A novel birnavirus identified as the causative agent of summer atrophy of pearl oyster (Pinctada fucata (Gould)).

The Akoya pearl oyster (Pinctada fucata (Gould)) is the most important species for pearl cultivation in Japan. Mass mortality of 0-year-old juvenile oysters and anomalies in adults, known as summer atrophy, have been observed in major pearl farming areas during the season when seawater temperatures exceed about 20 °C since 2019. In this study, we identified a novel birnavirus as the pathogen of summer atrophy and named it Pinctada birnavirus (PiBV). PiBV was first presumed to be the causative agent when it was detected specifically and frequently in the infected oysters in a comparative metatranscriptomics of experimentally infected and healthy pearl oysters. Subsequently, the symptoms of summer atrophy were reproduced by infection tests using purified PiBV. Infection of juvenile oysters with PiBV resulted in an increase in the PiBV genome followed by the atrophy of soft body and subsequent mortality. Immunostaining with a mouse antiserum against a recombinant PiBV protein showed that the virus antigen was localized mainly in the epithelial cells on the outer surface of the mantle. Although the phylogenetic analysis using maximum likelihood method placed PiBV at the root of the genus Entomobirnavirus, the identity of the bi-segmented, genomic RNA to that of known birnaviruses at the full-length amino acid level was low, suggesting that PiBV forms a new genus. The discovery of PiBV will be the basis for research to control this emerging disease.

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.

Mass mortality of pearl oyster (Pinctada fucata (Gould)) in Japan in 2019 and 2020 is caused by an unidentified infectious agent.

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.

Divergence in chondrogenic potential between in vitro and in vivo of adipose- and synovial-stem cells from mouse and human.

BACKGROUND: Somatic stem cell transplantation has been performed for cartilage injury, but the reparative mechanisms are still conflicting. The chondrogenic potential of stem cells are thought as promising features for cartilage therapy; however, the correlation between their potential for chondrogenesis in vitro and in vivo remains undefined. The purpose of this study was to investigate the intrinsic chondrogenic condition depends on cell types and explore an indicator to select useful stem cells for cartilage regeneration. METHODS: The chondrogenic potential of two different stem cell types derived from adipose tissue (ASCs) and synovium (SSCs) of mice and humans was assessed using bone morphogenic protein-2 (BMP2) and transforming growth factor-ß1 (TGFß1). Their in vivo chondrogenic potential was validated through transplantation into a mouse osteochondral defect model. RESULTS: All cell types showed apparent chondrogenesis under the combination of BMP2 and TGFß1 in vitro, as assessed by the formation of proteoglycan- and type 2 collagen (COL2)-rich tissues. However, our results vastly differed with those observed following single stimulation among species and cell types; apparent chondrogenesis of mouse SSCs was observed with supplementation of BMP2 or TGFß1, whereas chondrogenesis of mouse ASCs and human SSCs was observed with supplementation of BMP2 not TGFß1. Human ASCs showed no obvious chondrogenesis following single stimulation. Mouse SSCs showed the formation of hyaline-like cartilage which had less fibrous components (COL1/3) with supplementation of TGFß1. However, human cells developed COL1/3+ tissues with all treatments. Transcriptomic analysis for TGFß receptors and ligands of cells prior to chondrogenic induction did not indicate their distinct reactivity to the TGFß1 or BMP2. In the transplanted site in vivo, mouse SSCs formed hyaline-like cartilage (proteoglycan+/COL2+/COL1-/COL3-) but other cell types mainly formed COL1/3-positive fibrous tissues in line with in vitro reactivity to TGFß1. CONCLUSION: Optimal chondrogenic factors driving chondrogenesis from somatic stem cells are intrinsically distinct among cell types and species. Among them, the response to TGFß1 may possibly represent the fate of stem cells when locally transplanted into cartilage defects.

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.

Primary culture of mouse adipose and fibrous synovial fibroblasts under normoxic and hypoxic conditions.

Synovial fibroblasts have attracted considerable attention in studies of joint diseases. Although mice are useful and powerful in in vitro and in vivo experiments, primary cultures of mouse synovial fibroblasts are notoriously difficult because the mouse synovial tissues are much smaller and cell cycle arrests can be induced more easily in murine cells than in human cells. Here, we report a precise protocol for the isolation and culture of fibroblasts from mouse adipose and fibrous knee joint synovia. In both adipose and fibrous synovial fibroblasts, proliferation was decreased in addition to a higher rate of cellular senescence under normoxic conditions (20% O2); however, it was maintained over 20 days with low cellular senescence under hypoxic conditions (3% O2). The marker gene expression in adipose and fibrous synovial fibroblasts was not markedly altered after a 3-week culture. Both cells displayed similar potencies for chondrogenic, osteogenic, and adipogenic differentiation, and responses to a proinflammatory cytokine. The present method provides a sufficient amount of mouse synovial fibroblasts for in vitro and in vivo experiments in joint biology and the pathophysiology of osteoarthritis and rheumatoid arthritis.

Soft tunic syndrome in the edible ascidian Halocynthia roretzi is caused by a kinetoplastid protist.

An etiological study was conducted to clarify whether the flagellate-like cells found in histological preparations of the tunic of diseased Halocynthia roretzi (Drasche) were the causative agent of soft tunic syndrome in this ascidian. When pieces of softened diseased tunic were incubated overnight in sterile seawater, live flagellated cells, which were actively swimming in the seawater, were observed in 47 out of 61 diseased ascidians (77%), but not in moribund or abnormal individuals with normal tunics (n = 36) nor in healthy animals (n = 19). The flagellate was morphologically very similar to those observed in histological sections of the diseased tunic. By contrast, flagellates were not found in tunic pieces of healthy, moribund, and abnormal individuals that did not exhibit softening of the tunic. Light and electron microscopy revealed that the flagellate has polykinetoplastic mitochondria with discoidal cristae. The cytomorphologies of the flagellate were the same as those of the flagellate-like cells in the diseased tunic. We cultured the flagellate from the softened tunic in vitro and confirmed that the tunics of healthy ascidians, which were immersion-challenged with suspensions of the subcultured flagellates, became softened 17 d after exposure, including the final 12 d in aerated, running seawater. The occurrence of flagellates was also confirmed by incubating pieces of soft tunic from experimentally infected animals in seawater overnight. These results indicate that the flagellate is the causative agent of soft tunic syndrome.

Mass mortality of cultured ascidians Halocynthia roretzi associated with softening of the tunic and flagellate-like cells.

Since 2007, mass mortalities of cultured ascidians Halocynthia roretzi (Drasche) have occurred in Miyagi Prefecture, Japan. The mortalities occur from November through August, and the tunics of affected animals become abnormally weak and soft. The number of farming areas where mass mortalities have occurred has increased rapidly: 3 in 2007, 6 in 2008, and 14 in 2009. When an outbreak of the disease occurred, mortality reached 17 to 100%. Prominent histopathological changes in the diseased ascidians were found in the tunics; the tunics of affected animals were usually much thinner than those of healthy individuals, and the tunic matrix showed marked disintegration with irregular arrangements of fiber layers or the presence of hollow spaces. In addition, flagellate-like cells (10-14 microm x 2-3 microm) stained with hematoxylin were observed in the tunics of 31 out of 36 diseased animals (86%), but not in apparently healthy animals (n=38). Experimental infection with the disease was successfully conducted by immersing small pieces of tunic samples from diseased ascidians into aquaria with healthy ascidians. The flagellate-like cells were confirmed in the tunics of all the experimentally infected animals. These results indicate that the mass mortalities of ascidians accompanied by abnormally softened tunics were caused by an infectious agent, and suggest the involvement of the flagellate-like cells in the disease.

Mass mortality of giant abalone Haliotis gigantea caused by a Francisella sp. bacterium.

In February 2005, a mass mortality of giant abalone Haliotis (Nordotis) gigantea Gmelin, 1791 occurred on a private abalone farm in Shimane Prefecture, Japan. The cumulative mortality rate reached about 84%. In histological observations, bacteria-like spherical particles were found in affected animals, suggesting a bacterial infection. Many of the bacteria-like particles were found in the cells that were presumably host phagocytes. DNA was extracted from the hemolymph of a diseased abalone and a bacterial 16S rRNA gene was amplified by PCR. The bacterium was classified within the genus Francisella by gene sequence analysis. A bacterial isolate was obtained by spreading hemolymph of a diseased abalone on modified Eugon agar dissolved in 70% seawater containing 1% (w/v) hemoglobin. A gene fragment of the expected size was amplified from the bacterial isolate by PCR using specific primers for the 16S rRNA gene obtained from the diseased abalone. Experimental infections were carried out by intramuscular injection with the bacterial isolate or by immersion in the bacterial suspension using 2 species of abalone, the giant abalone and the Japanese black abalone Haliotis (Nordotis) discus discus Reeve, 1846. Most (98.6%) of the abalone challenged with the bacterial isolate died in experimental infections. These results suggest that the Francisella sp. isolate was the causative agent for the mass mortality of giant abalone. This is the first report of a pathogenic Francisella sp. isolate for mollusks.

X-cells in pseudotumors of yellowfin goby Acanthogobius flavimanus: a protistan organism distinct from that in flathead flounder Hippoglossoides dubius.

Yellowfin goby Acanthogobius flavimanus affected with X-cell pseudotumors were sampled from a river estuary in Tokyo Bay, Japan. We amplified the gene for small subunit ribosomal RNA (18S rRNA) of X-cells of the goby with PCR using universal primers. The gene that we obtained (DDBJ Accession no. AB451874) showed 91% sequence identity to that of the X-cells of the flathead flounder Hippoglossoides dubius. With in situ hybridization, the probes specific for the gene that we obtained hybridized with the goby X-cells but not with the flounder X-cells, whereas probes for the 18S rRNA gene of flounder X-cells hybridized with the flounder X-cells but not with goby X-cells. These findings indicate that, although the X-cells found in the goby are closely related to the protist found in flounder, the two are clearly distinct organisms.

Rapid coprecipitation technique using yttrium hydroxide for the preconcentration and separation of trace elements in saline water prior to their ICP-AES determination.

Yttrium hydroxide quantitatively coprecipitated Be(II), Ti(IV), Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Cd(II), and Pb(II) at pH 9.6 - 10.0 for seawater and pH 10.5 - 11.4 for a table-salt solution. The coprecipitated elements could be determined by inductively coupled plasma atomic emission spectrometry; yttrium was used as an internal standard element. The detection limits ranged from 0.001(6) microg (Mn(II)) to 0.22 microg (Zn(II)) in 100 mL of sample solutions. The operation time required to separate 11 elements was approximately 30 min.

Pathogenesis of experimentally induced bacterial cold water disease in ayu Plecoglossus altivelis.

Ayu Plecoglossus altivelis were experimentally infected with Flavobacterium psychrophilum, which is the causative agent of bacterial cold water disease (CWD). The fish infected by immersion usually died within an hour after they became moribund. The blood volume and haematocrit values of moribund fish were low, while those values of many infected fish that were not moribund were in the range of controls. Most of the affected fish in the immersion-infected groups had ulcerative lesions on their lower jaw. No histological evidence of haemolysis was observed. These results suggest that rapid bleeding occurred through ulcerative lesions, probably causing hypoxia which killed the fish. Ulcerative lesions developed on the dorsal skin when this area had been slightly abraded artificially prior to immersion challenge. Histologically, F. psychrophilum was initially found on the skin that had microscopic injuries, but not on normal skin. The bacterium then entered the dermis and migrated through connective tissues. The lesions subsequently expanded into the underlying musculature through the myosepta, developed necrotic myositis and formed externally open ulcers. Only in later stages of infection did mild lesions develop in the internal organs and the gill, probably caused by the bacterium migrating through blood vessels. This suggests that infection with CWD through the gill or digestive tract is unlikely. Virtually no open lesions were found in ayu challenged by intramuscular injections except at the injection sites. The results suggest that skin injuries are major portals of entry for F. psychrophilum in ayu, and the bacterium has affinity for collagenous connective tissues.

X-cells in fish pseudotumors are parasitic protozoans.

Bottom-dwelling teleosts, particularly flatfishes or cod living in temperate to cold seawater, sometimes develop tumor-like lesions on the body surface or in the branchial cavity. These lesions usually contain masses of so called 'X-cells' of unknown origin. We amplified a gene for small subunit ribosomal RNA (18S rRNA) from X-cell lesions of the flathead flounder Hippoglossoides dubius. Phylogenetic analysis clearly classified the obtained sequence as a protozoan, although the organism had no clear affinity with any known protistan groups. In situ hybridization showed that probes specific for the protozoan 18S rRNA hybridized only with X-cells, and not with the host-fish cells, indicating that X-cells harbor the protozoan rRNA. On the other hand, a probe specific for vertebrate 18S rRNA hybridized with the host-fish cells, but not with X-cells. This is conclusive evidence that X-cells are parasitic protozoans.

[A quantitative and qualitative study on effective information flow for infectious disease control in welfare facilities for the elderly].

OBJECTIVES: To clarify factors associated with effective information tranges among staff of welfare facilities for the elderly, and to propose measures for an appropriate information flow system in welfare facilities and public health centers, communication channels and methods, and encouraging factors and barriers were investigated in terms of a printed medium on the control and management of scabies infections. METHODS: A self-administered questionnaire survey and an interview survey were conducted with the staff of welfare facilities for the elderly where "Control and management manual of scabies infection" had been distributed by the Tama-Tachikawa Public Health Center in Tokyo. A self-administered questionnaire was sent to managers and chief practitioners of 66 facilities. Respondents were obtained from 66 managers and chief practitioners (response rate: 84.8%), and 831 practitioners (response rate: 53.1%). The questionnaire consisted of 20 items for managers and 18 items for chief practitioners, including experience of scabies epidemics in facilities, training experience for the use of "Control and management manual of scabies infection," measures for information gathering, and current information flow within the facility. A semi-structured interview survey was conducted with the manager and/or chief practitioner and practitioners in five facilities. The number of respondents was 10. The interview questions included job description, scabies control measures, dissemination of the manual to the staff, use of the manual, flows of health-related information, and factors associated with information flows. Summarized codes were extracted from the transcriptions from tape recording and were categorized repeatedly according to similarity. RESULTS: In the questionnaire survey, differences of Community information flow by types of facilities and professional backgrounds were found. Variation was detected in measures for information gathering and focuses in information management between managers/chief practitioners and practitioners. Practitioners wanted opportunities for information exchange while managers/chief practitioners mainly focused on prioritization of information collected. In addition, many respondents felt that information networks outside the facilities were poorly organized. From the interview survey, three major categories were extracted, that is, 'Information flow system,' 'Personal qualification,' and 'Factors related to the information flow system.' As factors related to the information flow system, the following 7 subcategories were extracted. 1. Interest in information; 2. Working style and workload; 3. Information networks outside the facility; 4. Information management in the facility; 5. Environment for information sharing; 6. Budget for the information system; and 7. Interpersonal communication. CONCLUSIONS: For an effective information system, welfare facilities for the elderly should work on staff training, building their own information flow systems and improving the environment for information sharing and networking with specialized agencies, such as public health centers. At the same time, public health centers should support networking, interpersonal two-way communication and training of welfare-facility workers.