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研究生:蔡宜倫
研究生(外文):Yi-Lun Tsai
論文名稱:台灣和金門地區蜱之鑑定
論文名稱(外文):The Identification of Ticks in Taiwan and Kinmen Island
指導教授:林子恩林子恩引用關係
指導教授(外文):James A. Lin
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:英文
論文頁數:100
中文關鍵詞:蜱(壁虱)人畜共通形態學鑑定粒線體單股構型多態性基因定序
外文關鍵詞:tickzoonosismorphological identificationmitochondriasingle strand conformation polymorphism (SSCP)sequence
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蜱種是人畜共通疾病的重要病媒,其中最為人所知的包括有tick-borne relapsing fever、Rocky Mountain spotted fever、Q fever 和 Lyme disease等。本研究目的,在建立台灣和金門地區蜱種形態學和分子鑑定的方法。在形態學鑑定之後,使用單股構型多態性(single-strand comformation polymorphism;SSCP)將12S rDNA、16S rDNA和cytochrome oxidase subunit I (COI)的粒線體基因片段進行分析,之後再將個別的聚合酶鏈鎖反應(polymerase chain reaction;PCR)產物進行序列分析,以確認單股構型多態性的分析結果和種系發生的分類。
形態學的鑑定結果,在台灣和金門地區共發現十種蜱種,包括:Rhipicephalus sanguineus Latreille, 1806, Rhipicephalus pumilio Schulze, 1935, Rhipicephalus haemaphysaloides Supino, 1897, Boophilus microplus Canestrini, 1887, Ixodes granulatus Supino, 1897, Amblyomma geoemydae Cantor, 1847, Haemaphysalis warburtoni Nuttall, 1912, Ixodes acutitarsus Karsch, 1899, Haemaphysalis phasiana Saito, Hoogstraal & Wassef, 1974 and Haemaphysalis campanulata Wargurton, 1908。
單股構型多態性的分析無法區分單一物種的序列差異(haplotype),無法明確的由16S和12S rDNA 的基因片段區分出R. sanguineus和R. pumilio,而且也無法明確的由12S rDNA的基因片段區分出R. sanguineus和R. haemaphysaloides。
依據43隻蜱的16S rDNA 基因片段序列的相似度和演化樹枝圖可知,在17隻蜱(包括8隻R. sanguineus和9隻R. pumilio)和文獻中R. sanguineus 的標準序列(NCBI)有95.1%~97.3%的相似度;在12隻蜱(包括2隻R. sanguineus和10隻R. haemaphysaloides)和文獻中R. sanguineus的標準序列有91.6%~93.8%的相似度;在11隻B. microplus和文獻中B. microplus的標準序列有95.2%~100%的相似度;在3隻I. granulatus和文獻中I. granulatus的標準序列有97.6%~98.3%的相似度。而18隻蜱的12S rDNA 基因片段序列的分析中,在3 隻R. haemaphysaloides中有98.8%的相似度;在8 隻蜱(包括3隻R. sanguineus and 5隻R. pumilio)中有98.7%的相似度;在4 隻B. microplus中有95%的相似度;在3 隻I. granulatus中有99.1%的相似度。
在本次研究中,台灣和金門地區的蜱種R. sanguineus,其序列的表現有顯著的差異。R. sanguineus和R. pumilio雖然可依形態學將之區分,但在16S、12S rDNA和COI的粒線體基因片段,以單股構型多態性分析並不能將之完全區分,而且在16S和12S基因片段的定序結果也非常相近,因此在未來的研究上,尋找可靠的分子標記以區分R. sanguineus和R. pumilio這兩種蜱種是相當必要的。
Ticks are important vectors of human and animal diseases. Among the best-known human diseases transmitted by ticks are tick-borne relapsing fever, Rocky Mountain spotted fever, Q fever and Lyme disease. The aim of this study was to establish morphological and molecular identification methods for ticks from Taiwan and Kinmen island. After morphological identification, single strand conformation polymorphism (SSCP) was used to screen the samples for genetic variation within segments of the 12S rDNA, 16S rDNA, and cytochrome oxidase subunit I (COI) mitochondrial genes. Sequencing of individual polymerase chain reaction (PCR) product was completed to verify SSCP designations and phylogenetically classify tick species.
By the results of morphological examination, ten species of tick were identified from Taiwan and Kinmen island including Rhipicephalus sanguineus Latreille, 1806, Rhipicephalus pumilio Schulze, 1935, Rhipicephalus haemaphysaloides Supino, 1897, Boophilus microplus Canestrini, 1887, Ixodes granulatus Supino, 1897, Amblyomma geoemydae Cantor, 1847, Haemaphysalis warburtoni Nuttall, 1912, Ixodes acutitarsus Karsch, 1899, Haemaphysalis phasiana Saito, Hoogstraal & Wassef, 1974 and Haemaphysalis campanulata Wargurton, 1908.
SSCP failed to differentiate sequence variants (haplotypes) within single taxa. Additionally, SSCP failed to clearly differentiate R. sanguineus and R. pumilio for both the 16S and 12S rDNA fragments examined. SSCP of the 12S fragment could also not clearly differentiate R. sanguineus and R. haemaphysaloides.
According to the sequence similarity and dendrogram constructed by 16S rDNA segments of 43 ticks, there are 95.1%~97.3% similarity between 17 ticks (including 8 R. sanguineus and 9 R. pumilio) and R. sanguineus reference strand (in Genbank of National Center for Biotechnology Information (NCBI)), 91.6%~93.8% similarity between 12 ticks (including 2 R. sanguineus and 10 R. haemaphysaloides) and R. sanguineus reference strand, 95.2%~100% similarity between 11 B. microplus and B. microplus reference strand, and 97.6%~98.3% similarity between 3 I. granulatus and I. granulatus reference strand. For the 12S rDNA analysis of 18 ticks, the similarity of 3 R. haemaphysaloides was 98.8%, the similarity of 8 ticks (including 3 R. sanguineus and 5 R. pumilio) was 98.7%, the similarity of 4 B. microplus was 95%, and the similarity of 3 I. granulatus was 99.1%
In this study, the ticks of R. sanguineus from Taiwan and Kinmen island represent significantly different sequence. Although R. sanguineus and R. pumilio could be identified by morphology, they could not be completely differentiated by SSCP of the 16S rDNA, 12S rDNA and COI genes. After sequencing, the fragments on 16S and 12S rDNA amplified from R. sanguineus and R. pumilio were very similar and in some cases identical. Future work is required to identify reliable molecular markers to differentiate between these two species.
1. Introduction
1.1 Systematic Relationships and Life Cycles of Ticks
1.1.1 Ticks and Their Relation to Other Arachnida
1.1.2 General Characteristics of Ticks
1.1.3 Life Cycles of Ticks
1.2 Significance of Ticks and Tick-borne Diseases
1.3 Ticks and Tick-borne Diseases in Taiwan
1.3.1 Ticks in Taiwan
1.3.2 Tick-borne Diseases in Taiwan
1.4 Morphological Identification
1.5 Molecular Identification
1.5.1 Molecular Techniques Used in Tick Genetics
1.5 2 Mitochondrial DNA of Ticks
1.5.3 Single Strand Conformation Polymorphism (SSCP)
2. Materials and Methods
2.1 Field Collection of Ticks
2.1.1 Tick Collection from Rats
2.1.2 Tick Collection from Other Animals
2.2 Processing of Ticks
2.3 Morphological Identification of Ticks
2.3.1 Identification Keys to Stages and sexes of Ticks
2.3.2 Identification Keys to Families
2.3.3 Identification Keys to Genera
2.3.4 Identification Keys to Species
2.4 Molecular Identification of Ticks
2.4.1 DNA Extraction
2.4.2 Mitochondrial DNA Amplification and Purification
2.4.3 SSCP Analysis of 16S, 12S, and COI PCR Product
2.4.4 Sequencing of 16S and 12S PCR Product
3. Results
3.1 Field Collection of Ticks
3.2 Morphological Identification
3.3 Molecular Identification
3.3.1 DNA Extraction
3.3.2 Mitochondrial DNA Amplification and Purification
3.3.3 SSCP Analysis of 16S, 12S, and COI PCR Products
3.3.4 Sequence Analysis of 16S and M13-12S PCR Products
4. Discussion
5. Appendices
6. Literature Cited
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