Takamitsu Sakai

The efficacy of five avirulent Edwardsiella tarda strains in a live vaccine against Edwardsiellosis in Japanese flounder, Paralichthys olivaceus.

We evaluated the tissue persistence and live vaccine efficacy of five avirulent Edwardsiella tarda strains (E22, SU100, SU117, SU138, and SU244) isolated from the Japanese eel (Anguilla japonica) and from the environment. The live vaccines, containing a single strain, were injected intraperitoneally into Japanese flounder (Paralichthys olivaceus). Viable bacteria from all the strains (excluding SU100) were recovered from trunk-kidney tissue 28 d post-injection. Japanese flounder inoculated with E22 had the highest relative percentage survival (RPS = 45%) in an artificial challenge with virulent E. tarda (NUF806). The serum of E22-vaccinated fish had a significantly higher agglutination titer against NUF806. In contrast, there was little or no increase in the agglutination titer of the fish that were inoculated with the remaining avirulent strains. Injection with avirulent E. tarda increased the expression of cytokine genes, including interleukin-1beta (IL-1beta), type 1 interferon (IFN), and IFN-gamma in head-kidney of the Japanese flounder.

Identification of Edwardsiella ictaluri and E. tarda by Species-Specific Polymerase Chain Reaction Targeted to the Upstream Region of the Fimbrial Gene

Phylogenetic analysis of nine strains of Edwardsiella ictaluri and eight strains of E. tarda (six typical motile strains and two atypical nonmotile strains) isolated from diseased fish was performed using the upstream region of the fimbrial gene cluster. Strains of E. ictaluri and E. tarda were significantly clustered into separate groups. Moreover, atypical E. tarda strains were clustered into a different group from the other strains. Three polymerase chain reaction (PCR) primer sets for differential detection of E. ictaluri as well as typical and atypical E. tarda were developed from the respective characteristic sequences. Strains of E. ictaluri, typical E. tarda, and atypical E. tarda were specifically detected by PCR using each primer set. No amplifications were observed after the use of these three primer sets with 25 other bacterial species, including fish pathogens. In addition, the three primer sets were able to detect the DNA of each target species from fish kidney and liver artificially infected with E. ictaluri or E. tarda.

Identification of a 19.3-kDa protein in MRHA-positive Edwardsiella tarda: putative fimbrial major subunit

The hemagglutinating properties of Edwardsiella tarda isolated from fish were investigated. Hemagglutination of E. tarda was not inhibited by D-mannose but was strongly inhibited by fetuin and N-acetylneuraminic acid. Extraction of hemagglutinating activity from bacterial cells was achieved using n-octyl-beta-D-thioglucoside (NOTG), and the NOTG extracts were fractionated by sucrose density gradient ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the fractions revealed that a 19.3-kDa protein band appeared in the fractions exhibiting highest hemagglutinating activity. In an immunoblot analysis of NOTG extracts from 18 strains of E. tarda, the 19.3-kDa protein was detected only in the extracts possessing hemagglutinating activity. The predicted amino acid sequence of a 534-bp gene encoding the 19.3-kDa protein was identical to fimbrial subunit (FimA) of E. tarda by FASTA homology search. These findings suggest that fimbriae are implicated in the hemagglutination of E. tarda.

Comparative genomics reveals that a fish pathogenic bacterium Edwardsiella tarda has acquired the locus of enterocyte effacement (LEE) through horizontal gene transfer.

BackgroundEdwardsiella tarda is an enterobacterium which causes edwardsiellosis, a fatal disease of cultured fishes such as red sea bream, eel, and flounder. Preventing the occurrence of E. tarda infection has thus been an important issue in aquaculture. E. tarda has been isolated from other animals and from many environments; however, the relationship between the genotype and evolutionary process of this pathogen is not fully understood. To clarify this relationship, we sequenced and compared the genomes of pathogenic and non-pathogenic E. tarda strains isolated from fish, human, and eel pond using next-generation sequencing technology.ResultsEight strains of E. tarda were sequenced with high accuracy (>99.9%) with coverages from 50- to 400-fold. The obtained reads were mapped to a public reference genome. By comparing single nucleotide and insertion/deletion polymorphisms, we found that an attenuated strain of E. tarda had a loss-of-function mutation in a gene related to the type III secretion system (T3SS), suggesting that this gene is involved in the virulence of E. tarda. A comprehensive gene comparison indicated that fish pathogenic strains possessed a type VI secretion system (T6SS) and pilus assembly genes in addition to the T3SS. Moreover, we found that an E. tarda strain isolated from red sea bream harbored two pathogenicity islands of T3SS and T6SS, which were absent in other strains. In particular, this T3SS was homologous to the locus of enterocyte effacement (LEE) in enteropathogenic and enterohemorrhagic Escherichia coli. Evolutionary analysis suggested that this locus, here named Et-LEE (E. tarda LEE), was introgressed into the E. tarda genome through horizontal transfer.ConclusionsWe found significant differences in the presence/absence of virulence-related genes among E. tarda strains, reflecting their evolutionary relationship. In particular, a single genotype previously proposed for fish-pathogenic strains may be further divided into two subgroups. Furthermore, the current study demonstrated, for the first time, that a fish pathogenic bacterium carried a LEE-like pathogenicity island which was previously reported only in zoonotic pathogenic enterobacteria. These findings will contribute to the exploration of strain-specific drug targets against E. tarda in aquafarms, while also shedding light on the evolution of pathogenesis in enterobacteria.

Full-Genome Sequencing and Confirmation of the Causative Agent of Erythrocytic Inclusion Body Syndrome in Coho Salmon Identifies a New Type of Piscine Orthoreovirus.

Erythrocytic inclusion body syndrome (EIBS) causes mass mortality in farmed salmonid fish, including the coho salmon, Onchorhynchus kisutchi, and chinook salmon, O. tshawytscha. The causative agent of the disease is a virus with an icosahedral virion structure, but this virus has not been characterized at the molecular level. In this study, we sequenced the genome of a virus purified from EIBS-affected coho salmon. The virus has 10 dsRNA genomic segments (L1, L2, L3, M1, M2, M3, S1, S2, S3, and S4), which closely resembles the genomic organization of piscine orthoreovirus (PRV), the causative agent of heart and skeletal inflammation (HSMI) in Atlantic salmon and HSMI-like disease in coho salmon. The genomic segments of the novel virus contain at least 10 open reading frames (ORFs): lambda 1 (λ1), λ2, λ3, mu 1 (μ1), μ2, μNS, sigma 1 (σ1), σ2, σ3, and σNS. An additional ORF encoding a 12.6-kDa protein (homologue of PRV p13) occurs in the same genomic segment as σ3. Phylogenetic analyses based on S1 and λ3 suggest that this novel virus is closely related to PRV, but distinctly different. Therefore, we designated the new virus ‘piscine orthoreovirus 2’ (PRV-2). Reverse transcription–quantitative real-time PCR revealed a significant increase in PRV-2 RNA in fish blood after the artificial infection of EIBS-naïve fish but not in that of fish that had recovered from EIBS. The degree of anemia in each fish increased as the PRV-2 RNA increased during an epizootic season of EIBS on an inland coho salmon farm. These results indicate that PRV-2 is the probable causative agent of EIBS in coho salmon, and that the host acquires immunity to reinfection with this virus. Further research is required to determine the host range of PRV species and the relationship between EIBS and HSMI in salmonid fish.

Identification of Major Antigenic Proteins of Edwardsiella Tarda Recognized by Japanese Flounder Antibody

Edwardsiella tarda is a fish pathogen that causes systemic infections in fresh water and marine fish. Determining the antigenic proteins is important for the development of an immunodiagnostic tests and a vaccine for effective infection control in fish. In the current study, antigens were detected by immunoblotting and affinity column chromatography using a Japanese flounder (Paralichthys olivaceus) antibody produced by experimental infection with E. tarda. GroEL, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), outer membrane protein A, filament protein, 30S ribosomal protein S6, 50S ribosomal protein L9, cold shock protein, and carbon storage protein were identified as antigens of E. tarda through biochemical analyses of the molecular weights, isoelectric points, and N-terminal amino-acid sequences. These proteins can be easily detected in flounder infected with E. tarda and are potential diagnostic markers.

Identification of novel putative virulence factors, adhesin AIDA and type VI secretion system, in atypical strains of fish pathogenic Edwardsiella tarda by genomic subtractive hybridization.

Edwardsiella tarda, which is known to be the causative agent of edwardsiellosis in freshwater and marine fish, has two motility phenotypes. Typical strains exhibiting motility are isolated mainly from freshwater fish and Japanese flounder. Atypical strains exhibiting non‐motility are isolated mainly from marine fish, with the exception of Japanese flounder. Subtractive hybridization was performed to identify genomic differences between these two phenotypes. Two fragments which showed homology to potential virulence factors were isolated from atypical strains: the autotransporter adhesin AIDA and a component of T6SS. We analysed DNA sequences of about 5 kbp containing these fragments and identified two partial ORF, and ORF encoding for other components of T6SS. The predicted amino acid sequences showed remarkably low homology to components of T6SS reported in the typical E. tarda strain PPD130/91. Furthermore, the organization of these ORF was different from the gene cluster of the typical E. tarda strain. AIDA and T6SS may therefore be associated with different pathogenicity in typical and atypical E. tarda hosts.

Molecular cloning and expression analysis of interferon gamma gene in Japanese flounder Paralichthys olivaceus

Interferons (IFNs) are secreted proteins produced by cells in response to viruses. They induce the antiviral state in cells and play a major role in the defense against virus infection. In contrast to IFN type I, which can be produced by most cell types, IFN-c is produced by T cells and natural killer (NK) cells, specifically by CD4 T helper 1 lymphocytes and CD8 cytotoxic T lymphocytes in response to antigens and mitogens [1]. IFN-c has multiple modulatory effects, including upregulation of pathogen recognition, antigen processing and presentation, the antiviral state, inhibition of cellular proliferation, and effects on apoptosis, activation of microbicidal effecter functions, immunomodulation, and leukocyte trafficking [1]. Since the first identification of an IFN-c homologue in fugu Takifugu rubripes [2], IFN-c genes were reported from several fish species [3–6]. Although a sequence for Japanese flounder Paralichthys olivaceus IFN has been reported [7], it had no similarity to the other IFNs but had more than 60% amino acid identity with the sequences from filamentous phage [8]. In this study, we report molecular cloning of a full-length complementary DNA (cDNA) encoding IFN-c from Japanese flounder. The expression profiles of the IFN-c gene in healthy and viral hemorrhagic septicaemia virus (VHSV) challenged fish were determined by real-time quantitative polymerase chain reaction (PCR). Total RNAs were extracted from the thymus of healthy Japanese flounder using TRIzole reagent (Invitrogen). A pair of degenerate primers for cloning of the IFN-c gene was designed based on the conserved regions of IFN-c sequences from T. rubripes (accession no. CAE82301) and Tetraodon nigroviridis (accession no. CAF95605, this sequence has been registered as unnamed protein product) (Table 1). To obtain the complete IFN-c gene sequences, 50 and 30 rapid amplification of cDNA ends (RACE) were performed with a SMART RACE cDNA Amplification kit (Clontech Laboratories), using primers based on the partial sequences obtained above. DNA sequencing was carried out on an ABI3730 genetic analyzer (Applied Biosystems). Amino acid sequence predictions and multiple alignments were carried out using the GENETYX (Genetic Information Processing Software) and MAFFT (F-INS-i) program of GenomeNet in combination with the GenBank databases for comparison with other known gene sequences. BLASTP sequence homology analyses were performed using the BLAST network server of the National Center for Biotechnology Information. Phylogenetic analysis was performed on the full-length amino acid sequences of known IFN-c using the neighbor-joining method [9]. Fifteen clones were sequenced for exclusion of PCR or sequence errors, and consensus sequence was obtained. Japanese flounder IFN-c nucleotide sequences were deposited in the GenBank under accession number AB435093. The nucleotide and the deduced amino acid sequence of IFNc are shown in Fig. 1. The IFN-c cDNA open reading frame (ORF) was 594 bp encoding 198 amino acids. As in mammalian, avian, and fish IFN-c genes [2–6], the 30untranslated region (30UTR) of the Japanese flounder IFN-c gene contains six multiple mRNA instability motifs (attta). A polyadenylation signal (aataaaa) is located 12 bp upstream from the poly (A) tail. A signal peptide consisting of 23 amino acids is T. Matsuyama (&) A. Fujiwara T. Sakai C. Nakayasu National Research Institute of Aquaculture, Fisheries Research Agency, Minami-Ise, Mie 516-0193, Japan e-mail: matsuym@fra.affrc.go.jp

Cloning and expression analyses of a unique IgT in ayu Plecoglossus altivelis

A unique heavy chain of immunoglobulin (Ig) T was cloned from ayu Plecoglossus altivelis, and its in vivo expression was compared with IgM and IgD expression using quantitative RT-PCR and in situ hybridization. Three immunoglobulin domains, corresponding to CH1, CH2, and CH4, in IgT of other teleost fish species were conserved in the heavy chain. IgT mRNA levels were higher in the spleen, head kidney, trunk kidney, and gill tissue than in the intestine, liver, and skin tissue. IgM transcripts were the most abundant transcripts in all tissues tested. In the in situ hybridization studies, IgT and IgM mRNA-positive cells were scattered across the trunk kidney of healthy fish, while IgD mRNA-positive cells were not detected. mRNA levels of IgT and IgM were significantly upregulated in the gills and trunk kidney after vaccination with Vibrio anguillarum bacterin, but the IgD mRNA level remained unchanged. These results suggest that this unique IgT is expressed in a small number of cells and that it plays a role in immune responses induced by immersion vaccination.

Antigenic proteins of Flavobacterium psychrophilum recognized by ayu Plecoglossus altivelis antisera

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis altivelis and is responsible for substantial economic losses in ayu culture in Japan. To develop effective vaccines for the disease, we identified antigenic proteins of F. psychrophilum using immunoglobulin from ayu that had recovered from BCWD. The whole protein extracted from the bacterium was separated using 2-dimensional polyacrylamide gel electrophoresis and was transferred to a polyvinylidene fluoride membrane. Subsequently, antigenic proteins of the bacterium were detected using western blotting and ayu antisera against F. psychrophilum. Each protein spot showing antigenicity was subjected to tandem mass spectrometry (MS/MS) analysis using a MALDI-QIT-TOF mass spectrometer. Protein identification based on the MS/MS data was performed using the genome database for F. psychrophilum JIP02/86, and the subcellular localization for each identified protein was predicted with web-based tools (LipoP and PSORTb). In total, 62 antigenic proteins were identified: of these, 46 were putative cytoplasmic proteins (e.g. elongation factor Tu and heat shock protein 90). The remaining 21 proteins were identified as putative membrane-bound or secreted proteins and are potential vaccine candidates. These proteins include OmpA, Omp 121, M13 family metallopeptidase, and M48 family metalloprotease.