Genetic identification of Borrelia burgdorferi spirochetes in Ixodes granulatus (Acari: Ixodidae) ticks and molecular phylogeny of I. granulatus ticks in the Taiwan area

為確認伯氏疏螺旋體菌（Borrelia burgdorferi）存在於台灣粒形硬蜱(Ixodes granulatus)，本研究針對伯氏疏螺旋體菌之體表蛋白A (OspA) 基因進行具種株專一性之聚合酶鏈反應分析，並依據增殖之基因序列分析存在於台灣粒形硬蜱體內之疏螺旋體菌的遺傳特質，結果顯示有B. burgdorferi-like 及 B. valaisiana-related兩種疏螺旋體菌株首次被偵測存在於台灣粒形硬蜱。遺傳演化分析結果則顯示其中六株B. burgdorferi-like疏螺旋體菌之基因序列相似度在B. burgdorferi sensu stricto基因種內具有90.5%~98.7%之高度同質性(homogeneity)，並可確認其遺傳上歸屬於B. burgdorferi sensu stricto基因種。然而其他七株B. valaisiana-related疏螺旋體菌，則在遺傳上被分類為新基因種(novel genospecies)，而非歸屬於已確認之B. valaisiana種株。而上述結果乃首次證實伯氏疏螺旋體菌株存在於台灣粒形硬蜱。此外，本研究亦首次確認粒形硬蜱在Ixodes屬之分子親緣關係，其遺傳演化關係則依據19個蜱種株(包含七種Ixodes屬及兩種外群屬)之粒線體16S及核醣體ITS2序列之比較分析，同時以鄰接法(neighbour-joining)及最大簡約法(maximum-parsimony)分析皆可呈現四組明顯區別之種群。若依據粒線體16S基因序列之比較分析，則顯示所有台灣粒形硬蜱在遺傳上歸屬於一個具有高度基因序列相似度(92.2%~99.3%)之單系群(monophyletic group)，並可以11.7%~30.8%之基因序列相異度而區別於其他Ixodes種及屬之硬蜱。而種株內遺傳分析(intraspecific analysis)結果亦顯示採集自台灣及馬來西亞之粒形硬蜱歸屬於兩種不同系譜(distinct lineages)。相對的，若依據核醣體ITS2序列之比較分析，雖可顯示粒形硬蜱與其他種屬硬蜱之間具有高度遺傳歧異性，但却顯示採集自台灣及日本之粒形硬蜱不具地理差異性。這些結果意含未來運用核醣體ITS2分子標的，做為亞洲地區粒形硬蜱種株專一性鑑定之可行性。To identify the Borrelia burgdorferi spirochetes existed in Ixodes granulatus ticks of Taiwan, the genetic identities of spirochetes detected in I. granulatus ticks were determined by analyzing the gene sequences amplified by a genospecies-specific polymerase chain reaction (PCR) assay based on the outer surface protein A (OspA) of B. burgdorferi sensu lato. Results indicate that B. burgdorferi-like and B. valaisiana-related spirochetes were detected for the first time in Ixodes granulatus ticks collected in Taiwan. The phylogenetic analysis reveals that the sequence similarity among six strains of B. burgdorferi-like spirochetes is highly homogeneous, ranging from 90.5% to 98.7%, within the genospecies of B. burgdorferi sensu stricto and confirms that these spirochetes were genetically related to the genospecies of B. burgdorferi sensu stricto. However, seven strains of B. valaisiana-related spirochetes were classified as novel genospecies of B. burgdorferi sensu lato rather than B. valaisiana and that was clearly distinguished from other genospecies of Borrelia spirochetes. These results provide the first evidence of B. burgdorferi spirochetes detected in I. granulatus ticks of Taiwan. In addition, molecular phylogeny of I. granulatus ticks among the Ixodes species was also determined for the first time in Taiwan. The phylogenetic relationships were analyzed by comparing the sequences of mitochondrial 16S and nuclear ITS2 rDNA genes obtained from 19 strains of ticks representing seven species of Ixodes and two outgroup species (Rhipicephalus sanguineus and Haemaphysalis inermis). Four major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. Based on the mitochondrial 16S rDNA sequences, all these I. granulatus ticks of Taiwan were genetically affiliated to a monophyletic group with high homology (92.2-99.3%) in sequence similarity and that can be discriminated from other Ixodes species and other genus of ticks with a sequence divergence ranging from 11.7% to 30.8%. Moreover, intraspecific analysis revealed that two distinct lineages are evident between the same species of I. granulatus ticks collected from Taiwan and Malaysia. In contrast, the phylogenetic analysis based on the nuclear ITS2 rDNA sequences among various tick species also revealed a high genetic heterogeneity between I. granulatus ticks and other species of ticks. However, geographical variation was not observed between the same species of I. granulatus ticks collected from Taiwan and Japan. These observations may imply the feasibility of ITS2-based molecular targeting for species-specific identification of I. granulatus ticks in Asia area.Table of contents ----------------------------------------Iist of tables -----------------------------------------IIIist of figures ------------------------------------------Vbstract -----------------------------------------------VIIbstract (in Chinese) -----------------------------------IX. Introduction ------------------------------------------1 1.1 Background description -----------------------------1 1.2 Heterogeneity and genetic identification of Borrelia burgdorferi spirochetes-----------------------------3 1.3 Molecular identification of Lyme disease vector ticks-----------------------------------------------6 1.4 Genetic analysis of Borrelia spirochetes and vector ticks in Taiwan-------------------------------------8 1.5 The objectives of this study -----------------------9. Materials and Methods --------------------------------11 2.1 Collection of tick specimens ----------------------11 2.2 Identification of tick specimens ------------------13 2.3 Detection of spirochetes by light and fluorescent microscopy ----------------------------------------15 2.4 DNA extraction from tick specimens ----------------15 2.5 DNA amplification for B. burgdorferi sensu lato spirochetes----------------------------------------16 2.6 DNA amplification for phylogenetic analysis of I. granulatus ticks ---- ------------------------------17 2.7 Sequence alignments and phylogenetic analysis -----18. Results ----------------------------------------------21 3.1 Collection of adult Ixodes granulatus ticks -------21 3.2 Ultrastructural features of adult I. granulatus of Taiwan --------------------------------------------23 3.3 Detection of B. burgdorferi spirochetes in adult I. granulatus ticks ----------------------------------27 3.4 Genetic identification of B. burgdorferi-like spirochetes detected in I. granulatus -------------29 3.5 Phylogenetic analysis of B. burgdorferi-like spirochetes detected in I. granulatus -------------30 3.6 Phylogenetic analysis of I. granulatus ticks collected on Taiwan area---------------------------39. Discussion and Conclusions ---------------------------58eferences ----------------------------------------------66ppendix ------------------------------------------------7