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研究生:歐沛祿
研究生(外文):Ashaolu, opeolu Joseph
論文名稱:犬隻中螺桿菌屬菌株的分子監測及變形蟲一氧化氮合酶基因的特性分析
論文名稱(外文):Molecular Surveillance of Helicobacter Species in Dogs & Characterization of Nitric Oxide Synthase Gene in Amoebas
指導教授:嵇達德
指導教授(外文):Ji, Dar-Der
口試委員:嵇達德夏干泰
口試委員(外文):Ji, Dar-DerHsia, Kan-Tai
口試日期:2022-10-13
學位類別:博士
校院名稱:國立陽明交通大學
系所名稱:公共衛生研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2022
畢業學年度:111
語文別:英文
論文頁數:145
中文關鍵詞:幽門螺桿菌小狗腸肝感染變形蟲
外文關鍵詞:H. pyloriDogsgastricEnterohepaticinfectionsAmoeba
相關次數:
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摘要
第一項研究的目的是確定臺北市寵物狗及流浪狗感染幽門螺桿菌屬的流行和多樣性。收集來自城市不同地區的 95隻狗的糞便檢體,並使用幽門螺桿菌屬的調查巢式 PCR檢驗這些糞便檢體。結果顯示95 份糞便檢體中有72 份 (75.79%) 的 PCR 檢測呈陽性。檢出的菌種包括H. pylori (26.39%)、H. canicola (18.06%)、H. bilis (13.89%)、H. canis (27.78%)、H. typhlonius (4.17%)、H. winghamensis (2.77%)、H. cinaedi (1.39%)、H. heilmannii (1.39%)、H. canadensis (1.39%) 和 2 種未分類的螺桿菌 (2.77%) 考慮到狗的感染風險因素,則使用多項邏輯回歸分析,我們發現居住地區(北,南,東或西),生活狀況(寵物或流浪)和品種類型(純種或混種)是與感染螺桿菌屬的相關危險因素。我們並在建立演化樹後發現,我們發現的菌株與GenBank中的標準參考菌株之間存在分子演化關係。我們的結果總體顯示,臺北市犬隻中存在多種螺桿菌屬的菌株種類,最重要的是這些檢測到的種類與之前一些在人類宿主中發現的菌株種類相似。因此,建議需對寵物狗及流浪狗給予特別關注,以防止螺桿菌屬的菌株在這些動物與人類飼主之間可能發生的交叉傳播。
同樣,關於變形蟲等真核原生動物中一氧化氮合酶 (NOS) 的遺傳信息尚不可得。因此,我們首先對 Naegleria gruberi 和 Acanthamoeba lenticulata 中的 NOS 基因進行基因組分析。結果顯示在兩種變形蟲中都存在 NOS 樣基因。雖然 A. lenticulata 中的 NOS 樣基因同時具有由 3200 bp ORF 編碼的加氧酶和還原酶結構域,但在 N. gruberi NOS樣基因1200 bp的ORF中卻僅具有加氧酶結構域,缺乏還原酶結構域的對應物。本研究的顯示,N. gruberi NOS 結構域與在細菌中發現的NOS 結構域較相似,但 A. lenticulata NOS 基因則與高等真核生物中完整 NOS 基因結構相似。瞭解變形蟲中 NOS 的活性機制可能揭示 NO 在微生物中的多種新功能,並幫助我們瞭解未來控制變形蟲的機制。



關鍵字:幽門螺桿菌、小狗、胃、腸肝、感染、變形蟲
Abstract
The purpose of the first study is to ascertain the prevalence and diversity of Helicobacter spp. infections in pet and stray dogs of Taipei city. Stool samples from 95 dogs from the different region of the city, using nested PCR, were used in the investigation of Helicobacter spp. in these stool samples.
Our result indicated that 72 (75.79%) of the 95 stool samples were positive to the PCR test. The species detected include H. pylori (26.39%), H. canicola (18.06%), H. bilis (13.89%), H. canis (27.78%), H. typhlonius (4.17%), H. winghamensis (2.77%), H. cinaedi (1.39%), H. heilmannii (1.39%), H. canadensis (1.39%) and 2 unclassified Helicobacter species (2.77%). Considering the risk factors associated with infection in the dogs, using multinomial logistic regression analysis, we found that region of residence (North, South, East or West), the living status (Pet or stray) and breed type (Pure or mixed bred) are the risk factors associated with being infected with Helicobacter spp. We also found after reconstructing a phylogeny tree, that, there exist a molecular phylogeny between our strains and standard reference strains from GenBank. Our results generally reveal that there exist variety of Helicobacter species in dogs of Taipei city, and that most importantly, the species detected are similar to those that have been reported in human host in some previous studies. Therefore, suggesting that special attention be given to these group of animals in order to prevent possible cross transmission between these animals and human hosts.
Likewise, the genetic information about nitric oxide synthase (NOS) in eukaryotic protozoan such as amoebas are not yet available, we therefore characterize NOS genes in Naegleria gruberi and Acanthamoeba lenticulata as a first step. The result shows an existence of NOS-like gene in both amoebas. While the NOS-like gene in A. lenticulata possess both the oxygenase and reductase domain coded by a 3200 bp ORF, that of N. gruberi possess only the oxygenase domain and lack the reductase counterpart by a 1200 bp ORF. The implication of this study is that, while N. gruberi possesses NOS domain similar to those found in bacteria, A. lenticulata possesses full NOS gene-like structure similar to those found in higher eukaryotes. Understanding the mechanism of NOSs activity in amoeba will presumably unveil a variety of new functions of NO in microbes and help us with their future control mechanisms.

Key words: H. pylori, Dogs, Gastric, Enterohepatic, Infections, Amoeba.
TABLE OF CONTENTS
Abstract------------------------------------------------------------------------------------------------------------- i
List of Figures ----------------------------------------------------------------------------------------------------vii
List of Table------------------------------------------------------------------------------------------------------viii
List of abbreviations----------------------------------------------------------------------------------------------ix
1.0 INTRODUCTION-------------------------------------------------------------------------------------------1
2.0 LITERATURE REVIEW----------------------------------------------------------------------------------4
2.1 Specie Characterization---------------------------------------------------------------------------------------4
2.1.1 Validated species--------------------------------------------------------------------------------------------8
2.1.2 Animals with Gastric Helicobacter spp.-----------------------------------------------------------------8
2.1.2.1 Pig-Associated Gastric Helicobacters -----------------------------------------------------------------8
2.1.2.2 Gastric Helicobacters Linked to Dogs and Cats ----------------------------------------------------11
2.1.2.3 Rabbit-Associated Gastric Helicobacter--------------------------------------------------------------13
2.1.2.4 Ferret-related gastric Helicobacter --------------------------------------------------------------------13
2.1.2.5 Hamster-Related Gastric Helicobacter ---------------------------------------------------------------15
2.1.2.6 Gastric Helicobacter Associated with Ruminants --------------------------------------------------16
2.1.2.7 Stomach-borne Helicobacter pertaining to horses -------------------------------------------------16
2.1.2.8 Gastric Helicobacters Associated with Non-Human Primates------------------------------------17
2.2.0 Enterohepatic Helicobacters spp.-----------------------------------------------------------------------18
2.2.1 Animals with Enterohepatic Helicobacter spp.-------------------------------------------------------20
2.3.0 Species-based classification -----------------------------------------------------------------------------21
2.3.1 Humans and Helicobacter Pylori ----------------------------------------------------------------------21
2.3.2 Humans with non-H. pylori Helicobacter species --------------------------------------------------23
2.3.3 Animals with non-H. pylori Helicobacter species --------------------------------------------------25
2.4.0 Enterohepatic Helicobacter spp. in Humans --------------------------------------------------------28
2.4.1 Gastroenteritis--------------------------------------------------------------------------------------------28
2.4.2 Inflammatory Bowel Disorder ------------------------------------------------------------------------28
2.4.3 Hepatobiliary Disorder ---------------------------------------------------------------------------------29
2.4.4 Infections Outside the Digestive Tract ---------------------------------------------------------------30
2.5.0 Genomics and Evolution of Non-Helicobacter pylori Helicobacters----------------------------31
2.6.0 Helicobacter spp. Pathogenesis-----------------------------------------------------------------------32
2.6.1 Pathogenesis of Non-H. pylori diseases -------------------------------------------------------------35
2.7.0 Helicobacter Infections’ Epidemiology--------------------------------------------------------------39
2.8.0 Detection and Diagnosis--------------------------------------------------------------------------------40
2.8.1 Invasive Tests---------------------------------------------------------------------------------------------40
2.8.2 Non-Invasive Tests---------------------------------------------------------------------------------------42
3.0 MATERIAL AND METHODS ------------------------------------------------------------------------44
3.1.0 Animal Samples ------------------------------------------------------------------------------------------44
3.2.0 DNA extraction from stool samples--------------------------------------------------------------------45
3.3.0 Semi-nested PCR-----------------------------------------------------------------------------------------46
3.4.0 Sequencing and Phylogenetic Analysis----------------------------------------------------------------48
3.5.0 Analyzing Statistical Data ------------------------------------------------------------------------------47
4.0 RESULTS---------------------------------------------------------------------------------------------------49
4.1.0 Helicobacter species and Prevalence------------------------------------------------------------------49
4.2.0 16S rRNA sequence phylogenetic analysis ----------------------------------------------------------53
4.3.0 Helicobacter Infection Risk Factors-------------------------------------------------------------------55
5.0 DISCUSSION----------------------------------------------------------------------------------------------56
Part 2
6.0 CHARACTERIZATION OF NITRIC OXIDE SYNTHASE GENE IN AMOEBAS-----70
6.1.0 INTRODUCTION-------------------------------------------------------------------------------------70
7.0 MATERIALS AND METHODS----------------------------------------------------------------------74
7.1.0 Cell culture-----------------------------------------------------------------------------------------------74
7.1.1 DNA and RNA extraction from the Amoebas ------------------------------------------------------74
7.1.2 Amplification of NOS Genes--------------------------------------------------------------------------75
7.1.3 Signal peptide, cleavage location, and domain annotation ----------------------------------------75
7.1.4 Sequence Alignments-----------------------------------------------------------------------------------76
7.1.5 Phylogenetic Tree---------------------------------------------------------------------------------------76
7.1.6 Alignment and NOS Tree Generation ---------------------------------------------------------------77
7.1.7 Bioinformatics search-----------------------------------------------------------------------------------77
8.0 RESULTS -------------------------------------------------------------------------------------------------78
8.1.0 Sequence and structural analysis of the NOS-like proteins from the Amoebas-----------------79
9.0 DISCUSSION----------------------------------------------------------------------------------------------82
10.0 SUMMARY & CONCLUSION----------------------------------------------------------------------87
11.0 PERSPECTIVES & FUTURE WORKS ----------------------------------------------------------88
12.0 REFERENCES------------------------------------------------------------------------------------------89
APPENDICES-------------------------------------------------------------------------------------------------93
Appendix I: Sequences from our Helicobacter samples------------------------------------------------128
Appendix II: AlNOS region domain and sequences--------------------------------------------------------------132
Appendix II: NgNOS region domain and sequences-------------------------------------------------------------134
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