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研究生:洪嘉偉
研究生(外文):Chia-Wei Hung
論文名稱:溶藻弧菌與腸炎弧菌對養殖鋸緣青蟹致病性及病理研究
論文名稱(外文):Studies on the Pathogenesis and Pathology of Scylla serrata infected by Vibrio alginolyticus and Vibrio parahaemolyticus
指導教授:劉秉忠
指導教授(外文):Ping-Chung Liu
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:86
中文關鍵詞:鋸緣青蟹弧菌症溶藻弧菌腸炎弧菌病理學
外文關鍵詞:Scylla serrataVibriosisVibrio alginolyticusVibrio parahaemolyticusPathology
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本論文探討弧菌(Vibrio)對台灣養殖鋸緣青蟹(Scylla serrata)之致病性,並進行病理學研究。於夏天高水溫期間,鋸緣青蟹易感染弧菌症而造成死亡,所造成的症狀主要包含了螯足泛黃、肌肉白濁糜爛及水漾化、血淋巴顏色異常偏黃褐色及無法正常凝固、肝胰臟糜爛等。自罹病之鋸緣青蟹分離出病原菌,其中以 Vibrio parahaemolyticus 及 Vibrio alginolyticus 出現率最高,致死率亦最強。
本實驗挑選出強毒株Vibrio parahaemolyticus(HP1)及Vibrio alginolyticus(YH5)進行菌體及細胞外產物(ECP)半致死劑量試驗(LD50)。V. parahaemolyticus(HP1)菌體及其ECP 之LD50 分別為 2.24 × 105 CFU / g 及 0.15 μg protein / g carb body weight;V. alginolyticus(YH5)菌體及其ECP 之LD50 分別為 3.16 × 104 CFU / g 及 0.42 μg protein / g carb body weight。兩種弧菌在致死性試驗中,不論是菌體或ECP 皆對鋸緣青蟹造成快速死亡; V. alginolyticus(strain YH5)個體死亡平均時間分別約為180 分鐘及60 分鐘,而V. parahaemolyticus(strain HP1)則分別約為140 分鐘及50 分鐘。實驗中各死亡個體呈現類似外觀症狀,起初是眼柄縮回、對外來刺激無反應及個體癱瘓,部分有自割現象(autotomy),將垂死或剛死之個體進行內部組織觀察,大部分個體肝胰臟有糜爛現象,此外並無其他明顯病徵。
經菌體及ECP 攻擊後之垂死鋸緣青蟹以Davidson’s solution 固定,個別染色後進行組織病理觀察,在V. alginolyticus(strain YH5)
III
及 V. parahaemolyticus(strain HP1)菌體攻擊中,均可見到肌纖維變性壞死,有萎縮現象;於部分組織亦發現細菌叢,兩者不同的是於strain HP1 可見粉紅色亮面異常物質沉積。肝胰臟均可見微管細胞變性壞死,細胞輪廓消失或模糊不清;均可觀察到菌叢及大小不一紅色亮面物質沉積。在V. alginolyticus(strain YH5)及V. parahaemolyticus(strain HP1)之ECP 攻擊中,兩者均可見肌纖維變性壞死。而在肝胰臟方面,strain YH5 及 strain HP1 處理組均可見微管細胞變性壞死及空洞化,微管腔內上皮細胞脫落,兩者不同的是於strain YH5 處理組可見粉紅色異常物質沉積。本論文研究結果顯示Vibrio parahaemolyticus 及 V. alginolyticus 為鋸緣青蟹之重要病原菌,確實對養殖之鋸緣青蟹造成感染,並產生組織病理上的變化,但其致病機制及主要毒素仍尚未瞭解,有待更進一步的研究分析。
This thesis investigated the pathogenicity of Vibrio on mud crab (Scylla serrata) cultured in Taiwan together with and pathological of seuration. Scylla serrata was susceptible to Vibriosis leading to death with the symptoms including yellowing of chelating feet, whitish and erosion of muscle, abnormal in the colour (often yellowish brown) and coagulation hemolymph, erosion in hepatopancreas and so on. Bacteria were isolated from diseased Scylla serrata, with Vibrio parahaemolyticus and V. alginolyticus as the two dominant pathogenic species. In this study, virulent Vibrio parahaemolyticus (HP1) and V. alginolyticus (YH5) were selected for virulence challenge tests. The LD50 values of V. parahaemolyticus (HP1) bacteria and its extracellular products (ECP) were 2.24 × 105 CFU/g and 0.15 μg protein/g crab, respectively; and for V. alginolyticus (YH5), the LD50 values of values of bacteria and its ECP were 3.16 × 104 CFU/g and 0.42 μg protein/g crab, respectively. In the virulence tests, both of the two Vibrio species caused rapid death in the mud crab challenged with bacteria and ECP. The average time of each individual mud crab died caused by of V. alginolyticus (YH5) and its ECP were 180 minutes and 60 mins, respectively, while V. parahaemolyticus (HP1) were 140 mins and 50 mins, respectively. Similar symptoms on the mud crabs could be observed with eyestalk retraction in the beginning, followed by no response to external stimuli and individual paralysis, and some individuals with self amputation (autotomy). Erosion of the hepatopancreas was observed in most of the moribund and freshly died mud crab with no other obvious symptom.
Tissues of moribund crab after challenge with bacteria and ECP were fixed with Davidson’s solution for further histopathological
V
observation. Mud crab challenged with V. alginolyticus(YH5) and V. parahaemolyticus(HP1) exhibited muscle fibres necrosis and atrophy, the presence of bacteria cluster in some tissues, and decrease of blood lymphocytes in necrotic tissues. The pathological difference between the two of bacterial strains was that abnormal material deposition visualized as bright pink could be observed only in muscle challenged with strain HP1. In the hepatopancreas, cell degeneration and necrosis of microtubules were observed, together with disappearance on blur of cell contours and the deposition of red light bacterial cells with different sizes. Necrosis of muscle fibres could be observed in crab treated with ECP of both two Vibrio strains. Visible necrosis and vacuolization of microtubules and together with loss of epithelial cells of luman, and bright pink abnormal material deposition could only be visualized in samples treated with strain HP1. This study showed that V. parahaemolyticus and V. alginolyticus were important pathogens of Scylla serrata, which could cause infection during the culture of Scylla serrata. Further studies are needed to understand the pathogenesis of these two Vibrio species in mud crab.
謝辭……………………………………………………………………...I
中文摘要..................................................................................................Ⅱ
英文摘要………………………………………………………………..Ⅳ
目錄..........................................................................................................Ⅵ 表目錄......................................................................................................Ⅹ 圖目錄..................................................................................................ⅩⅠ
第一章 前言..............................................................................................1
第二章 文獻整理......................................................................................3
第三章 材料方法....................................................................................14
3.1 試驗菌株與試驗生物之來源.....................................................14 3.2 細菌生理生化特性之鑑定分析.................................................14 3.2.1 格蘭氏染色..........................................................................15 3.2.2 細胞色素氧化酵素..............................................................15 3.2.3 葡萄糖的利用......................................................................15 3.2.4 MacConkey agar 之生長能力..............................................16 3.2.5 對 NaCl 之耐受性試驗......................................................16 3.2.6 對 pH 之耐受性試驗........................................................ 16 3.2.7 Kanagawa phenomenon 試驗............................................ ..16 3.2.8 藥物敏感性試驗..................................................................17 3.2.9 溶血能力測定......................................................................17 3.2.10 API 20E system...................................................................18 3.2.11 Biolog system......................................................................18
3.3 聚合酶連鎖反應(PCR)...........................................................18
3.3.1 萃取DNA...........................................................................18 3.3.2聚合酶連鎖反應...................................................................19 3.3.3 洋菜膠膠體電泳..................................................................20 3.3.4 定序......................................................................................20 3.4 細胞外產物(ECP)之製備及分析......................................... 20 3.4.1 菌株強化..............................................................................20 3.4.2 細胞外產物製備..................................................................21 3.4.3 蛋白質含量測定..................................................................21 3.4.4 蛋白質分解酵素活性測定..................................................21 3.4.5 Vibrio parahaemolyticus 溶血活性測定............................ 22 3.4.6 Vibrio parahaemolyticus 之 tdh 及 trh 基因測定........... 22 3.5 Vibrio parahaemolyticus 對鋸緣青蟹之攻擊試驗及組織病理觀察…………………………………………………...………..….23 3.5.1 菌體對鋸緣青蟹攻擊試驗及組織病理觀察......................23 3.5.2 細胞外產物(ECP)對鋸緣青蟹攻擊試驗及組織病理觀.察…………………………………...………………...…....24 3.6 Vibrio alginolyticus(strain YH5)對鋸緣青蟹之攻擊試驗及組織病理觀察..................................................................................24 3.6.1 菌體對鋸緣青蟹攻擊試驗之組織病理觀察......................24 3.6.2 細胞外產物(ECP)對鋸緣青蟹攻擊試驗及組織病理觀察……...………………………………………………...…25 第四章 結果............................................................................................26 4.1 養殖現場之採樣調查及鑑定.....................................................26 4.1.1 養殖現場調查及鑑定..........................................................26 4.1.2 PCR 鑑定.............................................................................26
4.1.3 菌株活體毒力試驗..............................................................27 4.2 試驗菌株生理生化特性.............................................................27 4.3 Vibrio parahaemolyticus 之溶血特性及tdh、trh 檢測...........28 4.4 鋸緣青蟹之毒力攻擊試驗.........................................................29 4.4.1 Vibrio parahaemolyticus 菌體對鋸緣青蟹之攻擊試驗…..29 4.4.2 Vibrio alginolyticus 菌體對鋸緣青蟹之攻擊試驗..............29 4.4.3 Vibrio parahaemolyticus 細胞外產物對鋸緣青蟹之攻擊試驗…………………………………………………………....30 4.4.4 Vibrio alginolyticus 細胞外產物對鋸緣青蟹之攻擊試驗..30 4.5 組織病理之觀察……………………………………………….30 4.5.1 毒性攻擊試驗之死亡個體外觀症狀……………………..30
4.5.2 Vibrio parahaemolyticus 菌體對鋸緣青蟹之組織病理觀察……………………………………………………...…….31 4.5.3 Vibrio alginolyticus 菌體對鋸緣青蟹之組織病理觀察.....31 4.5.4 Vibrio parahaemolyticus 細胞外產物對鋸緣青蟹之組織病理觀察…………………………………………...………...31 4.5.5 Vibrio alginolyticus 細胞外產物對鋸緣青蟹之組織病理觀察……………………………………………………...…...32 第五章 討論……………………………………………………...….....33 5.1 現場採樣結果分析……………………………………...……. 33 5.2 細菌基礎特性及鑑定………………………………………….35 5.3 Vibrio parahaemolyticus 溶血活性探討……………………….36 5.4 對鋸緣青蟹之攻擊試驗…………………………………….....38 5.5 病理組織觀察…………...……………………………………..39 參考文獻………………………………...……………………………...42
表………………………………………….…………………...............54 圖……………………………………….……………………...............65 附錄…………………………………..…………………………..........85
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