RESEARCH ARTICLE Spatial stratification of various Lyme disease spirochetes in a Central European site Dania Richter1, Boris Schro¨ der2,3, Niklas K. Hartmann3 & Franz-Rainer Matuschka1 1Abt. Parasitologie, Institut fu¨r Pathologie, Charite´ Universita¨ tsmedizin Berlin, Berlin, Germany; 2Institut fu¨r Erd- und Umweltwissenschaften, Universita¨ t Potsdam, Potsdam, Germany; and 3Bodenlandschaftsmodellierung, Leibniz-Zentrum fu¨r Agrarlandschaftsforschung (ZALF) e.V., Mu¨ncheberg, Germany Downloaded from https://academic.oup.com/femsec/article/83/3/738/596339 by guest on 29 September 2021 Correspondence: Dania Richter, Abt. Abstract Parasitologie, Institut fu¨r Pathologie, Charite´ Universita¨ tsmedizin Berlin, Malteserstraße To determine whether the genospecies composition of Lyme disease spirochetes 74-100, 12249 Berlin, Germany. Tel.: is spatially stratified, we collected questing Ixodes ricinus ticks in neighboring 49 30 838 70 372; fax: 49 30 776 2085; plots where rodents, birds, and lizards were present as reservoir host and com- e-mail: [email protected] pared the prevalence of various genospecies. The overall prevalence of spiro- chetes in questing ticks varied across the study site. Borrelia lusitaniae appeared Present addresses: Boris Schro¨ der, to infect adult ticks in one plot at the same frequency as did Borrelia afzelii in Landschaftso¨ kologie, Department fu¨r O¨ kologie und O¨ kosystemmanagement, the other plots. The relative density of questing nymphal and adult ticks varied Technische Universita¨ tMu¨ nchen, Freising, profoundly. Where lizards were exceedingly abundant, these vertebrates seemed Germany to constitute the dominant host for nymphal ticks, contributing the majority Niklas K. Hartmann, Lancaster Environment of infected adult ticks. Because lizards support solely B. lusitaniae and appear Centre, Lancaster University, Lancaster, UK to exclude other genospecies, their narrow genospecies association results in predominance of B. lusitaniae in sites where lizards are abundant, while limit- Received 16 July 2012; revised 13 September ing its spread to the host’s habitat range. To the extent that Central European 2012; accepted 5 October 2012. B. lusitaniae strains are nonpathogenic, the presence of numerous lizards Final version published online 12 November 2012. should locally decrease risk of infection for people. Evaluation of regional risk of infection by Lyme disease spirochetes should take the spatial effect of hosts DOI: 10.1111/1574-6941.12029 into consideration, which stratify the distribution of specifically infected ticks on a small scale. Editor: Julian Marchesi Keywords Lyme disease; Borrelia lusitaniae; Borrelia afzelii; stratification; lizard; zooprophylaxis. rochetes when feeding as a nymph on a bird (Matuschka Introduction & Spielman, 1992). Borrelia burgdorferi s.s. spirochetes, In Europe, a variety of Lyme disease spirochetes are on the other hand, are considered as generalists and may transmitted by Ixodes ricinus ticks. Although these vector thrive in both rodents and birds (Richter et al., 2000, ticks feed virtually indiscriminately on a wide array of 2004a). Whereas B. afzelii, B. garinii, B. valaisiana, and vertebrates, each of the Lyme disease genospecies appears B. burgdorferi s.s. are ubiquitously prevalent in questing to be associated with a particular group of hosts. Whereas ticks, others, such as Borrelia spielmanii and Borrelia lusi- Borrelia afzelii spirochetes appear to be perpetuated by taniae, which are closely associated with certain dormice rodent hosts, Borrelia garinii and Borrelia valaisiana are and lizards, respectively, may be limited in distribution to considered to be associated mainly with birds (Kurten- the habitats of their hosts (Richter et al., 2004b, 2006; bach et al., 2002; Hanincova´ et al., 2003). Hosts that are Richter & Matuschka, 2006a). At least, four species of liz- MICROBIOLOGY ECOLOGY MICROBIOLOGY reservoir competent for one Lyme disease genospecies ards support B. lusitaniae in nature. Sand lizards, Lacerta may even be zooprophylactic to another, because, for agilis, wall lizards, Podarcis muralis, green lizards, Lacerta example, a tick that has acquired B. afzelii from a rodent viridis, and Algerian sand racer, Psammodromus algirus, during its larval blood meal may be cleared of these spi- serve as reservoir hosts in Europe and the latter species ª 2012 Federation of European Microbiological Societies FEMS Microbiol Ecol 83 (2013) 738–744 Published by Blackwell Publishing Ltd. All rights reserved Spatial stratification of Lyme disease spirochetes 739 in Tunisia (Dsouli et al., 2006; Majla´thova´ et al., 2006; path. Ticks were collected per unit time by recording the Richter & Matuschka, 2006a; Amore et al., 2007). period of active flagging. Additional nymphal ticks col- Whereas B. lusitaniae constitutes the major, and in some lected at Plot A in April and May 2008 were included to sites, sole genospecies of Lyme disease spirochetes in the obtain sufficient nymphal ticks to determine the Mediterranean basin, it is only focally distributed in prevalence of spirochetal infection in this stage. Northern Europe (Richter & Matuschka, 2006a). To detect and identify the various spirochetes that may The composition of the diverse genospecies infecting be present in the ticks, the opisthosoma of each was opened, vector ticks varies and depends on the local host compo- and the contained mass of soft tissue dissected out in physi- sition. For relatively ubiquitous genospecies, such as ologic saline, transferred to a tube containing 180-lL lysis B. afzelii, B. garinii, and B. valaisiana, the proportion of buffer (ATL Tissue Lysis Buffer, Qiagen, Hilden, Germany) l À1 ° hosts competent for one or another of these genospecies and 20- L proteinase K (600 mAU mg ), and lysed at 56 C Downloaded from https://academic.oup.com/femsec/article/83/3/738/596339 by guest on 29 September 2021 likely may influence the relative genospecies mix in the overnight. DNA was extracted using the QIAamp DNA tick population. A genospecies with a narrow host associ- Mini Kit (Qiagen) according to the manufacturer’s instruc- ation, such as B. lusitaniae, which is perpetuated by non- tions, DNA of nymphal or adult ticks was eluted with 50- or ubiquitous hosts, as are lizards in Germany, may only 75-lL elution buffer, respectively, and stored at À20 °C occur where its host is present. As observed in other until PCR was performed. Borrelia genospecies were charac- host-parasite relationships (e.g. Heisswolf et al., 2009; terized by amplifying and sequencing a 600-nucleotide frag- Zurell et al., 2009), habitat parameters may particularly ment of the gene encoding the 16S rRNA. To increase the influence the distribution of those spirochetes that have a sensitivity for detection of spirochetal DNA in ticks, we limited range of reservoir hosts. used nested PCR as described previously (Richter & Matu- To determine whether the various Lyme disease geno- schka, 2006a). Each PCR amplification product was purified species are stratified or distributed evenly across a study using a QIAquick-Spin PCR column (Qiagen) according to site, we compared the prevalence of each genospecies in the manufacturer’s instructions. Amplified DNA fragments questing ticks collected in three neighboring plots, one of were directly sequenced in both directions using the inner which appeared to support lizard populations. In addi- primers by the dideoxynucleotide chain termination tion, we compared the relative density of nymphal and method on a Licor DNA4200 sequencer (Licor Biosciences, adult ticks. Bad Homburg, Germany). Each resulting sequence was compared with sequences of the same gene fragment repre- senting various spirochetal genospecies. The following Materials and methods sequences served for comparison: Accession numbers Our three plots were located at the top of a hill, 300 m X85196 and X85203 for B. burgdorferi s.s., X85190, X85192, above sea level, near the city of Heilbronn in the and X85194 for B. afzelii, X85193, X85199, and M64311 for southwestern German state of Baden-Wu¨rttemberg. The B. garinii, X98228 and X98229 for B. lusitaniae, X98232 southern slope of the hill serves as vineyard. Plot A was a and X98233 for B. valaisiana, AY147008 for B. spielmanii as sun-exposed brushy ecotone, 2–3 m in width, between an well as AY253149 for B. miyamotoi. A match, permitting no agricultural road and the forest, where sand lizards, more than two nucleotide changes, was required. Infections Lacerta agilis, and wall lizards, Podarcis muralis,were by more than one genospecies were recognized in the image exceedingly abundant (Richter & Matuschka, 2006a). Plot of the sequencing gel when simultaneous bands occurred at B was located at the edge of a forestry road cutting all signature sequences that differentiate coinfecting through the forest consisting mainly of beech trees. The genospecies from each other. shadowy Plot C was characterized by an abandoned path We used Fisher’s exact test for independence in m 9 n into the forest that had been virtually overgrown by bru- contingency tables to assess whether the prevalence of shy vegetation. Each plot was about 100 m long and each of the host-associated genospecies, B. afzelii, B. gari- about 50 m distant from the other two plots. nii, B. valaisiana, and B.lusitaniae, in questing nymphal Questing nymphal and adult ticks were collected or adult ticks is independent of the study plot (Fisher, monthly from April through October 2007 at all three 1935; Mehta & Patel,