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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).

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.

Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose 'Candidatus Maribrachyspira akoyae' gen. nov, sp nov., for this bacterium.

Temporal elevation of water temperature positively affects immune activity and disease resistance in poikilothermic teleost fish. The ayu, , an important fish species for Japanese freshwater fisheries, is usually produced under higher water temperatures than the natural conditions to facilitate rapid growth. However, it has been reported that rearing fish at higher water temperatures inhibits the development of the thymus, suggesting that resistance to infectious diseases is reduced in ayu reared at higher water temperatures. Here, we show that decreased resistance to bacterial cold-water disease and excessive inflammatory responses occurred in ayu reared at 22°C compared with those reared at lower temperatures. Ayu larvae were reared at 12°C, 15°C and 22°C for 77 days and fed 3% of their body weight. Thymus index and condition factor was calculated after the fish rearing. Then, ayu reared at the different temperatures were challenged with Flavobacterium psychrophilum and the fish were sampled for histopathology and gene expression analyses. Further, the fish were vaccinated with formalin-killed F. psychrophilum and continuously reared at the three different water temperatures. Serum antibody titer was determined by ELISA and cumulative mortality in each group was recorded after the bacterial challenge. Ayu reared at 22°C showed a significantly lower thymus index and higher condition factor than those reared at lower temperatures. Infiltrated leukocytes and many melanin pigments were frequently observed in the adipose tissues and spleens of ayu reared at 22°C, respectively, but not in those reared at 12°C. The gene expression levels of inflammatory cytokines such as IL-1β, IL-8 and TNFα in the spleen were significantly higher in the 22°C group than in the 12°C group. The cumulative survival rate after challenge with was 51.7%, 40.0% and 13.3% in the 12°C, 15°C and 22°C groups, respectively. The relative percent survival values of vaccinated fish reared at 15°C and 22°C groups were lower than those reared at 12°C. Moreover, the specific antibody titer of the vaccinated fish was the lowest in the 22°C group and the highest in the 12°C group. These results suggest that rearing the fish under high water temperature causes excessive inflammatory responses similar to metabolic inflammation in human obesity, resulting in a decrease of disease resistance. In addition, thymic involution induced by higher water temperature probably leads the poor response to vaccination. The present study provides insights into the physiological and immunological changes of fish under global warming.

Thrombocytes, nucleated hemostatic blood cells of non-mammalian vertebrates, are regarded as the functional equivalent of anucleated mammalian platelets. Additional immune functions, including phagocytosis, have also been suggested for thrombocytes, but no conclusive molecular or cellular experimental evidence for their potential ingestion and clearance of infiltrating microbes has been provided till date. In the present study, we demonstrate the active phagocytic ability of thrombocytes in lower vertebrates using teleost fishes and amphibian models. Ex vivo, common carp thrombocytes were able to ingest live bacteria as well as latex beads (0.5-3 μm in diameter) and kill the bacteria. In vivo, we found that thrombocytes represented nearly half of the phagocyte population in the common carp total peripheral blood leukocyte pool. Phagocytosis efficiency was further enhanced by serum opsonization. Particle internalization led to phagolysosome fusion and killing of internalized bacteria, pointing to a robust ability for microbe elimination. We find that this potent phagocytic activity is shared across teleost (Paralichthys olivaceus) and amphibian (Xenopus laevis) models examined, implying its conservation throughout the lower vertebrate lineage. Our results provide novel insights into the dual nature of thrombocytes in the immune and homeostatic response and further provide a deeper understanding of the potential immune function of mammalian platelets based on the conserved and vestigial functions.

Mass mortality of 0-year-old pearl oysters, Pinctada fucata (Gould), and anomalies in adults were observed in Japan’s major pearl farming areas in the summer of 2019 and 2020. Although adult oyster mortality was low, both adult and juvenile oysters underwent atrophy of the soft body, detachment of the mantle from nacre (the shiny inner surface of the valves), deposition of brownish material on the nacre, and loss of nacre luster. Infection trials were conducted to verify the involvement of pathogens in this phenomenon. Healthy adult pearl oysters were obtained from areas where this disease had not occurred to use as the recipients. The sources of infection were either affected adult oysters with atrophied soft bodies or batches of juveniles in which mortality had reached conspicuous levels. Transmission of the disease to the healthy oysters were tested either by cohabitation with affected oysters or by injections of the hemolymph of affected animals. The injection infection test examined the effects of filtration and chloroform exposure on the pathogen. Occurrence of the disease was confirmed by the appearance of brown deposits on the nacre and loss of nacre luster. The abnormalities of nacre were clearly reproduced in recipient shells in three out of four cohabitation trials with affected oysters. The disease was also reproduced in six out of six injection trails either with hemolymph filtered through 100 nm filter or with hemolymph treated with chloroform. In a serial passage with hemolymph injections, the disease was successfully transmitted through eight passages. These results suggest that the etiology of the disease is a non-enveloped virus with a diameter ≤100 nm.

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.

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

ABSTRACT 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.

Two monoclonal antibodies (MAbs: JFW1 and JFW10) were produced against peripheral blood leukocytes (PBL) in Japanese flounder. Additionally, MAbs against flounder immunoglobulin (Ig; JFW20 and JFW21) were generated for the surface marker of Igsup(+) leukocytes using purified serum Ig as an antigen. MAb JFW1 recognized the surface marker of granulocytes and monocytes and MAb JFW10 specifically bound to the surface antigen of thrombocytes. Flow cytometric analysis of PBL incubated with JFW1, JFW10, JFW20 and JFW21 revealed that 2.5-7.4, 23.7-50.1, 25.2-26.1 and 5.2-8.3% of all leukocytes were positive for these markers. Analysis of head kidney leukocytes (HKL) showed that JFW1, JFW10, JFW20 and JFW21 bound to 30.5-36.3, 1.9-2.8, 6.4-8.3 and 1.9-3.0% of all leukocytes, respectively. Western blot analysis after SDS-PAGE showed that JFW10 recognizes a protein of 115 kDa from lysed PBL. JFW20 recognized the 70 and 74 kDa proteins of the heavy chain of Ig from serum. No band was observed for either JFW1 or JFW21. These antibodies will be useful for the identification and isolation of Japanese flounder leukocyte subpopulations and will facilitate immunological studies of flounder.