臺灣博碩士論文加值系統

嗜水性產氣單胞菌(Aeromonas hydrophila)一般認為是屬於二次性感染原，或者是機緣性病原菌(opportunistic pathogen)，但對甲魚而言是重要病原菌。甲魚被本菌感染時，臨床上會造成食慾減退、衰弱伸頸、頸部腫大、口鼻出血、腹甲潮紅、肌肉水腫、出血性胃腸炎及敗血症等症狀而死亡，造成經濟上的損失。本研究以甲魚感染 A. hydrophila 引起嚴重之出血性胃腸炎和敗血症之菌株，進行分離純化、生化鑑定及其細胞外產物致病性之研究。首先自南區魚病中心民國八十八年至九十一年罹病甲魚所分離之 A. hydrophila 菌株中選出 10 株及參考菌株(ATCC 7966)以傳統之形態學、生化特性及 API 20NE、Biolog GN2 系統作鑑定之後，再選取臨床上引發出血性胃腸炎和敗血症之菌株，經由人工接種進行病原性之比較，選取病原性強之菌株 AR-01-359 進行試驗。首先進行以該菌株之菌體、細胞外產物及粗純化的細胞外產物進行腹腔內注射試驗，求得對甲魚之半致死劑量(LD50)分別為 1.04 × 104 CFU/ g (體重)、5.952μg/ g (體重)及 3.08 μg/ g (體重)。將 AR-01-359 菌株之細胞外產物經硫酸銨鹽析，再經疏水性層析法(hydrophobic interaction chromatography)及陰離子交換層析法(anion exchange chromatography)等步驟進行分離純化。純化蛋白分解酵素(protease)，由 SDS-PAGE 膠體電泳測得分子量約為 28 kDa。所得的蛋白分解酵素屬於熱安定性，不具有澱粉酶、明膠酶和磷脂酶等活性，而具有酪蛋白酶、乙醯膽鹼酶活性。本酵素可被EDTA-2Na、CaCl2、HgCl2、ZnCl2及KMnO4 等抑制，顯示蛋白分解酵素屬於金屬型蛋白分解酵素(metalloprotease)。本酵素在 pH 10 及 70 ℃時具有最大酵素活性，3.67 μg/ g (體重)時能使甲魚迅速死亡。本酵素對甲魚、吳郭魚、加州鱸、綿羊及紐西蘭兔血球之溶血活性分別為 109、218、435、218及871 units/ mg，證實本酵素對甲魚血球具有溶血效果。以兔子迴腸結紮試驗證實本酵素可造成腸管大量出血及積水之腸毒素病變，出血程度與供毒劑量成正相關性。本試驗成果可供甲魚 A. hydrophila 致病機制研究、疫苗開發及疾病防治之參考。

Although Aeromonas hydrophila has been considered as an opportunist or secondary pathogen in stressed animals or immunocompromised hosts, it is recognized as an important pathogenic microorganism for hemorrhagic septicemia of soft-shelled turtle.
The clinical signs of infected soft-shelled turtles are lack of appetite, swollen and weakness of neck, hemorrhages in oral and nasal cavities, breastplate, muscular edema, hemorrhagic gastro-enteritis and septicemia. Infection by this microorganism can lead into a serious mortality and thus causes a great economic loss in the soft-shelled turtle industry. Ten strains of A. hydrophila were randomly selected from the Southern Regional Fish Disease Center during 1999 to 2002, together with the type strain of A. hydrophila (ATCC 7966) for confirmation study. The conventional morphological and biochemical methods as well as API 20NE system and Biolog GN2 system were used to identify of these strains. In this study the virulence factor of A. hydrophila to soft-shelled turtle and its biochemical characterization were studied. The strain AR-01-359 of A. hydrophila was chosen as a candidate due to it produced the most serious symptoms and lesions by pathogenicity screening test. The 50 percent lethal dose (LD50) of the live bacterial suspension, the crude extracellular product (ECP), and the ammonium sulfate purified ECP were 1.04×104 CFU/ g (body weight, BW), 5.952μg/ g (BW) and 3.08μg/ g (BW) of soft-shelled turtles, respectively. The crude ammonium sulfate precipitated ECP was further purified by various steps of hydrophobic interaction chromatography and anion exchange chromatography. The molecular mass of the final purified product was 28 kDa as estimated by SDS-PAGE. The resultant product was identified as a protease with acetylcholinesterase and caseinase activities. It was inhibited by EDTA-2Na (ethylenediamine tetraacetic acid disodium salts), CaCl2, HgCl2, ZnCl2 and KMnO4, thus it was further concluded as a metalloprotease. This purified protease showed the maximal activity at pH 10 and 70 ℃, and was lethal to soft-shelled turtle with 3.67 μg protein/g (BW). The hemolytic activities of this protease against erythrocytes of soft-shelled turtle, tilapia, largemouth bass, sheep and rabbit were 109, 218, 435, 218 and 871 units/ mg protein, respectively. Results of the rabbit ileal loop assay were demonstrated that there were appositive relationship between the severity and the dosages in the hemorrhagic intestinal lesions.

目 錄
中文摘要………………………………………………………… Ⅰ
英文摘要………………………………………………………… Ⅲ
誌謝……………………………………………………………….. Ⅴ
目錄……………………………………………………………….. Ⅵ
圖表索引....…………………………………………………….. ⅩI
第1章 緒言…………………………………………………… 1
第2章 文獻回顧……………………………………………… 3
2.1中華鱉之簡介…………………………………………....……3
2.1.1台灣中華鱉養殖狀況….………………………………….. 3
2.1.2分類及型態….…………………………………………….. 3
2.1.3生理特性…………………………………………………... 4
2.2嗜水性產氣單胞菌之性質..…………………..….………… 5
2.2.1嗜水性產氣單胞菌之歷史沿革…………………………... 5
2.2.2Aeromonas 屬之分類...………………………………... 6
2.2.2.1外膜蛋白分類法………….…………………………. 7
2.2.2.2以 DNA 分類……………………………………….. 7
2.2.2.3血清學分類法……………………………………….. 8
2.2.2.4脂肪酸成分及比例鑑定法…………………………... 9
2.3嗜水性產氣單胞菌之形態、生理及生化特性……………… 9
2.4嗜水性產氣單胞菌之致病性…..…………............... 12
2.4.1嗜水性產氣單胞菌對養殖動物之病例與病徵...…….…. 13
2.4.2嗜水性產氣單胞菌對動物之病例與病徵………………... 14
2.4.3嗜水性產氣單胞菌對人類之病例與病徵………………... 14
2.4.4嗜水性產氣單胞菌之抗藥性……………………………... 15
2.5嗜水性產氣單胞菌之致病機制…………….....………... 16
2.5.1細胞表層結構致病因子…………………………………... 16
2.5.2細胞外毒素之致病因子…………………………………... 17
2.5.2.1溶血素(Hemolysin)...………………………….….…. 18
2.5.2.2蛋白酶(Protease)……………………………..………. 20
2.5.2.3磷脂酶(Phospholipase)...………….………..……… 21
2.5.2.4甘油磷脂質膽固醇醯基轉移酶(glycerophospholipid-cholesterol acyltransferase, GCAT)………………….…… 21
2.5.2.5乙醯膽鹼酯酶(Acetylcholinesterase, AcChe).…… 22
2.5.2.6腸毒素(Enterotoxin)………………….…….……... 22
2.5.2.7細胞毒素(Cytotoxin)………………….…...………. 23
第3章 材料與方法……………………………….…………...24
3.1菌株來源……………………………………………………... 24
3.1.1菌株的分離………………………………………………... 24
3.1.2菌株的活化及強化毒性…………………………………... 24
3.2供試甲魚來源……………………….……………………... 25
3.3菌株之生化特性鑑定………………….…………………... 25
3.3.1革蘭氏染色………………………………………………... 25
3.3.2細胞色素氧化酵素(Cytochrome oxidase)試驗………… 26
3.3.3觸酶(Catalase)試驗…………………………….....….. 26
3.3.4吲哚試驗(Indole test)...………………...………….. 26
3.3.5葡萄糖的氧化與發酵反應(O/F test)試驗………..……. 26
3.3.6MR-VP試驗………………………………………………. 27
3.3.7尿素酶試驗(Urease test)…..………………..……... 27
3.3.8檸檬酸鹽(Simmons Citrate)的利用…………...…….. 27
3.3.9七葉靈的水解能力(Esculin hydrolysis test)……….. 27
3.3.10溶血性測定(Haemolytic test)..……………..……... 28
3.3.11在MacConkey agar之生長能力………………………….. 28
3.3.12對NaCl耐受性試驗………………………………………. 28
3.3.13API 20NE system 之生化學鑑定………………………… 28
3.3.14BIOLOG GN2 system之生化學鑑定…………………….. 29
3.3.15API-ZYM system之酵素特性鑑定………….…………… 29
3.3.16菌株對甲魚的毒性試驗………………………………... 29
3.4細胞外產物之製備及基本特性分析………………………... 30
3.4.1細胞外產物(Extracellular products, ECP)之製備…. 30
3.4.2細胞外產物基本特性分析………………………..…….. 30
3.4.2.1蛋白質含量測定……..……………………….………...30
3.4.2.2蛋白分解酵素(Protease)活性測定…………..….…… 31
3.4.2.3溶血活性測定(Haemolytic activity test).…..…… 31
3.4.2.4乙醯膽鹼酵素測定……………………….…………... 31
3.4.2.5對酪蛋白(Casein)的水解能力…….…….…...…….. 32
3.4.2.6對明膠蛋白(Gelatin)的水解能力…….…...……….. 32
3.4.2.7對卵磷脂(Lecithin)的水解能力…………...………. 32
3.4.2.8細胞外產物對甲魚的毒性試驗………….…………... 34
3.5細胞外產物中蛋白分解酵素之純化與特性…….………... 34
3.5.1細胞外產物中蛋白分解酵素之純化分離…….………... 34
3.5.1.1硫酸銨沉澱法(Ammonium sulfate precipitation)... 34
3.5.1.2疏水性層析法(Hydrophobic interaction chromatography, HIC)………………..............……..…..……… 35
3.5.1.3陰離子交換層析法－Q Sepharose High Performance管柱層 析……………………….......……………………... 35
3.5.2電泳與染色………………………………………………... 35
3.5.3蛋白分解酵素之特性分析……………………….……... 37
3.5.3.1蛋白分解酵素之熱安定性試驗及最適溫度…….…... 37
3.5.3.2蛋白分解酵素之最適pH值………………….………. 37
3.5.3.3抑制劑對蛋白分解酵素之影響…………….………... 38
3.5.3.4乙醯膽鹼酵素測定…………………………….……... 38
3.5.3.5溶血素之活性測定(溶血力價、溶血活性)…….…… 38
3.5.3.6兔子迴腸結紮試驗………………………….………... 39
第4章結果………………………………………………………… 40
4.1菌株的生化特性…………………………………..……….. 40
4.1.1菌株的分離與保存………………………………………... 40
4.1.2.試驗菌株之生化特性…………………………………... 40
4.1.3API-ZYM system 之酵素特性鑑定……………………… 41
4.1.4.菌株對甲魚的毒性試驗………………………………... 41
4.2細胞外產物基本特性分析………………….………….….. 47
4.2.1細胞外產物對甲魚的毒性試驗……......……………... 47
4.3細胞外產物之純化製備及基本特性分析…….…………... 52
4.3.1硫酸銨沉澱法………....…………….………………….. 52
4.3.1.1粗純化的蛋白分解酵素對甲魚的毒性試驗…….….. 52
4.3.2疏水性層析(Hydrophobic interaction chromatography)52
4.3.3陰離子交換層析(Q Sepharose High Performance chromatography)………….……...………………………… 54
4.3.4分子量的測定……………………………………………... 54
4.3.5蛋白分解酵素之最適溫度、熱安定性及最適pH值……... 54
4.3.6抑制劑對蛋白分解酵素之影響…………………………... 58
4.3.7乙醯膽鹼酵素及溶血素之活性測定……………………... 58
4.3.8兔子迴腸結紮試驗………………………………………... 58
第5章討論………………………………………………………… 63
參考文獻………………………………………………………….. 69
作者簡介………………………………………………………….. 97
圖表索引
圖1.將純化菌株調製成懸浮液接種於甲魚，瀕死的甲魚經剖檢後可見腸道充出血，腸管漿膜面可見明顯潮紅……......……… 48
圖2.出血性壞死性腸炎，在小腸粘膜可見明顯出血及壞死，壞死的黏膜上皮脫落於腸腔中(H＆E stain, 100X) ……...…… 48
圖3.將細胞外產物接種於甲魚，瀕死的甲魚經剖檢後可見腸管嚴重出血性的病變.…………………….………………...……. 50
圖4.將細胞外產物接種於甲魚，瀕死的甲魚經剖檢後可見腸管漿膜面明顯潮紅……….………….………………………...…. 50
圖5.將細胞外產物接種於甲魚，肌肉可見到明顯的充出血及水腫變化(H＆E stain, 100X)…..………….……….………….... 51
圖6.將細胞外產物接種於甲魚，腸管漿膜面有大量纖維素性滲出物覆蓋(H＆E stain, 100X)……………………..………….. 51
圖7.在FPLC-疏水性層析法中以1 M ammonium sulfate in 50 mM phosphate buffer (pH 7)平衡管柱。1至0 M ammonium sulfate降低鹽梯度之方式進行分離純化………………........….. 53
圖8.在FPLC-陰離子交換層析法中以20 mM Tris (hydromethyl)methylamine buffer (pH 8)平衡管柱。以1M NaCl in 20 mM Tris buffer (pH 8)，拉高鹽度梯度方式由0 M NaCl 至1 M NaCl進行分離純化………….………………....................... 56
圖9.甲魚注射HIC管柱層析分離出第3個波峰之蛋白分解酵素，會迅速造成甲魚死亡，並造成注射部位、泄殖腔充出血………………………………………….………………….. 56
圖10.甲魚注射HIC管柱層析分離出第3個波峰之蛋白分解酵素，會迅速造成甲魚死亡，並造成腹腔出血及肌肉充出血........ 56
圖11.SDS-PAGE進行Coomassie brilliant blue R250 stain。M: marker protein (Invitrogen)。Lane1: 細胞外產物。Lane2: 經疏水性層析法具蛋白分解酵素活性且會造成甲魚死亡之細胞外產物。Lane3: 經陰離子交換層析法具蛋白分解酵素活性且會造成甲魚死亡之細胞外產物……………………... ............................57
圖12.蛋白分解酵素與HPA混合並溶於PBS中，分別於5、10、20、30、40、50、60、70、80℃等溫度作用10分鐘，測定各溫度殘存蛋白分解酵素活性………………………….................... 59
圖12-1.純化蛋白分解酵素分別於pH 範圍3.0-10.0之溶液，於37℃水浴槽中作用1小時，然後測定蛋白分解酵素活性…..….. 59
圖12-2.純化蛋白分解酵素分別於5、10、20、30、40、50、60、70、80℃等溫度作用30分鐘，然後移入4℃水浴槽內冷卻，測定各溫度殘存蛋白分解酵素活性……………………...........…... 59
圖13.兔子迴腸結紮試驗，將蛋白分解酵素以不同濃度184、92、46、23、11.5、5.75μg與生理食鹽水( 對照組 )，分別注入於迴腸腔內。取出腸管觀察出血與腸腔積液的情形，兔子的腸出血程度與攻毒劑量成正相關性……………...........……............... 62
圖14.兔子迴腸結紮試驗在病理組織切片下，1-3組可見到出血性壞死性腸炎病變(H＆E stain, 100X)………….....………….. 62
表1.Characteristics for differentiation of Aeromonas species….......................................… 11
表2.Pathogenic extracellular factors elaborated by A. hydrophila.................................... 19
表3.乙醯膽鹼酵素測定之標準曲線製作……………………... 33
表4.快速電泳系統之SDS-PAGE之製備………………………. 36
表5.A. hydrophila分離菌株AR-01-359及ATCC 7966株之生化特性…………………………………………………………... 42
表6.A. hydrophila分離菌株AR-01-359及ATCC 7966株API 20NE鑑定試驗…………………………………………….......…. 43
表7.A. hydrophila分離菌株AR-01-359及ATCC 7966株之耐鹽試驗…………….………………………………………….. 44
表8.A. hydrophila分離菌株AR-01-359及ATCC 7966株之API ZYM鑑定試驗………………………………………………............. 45
表9.A. hydrophila分離菌株AR-01-359之甲魚腹腔接種試驗… 46
表10.A. hydrophila分離菌株AR-01-359之細胞外產物之水解試驗……………………………………………....……………… 49
表11.A. hydrophila分離菌株AR-01-359之未純化細胞外產物之甲魚接種試驗……………….………………………...……….. 49
表12.A. hydrophila分離菌株AR-01-359之粗純化蛋白分解酵素之甲魚接種試驗……………………………………...………... 53
表13.A. hydrophila分離菌株AR-01-359之純化蛋白分解酵素結果…………………………………………….……………….. 55
表14.抑制劑對分離菌株AR-01-359純化蛋白分解酵素之作用… 60

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