Abstract

To determine whether the Lyme disease spirocheteBorrelia lusitaniae is associated with lizards, we compared the prevalence and genospecies of spirochetes present in rodent- and lizard-associated ticks at a site where this spirochete frequently infects questing ticks. Whereas questing nymphalIxodes ricinus ticks were infected mainly byBorrelia afzelii, one-half of the infected adult ticks harboredB. lusitaniae at our study site. Lyme disease spirochetes were more prevalent in sand lizards (Lacerta agilis) and common wall lizards (Podarcis muralis) than in small rodents. Although subadult ticks feeding on rodents acquired mainlyB. afzelii, subadult ticks feeding on lizards became infected byB. lusitaniae. Genetic analysis confirmed that the spirochetes isolated from ticks feeding on lizards are members of theB. lusitaniae genospecies and resemble type strain PotiB2. At our central European study site, lizards, which were previously considered zooprophylactic for the agent of Lyme disease, appear to perpetuateB. lusitaniae.

MATERIALS AND METHODS

Our study site was located at the ridge of Ranzenberg Mountain about 300 m above sea level, near the town of Heilbronn in the state of Baden-Württemberg, Germany. The southern slope of this mountain is used as a vineyard. Lizards, rodents, and ticks were obtained at the edge of the forest which was separated from the vineyard by an agricultural road.

Lizards were captured by hand in the early morning hours in April, June, and August 2005. They were identified to species and inspected by using a magnifying glass. Any ticks infesting them were carefully removed with forceps and identified to stage and species. The lizards were released promptly after ticks were removed. Rodents were captured in live traps (Longworth Scientific Instruments, Abingdon, United Kingdom) baited with apple, grain, and cotton. A total of 40 traps were placed during three consecutive nights in April and June 2005. Captured small rodents were taken to the laboratory, where they were identified and caged over water until all attached ticks had detached. The water was inspected twice daily, and the ticks were removed, identified, and counted. Ticks derived from hosts were confined in screened vials and stored at 22°C, until they were examined for spirochetes. Questing ticks were collected at the site in April 2005 by using a flannel flag, identified to stage and species, and preserved in 80% ethanol.

To detect and identify the various spirochetes that were present in questing and host-associated ticks, the opisthosoma of each tick was opened, and the mass of soft tissue was dissected in physiological saline, transferred to a tube containing 180 μl lysis buffer (ATL tissue lysis buffer; QIAGEN, Hilden, Germany) and 20 μl proteinase K (600 milli-activity units/mg), and lysed at 56°C overnight. DNA was extracted using a QIAamp DNA mini kit (QIAGEN) according to the manufacturer's instructions, and DNAs of nymphal and adult ticks were eluted with 50 and 75 μl of elution buffer, respectively, and stored at −20°C until 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 detection of spirochetal DNA in ticks, we used nested PCR. Aliquots of DNA suspensions (2 μl) were added to 48-μl mixtures containing each deoxynucleoside triphosphate at a concentration of 200 μM, 1.5 mM MgCl₂, 1 UTaq polymerase (QIAGEN), and 15 pmol of the outer primer pair and the PCR buffer supplied with theTaq polymerase. We used the following sequences of the 16S rRNA gene as outer primers (30) (5′-3′): CTA ACG CTG GCA GTG CGT CTT AAG C (16S1A) and AGC GTC AGT CTT GAC CCA GAA GTT C (16S1B) (positions 36 to 757). The mixture was placed in a thermocycler (PTC 200; MJ Research, Biozym, Germany), heated for 1 min at 94°C, and subjected to 30 cycles of 20 s of denaturation at 94°C, 20 s of annealing at 63°C, and 40 s of extension at 72°C, followed by a final extension for 2 min at 72°C. After the first amplification with the outer set of primers, 2 μl of the amplification product was transferred to a fresh tube containing 48 μl of the reaction mixture described above, except that 2.5 mM MgCl₂ and 20 pmol of the following inner primer pair (5′-3′) were used: AGT CAA ACG GGA TGT AGC AAT ACA (16S2A) and GGT ATT CTT TCT GAT ATC AAC AG (16S2B) (positions 66 to 720). This mixture was subjected to 35 amplification cycles using the cycle conditions described above, except that the annealing reaction was performed at 56°C and the extension reaction lasted 30 s. DNA was extracted, reaction vials were prepared for amplification, templates were added, and products were electrophoresed in separate rooms. As an additional precaution, the reaction mixtures were prepared in a designated PCR workstation (Labcaire Systems, North Somerset, United Kingdom) and templates were added to the mixtures in a second PCR workstation. Benches and equipment were wiped down with a DNA decontamination solution (DNA erase; MP Biomedicals, Eschwege, Germany) after each use. To each sixth reaction mixture, water was added instead of extracted DNA as a negative control. PCR products were detected by electrophoresis in a 1.5% agarose gel stained with ethidium bromide.

Each PCR amplification product was purified by using a QIAquick-Spin PCR column (QIAGEN) according to the manufacturer's instructions. Amplified DNA fragments were directly sequenced in both directions using the inner primers by the dideoxynucleotide chain termination method with a Licor DNA4200 sequencer (Licor Biosciences, Bad Homburg, Germany). Each resulting sequence was compared with sequences of the same gene fragment from various spirochete genospecies. The following sequences (identified by the accession numbers under which they were deposited) were used for comparison:X85196 andX85203 forB. burgdorferi sensu stricto;X85190,X85192, andX85194 forB. afzelii;X85193,X85199, andM64311 forB. garinii;X98228 andX98229 forB. lusitaniae;X98232 andX98233 forB. valaisiana;AY147008 forB. spielmanii; andAY253149 forB. miyamotoi. A complete match (no more than two nucleotide changes) was required for identification.

Spirochetes were cultured from nymphal or adult ticks that had derived from larvae or nymphs, respectively, feeding on lizards. The ticks were washed individually for 3 min in distilled water, for 3 min in 100% ethanol, and for 3 min in 1% benzalkonium chloride. After three washes in 0.9% NaCl, ticks were placed in Barbour-Stoenner-Kelly H medium (Sigma, Deisenhofen, Germany) and dissected using sterile forceps. Each midgut was transferred to 1 ml of Barbour-Stoenner-Kelly H medium supplemented with 6% heat-inactivated rabbit serum (Sigma) and amphotericin (2.5 μg/ml; Sigma). The suspensions were incubated at 32°C and checked for spirochete growth every third day by dark-field microscopy. Dense cultures of spirochetes were frozen and stored at −70°C.

To confirm the identities of theB. lusitaniae isolates, fragments of theospA, flagellin,hbb,groEL,recA, 16S rRNA, andrrf-rrl intergenic spacer genes were amplified from DNA of cultured spirochetes and sequenced by using previously described methods (3,6,7,23,24,35).

RESULTS

DISCUSSION

The geographic distribution ofB. lusitaniae differs from that of most other Lyme disease spirochetes. This species is most prevalent in Mediterranean countries, such as Portugal, Tunisia, and Morocco, where it generally constitutes the sole or major genospecies infecting vector ticks (Table5) (1,5,32,37). The first isolates ofB. lusitaniae were obtained fromI. ricinus ticks collected in Portugal, and the species name reflects this origin (16,22). In more northern or eastern countries,B. lusitaniae has been detected at only a few sites (30), at which it infects ticks less frequently than it does on the Mediterranean coast (8,11,36; Richter and Matuschka, submitted for publication). On the Turkish coast of the Black Sea, where infected ticks are rare, almost one-half of the infected ticks harboredB. lusitaniae (9). WhereasB. lusitaniae appears to be focally distributed and is rare compared to other genospecies in central or eastern Europe, which has a continental climate, it appears to be the predominant genospecies and to be widespread in countries with a Mediterranean climate.

At our Ranzenberg study site, subadult vector ticks feeding on sand lizards and common wall lizards acquired virtually onlyB. lusitaniae spirochetes.B. lusitaniae infection in these lizards was more prevalent thanB. afzelii infection in rodents. Generally, the smaller the larva-nymph ratio of ticks feeding on a competent host, the more frequently this host is exposed to infection. Indeed, spirochetal infection is more frequent in larger rodents, such as rats or dormice, than in mice and voles, because they feed more nymphal ticks (17,18). Although we detected no questing nymphal ticks infected byB. lusitaniae in our sample, lizards may be readily infected with this genospecies, because they fed up to eight times more nymphal ticks than did rodents. In addition, lizards live longer than mice and voles, and thus, spirochetal infection may accumulate throughout their life. The observation that these lizards appeared to contribute mainly adult ticks infected byB. lusitaniae at our study site may have been affected by seasonal variation; no questing ticks were obtained in the summer and fall. At another central European study site, Lembach, about 4 of 100 questing nymphs harboredB. lusitaniae (Richter and Matuschka, submitted). Also, every third nymph was infected by this genospecies at a Tunisian site (37), where different lizards may serve as hosts for vector ticks andB. lusitaniae. At our German study site,B. lusitaniae spirochetes appear to be perpetuated by at least two kinds of lizards.

Larval ticks attaching to hosts in nature may have inherited spirochetes from their mothers. WhereasB. miyamotoi spirochetes appear to be transmitted transovarially (33), this route of transmission has not been proven for any Lyme disease spirochete and, indeed, this possibility has been excluded forB. afzelii (21). However, even ifB. lusitaniae were transovarially transmitted, our field observations strongly suggest that lizards, but not rodents, permit survival in feeding ticks and subsequent transstadial transmission of the spirochetes. To further examine the reservoir competence of lizards forB. lusitaniae spirochetes, the susceptibility, intrinsic incubation period, and degree and duration of infectivity of lizards have to be analyzed experimentally in the laboratory.

Particular reservoir hosts may contribute differentially to the prevalence of diverse spirochetal genospecies at a site.B. spielmanii, for example, appears to be prevalent at sites at which garden dormice are abundant (29). At our Ranzenberg study site, virtually all subadult ticks that acquired spirochetes from lizards harboredB. lusitaniae, whereas the subadult ticks that became infected while feeding on rodents harboredB. afzelii. The lizard- and rodent-associated spirochetes mutually excluded each other when ticks were feeding on their permissive hosts. Interestingly, the genospecies detected in a tick following its nymphal blood meal reflected the latest host association. Spirochetes belonging to other genospecies to which a tick may have been exposed while feeding in its larval stage on another kind of host were rarely detected. Our observation that spirochetes from the latest host association appear to prevail in the tick supports the suggestion that the reservoir competence of a host for a particular genospecies results from the spirochete's resistance to serum complement of that host (13,14). Any spirochete that is not adapted to the host on which the vector feeds may thus be eliminated from the tick during the blood meal. Lizards appear to contributeB. lusitaniae spirochetes to the genospecies composition at our Ranzenberg study site.

In areas where the climate is continental, the distribution of lizards is focal and their home range (2) may not allowB. lusitaniae to spread from one lizard habitat to another if this spirochete is exclusively associated with reptiles. Recently, an association ofB. lusitaniae with birds was suggested, because numerous larval ticks obtained from birds captured during migration harbored this genospecies (26). If birds were regular reservoir hosts forB. lusitaniae, as they are forB. valaisiana andB. garinii,B. lusitaniae should be as widespread as these bird-associated spirochetes. The reservoir competence of birds and lizards forB. lusitaniae, however, needs to be determined experimentally. Because the distribution ofB. lusitaniae in areas with a continental climate is more focal than its distribution in areas with a Mediterranean climate, it may be that migratory birds help spread this spirochete by seeding foci of transmission where both lizards and vector ticks are present. The stress of migration may induce temporary reservoir competence forB. lusitaniae. Indeed, latent spirochetes may become reactivated in birds by migratory stress (10). Comparing the genetic diversity ofB. lusitaniae isolates derived from central and eastern European foci with the genetic diversity ofB. lusitaniae isolates of Mediterranean origin may help elucidate this suggestion. It appears thatB. lusitaniae is perpetuated by reptiles, but it may be distributed by avian hosts.

B. lusitaniae is not perpetuated at all sites where lizards and vector ticks are present. The abundance of sand lizards at a study site in Berlin resulted in an overall low prevalence of Lyme disease spirochetes in questing ticks, as determined by dark-field microscopy, compared to the prevalence at other sites where lizards were not present (19). Sand lizards that were experimentally exposed to infected ticks failed to become infectious for xenodiagnostic ticks. The spirochetes used in that study had been derived originally from a Berlin study site, had been perpetuated in rodents in the laboratory, and thus most likely wereB. afzelii and/orB. burgdorferi sensu stricto (20). On the Pacific coast of the United States, western fence lizards (Sceloporus occidentalis) and southern alligator lizards (Elgaria multicarinata) exert a similar zooprophylactic effect (15). Both spirochetal genospecies that are transmitted by the vector tickIxodes pacificus,B. burgdorferi sensu stricto andB. bissettii, are sensitive to bacteriolysis by the alternative complement pathway of these reptiles (12,34). In a tick-permissive habitat that supports lizards, the overall prevalence of Lyme disease spirochetes in questing ticks may be low, as long asB. lusitaniae spirochetes are absent.

Acknowledgments

This study was supported in part by a grant from the Landesstiftung Baden-Württemberg.

We thank Rainer Allgöwer for capturing lizards and rodents, and we thank Udo Bischoff, Mandy Pötter, and Andrea Schäfer for excellent technical assistance.

REFERENCES