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NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides.

Vaccination is widely recognized as an effective strategy for preventing various bacterial and viral diseases. In this study, protein, DNA, and egg yolk antibody (IgY) vaccines targeting the outer membrane protein VF14355 of Vibrio fluvialis (V. fluvialis) were administered to goldfish (Carassius auratus, C. auratus) subsequently challenged with V. fluvialis and Aeromonas hydrophila (A. hydrophila). The immune efficacy of the three VF14355 vaccines was evaluated through their immune activities, protective rates, anti-inflammatory and antioxidant effects, histopathology, and immunofluorescence, and the results indicated that the protective rates in the three immunized groups were significantly higher than those in the control group; furthermore, the number of kidney bacteria was significantly reduced in the immunized group compared to the control group. The ELISA results demonstrated an in vitro interaction between the bacteria and C. auratus serum. The plasma phagocytosis index and phagocytosis percentage were significantly increased in C. auratus, and their serum immune factor levels, including those of acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM), were increased, while those of serum antioxidant factors, such as superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA), were reduced in the immunized group; notably, the expression levels of inflammatory factors were also diminished in the immunized groups. Histopathological analyses further revealed that the organ structures of the immunized group remained intact, and immunofluorescence tests indicated significant reductions in apoptosis factor p53 and DNA damage factor γH2A.X in kidney tissues. Therefore, the protein, DNA, and IgY vaccines of VF14355 demonstrate the potential to confer resistance against various bacterial infections, positioning them as promising multivalent vaccine candidates for aquaculture.

The tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis. Vibrio vulnificus, an opportunistic pathogen, harbors three distinct tad loci. Among them, only tad1 locus was highly upregulated in in vivo growing bacteria compared to in vitro culture condition. To understand the pathogenic roles of the three tad loci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only the Δtad123 triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the Δtad123 mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. The Δtad123 mutant cells showed attenuated host cell adhesion, decreased biofilm formation, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. The Δtad123 mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Complement depletion by treating with anti-C5 antibody rescued the viable count of Δtad123 in infected mouse bloodstream to the level comparable to wild type strain. Taken together, all three tad loci cooperate to confer successful invasion of V. vulnificus into deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.

The increase in marine diseases, particularly in economically important mollusks, is a growing concern. Among them, the Pacific oyster ( Crassostrea gigas ) production faces challenges from several diseases, such as the Pacific Oyster Mortality Syndrome (POMS) or vibriosis. The microbial education, which consists of exposing the host immune system to beneficial microorganisms during early life stages is a promising approach against diseases. This study explores the concept of microbial education using controlled and pathogen-free bacterial communities and assesses its protective effects against POMS and Vibrio aestuarianus infections, highlighting potential applications in oyster production. We demonstrate that it is possible to educate the oyster immune system by adding microorganisms during the larval stage. Adding culture based bacterial mixes to larvae protects only against the POMS disease while adding whole microbial communities from oyster donors protects against both POMS and vibriosis. The efficiency of immune protection depends both on oyster origin and on the composition of the bacterial mixes used for exposure. No preferential protection was observed when the oysters were stimulated with their sympatric strains. Furthermore, the added bacteria were not maintained into the oyster microbiota, but this bacterial addition induced long term changes in the microbiota composition and oyster immune gene expression. Our study reveals successful immune system education of oysters by introducing beneficial microorganisms during the larval stage. We improved the long-term resistance of oysters against critical diseases (POMS disease and Vibrio aestuarianus infections) highlighting the potential of microbial education in aquaculture.

Outer membrane protein K (OmpK) has been proven to be an ideal vaccine candidate for broad-spectrum cross-prevention against Vibriosis. However, due to the extensive biological and genetic diversity of Vibrio species, current OmpK subunit vaccines can only target different strains of the same bacterial species or closely related species and have difficulty providing promising cross-immunoprotection against more diverse Vibrio infections. In recent years, the development of epitope-focused vaccines has been described as the latest stage in the development of vaccine formulations, providing new ideas for the development of broad-spectrum Vibrio vaccines. Interestingly, a cryptic epitope (K7) was identified in OmpK from Vibrio species, which is itself immunogenic but is not involved in the immune response to intact OmpK. Epitope K7 is a 15-residue hairpin structure in OmpK predicted to contain a 6-residue extracellular turn region. Interestingly, unlike other highly variable extracellular long loops, epitope K7 is the only conserved extracellular short turn in OmpK, with a similarity of 33 % to 93 %. K7 homologous peptides stimulated the production of specific antibodies, confirming their high immunogenicity. Cross-immunoreactivity between K7 homologous and K7-induced antibodies was evaluated by peptide-based ELISA, western blot, and cell-based ELISA. Flow cytometry and immunofluorescence assay further confirmed that the native epitope K7 in OmpK is surface-exposed and therefore an extracellular target that binds to antibodies. Moreover, an antibody-dependent and complement-mediated serum bactericidal assay suggested that epitope K7-induced antibodies have vibriocidal activity. In conclusion, we identified a conserved cryptic epitope with cross-immunoreactivity in OmpK from Vibrio species. Our results suggest that epitope K7 could be an ideal candidate for the design of epitope-focused vaccines against diverse Vibrio infections.

Probiotics offer significant health advantages as they enter the digestive system via diet or water intake, playing a crucial role in enhancing immunity, growth, gastrointestinal microbiota, and feed attribute. The main objective of study was to focus on the impact of probiotic functional feed (PFF) on Nile tilapia ( Oreochromis niloticus ) exposed to challenges from Vibrio harveyi and Vibrio parahaemolyticus . The investigation aims to analyze the genes linked to immunity, hemato-biochemical indices, and the immunological response in tilapia. PFF is a vital component of fish feed production, providing suitable nutrition for various ages and stages to promote healthy growth. The study comprises four treatments: CPF-1 (control group, diet included solely of basal fish feed), the 20% of PFF2 ( Rossellomorea marisflavi spp. (DAS-SCF02–1 × 104), PFF3 ( Agrococcus spp. (RKDAS1-1 × 106), and PFF4- (DAS-SCF02–1 × 104 + RKDAS1 (1 × 107). A total of 150 Nile tilapia juveniles, weighing 2.56 ± 1.26 g, were administered PFF in triplicates. Significant improvements were observed in hematological indices, encompassing white blood cells (WBC), hemoglobin (Hb), red blood cells (RBC), hematocrit (Htc), and blood performance (BP) in probiotic-treated groups compared to control. Biochemical analysis revealed lower levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in fish fed with probiotics, accompanied by increased total protein, albumin, and globulin levels. Furthermore, probiotic-fed fish exhibited heightened blood glucose, total cholesterol, and triglyceride levels. Immunological assessments demonstrated increased lysozyme activity, intracellular superoxide anion production, reactive nitrogen species synthesis, and myeloperoxidase activity in probiotic-fed groups. Immune gene expression analysis revealed up-regulation of stress response, cytokine signaling, and immune defense-related genes ( HSP70 , IL-1β , I C3 , IFN-α , IFN-γ , GF1 , GH , IL-1 , and Lyz ). In a Vibrio challenge study, probiotic-fed fish exhibited improved survival rates, underscoring the protective effects of probiotics against bacterial infections. Overall, this research underscores the multifaceted benefits of probiotic supplementation in enhancing the health and immunity of tilapia.

Vibrio vulnificus populates coastal waters around the world, where it exists freely or becomes concentrated in filter feeding mollusks. It also causes rapid and life-threatening sepsis and wound infections in humans. Of its many virulence factors, it is the V. vulnificus capsule, composed of capsular polysaccharide (CPS), that plays a critical role in evasion of the host innate immune system by conferring antiphagocytic ability and resistance to complement-mediated killing. CPS may also provoke a portion of the host inflammatory cytokine response to this bacterium. CPS production is biochemically and genetically diverse among strains of V. vulnificus, and the carbohydrate diversity of CPS is likely affected by horizontal gene transfer events that result in new combinations of biosynthetic genes. Phase variation between virulent encapsulated opaque colonial variants and attenuated translucent colonial variants, which have little or no CPS, is a common phenotype among strains of this species. One mechanism for generating acapsular variants likely involves homologous recombination between repeat sequences flanking the wzb phosphatase gene within the Group 1 CPS biosynthetic and transport operon. A considerable number of environmental, genetic, and regulatory factors have now been identified that affect CPS gene expression and CPS production in this pathogen.

are widespread in coastal environments, where they are being increasingly associated with mortality episodes of farmed bivalves. This is the case for strain r172 isolated from a mortality event of adult mussels in Spain in 2022. In this study, the infection dynamics and immune responses of adult challenged with r172 were investigated using different experimental approaches. First, a virulence assay by injection (1 x 108 CFU/100 µL) was performed at different temperatures (12, 18 and 24 °C). The results showed that mussel mortality (about 50 % within 8 days) was independent of increasing temperatures. Subsequently, an injection/cohabitation experiment was carried out placing together in the same tank -injected (Donors, 108 CFU/mL) with un-injected mussels (Recipients). The time course of infection was then followed by evaluating positivity to by PCR, responses of haemolymph components (haemocyte lysosomal membrane stability and serum lysozyme activity) and tissue histopathology (gills and digestive gland). The results showed a partial horizontal transfer of from infected to uninfected mussels, with transient effects on haemolymph responses and histopathological lesions in both groups. Finally, in order to mimic more realistic environmental conditions, a bath infection experiment was carried out, exposing mussels to in seawater (105 CFU/mL). This condition resulted in lower stress in haemocytes; moreover, no lysozyme release or histopathological alterations were observed. Overall, the results show that is able to cope with challenge with , indicating that this Vibrio species is moderately pathogenic to adult mussels under the established experimental conditions.

This study investigated the effects of replacement of fishmeal (FM) with poultry by-product (PBM) protein, supplemented with black soldier fly, Hermetia illucens (HI) larvae on growth, histomormhology, immunity and resistance to Vibrio harveyi in juvenile barramundi. Two hundred and twenty five barramundi averaging 3.51 ± 0.03 g were randomly allocated into three groups and fed isonitrogenous and isocalorific diets containing different levels of PBM supplemented with HI as follows: Control (FM based diet), 45PBM + HI (45% PBM supplemented with 10% HI), and 90PBM + HI (90% PBM supplemented with 10% HI) for 6 weeks. Results showed that dietary inclusion of 45PBM + HI significantly improved the growth performance than control whereas growth inhibition occurred in the 90PBM + HI. The 45PBM + HI groups demonstrated significant increases in histometric measurements (villus and enterocyte width, and microvilli height) and acidic mucins. The impaired growth in 90PBM + HI groups was further associated with multifocal necrosis in the liver, an upregulation of the stress related genes (HSP70 and HSP90) and increase in the levels of liver enzymes. When 45PBM + HI was fed, survival against V. harveyi increased significantly and also an increase in serum immunity and immune-related genes in the head kidney was observed after infection.

The interleukin-17 (IL-17) family consists of proinflammatory cytokines conserved during evolution. A comparative genomics approach was applied to examine IL-17 throughout evolution from poriferans to higher vertebrates. Cnidaria was highlighted as the most ancient diverged phylum, and several evolutionary patterns were revealed. Large expansions of the IL-17 repertoire were observed in marine molluscs and echinoderm species. We further studied this expansion in filter-fed Mytilus galloprovincialis , which is a bivalve with a highly effective innate immune system supported by a variable pangenome. We recovered 379 unique IL-17 sequences and 96 receptors from individual genomes that were classified into 23 and 6 isoforms after phylogenetic analyses. Mussel IL-17 isoforms were conserved among individuals and shared between closely related Mytilidae species. Certain isoforms were specifically implicated in the response to a waterborne infection with Vibrio splendidus in mussel gills. The involvement of IL-17 in mucosal immune responses could be conserved in higher vertebrates from these ancestral lineages.