Schabereiter-Gurtner Claudia, Lubitz Werner, Rölleke Sabine
Institute of Microbiology and Genetics, University of Vienna, Dr Bohr-Gasse 9, 1030 Vienna, Austria.
J Microbiol Methods. 2003 Feb;52(2):251-60. doi: 10.1016/s0167-7012(02)00186-0.
Ticks play an important role in the transmission of arthropod-borne diseases of viral, protozoal and bacterial origin. The present article describes a molecular-biological based method, which facilitated the broad-range analyses of bacterial communities in ixodid ticks (Ixodes ricinus). DNA was extracted both from single ticks and pooled adult ticks. Eubacterial 16S rRNA gene fragments (16S rDNA) were amplified by polymerase chain reaction (PCR) with broad-range ribosomal primers. Sequences spanning the hypervariable V3 region of the 16S rDNA and representing individual bacterial taxons were separated by denaturing gradient gel electrophoresis (DGGE). For phylogenetic identification, DGGE bands were exised, cloned and sequenced. In addition, we set up a 16S rDNA clone library which was screened by DGGE. Sequences were compared with sequences of known bacteria listed in the GenBank database. A number of bacteria were affiliated with the genera Rickettsia, Bartonella, and Borrelia, which are known to be pathogenic and transmitted by ticks. Two sequences were related to the yet to be cultivated Haemobartonella. To our knowledge, Haemobartonella has never been directly detected in I. ricinus. In addition, members of the genera Staphylococcus, Rhodococcus, Pseudomonas, and Moraxella were detected, which have not been identified in ticks so far. Two bacteria were most closely related to a rickettsial endosymbiont of an Acanthamoeba sp., and to an endosymbiont (Legionellaceae, Coxiella group) of the microarthropod Folsomia candida. The results prove that 16S rDNA genotyping in combination with DGGE analysis is a promising approach for the detection and identification of bacteria infecting ticks, regardless of whether these bacteria are fastidious, obligate intracellular or noncultivable.
蜱虫在传播病毒、原生动物和细菌引起的节肢动物传播疾病中起着重要作用。本文描述了一种基于分子生物学的方法，该方法有助于对蓖麻硬蜱（Ixodes ricinus）中的细菌群落进行广泛分析。从单个蜱虫和成年蜱虫群体中提取DNA。使用广谱核糖体引物通过聚合酶链反应（PCR）扩增真细菌16S rRNA基因片段（16S rDNA）。通过变性梯度凝胶电泳（DGGE）分离跨越16S rDNA高变V3区域并代表单个细菌分类群的序列。为了进行系统发育鉴定，将DGGE条带切下、克隆并测序。此外，我们建立了一个通过DGGE筛选的16S rDNA克隆文库。将序列与GenBank数据库中列出的已知细菌序列进行比较。许多细菌与立克次氏体属、巴尔通体属和疏螺旋体属相关，这些细菌已知具有致病性并由蜱虫传播。有两个序列与尚未培养的血巴尔通体有关。据我们所知，从未在蓖麻硬蜱中直接检测到血巴尔通体。此外，还检测到葡萄球菌属、红球菌属、假单胞菌属和莫拉克斯氏菌属的成员，这些细菌迄今为止尚未在蜱虫中鉴定出来。有两种细菌与棘阿米巴属的一种立克次氏体共生菌以及小型节肢动物念珠伪跳虫的一种共生菌（军团菌科，柯克斯氏体属）关系最为密切。结果证明，16S rDNA基因分型与DGGE分析相结合是一种有前景的方法，可用于检测和鉴定感染蜱虫的细菌，无论这些细菌是苛求菌、专性细胞内菌还是不可培养菌。
