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NO41 - 1 (2006)

Effect of Iron on Resuscitation of Tenacibaculum sp., the Causative Bacterium of Spotting Disease of Short-Spined Sea Urchin Strongylocentrotus intermedius, from the Viable but Non-culturable (VBNC) State

<>Tomohisa Yamase, Tomoo Sawabe, Kensi Kuma and Kenichi Tajima*<>Graduate School of Fisheries Sciences, Hokkaido University,
Hakodate, Hokkaido 041-8611, Japan
(Received July 4, 2005)
ABSTRACT裕enacibaculum sp., the causative bacterium of spotting disease of short-spined sea urchin Storongylocentrotus intermedius, has been known to enter into the viable but non-culturable (VBNC) state in 75% Herbst's artificial seawater at 5゚C. Previous studies indicated that elevation of water temperature or addition of iron induced the resuscitation of the bacterium from the VBNC state. The present study revealed that the resuscitation period from the VBNC state was further prolonged when Tenacibaculum sp. in the VBNC state was resuscitated under the presence of iron, catalase and the homogenate of the sea urchin. Environmental waters around a sea urchin hatchery in Hokkaido, Japan often contained iron at concentrations over 0.34 mg/L in summer. The infection experiment showed that the VBNC cells activated with iron produced the spotting disease to healthy sea urchins. These results suggest that the VBNC cells of Tenacibaculum sp. resuscitated during summer under iron-rich environment are associated with outbreaks of the disease.

Key words: spotting disease, Strongylocentrotus intermedius, short-spined sea urchin, Tenacibaculum sp., VBNC, resuscitation, iron

Atypical Aeromonas salmonicida Infection in Cultured Marbled Sole Pleuronectes yokohamae

<>Akira Kumagai1*, Kouiti Sugimoto2, Daisuke Itou2,3, Takashi Kamaishi4, Satoshi Miwa5 and Takaji Iida4<>1Miyagi Prefectural Freshwater Fisheries Experimental Station,Taiwa,
Miyagi 981-3625, Japan
2Miyagi Prefectural Fish Farming Center, Ishinomaki,
Miyagi 986-2402, Japan
3Miyagi Prefecture Fisheries Research and Development Center, Ishinomaki,
Miyagi 986-2135, Japan
4National Research Institute of Aquaculture, Fisheries Research Agency,
Minami-Ise, Mie 516-0193, Japan
5Inland Station, National Research Institute of Aquaculture, Fisheries Research
Agency, Tamaki, Mie 519-0423, Japan
(Received October 13, 2005)
ABSTRACT悠n spring of 2003, a mass mortality occurred in a population of marbled sole Pleuronectes yokohamae reared at a hatchery of Miyagi Prefecture, Japan. Since the first case, outbreak of the disease has been repeated every year in the hatchery. The affected fish weighing 1・ g showed hemorrhagic lesions on the body surface. The maximum cumulative mortality reached more than 80%. The prominent histopathological feature was hemorrhage and necrosis in the gill, muscle, liver, heart and kidney associated with extensive bacterial multiplication. A single species of bacterium was isolated from the kidneys of the diseased fish using trypticase soy agar containing 1% NaCl at 20゚C. The isolates were identified as atypical Aeromonas salmonicida by biochemical, serological and molecular phylogenetic analyses. Experimental infections confirmed that an isolate was highly pathogenic not only to marbled sole, but also to Japanese flounder Paralichthys olivaceus and spotted halibut Verasper variegatus, with LD50 of less than 102 CFU/fish (1.4・.8 g body weight) by intramuscular injection.

Key words: atypical Aeromonas salmonicida, Pleuronectes yokohamae, marbled sole

Adhesion of Tenacibaculum sp. to Short-spined Sea Urchin Strongylocentroutus intermedius and Control of Spotting Disease by Inhibiting Adhesion Using Carbohydrate

<>Rumi Taniguchi, Tomoo Sawabe and Kenichi Tajima*<>Graduate School of Fisheries Science, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan
(Received October 20, 2005)
ABSTRACT邑e examined the adhesion of Tenacibaculum sp., the causative bacterium of spotting disease of short-spined sea urchin Stronglycentroutus intermedius, to the host. The number of adhesive cells of Tenacibaculum sp. strain F-2 isolated from diseased sea urchin was about 30 times more than those of two stains of non-pathogenic marine Cytophaga sp. isolated from healthy sea urchins. The adhesion of Tenacibaculum sp. F-2 to the sea urchins was inhibited by about 90% when the sea urchins were pre-treated with 0.1% D-galactose or D-xylose for 1 h. With this treatment, all sea urchins remained asymptomatic and were still alive at 7th day after being immersed with 106 or 107 CFU/mL Tenacibaculum sp. F-2 for 1h at 23゚C, while mortality of control reached 100%. These results indicate that carbohydrate treatment of the sea urchin is useful to control the disease.

Key words: Strongylocentrotus intermedius, spotting disease, Tenacibaculum sp., adhesion, carbohydrate inhibition, disease control, short-spined sea urchin

Sensitive and Rapid Detection of Koi Herpesvirus by LAMP Method

<>Manabu Yoshino*, Hajime Watari, Tadashi Kojima and Masanari Ikedo<>Eiken Chemical Co., Ltd. Biochemical Research Laboratory, Nogi-Machi,
Tochigi 329-0114, JAPAN
(Received December 2, 2005)
ABSTRACT邑e designed a LAMP (loop-mediated isothermal amplification) primer set comprised with six primers from koi herpesvirus (KHV, CyHV-3) specific PCR amplicon (accession number: AF411803) and developed a rapid and sensitive detection method for KHV. The target sequence was amplified under isothermal condition at 65゚C within 60 min, and the detection limit was at least six copies of the plasmid inserted KHV specific sequence. Cross-reactivity with other fish-pathogenic viruses and bacteria was not observed. The reaction was monitored in real-time by an increase in the turbidity of a large amount of by-product, pyrophosphate. As an alternative method, a visual detection at end-point of reaction could be achieved by an increase of turbidity in reaction mixture with naked eyes. The LAMP method was applicable to amplify the target sequence from crudely extracted samples of gills by a simple procedure.

Key words: koi herpesvirus, loop-mediated isothermal amplification, KHV, LAMP, detection

Induction of Hemagglutinating Activity of Edwardsiella tarda by Sodium Chloride

<>Hideki Yasunobu1, Yoko Arikawa2, Kaori Furutsuka-Uozumi1,5, Munehiko Dombo3, Takaji Iida4, Mahmoud Mostafa Mahmoud2, Jun Okuda2 and Toshihiro Nakai2*<>1Fisheries Technology Institute, Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries, Akashi, Hyogo 674-0093, Japan
2Graduate School of Biosphere Science, Hiroshima University,
Higashi-Hiroshima 739-8528, Japan
3Research and Development Center, Unitika Ltd., Uji, Kyoto 611-0021, Japan
4National Research Institute of Aquaculture, Fisheries Research Agency,
Minami-Ise, Mie 516-0193, Japan
5Present address; Fishing Port Management Division, Agriculture, Forestry &
Fisheries Bureau, Hyogo Prefectural Government, Kobe 650-8567, Japan
(Received December 28, 2005)
ABSTRACT裕he hemagglutinating activity (HA) of Edwardsiella tarda, which had been isolated from cultured fish and culture environments, was investigated in relation to NaCl concentration of the growth medium. E. tarda cells were cultured in a peptone-yeast extract broth supplemented with 3% NaCl (3%-NaCl culture) and without NaCl (0%-NaCl culture). Hemagglutination assays with guinea pig erythrocytes classified the strains into three HA patterns. Seventeen strains exhibited HA only with the 3%-NaCl culture (type A). A more frequent type (35 strains) displayed HA in both 0%- and 3%- NaCl cultures but the 3%-NaCl culture showed higher HA activity than the 0%-NaCl culture (type B). No HA was detected in both cultures of the other three strains (type C). The NaCl-induced HA was well correlated with the expression of a 19.3 kDa protein, a fimbrial major subunit (FimA). Infection experiments with a selected strain (type A) of E. tarda revealed that the 3%-NaCl culture was more virulent to Japanese flounder Paralichthys olivaceus than the 0%-NaCl culture, when fish were challenged by an oral route. This induction of the fimbrial protein under higher NaCl concentration may play an important role in the virulence of E. tarda in marine environments.

Key words: hemagglutination, Edwardsiella tarda, pathogenicity, fimbriae, Paralichthys olivaceus

Lagenidium thermophilum Isolated from Eggs and Larvae of Black Tiger Shrimp Penaeus monodon in Thailand

<>Yasunori Muraosa1, Ong-ard Lawhavinit2 and Kishio Hatai1*<>1Division of Fish Diseases, Nippon Veterinary and Animal Science University,
Tokyo 180-8602, Japan
2Faculty of Veterinary Medicine, Kasetsart University,
Bangkok, Thailand
(Received January 15, 2006)
ABSTRACT輸 fungal infection was found in eggs and larvae of black tiger shrimp Penaeus monodon at a hatchery in Chachensao Province, Thailand in August 2000. Fungi were isolated from eggs and larvae with fungal infection, and studied on the morphological and biological characteristics. When it was transferred from PYGS broth to artificial seawater, discharge tubes developed from the mycelia, and a vesicle for zoospore formation was produced at the top of each
discharge tube. The characteristic feature of an asexual reproduction of the fungus was that zoospores swam away in seawater after the vesicle separated from the discharge tube. Based on these morphological characteristics, the fungus was identified as Lagenidium thermophilum.
Some biological characteristics of the selected isolate NJM 0031 were compared with the other species in the genus Lagenidium isolated from some crustaceans. As a result, the isolate NJM 0031 showed similar characteristics to those of L. thermophilum ATCC 200318 isolated from mangrove crab Scylla serrata. The isolate was demonstrated to be pathogenic to larvae of black tiger shrimp by artificial infection. This is the first report of L. thermophilum infection in black tiger shrimp in Thailand.

Key words: Lagenidium thermophilum, fungal infection, Penaeus monodon, black tiger shrimp