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Two liver-expressed antimicrobial peptide 2 (LEAP2) isoforms were characterized in a primitive chondrostean sturgeon species, Acipenser baerii (Acipenseriformes). A. baerii LEAP2 isoforms represented essentially common structures shared by their vertebrate orthologs at both genomic (i.e., tripartite organization) and peptide (two conserved disulfide bonds) levels. A. baerii LEAP2 isoforms (designed LEAP2AB and LEAP2C, respectively) phylogenetically occupy the most basal position in the actinopterygian lineage and represent an intermediate character between teleostean and tetrapodian LEAP2s in the sequence alignment. Molecular phylogenetic analysis including LEAP2s from extant primitive fish species indicated that the evolutionary origin of ancestral LEAP2 in vertebrate groups should date back to earlier than the actinopterygian-sarcopterygian split. Gene expression assays under both basal and stimulated conditions suggested that A. baerii LEAP2 isoforms have undergone substantial subfunctionalization in tissue distribution pattern, developmental/ontogenetic expression, and immune responses. LEAP2AB showed a predominant liver expression, while LEAP2C exhibited the highest level of expression in the intestine. LEAP2C was a more dominantly expressed isoform during embryonic development and prelarval ontogeny. The LEAP2AB isoform is more closely associated with innate immune response to microbial invasion, compared with LEAP2C, as evidenced by results from LPS, poly(I:C) and Aeromonas hydrophila challenges. Synthetic mature peptides of LEAP2AB displayed a more potent antimicrobial activity than did LEAP2C. Data from this study could be useful not only to provide deeper insights into the evolutionary mechanism of LEAP2 in the actinopterygian lineage but also to better understand the innate immunity of this commercially important chondrostean species., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In the present study, we have developed short interfering RNA (siRNA) expression vector utilizing rock bream beta-actin promoter and examined the possible use for the inhibition of highly pathogenic fish virus, rock bream iridovirus (RBIV), replication in vitro. Initially, in order to express siRNA effectively, we added several modifications to wild-type rock bream beta-actin promoter. Next, we succeeded in knocking down the expression of enhanced green fluorescent protein reporter gene expression in fish cells using newly developed vector more effectively than the fugu U6 promoter-driven vector we described previously. Finally, we could observe that cells transfected with modified rock bream beta-actin promoter-driven siRNA expression vector targeting major capsid protein (MCP) gene of RBIV exhibited more resistance to RBIV challenge than other control cells. Our results indicate that this novel siRNA expression vector can be used as a new tool for therapeutics in virus infection in fish species.

For the production of ghost bacteria vaccine to prevent the streptococcal disease in aquaculture fish species, a double cassettes vector was constructed and cloned in Escherichia coli DH5alpha. Ghost bacteria vaccine production from Escherichia coli DH5alpha/pHCE-InaN-GAPDH-Ghost 37 SDM (SIG) was maximized at a glucose concentration of 1 g/l, agitation of 300 rpm, and aeration of 1 vvm. The maximal efficiency of ghost bacteria formation was obtained at the mid-exponential phase (OD600=2.0) with the concentration of 0.77 g/l for SIG. The molecular mass of GAPDH was detected at 67 kDa with the insoluble fraction, by SDS-PAGE and Western blot. The protective efficacy of ghost bacteria vaccine was evaluated by challenge test using olive flounder. The cumulative mortalities of the positive control, formalin-killed cell (FKC) vaccine, and SIG vaccine immunized groups were 91% , 74% , and 57% , respectively. These results suggest that SIG vaccine showed efficacy as a vaccine and had a higher potential to induce protective antibodies than did FKC vaccine.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been recognized as an invariant internal control for various gene expression studies due to its classical housekeeping role in the glycolytic metabolism. However recently, this enzyme has been proven to be a multifunctional protein involved in diverse non-glycolytic activities. In the present study, two paralogue isoforms of GAPDH mRNAs were characterized from a marine teleost species, rockbream (Oplegnathus fasciatus; Perciformes) and their transcriptional responses to bacterial and viral infections were examined. Rockbream GAPDH1 and GAPDH2 cDNAs encoded 333 and 335 amino acids, respectively, and the amino acid sequence identity between those two isoforms was 74%. Two isoform GAPDH mRNAs were detected ubiquitously in all of tissues examined, but their expression levels were quite variable among tissues. Based on the real-time RT-PCR analysis, the transcription of rbGAPDH1 was affected by neither bacterial (Escherichia coli, Edwardsiella tarda, Vibrio anguillarum or Streptococcus iniae) nor viral (rockbream iridovirus; RBIV) challenges. However on the contrary, the mRNA expression of rbGAPDH2 was significantly up-regulated in liver resulting from the bacterial infections (up to 25-fold), and in both liver (more than sixfold) and kidney (up to fivefold) from the viral infection. Results in the present study suggest that teleost GAPDH isoforms may also be potentially involved in immune modulations especially with respect to inflammatory responses, which is distinct from its classical glycolytic function.

A dual vector expressing the ghost-inducing PhiX174 lysis E gene and the bacterial DNA degrading staphylococcal nuclease A (SNA) gene was constructed to solve the problem of remnant antibiotic resistance genes and genomic DNA with intact pathogenic islands in the final product of Edwardsiella tarda ghosts (ETG). The SNA (devoid of secretion signal sequence and the nuclease B amino terminus sequence), fused with the 26 amino acid N-terminal sequence of the lambda phage Cro gene, showed successful degradation of bacterial nucleic acids. Furthermore, the nuclease activity of SNA in E. tarda was enhanced by codon optimization of the SNA gene using site-directed mutagenesis. ETG were generated via coexpression of the SNA gene and lysis gene E under the control of each lambdaP(R) promoter. The ghost bacteria generation system we describe is advantageous as it allows the use of a single plasmid, improves safety and vaccine purity by limiting residual genetic content from the ghost bacteria, and reduces production costs through cheap means of induction that use only temperature shifts.

The vaccine potential of Edwardsiella tarda ghosts produced by gene E mediated lysis was investigated using tilapia (Oreochromis mosambicus). Tilapia immunized with E. tarda ghosts (ETG) and formalin killed E. tarda (FKC) vaccines showed significantly higher serum agglutination titers than control fish. Fish immunized with ETG showed no significant differences with fish immunized with FKC in serum agglutination titers, but showed significantly higher bactericidal activity than fish immunized with FKC. Furthermore, fish immunized with ETG showed higher protection than fish immunized with FKC. As this promising type of a non-living whole cell envelope preparation seems to be favorable over conventional vaccines, we suggest E. tarda ghosts as a new vaccine candidate.

In the present study, Pseudocohnilembus persalinus was first reported as a species causing scuticociliatosis in olive flounder Paralichthys olivaceus. Based on the stained specimens, P. persalinus was clearly differentiated from Uronema marinum, which has been shown to be a cause of scuticociliatosis in farmed olive flounder in Korea from its characteristic oral infraciliature structure. The 1754 bp small subunit ribosomal RNA (SS rRNA) gene sequence of P. persalinus showed 95% homology with the partial sequence of P. hargisi SS rRNA. Moreover, multiplex PCR based on the species-specific amplification of the SS rRNA gene sequence enabled us to distinguish P. persalinus from U. marinum in a simple and rapid manner. P. persalinus was clearly differentiated from U. marinum even when the host was infected simultaneously with both species. These data suggest that the multiplex PCR procedure would make it possible to avoid the cumbersome and time-consuming procedures of morphological analysis for the definitive identification of ciliates.

In the course of identifying scuticociliates recently obtained from systemically infected olive flounder Paralichthys olivaceus in Korea, we found a scuticociliate species whose small subunit ribosomal RNA (SS rRNA) gene was not amplified by species-specific primers previously designed for Uronema marinum and Pseudocohnilembus persalinus. By studying morphological characteristics of wet-mounted and stained specimens, we identified the species as Philasterides dicentrarchi, which has been reported to cause systemic infection in the European sea bass Dicentrarchus labrax and turbot Scophthalmus maximus. In this study, we compared morphological characteristics of our specimens with previously reported Philasterides species, including P. dicentrarchi, and sequenced the SS rRNA gene in order to design P. dicentrarchi specific primers. This is the first report on scuticociliatosis caused by P. dicentrarchi from marine fish in Asia.

Little is known about the effects of bile acids in relation to infectivity on the biological characteristics of Uronema marinum, a serious opportunistic parasite of farmed olive flounder Paralichthys olivaceus. In this study, we examined the effects of bile acids on the proliferation of U. marinum and on proteinase production in vitro. Proliferation of U, marinum was significantly enhanced by lithocholic acid (LCA) at 30 and 60 pmol, and by chenodeoxycholic acid (CDCA) at 0.06 pmol. In contrast, a significant decrease in proliferation was observed with cholic acid (CA) at 30 and 60 micromol, and with deoxycholic acid (DCA) at all amounts used. Proteinase production from live U. marinum was significantly increased by LCA, whereas CA significantly decreased proteinase production. CDCA and DCA had no effect on proteinase production. Although the types and concentrations of bile acids in the faeces of olive flounder are not known, the present results suggest that bile acids in the culturing water might influence the proliferation and production of proteinases in U. marinum, resulting in an increased possibility of scuticociliatosis in olive flounder farms.