish pathogens-Aeromonas hydrophila (10mm), Aeromonas caviae (22mm), Edwardsiella tarda (17mm), Vibrio harveyi (22mm) and Vibrio anguillarum (12mm) using the agar plug method. Optimization of this potent strain involved with various factors, including pH, temperature, carbon source and salt condition to enhance both yield production and antibacterial efficacy. In anti-biofilm assay shows the maximum percentage of inhibition while increasing concentration of TMA13 extract. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) assays with TMA13 crude extract demonstrated potent activity against fish pathogens at remarkably low concentrations. Time-kill kinetics assay showcased growth curve variations over different time intervals for all fish pathogens treated with a 100µg/ml concentration of crude extract, indicating a decline in cells viability and progression into the death phase. Additionally, fluorescence microscopic visualization of bacterial cells exposed to the extracts emitting green and red fluorescence, enabling live-dead cell differentiation was also studied. Further characterization of the crude extract through GC-MS and FT-IR analyses performed and identified secondary metabolites with functional groups exhibiting significant antibacterial activity. This study elucidates the capacity of Streptomyces argenteolus TMA13 to enhance the production of antibiotic compounds effective against fish pathogens.

Insights into the connection between pathogen water eDNA and disease progression in zebrafish infected with Vibrio anguillarum

The host-pathogen interactions are tripartite and include the host, the pathogen, and the environment. For a long time, attention has mainly been addressed to the host's immune mechanisms and the pathogen's virulence factors while the environmental factor has received less interest. About a decade ago, studies on environmental DNA (eDNA) started to emerge as this approach is versatile for numerous research fields and interest is persistently growing. In the present study, the relationship between disease progression and pathogen water environmental DNA level was investigated. To perform this investigation, an infection model was setup using zebrafish and the gram-negative bacteria Vibrio anguillarum. Two different fish densities and bacterial concentrations were used to determine different patterns of pathogen eDNA in the fish tanks. Water sampling to collect eDNA was conducted alongside with observation of the fish Clinical Signs, mortalities, and immune responses. Water eDNA relative abundance of the pathogen could be determined directly after fish challenge, when Clinical Signs occurred, and when mortality started. Moreover, immune-relevant gene expression in zebrafish gills showed that il-1β, il-8, il-10, nf-κb and saa correlated to the pathogen eDNA signal, depending on fish density and bacterial concentration. In conclusion, this study demonstrated that water eDNA is a promising non-invasive tool to state disease progression and immune response in zebrafish infected with V. anguillarum.

Development and use of a Chinook salmon spleen stromal-like cell line to study the cellular immune response to Vibrio anguillarum

Despite increased interest in Chinook salmon aquaculture, there is inadequate understanding regarding the bacterial immune defenses of this species. This study describes the establishment and characterization of a continuous stromal-like cell line derived from Chinook salmon spleen, CHST, and its response to a heat-killed bacterial exposure challenge. Optimal growth of CHST was seen at 18 °C when grown in Leibovitz's L15 media supplemented with 20 % fetal bovine serum. DNA analyses confirmed that CHST did originate from Chinook salmon tissue. Vibrio anguillarum, a common pathogen of marine aquaculture and the causative agent of an acute hemorrhagic septicaemia known as vibriosis was used for exposure studies. V. anguillarum was heat-killed and CHST was challenged so that pro-inflammatory (IL-1β, IL-6 and TNFα) as well as anti-inflammatory (IL-10) cytokine transcript levels could be measured at 1, 4, 12, 24 and 96 hour (h) post-exposure. The heat-killed bacteria were observed to significantly stimulate the expression of all three pro-inflammatory cytokines at 4, 12, 24 and 96 h post-exposure with the peak in expression occurring at 12 and 24 h. Meanwhile, IL-10 was not observed to significantly increase until 96 h post-stimulation, which was also the time when the inflammatory cytokine expression was decreasing. The establishment and characterization of CHST provides a valuable model for studying the immune response of Chinook salmon stromal-like cells in response to stimuli, including those of bacterial origin.

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, β-ar, hsp70, c3, lysozyme, α-enolase, nadph oxidase, il1β, il6, tnfα, il10 and tgfβ1) 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.

Manipulated C5aR1 over/down-expression associates with IL-6 expression during bacterial inflammation in half-smooth tongue sole (Cynoglossus semilaevis)

The complement component 5a/complement component 5 receptor 1 (C5a/C5aR1) pathway plays a crucial role in the onset and development of inflammation, but relevant studies in fish are lacking. In this study, we successfully characterized the relationship between half-smooth tongue sole (Cynoglossus semilaevis) C5aR1 (CsC5aR1) and bacterial inflammation. First, we showed that the overexpression of CsC5aR1 significantly increased bacterial pathological damage in the liver and intestine, whereas inhibition attenuated the damage. The in vitro experiments suggested that CsC5aR1 was able to positively regulate the phagocytic activity and respiratory burst of tongue sole macrophages. In terms of both transcriptional and translational levels, overexpression/inhibition of CsC5aR1 was followed by a highly consistent up-regulation/decrease of its downstream canonical inflammatory factor interleukin-6 (CsIL-6). Furthermore, we stimulated macrophages by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and found a broad-spectrum response to bacterial infections by the C5a/C5aR1 complement pathway together with the downstream inflammatory factor CsIL-6. Subsequently, we directly elucidated that CsIL-6 is an indicator of C5a/C5aR1-mediated inflammation at different infection concentrations, different infectious bacteria (Vibrio anguillarum and Mycobacterium marinum), and different detection levels. These results might provide a new inflammation bio-marker for early warning of bacteria-induced hyperinflammation leading to fish mortality and a promising target for the treatment of bacterial inflammation in teleost.