The multiplication of red sea bream iridovirus (RSIV : KM99 from red sea bream) was investigated in the two groups of grouper Epinephelus malabaricus (average body weight, 7.7 g and 84.3 g) intraperitoneally inoculated with the virus at 28°C. Dead fish were observed from 8th day post-inoculation, and the cumulative mortality was 90% in fish of both sizes. Viral antigen-positive cells were detected with an indirect immunofluorescence test using a monoclonal antibody in the spleen and head kidney of the infected fish from 2nd day post-inoculation and in the liver and body kidney from 6th day post-inoculation. The number of positive cells in the spleen and head kidney was higher than that in the liver and body kidney. Transmission electron microscopy showed that the multiplication of the virus occurred in the enlarged cells in the fish at 4th day post-inoculation. In the dead fish, virus particles were found around the disrupted enlarged cells and small blood vessels. These findings suggest that the enlarged cells form primarily in the spleen and head kidney of the infected grouper as a result of viral multiplication and that the cells move to other organs via blood vessels and cause disruption by releasing progeny viruses.
Physiological properties and pathogenicity of three isolates of viral hemorrhagic sepicemia virus (VHSV), Obama25 isolated from wild Japanese flounder Paralichthys olivaceus in 1999, JF00Ehi1 from farmed diseased Japanese flounder in 2000 and KRRV9601 from farmed diseased Japanese flounder in 1996, were studied. The former two isolates belong to American genotype (genogroup I) and the latter one to European genotype (genogroup III). Among three fish cell lines tested, the three isolates multiplied best in FHM cells, moderately in EPC cells but hardly in RTG-2 cells. In FHM cells, these isolates multiplied at 10, 15, and 20°C but not at 25°C. The optimum temperature was 15°C for KRRV9601 and 20°C for the other two isolates. In in vitro stability tests in different waters at different temperatures, the viral infectivity decreased rapidly in non-treated seawater with increasing water temperature. The two American genotype isolates, Obama25 and JFOOEhi1, exhibited a similar level of virulence in flounder at 13°C causing disease signs similar to those observed in naturally affected flounder. On the other hand, no mortality was produced by KRRV9601, though the virus was re-isolated from surviving fish. These results in the pathogenicity test support the fact that the American genotype of VHSV has been prevailing among wild and farmed flounder in Japan.
Virucidal activities of methanol, ethanol, 1-propanol, phenol, cresol, chlorine, iodophor and 3 kindsof invert soaps against viral hemorrhagic septicemia virus (VHSV) isolated from Japanese flounder Paralichthys olivaceus were examined. The disinfectants were diluted with PBS (-) or artificial seawater. The virus (VHSV : JF00Ehi1) was treated with each disinfectant at different concentrations for different periods. The reaction mixtures contained fetal calf serum at a final concentration of 1%. The treated virus was then inoculated to the cultured cell line, FHM, to determine virucidal activities of the disinfectants. When the disinfectants were diluted with PBS, the virus was easily inactivated by 1-propanol, cresol, chlorine, iodophor or all invert soaps, whereas methanol, ethanol and phenol had less activities. However, when the disinfectants were diluted with artificial seawater virucidal activities of cresol and chlorine apparently decreased.
Eight antimicrobial compounds were examined to evaluate their therapeutic effects against experimentally or naturally induced vibriosis of the Pacific oysterCrassostrea gigas. In experimental infections with a strain ofVibrio splendidusbiovar II, a causative agent of bacillary necrosis of cultured triploid oyster larvae, chloramphenicol (CP) exhibited complete protection against challenges at 105or 106CFU/mL, and erythromycin (EM), novobiocin (NB), gentamicin and streptomycin (SM) were effective to reduce the mortality, but nalidixic acid or oxytetracycline was not. CP and EM were also highly effective against experimental infections with other six strains ofVibriospecies (V. splendidusbiovar ll, V. pelagiusI, V. campbellii, and V. tubiashii) which had been isolated from oyster larvae or the rearing water, but NB and SM were less effective. On the other hand, not only CP and EM but also NB and SM exhibited higher protection against the natural infection.
A survey of virus isolation was conducted on 160 samples of wild Japanese flounder Paralichthys olivaceus and 366 samples of other 9 wild fish species collected in 8 coastal areas of Japan in 2001. Aquabirnavirus (ABV) was isolated from flounder (15%), Japanese horse mackerel Trachurus japonicus (23%), and dark banded rockfish Sebastes inermis (4%). Viral hemorrhagic septicemia virus (VHSV) was isolated from flounder (10%) and sand lance Ammodytes personatus (2%) while the other 6 fish species were virus-negative. Concurrently, the distribution of viruses was also examined in 200 flounder collected from different hatcheries and grow-out farms. Only 1.5% of the farmed flounder were ABV-positive and none was VHSV-positive.
One hundred spat of Crassostrea gigas obtained from a coastal area in north-eastern Japan were examined for the protistan parasite Haplosporidium nelsoni. Haplosporidium-like plasmodia were histologically observed in two spat and positively reacted with a H. nelsoni-specific probe in in situ hybridization. Four spat including the two spat in which the plasmodia were found showed positive reaction in PCR analysis for the detection of H. nelsoni.The small subunit ribosomal RNA sequence amplified from the spat was virtually identical (99.7%) to the sequence of H. nelsoni previously reported. These results demonstrate that H. nelsoni is distributed in Japan.
A survey of koi herpesvirus (KHV) and carp edema virus (CEV) was made in cultured colorcarp Cyprinus carpio. Using polymerase chain reaction (PCR), 205 fish collected from 20 culture farms located in Niigata Prefecture were examined for the presence of KHV (205 fish) and CEV (35 fish showing signs of so-called “sleeping disease”). Since the result of PCR amplification for KHV was negative in all samples, it is considered that koi herpesvirus disease does not exist in Niigata Prefecture. On the other hand, CEV genome was detected in 87.5% of “sleeping disease”-affected fish. This indicates the involvement of CEV in “sleeping disease”, though the direct cause-effect relationship has not been established.
Treatment of supplied water with a high quality ultraviolet (UV) lamp was examined for prevention of scuticociliatosis of farmed juvenile Japanese flounder (Paralichthys olivaceus).In an examination for the ciliate-cidal effect of UV irradiation, scuticociliates showed low susceptibility to UV compared with fish pathogenic viruses or bacteria, the minimal killing dosage being 2.0×105μW·sec/cm2.UV treatment of supplied water to the tank was performed in a flounder farm in southern Hokkaido, where scuticociliatosis frequently occurred.It was revealed that the UV treatment at 3.0×105μW·sec/cm2 was effective to prevent scuticociliatosis.
Yellowtail Seriola quinqueradiata were placed in an enzootic area of blood fluke infestation. Subsequently, the fish were challenged with the bacterial fish pathogen Lactococcus garvieae. The final cumulative mortality by L. garvieae was significantly higher in the blood fluke-infested fish than in the uninfested fish. The rate of the number of the gill filaments harboring the parasite eggs in the dead fish was significantly higher than that of the surviving fish, and among the dead fish, the fish with the higher rate died in a shorter time. These results suggest that the blood fluke infestation promotes the mortality by L. garvieae infection in yellowtail.
The Japanese Society of Fish Pathology held a symposium entitled “Current Status and Future Prospect of the Research on Fish Diseases in Japan” in Kochi on September 14th, 2002. This symposium was aimed to overview the status of researches on fish diseases and its future prospect in Japan. Keynote lecturers from the Japanese Society of Fish Pathology, Japanese Government, a fisheries association and Korea presented reports on fish disease problems. Opinions of representative speakers from fish farmers, researchers in charge of clinical fish management, and employees of fish feed / pharmaceutics / vaccine companies were also presented. These are summarized as the necessity of: combination of basic and applicable researches; interchange of researches and researchers on fish diseases with those of other research fields; encouragement of young researchers.