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NO39 - 4

Short Term in vitro Culture of Cryptocaryon irritans,a Protozoan Parasite of Marine Fishes

<>Apolinario V. Yambot1,3 and Yen-Ling Song 1,2* <>1 Institute of Zoology, National Taiwan University, Taipei 106, Taiwan, ROC
2 Department of Life Science, National Taiwan University,
Taipei 106, Taiwan, ROC
3 Present address: College of Fisheries-Freshwater Aquaculture Center,
Central Luzon State University, Philippines
(Received March 19, 2004)
ABSTRACT輸ttempts were made to cultivate Cryptocaryon irritans in vitro at 23-25゚C.
Attachment of theronts and subsequent enlargement into trophonts were achieved in two experiments using strips of trypticase soy agar (TSA, supplemented with 3% NaCl) as an attachment substrate in filtered seawater. In the third experiment, transformation of theronts into trophonts was achieved in an enriched liquid medium composed of 50% filtered seawater, 30% Leibovitz L-15 and 20% fetal calf serum without attachment onto the TSA. Sizes (mean ± SD) of the trophonts, 114.6 ± 57.9 µm to 295.9 ± 130 µm, were from a recorded size range (50 to 700 µm) of the parasite in vivo. Although only limited numbers of theronts (0.28-1.71%) transformed into trophonts, these results showed that the in vitro culture of C. irritans is potentially feasible as evidenced by the
enlargement of the trophonts within the in vivo size range using either a solid medium as an attachment substrate or a liquid medium without attachment. There is a need, however, to determine essential factors that influence the transformation of the trophonts into viable tomonts capable of producing theronts.

Key words: Cryptocaryon irritans, in vitro culture, theront, trophont, culture media

No Evidence for the Presence of Flavobacterium psychrophilum within Ayu Eggs

<>Akira Kumagai 1,3* , Chihaya Nakayasu 1 and Norihisa Oseko 2 <>1 Inland Station, National Research Institute of Aquaculture, Fisheries Research Agency,
Tamaki, Mie 519-0423, Japan
2 National Research Institute of Aquaculture, Fisheries Research Agency,
Nansei, Mie 516-0193, Japan
3 Present address:Miyagi Prefectural Freshwater Fisheries Experimental
Station, Taiwa, Miyagi 981-3625, Japan
(Received May 20, 2004)
ABSTRACT裕his study was made to clarify a possibility of vertical transmission of bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis. The presence of Flavobacterium psychrophilum on the surface and in the contents of unfertilized and fertilized eggs, which were obtained from ayu naturally carrying F. psychrophilum, was examined by a culture method. The bacterium was detected on the egg surface of 19 out of 65 unfertilized egg samples and of 5 out of 30 fertilized egg samples (300 eggs/sample). On the other hand, F. psychrophilum was not
detected from the surface of eggs disinfected by povidone-iodine (5 ppm, 10 min) or hydrogen peroxide (150 ppm, 30 min). The bacterium was not detected in the contents of these surface-disinfected eggs. These results suggest that F. psychrophilum is not transmitted through intra-ovum infection in ayu eggs. Therefore, disinfection of eggs is useful to prevent the vertical transmission of BCWD in ayu hatcheries.

Key words: Flavobacterium psychrophilum, bacterial coldwater disease, intra-ovum infection, disinfection of eggs, Plecoglossus altivelis, BCWD

Characterization of Expressed Genes in Kidney Cells of Japanese Flounder Paralichthys olivaceus Following Treatment with ConA/PMA and LPS

<>Nur Rahmawaty Arma, Ikuo Hirono and Takashi Aoki*<>Graduate School of Marine Science and Technology, Tokyo University of Marine Science and
Technology, Tokyo 108-8477, Japan
(Received June 21, 2004)
ABSTRACT裕wo cDNA libraries were prepared from Japanese flounder Paralichthys olivaceus kidney cells that had been treated with LPS or a combination of ConA and PMA. From these two libraries, we sequenced 373 and 379 clones, respectively. Of these 752 clones, 109 clones were characterized as immune-related. Among the immune-related cDNAs, the following have not been previously reported in fish: NK-lysin, perforin, complement C1q, CD9 antigen, CD63 antigen, CD82 antigen, ISGF-3, IP-30, G-CSF, equistatin precursor, TLS-CHOP, fas ligand and CARD4. This study highlights the success of using ConA/PMA and LPS as mitogens to increase the number of immune-related genes expressed in Japanese flounder and demonstrates that different stimuli
induce the expression of different immune-related genes.

Key words: expressed sequence tag, Paralichthys olivaceus, Japanese flounder, biodefense gene, cDNA sequence, kidney

Infection Dynamics of Neoheterobothrium hirame in Japanese Flounder in the Joban Sea,Eastern Japan

<>Takeshi Tomiyama * , Masato Watanabe and Ken-ichi Ebe<>Fukushima Prefectural Fisheries Experimental Station, Onahama,
Iwaki 970-0316, Japan
(Received June 30, 2004)
ABSTRACT邑e investigated the infection dynamics of adult Neoheterobothrium hirame in young Japanese flounder Paralichthys olivaceus (20-50 cm in total length) in the Joban Sea, eastern Japan. The prevalence and abundance of the parasite, which were statistically irrelevant to host factors namely both wild/released and male/female, showed seasonal fluctuations and peaked in winter. The oldest record of the parasite in the Joban Sea was found in 1997. Although the infection level was high from 1998 to 2002, it decreased dramatically in 2003. The factors influencing the adult parasite infection level were unclear, but one of the possible factors would be the continuous low water temperature from August 2002 to February 2004, especially in the summer of 2003, when new infections should have occurred. The low temperature seems to have decreased the egg-laying rate of N. hirame and consequently lowered its infection level.

Key words: Neoheterobothrium hirame, Monogenea, water temperature, Paralichthys olivaceus

Tissue Distribution of the Agent of Akoya Oyster Disease in Japanese Pearl Oyster Pinctada fucata martensii

<>Chihaya Nakayasu 1* , Hideo Aoki 2 , Maki Nakanishi 2 , Hirofumi Yamashita 3 , Masanori Okauchi 4 , Norihisa Oseko 4 and Akira Kumagai 1, 5 <>1 Inland Station, National Research Institute of Aquaculture, Tamaki, Mie 519-0423, Japan
2 Mie Prefectural Science and Technology Promotion Center, Hamajima,
Mie 517-0404, Japan
3 Ehime Prefectural Fisheries Experimental Station, 5516 Shitaba,
Uwajima, Ehime 798-0104, Japan
4 National Research Institute of Aquaculture, Nansei, Mie 516-0193, Japan
5 Present address: Miyagi Prefectural Freshwater Fisheries Experimental
Station, Taiwa, Miyagi 981-3625, Japan
(Received July 9, 2004)
ABSTRACT柚ass mortalities caused by akoya oyster disease have been occurring in cultured Japanese pearl oyster Pinctada fucata martensii in western Japan. Although the involvement of some causative agents was suggested, the cause of the disease is still unidentified. In this study, an experimental infection was performed by cohabitation of the diseased and healthy pearl oysters. A high mortality and characteristic disease conditions were observed in the test group. This result reconfirmed that this disease is caused by a certain infectious agent. To clarify the tissue distribution of the causative agent in diseased pearl oysters, two experimental infections were performed by transplantation of pieces of various tissues or injection of homogenates of various tissues from diseased pearl oysters into healthy pearl oysters. The hemolymph, mantle and adductor muscle showed high infectious titers compared with those of the heart, digestive gland and hemocytes. The mantle had the highest infectious titer. Moreover, injection of the supernatant of a mantle homogenate also transmitted the disease to test pearl oysters. These results suggest that the causative agent is concentrated in the mantle.

Key words: Pinctada fucata martensii, Japanese pearl oyster, akoya oyster disease, mass mortality, experimental infection, tissue distribution, mantle

Detection of Flounder Herpesvirus (FHV) by Polymerase Chain Reaction

<>Yoshisuke Iida * and Takahiro Nagai<>Hiroshima Prefectural Fisheries Experiment Station, Ondo, Hiroshima 737-1207, Japan
(Received September 21, 2004)
ABSTRACT邑e developed a polymerase chain reaction (PCR) method to detect flounder herpesvirus (FHV), the causative agent of viral epidermal hyperplasia of larval Japanese flounder Paralichthys olivaceus. The developed PCR method was specific to FHV and no PCR products were obtained in other fish-pathogenic herpesviruses and flounder-pathogenic viruses tested. FHV was detected by the PCR test with a sample containing 100 or more epidermal cells, which were derived from flounder larvae with epidermal hyperplasia. Four samples of diseased flounder larvae, which were collected from natural mass mortalities and diagnosed as viral epidermal hyperplasia by conventional methods, were positive by the PCR test for FHV. These results show that the present PCR method is useful for rapid and reliable diagnosis of fish clinically or subclinically infected with FHV.

Key words: flounder herpesvirus, PCR, viral epidermal hyperplasia, Paralichthys olivaceus, FHV

Partial Purification of Kuchijirosho Causative Agent by Sodium Iotalamate Density Gradient Centrifugation

<>Toshiaki Miyadai 1,2* , Emi Hashimoto 2 , Kumiko Hashimoto 2 , Tomomi Watari 3 , Maki Ohtani 1,4 and Daisuke Tahara 2 <>1 Research Center for Marine Bioresources, Faculty of Biotechnology, Fukui Prefectural University,
Obama, Fukui 917-0116, Japan
2 Laboratory of Marine Biotechnology, Department of Marine Bioscience, Fukui Prefectural University,
Obama, Fukui 917-0003, Japan
3 Resource Center for Freshwater Fisheries of Fukui
Prefecture, Nakanogou-cho,
Fukui 910-0816, Japan
4 Laboratory of Marine Ecological Metabolism,
Department of Marine Bioscience, Fukui
Prefectural University, Obama,
Fukui 917-0003, Japan

(Received May 24, 2004)

ABSTRACT裕o purify the kuchijirosho causative agent
derived from brains of kuchijirosho-affected tiger puffer Takifugu rubripes, the method of density gradient centrifugation using iodinated contrast medium, Angio-Conray (solium iotalamata solution), was used. The extracts of kuchijirosho-affected brains were loaded on 3-55% gradient of Angio-Conray, and centrifuged at 280,000 ∞ g for 2 h.
The lethal activity to grass puffer Takifugu niphobles was found in a fraction with a density around 1.096 g/cm3. The infectivity of the extracts of kuchijirosho-affected brains was lost by treatment with ether, UV-irradiation, ß-propiolacton or proteinase K.

Key words: kuchijirosho, sodium iotalamate, density gradient centrifugation, Takifugu rubripes, snout ulcer disease

Effect of Water Temperature on the Egg Production and Egg Viability of the Monogenean Heterobothrium okamotoi Infecting Tiger Puffer Takifugu rubripes

<>Naoko Yamabata, Tomoyoshi Yoshinaga * and Kazuo Ogawa<>Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of
Tokyo, Tokyo 113-8657, Japan

(Received August 25, 2004)

ABSTRACT裕iger puffer experimentally infected with Heterobothrium okamotoi was given gradual changes of
water temperature, and the effect of temperature on the production and viability of the parasite eggs was examined.
Among 10, 15, 20, 25 and 30゚C, the highest egg production rate was observed at 25゚C. While most of eggs produced at 10 or 20゚C hatched at 20゚C-incubation, those produced at 30゚C decreased in hatching rate at 20゚C- or 30゚C-incubation. Eggs produced at 26゚C or more contained morphologically abnormal ones. These results indicate that the optimal temperature for the egg production of H. okamotoi is approximately 25゚C.

Key words: Heterobothrium okamotoi, egg production, egg viability, Takifugu rubripes, tiger puffer