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NO40 - 2

Discrepancy between in vitro Non-specific Defense Activities of Peripheral Blood Phagocytes and Resistance against Vibriosis in Rainbow Trout under Temperature Stress

<>Shuji Aoshima1*, Yasuharu Ishitaka2 and Nobuaki Okamoto2<>1Fuji Trout Culture Brunch, Shizuoka Prefectural Fisheries Experiment Station, Fujinomiya,
Shizuoka 418-0108, Japan
2Faculty of Marine Science, Tokyo University of Marine Science and Technology,
Minato, Tokyo 108-8477, Japan
(Received December 22, 2004)
ABSTRACT悠n this study, we examined the effect of temperature shock on non-specific defense responses and disease resistance of rainbow trout Oncorhynchus mykiss. Water temperature was changed from 10゚C to 23゚C in 24 h, maintained at 23゚C for 7 days, and again decreased to 10゚C in 1 h. Percentage of phagocytosing phagocytes, NBT reduction activity, potential killing activity and plasma bacteriolysis activity were measured before, during and after the temperature changes. Just before the temperature was decreased to 10゚C, fish were challenged with Vibrio anguillarum. Exposure to the temperature stress significantly increased mortality caused by vibriosis, but the percentage of phagocytosing phagocytes and potential killing activity significantly increased. The present data shows that increased non-specific defense responses in vitro do not always reflect increased disease resistance.

Key words: Oncorhynchus mykiss, Vibrio anguillarum, phagocyte activity, disease resistance, temperature stress, rainbow trout

An Enzyme Linked Immunosorbent Assay (ELISA) for Detection of Antibodies to the Koi Herpesvirus (KHV) in the Serum of Koi Cyprinus carpio

<>Mark A. Adkison1, Oren Gilad2 and Ronald P. Hedrick1*<>1Department of Medicine and Epidemiology, School of Veterinary Medicine,
University of California, Davis, CA 95616, USA
2Cancer Center, University of California, Sacramento, CA 95817, USA
(Received January 26, 2005)
ABSTRACT輸n enzyme linked immunosorbent assay (ELISA) detected the presence of anti-koi herpesvirus (KHV) antibodies in the serum of koi or colored carp (Cyprinus carpio) following either natural or experimental exposures to KHV. Concentrations of anti-KHV antibodies were detected at serum dilutions as great as 1:62,500 in a population of koi kept in virus-free water for 1 year following a naturally occurring outbreak due to KHV. At serum dilutions less than 1:2,500 cross reactions with a second herpes-like viral agent Cyprinid herpesvirus 1 (CyHV-1) was detected in serum from both experimentally and naturally KHV exposed koi. Passive immunization by administration of anti-KHV antibodies from koi recovered from previous virus infections to naive koi provided only partial and transient protection to waterborne challenges with KHV. Koi that maintained high levels of serum anti-KHV antibodies after 1 year in virus-free water are deemed as suspect carriers of the virus. The identification of suspect carriers by screening of koi and common carp populations, including potential broodstocks, with the KHV ELISA should improve the ability to control this important viral pathogen.

Key wards: koi herpesvirus, KHV, Cyprinus carpio, ELISA, antibody, koi

A Practical Method to Distinguish between Neobenedenia girellae and Benedenia seriolae

<>Ryuhei Kinami1, Junko Miyamoto1, Tomoyoshi Yoshinaga1*, Kazuo Ogawa1 and Yoshitomo Nagakura2<>1Laboratory of Fish Diseases, Department of Aquatic Bioscience, Graduate School of Agricultural
and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
2Goto Station, National Center for Stock Enhancement, Fisheries Research Agency,
Tamanoura, Nagasaki 853-0508, Japan
(Received February 2, 2005)
ABSTRACT輸 practical method to distinguish two pathogenic monogeneans of fish, Neobenedenia girellae and Benedenia seriolae, is described. N. girellae and B. seriolae were obtained from experimentally infected olive flounder Paralichthys olivaceus and yellowtail Seriola quinqueradiata, respectively, by freshwater bathing and fixed in 10% formalin. The two species were found to have a different shape at the anterior end in the midline of the body and are clearly distinguishable by this difference. Using this method, the ratio of these two parasite species was monitored in greater amberjack Seriola dumerili cultured in net cages at Amami-Oshima Island for one year. A seasonal shift of abundance between the two species was observed.

Key words: Neobenedenia girellae, Benedenia seriolae, morphological distinction, seasonal succession, Seriola dumerili, greater amberjack

Identification and Pathogenicity of Intracellular Francisella Bacterium in Three-line Grunt Parapristipoma trilineatum

<>Takashi Kamaishi1*, Yutaka Fukuda2, Masaru Nishiyama3, 5, Hidemasa Kawakami4, Tomomasa Matsuyama1, Tomoyoshi Yoshinaga1, 6 and Norihisa Oseko1<>1National Research Institute of Aquaculture, Fisheries Research Agency, Nansei, Mie 519-0193, Japan
2Fisheries Research Institute, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Kamiura, Oita 879-2602, Japan
3Kochi Prefectural Sukumo Fisheries Guidance Station, Sukumo, Kochi 788-0265, Japan
4Ehime Prefectural Fish Disease Control Center, Uwajima, Ehime 798-0087, Japan
5Present address: Kochi Prefectural Government Fisheries Promotion Section,
Marunoushi, Kochi 780-0850, Japan
6Present address: Department of Aquatic Bioscience, Graduate School of Agricultural
and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
(Received February 3, 2005)
ABSTRACT唯acteriological examination was conducted on the disease of cultured marine fish, three-line grunt Parapristipoma trilineatum (Japanese common name: isaki). The affected fish characteristically showed white spots of granulomas containing an intracellular bacterium in the kidney and spleen. Total DNA was extracted from the kidney of the diseased fish. The coding region of small subunit ribosomal RNA (16S rDNA) of eubacteria was amplified from the DNA by PCR, and nucleotide sequence was determined. The sequenced 16S rDNA revealed high similarity (97.3-98.5%) to those of Francisella species. On the basis of phylogenetic analysis, the closest relative organism was revealed to be Francisella philomiragia. A bacterium was isolated from the spleen of one affected fish using cystine heart agar medium with 1% hemoglobin. The 16S rDNA sequence of the isolated bacterium was consistent with that determined from the kidney tissue of the affected fish. An experimental infection with the isolate exhibited the same disease signs in the injected fish, and the bacterium was reisolated from these fish. These results demonstrate that the intracellular bacterium is pathogenic and is identified to the genus Francisella. This is the first report of pathogenic Francisella from marine fish.

Key words: granuloma, intracellular bacterium, 16S rDNA, Francisella, Parapristipoma trilineatum

Cloning and Characterization of Two Types of tonB Genes, tonB1 and tonB2, and Ferric Uptake Regulator Gene, fur from Photobacterium damselae subsp. piscicida

<>Hiroaki Naka, Ikuo Hirono and Takashi Aoki*<>Laboratory of Genome Science, Graduate School of Marine Science and Technology,
Tokyo University of Marine Science and Technology, Minato,
Tokyo 108-8477, Japan
(Received February 19, 2005)
ABSTRACT裕here is some evidence that the virulence of some pathogenic bacteria is related to their iron uptake system. To better understand this system, three genes involved in iron uptake of Photobacterium damselae subsp. piscicida (tonB1, tonB2 and iron uptake regulator or fur) were cloned from a genomic library and sequenced. The amino acid sequence identities of tonB1 and tonB2 with the corresponding sequences of Vibrio spp. range from 32.4 to 39.5% and from 43.6 to 50.5%, respectively. The amino acid sequence identities of fur with the corresponding sequences of other Vibrio spp. are over 85%. We were able to construct a fur gene knock-out mutant using the marker exchange method. Using this knock-out mutant, we showed that tonB1, but not tonB2, is regulated by fur in P. damselae subsp. piscicida. The fur gene is also important for iron regulation since it has an influence on the expression of the heme receptor gene, hutA. The fur gene knock-out mutant generated in this study will be helpful in future studies of determining the role of iron in P. damselae subsp. piscicida pathogenesis and virulence.

Key wards: Photobacterium damselae subsp. piscicida, iron acquisition, TonB, ferric up take regulator, fur, tonB

Control of Water Mold Infection in Rainbow Trout Eggs by Using Copper Fiber

<>Masayuki Miura1, 3*, Heisuke Oono2, Nana Tuchida1, Kishio Hatai2 and Toru Kiryu1, 3*<>1Oshino Trout Hatchery, Yamanashi Fisheries Technology Center, Shibokusa,
Yamanashi 401-0511, Japan
2Division of Fish Diseases, Nippon Veterinary and Animal Science University,
Musashino, Tokyo 180-8602, Japan
3Yamanashi Fisheries Technology Center, Kai, Yamanashi 400-0121, Japan
(Received March 28, 2005)
ABSTRACT裕he antifungal efficacy of copper fiber against water mold infection in rainbow trout Oncorhynchus mykiss eggs was evaluated by the percentage of eggs with fungal infection. Copper fiber was put into the upstream of an egg breeding tank and rainbow trout eggs were soaked into the downstream of the same tank. Water mold infection in eggs was lower in the groups with copper fiber set in the water than that in the control group. Effects of copper on the embryonic development of the eggs were also examined for two weeks with the copper fiber soaked in the water. The concentration within the range from 0.006 to 0.020 ppm did not showed apparently adverse effects on the rate of eyed eggs or, after transferring eggs in the water without copper, on the rates of hatching or deformity. The solution prepared from copper nitrate reagent prevented the zoospore germination of Saprolegnia diclina at the concentration of 0.006 ppm in vitro.

Key words: copper fiber, water mold infection, Oncorhynchus mykiss, egg, Saprolegnia diclina, rainbow trout