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NO37 - 4

Multiplication of Red Sea Bream Iridovirus (RSIV) in the Experimentally Infected Grouper Epinephelus malabaricus

<>Motohiko Sano1,3, Megumi Minagawa1,4 and Kazuhiro Nakajima2,5<>1 Ishigaki Tropical Station, Seikai National Fisheries Research Institute, Ishigaki,
Okinawa 907-0451, Japan
2 Inland Station, National Research Institute of Aquaculture,
Tamaki, Mie 519-0423, Japan
3 Present address: Inland Station, National Research Institute of Aquaculture,
Tamaki, Mie 519-0423, Japan
4 Present address: Seikai National Fisheries Research Institute,
Nagasaki 850-0951, Japan
5 Present address: Headquarters, Fisheries Research Agency,
Fukuura, Kanazawa-ku, Yokohama 236-8648, Japan
(Received March 8, 2002)

ABSTRACT裕he 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.

Key words: viral multiplication, red sea bream iridovirus, Epinephelus malabaricus, immunofluorescence, TEM, RSIV, grouper

Properties of Viral Hemorrhagic Septicemia Virus (VHSV) Isolated from Japanese Flounder Paralichthys olivaceus

<>Koh-ichiro Mori 1, Hideki Iida 2, Toyohiko Nishizawa 2, Misao Arimoto 1, Kazuhiro Nakajima 3 and Kiyokuni Muroga 2<>1 Kamiura Station of Japan Sea-Farming Association, Kamiura, Oita 879-2602, Japan
2 Faculty of Applied Biological Science, Hiroshima University, Higashihiroshima 739-8528, Japan
3 National Research Institute of Aquaculture, Tamaki, Mie 519-0423, Japan
(Received May 7, 2002)

ABSTRACT猶hysiological 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 JF00Ehi1, 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.

Key words: viral hemorrhagic septicemia virus, Paralichthys olivaceus, physiological property, virulence, temperature effect, Japanese flounder , VHSV

Virucidal Effects of Various Disinfectants on Viral Hemorrhagic Septicemia Virus (VHSV) Isolated from Japanese Flounder

<>Jun Kurita 1, Yoshisuke Iida 2, Kazuhiro Nakajima 3 and Kiyoshi Inouye 4<>1 Inland Station, National Research Institute of Aquaculture, Fisheries Research Agency,
Tamaki, Watarai, Mie 519-0423, Japan
2 Hiroshima Prefectural Fisheries Experiment Station, Ondo, Aki,
Hirosima 737-1207, Japan
3 Headquaters, Fisheries Research Agency, Fukuura, Kanazawa, Yokohama,
Kanagawa 236-8648, Japan
4 National Research Institute of Aquaculture, Fisheries Research Agency,
Nansei, Watarai, Mie 516-0193, Japan
(Received June 14, 2002)

ABSTRACT-Virucidal activities of methanol, ethanol, 1-propanol, phenol, cresol, chlorine, iodophor and 3 kinds of 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.

Key words: disinfectants, viral hemorrhagic septicemia, Paralichthy olivaceus, virucidal effect, Japanese flounder, VHSV

Therapeutic Effects of Antimicrobial Compounds against Bacillary Necrosis of Larval Pacific Oyster

<>Danji Matsubara 1, Makoto Tanaka 1, Yoshie Soumyou 1, Kohji Hirakawa 1, Ryuji Doi 2 and Toshihiro Nakai 2<>1 Hiroshima Prefectural Farming Fisheries Association, Takehara 729-2313, Japan
2 Graduate School of Biosphere Science, Hiroshima University,
Higashihiroshima 739-8528, Japan
(Received July 25, 2002)

ABSTRACT-Eight antimicrobial compounds were examined to evaluate their therapeutic effects against experimentally or naturally induced vibriosis of the Pacific oyster Crassostrea gigas. In experimental infections with a strain of Vibrio splendidus biovar II, a causative agent of bacillary necrosis of cultured triploid oyster larvae, chloramphenicol (CP) exhibited complete protection against challenges at 105 or 106 CFU/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 of Vibrio species (V. splendidus biovar II, V. pelagius I, 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.

Key words: Crassostrea gigas, chemotherapy, bacillary necrosis, Vibrio splendidus, Pacific oyster

Isolation of Aquabirnavirus and Viral Hemorrhagic Septicemia Virus (VHSV) from Wild Marine Fishes

<>Leo Watanabe 1, Rolando Pakingking Jr. 1, Hideki Iida 1, Toyohiko Nishizawa 1,4, Yoshisuke Iida 2, Misao Arimoto 3 and Kiyokuni Muroga 1<>1 Faculty of Applied Biological Science, Hiroshima
University, Higashi-hiroshima 739- 8528, Japan
2 Hiroshima Prefectural Fisheries Experiment Station,
Ondo, Hiroshima, 737-1207, Japan
3 Kamiura Station of Japan Sea-Farming Association,
Kamiura, Oita 879-2602, Japan
4 Presend address: Graduate School of Fisheries
Sciences, Hokkaido University, Hakodate,
Hokkaido, 041-8611, Japan

(Received April 9, 2002)

ABSTRACT-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.

Key words: aquabirnavirus, viral hemorrhagic septicemia virus, wild marine fish, Paralichthys olivaceus, Ammodytes personatus, yellowtail ascites
virus, VHSV

Detection of Haplosporidium nelsoni in Pacific oyster Crassostrea gigas in Japan

<>Takashi Kamaishi 1 and Tomoyoshi Yoshinaga 1,2<>1 National Research Institute of Aquaculture, Fisheries Research Agency, Nansei, Mie 516-0193, Japan
2 Present address: Department of Aquatic Biosciences, Granduate School of Agricultural and Life Sciences,
The University of Tokyo, Yayoi, Bunkyo,
Tokyo 113-8657, Japan

(Received May 9, 2002)

ABSTRACT-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.

Key words: Haplosporidium nelsoni, MSX, Crassostrea gigas, SrRNA, Pacific oyster

A survey of Koi Herpesvirus and Carp Edema Virus in Colorcarp Cultured in Niigata Prefecture, Japan

<>Kenjiro Amita 1, Mitsuhiro Oe 1, Hisato Matoyama 1,3, Noriko Yamaguchi 2 and Hideo Fukuda 2<>1 Niigata Prefectural Inland-water Fisheries Experimental Station, Okawara-machi 2650,
Nagaoka 940-1137, Japan
2 Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan 4-5-7, Minato-ku,
Tokyo 108-8477, Japan
3 Present adress: Fisheries Division Niigata Prefectual Government, Shinkocho 4-1,
Niigata 950-8570, Japan

(Received May 13, 2002)

ABSTRACT-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 isleeping diseasei). 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 isleeping diseasei-affected fish. This indicates the involvement of CEV in isleeping diseasei, though the direct cause-effect relationship has not been established.

Key word: koi herpesvirus, pathogen detection, carp edema virus, Cyprinus carpio, KHV, CEV

Prevention of Scuticociliatosis in Japanese flounder by Treatment of Water-supply with a High Quality UV Lamp

<>Hisae Kasai 1, Shuichi Osawa 2, Tadashi Kobayashi 2 and Mamoru Yoshimizu 1<>1 Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, 041-8611, Japan
2 Fukushima Yoshioka Fisheries Cooperative, Yoshioka, Fukushima-cho, Hokkaido, 049-1453, Japan

(Received June 7, 2002)

ABSTRACT-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 x 105 µW ・ 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 x 105 µW ・ sec/cm2 was effective to prevent scuticociliatosis.

Key words: UV, disinfection, scuticociliate, scuticociliatosis, Paralichthys olivaceus, Japanese flounder

Blood Fluke Promotes Mortality of Yellowtail Caused by Lactococcus garvieae

<>Mami Kumon 1, Takaji Iida 2, Yutaka Fukuda 3, Misao Arimoto4 and Ken Shimizu 4<>1 Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai-Nishi, Miyazaki 889-2192, Japan
2 Pathology Division, National Research Institute of Aquaculture, Fisheries Research Agency, Nansei, Mie 516-0193, Japan
3 Oita Institute of Marine and Fisheries Science, Kamiura, Oita 879-2602, Japan
4 Kamiura Station of Japan Sea-Farming Association, Kamiura, Oita 879-2602, Japan

(Received June 21, 2002)

ABSTRACT-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.

Key words: blood fluke, Lactococcus garvieae, Seriola quinqueradiata, mortality, yellowtail