Relapsing fever-like spirochetes infecting European vector tick of Lyme disease agent - Research

Emerging Infectious Diseases, June, 2003 by Dania Richter, Daniela B. Schlee, Franz-Rainer Matuschka

To determine whether relapsing fever--like spirochetes associated with hard ticks may infect Ixodes ricinus ticks in central Europe, we screened questing ticks for 16S rDNA similar to that of Asian and American relapsing fever--like spirochetes. We compared the prevalence of these spirochetes to that of Lyme disease spirochetes transmitted by the same vector. Relapsing fever--like spirochetes infect 3.5% of questing vector ticks in our three central European sites near the Rhein Valley. These spirochetes differ genetically from their American and Asian analogs while being relatively homogeneous in the region we sampled. The Lyme disease genospecies most commonly detected in central Europe are distributed broadly, whereas those that are less frequently found appear to be place-specific. The absence of co-infected ticks suggests that relapsing fever--like and Lyme disease spirochetes may not share hosts. Exposure risk for relapsing fever--like spirochetes is similar to that of certain Lyme disease genospecies. Although many persons may be bitten by ticks infected by relapsing fever--like spirochetes, health implications remain unknown.

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Diverse pathogens infect ticks of the Ixodes ricinus complex in North America, Asia, and Europe. Viral agents include those of tick-borne encephalitis, Russian spring-summer encephalitis, louping ill, Powassan encephalitis (1), and its variant known as deer tick virus (2). The piroplasms that infect these ticks include Babesia divergens, B. microti, B. odocoilei, and B. gibsoni (3). Other infectious agents include Ehrlichia (Anaplasma) phagocytophila, Rickettsia helvetica, possibly Bartonella quintana, and a nonpathogenic endosymbiontic rickettsia (4-6). Entomophagic fungal, nematode, and wasp pathogens are also found in these ticks (7). The diverse array of Lyme disease spirochetes infecting ticks in the I. ricinus complex has been subdivided into at least six known genospecies, Borrelia burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana, B. bissettii, and B. lusitaniae (8,9). More recently, a relapsing fever--like spirochete, B. miyamotoi (10), has been added to this array of parasitic microbes.

B. miyamotoi was originally described from I. persulcatus ticks sampled in Japan. Its flagellin and 16S rRNA sequences resemble those of the relapsing fever spirochetes more closely than those of any of the known Lyme disease spirochetes (10,11). A closely related agent, designated as Borrelia nov. sp., has recently been discovered in North America (12). The primers used to detect infection by Lyme disease spirochetes generally fail to amplify DNA from these organisms. Although Borrelia nov. sp. infects approximately 2% of the American I. scapularis ticks, Lyme disease spirochetes are found in approximately 12%. The prevalence of B. miyamotoi infection in Japan, however, has not been determined. Whether these potential threats to human health are present in Europe remains unknown.

Relapsing fever--like spirochetes associated with hard ticks may infect European I. ricinus ticks. To determine whether such organisms are present in Europe, we screened questing ticks for DNA similar to that of the American and Asian relapsing fever--like spirochetes. In particular, we amplified a fragment of the 16S rRNA gene found in the various genospecies of Lyme disease spirochetes as well as in the Asian and American relapsing fever--like spirochetes and estimated how prevalent such infections are in a region in central Europe.

Materials and Methods

We sampled questing I. ricinus ticks from three sites located near the Rhein Valley in Germany and France. The German site was near the town of Maikammer and the French site was near the town of Lembach, approximately 40 km southwest of Maikammer. A second French site, Petite Camargue Alsacienne, was situated 160 km farther south, near the German and Swiss borders (13). Ticks were collected in April 2001 by passing a flannel flag over vegetation. Ticks were confined in screened vials and stored at 10[degrees]C [ or -] 1[degrees]C, and all nymphs and adults were identified to species.

To detect and identify the various Lyme disease spirochetes and relapsing fever--like spirochetes in these ticks, the opisthosoma of each was opened in a drop of physiologic saline, and the midgut was obtained and transferred to a tube containing 180 [micro]L lysis buffer (ATL Tissue Lysis Buffer, Qiagen GmbH, Hilden, Germany) and 20 [micro]L proteinase K (600 mAU/mg). Midguts were lysed at 56[degrees]C overnight. DNA was extracted by using the QIAamp DNA Mini Kit (Qiagen GmbH) according to the manufacturer's instructions. DNA of nymphal or adult ticks was eluted with 50 [micro]L or 75 [micro]L elution buffer, respectively, and stored at -20[degrees]C until polymerase chain reaction (PCR) was performed.

Borrelia genospecies were characterized by amplifying and sequencing a 600-nucleotide fragment of the gene encoding the 16S rRNA. To increase the sensitivity for spirochetal DNA detection in ticks, we used nested PCR. Aliquots of DNA suspensions (2 [micro]L) were diluted to 50 [micro]L by using 200 [micro]M of each deoxynucleoside triphosphate, 1.5 mM Mg[Cl.sub.2], 0.5 U Taq polymerase (Qiagen GmbH) as well as 15 pmol of the outer primer pair and PCR-buffer supplied with the Taq polymerase. We used the following primer sequences of the 16S rRNA gene as outer primers (14): 16S1A 5'-CTA ACG CTG GCA GTG CGT CTT AAG C-3' and 16S1B 5'-AGC GTC AGT CTT GAC CCA GAA GTT C-3' (positions 36-757). The mixture was placed in a thermocycler (PTC 200, MJResearch, Biozym Diagnostic, Hess. Oldendorf, Germany), heated for 1 min at 94[degrees]C, and subjected to 30 cycles of 20 s denaturation at 94[degrees]C, 20 s each for the first annealing reaction at 63[degrees]C with a 40 s extension at 72[degrees]C and a final extension for 2 min at 72[degrees]C. After the first amplification with the outer set of primers, 2 [micro]L of the amplification product was transferred to a fresh tube containing 48 [micro]L of the reaction mixture previously described, except that 2.5 mM Mg[Cl.sub.2] and 20 pmol of the inner primer pair was used 16S2A 5'-AGT CAA ACG GGA TGT AGC AAT AC-3' and 16S2B 5'-GGT ATT CTT TCT GAT ATC AAC AG-3' (positions 66-720). This mixture was subjected to 35 amplification cycles by using the cycle conditions described previously, except that the annealing reaction was performed at 56[degrees]C and the extension reaction lasted 30 s. DNA was extracted, reaction vials were prepared for amplification, templates were added, and products underwent electrophoresis in separate rooms. In each sixth reaction mix, water was added instead of extracted DNA to serve as negative control. PCR products were detected by electrophoresis in a 1.5% agarose gel stained with ethidium bromide.