碩士 / 國立海洋大學 / 水產養殖學系 / 87 / This study attempts to investigate the characteristics and pathogenicity of vibrio strains RGSW and RG3 isolated from disease fry of yellowfin seabream with a syndrome of red gut.
RGSW and RG3 strains were identified to be Vibrio alginolyticus and V. harveyi according to a series of standard biochemical and physiological tests, and biochemical tests using API 20E and BIOLOG GN systems. RGSW strain was resistant to ampicillin、chloramphenicol、novobiocin、oxytetracycline、penicillin G、streptomycin、sulphonamides、vancomycin，however it was sensitive to furazolidone、nalidixic acid and oxolinic acid. RG3 strain was resistant to ampicillin、penicillin G and vancomycin. While sensitive to the rest of antibiotics tested. RGSW was able to grow in 11 % NaCl , but RG3 strain couldn’t.
The extracellular products (ECP) of both strains possessed low haemolytic activity. The proteolytic activities of RGSW and RG3 ECP were 4007.46 and 1640.81 HPA units / mg protein, respectively. Both of the two ECP exhibited caseinase and gelatinase activities. The proteolytic activities of both ECP were demonstrated to be heat labile and inactive after incubation at over 50℃ for 30 minutes, but exhibited different activities under different pH conditions. Serine protease inhibitors─PMSF, TLCK, TPCK and 3,4-dichloroisocoumarin were found to inhibit proteolytic activities of ECP. The present results indicated that the major protease in both ECP might be serine protease.
In lethal test, yellowfin seabream were killed after intraperitoneal (i.p.) injection of the bacteria with a dose of 105 colony forming units（CFU）/ g fish body weight. The LD50 of ECP of RGSW and RG3 strains were 0.124 and 0.975 g protein / g fish body weight for fish weighting about 40 gram, respectively. In crossed immunoelectrophoresis（CIE） gels analysis, some of the unknown serum components in the moribund yellowfin seabream injected with bacteria and ECP were affected. In vaccination trial , the fish vaccinated with formalinized-bacteria or -ECP by injection could provide protection against bacteria or ECP challenge.
A 22 kDa protease, determined in SDS-PAGE, was partially purified from the ECP of V. harveyi strain RG3 strain using 50~70% ammonium sulfate precipitation, HIC-FPLC, Q HP-FPLC, and Mono Q-HPLC chromatography. The partially purified protease were lethal to yellowfin seabream with an LD50 value of 3.75 g protein / g fish body weight for fish weighting about 5 gram. The optimum pH of the protease was 7 , and it was heat labile. The proteolytic activities were almost inhibited by serine protease inhibitor─PMSF , TLCK, TPCK and 3,4-dichloroisocoumarin, and partially inhibited by cystein protease inhibitor─L-cystein, Iodoacetic acid and metallo-protease inhibitor ─ 1,10-phenanthroline monohydrate. The proteolytic activities also inhibited by Cu2+, Hg2+, Mn2+, and Zn2+ The partially purified protease also affected the serum components of yellowfin seabream ( in vivo and in vitro).
| Identifer | oai:union.ndltd.org:TW/087NTOU0086013 |
| Date | January 1999 |
| Creators | Yeh Yu-Ping, 葉玉萍 |
| Contributors | Lee Kuo-Kau, 李國誥 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 75 |
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