Characterization of a specific monoclonal antibody against immunoglobulin light kappa/L1 chain in olive flounder (Paralichthys olivaceus).

Immunoglobulins (Ig) are heterodimeric proteins that play critical roles in the adaptive immune system of vertebrates. Because of their plasticity, teleostean Igs are more diverse, and thus do not conform to mammalian classifications. Because of this, mammalian-based Ig cell markers cannot be used successfully to study immune responses in fish. There is therefore a need to produce Ig-specific cell markers for fish. Here, we attempted to identify the specific isotype detected by an Ig light chain-specific monoclonal antibody (anti-olive flounder IgL-mAb: M7C3-4) that we had previously produced [11]. Three newly identified sequences of the Ig light chain from olive flounder were classified according to their isotypes. Subsequent analyses revealed that M7C3-4 was able to specifically detect lymphocytes expressing one of the κ chains (Igκ-a) in olive flounder. Interestingly, Igκ-a+ B cells were more abundant in spleen and trunk-kidney than in peripheral blood, indicating a distribution different from that of IgM+ B cells. Our work reveals interesting aspects of B cell distribution and differentiation, and may aid in the production of suitable and effective cell markers for olive flounder.

Development of an immunochromatography assay kit for rapid detection of ranavirus.

Ranaviruses are large, double-stranded DNA viruses of the family Iridoviridae and are known to be primary pathogens in frogs, fish and other amphibians. These viruses have been shown to be highly adaptable and have the ability to cross species barriers, making them a potent threat to global biodiversity. There is therefore, a need for rapid and efficient diagnostic methods to control the spread of these viruses. To address this, monoclonal antibodies (MAbs) were developed against ranavirus strain FV-3 (standard frog virus 3) to detect the major capsid protein and FV-3gorf19R related hypothetical protein in both the FV-3 and KRV-1 (Korean ranavirus) strains. The antibodies were then applied on a colloidal gold-immunochromatographic assay (GICA) as a kit for the detection of ranaviruses. The kit was able to detect low concentrations of the virus (10(1)TCID50/ml) and showed analytical specificity when tested against other viral pathogens, including those belonging to the same family. It was possible to detect ranavirus in experimentally infected frogs within 30 min using the kit. The kit described here is expected to be a valuable and informative tool for on-site detection of ranavirus in frog.

Combination treatment against scuticociliatosis by reducing the inhibitor effect of mucus in olive flounder, Paralichthys olivaceus.

The olive flounder, Paralichthys olivaceus, is an economically important food fish in Japan and Korea. Scuticociliatosis is a major parasitic disease, and fatal infection with scuticociliates, or mixed infections with scuticociliates and other pathogenic agents (e.g., Vibrio spp.) cause severe mortalities in farmed olive flounders. To date, however, effective chemotherapeutic treatment of scuticociliatosis has only been reported at the in vitro level. In this study, we employed combination treatment, using benzalkonium chloride (to remove excess mucus from the body surface) and bronopol (to kill the parasites), to overcome the protective effect of mucus by some medicine to the scuticociliates. In the presence of the mucus mixture, the higher dose of bronopol (156 ppm) yielded morphologies and motilities similar to those of ciliates treated with the lower dose of bronopol (80 ppm) in the absence of mucus. We also investigated the in vivo effects of this treatment in field trials involving a total of 15,025 naturally infected flounders. We observed that short-term bath treatments with benzalkonium chloride (50 ppm) followed by bronopol (500 ppm) were effective, assessed by the relative percentage mortality (RPS) value. Thus, this study provides a notable therapeutic strategy by removing the mucus to treat scuticociliatosis in olive flounders at the aquaculture field level.

Development of a multiplex PCR assay to detect Edwardsiella tarda, Streptococcus parauberis, and Streptococcus iniae in olive flounder (Paralichthys olivaceus).

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.

Comparative genomic characterization of three Streptococcus parauberis strains in fish pathogen, as assessed by wide-genome analyses.

Streptococcus parauberis, which is the main causative agent of streptococcosis among olive flounder (Paralichthys olivaceus) in northeast Asia, can be distinctly divided into two groups (type I and type II) by an agglutination test. Here, the whole genome sequences of two Japanese strains (KRS-02083 and KRS-02109) were determined and compared with the previously determined genome of a Korean strain (KCTC 11537). The genomes of S. parauberis are intermediate in size and have lower GC contents than those of other streptococci. We annotated 2,236 and 2,048 genes in KRS-02083 and KRS-02109, respectively. Our results revealed that the three S. parauberis strains contain different genomic insertions and deletions. In particular, the genomes of Korean and Japanese strains encode different factors for sugar utilization; the former encodes the phosphotransferase system (PTS) for sorbose, whereas the latter encodes proteins for lactose hydrolysis, respectively. And the KRS-02109 strain, specifically, was the type II strain found to be able to resist phage infection through the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system and which might contribute valuably to serologically distribution. Thus, our genome-wide association study shows that polymorphisms can affect pathogen responses, providing insight into biological/biochemical pathways and phylogenetic diversity.

Phylogenomic network and comparative genomics reveal a diverged member of the ΦKZ-related group, marine vibrio phage ΦJM-2012.

Bacteriophages are the largest reservoir of genetic diversity. Here we describe the novel phage ΦJM-2012. This natural isolate from marine Vibrio cyclitrophicus possesses very few gene contents relevant to other well-studied marine Vibrio phages. To better understand its evolutionary history, we built a mathematical model of pairwise relationships among 1,221 phage genomes, in which the genomes (nodes) are linked by edges representing the normalized number of shared orthologous protein families. This weighted network revealed that ΦJM-2012 was connected to only five members of the Pseudomonas ΦKZ-like phage family in an isolated network, strongly indicating that it belongs to this phage group. However, comparative genomic analyses highlighted an almost complete loss of colinearity with the ΦKZ-related genomes and little conservation of gene order, probably reflecting the action of distinct evolutionary forces on the genome of ΦJM-2012. In this phage, typical conserved core genes, including six RNA polymerase genes, were frequently displaced and the hyperplastic regions were rich in both unique genes and predicted unidirectional promoters with highly correlated orientations. Further, analysis of the ΦJM-2012 genome showed that segments of the conserved N-terminal parts of ΦKZ tail fiber paralogs exhibited evidence of combinatorial assortment, having switched transcriptional orientation, and there was recruitment and/or structural changes among phage endolysins and tail spike protein. Thus, this naturally occurring phage appears to have branched from a common ancestor of the ΦKZ-related groups, showing a distinct genomic architecture and unique genes that most likely reflect adaptation to its chosen host and environment.

Heat shock protein profiles on the protein and gene expression levels in olive flounder kidney infected with Streptococcus parauberis.

Heat shock proteins (HSPs) have been observed in cells exposed to a variety of stresses, including infectious pathogens. This study used a label-free, quantitative proteomic approach and transcriptional gene expression analysis to investigate infection-related HSP proteins and their encoding genes in whole kidneys from olive flounder (Paralichthys olivaceus). During Streptococcus parauberis infection in the flounder, the genes encoding Hsp10, Hsp40A4, Hsp40B6, Hsp40B11, Hsp60, Hsp70, glucose regulated protein 78 (Grp78), Hsp90α, Hsp90ß and Grp94 were induced, and the protein levels of Hsp60, Hsp70, Hsp90α, Hsp90ß and Grp94 were differentially regulated over time. Subsequent results also revealed that Hsp60, Hsp70, Hsp90α, Hsp90ß and Grp94 appear to be the dominant and critical HSPs in olive flounder during bacterial infection. This is the first estimation of the differential involvement of HSPs in the immune response of olive flounder exposed to bacterial infection.

Complete genome sequence of the bacteriophages ECBP1 and ECBP2 isolated from two different Escherichia coli strains.

Escherichia coli is recognized as one of the most abundant avian bacterial pathogens. In this study, we report the sequencing by the traditional Sanger method of ECBP1 and ECBP2: bacteriophages that infected two different E. coli strains which might be used as therapeutic agents in combination with alternative antibiotics.

Cathepsins in the kidney of olive flounder, Paralichthys olivaceus, and their responses to bacterial infection.

Cathepsin activities are responsible for mediating various pathways involved in immune response, including the apoptosis pathway, toll-like receptor (TLR) signaling, cytokine induction and activation of granule serine proteases. In the present study, we investigated cathepsin responses in the kidneys of olive flounder infected with Streptococcus parauberis, analyzing cathepsin expression using a label-free, quantitative proteomic approach in conjunction with quantitative real-time polymerase chain reaction (qRT-PCR). In proteomic analyses, we detected cathepsin B, D, L and S proteins, noting significant decreases and increases in cathepsins B and L, respectively, with infection. Taken together with an evaluation of cathepsin B, D, F, K, L, S and X gene expression in normal and infected kidneys by qRT-PCR, our results indicate that cathepsins B, D, L and S are the dominant lysosomal proteases in the immune system of the teleostei, olive flounder. Cathepsins F, K and X were regarded as minor cathepsins.

Seasonal variation and comparative analysis of non-specific humoral immune substances in the skin mucus of olive flounder (Paralichthys olivaceus).

The epidermal secretion of fish contains various non-specific immune substances that act as the first line of defense against invading pathogens. The present study investigated the level of mucosal antibodies, the activities of hemagglutinin and protease, and other enzymes in the skin mucus of farm reared olive flounder (Paralichthys olivaceus) for 1 year, in order to gain an insight into the relationship between these mucosal immune substances and their seasonal variation. These levels varied significantly during different months of sample collection. The present study showed a positive correlation between water temperature and the level of mucosal antibodies, and an inverse relationship between the level of mucosal antibodies and the activity of mucosal hemagglutinin and protease, but no relationship between lysozyme activity and other innate immune substances. This relationship is thought to be a compensatory response in olive flounder to protect itself against pathogenic microorganisms which are inherently present in the aquatic environment.

Kidney proteome responses in the teleost fish Paralichthys olivaceus indicate a putative immune response against Streptococcus parauberis.

The proteomic response to bacterial infection in a teleost fish (Paralichthys olivaceus) infected with Streptococcus parauberis was analyzed using label-free protein quantitation coupled with LC-MS(E) tandem mass spectrometry. A total of 82 proteins from whole kidney, a major lymphoid organ in this fish, were found to be differentially expressed between healthy and diseased fish analyzed 6, 24, 72 and 120 h post-infection. Among the differentially expressed proteins, those involved in mediating immune responses (e.g., heat shock proteins, cathepsins, goose-type lysozyme and complement components) were most significantly up-regulated by infection. In addition, cell division cycle 48 (CDC48) and calreticulin, which are associated with cellular recovery and glycoprotein synthesis, were up-regulated in the universal protein group, whereas the other proteins in that group were down-regulated. There was continuous activation of expression of immune-associated proteins during infection, but there was also loss of expression of proteins not involved in immune function. We expect that our findings regarding immune response at the protein level would offer new insight into the systemic response to bacterial infection of a major immune organ in teleost fish.

Recombinant interferon-γ activates immune responses against Edwardsiella tarda infection in the olive flounder, Paralichthys olivaceus.

Interferon gamma (IFN-γ) is a cytokine that plays a very important role in defining Th1 immune response in all vertebrates. In this study, recombinant IFN-γ (rIFN-γ) from the olive flounder (Paralichthys olivaceus) was produced in an Escherichia coli system using a pET expression vector. Stimulation of whole kidney leukocytes (immune-related cells) in vitro with the resulting rIFN-γ significantly induced the gene expression of interleukin-1ß (IL-1ß), signal transducer and activator of transcription 1 (STAT1), CXCL13-like chemokine (CXCL13), and IFN-γ. rIFN-γ also weakly induced the expression of IL-1ß, tumor necrosis factor-α (TNF-α), CXCL13, and IFN-γ in olive flounder-derived HINAE (non-immune) cells. The effects of rIFN-γ against Edwardsiella tarda infection in vivo were assessed by intraperitoneally injecting a mixture of rIFN-γ (100 ng) and E. tarda (1 × 10(5) CFU/ml) into the olive flounder. The survival rate in the rIFN-γ-injected group was 60% compared to 0% in the group treated with E. tarda only, demonstrating that olive flounder IFN-γ is effective in reinforcing immune responses and preventing against edwardsiellosis.

Molecular cloning and functional analysis of nucleotide-binding oligomerization domain 1 (NOD1) in olive flounder, Paralichthys olivaceus.

The gene encoding nucleotide-binding oligomerization domain 1 (NOD1) was cloned from olive flounder (Paralichthys olivaceus) and the role played by NOD1 during Edwardsiella tarda infection was evaluated. The complete open reading frame of NOD1 was 2820 bp in length, encoding a 939-amino acid polypeptide. The NOD1 protein contains three conserved domain structures including C-terminal LRRs, a central NACHT motif, and an N-terminal CARD domain, which show similarities of 49-74% to those of other vertebrate counterpart proteins. NOD1 expression was observed in all fish tissues examined, and the levels increased in olive flounder infected with E. tarda, Streptococcus iniae, or viral hemorrhagic septicemia virus (VHSV). When hirame natural embryo (HINAE) cells over-expressing NOD1 were infected with E. tarda, bacterial growth was inhibited, and the IL-1ß transcript level increased compared to that of the control. These findings imply that NOD1 plays an important role in response to E. tarda infection of olive flounder.

RNA-seq-based metatranscriptomic and microscopic investigation reveals novel metalloproteases of Neobodo sp. as potential virulence factors for soft tunic syndrome in Halocynthia roretzi.

Bodonids and trypanosomatids are derived from a common ancestor with the bodonids being a more primitive lineage. The Neobodonida, one of the three clades of bodonids, can be free-living, commensal or parasitic. Despite the ecological and evolutionary significance of these organisms, however, many of their biological and pathological features are currently unknown. Here, we employed metatranscriptomics using RNA-seq technology combined with field-emission microscopy to reveal the virulence factors of a recently described genus of Neobodonida that is considered to be responsible for ascidian soft tunic syndrome (AsSTS), but whose pathogenesis is unclear. Our microscopic observation of infected tunic tissues suggested putative virulence factors, enabling us to extract novel candidate transcripts; these included cysteine proteases of the families C1 and C2, serine proteases of S51 and S9 families, and metalloproteases grouped into families M1, M3, M8, M14, M16, M17, M24, M41, and M49. Protease activity/inhibition assays and the estimation of expression levels within gene clusters allowed us to identify metalloprotease-like enzymes as potential virulence attributes for AsSTS. Furthermore, a multimarker-based phylogenetic analysis using 1,184 concatenated amino acid sequences clarified the order Neobodo sp. In sum, we herein used metatranscriptomics to elucidate the in situ expression profiles of uncharacterized putative transcripts of Neobodo sp., combined these results with microscopic observation to select candidate genes relevant to pathogenesis, and used empirical screening to define important virulence factors.

Complete genome sequence and immunoproteomic analyses of the bacterial fish pathogen Streptococcus parauberis.

Although Streptococcus parauberis is known as a bacterial pathogen associated with bovine udder mastitis, it has recently become one of the major causative agents of olive flounder (Paralichthys olivaceus) streptococcosis in northeast Asia, causing massive mortality resulting in severe economic losses. S. parauberis contains two serotypes, and it is likely that capsular polysaccharide antigens serve to differentiate the serotypes. In the present study, the complete genome sequence of S. parauberis (serotype I) was determined using the GS-FLX system to investigate its phylogeny, virulence factors, and antigenic proteins. S. parauberis possesses a single chromosome of 2,143,887 bp containing 1,868 predicted coding sequences (CDSs), with an average GC content of 35.6%. Whole-genome dot plot analysis and phylogenetic analysis of a 60-kDa chaperonin-encoding gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding gene showed that the strain was evolutionarily closely related to Streptococcus uberis. S. parauberis antigenic proteins were analyzed using an immunoproteomic technique. Twenty-one antigenic protein spots were identified in S. parauberis, by reaction with an antiserum obtained from S. parauberis-challenged olive flounder. This work provides the foundation needed to understand more clearly the relationship between pathogen and host and develops new approaches toward prophylactic and therapeutic strategies to deal with streptococcosis in fish. The work also provides a better understanding of the physiology and evolution of a significant representative of the Streptococcaceae.

Identification and determination of antigenic proteins of Korean ranavirus-1 (KRV-1) using MALDI-TOF/TOF MS analysis.

Ranaviruses are serious pathogens of fish, amphibians, and reptiles, and pose a major threat to global biodiversity. A ranavirus isolated from tissues of diseased tadpoles and frogs in Gangwon province, Korea, in 2006 and 2007, was designated Korean ranavirus-1 (KRV-1) and was infectious in a variety of fish cell lines with highest titers (10(10)TCID(50)/ml) in Epithelioma papulosum cyprini cells (EPCs) and baby hamster kidney-21 (BHK-21) cells. Bullfrog (Rana catesbeiana) tadpoles challenged by immersion in 10(5)TCID(50)/ml of KRV-1 showed 60% mortality within 10 days. SDS-PAGE of frog virus 3 (FV3) and KRV-1 proteins yielded several bands 35-49kDa in size, which were identified as major capsid proteins (MCPs) by MALDI-TOF MS. Immunoblotting of FV3 proteins showed antigenic bands 34kDa and 93kDa in size which were identified by MALDI-TOF/TOF MS as MCP and neurofilament triplet H1-like protein (NF-H1), respectively. In KRV-1, antigenic bands at 32kDa, 69kDa, and 72kDa were identified as MCP, Hypothetical protein, and NF-H1, respectively. The genes encoding these KRV-1 proteins were sequenced. KRV-1 appeared to be closely related to the soft-shelled turtle iridovirus (STIV), based on alignments of amino acid sequences from various ranaviruses. Variability in ranavirus antigenic proteins was apparent in an earlier study. It is expected that use of the methods employed here, together with the results of the present work, will contribute to an understanding of the pathogenesis of ranaviruses, and will further the development of DNA- or protein-based bait vaccines for conservation of natural habitats.

Outer membrane vesicles as a candidate vaccine against edwardsiellosis.

Infection with Edwardsiella tarda, a gram-negative bacterium, causes high morbidity and mortality in both marine and freshwater fish. Outer membrane vesicles (OMVs) released from gram-negative bacteria are known to play important roles in bacterial pathogenesis and host immune responses, but no such roles for E. tarda OMVs have yet been described. In the present study, we investigated the proteomic composition of OMVs and the immunostimulatory effect of OMVs in a natural host, as well as the efficacy of OMVs when used as a vaccine against E. tarda infection. A total of 74 proteins, from diverse subcellular fractions, were identified in OMVs. These included a variety of important virulence factors, such as hemolysin, OmpA, porin, GAPDH, EseB, EseC, EseD, EvpC, EvpP, lipoprotein, flagellin, and fimbrial protein. When OMVs were administrated to olive flounder, significant induction of mRNAs encoding IL-1ß, IL-6, TNFα, and IFNγ was observed, compared with the levels seen in fish injected with formalin-killed E. tarda. In a vaccine trial, olive flounder given OMVs were more effectively protected (p<0.0001) than were control fish. Investigation of OMVs may be useful not only for understanding the pathogenesis of E. tarda but also in development of an effective vaccine against edwardsiellosis.

Enhanced reliability of avian influenza virus (AIV) and Newcastle disease virus (NDV) identification using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS).

In-solution enzymatic and nonenzymatic digestion methods have been successfully implemented in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS)-based virus identification, extending to typing/subtyping of deadly influenza viruses. However, these methods are inefficient in obtaining more precise information on surface proteins of myxovirus particles, not only the hemagglutinin and neuraminidase of influenza virus but also the hemagglutinin-neuraminidase of Newcastle disease virus (NDV). Imbalances in viral protein composition cause ion suppression of tryptic fragments from low-abundant target proteins (surface proteins), adversely affecting reproducibility of mass spectra. Additionally, the coexistence of tryptic peptides from several proteins requires sophisticated statistical solutions for precise result interpretations. To circumvent these, we apply detergent-based (gel-free) partitioning of whole viruses into soluble surface proteins and insoluble virus materials, using differential centrifugation. MALDI-TOF or MALDI-TOF/TOF MS was applied to analyze tryptic peptides from separated viral proteins. In this study, we achieved type/subtype of avian influenza virus (AIV) within 5 h, based on 4 major proteins, by significantly reducing ion suppression and signal overlap from various protein sources. Hence, our approach can both yield dependable results and allow Web-based search engines to be directly employed, obviating the need for additional statistical strategy. Additionally, we demonstrate the utility of the method using NDV.

Innate immune response in the hemolymph of an ascidian, Halocynthia roretzi, showing soft tunic syndrome, using label-free quantitative proteomics.

Soft tunic syndrome of Halocynthia roretzi manifests as soft, weak, and rupturable tunics, causing mass mortality. Utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS), innate immune response was established by comparing hemolymph protein profiles of ascidians with healthy or softened tunics. Of 100 proteins in each individual ascidian, 59 proteins from healthy and 56 proteins from diseased ascidians were functionally classified. Proteins found only in diseased individuals included trypsin inhibitor and Hr-29, and with high exponentially modified protein abundance index (emPAI) values. From 41 proteins identified to be common to both healthy and diseased ascidians, 15 were associated with innate immune response. Ficolin 3, a component of the lectin-complement system, was significantly decreased in diseased ascidians, but a cell surface protein, type II transmembrane serine protease-1 (TTSP), was considerably elevated. These results suggest that trypsin inhibitor, ficolin 3, and TTSP are probably involved in the innate immune response related to this tunic disease. Beside, Hr-29 could be suggested as a biomarker for soft tunic syndrome.

Comparison of antigenic proteins from Lactococcus garvieae KG- and KG+ strains that are recognized by olive flounder (Paralichthys olivaceus) antibodies.

Lactococcus garvieae is an important etiological agent of lactococcosis in various fish species including olive flounder (Paralichthys olivaceus). In this study, proteomic and immunoproteomic analyses were employed to compare the antigenic profiles of strains KG9408, MS93003, and NSS9310 strains of L. garvieae. Proteomic analysis using two-dimensional gel electrophoresis (2-DE) revealed differences in five protein spots among the different L. garvieae strains. In immunoproteomic analysis, there was a significant difference in the 2-DE immunoblot profiles of the L. garvieae strains using sera collected from fish surviving infection with either L. garvieae strains KG9408 or NSS9310. These sera reacted with 8 and 7 unique antigenic protein spots, respectively. Heat shock protein (HSP) 70 and DNA-directed RNA polymerase were among the specific antigens recognized by the anti-NSS9310 serum. In addition, the anti-NSS9310 and anti-KG9408 olive flounder sera reacted with 25 common antigenic protein spots of all the L. garvieae strains, which included elongation factor (EF)-Tu, arginine deiminase (AD), inosine-5'-monophosphate dehydrogenase (IMPD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphomannomutase (PMM), L-lactate dehydrogenase (L-LDH), 6-phosphofructokinase and UDP-galactose 4-epimerase (UDP-galactose). Based on the present results, the 8 antigens recognized by the anti-KG9408 serum and the 25 common antigens recognized by both sera may serve as potential markers for developing an effective vaccine against this bacterium.