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NO38 - 4

A Review: Gaps in Our Knowledge on Myxozoan Parasites of Fishes

<>Hiroshi Yokoyama<>Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
(Received June 23, 2003)
ABSTRACT祐ince the epoch-making discovery of the life-cycle of Myxobolus cerebralis by Wolf and Markiw (1984), studies on myxozoan parasites of fishes have continued to progress rapidly. Molecular analyses of myxozoan SSUrDNA have been successful in clarifying its systematic position not as protozoans but as metazoans. However, there remain gaps in our understanding of their biology and pathology. Alternate myxosporean and actinosporean stages in the life-cycles of more than 25 species of freshwater myxozoan have been demonstrated, but many unresolved questions remain, e.g., the relationship between the parasite and its alternate invertebrate hosts and the mode of transmission in marine myxozoans. These unsolved questions have made it difficult to design an effective control strategy for myxozoan diseases. Recent studies on countermeasures have been directed towards the introduction of resistant fish strains and controlling oligochaete populations.

Key words: Myxozoa, myxosporean, actinosporean, parasite, taxonomy, biology, control strategy

The Kinetics of Leukocytes and Histopathology of Red Sea Bream in Artificial Infection of Edwardsiella tarda

<>Shiro Toida1, Kinya Kanai2 and Kazuma Yoshikoshi2*<>1Graduate School of Marine Science and Engineering, Nagasaki University, Nagasaki 852-8521, Japan
2Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan
(Received March 3, 2003)
ABSTRACT裕o explain the involvement of leukocytes in defense system of the red sea bream Pagrus major, the kinetics of leukocytes in the peripheral blood and the sequential histopathological changes in the inflammatory foci during artificial infection of Edwardsiella tarda were investigated. In peripheral blood, the lymphocyte count declined and the granulocyte count increased at 1 day post-inoculation (dpi). The monocyte count started to increase from 5 dpi to go to a peak at 15 dpi. In inflammatory foci, the formation of granulomas was characteristic. In the foci, prior to the formation of granulomas, lymphocytes and granulocytes infiltrated, followed by monocytes infiltration. There was strong correlation between the progression of granulomas and the kinetics of leukocytes in the peripheral blood.

Key words: leukocyte, histopathology, Edwardsiella tarda, inflammation, kinetics, Pagrus major, red sea bream, granulocyte

Histopathological Changes Probably Associated with a Virus in Larval and Juvenile Marbled Sole Pleuronectes yokohamae

<>Tadashi Isshiki1*, Taizou Nagano1, Masaaki Abe1,3 and Teruo Miyazaki2<>1Kagawa Prefectural Fisheries Experiment Station, Takamatsu, Kagawa 761-0111, Japan
2Faculty of Bioresouces, Mie University, Tsu, Mie 514-8507, Japan
3Present address: Fisheries Division, Agriculture and Fisheries Department,
Kagawa Prefectural Government, Takamatsu, Kagawa 760-8570, Japan
(Received May 23, 2003)
ABSTRACT裕he seed production of marbled sole Pleuronectes yokohamae has been attempted at a hatchery in Kagawa Prefecture, Japan since 1989, while a subacute disease with a considerable amount of mortality occurred in larval and juvenile fish (24・9 days old) in 1989, 1990, 1991, 1997 and 2001. Diseased fish displayed edema, an abdominal distension due to ascites, and exophthalmia. The outbreak of the disease usually continued for 1 month or longer and the cumulative mortality was often over 40%. The evident histopathological features were the formation of multinuclear syncytia and following necrosis in the liver, renal tubules, pancreas and intestinal epithelium, and hemorrhage in the brain, choriocapillaries, stomach and skin. Electron microscopy of affected hepatocytes revealed formation of inclusion bodies consisting of crystalline arrays of virus particles (75・0 nm in diameter). These results suggest that the pathognomonic sign of the present disease is necrosis accompamied by the syncytial formation which would be associated with a virus infection.

Key words: Pleuronectes yokohamae, marbled sole, histopathology, syncytial formation, necrosis

Virulence Properties of a Newly Identified Species Aeromonas sp. T8 Group Isolated from EUS-affected Fish

<>M. M. Rahman1, T. Somsiri2, K. Tajima1* and Y. Ezura1<>1Laboratory of Microbiology, Graduate School of Fisheries Sciences, Hokkaido University,
Hakodate, Hokkaido 041-8611, Japan
2Aquatic Animal Health Research Institute, Kasetsart University Campus,
Jatujak, Bangkok 10900, Thailand
(Received May 30, 2003)
ABSTRACT裕he present study was conducted to determine the virulence and virulence properties of 4 bacterial strains belonging to a newly identified species Aeromonas sp. T8 group, isolated from epizootic ulcerative syndrome (EUS)-affected fish of Southeast Asian countries. Virulence of a representative strain T8 to fish was investigated through intramuscular injection to silver barb Barbodes gonionotus, striped snakehead Channa striatus and tilapia Oreochromis sp., where the LD50 value was determined to be 1.5 ララ 105, 3.2ラ105 and 2.9ラ105 CFU/fish, respectively.
However, all strains were found non-virulent for mice upon intraperitoneal or intravenous injections at a concentration of 3 ラ 107 CFU/mouse. Live bacterial cells of these strains showed lipase, protease and hemolytic activities in agar plate assays. The hemolytic activity of the extracellular products (ECPs) was found to be higher for fish blood cells than for mammalian blood cells. Heat stability, EDTA and PMSF sensitivities of b-hemolytic activity of the ECPs differed from that of A. hydrophila and A. caviae reference strains. ECP of only one strain (P2) showed cytotoxicity against both fish (RTG-2, FHM, EPC, BF-2 and SNN-1) and human (HeLa) cell lines, while none of the strains exhibited enterotoxicity to suckling mice. All of the strains also failed to amplify aerolysin and cytolytic enterotoxin (AHCYTOEN) genes by PCR.

Key words: Aeromonas sp. T8, virulence factor, protease, hemolysin, cytotoxin, enterotoxin, EUS

DNA Probes for Detection of Marteilioides chungmuensis from the Ovary of Pacific Oyster Crassostrea gigas

<>Naoki Itoh1, Tadashi Oda2,3, Tomoyoshi Yoshinaga1 and Kazuo Ogawa1*<>1Laboratory of Fish Diseases, Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
2Fisheries Experiment Station of Okayama Prefecture, Ushimado,
Okayama 701-4303, Japan
3Present address: Farming Fisheries Center of Okayama Prefecture, Ushimado,
Okayama 701-4303, Japan
(Received June 2, 2003)
ABSTRACT裕he detection of the paramyxean parasite Marteilioides chungmuensis has been achieved only by using standard histological techniques or gross observations, which have a limited sensitivity for early diagnosis. In the present study, small subunit ribosomal DNA of the parasite was sequenced, and polymerase chain reaction (PCR) primers designed accordingly. It has been demonstrated that both the PCR and in situ hybridization techniques are specific for M. chungmuensis. Moreover, compared to traditional histological techniques and gross observation, the PCR diagnosis showed a far greater sensitivity. These new diagnostic tools based on molecular techniques can also be used for the elucidation of the parasite痴 life cycle and the identification of possible intermediate hosts.

Key words: Marteilioides chungnmuensis, parasite, detection, Crassostrea gigas, Pacific oyster, PCR, in situ hybridization, Paramyxea,

Water-soluble Adjuvants Enhance the Protective Effect of Flavobacterium psychrophilum Vaccines in Ayu Plecoglossus altivelis

<>M. Habibur Rahman1, Mitsuru Ototake1* and Teruyuki Nakanishi2<>1Bio-defence group, Aquatic Animal Health Division, National Research Institute of Aquaculture,
Tamaki, Mie 519-0423, Japan
2Laboratory of Fish Pathology, Department of Veterinary Medicine,
Nihon University, Fujisawa, Kanagawa 252-8510, Japan
(Received June 19, 2003)
ABSTRACT唯acterial coldwater disease is considered to be one of the most serious diseases in ayu Plecoglossus altivelis in Japan. We evaluated the efficacy of three highly water-soluble adjuvants (Montanidae IMS-1311, IMS-1312 and IMS-2212) combined with formalin-killed bacterin of Flavobacterium psychrophilum (FKB) in ayu, in comparison with a minimally water-soluble adjuvant (Montanidae ISA-763A). The retention period and toxicity of these adjuvants in ayu were also examined. Experimental challenge 4 wk after vaccination showed that FKB adjuvanted with IMS-1311 and IMS-1312 had higher potency than FKB alone and significantly (p < 0.05) higher potency than the control with sterile distilled water. Residues of the highly water-soluble adjuvants in ayu remained for shorter periods than that of the minimally water-soluble adjuvant, although the former adjuvants were acutely toxic at doses higher than 25 µL/fish(1.7 ア 0.5 g). The high level of protection, short retention period and lack of toxicity indicate that a dose of 5 µL of IMS-1311 or IMS-1312 combined with 5 µL of FKB makes a promising vaccine against bacterial coldwater disease in ayu.

Key words: adjuvant, vaccine, bacterial coldwater disease, Flavobacterium psychrophilum, toxicity, Plecoglossus altivelis, ayu

Comparison of Genomic Sequence of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) between Taiwan and Other Geographical Isolates

<>Hui-Lan Hsia, Li-Li Chen, Shao-En Peng, Hon-Tsen Yu, Chu-Fang Lo, Guang-Hsiung Kou*<>Graduate Institute of Zoology, National Taiwan University,
Taipei 106, Taiwan R.O.C.

(Received May 6, 2003)

ABSTRACT友ive isolates of infectious hypodermal and hematopoietic necrosis virus (IHHNV) (three from Taiwan, one from Thailand and one from Ecuador) were sequenced and compared with two other previously sequenced isolates, one from Hawaii and one from the Gulf of California, Mexico. An unrooted neighbor-joining (NJ) phylogenetic tree analysis and a pairwise comparison both show that the variation among these seven different isolates was low. Despite this sequence similarity, our data suggest that two of the three Taiwan isolates may have been imported from the Americas, while the other isolate and the Thailand isolate may be instances of a local (South East Asian) strain. This has implications for the epidemiology of IHHNV, and suggests that geographic isolation has allowed the virus to evolve into two distinct groups.

Key Words: Infectious hypodermal and hematopoietic
necrosis virus, IHHNV, geographic variation, genomic sequence

First Isolation of Edwardsiella ictaluri from Cultured Striped Catfish Pangasius hypophthalmus in Indonesia

<>Kei Yuasa1, Edy Barkat Kholidin2, Novita Panigoro2 and Kishio Hatai3*<>1Fisheries & Aquaculture International Co., Ltd., No.7
Kohji-machi Bldg, 4-5 Khoji-machi, Chiyoda,
Tokyo 102-0083, Japan
2Jambi Freshwater Aquaculture Development Center, Sungai Gelam, Kumpeh Ulu,
Muara Jambi, Indonesia
3Division of Fish Diseases, Nippon Veterinary and
Animal Science University, Musashino,
Tokyo 180-8602, Japan

(Received May 8, 2003)

ABSTRACT悠n February 2002, mass mortalities were first observed among striped catfish Pangasius hypophthalmus reared in freshwater ponds of central Sumatra, Indonesia. The affected fish showed pale gills, abdominal swelling and hemorrhage of the skin, as well as swelling of internal organs with white patches. Edwardsiella ictaluri was isolated from the internal organs of the diseased fish. The isolates showed pathogenicity to P. hypophthalmus by an artificial infection. However, the isolate preserved in a semi-solid BHI agar for 6 months lost pathogenicity.

Key words: Edwardsiella ictaluri, Pangasius hypophthalmus, pathogenicity, Indonesia

Virucidal Effect of Disinfectants against Oncorhynchus masou virus (OMV)

<>Shuichi Hatori1, Akira Motonishi2, Toyohiko Nishizawa1 and Mamoru Yoshimizu1*<>1Graduate School of Fisheries Sciences, Hokkaido
University, Hakodate, Hokkaido 041-8611, Japan
2Suwa Branch of Nagano Prefectural Fisheries
Experimental Station, Shimosuwa,
Nagano 393-0034, Japan

(Received May 16, 2003)

ABSTRACT遊irucidal effects of six kinds of disinfectants were examined against Oncorhynchus masou virus (OMV), and infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV) and hirame rhabdovirus (HIRRV). At 15°C for 20 min, minimum concentrations showing 100% plaque reduction of OMV by iodophor, sodium hypochlorite solution, benzalkonium chloride solution, saponated cresol solution, formaldehyde solution and potassium permanganate solution were 40, 50, 100, 100, 3,500 and 16 ppm, respectively. Susceptibility of OMV against these disinfectants was higher than that of IPNV, IHNV and HIRRV, suggesting that the usage of disinfectants against IPNV and/or IHNV is also effective against OMV.

Key words: virucidal effect, Oncorhynchus masou virus, disinfectant, OMV, fish virus

Symposium 撤rogress in Fish and Shellfish Immunology and Contribution to the Fisheries・>

Yukinori Takahashi<>National Fisheries University, Shimonoseki, Yamaguchi 759-6595, Japan
ABSTRACT輸 symposium entitled 撤rogress in fish and shellfish immunology and contribution to the fisheries・was held by the Japanese Society of Fish Pathology in Shimonoseki on 4th October, 2003. This was aimed to have an overview of the progress in fish and crustaceans immunology and its contribution to the development of aquaculture. The symposium was composed of six categories: (1) Prevention of infectious diseases of crustaceans by activation of immunodefense system, (2) Current status and prospects in fish immunology, (3) Immune relevant genes and their functions in fish, (4) Prevention of infectious diseases in fish by inducing cytokines, (5) Current status and prospects of development of fish vaccines, and (6) Potential application of recombinant cytokines and new gene ration vaccine for the control of infectious diseases in domestic animals.
Outbreaks of infectious diseases, causing serious damage to cultured fish and shrimp, have been occurring in Japan. The advanced knowledge in fish and shellfish immunology will contribute to the control of infectious diseases in aquaculture.