海水養殖面臨的疾病問題和流行疫情需要有系統的監測和研究來協助解決本文就海水養殖魚病進行病徵之研究。罹病之海鱺脾臟腫大並伴隨著白色結節，以組織病理切片技術觀察可見肉芽腫病變的生成。根據病徵推斷將細菌感染之器官，利用連續稀釋法(serial dilutions method)及藥物紙錠擴散法(disc diffusion method)進行細菌分離，再以MacConkey、TCBS及血液培養基三種培養基進行分析。以連續稀釋法分離菌株，在澎湖海鱺養殖場(THOD)分離出的革蘭氏陰性菌佔93% (119/128)，其中以弧菌檢出的比率佔57% (73/128)；小琉球海鱺養殖場(HDSB)分離出的革蘭氏陰性菌佔60% (54/90)，其中以弧菌檢出的比率僅12.2% (11/90)。恆春海鱺養殖場(EMD)分離出的革蘭氏陰性菌佔59% (61/104)，其中以弧菌檢出的比率佔70.2% (77/90)。而在不同時間分析石斑魚養殖場(PCG)之菌相，2003年11月主要分離出的革蘭氏陰性菌佔75% (104/139)，其中以弧菌檢出的比率最高有88% (123/128)；2004年11月主要分離出的革蘭氏陰性菌佔55% (24/44)，其中以弧菌檢出的比率佔73% (32/44)。另外以藥物紙錠擴散法進行分析，EMD海鱺養殖場之抗藥性菌株，以革蘭氏陰性菌最多，佔88% (66/75)，其中弧菌屬檢出的比率最高佔97% (73/75)。PCG石斑魚養殖場之抗藥性菌株，以革蘭氏陽性菌佔30% (9/31)，其中弧菌屬檢出的比率僅佔26% (8/31)。另外以變性梯度膠體電泳技術(denaturing gradient gel electrophoresis, DGGE)進行菌相分析，三場不同養殖場分出六大群，其中J6、R13、T29與FI02具有100%的相似度。以Quantity One Version 4.5 (Bio-Rad)分析兩種分離法之菌相，可分出六大群，其中F群幾乎都是利用連續稀釋法所篩選之菌株，而B群及E群皆由藥物紙錠擴散法所篩選出的菌株。在藥物敏感性實驗，對β-lactam類的抗生素已經有抗藥性菌的產生，對於quinolones類的抗生素仍具有感受性。

The fish disease epidemiology is urgent to be investigated for the surveillance and prevention. The diseased fish showed splenomegaly with diffusion of white nodules and microscopical granulomatous formation. It is important to develop a method of pathogens isolated from clinical samples with serial dilution method and disc diffusion method. Representative colonies were selected from diseased cobia on BHIA plate and were inoculated onto MacConkey agar, TCBS agar, and blood agar. The cage-culture of the different bacterial groups detected in the survey of bacteria isolated from THOD, HDSB, EMD with serial dilution method. 119 from 128 isolated strains were Gram’s negative (93%), including pathogenic Vibrio spp. 57% (73/128) in THOD. 54 from 90 isolated strains were Gram’s negative (60%), including pathogenic Vibrio spp. 12.2% (11/90) in HDSB. 61 from 104 isolated strains were Gram’s negative (59%), including pathogenic Vibrio spp. 70.2% (77/90) in EMD. In different times diseased grouper, 104 from 139 isolated strains were Gram’s negative (75%), including pathogenic Vibrio spp. 88% (123/139), in 2003. While 24 from 44 isolated strains were Gram’s negative (55%), including pathogenic Vibrio spp. 73% (32/44), in 2004. 66 from 75 isolated strains were Gram’s negative (88%), including pathogenic Vibrio spp. 97% (73/75) by disc dilution method in EMD. 9 from 31 isolated strains were Gram’s negative (30%), including pathogenic Vibrio spp. 26% (8/31), by disc dilution method of grouper in PCG. A DGGE (denaturing gradient gel electrophoresis) technique can identify six groups of bacteria from cobia, and J6, R13, T29 have similarity 100%. Quantity One Version 4.5 (Bio-Rad) can identify six groups of bacteria from diffusion methods that F group diluted the bacterial strain from serial dilution method. B group and E group diluted the bacterial strain from disc diffusion method. Higher resistance rates of the different bacterial strains isolated from cobia and were β-lactam and susceptible were observed in quinolones.

目錄

致謝 --------------------------------------------------------------------------- I
中文摘要 --------------------------------------------------------------------- II
Abstract ------------------------------------------------------------------------ III
目錄 --------------------------------------------------------------------------- IV
表目錄 ------------------------------------------------------------------------ VII
圖目錄 ------------------------------------------------------------------------ VIII
附錄 --------------------------------------------------------------------------- X
壹、前言
一、台灣海鱺箱網養殖概況------------------------------------------------------- 1
二、台灣養殖石斑魚概況---------------------------------------------------------- 4
三、魚類之細菌性疾病------------------------------------------------------------ 5
(一) 海水魚之弧菌（Vibrio spp.）----------------------------------------------- 6
(二) 產氣單胞菌（Aeromonas spp.）--------------------------------------------- 8
(三) 巴斯德桿菌（Photobacterium damsela spp. piscicida）---------------- 11
(四) 愛德華氏菌（Edwardsiella spp.）------------------------------------------ 12
(五) 鏈球菌（Streptococcus spp.）---------------------------------------------- 13
四、魚類細菌性疾病的檢驗診斷方法------------------------------------------- 14
(一) 生物型（biotype）之鑑定方法----------------------------------------------- 16
(二) 血清型（serotyping）之鑑定方法------------------------------------------- 17
(三) 基因型(genotype)之鑑定方法---------------------------------------------- 17
(四) 分子生物學鑑定方法-------------------------------------------------------- 18
五、細菌性疾病的處理及控制---------------------------------------------------- 20
(一) 抗生素之種類----------------------------------------------------------------- 21
(二) 抗生素之抑菌作用----------------------------------------------------------- 23
(三) 細菌的抗藥性----------------------------------------------------------------- 23
六、研究目的 ----------------------------------------------------------------------- 25
貳、材料與方法
一、病例收集及檢驗處理-------------------------------------------------------- 27
二、細菌分離------------------------------------------------------------------------ 27
(一) 連續稀釋法 ------------------------------------------------------------------ 27
(二) 藥物紙錠擴散法之建立----------------------------------------------------- 28
(三) 培養基之分析法-------------------------------------------------------------- 29
三、組織病理檢驗技術------------------------------------------------------------- 30
四、分離細菌對吳郭魚之人工感染試驗之致病性---------------------------- 31
(一) 實驗用魚之飼養-------------------------------------------------------------- 31
(二) 菌液之製備-------------------------------------------------------------------- 31
(三) 肌肉注射之人工感染實驗-------------------------------------------------- 31
(四) 不同劑量之人工感染實驗-------------------------------------------------- 32
(五) 組織病理學變化分析-------------------------------------------------------- 32
五、細菌型態及生理特性之分析 ---------------------------------------------- 33
(一) 細菌外形之觀察-------------------------------------------------------------- 33
(二) 革蘭氏染色-------------------------------------------------------------------- 3
(三) 細菌之耐鹽性----------------------------------------------------------------- 33
(四) 運動性-------------------------------------------------------------------------- 33
(五) 溫度耐受性實驗-------------------------------------------------------------- 33
(六) API 20E ---------------------------------------------------------------------- 34
(七) 細菌生長之情況-------------------------------------------------------------- 34
(1) 混濁度之測定法（OD600 nm）----------------------------------------------- 34
(2) 顯微鏡之觀察--------------------------------------------------------------- 34
(3) 菌落之形成------------------------------------------------------------------ 35
六、分子生物學之檢測------------------------------------------------------------- 35
(一) 抽取細菌DNA---------------------------------------------------------------- 35
(二) Genomic DNA濃度與純度測定------------------------------------------ 35
(三) 聚合酶連鎖反應 (Polymerase Chain Reaction) ------------------------ 36
(四) 引子 (primer)的設計-------------------------------------------------------- 37
(五) PCR-16S rDNA片段瓊脂膠體電泳--------------------------------------- 38
(六) PCR-16S rDNA片段純化與濃縮------------------------------------------- 39
七、變性梯度膠體電泳------------------------------------------------------------ 40
(一) 變性梯度膠體30%∼70%製作-------------------------------------------- 40
(二) 進行變性梯度膠體電泳----------------------------------------------------- 41
(三) SYBR greenⅠ螢光染色----------------------------------------------------- 41
(四) DGGE Fingerprinting pattern 統計分析----------------------------------- 42
八、藥物敏感性試驗---------------------------------------------------------------- 43
參、結果
一、病徵觀察及組織病理檢驗--------------------------------------------------- 44
二、內臟菌相分析------------------------------------------------------------------- 46
(一) 以培養基分析石斑魚場之菌相-------------------------------------------- 46
(二) 以培養基分析不同海鱺養殖場之菌相----------------------------------- 47
(三) 以培養基分析不同時間下石斑魚場之菌相----------------------------- 47
(四) 以培養基分析不同魚種之菌相-------------------------------------------- 47
(五) 以藥物紙錠擴散法分析抗藥性菌----------------------------------------- 48
三、細菌之致病性------------------------------------------------------------------- 48
(一) 致病菌之確認----------------------------------------------------------------- 48
(二) 致病劑量----------------------------------------------------------------------- 49
(三) 組織病理----------------------------------------------------------------------- 50
四、致病細菌之鑑定-------------------------------------------------------------- 51
(一) 細菌型態及生理特性-------------------------------------------------------- 51
(二) 生長情況----------------------------------------------------------------------- 52
(三) API 20E套組鑑定------------------------------------------------------------- 53
五、分子生物學方法的檢測------------------------------------------------------ 54
(一) 細菌16S rDNA序列分析--------------------------------------------------- 54
(二) 以DGGE進行不同養殖場菌相之檢測----------------------------------- 55
(三) 以DGGE進行不同魚種抗藥性菌相之檢測----------------------------- 56
(四) 以DGGE進行三場不同海鱺養殖場菌相之檢測----------------------- 57
(五) 以DGGE進行不同分離法菌相之檢測----------------------------------- 57
六、藥物敏感性試驗---------------------------------------------------------------- 58
(一) 不同海鱺養殖場抗藥性情形----------------------------------------------- 58
(二) 抗藥性菌之產生情況-------------------------------------------------------- 59
肆、討論
一、組織病理之檢驗診斷---------------------------------------------------------- 60
二、病徵病理的再現性------------------------------------------------------------ 62
三、細菌篩檢方法之建立---------------------------------------------------------- 65
四、PCR-DGGE應用在細菌檢測的探討---------------------------------------- 66
五、箱網養殖場之抗生素使用與抗藥性菌的產生---------------------------- 68
六、未來展望------------------------------------------------------------------------- 69
伍、參考文獻----------------------------------------------------------------------------- 71
陸、圖表 ---------------------------------------------------------------------------------- 89
柒、附錄 ---------------------------------------------------------------------------------- 131

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