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NO47 - 1 (2012)

Infiltration of Bacteriophages from Intestinal Tract to Circulatory System in Goldfish

Yasuhiko Kawato and Toshihiro Nakai*
Graduate school of Biosphere Science, Hiroshima University, Hiroshima 739-8528, Japan (Received September 26, 2011)

ABSTRACT―We introduced eight fish pathogen phages and three Escherichia coli phages (T4, T7 and λ) into the intestinal tract of goldfish Carassius auratus by anal intubation (108.3 PFU/fish). All phages appeared in the kidney at mean PFUs ranging from 102.7 to 104.3 PFU/g 3 h after intubation. Anal intubation of a representative Pseudomonas plecoglossicida phage (PPpW-4) into goldfish revealed that the introduced phages transfer to the blood and kidney within 10 min and are retained there for at least 12 h. An intubation dose of 107.3 PFU/fish or higher is required for constant presence of the phages in the circulatory system. Our results indicate that phages have the ability to pass through the intestinal wall and spread to the circulatory system, suggesting the potential application of phage therapy with oral administration.

Key words: bacteriophage, pharmacokinetics, phage therapy, Carassius auratus, Pseudomonas plecoglossicida

Localization Analysis of Fluorescent-labeled Aeromonas veronii in the Intestinal Tract of Carp using an in vivo Imaging System

Aki Namba1, Nobuhiro Mano2*, Hitomi Hirose2 and Teruyuki Nakanishi1
1Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Kanagawa 252-0880, Japan
2Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Kanagawa 252-0880, Japan(Received September 27, 2011)

ABSTRACT―Aeromonas veronii is a Gram-negative opportunistic pathogen in fish. We have previously investigated the intestinal microbiota of carp Cyprinus carpio and revealed that most bacterial isolates with high mucus-binding capability were A. veronii. With regard to infection, mucosal adhesion of bacteria is regarded as a prerequisite for colonization and invasion. In the present study, we selected three A. veronii strains with different mucus-binding capability and pathogenicity, and compared the colonization capability and localization of these strains in the intestinal tract of carp, using an in vivo imaging system. We showed that only a pathogenic strain was mainly visible around the intestinal bulb and rectum of the intestinal tract at 48 h after administration. Frozen sections with fluorescence signals revealed that the administered bacteria were localized on the mucosal surfaces and in the lamina propria of the intestinal bulb, whereas they were mainly detected in the remaining intestinal contents in the rectum. These results suggest that the intestinal bulb is the primary region for A. veronii to invade and proliferate in healthy carp.

Key words: Aeromonas veronii, Cyprinus carpio, CFDA/SE, intestinal tract, in vivo imaging

Analysis of Immune Response in Yellowtail upon Vaccination with a Bivalent Vaccine of Vibrio anguillarum and Lactococcus garvieae

Nao Nakajima1,3*, Mayumi Kijima1, Michiko Kawanishi1, Goushi Katou2, Kunihiko Futami3, Takayuki Katagiri3, Makoto Endo3 and Masashi Maita3
1National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Tokyo 185-8511, Japan
2Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan
3Laboratory of Fish Health Management, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan
(Received October 4, 2011)

ABSTRACT―We inoculated yellowtail Seriola quinqueradiata with a commercial bivalent vaccine containing inactivated Vibrio anguillarum and Lactococcus garvieae to better understand the immunological responses to the vaccination. Serum samples were collected from vaccinated and control (PBS-injected) fish at 1, 3, 5, 7, 10, and 14 days post-vaccination (dpv). Compared to the controls, the serum from the vaccinated fish inhibited the proliferation of V. anguillarum from 7 dpv. However, vaccination had no effect on L. garvieae proliferation. The antibody titer against V. anguillarum obviously increased from 7 dpv, but no such increase was detected for L. garvieae. We confirmed that the protection conferred by the V. anguillarum vaccine was a result of the antibodies against the pathogen. However, humoral factors related to protection were not observed for L. garvieae. The expression of immune-related genes in the head kidney was profiled by microarray analysis. Twenty-two immune-related genes were up-regulated during the experimental period. Up-regulation of cellular immune-related genes, such as tumor necrosis factor, CC chemokine, and macrophage colony-stimulating factor receptor, was mostly observed from 1 to 3 dpv, whereas that of humoral immune-related genes was observed from 3 to 14 dpv. Notably, immunoglobulin-related genes showed continuous up-regulation during the 2-week experimental period.

Key words: Seriola quinqueradiata, Yellowtail, Vaccine, Immune response, Gene expression, Vibrio anguillarum, Lactococcus garvieae

Broussonetia kazinoki as a Feed Additive Enhances Disease Resistance against Streptococcus parauberis in Paralichthys olivaceus

Ju-Sang Kim1†, Ramasamy Harikrishnan1†, Man-Chul Kim1, Chellam Balasundaram2 and Moon-Soo Heo1*
1Department of Aquatic Biomedical Sciences & Marine and Environmental Research Institute, Jeju National University, Jeju 690-756, South Korea
2Department of Animal Science, Bharathidasan University, Tamilnadu, India
(Received April 21, 2011)

ABSTRACT―We report the effect of Broussonetia kazinoki as a feed additive on disease resistance in olive flounder Paralichthys olivaceus, against Streptococcus parauberis. The cumulative mortalities were lower in fish fed with diets supplemented with 1.0% and 2.0% than in those fed with 0% and 0.1% diets. Phagocytic, complement and lysozyme activities were significantly enhanced by 0.1%, 1.0% and 2.0% B. kazinoki diets. Respiratory burst activity was significantly enhanced by 1.0% and 2.0% diets. This study suggests that diets supplemented with 1.0% and 2.0% B. kazinoki enhance the immune parameters concerning resistance against S. parauberis infection in P. olivaceus.

Key words: Broussonetia kazinoki, innate immune response, mortality, Paralichthys olivaceus, Streptococcus parauberis

Sciadicleithrum variabilum (Dactylogyridae: Monogenea) Infection in Symphysodon discus: a Case Report

Giovanni Aquaro1, Cristian Salogni2, Paolo Galli1, Lucia Gibelli2 and Daniela Gelmetti2*
1Department of Biotechnology and Bioscience, University of Milan Bicocca, Piazza della Scienza 2, 20134 Milan, Italy
2Zooprofilattico  Sperimentale della Lombardia ed Emilia Romagna, via Bianchi 7/9, 25124 Brescia, Italy
(Recieved May 28, 2011)

ABSTRACT―During the winter of 2009, we observed an outbreak of severe respiratory disease in Symphysodon discus in a fish farm. The disease began with anorexia, respiratory distress and behavioral disorders and caused the death of all affected juveniles (80%) within a few days. Gill filaments were inflamed, hyperplastic and necrotic, and were infected with Sciadicleithrum variabilum. We amplified the cytochrome oxidase I (COI) gene of the monogenean using PCR and sequenced the gene as a molecular marker to rapidly identify the species. We demonstrated successful sequencing of the COI gene for S. variabilum identification, together with the need to monitor this pathogen in fish farms.

Key words: pathology, Sciadicleithrum variabilum, PCR, Monogenea, cytochrome oxidase I, Sympysodon discus

Zanthoxylum piperitum-supplemented Diet Enhances Disease Resistance in Epinephelus bruneus against Vibrio carchariae

Ramasamy Harikrishnan1, Ju-Sang Kim1, Man-Chul Kim1, Chellam Balasundaram2 and Moon-Soo Heo1*
1Department of Aquatic Biomedical Sciences & Marine and Environmental Research Institute, Jeju National University, Jeju 690-756, South Korea
2Department of Animal Science, Bharathidasan University, Tamilnadu, India
(Received July 26, 2011)

ABSTRACT―We studied the effects of diet supplemented with 0% (control), 0.1%, 1.0% and 2.0% Zanthoxylum piperitum on the immune response and disease resistance in kelp grouper Epinephelus bruneus against Vibrio carchariae. The phagocytic and lysozyme activities were significantly enhanced in fish fed with diets supplemented with 1.0% and 2.0% Z. piperitum. The cumulative mortality of fish fed with 1.0% and 2.0% supplemented diets was lower than that of the controls. Therefore, it was suggested that diets supplemented with 1.0% and 2.0% Z. piperitum positively enhanced the innate immune system in E. bruneus against V. carchariae.

Key words: Epinephelus bruneus, innate immune response, mortality, Vibrio carchariae, Zanthoxylum piperitum

Inhibition of Siniperca chuatsi Rhabdovirus by RNA Interference in a Fish Cell Line

Guang-Zhou Zhou1,2*, Rong Zhu1, Jian-Fang Gui1 and Qi-Ya Zhang1*
1State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
2College of Bioengineering, Henan University of Technology, Zhengzhou 450001, China
(Received September 17, 2011)

ABSTRACT―We evaluated the antiviral activity of several small interfering RNAs (siRNAs) corresponding to the Siniperca chuatsi rhabdovirus (SCRV) nucleoprotein (N) gene against the SCRV in a fish cell line, EPC. Among the siRNAs we examined, si983 was most effective at reducing virus titer compared to the mock group. Western blot analysis suggested that si983 inhibited N gene expression and virus replication in a dose-dependent manner. These results indicate that N gene 983 sites are potential targets for antiviral therapy. Furthermore, RNAi targeting of the N gene may represent a viable method of treating against SCRV infection.

Key words: Mandarin fish, rhabdovirus, nucleoprotein, RNA interference, SCRV

Larval Cestodes Found in the Skeletal Muscle of Cultured Greater Amberjack Seriola dumerili in Japan

Kazuo Ogawa1*, Takashi Iwaki1, Naoki Itoh2 and Taizo Nagano3
1Meguro Parasitological Museum, Tokyo 153-0064, Japan
2Laboratory of Aquacultural Biology, Graduate School of Agricultural Science, Tohoku University,
 Miyagi 981-8555, Japan
3Kagawa Prefectural Fisheries Experimental Station, Kagawa 761-0111, Japan
(Received October 21, 2011)

ABSTRACT―A long, tubular "parasite" was found in the muscle of greater amberjack Seriola dumerili cultured in Kagoshima and Miyazaki prefectures. Infection with this parasite was very rare, only occurring in two amberjacks from Kagoshima and one from Miyazaki. Molecular analysis indicates the parasite is a cestode of the order Trypanorhyncha, most closely related to the subfamily Grillotiinae, family Lacistorhynchidae. The tubular structure was a blastocyst. There was no evidence of the scolex in the blastocysts, which may not have been sampled intact, making it impossible to identify the parasite. This is the first report of Trypanorhyncha infection in greater amberjack in Japan.

Key words: Trypanorhyncha, cestode, Seriola dumerili, blastocyst