臺灣博碩士論文加值系統

短螺旋體 (Brachyspira species) 造成豬赤痢與豬螺旋體性腸炎，主要的病原分別為Brachyspira hyodysenteriae與Brachyspira pilosicoli，主要感染生長至肥育期豬隻，此時乃是豬隻重要的增重階段，豬隻因發病導致採食量下降與飼料換肉率提高，並增加藥物治療費用而造成嚴重的經濟損失。過去研究中，豬赤痢所選用的第一線常用藥物如tylosin及lincomycin已具有80% 以上的抗藥性，而較為後線之抗菌劑如tiamulin感受性亦有下降的趨勢。但台灣尚無豬短螺旋體抗微生物藥物感受性試驗之研究資料，臨床治療時往往盲目投藥，為了治療與預防疾病及減緩病原菌抗藥性的產生，建立台灣短螺旋體對藥物感受性之基礎值有其必要性。
本研究首先參考其他文獻中培養短螺旋體的不同方法與溫度進行分離方法的最佳化，將14個以聚合酶鏈鎖反應檢測為豬赤痢陽性之樣本，以不同4種培養基、有無進行前處理及37℃或42℃下進行培養，共16種組合進行細菌分離，並統計分離率，同時設計一評分方法量化分離方法的優劣，結果CVSR培養基於42℃下培養，分離率最佳 (100%)，前處理有無可增加分離方法計分，降低分離的失誤率，但可能導致低病原量之樣本無法分離出細菌。
以前述實驗結果方法進行豬短螺旋體之分離與收集，病材來自國立嘉義大學雲嘉南動物疾病診斷中心之送檢病例及屠宰場收集之廢棄結腸，經細菌分離及菌種鑑定後，共分離55株B. hyodysenteriae、5株B. pilosicoli、11株B. murdochii，B. innocens、B. intermedia與未知Brachyspira sp. 各1株，挑選其中56株短螺旋體以21種抗微生物藥物進行最小濃度抑制試驗 (minimal inhibitory concentration)，以其結果評估及擬定治療藥物以控制豬赤痢，建議臨床上使用lincomycin及tiamulin作為首選抗菌劑，若其無效則嘗試以oxytetracycline與doxycycline來進行治療，或以gentamicin與離子型球蟲藥如monensin及salinomycin，但後兩類藥物者分別須注意其腎毒性與肌肉毒性而審慎使用。

Brachyspira species cause swine dysentery (SD) and porcine intestinal spirochetosis (PIS/PCS). The major pathogens are Brachyspira hyodysenteriae and Brachyspira pilosicoli, these diseases usually occur in growing to finishing pigs, the most important period of weight gaining. Severe economical losses caused by decrease of feed input and feed conversion ratio, costs of treatment and death. Using antibacterial agents can control the epidemic, but Brachyspira have been reported having high level resistance to commonly used first-line antibiotics such as tylosin and lincomycin in most international research. Moreover, the susceptibility of second-line antibiotics such as tiamulin has been decline in some countries. There isn’t any antimicrobial susceptibility and isolation information of swine Brachyspira in Taiwan at present and reluctantly blind dosing often make the epidemic situation worse.
This study was aimed to find out the optimal isolation method of Brachyspira in Taiwan. Using 4 different medium from references, with or without pre-treatment, and culture under 37℃ or 42℃ respectively. Total 16 different isolation methods were used to isolate Brachyspira from 14 PCR positive samples of Brachyspira hyodysenteriae. All of these methods, CVSR medium culture under 42℃ seems to be the best method which the isolative rate is 100%. Isolation after pre-treatment could reduce the probability of mistake, but may cause the false negative of some low bacterial quantity samples.
55 Brachyspira hyodysenteriae and 19 other species of Brachyspira be isolated, and 56 of them were used for antimicrobial susceptibility test. 21 antimicrobial agents were used in antimicrobial minimal inhibitory concentration test with agar dilution method. According to the result, using lincomycin and tiamulin as the first-line antibiotic to treat SD recommendly. If the treatment failed, administration of oxytetracycline, doxycycline, gentamicin and ionophores may be effective.

摘　　要 i
Abstract ii
目　　次 iii
表　　次 1
圖　　次 vii
第一章 緒論 1
第二章 文獻探討 2
第一節 豬短螺旋體的介紹 2
2.1.1 型態、生化特性及種類 2
2.1.2 疾病介紹 3
2.1.3 豬短螺旋體盛行率 4
2.1.4 短螺旋體診斷及鑑定 5
2.1.5 豬赤痢及豬螺旋體性腸炎的預防與控制 6
第二節 短螺旋體分離與抗微生物藥物感受性試驗 7
2.2.1 短螺旋體分離方法 7
2.2.2 短螺旋體的抗微生物藥物感受性試驗方法 8
2.2.3 赤痢短螺旋體及其他短螺旋體之藥物感受性試驗及抗藥性研究 9
第三節 抗微生物藥物對豬短螺旋體之藥效與藥動學 10
2.3.1 抗微生物藥物對豬短螺旋體之最小抑制濃度閾值 10
2.3.2 豬短螺旋體治療抗菌劑及其藥物動力學 10
第三章 材料與方法 12
第一節 豬短螺旋體分離方法最佳化 12
3.1.1 樣本 12
3.1.2 培養基製備 12
3.1.3 培養流程與多項因子培養 13
3.1.4 分離方法計分： 13
第二節 豬短螺旋體分離與鑑定 14
3.2.1 樣品來源 14
3.2.2 細菌分離 14
3.2.3 分子生物學鑑定 14
3.2.4 細菌保存 15
第三節 抗微生物藥物感受性試驗 15
3.3.1 試驗菌株 15
3.3.2 試驗之抗微生物藥物 16
3.3.3 培養基製備 16
3.3.4 細菌接種 16
第四節 統計與分析 17
3.4.1 豬短螺旋體分離最佳化 17
3.4.2 抗微生物藥物感受性試驗 17
第四章 結果 18
第一節 豬短螺旋體分離方法最佳化 18
4.1.1 分離率 18
4.1.2 分離方法計分 18
第二節 細菌分離與鑑定 18
第三節 抗微生物藥物感受性試驗 19
第五章 討論 21
第一節 豬短螺旋體分離方法 21
第二節 抗微生物藥物最小濃度抑制試驗方法 22
第三節 赤痢豬短螺旋體、結腸菌毛樣短螺旋體及其他豬短螺旋體抗微生物藥物 感受性試驗結果差異 23
第四節 豬短螺旋體對抗微生物藥物之抗藥性產生與感受性下降 23
5.4.1 巨環類藥物、林可黴素及泰妙素 24
5.4.2 健他黴素 26
5.4.3 四環素類藥物 27
5.4.4 青黴素類藥物 28
5.4.5 其他類藥物 29
第五節 藥物動力學與藥效學探討 31
第六章 結論 34
參考文獻 35
附錄 56
Appendix 1 Differentiation of six Brachyspira species by their hemolyis pattern on trypticase soy blood agar, biochemical reactions, and utilization of sugars. 56
表　　次
Table 1 Unofficial MIC break points of antimicrobial agents against Brachyspira spp. used from reference. 46
Table 2 Primers used to identify Brachyspira species in this study. 47
Table 3 MIC values of Brachyspira hyodysenteriae ATCC 27164 against 20 antimicrobial agents. 48
Table 4 Results of antimicrobial suspectibility testing of 56 Brachyspira spp. with 20 antimicrobial agents. 49
Table 5 Results of antimicrobial suspectibility testing of 37 B. hyodysenteriae with 20 antimicrobial agents. 50
Table 6 Results of antimicrobial suspectibility testing of 19 WBHIS with 20 antimicrobial agents. 51
Table 7 Correlation of antimicrobial resistance between macrolides and lincomycin in some specific isolates. 52
Table 8 Correlation of antimicrobial resistance between tiamulin and lincomycin in some specific isolates. 53
圖　　次
Fig. 1 Phylogenetic trees of Brachyspira spp. partial nox gene nucleotide sequences. 54
Fig. 2 Box-plot of 16 difference isolation method scores. 55

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