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In this study, we have isolated four strains of Vibrio anguillarum, revealing that they share the same serotype of O1, biochemical characteristics and virulence factor genes. However, there were differences in haemolytic activity among the bacterial strains; a strain with lower pathogenicity showed γ‐haemolytic activity, whereas other virulent strains showed α‐haemolytic activity on blood agar and higher empA gene expression in RTG‐2 cell line. The most virulent strain was V. anguillarum RTBHR from diseased masu salmon (Oncorhynchus masou), which resulted in mortality of 100% and 93.3% when injected intraperitoneally at concentrations of 9 × 105 and 6.3 × 105 colony‐forming units/fish in rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch), respectively. A formalin‐inactivated vaccine of V. anguillarum RTBHR induced a protective and specific immunity in rainbow trout as the vaccinated fish exhibited low cumulative mortality in a challenge test and a high specific antibody response in enzyme‐linked immunosorbent assay at 8 weeks post‐vaccination. The produced antibody was bound to bacterial proteins of 30–37 kDa in size. This adaptive immune response was detected as early as day 1, with quantitative polymerase chain reaction analysis revealing the upregulated expression of genes encoding for TCRα, T‐bet, mIgM and sIgM in rainbow trout. This suggested that the vaccine induced T (probably a more dominant Th1 response) and B cell responses. In conclusion, the vaccine successfully protected fish from V. anguillarum infection by eliciting cellular and humoral immune responses. [ABSTRACT FROM AUTHOR]

Streptococcus parauberis , a gram-positive cocci, causes bacterial disease in farmed fish. The recent increase in S. parauberis infection in aquatic farms in South Korea has justified the importance of vaccine development for the prevention of this disease. In this study, we evaluated the effect of subunit vaccines prepared from recombinant M-like protein (SimA) and fibrinogen-binding protein (FBP) candidates with an aluminum hydroxide adjuvant against S. parauberis infection in olive flounder Paralichthys olivaceus. For the in vivo experiment, fish (average length, 7.18 cm; average weight, 3.5 g) were injected intraperitoneally with: phosphate buffer saline (PBS, group 1), PBS/aluminum hydroxide (group 2), FBP/aluminum hydroxide (group 3), SimA/aluminum hydroxide (group 4), and SimA/FBP/aluminum hydroxide (group 5). After 3 weeks, the fish in each group were boosted using PBS (group 1 and 2), FBP (group 3), SimA (group 4), and SimA/FBP (group 5) without adjuvant. We found that the relative percent survival of fish after S. parauberis exposure in group 2, 3, 4, and 5 was 6.25%, 18.75%, 50%, and 12.5%, respectively, whereas the mortality in groups 1 was 80%, respectively. We performed Western blot, ELISA, and quantitative real time RT-PCR (qRT-PCR) after vaccination to investigate the further efficacy of the vaccine. Western blot and ELISA of vaccinated fish serum confirmed the production of specific antibodies against SimA and FBP. Furthermore, results of qRT-PCR showed that recombinant protein SimA induced a remarkably specific-antibody response compared with that in FBP or control and increased the expression of various immune response-related genes including interleukin-8 (IL-8), toll-like receptor 2 (TLR2), tumor necrosis factor-α (TNF-α), CD4-1, and MHC II. Thus, these results indicate that SimA is a potent vaccine candidate for protection against S. parauberis infection. • SimA based vaccine against Streptococcus parauberis in fish prevents infection. • Aluminum hydroxide serves as an effective adjuvant. • SimA/adjuvant-vaccinated fish showed extended survival rate. • In addition expressed immune response-related genes and elevated antibody titer. [ABSTRACT FROM AUTHOR]

Interleukin-1 beta (IL-1β) is transcribed by monocytes, macrophages, and dendritic cells in response to activation of toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) or cytokine signalling and causes a rapid inflammatory response to infection. IL-8, also known as chemokine C-X-C motif ligand (CXCL)-8, is regulated by IL-1β and affects the chemotaxis of macrophages and neutrophils upon pathogen infection. In healthy red sea bream, rsbIL-1β is most highly distributed in the liver, and rsbIL-8 is most highly distributed in the head kidney. In response to RSIV infection, rsbIL-1β and rsbIL- 8 mRNA are significantly upregulated in the kidney and spleen. This may be because the primary infection targets of RSIV are the kidney and spleen. In the gills, both genes were significantly upregulated at 7 days after RSIV infection and may be accompanied by a cytokine storm. In the liver, both genes were significantly downregulated at most observation points, which may be because the immune cells such as macrophages and dendritic cells expressing rsbIL-1β or rsbIL-8 migrated to other tissues because the degree of RSIV infection was relatively low. Using a GFP fusion protein, it was confirmed that rsbIL-1β and rsbIL-8 were localized to the cytoplasm of Pagrus major fin (PMF) cells. RsbIL-1β overexpression induced the expression of interferon gamma (IFN-γ), myxovirus-resistance protein (Mx) 1, IL-8, IL-10, TNF-α, and MyD88, while rsbIL-8 overexpression induced the expression of IFN-γ, Mx1, rsbIL-1β and TNF-α. In addition, overexpression of both genes significantly reduced the genome copies of RSIV and significantly reduced the viral titers. Therefore, rsbIL-1β and rsbIL-8 in red sea bream play an antiviral role against RSIV through their normal signalling. • RsbIL-1β and rsbIL-8 are ubiquitously distributed in healthy red sea bream. • RsbIL-1β and rsbIL- 8 mRNAs are significantly upregulated in the kidney and spleen. • RsbIL-1β and rsbIL-8 were localized to the cytoplasm of Pagrus major fin (PMF) cells. • RsbIL-1β induced IFN-γ, Mx1, IL-8, IL-10, TNF-α, and MyD88, and rsbIL-8 induced IFN-γ, Mx1, rsbIL-1β and TNF-α. • RsbIL-1β and rsbIL-8 significantly reduced the genome copies of RSIV and significantly reduced the viral titers. [ABSTRACT FROM AUTHOR]

Thioredoxin-like protein 1 (TXNL1) is a redox-active protein belonging to the thioredoxin family, which mainly controls the redox status of cells. The TXNL1 gene from Amphiprion clarkii (AcTXNL1) was obtained from a pre-established transcriptome database. The AcTXNL1 is encoded with 289 amino acids and is predominantly localized in the cytoplasm and nucleus. The TXN domain of AcTXNL1 comprises a34CGPC37 motif with redox-reactive thiol (SH-) groups. The spatial distribution pattern of AcTXNL1 mRNA was examined in different tissues, and the muscle was identified as the highest expressed tissue. AcTXNL1 mRNA levels in the blood and gills were significantly increased in response to different immunostimulants. In vitro antioxidant capacity of the recombinant AcTXNL1 protein (rACTXNL1) was evaluated using the ABTS free radical-scavenging activity assay, cupric ion reducing antioxidant capacity assay, turbidimetric disulfide reduction assay, and DNA nicking protection assay. The potent antioxidant activity of rAcTXNL1 exhibited a concentration-dependent manner in all assays. Furthermore, in the cellular environment, overexpression of AcTXNL1 increased cell viability under H 2 O 2 stress and reduced nitric oxide (NO) production induced by lipopolysaccharides (LPS). Collectively, the experimental results revealed that AcTXNL1 is an antioxidant and immunologically important gene in A. clarkii. • TXNL1 from A. clarkii was molecularly and functionally characterized. • AcTXNL1 was ubiquitously expressed in all tissues and significantly inducible upon immunostimulants. • Recombinant AcTXNL1 showed potent antioxidant and DNA protection activities. • Overexpression of AcTXNL1 enhanced cell viability under oxidative stress and reduced NO production in macrophages. [ABSTRACT FROM AUTHOR]

• ANK-based DNA vaccine induced a protective immunity in RBIV-infected rock bream. • ANK gene expression displayed a correlation with fish mortality. • TLR3, TLR9, MyD88, Mx, ISG15, PKR, MHC class I, perforin, Fas, Fas ligand and caspases were highly expressed in ANK-based vaccinated group. • IL1β, IL8 and TNFα were not enhanced in ANK-based vaccinated group. Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream (Oplegnathus fasciatus) and remains an unsolved problem in Korea aquaculture industry. In this study, we assessed the potential of ankyrin repeat (ANK)-containing proteins to induce protective immunity in RBIV-infected rock bream. Rock bream administered with ankyrin repeat-containing protein-based DNA vaccine (200 ng/fish) exhibited significant protection against at 4 and 8 weeks post vaccination to infected with 6.7 × 105 RBIV at 23°C; relative percent survival (RPS) of 60.04% and 40.1%, respectively. Furthermore, survivors from the first infection were strongly protected from RBIV (1.1 × 107) re-infection at 70 days post infection, as 100% RPS was observed and without clinical signs of RBIV diseases. Moreover, TLR3 (9.5-fold), TLR9 (5.2-fold), MyD88 (15.9-fold), Mx (55.5-fold), ISG15 (19.0-fold), PKR (24.2-fold), MHC class I (5.1-fold), perforin (6.5-fold), Fas (6.4-fold), Fas ligand (7.1-fold), caspase8 (5.0-fold), caspase9 (12.5-fold), and caspase3 (6.3-fold) responses were significantly elevated in the muscle (vaccine injection site) of ANK-based DNA vaccinated fish at 7 days post vaccination. However, inflammatory cytokines (IL1β, IL8, and TNFα) were not enhanced in the vaccinated rock bream. Moreover, ANK gene may be a good candidate to detect RBIV infection or in revealing specific information to elucidate the pathogenic mechanisms underlying RBIV infection. In summary, ANK-based DNA vaccination in rock bream induced TLR- and IFN-mediated or apoptosis-related immune responses and suggest efficient preventive measures against RBIV. [ABSTRACT FROM AUTHOR]