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NO46 - 1 (2011)

Genetic Determinants of Virulence in the Marine Fish Pathogen Vibrio anguillarum

<>Hiroaki Naka* and Jorge H. Crosa<>Department of Molecular Microbiology and Immunology, Oregon Health and Science University,
Portland, Oregon 97239, USA
(Received December 15, 2010)

ABSTRACT―One of the most studied fish pathogens is Vibrio anguillarum. Development of the genetics and biochemistry of the mechanisms of virulence in this fish pathogen together with clinical and ecologic studies has permitted the intensive development of microbiology in fish diseases. It is the intention of this review to compile the exhaustive knowledge accumulated on this bacterium and its interaction with the host fish by reporting a complete analysis of the V. anguillarum virulence factors and the genetics of their complexity.

Key words: Vibrio anguillarum, virulence factor, genetics

Long-term Culture of Japanese Flounder Paralichthys olivaceus Leukocytes

<>Tomoki Ozaki, Hitoshi Hatakeyama, Shinpei Wada and Osamu Kurata*<>Laboratory of Fish Diseases, Department of Veterinary Science, Nippon
Veterinary and Life Science University, Tokyo 180-8602, Japan
(Received December 22, 2010)

ABSTRACT―We developed a long-term culture system for Japanese flounder Paralichthys olivaceus leukocytes supported by JFF07-1 feeder cells established from Japanese flounder fin tissue. Kidney leukocytes were seeded onto a monolayer of the feeder cells in enriched RDF medium supplemented with 20% fetal bovine serum and 2.5% flounder serum. Several colonies adhered to the feeder cells after 7 days of cultivation, demonstrating leukocyte proliferation. Increasing numbers of floating cells, which signified colony growth, were observed as the length of the culture period increased. The optimum culture conditions consisted of an incubation temperature of 25ーC, the addition of 2.5% flounder serum to the medium and the inoculation of kidney leukocytes at a density of 2 x 106 cells/mL. The proliferated cells were grouped into three types based on May-Grunwald Giemsa staining: basophilic cytoplasmic cells (65%), neutrophilic cytoplasmic cells (30%) and large cells containing many vacuoles (5%). The cells showed acid-phosphatase activity (90%), peroxidase activity (31%) and non-specific esterase activity (57%). Electron microscopy revealed that many of the cells contained endoplasmic reticula and mitochondria, but not specific granules with the fibrillar structure that characterizes flounder granulocytes. A monocyte lineage thus appeared to be the dominant population among the proliferated cells in the culture system. The composition of growing cells was also kept after 20 passages.

Key words: feeder cell, intracellular enzyme, kidney leukocyte, monocyte, Paralichthys olivaceus

Immunoprotection of Japanese Flounder Paralichthys olivaceus by Recombinant Vibrio anguillarum Hemolysin

<>Hong-Bin Wang1,2, Yu-Liang Jiao2*, Jia-Tao Xu2, Xin-Shu Li2, Bing Xu2, Dan Dong2 and Bin-Lun Yan1<>1Jiangsu Key Laboratory of Marine Biotechnology, Lianyungang, 222005, People's Republic of China
2College of Marine Sciences, HuaiHai Institute of Technology, Lianyungang, 222005, People痴
Republic of China
(Received May 11, 2010)

ABSTRACT―The gene vah4 encoding Vibrio anguillarum hemolysin was cloned and hetelogously expressed. The purified recombinant VAH4 protein was then injected into Japanese flounder Paralichtys olivaceus as an immunogen. Activities of superoxide dismutase, catalase, alkaline phosphatase and acid phosphatase in liver became higher in the immunized group. However, these improvements vanished in 72 h after the injection. VAH4-specific antibodies were detected at a titer of 1:10,240 by ELISA on the 10th day after the injection. In a separate experiment, fish were immunized with VAH4 twice and challenged with V. anguillarum. The immunization with the VAH4 protected fish from V. anguillarum infection.

Key words: Vibrio anguillarum, vah4 gene, hemolysin, Paralichthys olivaceus

Detection of Japanese Flounder Antibody against Fimbrial Major Protein of Edwardsiella tarda

<>Takamitsu Sakai1*, Yasuyuki Miyoshi3, Tomomasa Matsuyama2, Chihaya Nakayasu1, Takashi Kamaishi2, Yutaka Fukuda3 and Takaji Iida2<>1Tamaki Station, National Research Institute of Aquaculture, Fisheries Research Agency,
Mie 519-0423, Japan
2National Research Institute of Aquaculture, Fisheries Research Agency, Mie 516-0193, Japan
3Fisheries Research Institute, Oita Prefectural Agriculture, Forestry and Fisheries Research Center,
Oita 879-2602, Japan
(Received October 21, 2010)

ABSTRACT―A recombinant fimbrial protein of Edwardsiella tarda was designed to detect the specific antibody in Japanese flounder Paralichthys olivaceus. The specific antibody was detected from the fish intraperitoneally injected with E. tarda expressing the fimbria on LB agar containing 3% NaCl, but not from those injected with E. tarda without the fimbria, Streptococcus iniae, S. parauberis or viral hemorrhagic septicemia virus. In a flounder farm suffered with edwardsiellosis, the antibody-positive fish increased in ration with the occurrence of mortality. These results suggested that E. tarda with the fimbria was important for epidemics of edwardsiellosis at flounder farms.

Key words: Edwardsiella tarda, fimbrial protein, immuno-dot-blot test, Paralichthys olivaceus

Pathogenic Potential of Edwardsiella tarda Strains Isolated from Turbot

<>Nuria Castro*, Beatriz Magarinos, Soledad Nunez, Juan Luis Barja and Alicia E. Toranzo<>Microbiology and Parasitology Department, Faculty of Biology-CIBUS and Aquaculture Institute,
University of Santiago de Compostela, Santiago de Compostela, 15782 Spain
(Received November 3, 2010)

ABSTRACT―In this study, pathogenicity of Edwardsiella tarda isolates from turbot Scophthalmus maximus as well as other strains from different hosts were tested against turbot, sole Solea senegalensis and sea bass Dicentrarchus labrax. In addition, the influence of challenge route and temperature in the pathogenic potential of E. tarda strains from turbot were also examined. The results obtained showed that E. tarda did not have host specificity in virulence and that the turbot isolates were highly virulent regardless of the inoculation route and temperature. Moreover, inoculation experiments performed in mice suggest that these isolates are virulent for mammals. All these findings suggest that this emergent turbot pathogen constitutes an important risk factor for the marine aquaculture.

Key words: Edwardsiella tarda, Scophthalmus maximus, turbot, virulence

Importance of Gills for Development of Psudotuberculosis at Early Phase of Infection in Amberjack

<>Ichiro Nagano1, Syun-ichirou Oshima1 and Kenji Kawai2*<>1Graduate School of Kuroshio Science, Kochi University, Kochi 783-8502, Japan
2Department of Aquaculture, Kochi University,
Kochi 783-8502, Japan
(Received November 25, 2010)

ABSTRACT―Our previous suggestion that the gill was a key organ for the development of pseudotuberculosis at the early phase of infection was evaluated in natural infection. From naturally infected amberjack Seriola dumerili groups at two fish farms, total 15 dead fish and total 18 apparently healthy fish were collected. In the dead fish, bacterial isolation rate from the kidney and the detection rates of the pathogen in the gills, kidney and spleen by fluorescent antibody method were 87, 100, 100 and 100%, respectively. In the apparently healthy fish, those rates were 6, 72, 33 and 33%, respectively. The quite high detection rate of the bacteria at the gills of the apparently healthy fish may confirm the suggestion.

Key words: Photobacterium damselae subsp. piscicida, pseudotuberculosis, Seriola dumerili

Resistance to Loma salmonae in Rainbow Trout Oncorhynchus mykiss Subsequent to Recovery from an Initial Low-dose Cohabitation Exposure

<>Jennifer E. Harkness* and David J. Speare<>Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward
Island, Charlottetown, Prince Edward Island, C1A 4P3, Canada
(Received December 16, 2010)

ABSTRACT―Protective response was investigated in rainbow trout Oncorhynchus mykiss that had recovered from a primary low-dose cohabitation challenge of Loma salmonae and were re-challenged via a high dose oral exposure under experimental conditions. Compared to uninfected control fish, the previously exposed trout had 82.6% and 86.0% fewer xenomas, and many of the recovered fish (47% and 55%) showed no signs of infection whereas 95% and 100% of the control fish developed xenomas on their gills in two experimental tanks. From the results we conclude that rainbow trout develop protective immunity following low-dose exposure to L. salmonae acquired through cohabitation. The relevance of this with respect to unexpected patterns of disease presentation at commercial salmon farming sites is discussed.

Key words: Loma salmonae, immunity, Oncorhynchus mykiss, rainbow trout, cohabitation

Development of a Sensitive Method for the Detection of Young Larvae of the Parasitic Pycnogonid Nymphonella tapetis in Manila Clam Ruditapes philippinarum

<>Tomoyoshi Yoshinaga1*, Yutaka Kobayashi2, Mitsuharu Toba2 and Yoshifumi Miyama2<>1Department of Aquatic Bioscience, Graduate School of Agricultural Life Sciences, The University of Tokyo,
Tokyo 113-8657, Japan
2Tokyo Bay Fisheries Laboratory, Chiba Prefectural Fisheries Research Center,
Chiba 293-0042, Japan
(Received December 21, 2010)

ABSTRACT―We developed a sensitive method for the detection of Nymphonella tapetis (sea spider) larvae in Manila clam Ruditapes philippinarum. The gills were removed from infected clams and lysed in 2N NaOH at 60ーC. The lysate was centrifuged and the pellet was stained in 0.2% Uvitex 2B solution and observed under a fluorescent microscope. Using this method, we were easily able to enumerate the larvae. The majority of larvae were found in the gills, which were indicated as the primary site of infection for N. tepetis larvae.

Key words: Nymphonella tapetis, Ruditapes philippinarum, detection, Uvitex 2B