Tomokazu Takano

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.

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.

Linkage Mapping of Toll-Like Receptors (TLRs) in Japanese Flounder, Paralichthys olivaceus

Toll-like receptors (TLRs) are responsible for the recognition of specific pathogen-associated molecular patterns and consequently activate signal pathways leading to inflammatory and interferon responses. The region surrounding several TLRs was previously found to be associated with resistance to specific disease. Hence, we determined the location of 11 TLRs in Japanese flounder (Paralichthys olivaceus) using polymorphic microsatellite markers. TLR1 and TLR3 were located on linkage group (LG) 21 and 7, respectively. Membrane TLR5 and soluble TLR5 were mapped to LG22. TLR7 and TLR8 were mapped to LG3. TLR9 was found on LG1 and TLR14 and TLR21 were located on the same linkage group, LG10. TLR22 was found on LG8. Interestingly, TLR2 was mapped with the previously reported Poli9-8TUF microsatellite marker which is tightly associated with lymphocystis virus disease resistance. Therefore, TLR2 is a candidate gene for resistance to lymphocystis disease. These results imply that the location of a TLR associated with a particular disease may be valuable for the research on the relationship between host immune response and disease resistance.

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.

Differential gene expression profiles in Japanese flounder (Paralichthys olivaceus) with different susceptibilities to edwardsiellosis.

Edwardsiellosis, which is caused by Edwardsiella tarda, a Gram-negative bacterium, is one of the most serious problems in Japanese flounder (Paralichthys olivaceus) culture. In this study, we examined the immune responses at the molecular level of genetic groups of Japanese flounder that are either susceptible or moderately susceptible to E. tarda infection using a cDNA microarray which was spotted with approximately 2000 different genes. Four different genetic groups of Japanese flounder (groups A, B, C and D) were infected with E. tarda by immersion. Mortality was 100% in groups A and C but only about 50% in groups B and D. Microarray analyses revealed 36 genes that were differentially expressed between the susceptible (A and C) and resistant (B and D) groups before E. tarda infection. Three days after the challenge, the resistant groups highly expressed MHC class I antigen processing and presentation-related genes, while the susceptible groups highly expressed genes involved in innate immune responses. The microarray results could be useful for selective breeding to enhance disease resistance of Japanese flounder against E. tarda and for studying strategies for controlling edwardsiellosis.

DNA vaccine-mediated innate immune response triggered by PRRs in teleosts

Aquacultured fish are threatened by many pathogens, often with serious consequences. Vaccination is one of the most effective tools for enhancing host immunity and protecting fish from infections. Vaccination with DNA vaccine is based on the administration of the gene encoding a vaccine antigen. Several effective DNA vaccines that encode viral or bacterial antigenic proteins have already been shown to be effective for cultured fish. This review summarizes current knowledge on fish DNA vaccines, and the mechanism of interaction between the DNA vaccines and host immunity, especially focusing on the enhancement of innate immunity mediated through direct-recognition of DNA vaccine by pattern recognition receptors (PRRs). To date, numerous fish PRR genes have been identified, and the primordial functions of PRRs involved in the innate immune response to viral and bacterial nucleic acids have been increasingly clarified. The evolutionary conservations and divergences in the PRR mechanisms of teleosts and mammals are focused on their molecular features and the recognition of DNA vaccine mediated by TANK binding kinase 1. In addition, the mechanism of type I interferon production in teleosts, which is enhanced after the recognition of cytosolic nucleic acids and current topics on DNA sensing by PRRs are also introduced.

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.

Ontogeny of anti-viral hemorrhagic septicemia virus (VHSV) immunity in developing Japanese flounder

We examined the ability of developing Japanese flounder (Paralichthys olivaceus) to acquire protective immunity after exposure to viral hemorrhagic septicemia virus (VHSV). Juveniles measuring 9.8 cm average body length were not susceptible to infection with VHSV at 20 °C, while the smaller fish were susceptible. Mortality was not observed after secondary infection at 15 °C in the 9.8 cm cohort that had previously been exposed to the virus at 20 °C, while the smaller fish were susceptible to secondary infection. The expression of interferon (IFN)-related genes was shown to be better developed in larger fish upon virus infection and basal expression levels of the virus recognition proteins were higher in larger fish. Virus-specific antibody was detected in the larger fish, but not in smaller fish. These data indicate that the largest juvenile (9.8 cm) acquired immunity against VHSV infection at the first virus challenge, but smaller fish did not. The anti-viral immune system in the Japanese flounder matures when juveniles reach approximately 10 cm.

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.

Complete genome sequence of Photobacterium damselae subsp. piscicida strain OT-51443 isolated from yellowtail (Seriola quinqueradiata) in Japan

ABSTRACT Pseudotuberculosis caused by infection of Photobacterium damselae subsp. piscicida has caused serious economic damages to aquaculture farms worldwide. Here, the whole-genome sequence of P. damselae subsp. piscicida strain OT-51443, isolated in Japan, was determined and suggests that this genome consists of two chromosomes and five plasmids.