天蠶素防治海鱺巴斯德桿菌症之適用性評估

Abstract

巴斯德桿菌 （Photobacterium damsela subsp. piscicida） 是台灣箱網養殖海鱺 （Rachycentron canadum）主要的細菌性病原菌，感染力與致死率均高，以抗生素為主的治療方式已出現不少抗藥性的案例。天蠶素是一種發現已久的抗菌胜肽，由37個胺基酸組成簡單的雙極性阿法螺旋結構，具有廣效的殺菌作用。目前普遍認為其殺菌機制為膜電位喪失、裂解細菌細胞膜、滲透壓失調或胞質流失，是一種細菌不易產生抗性的殺菌機制。 天蠶素對巴斯德桿菌的最低抑菌濃度約為0.7 μM；天蠶素對巴斯德桿菌的最低殺菌濃度與半致死劑量經平板計數測定分別為0.9 μM和0.7 μM；天蠶素殺死巴斯德桿菌所需的時間經平板計數發現會隨著天蠶素濃度增加而縮短。利用錐藍排開法測定64 μM的天蠶素對海鱺肌肉細胞作用8小時才會產生細胞毒性；利用血紅素吸光值測定天蠶素對海鱺紅血球的半溶血效應濃度（effective concentration, EC50）遠大於1000 μM；上述結果顯示天蠶素對海鱺細胞傷害力低，會差異性選擇巴斯德桿菌而裂解。 以抑菌圈分析法發現海鱺胃粗萃取液不會影響天蠶素抑菌活性，但前腸的粗萃取液會使天蠶素活性與時遞減，作用兩小時後已偵測不出，顯示天蠶素在海鱺腸道中不穩定。天蠶素在血液中亦不穩定，推測可能是遭血中蛋白酵素的分解，這使得天蠶素將無法藉由血液送至表皮等感染點。 將天蠶素與巴斯德桿菌同時注射入海鱺腹腔，12小時後追加天蠶素，七天內海鱺存活率為100%，未注射天蠶素的感染組存活率為25%，統計分析顯示兩組存活率有顯著差異（p < 0.05）。海鱺同時注射天蠶素與巴斯德桿菌但未追加注射天蠶素組存活率為44%，未注射天蠶素的感染組存活率為17%，兩組存活率無顯著差異。天蠶素與巴斯德桿菌同時灌食，僅每尾魚（體重20 g）灌食2 mg天蠶素組的存活率（75%）與感染組存活率（0%）比較才有顯著差異（p < 0.05）。建議海鱺遭受巴斯德桿菌感染時，需即時給予高劑量天蠶素才能徹底殺菌、顯著減少死亡。包裹之天蠶素即便在腸道中釋放亦沒有預期殺死巴斯德桿菌的效果。 總結，天蠶素在離體實驗上殺菌劑量低且需時短，對巴斯德桿菌十分有效；但在活體實驗上對酵素不穩定易失去活性。除非受感染當下即時注射並追加天蠶素、或餵食高劑量天蠶素才可顯著降低海鱺被巴斯德桿菌感染所造成的死亡率。

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.