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Visualization of Borrelia burgdorferi sensu lato by fluorescence in situ hybridization (FISH) on whole-body sections of Ixodes ricinus ticks and gerbil skin biopsies

Institut für Mikrobiologie und Hygiene, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Dorotheenstraße 96, D-10117 Berlin, Germany¹
Institut für Biologie, Angewandte Zoologie/Ökologie der Tiere, Freie Universität Berlin, Haderslebener Straße 9, D-12163 Berlin, Germany²
Zoologisches Institut und Museum, Ernst-Moritz-Arndt-Universität, J.-S.-Bachstraße 11/12, D-17487 Greifswald, Germany³

Author for correspondence: Bettina Hammer. Tel: +49 30 450 524074. Fax: +49 30 450 524902. e-mail: bettina.hammer{at}charite.de

The objective of this study was to visualize borreliae directly in whole-body sections of Ixodes ricinus by fluorescence in situ hybridization (FISH). Borrelia afzelii mono-infected or Borrelia burgdorferi sensu stricto (ss)/B. afzelii double-infected nymphs were fixed, embedded in cold polymerizing resin and sectioned. The same sample processing was applied to skin biopsies taken from a Mongolian gerbil after an infectious tick-bite. FISH was carried out using 16S-rRNA-directed, fluorescence-labelled oligonucleotide probes specific for the genus Borrelia and specific within the group of Lyme borreliosis-associated genospecies B. afzelii, B. burgdorferi ss, Borrelia garinii and Borrelia valaisiana. Sensitivity and specificity of the newly designed probes were evaluated using PCR, dot-blot hybridizations and FISH. Despite significant autofluorescence of certain tick tissues (which allowed good histological orientation within the sections), borreliae showing the typical spirochaetal morphotype were clearly visible in five out of six putatively infected ticks. These findings were confirmed by electron microscopy of ticks from the same infected batch as used for FISH. Attempts to produce ticks infected by two different Borrelia genospecies were not successful. FISH on whole-body sections of resin-embedded ticks offers the possibility of visualizing and identifying borreliae within tick tissues. This technique has great potential for the investigation of the transmission of bacteria or other micro-organisms by arthropod vectors. Furthermore, clear visualization of single spirochaetes distributed along subcutaneous fat cell membranes in gerbil skin biopsies suggests that FISH might also be suitable for the detection of borreliae in clinical tissue specimens.

Keywords: Lyme borreliosis, arthropods, mixed infection, in situ hybridization, FISH

Abbreviations: EM, electron microscopy; FISH, fluorescence in situ hybridization; IFA, indirect immunofluorescence assay; LB, Lyme borreliosis; sl, sensu lato; ss, sensu stricto

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