Spatial Stratification of Various Lyme Disease Spirochetes in a Central

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 spirochetes 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, 1986). plots by means of a flannel flag by the same person. Ticks We performed nine comparisons and to account for were taken to the laboratory, preserved in 80% ethanol, multiple comparisons, we report p-values adjusted by and microscopically identified to stage and species. We Holm’s correction, which strictly controls the family-wise determined the relative density of questing nymphal and error-rate under arbitrary assumptions (Holm, 1979; Son- adult ticks in each of our three plots by dragging a flan- nemann, 2008). All calculations were performed with R nel flag (1.0 by 1.5 m) for a specified time span over the (version 2.13.2; R Development Core Team, 2011, http:// vegetation growing immediately adjacent to the road or www.r-project.org).

Results First, we determined the overall rate of infection in one

questing ticks collected in our three neighboring study > genospecies plots. A quarter to a third of the nymphs and a third to half of the adult ticks in these three plots were infected by spirochetes (Table 1). Whereas infection rates in nymphal (26.1%) and adult ticks (28.4%) did not vary

within Plot C, one and a half as many adults (49.3%) as (%) nymphs (31%) and twice as many adults (49.3%) as gar val miy

nymphs (24.3%) were infected in Plots A and B, respec- Borrelia tively. The non-Lyme disease spirochete, B. miyamotoi, Downloaded from https://academic.oup.com/femsec/article/83/3/738/596339 by guest on 29 September 2021 was present in about 7.5% of all infected ticks (range SV1 3.7–12.5%), independent of tick stage or plot. Whereas in Plots B and C, about 5% of all infected adult ticks harbored more than one kind of spirochete, twice as many adults were coinfected in Plot A. No nymphal tick was coinfected. In seven of the nine coinfected adult 1.8 18.0 0.0 0.0 0.9 2.7 0.9 0.0 4.21.1 23.9 19.6 2.8 1.1 4.2 0.0 11.3 2.8 0.0 2.8 1.1 2.8 3.3 0.0 0.0 15.6 2.8 3.5 2.1 3.5 2.1 1.4 ticks, B. miyamotoi paired with a Lyme disease genospe- lus afz bur cies. In Plot A, two adults were coinfected by B. miyamotoi and B. valaisiana, one by B. miyamotoi and miyamotoi. B. garinii, one by B. afzelii and SV1 as well as one by , miy one

B. afzelii and B. garinii; in Plots B and C, two adults ; > genospecies each were coinfected by B. miyamotoi and B. afzelii. The overall prevalence of spirochetes in questing ticks varied valaisiana miy across the study site. , val

For each genospecies, we determined its proportion ; §

in infected ticks across the three plots of our study site. val garinii

The proportion of infected adult ticks harboring B. lusi- , § (%) Examined ticks infected with taniae was largest in the lizard-inhabited Plot A gar gar (57.8%), less than a seventh of that in Plot B (8.6%)

and no B. lusitaniae-infected ticks were detected in Plot Borrelia SV1 C (Fisher’s exact test, Holm corrected: Pcorr. < 0.0001).

Adult ticks in Plot A were almost eight times more ‡ ticks collected at our study plots, displayed relative to other spirochetes and as absolute prevalence. Plot A was inhabited by likely to be infected by B. lusitaniae than were nymphal bur

ticks (7.4%; Pcorr. = 0.0002), and infection of nymphs (Casjens et al., 2011); = † did not vary signiﬁcantly between plots (Pcorr. 1). afz Whereas B. afzelii infected a similar proportion of Ixodes ricinus * 7.4 74.1 0.0 0.0 3.7 11.1 3.7 0.0 8.64.2 48.6 75.0 5.7 4.2 8.6 0.0 22.9 0.0 5.7 4.2 5.7 12.5 5.7 0.0 0.0 55.0 10.0 12.5 7.5 12.5 7.5 5.0 infected nymphal ticks in all three plots (averaging isolate SV1 lus , 75.6%; Pcorr. = 1), it was least abundant in adult ticks SV1

questing in Plot A. Less than a ﬁfth of the infected .; adults (17.7%) there harbored B. afzelii, but about half of the adult ticks (48.6 and 55%) did so in the other 48.424.3 57.8 17.7 0.0 4.4 8.9 13.3 8.9 11.1 28.0 8.6 0.0 2.2 4.3 6.5 4.3 5.4 49.3 26.1 28.4 plots (Pcorr. = 0.0043). The proportional prevalence of

B. burgdorferi s.s. in adult ticks appeared somewhat sim- burgdorferi s.s ilar to that of B. afzelii across the three plots. A spiro- , bur chete, genotypically related to the recently described ; isolate SV1 (Casjens et al., 2011) was detected solely in adult ticks; its proportion was largest in Plot C, where it afz, afzelii infected as much as an eighth of the infected adult ticks ; (12.5%) and smallest in Plot A infecting 4.4% of the Prevalence of various spirochetes in questing Ticks Infected ticks infected with Adult 93 Adult 71 Adult 141 Stage no. examined % infected infected adult ticks. The proportions of B. valaisiana lusitaniae ,

and B. garinii (B. garinii serotype 4, now designated as Associated mainly with rodents. Associated with birds and rodents. Associated mainly with birds. B Nymph 111 *Associated with lizards. numerous lizards, whereas none were observed in the neighboring Plots B and C Plot A Nymph 87 31.0 7.4 77.8 0.0 0.0 7.4 0.0 7.4 0.0 2.3 24.1 0.0 0.0 2.3 0.0 2.3 0.0 C Nymph 92 lus Table 1. † ‡ §

Table 2. Density of questing Ixodes ricinus ticks in our study plots ages of about 120 and 140 ticks were collected per hour determined by ﬂagging the vegetation, averaged across the season, in Plots A and B, respectively, more than 270 ticks were and the rate of nymphs questing during the month of April. Plot A sampled per hour in the shadowy Plot C (Table 2, was inhabited by numerous lizards, whereas none were observed in Fig. 1). Nymphal activity appeared low in all plots during the neighboring Plots B and C June, while adult ticks were particularly abundant. In Plot Tick density per hour A, almost ﬁve times more adult than nymphal ticks were Plot Nymphs Adults % Nymphs questing in April found questing (21.3 nymphs vs. 102.2 adults per hour). In contrast, three times more nymphs than adults were A 21.3 102.2 73.0 B 106.6 38.0 34.7 collected in Plot B (106.6 nymphs vs. 38 adults per hour). C 109.5 163.1 29.8 Both stages were almost similarly abundant in Plot C. Of

the few nymphs that were collected throughout the tick Downloaded from https://academic.oup.com/femsec/article/83/3/738/596339 by guest on 29 September 2021 season in Plot A, three quarters (73%) were collected in B. bavariensis (Margos et al., 2009), was not detected in April. During that month, only a third of all nymphs this study) infecting questing adult ticks appeared not to were collected in Plots B and C (34.7% and 29.8%, = vary significantly across the study site (Pcorr. 0.7289 for respectively). The relative density of questing nymphal = B. garinii in adults; Pcorr. 1 for B. valaisiana in adults and adult ticks varied in each of our three study plots. and both genospecies in nymphs). No ticks harbored B. spielmanii spirochetes. Thus, the proportions of at least two of the six different genospecies prevalent in our study Discussion site appeared to be distinctly stratified in questing adult The various genospecies of Lyme disease spirochetes ticks, with B. lusitaniae infecting adult ticks in Plot A at appear not to be evenly distributed within our study site. the same frequency as did B. afzelii in the other plots. Although our three study plots lie in close vicinity of each We determined the relative density of nymphal and other in an area smaller than an acre, the ratio of particu- adult ticks questing in our plots. Whereas seasonal aver- lar genospecies infecting adult ticks varied significantly among them. Whereas B. lusitaniae predominated in questing adult ticks in Plot A, B. afzelii did so in the (a) other plots. In contrast to Plots B and C, Plot A was inhabited by numerous lizards, considered to be the main reservoir host of B. lusitaniae (Dsouli et al., 2006; Maj- la´thova´ et al., 2006; Richter & Matuschka, 2006a; Amore et al., 2007). Our observation that more than half of all infected adult ticks harbored this spirochete, suggests that at least this fraction of the nymphal population had com- pleted their blood meal on lizards. Although host composition in a plot may vary between years, the rate of infected adults harboring B. lusitaniae did not differ significantly from that in the previous year (27 of 59 infected adults harbored B. lusitaniae in 2006 and 26 of (b) 45 infected adults did so in 2007). In a site where lizards are exceedingly abundant, these vertebrates seem to constitute the dominant host for nymphal ticks, contributing the majority of infected adult ticks. The relative density of questing nymphal and adult ticks appeared also to be spatially stratified in our study site. The presence or absence of lizards may similarly affect the relative abundance of tick stages questing there. In the lizard-inhabited Plot A, we found few nymphs questing and did so mainly when ticks first became active in early spring. At this time, few alternative hosts

Fig. 1. Seasonal activity of questing nymphal (a) and adult (b) Ixodes appeared to deplete the population of questing nymphs. ricinus ticks in our study plots determined by ﬂagging the vegetation. Once lizards emerge from their hibernation and with Plot A was inhabited by numerous lizards, whereas none were their increasing activity throughout the spring and sum- observed in the neighboring Plots B and C. mer months, they may be the most readily available host

FEMS Microbiol Ecol 83 (2013) 738–744 ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 742 D. Richter et al. in this plot. They appear to serve as hosts for at least a ever, the presence of hosts that are competent for quarter of all questing nymphs, as inferred by the rate of B. spielmanii or B. lusitaniae profoundly affects the local infection with the lizard-associated B. lusitaniae in adult composition of genospecies in questing ticks (Richter ticks. The relative abundance of questing tick stages may et al., 2004b; Richter & Matuschka, 2006a). Because be inﬂuenced by various habitat conditions, such as avail- lateral movement of questing Ixodes ricinus-like ticks able hosts and microclimate, and may vary between years. rarely exceeds a few meters (Carroll & Schmidtmann, Infection with a particular genospecies appears to be 1996), our observations in this study suggest that the associated with the tick stage. Far more adult ticks har- presence of ticks infected with specialist spirochetes is bored B. lusitaniae in our lizard plot than did nymphs, restricted to the habitat range of its particular host ani- whereas B. afzelii was more likely to infect nymphal than mal. The speciﬁc habitat requirements of lizards, for

adult ticks. This observation may also reflect the feeding example, sand and wall lizards, in Central European sites Downloaded from https://academic.oup.com/femsec/article/83/3/738/596339 by guest on 29 September 2021 preference of larval and nymphal ticks. Generally, larvae (Bischoff, 1984; Gruschwitz & Bo¨hme, 1986) appear to outnumber nymphs infesting rodents by a factor of 20 or result in a focal distribution of B. lusitaniae-infected ticks. more, whereas both stages appear to infest sand and wall Our observation that the genospecies composition signifi- lizards almost equally (Matuschka et al., 1991; Richter & cantly varies across distances as short as a few dozen Matuschka, 2006a; Amore et al., 2007). Larvae feeding on meters suggests that the presence of hosts with a specific lizards sometimes fail to develop (Dsouli et al., 2006; genospecies association impacts the composition of geno- nonpublished observation). This is paralleled by a similar species across a relatively small area. The distribution of observation for larvae feeding on pheasants (Kurtenbach particular Lyme disease genospecies is spatially stratified. et al., 1998). Sufficient larval ticks, however, appear to The proportion of B. lusitaniae relative to other spiro- engorge successfully on lizards and acquire B. lusitaniae chetes appears to vary in lizard-inhabited sites and may from them to subsequently transmit them in their nym- affect risk of infection. In Mediterranean sites, B. lusita- phal stage to hitherto noninfected lizards. This together niae frequently constitutes the sole spirochete in questing with these lizards’ life expectancy ensures that three quar- vector ticks (De Michelis et al., 2000; Younsi et al., 2001; ters of the lizard population in our study site are infected Sarih et al., 2003). But even in particular Central Euro- by B. lusitaniae (Richter & Matuschka, 2006a), thereby pean sites, B. lusitaniae can largely displace other geno- efficiently perpetuating this spirochete. species. In a German site, about 100 km south of our In contrast to B. lusitaniae, other Lyme disease geno- study site and inhabited by numerous sand lizards, species are able to thrive in a wider host array. Borrelia B. lusitaniae infected more than two-thirds of the infected burgdorferi s.s., for example, may be perpetuated in nat- nymphal ticks and almost all infected adult ticks (non- ure by both rodents and birds (Richter et al., 2000, published observation). Because lizards appear incompe- 2004a). Whereas it is the sole Lyme disease genospecies in tent for all other Lyme disease spirochetes (Richter & northeastern U.S., its prevalence in Central Europe is gen- Matuschka, 2006a), those appear to be displaced where erally lower than that of other genospecies. We demon- lizards are the main hosts for vector ticks. Despite the strated experimentally that specialist spirochetes, such as high relative proportion of B. lusitaniae in particular B. afzelii, appear to be more readily transmitted between Central Europe sites, it has not been associated with rodents and ticks and vice versa than are generalist spiro- human cases in this region. Although two human cases chetes, such as B. burgdorferi s.s. (Richter et al., 2004a). apparently related to B. lusitaniae infection have been Specialists appear to thrive efficiently where their closely described in Portugal (Collares-Pereira et al., 2004; De associated host is abundant. One such specialist is B. lusi- Carvalho et al., 2008), they may be explained by variable taniae (Richter & Matuschka, 2006a; Amore et al., 2007). pathogenicity, as has been observed for B. burgdorferi s.s. Because lizards support solely B. lusitaniae and appear to strains in Northern America (Wormser et al., 2008). An exclude all other genospecies, their narrow genospecies abundant tick host, which supports a nonpathogenic association, and possibly also their life expectancy, result genospecies while excluding pathogenic genospecies, may in predominance of B. lusitaniae in sites where lizards are enhance the proportion of its associated spirochete in the abundant, while simultaneously limiting its spread to the local tick population and may thereby act zooprophylacti- habitat range of its host. cally. To the extent that Central European B. lusitaniae Because rodents and birds, serving as reservoir hosts strains are nonpathogenic, the presence of numerous liz- for B. afzelii, B. burgdorferi and B. garinii, B. valaisiana, ards should locally decrease risk of infection for people. respectively, are ubiquitous, these spirochetes infect quest- Our study demonstrates that ecological differences may ing ticks throughout Northern and Central Europe. The induce spatial stratification in the relative prevalence of genospecies composition varies somewhat with the pre- Lyme spirochete genospecies even on small spatial scales, ponderance of rodents or birds. In particular sites, how- in our case less than one acre. Although this study reports

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 743 on observations in one site, it may serve to initiate more Europas, 2/I (Bo¨hme W, ed), pp. 23–68. Aula-Verlag, extensive studies on the stratification of the diverse Lyme Wiesbaden. disease genospecies in Central Europe. The genospecies Carroll JF & Schmidtmann ET (1996) Dispersal of black- stratification observed in our study site seemed to parallel legged tick (Acari: Ixodidae) nymphs and adults at the 33 – a linear trend from a sun exposed to a shadowy location, woods-pasture interface. J Med Entomol : 554 558. from Plot A to Plot C, in accordance with the habitat Casjens SR, Fraser-Liggett CM, Mongodin EF, Qiu WG, Dunn JJ, Luft BJ & Schutzer SE (2011) Whole genome sequence of choice of lizards. The diversity of bacterial communities an unusual Borrelia burgdorferi sensu lato isolate. J Bacteriol infecting I. ricinus ticks varies between and even within 193: 1489–1490. particular forest habitat types (van Overbeek et al., 2008) Collares-Pereira M, Couceiro S, Franca I, Kurtenbach K, and is likely associated with the composition and abun- Scha¨fer SM, Vitorino L, Goncalves L, Baptista S, Vieira ML dance of different tick hosts which in turn depend on the & Cunha C (2004) First isolation of Borrelia lusitaniae from Downloaded from https://academic.oup.com/femsec/article/83/3/738/596339 by guest on 29 September 2021 local habitat structure. In contrast, the non-Lyme disease a human patient. J Clin Microbiol 42: 1316–1318. spirochete, B. miyamotoi, which is vertically transmitted De Carvalho IL, Fonseca JE, Marques JG, Ullmann A, (Richter et al., 2012) and appears to be independent of Hojgaard A, Zeidner N & Nu´ ncio MS (2008) Vasculitis-like reservoir hosts, is evenly distributed across our study site syndrome associated with Borrelia lusitaniae infection. Clin and thus may serve to demonstrate the uniformity of our Rheumatol 27: 1587–1591. sample. Concurrent and detailed characterization of local De Michelis S, Sewell H-S, Collares-Pereira M, Santos-Reis habitat conditions and host arrays may further strengthen M, Schouls LM, Benes V, Holmes EC & Kurtenbach K subsequent studies on the stratifying associations of Lyme (2000) Genetic diversity of Borrelia burgdorferi sensu lato 38 disease genospecies with particular ecological parameters. in ticks from mainland Portugal. J Clin Microbiol : – However, the comparison of abundances of tick hosts of 2128 2133. 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Therefore, evaluation of regional Aula-Verlag, Wiesbaden. risk of infection by Lyme disease spirochetes should take Hanincova´ K, Taragelova´ V, Koci J, Scha¨fer SM, Hails R, the spatial effect of hosts into consideration, which strat- Ullmann AJ, Piesman J, Labuda M & Kurtenbach K (2003) ify the distribution of specifically infected ticks on a small Association of Borrelia garinii and B. valaisiana with 69 – scale. songbirds in Slovakia. Appl Environ Microbiol : 2825 2830. Heisswolf A, Reichmann S, Poethke H-J, Schro¨der B & Acknowledgements Obermaier E (2009) Habitat quality matters for the distribution of an endangered leaf beetle and its egg We thank Andrea Scha¨fer for excellent technical assis- parasitoid in a fragmented landscape. J Insect Conserv 13: tance. This study was partially supported by the Baden- 165–175. Wu¨rttemberg-Stiftung. None of the authors has a conflict Holm S (1979) A simple sequentially rejective multiple test of interest to declare. procedure. 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