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NO53 - 1

Extracellular Proteinases of Miamiensis avidus Causing Scuticociliatosis are Potential Virulence Factors

Yukie Narasaki1,2, Yumiko Obayashi2, Sayami Ito2,3, Shoko Murakami2, Jun-Young Song4, Kei Nakayama1,2,3 and Shin-Ichi Kitamura1,2,3*

1Graduate School of Science and Engineering, Ehime University, Ehime 790-8577, Japan
2Center for Marine Environmental Studies (CMES), Ehime University, Ehime 790-8577, Japan
3Department of Biology, Faculty of Science, Ehime University, Ehime 790-8577, Japan
4Pathology Division, National Fisheries Research and Development Institute, Busan 619-902, Korea

ABSTRACTMiamiensis avidus is the causative agent of scuticociliatosis in various marine fish species. The virulence factors of the parasite have not been identified, so far. In this study, we examined M. avidus extracellular proteinases (ECPs) as potential virulence factors, using culture supernatants as an ECPs source. We investigated the substrate specificity of ECPs using artificial peptides, and the cytotoxicity of the ECPs was examined using CHSE-214 cells. To elucidate the role of ECPs in ciliate growth, M. avidus was cultured on CHSE-214 cells in the presence of proteinase inhibitors. We detected proteinase activities from the supernatant of M. avidus. Viable CHSE-214 cells decreased significantly in number, when incubated in a medium supplemented with the culture supernatant of M. avidus. The growth of ciliates on CHSE-214 cells was delayed in the presence of PMSF (serine proteinase inhibitor) and E-64 (cysteine proteinase inhibitor). These results suggested that the culture supernatant contained ECPs showing cytotoxicity, and the proteinases facilitated nutrient uptake by the ciliates. Thus, ECPs may be responsible for virulence factors of M. avidus.

Key words: scuticociliatosis, Miamiensis avidus, extracellular proteinase, virulence factor

Percentage Dynamics of CD4-1+ and CD8α+ T Cells in Different Tissues at Various Developmental Stages in Ginbuna Crucian Carp Carassius auratus langsdorfii

Mohamed A. Khallaf1, Shino Ogawa2, Hideaki Toda2, Yuta Matsuura2, Ryuichiro Miyazawa2 and Teruyuki Nakanishi2*

1Faculty of Veterinary Medicine, Sadat City University, Sadat City 32897, Egypt
2Department of Veterinary Medicine, Nihon University, Kanagawa 252-0880, Japan

ABSTRACT―Determination of numbers of T cell subsets in tissues is important to assess the functional maturity of the immune system. The distribution of T cells in lymphoid tissues during ontogenic development has been examined in several fish species using pan-T cell markers. However, information on the age-related changes in the number of T cell subsets in tissues is limited due to the lack of antibodies against CD4 and CD8 in fish. In the present study we examined the percentage dynamics of CD4-1+ and CD8α+ cells in various tissues accompanied with age of ginbuna crucian carp using flow cytometry. We found rapid increases in both CD4-1+ and CD8α+ T cells up until 6 months post-hatch (mph). CD4 and CD8 double-positive cells (DP cells) are present only in the thymus and DP cells increased, while double negative cells decreased, in fish up to 3-6 mph. The percentages of CD4-1+ T cells were always higher than those of CD8α+ T cells in the various tissues examined, except for the thymus and intestine. In contrast, significantly higher percentages of CD8α+ T cells than CD4-1+ T cells were found in the intestine during the period between 6 mph and 2 years post-hatch.

Key words: CD4, CD8, T lymphocytes, ontogeny, Carassius auratus langsdorfii, ginbuna crucian carp, thymus, flow cytometry

Variation in the Integration Site for Capsule Gene Cluster in the Genome among Strains of Lactococcus garvieae

Kinya Kanai1*, Toshio Honma2, Akane Souda2, Kimihiro Shutou2 and Yukitaka Sugihara3

1Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki 852-8521, Japan
2Bio Science Co, Ltd, Tokushima 779-1292, Japan
3Nagasaki Prefectural Institute of Fisheries, Nagasaki 851-2213, Japan

ABSTRACTLactococcus garvieae is known as a pathogen of freshwater and marine fish species worldwide. L. garvieae isolates of serotype I have been divided into two serological phenotypes, namely KG- and KG+, which are differentiated by the presence or absence of polysaccharide capsule, and a phenotypic change from KG- to KG+ occurs during repeated subculturing. When we subcultured laboratory collections of L. garvieae KG- strains repeatedly, they were divided into two groups. One group consisted of the strains which changed phenotypically after a relatively small number of times of subculturing, and the other group consisted of those which hardly changed. Genetic analyses revealed that the capsule gene cluster of the strains in the former group was integrated in a plasmid (CPS plasmid) and that in the latter group it was integrated in the chromosome or in both CPS plasmid and chromosome simultaneously. In the present study, we found three novel CPS plasmids. These plasmids were similar in structure to the CPS plasmid pBSLG13015 which was previously found in L. garvieae filefish isolates. In addition, we found a novel integration site of chromosomal capsule gene cluster. The majority of strains isolated before 1991 were those whose capsule gene cluster was integrated in the CPS plasmids.

Key words: Lactococcus garvieae, capsule gene cluster, CPS plasmid, KG-, KG+, phenotypic change

Comparative Proteomic Analysis Between Virulent and Less Virulent Strains of Edwardsiella ictaluri Isolated from Ayu Plecoglossus altivelis

Takamitsu Sakai1*, Tomonori Kuwada2, Yoshinori Muto2, Tomokazu Takano3, Kei Yuasa3 and Norihisa Oseko3

1Tamaki Laboratory, National Research Institute of Aquaculture, Fisheries Research and Education Agency, Mie 519-0423, Japan
2Gifu Prefectural Research Institute for Fisheries and Aquatic Environments, Gifu 501-6021, Japan
3Nansei Main Station, National Research Institute of Aquaculture, Fisheries Research and Education Agency, Mie 516-0193, Japan

ABSTRACT―Outbreaks of disease caused by the infection of Edwardsiella ictaluri in ayu Plecoglossus altivelis have been reported in rivers in Japan since 2007. In this study, we performed comparative proteomic analysis between the virulent strain FPC1091 and the less virulent strain FPC1214 to identify virulence factors of E. ictaluri to ayu. In the experimental infection, cumulative mortalities of the ayu intraperitoneally injected with FPC1091 (1.2 × 107 CFU/fish) and FPC1214 (2.2 × 107 CFU/fish) were 100% and 26.7%, respectively. The genetic fingerprint profile of FPC1214 was identical to that of FPC1091 in the amplified-fragment length polymorphism (AFLP) analysis. The analysis of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry between FPC1091 and FPC 1214 revealed 23 proteins that were either specific to or produced in greater amounts in the virulent strain FPC1091. These proteins included components of the type III and type VI secretion systems, and the proteins that seem to be involved in the resistance against oxidative stress by host phagocytic cells, suggesting that these proteins are the virulence factors of E. ictaluri to ayu.

Key words: Edwardsiella ictaluri, Plecoglossus altivelis, 2D-PAGE, Proteomic analysis

Development of Anti-atypical Aeromonas salmonicida Monoclonal Antibodies for Diagnosis of "New Ulcer Disease" in Koi Carp

Tomonori Somamoto1*, Shinpei Maruyama1, Takahiro Nagasawa1, Miki Nakao1, Atsushi Sato2, Hajime Hatta3, Mitsuru Sato4, Yukie Murakami-Yamaguchi5, Kumiko Kizu-Mori5, Yuki Hirakawa3 and Hiroshi Narita3

1Laboratory of Marine Biochemistry, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan
2Kyorin Co. Ltd., Hyogo 670-0902, Japan
3Department of Food Nutrition, Kyoto Women's University, Kyoto 605-8501, Japan
4Animal Bioregulation Unit, Division of Animal Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Ibaraki 305-8634, Japan
5Kyoto College of Nutritional & Medical Sciences, Kyoto 616-8376, Japan

ABSTRACT―To develop an accurate diagnosis of "new ulcer disease" in koi carp, we produced four monoclonal antibodies (mAbs) against a strain of atypical Aeromonas salmonicida isolated from koi carp. These mAbs did not cross-react with an isolate of atypical A. salmonicida from Japanese flounder and other pathogenic bacteria. Re-isolation from artificially infected koi carp was achieved by selecting the blue colonies on agar medium containing Coomassie brilliant blue, and some of the colonies were detected by immunofluorescent staining using the mAbs. These results suggested that the mAbs can distinguish atypical A. salmonicida from koi carp from resident aeromonads.

Key words: atypical Aeromonas salmonicida, monoclonal antibody, "new ulcer disease", koi carp

Asymptomatic Infection of Renibacterium salmoninarum in Hatchery-reared Juvenile Chum Salmon Oncorhynchus keta Resulted in Mass Mortalities after Long-term Rearing

Kunio Suzuki1*, Shinya Mizuno1, Yoshitomo Katsumata1, Naoyuki Misaka2, Mahito Miyamoto1 and Yoshitaka Sasaki1

1Salmon and Freshwater Fisheries Research Institute, Hokkaido Research Organization, Hokkaido 061-1433, Japan
2Mariculture Fisheries Research Institute, Hokkaido Research Organization, Hokkaido 051-0013, Japan

ABSTRACT―Mass mortalities due to bacterial kidney disease (BKD) occurred after 10-15 months rearing in two groups of chum salmon Oncorhynchus keta which had been raised from asymptomatically infected eggs and fry transferred from private hatcheries. Mortality reached 56.1% and 19.0% in the two groups, respectively, 146 days after the first death. A Renibacterium salmoninarum-specific gene was detected from various organs of the subclinical and dead fish. In the dead fish, the number of gene copies was higher than those in the subclinical fish in all the eight organs examined. These results suggested that the bacterium distributed in the bodies of asymptomatic fish proliferated systemically, resulting in an outbreak of BKD.

Key words: bacterial kidney disease, Renibacterium salmoninarum, subclinical infection, Oncorhynchus keta, outbreak

Mass Mortalities of Goldfish Carassius auratus Infected with Clinostomum Metacercariae, Associated with Elevated Water Temperature

Shinya Yasumoto*, Tsukasa Kabayama, Masakazu Kondo and Yukinori Takahashi

Department of Applied Aquabiology, National Fisheries University, Japan Fisheries Research and Education Agency, Yamaguchi 759-6595, Japan

ABSTRACT―Mass mortalities of goldfish Carassius auratus occurred at some farms and wholesalers in Japan, where fish were held at around 33°C to control herpesviral hematopoietic necrosis. All dead fish showed light to moderate Clinostomum infection. Cysts containing metacercariae were accompanied by hemorrhage and congestion, and metacercariae were activated and excysted during the high temperature water treatment. When infected fish were experimentally treated at 20°C, 31°C, 33°C and 35°C, mortality rates reached to 0%, 5%, 40% and 100%, respectively. The experimental results indicated that the elevation of water temperature against the virus infection caused the mass mortalities in Clinostomum-infected goldfish.

Key words: goldfish, Clinostomum, mass mortality, herpesviral hematopoietic necrosis, high temperature water treatment