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NO35 - 3

Effect of Lipopolysaccharide on In Vitro Phagocytosis by Hemocytes from Giant Freshwater Prawn (Macrobrachium rosenbergii )

January 28, 2000<>Hung-Hung Sung*, Pei-An Kuo and Wen-Yi Kao<>Department of Microbiology, Soochow University, Taipei, Taiwan 111, Republic of China

ABSTRACT ・When separate batches of Macrobrachium rosenbergii hemocytes were cultured in different media at 25コC for 40 min, the percentage of viable hemocytes cultured in modified M-199 medium (M-199; 80%) was significantly higher than the percentage cultured in modified crayfish saline (M-CFS; 65%), although wide individual variation was noted. The percentage of phagocytic cells increased significantly with lipopolysaccharide (LPS) concentrations up to 16 mg/mL, but no further significant increases were found at concentrations from 16 to 100 mg/mL. Microscopy of phagocytosis in hemocytes cultured in M-199 and induced with LPS at a final concentration of 20 mg/mL showed that target zymosan particles (1ミ4 mm) were engulfed by granulocytes (GC) and semigranulocytes (SGC), but were only attached to and not engulfed by hyaline cells (HC). However, smaller sized fluorescent-labeled beads (0.5 mm) were primarily phagocytosed by HC. This suggests that all three types of hemocytes from M. rosenbergii are able to phagocytose foreign particles. Microscopy showed that LPS significantly altered hemocyte morphology by increasing pseudopodia stretching and hemocyte size and it also increased the number of engulfed particles per phagocyte. The percentage of HC positive for two lysosomal enzymes (acid phosphatase and a-naphthyl acetate esterase) related to phagocytosis was higher than that of positive SGC or GC.

Natural and Experimental Infections of Campylobacter cryaerophila in Rainbow Trout: Gross Pathology,Bacteriology, Clinical Pathology and Chemotherapy

January 28, 2000<>Seyit Aydin*, Nejdet G・tepe and Harun Yildiz<>Fisheries Faculty, Canakkale Onsekiz Mart University, Karacaen, Canakkale, Turkey

ABSTRACT ・Campylobacter cryaerophila was isolated from naturally infected rainbow trout (Oncorhynchus mykiss), and its pathogenicity was tested by intramuscular injection using 1-year-old rainbow trout and scattered mirror carp (Cyprinus carpio). C. cryaerophila did not induce
experimental infection in scattered mirror carp. Natural and experimental infections caused mortalities in rainbow trout with gross clinical abnormalities such as exophthalmia, damage of liver, bloody kidney and haemorrhagic heart and swollen intestine. Glucose, cholesterol, triglyceride and haematocrit levels in blood of both naturally and experimentally infected fish were significantly decreased as compared to healthy fish. Significant decreases were observed in the serum glutamate oxalacetate transaminase level of experimentally infected fish and serum total protein value of naturally infected fish. Albumin levels of serum were not significantly different among the three treatments. Sensitivities of three isolates of C. cryaerophila against 51 chemotherapeutants were determined. Minimum inhibitory concentrations of formalin and enrofloxacin to the isolates were between 3.5D~4.5 mL/mL and 0.025D~1 mL/mL, respectively. Oral applications of enrofloxacin after bath disinfections with formalin controlled the natural infections.

In Vitro Degranulation of Tilapia Eosinophilic Granular Cells and its Effect on Neutrophil Migration

April 10, 2000<>Tomomasa Matsuyama1 and Takaji Iida2*<>1 United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan
2 Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai-Nishi 1-1, Miyazaki 889-2192, Japan

ABSTRACT ・Migration of neutrophils toward the supernatant of degranulated eosinophilic granular cells (EGCs) was examined using a modified Boyden chamber. EGCs of tilapia (Oreochromis niloticus) were isolated from the swim bladder membrane, and degranulated by incubation with substance P (neurotransmitter) or compound 48/80. The degranulation of the EGCs was also
observed after incubation with normal tilapia sera containing zymosan, but not with heat-inactivated sera. Additionally, significantly higher neutrophil migration was observed when the degranulation of the EGCs occurred in the lower compartment of a Boyden chamber. High-speed centrifugation inhibited the degranulation of the EGCs stimulated by substance P or zymosan plus normal tilapia sera, and migration of neutrophils was inhibited. The results obtained in the present study suggest that neutrophil migration stimulating factor(s) are released by degranulation of EGCs in tilapia.

Hematology, Histopathology and the Monogenean Neoheterobothrium hirame Infection in Anemic Flounder

May 10, 2000<>Tomoyoshi Yoshinaga1*, Takashi Kamaishi1, Isao Segawa1, Akira Kumagai2,Chihaya Nakayasu2, Keisuke Yamano1, Terufumi Takeuchi3and Minoru Sorimachi2 <>1 Inland Station, National Research Institute of Aquaculture, Tamaki, Mie 519-0423, Japan
2 National Research Institute of Aquaculture, Nansei, Mie 516-0193, Japan
3 Fisheries Farming Experimental Station, Wakayama Research Center of Agriculture, Forestry and Fisheries, Tanabe, Wakayama 646-0053, Japan

ABSTRACT ・Recently, severe anemia of unknown etiology has been observed frequently in both wild and cultured Japanese flounder, Paralicthys olivaceus, in Japan. Hematological and parasitological examinations and histopathlogical examinations on the hematopoietic organs were carried out in anemic flounder. The anemia was hematologically characterized by the appearance of many immature erythrocytes and abnormal staining in the cytoplasm (vacuolation or weak staining) of erythrocytes. Histopathologically, deposition of hemosiderin or necrosis was rarely observed in the kidney or spleen of the anemic flounder. The blood-feeding monogenean, Neoheterobothrium hirame, was observed at high prevalences in flounder groups in which many anemic fish were contained. These observations suggest that the anemia was caused by the hematophagia by the parasite.

An Investigation into the Larval Energetics and Settlement of the Sea Louse, Lepeophtheirus salmonis, an Ectoparasitic Copepod of Atlantic salmon, Salmo salar

May 12, 2000<>Carl S. Tucker*, Christina Sommerville and Rodney Wootten<>Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, Scotland, UK

ABSTRACT ・This study was undertaken to investigate the energy levels of Lepeophtheirus salmonis larvae and test the hypothesis that aged lecithotrophic copepodids would have reduced settlement ability. Calculated energy levels for all pre-settlement stages were determined following Carbon: Hydrogen: Nitrogen analysis. The calculated energy levels of winter parasitic copepodid larvae (approx. 7800 cal/g dry weight) were similar to those of winter free-living copepod larval stages. Energy levels of copepodids were found to decline sharply between those aged day 1 and 2 post moult and between those aged day 5 and 7. Settlement experiments with aged copepodids, at summer and winter sea water temperatures showed a statistically significant difference in settlement ability between copepodids aged 7 days and those aged 1 and 3 days. However, once attached and settled on the host the rate of development and initial survival was found not to be statistically different from that of the other age groups that were examined. Copepodid durability, as a free swimming stage, and its ability to infect the host in appreciable numbers one week after moulting to the infective stage will have important implications for the salmon culture industry.

Myxosporeans and Their Hyperparasitic Microsporeans in the Intestine of Emaciated Tiger Puffer

June 9, 2000<>Tin Tun, Hiroshi Yokoyama*, Kazuo Ogawa and Hisatsugu Wakabayashi<>Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan

ABSTRACT ・Three myxosporeans and 2 hyperparasitic microsporeans were found in the intestine of emaciated cultured tiger puffer Takifugu rubripes in Kyushu district of Japan. Morphology of the 3 myxosporeans were examined and described as Myxidium fugu n. sp., Leptotheca fugu n. sp. and unidentified Myxidium sp. Spores of M. fugu n. sp. having 2 club-shaped polar capsules were bean-shaped, 14.0 (13.5ミ15.5) mm in length and 9.0 (8.0ミ10.0) mm in width. Spores of L. fugu n. sp. having 2 spherical polar capsules were trapezium-shaped, 9.0 (8.3ミ9.5) mm in length and 14.0 (13.0ミ15.0) mm in thickness. Their piscine intestinal developmental stages were illustrated and arranged in a hypothetical developmental sequence. Plasmodia of M. fugu n. sp. were located on the epithelium of intestine, whereas those of L. fugu n. sp. and Myxidium sp. were found intercellularly in the intestinal epithelium. Only a few mature spores could be observed in these myxosporeans, but the differences in stainability by Diff-Quik and Uvitex 2B and in location of development made it possible to differentiate the species. Two hyperparasitic microsporeans were often found in the plasmodia of M. fugu n. sp. and L. fugu n. sp, but their pathogenic effects to host myxosporeans were unclarified.

Polymerase Chain Reaction and Indirect Fluorescent Antibody Technique for the Detection of Kudoa amamiensis (Multivalvulida: Myxozoa) in Yellowtail Seriola quinqueradiata

June 23, 2000<>Hiroshi Yokoyama1*, Daisuke Inoue1, Akihiro Sugiyama2 and Hisatsugu Wakabayashi1<>1 Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan
2 Okinawa Prefectural Fisheries Experimental Station, 1-3-1, Nishizaki, Itoman, Okinawa 901-0305, Japan

ABSTRACT ・Polymerase chain reaction (PCR) and indirect fluorescent antibody technique (IFAT) were developed for the detection of Kudoa amamiensis (Multivalvulida: Myxozoa), which produces pseudocysts in the skeletal musculature of yellowtail Seriola quinqueradiata. A nested PCR derived from the 18SrDNA sequences of K. amamiensis proved to be highly specific and sensitive, detecting a single spore in a 20 mL reaction mixture. In a periodic monitoring of yellowtail, the single-round and nested PCRs were conducted as well as conventional diagnostic techniques (visual inspection, wet-mount observation and histological examination). After one month culture of yellowtail in an endemic area, neither pseudocysts nor spores could be detected visually, whereas 53% of the examined fish were positive for infection by the nested PCR. After 3 and 5 months, macroscopic pseudocysts were detected in 27% and 60% of the fish, respectively, while the nested PCR was more sensitive (93% and 70% were positive, respectively). Histologically, intracellular organisms with several internal cells were detected only in the muscle fibers of the samples which were positive by PCR tests. By the IFAT using a rabbit antiserum to sonicated K. amamiensis spores, both pre-spore and spore stages reacted, and intracellular plasmodia were positively identified as presporogonic stages of K. amamiensis. In conclusion, we developed the IFAT and the nested PCR for the early diagnosis of K. amamiensis, and described for the first time its intracellular presporogonic stage in the muscle fiber of yellowtail.

Primary Culture of Tilapia Capillary Endothelial Cells

May 12, 2000<>Tomomasa Matsuyama1 and Takaji Iida2*<>1 United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan
2 Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai-Nishi 1-1, Miyazaki 889-2192, Japan

Transfer of Fluorescent Microspheres from the Integumental Tissues to the Kidney and Spleen in Rainbow Trout

May 18, 2000<>Ikunari Kiryu1,2*, Mitsuru Ototake2, Teruyuki Nakanishi3 and Hisatsugu Wakabayashi1<>1 Department of Aquatic Bioscience, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Yayoi-1-1-1, Bunkyo, Tokyo 113-8657, Japan
2 Inland Station, National Research Institute of Aquaculture, 224-1 Hiruta, Tamaki, Mie 519-0423, Japan
3 Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8570, Japan