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研究生:鄭孝全
研究生(外文):Hsiao-Chuan Cheng
論文名稱:犬正常齒齦與牙周病齒齦和齒石之菌相調查
論文名稱(外文):Investigation of Cultivable Bacteria from Normal and Periodontal-Diseased Gingivae, and Dental Calculus in Dogs
指導教授:楊志寰
指導教授(外文):Chih-Huan Yang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:165
中文關鍵詞:細菌牙周病正常齒石齒齦
外文關鍵詞:bacteriaperiodontal diseasenormaldental calculusgingiva
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牙周病和齒石形成是常見於犬的口腔疾病,而口腔內細菌在形成齒石與牙周病的過程中扮演重要角色。因此,進行齒齦和齒石之菌相調查,應有助於瞭解正常齒齦至形成牙周病和齒石之口腔菌相變化,進而做為預防和治療之參考。本研究的目的,在調查正常齒齦和患有牙周病且有齒石形成犬之齒齦和齒石的菌相分布,期能比較彼此菌相之異同。所有採集之齒齦拭子和齒石樣本經處理和培養後,皆利用形態學、傳統和自動生化學鑑定等方法,進行菌種鑑定。未能由上述方式鑑定出之菌種,則以細菌16S rRNA基因定序方式做比對判定。本研究計整理18隻具正常齒齦犬和20隻患牙周病和齒石形成犬之齒齦或/和齒石樣本,分別鑑定出75株菌(含25屬48種)、99株菌(含33屬56種)和83株菌(含30屬55種)。由正常齒齦分離之好氣菌70株(93.3%)中,以革蘭氏陰性(G-)菌40株(57.1%)為多數,其中以巴士德桿菌(Pasteurella)17株(42.5%)為最多。鏈球菌(Streptococcus)10株(33.3%)為陽性(G+)菌(30株)最多者。5株厭氣菌是以梭菌(Clostridium)(3株)為主。自牙周病犬齒齦分離之好氣菌79株(79.8%),包括G-菌45株(57%)和G+菌34株(43%),分別以巴士德菌21株(46.7%)和鏈球菌8株(23.5%)為最多。厭氣性G-菌(10株)和G+菌(10株),各以Prevotella 7株(70%)和梭菌4株(40%)為代表。分離自齒石之好氣菌69株(83.1%),以巴士德菌10株(24.4%)佔G-菌(41株,59.4%)和屬G+菌(28株,40.6%)之棒狀桿菌(Corynebacterium)(10株,35.7%)的比例為最高。齒石厭氣菌(14株,16.9%)之G-菌(8株,57.1%)係以Prevotella 5株(62.5%)為多數。其厭氣性G+菌(6株)以梭菌(2株)和Lactobacillus fructivorans 2株(33.3%)為主。由本研究的結果顯示,三種樣本之好氣性G-菌,皆以巴士德菌為優勢菌屬,主要是Pasteurella dagmatis、P. canis/stomatis和P. pneumotropica等。G+菌之優勢菌屬除牙周病齒石以棒狀桿菌為主外,其餘皆為鏈球菌。而且,由分離之菌種比例可知,牙周病犬之齒齦和齒石之厭氣菌有明顯增多的趨勢,並以Prevotella為優勢菌屬。因此,犬口腔細菌的菌相消長和厭氣菌的比例增加,對犬牙周病和齒石形成之致病性值得進一步探討。
Periodontal disease and dental calculus buildup are common dental problems in dogs, and oral bacteria play an important role in the processing of periodontal disease and forming dental calculus. Therefore, the investigation of cultivable bacteria from the gingiva and calculus would be helpful for understanding the microbial population which shifts from normal to develop periodontal disease and buildup dental calculus, and could also be the reference for prevention and treatment of periodontal disease. The purpose of the study is to investigate and compare the distribution of bacteria in normal, and periodontal-diseased gingivae, with dental calculus of the dogs. After processing and culturing all of the gingival swabs and dental calculi, the cultivable bacteria were identified by morphology, traditional biochemistry methods and Biolog automated system. The bacteria, which could not be identified by the methods mentioned above, were tested by 16S rRNA gene sequencing method. Samples from 18 dogs that had normal gingival tissue, and 20 dogs suffering from periodontal disease with dental calculus buildup were collected. Totally, 75 (including 25 genera and 48 species), 99 (including 33 genera and 56 species), and 83 (including 30 genera and 55 species) bacterial strains were identified from normal gingiva, gingiva of periodontal disease, and dental calculus, respectively. In 70 (93.3%) normal gingival cultivable aerobes, Gram-negative (G-) bacteria are the main isolates in which Pasteurella spp. are the superior bacteria. Streptococcus spp. (10 strains, 33.3%) are the primary strains of Gram-positive (G+) aerobes. Clostridium (3 strains) is the main bacteria of 5 anaerobes from normal gingiva. 79 strains (79.8%) of aerobic bacteria are isolated from periodontal-diseased gingiva, including 45 (57%) G- and 34 (43%) G+ strains, in which Pasturella (21 strains, 46.7%) and Streptococcus (8 strains, 23.5%) are the most common isolates, respectively. Prevotella (7 strains, 70%) and Clostridium (4 strains, 40%) are the representatives of G- (10 strains) and G+ (10 strains) bacteria. The highest ratio of G- (41 strains, 59.4%) and G+ (28 strains, 40.6%) bacteria belongs to Pasturella (10 strains, 24.4%) and Corynebacterium (10 strains, 35.7%), respectively, from 69 aerobic isolates of dental calculus. In anaerobes (14 strains) from calculi, Prevotella (5 strains, 62.5%) of G- bacteria (8 strains) is the main type. The primary anaerobic G+ bacteria (6 strains) of dental calculus are Clostridium (2 strains) and Lactobacillus fructivorans (2 strains). The result of the study has shown that Pasturella is the superior genus of three studied groups in aerobic G- bacteria, and its most cultivable species are P. dagmatis, P. canis/stomatis and P. pneumotropica. Streptococcus is the superior aerobic G+ genus in the studied samples, except in the dental calculus, in which Corynebacterium is predominated. In addition, from the result of the ratio of the isolated species, the anaerobes in the gingiva and dental calculus of the periodontal-diseased dogs are increased significantly. Therefore, the shifts of oral bacterial populations and the increased ratio of anaerobes in dogs are worth for the further study on the pathogenesis of periodontal disease and dental calculus buildup.
誌謝…………………………………………………………………………Ⅰ
目錄…………………………………………………………………………Ⅱ
表次…………………………………………………………………………Ⅵ
圖次…………………………………………………………………………Ⅷ
附錄次………………………………………………………………………Ⅸ
中文摘要……………………………………………………………………Ⅹ
第一章 諸言…………………………………………………………………1
第二章 文獻探討……………………………………………………………4
第一節 口腔環境與細菌……………………………………………………4
一、 犬口腔之介紹…………………………………………………………4
(一) 犬之口腔結構與功能…………………………………………………4
(二) 犬之牙齒發生…………………………………………………………7
(三) 犬之牙周構造…………………………………………………………8
二、 口腔生態系……………………………………………………………12
(一) 口腔之菌種生態………………………………………………………12
(二) 口腔菌相之建立………………………………………………………14
(三) 影響口腔細菌生態之因素……………………………………………15
三、 牙科學之重要菌種……………………………………………………18
(一) 革蘭氏陽性菌…………………………………………………………18
(二) 革蘭氏陰性菌…………………………………………………………22
(三) 螺旋體…………………………………………………………………25
第二節 口腔細菌和牙菌斑形成之關係……………………………………25
一、 牙菌斑和齒石的形成…………………………………………………25
(一) 牙菌斑的組成…………………………………………………………26
(二) 牙菌斑形成的過程……………………………………………………28
(三) 影響牙菌斑堆積的原因………………………………………………30
(四) 齒石的形成……………………………………………………………31
第三節 牙周病之探討………………………………………………………34
一、 牙周病的形成和影響…………………………………………………34
(一) 牙周病之發生原因……………………………………………………34
(二) 牙周病之影響…………………………………………………………36
二、 口腔細菌在牙周病所扮演的角色……………………………………38
(一) 口腔細菌與牙周病……………………………………………………38
(二) 口腔細菌之致病產物…………………………………………………39
(三) 口腔細菌引起宿主之防衛反應………………………………………41
三、 牙周病之治療…………………………………………………………42
(一) 牙周病之外科手術治療………………………………………………42
(二) 牙周病之藥劑治療……………………………………………………43
四、 牙周病之預防…………………………………………………………45
(一) 犬口腔之居家照顧……………………………………………………45
(二) 犬口腔之專業牙科預防………………………………………………48
第四節 口腔細菌之鑑定方法………………………………………………49
一、 形態學觀察……………………………………………………………50
(一) 肉眼觀察………………………………………………………………50
(二) 顯微鏡之利用…………………………………………………………52
二、 生化學鑑定……………………………………………………………54
(一) 培養基之選擇…………………………………………………………55
(二) 生化試驗………………………………………………………………57
(三) O2之耐受性試驗………………………………………………………59
(四) Biolog自動菌種鑑定系統……………………………………………60
三、 免疫學診斷……………………………………………………………60
(一) 免疫螢光法……………………………………………………………61
(二) 酵素連結免疫吸附法…………………………………………………61
四、 分子生物學方法………………………………………………………62
(一) 核酸探針………………………………………………………………62
(二) 核糖體16S核糖核酸基因定序法……………………………………63
五、 厭氧菌之快速鑑定方法………………………………………………64
(一) 常用之厭氣菌選擇性培養基…………………………………………64
(二) 其它之厭氣菌初步鑑定法……………………………………………65
第三章 實驗材料與方法……………………………………………………68
一、 實驗材料………………………………………………………………68
二、 實驗方法………………………………………………………………68
(一) 預備實驗………………………………………………………………68
(二) 正式實驗………………………………………………………………70
(三) 菌種鑑定方法…………………………………………………………72
(四) 細菌16S rRNA基因序列定序方法……………………………………75
(五) 菌種保存方法…………………………………………………………78
第四章 實驗結果……………………………………………………………79
一、 預備實驗………………………………………………………………79
二、 正式實驗………………………………………………………………86
(一) 正常齒齦犬之菌相結果………………………………………………87
(二) 牙周病犬之齒齦菌相結果……………………………………………91
(三) 牙周病犬之齒石菌相結果……………………………………………95
(四) 16S rRNA基因序列定序法……………………………………………98
(五) 三種犬口腔來源之菌相和優勢菌屬…………………………………99
第五章 討論………………………………………………………………103
英文摘要……………………………………………………………………113
參考文獻……………………………………………………………………115
附錄…………………………………………………………………………132
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