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NO37 - 1 (2002)

Effects of Cecropin Peptides on Aquatic Animal Pathogens and Shrimp Hemocytes

<>Shih-Hu Ho1, Chia-Zong Lin2, Yu-Chih Chen1 and Yen-Ling Song1*<>1Department of Zoology and
2Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan, 106, Republic of China

ABSTRACT悠n this paper, the effects of the well-characterized antimicrobial amphipathic peptides (cecropins A and P1) on pathogens of aquatic organisms were assessed. Results showed that cecropins inhibited growth of 15 strains of bacteria, including Gram-negative bacteria: Aeromonas hydrophila, A. salmonicida, Photobacterium damselae subsp. damselae, Pseudomonas anguilliseptica, Vibrio alginolyticus, V. anguillarum, V. carchariae, V. harveyi, V. ordalii, V. parahaemolyticus, V. tubiashii and V. vulnificus biogroup I. Cecropins also inhibited growth of two strains of the yeast Debaryomyces hanseni, pathogenic to freshwater prawn Macrobrachium rosenbergii. Minimum inhibitory concentration (MIC) of cecropin A ranged from 0.98 to 7.81 mM and minimum bactericidal concentration (MBC) ranged from 3.90 to 15.59 mM against the most prevalent vibrios pathogenic to cultured shrimp. A higher level of cecropin was required in a high salt environment to exhibit the effect against the pathogens. Influence of NaCl on bactericidal activity of cecropin A was also assayed. 51Cr-release assay showed a non-cytotoxic effect of cecropin A on shrimp (Penaeus monodon) hemocytes at MBC, but cytotoxic effect at 50 mM (3.2-fold MBC) or higher concentration. This suggests that cecropin peptides may be difficult to be applied in vivo to treat infected marine shrimp.

A Galactose-Binding Protein Revealed as a Hemagglutinin in Aphanomyces piscicida

<>Osamu Kurata*, Kayoko Sanpei, Kazuyuki Hikiji and Kishio Hatai<>Division of Fish Diseases, Nippon Veterinary and Animal Science University, 1-7-1 Kyonan-cho, Musashino, Tokyo 180-8602, Japan

ABSTRACT輸phanomyces piscicida is a water mold that is pathogenic to several species of fish. Here we describe that a galactose-binding protein (GBP) purified from A. piscicida has a hemagglutinating activity. The GBP was separated from the supernatant of homogenized hyphae of A. piscicida by affinity chromatography using an immobilized D-galactose agarose gel. The GBP agglutinated goldfish erythrocytes. The molecular weight of the GBP was estimated at approximately 40 kDa by SDS-PAGE. A specific antiserum was produced against the GBP and the 40 kDa protein reacting with the antiserum was observed in only Aphanomyces strains isolated from diseased fish. These results suggest that the 40 kDa GBP is closely associated with Aphanomyces infections, such as mycotic granulomatosis, epizootic ulcerative syndrome, red spot disease and ulcerative mycosis.

Inhibitory Effect of Cortisol on the Defense Activities of Tilapia Neutrophils in Vitro

<>Junko Kurogi1 and Takaji Iida2*<>1The United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan
2Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai-nishi 1-1, Miyazaki 889-2192, Japan.

ABSTRACT涌n the premise that fish neutrophil activities are depressed under stressed conditions, this study investigated the direct effect of cortisol on the in vitro activity of neutrophiles collected from tilapia Oreochromis niloticus. Neutrophils were obtained from swim bladders and exposed to different levels of cortisol (0・000 ng/mL), and their chemotactic, phagocytic and respiratory burst activities were assessed. This treatment suppressed neutrophil activities as seen in the reduction of chemotactic, phagocytic and respiratory burst activities. Neutrophil activities appeared to be suppressed in a dose-dependent manner leading to a considerable reduction in neutrophil activities with high concentrations of cortisol. Nevertheless, we showed that the respiratory burst activity was able to recover from the observed effect after cortisol was removed. The results in this study strongly suggest that cortisol has a directly adverse effect on fish neutrophil defense activities, but that the effect is reversible.

Impaired Neutrophil Defense Activities and Increased

<>Junko Kurogi1 and Takaji Iida2*<>1The United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan
2Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai-nishi 1-1, Miyazaki 889-2192, Japan.

ABSTRACT悠n order to reveal the effect of cortisol on tilapia neutrophil defense activities in vivo, fish were given intraperitoneal injections of cortisol mixed with coconut oil as an 妬mplant・(5 mg/0.5 mL/fish). Fish were injected with Escherichia coli into their swim bladders before 24 h prior to collecting neutrophils, which were then assessed for their chemotactic, phagocytic and respiratory burst activities. In addition, concentrations of plasma cortisol and glucose were measured. Cortisol-implanted fish showed considerably higher levels of cortisol and their neutrophil activities were significantly suppressed compared to the control group at 1 day post-implantation. Both groups of fish were challenged with Edwardsiella tarda (1 mg/fish). Cortisol-implanted fish started to die 6 days after the challenge, whereas no deaths were observed in the controls. After the challenge experiment, E. tarda was recovered from more than half of the cortisol-treated fish group and from the only one of the controls. Results from this study have demonstrated that elevated levels of cortisol in fish bodies, similar to that observed during stressful experiences, decrease neutrophil activities and increase susceptibility to bacterial pathogens.

Identification and Development of a Paramyxean Ovarian Parasite in the Pacific OysterCrassostrea gigas

<>Naoki Itoh1*, Tadashi Oda2, Kazuo Ogawa1 and Hisatsugu Wakabayashi1,3<>1Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan
2Fish Propagation Section, Fisheries Experiment Station of Okayama Prefecture, Ushimado, Okayama 701-4303, Japan
3Fish Health Laboratory, Sendagi 3-22-11-717, Bunkyo, Tokyo 113-0022, Japan

ABSTRACT裕he intracellular ovarian parasite causing unaesthetic appearance in the ovary of Pacific oysters Crassostrea gigas in Japan has remained unidentified since the first report of its occurrence in the 1930s. The parasite was observed through transmission electron microscopy, and identified as the paramyxean Marteilioides chungmuensis, which was first reported from Pacific oysters in Korea. In order to obtain information on the development of the parasite, the diameters of both the parasite and the infected ova were measured. The growth of the parasite was highly correlated with the growth and maturation of host gonad cells. Histological observations suggest that M. chungmuensis invades immature ova, which move to the center of the follicle along with its development, and infected ova are finally released outside through the genital canal.

Fish-to-fish Transmission of Myxidium spp. (Myxozoa) in Cultured Tiger Puffer Suffering from Emaciation Disease

<>Hiroshi Yasuda1, Tsuyoshi Ooyama1, Kazuo Iwata1, Tin Tun2, Hiroshi Yokoyama2* and Kazuo Ogawa2<>1Miyazaki Prefectural Fisheries Experimental Station, Aoshima 6-16-3, Miyazaki, 889-2162, Japan
2Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan

ABSTRACT勇xperimental transmission of Myxidium fugu and Myxidium sp. TP (formerly described as Myxidium sp. in tiger puffer Takifugu rubripes) among tiger puffer was successful by the following 3 methods; 1) feeding infected gut tissue, 2) cohabitation with infected fish, 3) exposure to effluent from a tank containing infected fish. Regardless of the transmission methods, prevalences of infection in the intestine with M. fugu reached 100%. However, those with Myxidium sp. TP, which is suspected of an etiological agent of the emaciation disease, varied among experiments conducted in different months, suggesting that development of Myxidium sp. TP was strongly influenced by the ambient temperature. It was evident that trophozoites of Myxidium sp. TP excreted from the infected fish were transmittable directly to other fish. Mortalities and morbidities in fish experimentally infected with Myxidium sp. TP seemed to be associated with the parasiteユs development which might be promoted by higher temperature. The present study suggests that fish-to-fish transmission of Myxidium spp. occurs in sea cages, inducing a rapid spread of the emaciation disease among farmed tiger puffer.

Detection of Caspase Activities in Cultured Fish Cells Infected with Viral Deformity Virus

<>Satoshi Miwa1*, Nobuhiro Mano1,2 and Futoshi Aranishi1<>1National Research Institute of Fisheries Science, Fukuura 2-12-4, Kanazawa, Yokohama 236-8648, Japan
2Present address: Department of Marine Science and Resources, Nihon University, Kameino 1866,
Fujisawa, Kanagawa 252-8510, Japan

ABSTRACT裕o know whether caspases are involved in virus-induced apoptosis in fish cells, CHSE-214 cells were infected with viral deformity virus (VDV), which is a member of Birnaviridae. Some of the infected cells exhibited morphological features that are typical of apoptosis, although the ratio of these cells was less than 10% throughout the experiment. Caspase activities, especially that of caspase 6, were detected in infected cells, but not in uninfected cells. Activity for caspases 3 or 7 peaked later than those for the other caspases. These observations suggest the involvement of caspase cascade in virus-induced apoptosis in fish cells and functional differentiation among caspases.

Hematological Characterization of Anemia Recently Prevailing in Wild Japanese Flounder in Japan

Chihaya Nakayasu1*, Tomoyoshi Yoshinaga2 and Akira Kumagai1
1Inland Station, National Research Institute of Aquaculture, Fisheries Research Agency, Tamaki, Mie 519-0423, Japan
2National Research Institute of Aquaculture, Fisheries Research Agency, Nansei, Mie 519-0193, Japan

ABSTRACT幽ematological characteristics of the anemia prevailing in wild Japanese flounder Paralichthys olivaceus were examined in detail by cell counting and microscopy.
The erythrocyte counts, MCH, and MCHC decreased correlatively with the decrease in the hemoglobin concentration. Moreover, the reduction of erythrocyte volume was also observed in many anemic fish. Light microscopy showed erythrocytes with structural abnormalities, such as vacuolated, weakly stained cytoplasm and deformed outlines. In the TEM observations of erythrocytes, an insufficient accumulation of hemoglobin was observed as well. These results indicate that the anemia can be identified as a hypochromic microcytic anemia accompanied with hypoglobulia and a structural abnormality of the erythrocytes.

Effects of Water Temperature on the Egg-laying of the Monogenean Neoheterobothrium hirame

<>Nobuyuki Tsutsumi1, Keiichi Mushiake2, Koh-ichiro Mori2, Misao Arimoto2, Tomoyoshi Yoshinaga3 and Kazuo Ogawa1*<>1Laboratory of Fish Diseases, Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo,Yayoi, Bunkyo, Tokyo 113-8657, Japan
2Kamiura Station of Japan Sea-Farming Association, Kamiura, Minamiamabe, Oita 879-2602, Japan
3National Research Institute of Aquaculture, Fisheries Research Agency, Nansei, Mie 519-0193, Japan

ABSTRACT裕he egg-laying rates of the monogenean Neoheterobothrium hirame, a parasite of Japanese flounder, were measured at different water temperatures. The daily egg-laying rates per worm were 203, 578, 781 and 651 eggs at 10, 15, 20 and 25ーC, respectively. Thus the egg-laying rate increases progressively with temperature from 10 to 20ーC, but no significant difference was detected between values at 20 and 25ーC or between 15 and 25ーC. While all the eggs obtained below 20ーC appeared normal with straight appendages at both ends, 1.4% of the eggs obtained at 25ーC appeared abnormal, suggesting that 25ーC is at or near the upper limit for the normal egg-laying of this parasite.

Cultured Fish Species Affected by Red Sea Bream Iridoviral Disease from 1996 to 2000

<>Hidemasa Kawakami1 and Kazuhiro Nakajima2<>2Inland Station, National Research Institute of Aquaculture, Fisheries Research Agency ,
Tamaki, Mie 519-0423, Japan

ABSTRACT涌ccurrences of red sea bream iridoviral disease (RSIVD) in cultured marine fish were investigated by our diagnostic works and by collecting information from prefectural fisheries research stations. The infection was identified by using indirect immunofluorescence test with a monoclonal antibody against red sea bream iridovirus (RSIV). In total, RSIVD was confirmed in 29 cultured marine fish species from 1996 to 2000. After 1995, 11 fish species were newly confirmed as susceptible hosts to RSIV. Since the first occurrence in 1990, RSIVD has been recorded in a total of 31 cultured marine fish species in 18 prefectures located in south-western part of Japan.