台灣養殖蝦類病原細菌Vibrio damsela 之分離與鑑定

Abstract

從1988年3月至10月，調查全省沿海蝦類養殖場，從中腸腺（hepatopanceas）蒼白，萎縮，黑變，結癤或肌肉白濁的草蝦（Penaeus monodon Fabricius）及砂蝦（Metapenaeus ensis）分離出弧菌（Vibrio)12株，經型態，生理，生化特性及遺傳物質（DNA）的分析鑑定為V.damsela。此菌為革蘭氏染色陰性短桿菌，具有端單鞭毛及無數的菌毛，可運動。鹽度可適應0.5－5. 5% NaCl。在室溫及37℃生長。有細胞色素氧化酵素。在無氧狀況下能醱酵葡萄糖產生氣體。有精氨酸去羧酸酵素（arginine de-carboxylase）。能水解酪蛋白（casein）。還原硝酸。分解幾丁質。無氧狀況下能分解右旋果糖(D－fructose），右旋半乳糖（D－galactose）及麥芽糖（maltose）並產生酸。對抗生素：Novobiocin,Chloramphenical,Tetracycline及0/129敏感。Voges-Proskauer試驗為陽性反應。不產生Indole。不利用檸檬酸。無法分解酪氨酸，明膠，澱粉。無氧狀況下不利用阿拉伯糖（arabinose) ，楊梅甘（arbutin) , 肌醇（inositol) ，乳糖（lactose) ，蘋果糖（malibiose) ，鼠李糖（rhamnose) ，水楊甘（salicine) ，山梨糖醇 (sorbitol) ，蔗糖（sucrose）並且不產生酸。 和V.damsela標準菌株（ATCC 33539）之DNA鹽基(bases）配對時；在61℃ 進行雜交結合反應（DNA hybridization），有64-93%的相似性，在73℃反應則有60–92%的相似性，將本地菌株M2G831利用導管從肛門插入至頭胸甲與腹節交接處做人工感染試驗顯示，5X10^5個菌落形成單位（濃度10^7CFU/ml，菌液注射50ul）就能使草蝦之中腸腺變蒼白產生萎縮現像，並可從該蝦之中腸腺再分離出該菌。組織病理顯示實驗感染之症狀與自然發病之症狀相似，腸道周圍浸潤多數的血球細胞，盲管組織細胞崩解並剝落於管腔中，繼而發展出膿瘍。本實驗証明V. damsela是引起草蝦及砂蝦病變之一種病原菌。

A Vibrio infection was found as the cause of mortality (91%) among the cultured tiger shrimp (Penaeus monodon Fabricius) and sand shrimp (Merapenaeus ensis) in Taiwan from March to October,1988. Affected shrimp displayed nonspecific signs including poor growth rate, anorexia, rough shell and milky musculature. In the hepatopancreas large numbers of short, curved bacterial rods were obserced , giving the injected tissue an pale and atrophic appearance. The 12 isolates of Vibrio were characterized by morphological, physiological and biochemical properties. They were found to represent very homogeneous group of bacteria thought to beolng to a single species. The cells were Gram negative. Single polar flagellum and numerous pili were observed. Visible colonies developed at 25 and 37℃. The NaC1 range for growth was from 0.5 to 5.5%. All isolates were positive for oxidase and arginine decarboxyease , but not tyrosinase. Gas was produced from glucose fermentatively . They were also unable to utilize citrate as a sole carbon source. All strains were proteolitic by degradation of casein but not gelatin. Nitrate was reduced to nitrite under anaerobic Condition .They were able to utilize some carbohydrates , for example , galactose , maltose , fructose and chitin , but not starch , arabinose lactose , malibiose ,rhamnose and sucrose - Growth of cells was inhibited by the prerence of antibiotics such as novobiocin , chloramphenical and tetracycline, so does 0/129. All isolates were positive for Voges-Proskauer test, but negative for tryptophanase through failure to produce indole. They were unable to utilize arbutin , inositol , sorbitol and salicine. DNA relatedness to reference strain (ATCC 33539) confirmed that the isolates are V. damsela. Experimental infections by anus intubation were produced in tiger and sand shrimps. It showed that 5 X 10 CFU/shrimp bacteria can induce hepatopancreatic pathology. Hemocytes infiltrated around gastrointestine . Hepatopancreatic tubules wrer necrotic and cell debris were observed in the lumen .This result suggest that V. demsela is one of the etiological agent of shrimp infections.