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NO40 - 4

PCR-RFLP Analysis for Identification of Aeromonas Isolates Collected from Diseased Fish and Aquatic Animals

<>Md. Mahbubur Rahman1,2*, Temdoung Somsiri3,Reiji Tanaka1, Tomoo Sawabe1 and Kenichi Tajima1<>1Laboratory of Microbiology, Graduate School of Fisheries Sciences, Hokkaido University,
Hakodate, Hokkaido 041-8611, Japan
2Department of Biotechnology, Shah Jalal University of Science and Technology,
Sylhet-3114, Bangladesh
3Aquatic Animal Health Research Institute, Kasetsart University campus,
Jatujak, Bangkok 10900, Thailand
(Received February 7, 2005)

ABSTRACT裕he study was conducted to develop a PCR and an improved PCR-RFLP analysis method for rapid species-identification of Aeromonas genospecies. The forward and reverse primers for PCR were designed from the complementary sequences of the 16S rDNA of all 15-recognized Aeromonas genospecies to amplify a 1206-bp PCR product. The PCR amplified the expected product from the DNA template of type or reference strains of all recognized Aeromonas genospecies as well as 106 Aeromonas strains, while no PCR product was obtained from any of the non-Aeromonas strains tested. The PCR-RFLP analysis using the restriction enzymes (AluI, MboI, PvuII, PstI and NarI) provided identification of almost all Aeromonas species. However, the Aeromonas sp. T8 group and A. caviae exhibited a similar RFLP pattern. Some selected biochemical tests were found to be helpful for differentiation of the two species.

Key words: PCR-RFLP, species-identification, Aeromonas genospecies

Histopathological Study on Oncorhynchus masou Virus Disease (OMVD) of Cultured Rainbow Trout in Natural Outbreaks and Artificial Infection

<>Mitsuru Furihata1, 2*, Kana Suzuki3, Akira Hosoe1 and Teruo Miyazaki3<>1Nagano Prefectural Fisheries Experimental Station, Azumino, Nagano 399-7102, Japan
2Graduate School of Fisheries Sciences, Hokkaido University, Hakodate,
Hokkaido 041-8611, Japan
3Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan
(Received June 27, 2005)
ABSTRACT涌ncorhynchus masou virus (OMV) was isolated from naturally diseased rainbow trout Oncorhynchus mykiss cultured in Nagano Prefecture. In the present study, histopathological and electron microscopic features were performed on spontaneously and experimentally infected fish with OMV. The definite change was necrosis of OMV-infected cells, which were observed in the spleen, hematopoietic tissues in the kidney, liver, intestine, heart, gill filaments, epidermis and lateral musculature. In particular, the intestine showed severe necrosis and hemorrhage in the epithelium and underlying tissues, which is the first description of OMV disease.

Key words: Oncorhynchus masou virus disease, salmonid herpesvirus 2, histopathology, Oncorhynchus mykiss, rainbow trout, OMV

Experiments on the Evaluation of the Blood Feeding of Heterobothrium okamotoi (Monogenea: Diclidophoridae)

<>Kazuo Ogawa*, Masayoshi Yasuzaki and Tomoyoshi Yoshinaga<>Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
(Received July 25, 2005)
ABSTRACT唯lood feeding of the monogenean Heterobothrium okamotoi from tiger puffer Takifugu rubripes was examined. Physiological saline containing fluorescent microspheres (1 µm in diameter) was injected into the hepatic vein of one-year-old tiger puffer experimentally or naturally infected with H. okamotoi, and blood was taken from the host at 3 and 12 h post-injection (PI). Parasites were collected at 12 h PI and individually digested with KOH. Microspheres in the host blood and parasite body were counted by fluorescence microscopy. The amount of blood taken by individual parasites was calculated by the number of microspheres in the parasite body divided by the mean value of the microsphere concentration in the host blood at 3 and 12 h PI. The results show that the amount of blood increased as the parasite developed and that an adult fed 0.69 ア 0.93 µL of host blood for 12 h (= 42 µL/ month). This is the first report of a quantitative study on the blood feeding of polyopisthocotylean monogeneans.

Key words: Heterobothrium okamotoi, Takifugu rubripes, blood feeding, Monogenea, tiger puffer

Occurrence of the Myxosporean Emaciation Disease Caused by Enteromyxum leei in Cultured Japanese Flounder Paralichthys olivaceus

<>Hiroshi Yasuda1, Tsuyoshi Ooyama1, AtsushiNakamura1, Kazuo Iwata1, Oswaldo Palenzuela2 and Hiroshi Yokoyama3*<>1Miyazaki Prefectural Fisheries Experimental Station, Miyazaki, 889-2162, Japan
2Instituto de Acuicultura de Torre de la Sal (CSIC), 12595 Ribera de Cabanes, Castellon, Spain
3Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Tokyo 113-8657, Japan
(Received July 29, 2005)
ABSTRACT裕he myxosporean emaciation disease has been recently found in Japanese flounder Paralichthys olivaceus cultured in land-based tanks in Miyazaki Prefecture, Japan. Affected fish showed external signs of distended abdomen, rectal prolapse, sunken eyes and a typical bony ridge on the skull. Parasitological and histopathological examinations revealed that the developmental stages of Enteromyxum leei parasitized the intestinal epithelium of diseased fish. No other enteric myxozoans were observed in the affected fish. A molecular analysis of SSU rDNA confirmed that the causative myxozoan was identified as E. leei. Experimental transmission of E. leei was achieved to naive Japanese flounder and tiger puffer Takifugu rubripes by feeding with gut tissues taken from infected Japanese flounder. The present study indicates that the myxosporean emaciation disease of Japanese flounder is caused by E. leei.

Key words: Enteromyxum leei, Myxozoa, emaciation disease, Paralichthys olivaceus, Japanese flounder, Takifugu rubripes, tiger puffer

Effectiveness of Low Rearing Density against Red Sea Bream Iridoviral Disease in Red Sea Bream Pagrus major

<>Shinji Tanaka1* and Misa Inoue1,2<>1Owase Fisheries Laboratory, Fisheries Research Division, Mie Prefectural Science and
Technology Promotion Center, Owase, Mie 519-3602, Japan
2Present addressナFMie Prefecture Kihoku Branch Office, Department of Agriculture,
Fisheries, Commerce and Industry, Owase, Mie 519-3695, Japan
(Received August 11, 2005)
ABSTRACT勇ffectiveness of low rearing density was examined against red sea bream iridoviral disease (RSIVD) in juvenile red sea bream Pagrus major. In an experimental challenge test, in which the number of fish and the volume of rearing space were set in three levels, low-density groups (2.1 or 2.2 kg/m3) showed significantly lower mortalities than medium- (4.2 kg/m3) and high-density groups (8.4 kg/m3). In a field trial, fish were reared in neighboring net pens (6.4 m3) at three densities (0.8, 2.8, 9.6 kg/m3) for 73 days. The mortality due to natural infection with RSIV showed a clearly inverse correlation with the rearing density. Additionally, hemoglobin content and plasma component levels were measured in the before-mentioned field trial and an indoor experiment, in which the number of fish and the aquarium size were set in three levels (2.8, 5.3 or 5.8, 11.3 or 11.6 kg/m3). The levels of plasma total cholesterol and phospholipid in high-density groups were significantly lowered than those of low-density groups. Low rearing density was effective against RSIVD in red sea bream, and plasma lipid components may play a role in the disease resistance.

Key words: red sea bream iridoviral disease, rearing density, Pagrus major, iridovirus, RSIVD, red sea bream, plasma lipid component, disease resistance

Antimicrobial Activity of Medicinal Herbs in vitro against Fish Pathogen, Aeromonas hydrophila

<>Ramasamy Harikrishnan* and Chellam Balasundaram<>Fish Disease Management Unit, Department of Animal Science, Bharathidasan University,
Tiruchirapalli ・620024,
Tamil Nadu, India

(Received May 30, 2005)

ABSTRACTャ・This study evaluates the potency of aqueous and ethanolic decoction (individual extract) and concoction (mixed extract) of three common medicinal herbs i.e. Curcuma longa, Ocimum sanctum and Azardirachta indica to inhibite the in vitro growth of A. hydrophila. Among the decoctions A. indica exhibited the most potent antibacterial property against A. hydrophila. Among the concoctions both the aqueous and ethanolic tri-herbal extracts mixed in the ratio of 1:1:1 have the highest antibacterial activity even at the lowest concentration than the other concoctions and decoctions.

Key Words: Aeromonas hydrophila, Curcuma longa, Ocimum sanctum, Azardirachta indica, antimicrobial activity, herb

Long-Term Maintenance of Primary Cell Cultures from Ovarian Tissue of Kuruma Prawn Penaeus japonicus

<>Minoru Maeda1*, Ken-ichi Kusumoto2, Eiichi Mizuki2, Toshiaki Itami3 and Michio Ohba4<>1Kyushu Medical Co., Ltd., Fukuoka Bio Incubation Center, Kurume, Fukuoka 839-0864, Japan
2Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Kurume, Fukuoka 839-0861, Japan
3Department of Biological Production and Environmental Science, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
4Graduate School of Agriculture, Kyushu University, Fukuoka 812-8581, Japan

(Received July 25, 2005)

ABSTRACT裕he long-term maintenance of primary cell cultures from ovarian tissue of kuruma prawn Penaeus japonicus was examined in modified Leibovitz-15 medium supplemented with four growth factors (epidermal growth factor, basic-fibroblast growth factor, insulin and ciliary neurotrophic factor) and an antioxidant (2-mercaptoethanol; 2-ME). This medium allowed the primary-cultured cells to survive for a long period up to six months, while only for 45 days in the absence of the five supplements. Efficacy assessment for each supplement revealed that only 2-ME facilitated long-term maintenance of cell sheets.

Key words: Penaeus japonicus, primary cell culture, ovary, mercaptoethanol, growth factor

First Case of Fusarium oxysporum Infection in Cultured Kuruma Prawn Penaeus japonicus in Japan

<>Le Van Khoa1,2 and Kishio Hatai1*<>1Division of Fish Diseases, Nippon Veterinary and Animal Science University, Musashino, Tokyo 180-8602, Japan
2Research Institute for Aquaculture No.1, Bac Ninh City, Hanoi, Vietnam

(Received August 19, 2005)

ABSTRACT勇ight Fusarium strains were isolated from five prawns showing black gills at a farm in Kagoshima Prefecture, Japan in December 2001. On the other hand, no fungi were isolated from five prawns without black gills. Two out of the eight strains were morphologically identified as F. oxysporum and the other six strains as F. solani. Morphological characteristics of F. oxysporum were described and illustrated. The fungus showed pathogenicity when injected to juvenile kuruma prawns. This is the first case of F. oxysporum infection of kuruma prawn in Japan.

Key words: Fusarium oxysporum, morphology, Fusarium solani, pathogenicity, Penaeus japonicus

A PCR Method to Detect Myxobolus acanthogobii (Myxozoa: Myxosporea),the Causative Agent of Skeletal Deformities of Marine Fishes

<>Seiji Miyajima1, Hiroshi Yokoyama1*, Yutaka Fukuda2, Kumiko Okamoto2 and Kazuo Ogawa1<>1Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
2Fisheries Research Institute, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Oita 879-2602, Japan

(Received August 22, 2005)

ABSTRACT輸 polymerase chain reaction (PCR) method was developed to detect Myxobolus acanthogobii, a myxozoan parasite causing skeletal deformities in marine fishes. The PCR system targeting SSU rDNA of M. acanthogobii was confirmed to be species-specific; the detection limits of a single- and a nested-PCR were 1 and 0.01 pg DNA, respectively. Nested-PCR analyses were applied to epizootiological surveys of M. acanthogobii in cultured Japanese mackerel Scomber japonicus and in feral fishes (37 species). Three wild fishes, forksnout searobin Lepidotrigla alata, crimson seabream Evynnis japonica and scribbled toby Canthigaster rivulata, were found to be the hosts for M. acanthogobii.

Key words: Myxobolus acanthogobii, diagnosis, PCR, skeletal deformity

Symposium The Forefront of Studies on Koi Herpesvirus Disease

<>Teruo Miyazaki<>Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan
ABSTRACT 揖oi herpesvirus disease (KHVD) is an emergent cyprinid herpesvirus disease causing mass mortalities in koi and common carp Cyprinus carpio worldwide since 1998. In Japan, KHVD broke out in Autumn 2003 and has caused mass mortalities in farmed fish as well as wild fish all over Japan. Establishment of control measures against KHVD is the urgent theme, and in order to address this issue many scientists belonging to universities, national and prefectural research institutes, and pharmaceutical companies have studied on KHVD and produced excellent results. Not only scientists but also fish farmers are eager to know the results. This context motivated the author to organize a symposium in Mie. The symposium focused on the up-to-date results of studies on KHVD: KHV genome analysis, methods to inactivate KHV in the environmental water, ways to coexist with KHV, development of a delivery system for oral vaccination using liposome, and successful oral vaccination with liposomes entrapping KHV antigens.

Key words: koi herpesvirus, genome analysis, oral vaccination, disinfection, KHV