- 學位論文
貝里斯蜱媒介立克次體菌感染之分子檢測及其病媒蜱種株鑑定
Molecular Detection of Tick-borne Rickettsia infection and Species Identification of Hard Ticks (Acari: Ixodidae) Collected from Belize
摘要
中文摘要 研究背景：貝里斯乃位於中美洲之國家，極少有關蜱種及蜱媒介病原體之研究，而立克次體(Rickettsia)為已確認具醫學及獸醫學之重要性，並可區分為斑點熱(Spotted Fever Group, SFG)及斑疹熱(Typhus Group, TG) 之兩主群，此種藉由蜱叮咬而傳播之立克次體，若未適切治療將導致死亡。 研究目的：本研究目的在於鑑定採集自貝里斯Cayo District之硬蜱種株，並以分子檢測方法確認存在於這些硬蜱體中之立克次體感染狀況。 研究方法：所有硬蜱樣本於2019年夏季採集自Cayo District之六個地點，隨後這些採得硬蜱被運送來台灣，並依其形態特徵進行蜱種株確認。全基因體核酸則使用核酸萃取試劑組(Qiagen DNeasy kit)自每隻硬蜱勻質液萃取，而立克次體檢測則針對檸檬酸合成酶 (citrate synthase gene; gltA)及體表蛋白(outer membrane protein B; ompB)基因進行巢式聚合酶鏈反應試驗(nested-PCR test)。PCR反應產物則以凝膠電泳檢視及將陽性產物送往廠商定序(sequencing)，而立克次體之基因種株確認則以記錄在GenBank已知菌株做為比對。 研究結果：自貝里斯Cayo District之六個地區總共採集得338隻硬蜱，其中包括153 隻(45.3%) 扇頭蜱(Rhipicephalus spp.)、95隻 (28.1%)花蜱(Amblyomma spp.)及90隻(26.6%) 革蜱(Dermacentor spp.)，針對立克次體之檸檬酸合成酶 (gltA)及體表蛋白(ompB)基因進行巢式聚合酶鏈反應之分子檢測，則顯示整體有12.4% (42/338)陽性率。而檢測到之立克次體菌株在遺傳上則被歸屬為Rickettsia amblyommatis,、R. parkeri、 R. felis 及 R. marmionii。 結論：本研究確認先前有關R. amblyommatis及 R. parkeri在貝里斯發現之報告(Polsomboon, et al., 2017)，而R. marmionii乃首次提供分子檢測證據，此菌株乃首先於澳洲發現之新菌株(Unsworth et al., 2007)，在遺傳上歸屬於R. honei，並是導致Flinders Island spotted fever之致病原，R. felis則是首次在貝里斯發現。已有研究顯示R. amblyommatis, R. felis and R. parkeri 皆能造成人類斑點熱，若未能適切治療將導致死亡。因此本研究突顯持續進行蜱種及蜱媒介病原體研究在貝里斯之重要性，由於人類與家飼動物之親密接觸，蜱媒介病原體將可輕易的藉由蜱叮咬而傳播。
並列摘要
ABSTRACT Background. Belize is a country located in Central America where little research has been carried out on tick species and tick-borne pathogens. Rickettsia species is identified as a major tick-borne pathogen of medical and veterinary importance divided into two major groups; Spotted Fever Group (SFG) and Typhus Group (TG). The transmission of Rickettsia through tick bites may cause death if the infection is not properly treated. Aim. The aim of this study is to identify the genus of Belizean hard ticks (Acari: Ixodidae) collected from different areas in the Cayo District and molecular detection of Rickettsia species carried by these vector ticks. Methods. During the summer months of July and August 2019, ticks were collected from six different areas in the Cayo District, Belize. Dead ticks were then transported to Taiwan, where they were identified to the genus level using pictorial keys. Total genomic DNA was extracted from each individual tick using Qiagen DNeasy extraction kit. For molecular detection of Rickettsia species, nested-PCR assay was carried out targeting the citrate synthase (gltA) and outer membrane protein (ompB) genes. Gel electrophoresis was used to show the positive results of expected size band and PCR products were sent for sequencing. The Rickettsia species was identified by matching to the well-known species of GenBank. Results. A total of 338 ticks were collected from horses and dogs from 6 different areas of the Cayo District in Belize. Out of which, 153 (45.3%) were Rhiphacephalus, 95 (28.1%) were Amblyomma and 90 (26.6%) were Dermacentor. Molecular detection through nested-PCR assay targeting the gltA and ompB genes showed the total positive rate of 12.4% (42/338) in tick samples. The Rickettsia species found were genetically affiliated to the species of Rickettsia amblyommatis, R. parkeri, R. felis and R. marmionii. Conclusion. This study confirms the presence of R. amblyommatis and R. parkeri, which had previously been described in Belize (Polsomboon et al., 2017). The detection of R. marmionii is first time to be described in Belize, and it is a novel bacteria described by Unsworth et al. in 2007 in Australia. It is genetically related to and described as a strain of R. honei, the causative agent of Flinders Island spotted fever. R. felis had not been reported in Belize previously. Studies have also shown that R. amblyommatis, R. felis and R. parkeri can cause spotted fever in humans. These infections can cause death if not treated promptly. Thus, this study shows the importance of continuous research on ticks and tick-borne pathogens in Belize. Because of the close contact of domestic animals with humans, tick-borne pathogens can easily be transmitted to humans through tick bites.
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