Clinical symptoms and histopathological changes in coho salmon affected by the erythrocytic inclusion body syndrome (EIBS) are caused by the infection of piscine orthoreovirus 2 (PRV-2).

The relationship of histopathological changes and the infection of Piscine orthoreovirus 2 (PRV-2) was investigated in coho salmon that were suffering from the erythrocytic inclusion body syndrome (EIBS). Immunohistochemical observations revealed abundant σ1 protein of PRV-2 in the spongy layer of the ventricle of the heart, where severe myocarditis was observed. In the spleen, the virus protein was detected in many erythrocytes, some of which were spherical-shaped and apparently dead. The number of erythrocytes was decreased in the spleen compared to the apparently healthy fish. The virus protein was also detected in some erythrocytes in blood vessels. The viral protein was often detected in many macrophages ingesting erythrocytes or dead cell debris in the spleen or in the kidney sinusoids. Large amounts of the viral genomic segment L2 were also detected in these organs by RT-qPCR. Many necrotic foci were found in the liver, although the virus protein was not detected in the hepatocytes. These results suggest that the primary targets of PRV-2 are myocardial cells and erythrocytes and that clinical symptoms such as anaemia or jaundice and histopathological changes such as myocarditis in EIBS-affected coho salmon are caused by PRV-2 infection.

Comparison of portal vein hemodynamics with ultrasound-based elastography for the prediction of liver fibrosis in patients with chronic liver disease.

Chronic liver disease includes nonalcoholic fatty liver disease, progresses from steatosis and hepatitis to fibrosis and cirrhosis, with hemodynamic changes in portal blood flow. This study aimed to compare portal vein hemodynamics with liver stiffness (LS) and steatosis and included 28 subjects with chronic liver disease, in whom LS and steatosis were evaluated in the same image employing two elastography techniques: transient elastography (TE) with controlled attenuation parameter (CAP) using a FibroScan and two-dimensional shear-wave elastography (2D-SWE) with attenuation imaging (ATI). Additionally, peak maximum velocity (Vmax) of the right portal vein and spleen stiffness with 2D-SWE were evaluated. A strong positive correlation was present between LS values obtained with TE and 2D-SWE and between the attenuation coefficients of steatosis obtained with CAP and ATI. Additionally, a negative correlation was present between LS values and the Vmax of the right portal vein (r = 0.415, p = 0.031). The optimal Vmax cutoff value for discriminating liver fibrosis with an LS value of > 5 kPa was < 17 cm/s; the ability of Vmax to predict fibrosis was comparable to that of the FIB4-index. Low Vmax of the right portal vein was useful for identifying liver fibrosis in patients with chronic liver disease.

Isolation and characterization of hirame aquareovirus (HAqRV): A new Aquareovirus isolated from diseased hirame Paralichthys olivaceus.

We isolated a novel Aquareovirus (hirame aquareovirus: HAqRV) from Japanese flounder Paralichthys olivaceus suffering from reovirus-like infection. In electron microscopy, the spherical virion (75 nm in diameter) was observed with multi-layered capsid structure. The viral genome consisted of 11 segments and regions encoding 7 virion structural proteins and 5 non-structural proteins were predicted. The deduced amino acid sequences of those proteins were highly similar to those of the aquareoviruses. However, the similarity of complete genome sequence between the HAqRV and other aquareoviruses was less than 60%. Phylogenetic analyses based on the deduced amino acid sequences suggested that the HAqRV is not classified into the known species of Aquareovirus. Pathogenicity of HAqRV was clearly demonstrated in accordance with Koch's postulates by experimental infection using Japanese flounder. The results suggest that the HAqRV is a new Aquareovirus species which is highly virulent for the Japanese flounder at early life stages.

Development of a method to quantify endogenous IFNγ protein in amberjack species.

Interferon (IFN)γ is a pivotal cytokine that promotes and orchestrates innate cellular and adaptive cell-mediated immunity against intracellular pathogens. The capacity of T cells in mammals to produce IFNγ has been measured using specific antibodies in order to analyze cell-mediated immune responses against infection or immuno-stimulants. In fish, however, measurement of IFNγ protein levels has not been possible due to a lack of research tools. In the present study, therefore, we established antibodies that react with endogenous amberjack IFNγ. An enzyme-linked immunosorbent assay (ELISA) for IFNγ in amberjack species was developed using these antibodies. The ELISA could detect endogenous IFNγ at concentrations less than 100 pg/mL in PMA/ionomycin-stimulated leukocytes culture supernatant. IFNγ production was enhanced and lasted a long time following intracellular bacterial infection with Nocardia seriolae, which is thought to be targeted by cell-mediated immunity. These results demonstrate that quantification of IFNγ using the reported ELISA can be used to estimate the status of cell-mediated immunity in amberjack species.

Author Correction: A novel Asfarvirus-like virus identified as a potential cause of mass mortality of abalone.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A novel Asfarvirus-like virus identified as a potential cause of mass mortality of abalone.

A novel Asfarvirus-like virus is proposed as the etiological agent responsible for mass mortality in abalone. The disease, called abalone amyotrophia, originally was recognized in the 1980s, but efforts to identify a causative agent were unsuccessful. We prepared a semi-purified fraction by nuclease treatment and ultracentrifugation of diseased abalone homogenate, and the existence of the etiological agent in the fraction was confirmed by a challenge test. Using next-generation sequencing and PCR-based epidemiological surveys, we obtained a partial sequence with similarity to a member of the family Asfarviridae. BLASTP analysis of the predicted proteins against a virus database resulted in 48 proteins encoded by the novel virus with top hits against proteins encoded by African swine fever virus (ASFV). Phylogenetic analyses of predicted proteins of the novel virus confirmed that ASFV represents the closest relative. Comparative genomic analysis revealed gene-order conservation between the novel virus and ASFV. In situ hybridization targeting the gene encoding the major capsid protein of the novel virus detected positive signals only in tissue from diseased abalone. The results of this study suggest that the putative causative agent should be considered a tentative new member of the family Asfarviridae, which we provisionally designate abalone asfa-like virus (AbALV).

Comparative outcomes between palliative ileostomy and colostomy in patients with malignant large bowel obstruction.

OBJECTIVES: Palliative stoma creation should be considered in patients at high risk of colonic metallic stent failure. However, it is unclear whether ileostomy or colostomy is superior. This study compared short-term outcomes between palliative ileostomy and colostomy. METHODS: We identified 82 patients with malignant large bowel obstruction, caused by various advanced cancers, between January 2005 and December 2016. We compared short-term outcomes between the ileostomy group (n = 33) and the colostomy group (n = 49). RESULTS: For all 82 patients, clinical success was achieved. Three patients with ileostomy died within 30 days of ostomy formation. The ileostomy group had statistically significant differences in median operative time (113 vs. 129 minutes, p = 0.045) and blood loss (8 vs. 40 g, p = 0.037) in comparison with the colostomy group. No statistically significant differences were observed in the surgical complications (30.3 vs. 38.8%, p = 0.431), in the median period to oral intake (3 vs. 4 days, p = 0.335) and in the hospital stay after surgery (32 vs. 27 days, p = 0.509) between the two groups. Overall stoma-related complications occurred in 27 (32.9%) patients. Stoma-related complications occurred more frequently in the ileostomy group (16/33 vs. 11/49 patients, p = 0.014). High output stoma (6 patients) and irritation (5 patients) occurred more frequently in the ileostomy group. CONCLUSIONS: Palliative colostomy is superior to ileostomy due to fewer stoma-related complications. When ileostomy is required, aggressive interventions for high output stomas should be implemented.

Antibody-mediated bacterial killing of Ichthyobacterium seriolicida in Japanese amberjack.

Ichthyobacterium seriolicida is the causative agent of bacterial hemolytic jaundice (BHJ) in Japanese amberjack, Seriola quinqueradiata. Fish recovering from BHJ acquire protective immunity against reinfection. In this study, fish were passively immunized to determine whether serum antibody is involved in protection against BHJ. The susceptibility of I. seriolicida to the bactericidal activity of Japanese amberjack serum was also investigated. In passive immunization tests, significantly lower mortality was noted in fish that received convalescent serum. Bacteria were killed when exposed to convalescent serum but not serum from naïve fish. Electron microscopic analyses showed that I. seriolicida cells were morphologically altered by reaction with convalescent serum. Naïve fish serum became bactericidal upon addition of purified IgM from convalescent serum. Involvement of the classical complement pathway in the bactericidal mechanism was confirmed because bactericidal activity was lost upon heating convalescent serum or chelation treatment using EDTA. Convalescent fish serum thus protects against reinfection by I. seriolicida via humoral immunity mediated by activation of the classical complement pathway.

Antibody profiling using a recombinant protein-based multiplex ELISA array accelerates recombinant vaccine development: Case study on red sea bream iridovirus as a reverse vaccinology model.

Predicting antigens that would be protective is crucial for the development of recombinant vaccine using genome based vaccine development, also known as reverse vaccinology. High-throughput antigen screening is effective for identifying vaccine target genes, particularly for pathogens for which minimal antigenicity data exist. Using red sea bream iridovirus (RSIV) as a research model, we developed enzyme-linked immune sorbent assay (ELISA) based RSIV-derived 72 recombinant antigen array to profile antiviral antibody responses in convalescent Japanese amberjack (Seriola quinqueradiata). Two and three genes for which the products were unrecognized and recognized, respectively, by antibodies in convalescent serum were selected for recombinant vaccine preparation, and the protective effect was examined in infection tests using Japanese amberjack and greater amberjack (S. dumerili). No protection was provided by vaccines prepared from gene products unrecognized by convalescent serum antibodies. By contrast, two vaccines prepared from gene products recognized by serum antibodies induced protective immunity in both fish species. These results indicate that ELISA array screening is effective for identifying antigens that induce protective immune responses. As this method does not require culturing of pathogens, it is also suitable for identifying protective antigens to un-culturable etiologic agents.

Complete Genome Sequence of Ichthyobacterium seriolicida JBKA-6T, Isolated from Yellowtail (Seriola quinqueradiata) Affected by Bacterial Hemolytic Jaundice.

Ichthyobacterium seriolicida is a fish bacterial pathogen that causes hemolytic jaundice in farmed yellowtail in Japan. To understand more about the characteristics of this bacterium, we determined its complete genome sequence. Two hemolysin genes which may be important for its pathogenicity were identified in the I. seriolicida genome.

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.

Asymptomatic costal exostosis with thickening in the pericardium: a case report.

BACKGROUND: Costal exostosis is a benign condition that sometimes requires emergent surgery because of associated hemothorax; in addition, there have been cases with malignant transformation to chondrosarcoma. Here, we describe an asymptomatic patient who underwent thoracoscopic resection for primary costal exostosis. CASE PRESENTATION: A 16-year-old male was found to have a bow-shaped shadow on a chest X-ray. Chest computed tomography revealed a rod-like mass with a soft tissue shadow adjacent to the left fifth rib. A thoracoscopic partial resection of the left fifth rib was performed. Intraoperative findings included thickening of the pericardium near the tip of the growth and erosion of the visceral pleura of the left lung. The resected specimen was diagnosed as a primary costal exostosis based on histopathological findings. CONCLUSIONS: We review the published literature on costal exostosis and discuss the surgical indications of asymptomatic cases.

Ichthyobacterium seriolicida gen. nov., sp. nov., a member of the phylum 'Bacteroidetes', isolated from yellowtail fish (Seriola quinqueradiata) affected by bacterial haemolytic jaundice, and proposal of a new family, Ichthyobacteriaceae fam. nov.

A novel Gram-stain-negative, rod-shaped (0.3 × 4-6 µm), non-flagellated, aerobic strain with gliding motility, designated JBKA-6T, was isolated in 1991 from a yellowtail fish, Seriola quinqueradiata, showing symptoms of bacterial haemolytic jaundice. 16S rRNA gene sequence analysis showed that strain JBKA-6T was related most closely to members of the family Flavobacteriaceae in the phylum 'Bacteroidetes'. Furthermore, based on gyrB gene sequence analysis, JBKA-6T was classified into a single clade within the order Flavobacteriales, which was distinct from the known clades of the families Flavobacteriaceae, Blattabacteriaceae and Cryomorphaceae. The predominant isoprenoid quinone was identified as MK-6 (97.9 %), and the major cellular fatty acids (>10 %) were C14 : 0 and iso-C15 : 0. The main polar lipids were phosphatidylethanolamine, three unidentified phospholipids, two unidentified aminophospholipids and two unidentified polar lipids. The DNA G+C content of JBKA-6T, as derived from its whole genome, was 33.4 mol%. The distinct phylogenetic position and phenotypic traits of strain JBKA-6T distinguish it from all other described species of the phylum 'Bacteroidetes', and therefore it was concluded that strain JBKA-6T represents a new member of the phylum 'Bacteroidetes', and the name Ichthyobacterium seriolicida gen. nov., sp. nov. is proposed. The type strain of Ichthyobacterium seriolicida is JBKA-6T ( = ATCC BAA-2465T = JCM 18228T). We also propose that Icthyobacterium gen. nov. is the type genus of a novel family, Ichthyobacteriaceae fam. nov.

Expressed sequence tag analyses of three leukocyte subpopulations in ayu Plecoglossus altivelis altivelis, separated by monoclonal antibodies.

Ayu Plecoglossus altivelis altivelis are one of the most economically important fish for freshwater aquaculture in Japan. We conducted expressed sequence tag analyses of three leukocyte subpopulations, thrombocytes, neutrophils, and B lymphocytes in ayu using a next generation sequencer. The sequencing and de novo assembly yielded 22,494, 22,733, and 16,505 contigs from the thrombocyte, neutrophil, and B lymphocyte cDNA libraries, respectively. Pathways involving endocytosis, phagosomes, and lysosomes, were found in all three cDNA libraries using pathway analysis. The thrombocyte cDNA library contained 2894 unique sequences, including CXC chemokine receptor 4 and MHC class II. Cytokine and cytokine receptor genes such as interleukin (IL)-1ß, IL-8, IL-1 receptor (IL-1R), IL-8RA, and IL-8RB were found among the 3056 unique sequences of the neutrophil cDNA library. Typical B lymphocyte related genes such as B cell linker protein, immunoglobulin (Ig) M, IgD and transforming growth factor ß were found in the 1590 unique sequences of the B lymphocyte cDNA library. In summary, a large number of immune-related genes were identified from the three leukocyte cDNA libraries. Our results represent a valuable sequence resource for understanding the immune system function in ayu.

Protective efficacies and immune responses induced by recombinant HCD, atpD and gdhA against bacterial cold-water disease in ayu (Plecoglossus altivelis).

Protective efficacies of three antigenic proteins (3-hydroxyacyl-CoA dehydrogenase (HCD), ATP synthase beta subunit (atpD), and glutamate dehydrogenase (gdhA)) against Flavobacterium psychrophilum were investigated in ayu (Plecoglossus altivelis). Recombinant proteins of HCD, atpD, and gdhA were expressed in Escherichia coli BL21 cells. Ayu were then vaccinated with inactivated cells via the intraperitoneal route. Compared with the empty BL21- and PBS-injected groups, the vaccinated group had a significantly longer survival time after challenge with F. psychrophilum. The antibody titers against each recombinant protein were significantly higher in serum from vaccinated fish, compared with serum from control fish. Results of indirect immunofluorescence assays using serum indicated that the HCD, atpD, and gdhA proteins are located on the surface of F. psychrophilum. These results suggest that these three surface proteins are protective antigens and are good candidates for development of vaccines against bacterial cold-water disease in ayu.

N-Terminal region is responsible for chemotaxis-inducing activity of flounder IL-8.

The objective of this study was to locate the functional region responsible for the chemotaxis-inducing activity of flounder interleukin 8 (IL-8), which lacks the glutamic acid-leucine-arginine (ELR) motif essential for the induction of neutrophil migration by mammalian IL-8. Using a human cell line, we produced a secretory recombinant protein of flounder IL-8, and analyzed its chemotaxis-inducing activity on leukocytes collected from the flounder kidney. The recombinant IL-8 induced significant migration in neutrophils, which were morphologically and functionally characterized. Using the Edman degradation method, the N-terminal amino acid sequence of rIL-8 was identified as VSLRSLGV. To examine the significance of the N-terminal region for the bioactivity of flounder IL-8, we prepared several recombinant proteins that containing mutations at the N-terminus. Modification of three residues (residues 9-11: serine-leucine-histidine) corresponding in position to the ELR motif in mammalian IL-8 did not reduce its chemotaxis-inducing activity. However, deletion of the first six or more residues significantly reduced its chemotaxis-inducing activity. We propose that residue 6 (leucine) at the N-terminus is important for the chemotaxis-inducing activity of flounder IL-8.

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.

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

BACKGROUND: Edwardsiella 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. RESULTS: Eight 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. CONCLUSIONS: We 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.

Vibrio anguillarum bacterin uptake via the gills of Japanese flounder and subsequent immune responses.

The mucosal surfaces of fish allow for the introduction of foreign substances, including antigens, from the surrounding environment. In this study, uptake of Vibrio anguillarum J-O-3 serotype bacterin by Japanese flounder, and the subsequent immune responses were investigated. Immunohistochemistry revealed that the bacterin was taken up through the epithelial cells of gills. The transcription levels of inflammatory cytokines such as interleukin (IL)-1ß, IL-6 and tumor necrosis factor α were significantly up-regulated in the gills at 3 days following exposure to the bacterin. There was also a corresponding increase in IL-8 receptor, CD4-1, CD4-2 and CD8α transcript levels in the gills. Our findings suggest that the gills play a major role in the uptake of V. anguillarum bacterin and induction of inflammation, which results in an activation of the adaptive immune response in teleost fish.