碩士 / 國立臺灣大學 / 動物學研究所 / 97 / The major bacterial pathogen of cage-cultured cobia (Rachycentron canadum) in Taiwan is Photobacterium damsela subsp. piscicida, which is highly infectious and causes massive mortality. Resistance of the bacteria in field against antibiotics had reported. Cecropin A is a wide-spectrum antimicrobial peptide with 37 a.a., and structured as a simple amphipathic alpha-helix. The killing mechanism of cecropin is thought to lyse bacterial cell membrane, causing permeability unbalanced or cytoplasm loss. Acquisition of resistance may thus involve alternation of bacterial membrane composition, which may be more complex than a single step mutation.
In the present study, the minimal inhibition concentration of cecropin to Ph. damsela piscicida was demonstrated to be nearly 0.7 μM. Measured by plate counting, minimal bactericidal concentration and LD50 were 0.9 and 0.7 μM respectively. Killing Ph. damselae piscicida by cecropin was concentration-dependent measured by plate count. Evaluated by trypan blue exclusion, there was a significant cytotoxicity of cecropin to cobia muscle cells until 64 μM after 8 hours treatment. Hemolysis effective concentration (EC50) of cecropin to cobia erythrocytes was more than 1000 μM. Conclusively, cecropin is showed considerably selective for Ph. damselae subsp. piscicida over cobia cells.
With inhibition zone assay, the observed bacteriostatic activity of cecropin was not changed after 4 hours incubation with cobia stomach crude extraction, but the bacteriostatic activity disappeared gradually within 2 hours incubation with intestine crude extraction. This suggests that cecropin is not stable in cobia intestine. Antibacterial activity of cecropin was unstable in cobia blood matrice especially whole blood and plasma. Loss of antibacterial activity may be due to protease digestion. This instable property limits efficient transportation of cecropin to infected area, such as skin, in blood.
To test the protection in an in vivo experiment, we introduced Ph. damselae subsp. piscicida and cecropin concurrently by intraperitoneal (i.p.) inoculation. Survival rate was 100% when we boost the same dose cecropin 12 hr after infection. In the infection control without giving any cecropin, survival rate was 25%. Survival rate in boost group compared with infection control group was significantly higher (p < 0.05). But the survival rates of just one time i.p. injection of cecropin and infection control were 44% and 17%, and there was no significantly increase.
To test the protection in the feeding experiment, we introduced pre-mixed Ph. damselae subsp. piscicida and cecropin (or encapsulated cecropin) into esophagus by plastic tube. The survival rate in which every 20 g fish fed 2 mg cecropin was 75%, significantly higher than infection control (0%, p < 0.05). We suggested inoculating high dose cecropin concurrently to cobia infected by Ph. damselae subsp. piscicida can completely kill bacteria and significantly reduce death rate. The bactericidal activity of released cecropin from capsulated particle was not appeared as our purpose.
The bactericidal activity of cecropin to Ph. damselae subsp. piscicida was powerful in vitro, with low working concentration and rapid effect. But cecropin was not stable to enzymatic digestion and easily to lost bactericidal activity in vivo. The death rate can significantly reduce unless we boost cecropin by i.p. injection or feed high dose cecropin concurrently to cobia infected by Ph. damselae subsp. piscicida.
| Identifer | oai:union.ndltd.org:TW/097NTU05312003 |
| Date | January 2009 |
| Creators | Ruei-Gu Chen, 陳瑞谷 |
| Contributors | 宋延齡 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 55 |
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