During a routine survey of the Pacific oyster Crassostrea gigas in Tongyoung (previously Chungmu) on the southern coast of Korea, basophilic inclusions were observed in the gonadal tissues. They were detected from March to May at a prevalence rate of 3.3 to 7.1%. The inclusion bodies were Feulgen-positive and stained orange-red with phloxine tartrazine. Electron microscopic observation revealed non-enveloped, icosahedral particles 40 to 45 nm in diameter. These morphological characteristics resemble those of papova virus-like inclusions previously described from Pacific and eastern (American) oysters C. virginica in North America. Although many mitochondrial bodies and intact sperm cells were observed around the inclusion body, no host reaction, such as hemocytic infiltration, was detected. KEY WORDS: Pacific oyster · Crassostrea gigas · Viral gametocytic hypertrophy · Papova-like virus · Seminal glandResale or republication not permitted without written consent of the publisher embedding and cut into 4 µm thick sections. Some sections were stained with Harris's hematoxylin and eosin (H&E), some with Feulgen stain and some with phloxine tartrazine, for bright field light microscopy (Olympus Vanox AHBS3) examination.Ultrastructural examination. Samples showing inclusion bodies within the gonadal epithelia were selected for processing for transmission electron microscopy. Tissue specimens from the same oysters, preserved in Carson's fixative solution were rinsed in 0.2 M cacodylate buffer at pH 7.2 for 48 h at 4°C before post-fixing in 2.5% glutaraldehyde solution, and 1% osmium tetroxide. After dehydration through graded alcohols, the tissues were embedded in Epon resin compound (Ouken, Japan). Hardening was carried out at 35°C for 12 h, 45°C for 12 h, and 60°C for 48 h. Semi-thin sections (200 nm) were stained with toluidine blue, and ultra-thin sections (60 nm) were stained with uranyl acetate and lead citrate. Ultra-thin sections were examined with a JEOL 1200 EX-2 transmission electron microscope (TEM) at 80 kV. RESULTSBasophilic inclusions in gonoducts were observed in specimens from all sites except Stn 4. Infections were observed from March to May at prevalences of 3.3 to 7.1%. Prevalence at Stn 3 was higher than at the other sites ( Table 1). The heaviest infection included 62 inclusion bodies in a single tissue section; however, no host reaction was observed. The longest axis of the inclusion bodies varied from 15 to 60 µm, and the shape was oval to spherical. Some inclusion bodies had dense staining margins (Figs. 2 & 3). Feulgen staining confirmed the presence of dense nucleic acid (Fig. 4), and orange-red staining with phloxine tartrazine (Fig. 5) demonstrated a deoxyribonucleic acid (DNA) composition. Advanced infections led to disruption of the nuclear membrane and putative release of the viral particles (Fig. 4, arrowhead).Non-enveloped, icosahedral viral particles, 40 to 45 nm in diameter, filled the inclusion bodies (Figs. 6 & 7). These morphological characteristics resemble those of Pa...
Since the late 1980s, a birnaviral gill disease has been occurring in Japanese eels Anguilla japonica reared in warmwater ponds in western regions in Japan. Diseased eels mostly displayed marked formations of aneurysmal hematomas within gill lamellae and high mortalities. Histological examination revealed necrosis of pillar cells and subsequent aggregation of erythrocytes inside the lamellar capillaries, and proliferation of interlamellar epithelia onto the lamellae. Gastric gland cells were also necrotized. Electron microscopy revealed birnavirus infection in lamellar pillar cells. The causative birnavirus was isolated and cultured in fish cell lines and was found to be related to an infectious pancreatic necrosis virus (IPNV) Sp serotype by neutralization tests. The viral pathogenicity was confirmed by the results of histopathological examinations and infectivity experiments.
Mass mortality occurred at an Anguilla japonica eel farm equipped with a recirculating aquaculture system in Gimcheon, Korea, from late spring to early summer 2015. The cumulative 3-month mortality was 16% (approximately 24,300-150,000 fish). The majority of affected fish displayed ulcerative lesions that progressed to petechial haemorrhages and small white granulomas in the major organs. A Gram-positive, acid-fast, nonmotile bacterium was isolated from internal organ lesions. Phylogenetic analysis of 16S rRNA identified the species as Nocardia seriolae and the strain was designated EM150506. Afterwards, naïve eels were injected with 1.8 × 10 colony-forming units per fish to confirm the strain's pathogenicity, which resulted in a 20% mortality rate within 4 weeks. However, surviving fish still exhibited white N. seriolae colonies in internal organs. To our knowledge, this is the first report of a N. seriolae infection in cultured eel.
We prepared monoclonal antibodies (mAbs) against a recombinant tethered follicle-stimulating hormone (rec-FSH) from Japanese eel Anguilla japonica that was produced in Escherichia coli. Positive hybridomas (clones eFA-C5, eFA-C10, eFA-C11, eFA-C12, eFA-C13, and eFB-C14) were selected by using the eel FSH antigen in ELISA, and anti-eel FSH mAbs were purified from culture supernatants by performing affinity chromatography. Three of the 6mAbs were characterized and their isotypes were identified as IgG2b (eFA-C5 and eFA-C11) and IgG1 (eFB-C14). In western blotting assays, the mAbs recognized the antigen as a 24.3-kDa band, and further detected bands of 34 and 32kDa in the supernatants of CHO cells transfected with cDNA encoding tethered eel FSHβ/α and LHβ/α, respectively. PNase F-mediated deglycosylation of the recombinant proteins resulted in a drastic reduction in their molecular weight, to 7-9kDa. The mAbs eFA-C5 and eFA-C11 recognized the eel FSHα-subunit that is commonly encoded among glycoprotein hormones, whereas eFB-C14 recognized the eel FSHβ-subunit, and immunohistochemical analysis revealed that the staining by these mAbs was specifically localized in the eel pituitary. We also established an ELISA system for detecting rec-tethered FSHβ/α and LHβ/α produced from CHO cell lines. Measurement of biological activities in vitro revealed that only weak activity of rec-FSHβ/α was detected. The activity of rec-LHβ/α was found to be increased in a dose-dependent manner for eel oocyte maturation.
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