| Summary: | 碩士 === 國立海洋大學 === 水產養殖學系 === 83 === This study attempts to investigate the pathogenicity of vibrio strains V. al and Sw.y isolated from tiger prawn (Penaeus monodon) and kuruma prawn (P. japonicus) suffering from vibriosis with a syndrome of white spots in carapace.
Both of the strains isolated in the present study were identified to be Vibrio alginolyticus according to various biochemical tests of API 20E and IBOLOG systems. Both of the strains were sensitive to antibiotics such as chloramphenicol, ciprofloxacin, doxycycline hydrochloride, nalidixic acid, oxolinic acid and oxytetracycline, and demonstrated to be able to grow in the serum of tiger prawn.
The extracellular products (ECP) of both strains possesses low haemolytic activity and high proteolytic activity. The hide powderazure digestibility of ECPs of strains V.al and Sw.y was 2284 and 4206 units/mg protein, respectively. The highest yield of proteolytic activities in ECPs of strains V.al and Sw.y were observed when supplemented with 3% and 1% NaCl, respectively, and incubated for 24 hours at 25 ℃. The proteolytic activities of both ECPs were demonstrated to be heat labile and inactivated after incubation at 56℃ for 10 minutes, and exhibited different activitities when incubated in different pH values. The relative proteolytic activities of ECPs from strains V.al and Sw.y were 23% and 2%, respectively, when incubated with 5mM PMSF. The present results may suggest that the major protease in both ECPs is serine protease(s). The proteolytic activities of ECPs from strains V.al and Sw.y may be significantly inhibited by Fe2+ (10 mM) and partially inhibited by 10 mM of Zn2+, Mn2+, Co2+, Ca2+ or u2+. The relative proteolytic activities of ECPs from strains V.al and Sw.y were 18% and 16%, respectively, when incubated with 10 mM Fe2+.
The LD50 of strain V.al and its ECP in tiger prawn and kuruma prawn were 1.13×105 CFU、0.23μg protein and 2.46×105 CFU、0.63μg protein/g body weight, respectively. The LD50 of strain Sw.y and its ECP in tiger prawn and kuruma prawn were 1.57×105 CFU, 0.23μg protein and 4.43×104 CFU, 0.30μg protein/g body weight, respectively.
A 33 KDa protease, determined in both of SDS-PAGE and gel filtration, was partially purified from the ECP of strain Sw.y using HIC-FPLC and gel filtration-FPLC chromatography and still possessed strong proteolytic activity in SDS-PAGE. The protease was demonstrated to be toxic after intramuscularly injection into kuruma prawn. The LD50 of the protease is 0.27μg protein/g body weight. The optimum pH of the protease is 10. The proteolytic activity and toxicity to tiger prawn and kuruma prawn were almost inhibited by PMSF, the relative proteolytic activity was 8.1% when incubated with 10 mM Fe2+. In CIE gel, the precipitation arc with proteolytic activity was the closest origin point.
The incubation of plasma and serum from both tiger prawn and kuruma prawn revealed that the protease affected coagulogen in CIE gels. However, studies related to the mechanism of the interactions between the bacterial protease and haemolymph needs further investigations.
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