Extension of Ixodes ricinus ticks and agents of tick ...

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International Journal of Medical Microbiology 296 (2006) S1, 48–53 www.elsevier.de/ijmm

Extension of Ixodes ricinus ticks and agents of tick-borne diseases to mountain areas in the Czech Republic Vlasta Danielovaá,, Natalia Rudenkob, Milan Daniela,c, Jaroslava Holubovaá, Jan Maternad, Maryna Golovchenkob, Lucie Schwarzovaá National Institute of Public Health, Sˇroba´rova 48, CZ-10042 Prague 10, Czech Republic Institute of Parasitology, Academy of Sciences of the Czech Republic, Cˇeske´ Bude˘jovice, Czech Republic c School of Public Health, Institute for Postgraduate Medical Education, Prague, Czech Republic d Krkonosˇe National Park Authority, Krkonosˇe Museum, Vrchlabı´, Czech Republic a

b

Abstract Along with the shift of the hard tick Ixodes ricinus to higher altitudes observed in the Czech Republic a corresponding shift of tick-borne infections to higher altitudes has been expected. Therefore, I. ricinus ticks, mainly nymphs, were investigated for the presence of tick-borne viruses, tick-borne encephalitis (TBE), Tribec and Eyach, and the spirochaete Borrelia burgdorferi sensu lato in the Sˇumava and Krkonosˇ e Mountains (Czech Republic). The TBE virus and different genospecies of B. burgdorferi s.l. were detected by RT-PCR and PCR, respectively. TBE virus was detected in ticks at 620 and 720 m above sea level (a.s.l.), B. burgdorferi s.l. was detected in ticks up to 1065 m a.s.l. Four genospecies of B. burgdorferi s.l. were identified, B. afzelii, B. garinii, B. burgdorferi sensu stricto, and B. valaisiana. Some nymphs carried multiple Borrelia infections. The conditions of tick-borne agents’ distribution and potential epidemiological consequences are discussed. r 2006 Elsevier GmbH. All rights reserved. Keywords: Tick-borne encephalitis; Lyme borreliosis; Borrelia burgdorferi genospecies; Ixodes ricinus; Mountain areas; Epidemiological impact

Introduction In the early 1990s, the incidence of tick-borne encephalitis (TBE) rose sharply in Europe and has remained high since, with some slight fluctuation (for a synopsis see http://www.tbe-info.com/epidemiology/index.html). In the Czech Republic, the emergence of TBE in new areas including higher altitudes has been observed (Daniel et al., 2003). Research in the field revealed that the major factor is climate-related changes Corresponding author. Tel.: +420 267 082 472; fax: +420 267 082 370. E-mail address: [email protected] (V. Danielova´).

1438-4221/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2006.02.007

(Daniel et al., 2004). The influence of climate change on Ixodes ricinus distribution in Sweden was documented (Lindgren and Gustafson, 2001). This phenomenon was observed also in Denmark (Skarphe´dinsson et al., 2005) and in Norway (Skarpaas et al., 2004). The vertical limit of I. ricinus distribution in European countries differs according to geographical position and it increases with decreasing geographical latitude (Filippova, 1977). In former time, the vertical limit of I. ricinus occurrence in the Czech Republic was considered 700–800 m above sea level (a.s.l.) based on the research which covered almost the whole territory of the Czech Republic in 1960–1962. The research was coordinated on the governmental level (Cˇerny´ et al.,

ARTICLE IN PRESS V. Danielova´ et al. / International Journal of Medical Microbiology 296 (2006) S1, 48–53

1965). In 1959, we monitored ectoparasites on small mammals (included ticks) in the eastern part of Sˇumava Mts. Results confirmed the above-mentioned altitudinal limit (unpublished data). In 1981–1983, that limit was further confirmed in a long-term field experiment in the Krkonosˇ e Mountains (Giant Mts.) (Daniel et al., 1988; Daniel, 1993). The altitudinal limit of I. ricinus occurrence was consequently accepted as the limit of risk of acquiring tick-borne diseases. At that time, the incidence of TBE cases was consistent with this conclusion. Thus, there is sufficient historical data to make a comparison with the situation today. In 2001 and 2002, attention was directed to the Sˇumava Mts. at the southern boundary of the Czech Republic with Germany and Austria, because there human cases of TBE had been identified in places exceeding the known uppermost altitude of risk (Daniel et al., 2003). Also the healthcare and forestry staff of the Sˇumava National Park observed I. ricinus to occur in localities at higher altitudes than had previously been found. In the years 2002 and 2003, the upper limits of I. ricinus distribution were studied in detail in the Krkonosˇ e National Park in the highest Czech mountain range at the Polish border. In both cases, a marked vertical shift to higher altitudes (by up to 500 m) was found. This study attempted to answer the question, whether or not there has also been a shift to higher altitudes in the distribution of the tick-borne disease agents, TBE virus and Borrelia burgdorferi sensu lato.

Material and methods Two territories under study The two mountain ranges differ due to their different geological origin. The Sˇumava Mts. of Archean origin are characterized by extensive so-called Sˇumava plains in an altitude around 1000 m a.s.l., covering approximately 40% of the whole mountain extent, with single rounded summits over 1200 m, and with rough climatic conditions. In the central part of the Plains (Kvilda, 1050 m a.s.l.), a mean annual temperature of +2 1C, a minimum temperature of 41.6 1C (January 30, 1987), and a maximum of 30.8 1C (August 9, 1992) were recorded in the period 1986–2000. The predominant type of forest growths in the Sˇumava Mts. at altitudes of 600–1100 m a.s.l. were formerly floriferous beech woods, now preserved mainly in the south-eastern part (Chocholousˇ kova´ and Gutzerova´, 2003). Natural spruce growths occurred only above 1200 m a.s.l. in the central part of the Sˇumava Mts. The Krkonosˇ e Mts. reaching 1602 m a.s.l. are of primary origin. Many of their summits surpass the timberline, which is situated at approximately 1250 m a.s.l. (annual mean temperature at 1360 m a.s.l.: +1.9 1C). The natural conditions were described by Materna et al. (2005). I. ricinus monitoring was carried out in spring/summer 2002 and three times per season at each locality from the end of May to September in 2003. The ticks were collected by the standard flagging method in

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forest ecotones, by tick findings on forestry staff dogs, and on the basis of prognostic maps with places of expected I. ricinus occurrence (Daniel and Krˇ ı´ z˘, 2002). I. ricinus abundance was calculated as the number of ticks collected by one person per 60 min. The time of actual flagging was at least 1 h at each locality and each collecting visit. Collected ticks were determined to the species and stored at 80 1C for further processing.

Laboratory investigation In 2002, the presence of TBE, Tribec, and Eyach viruses in I. ricinus was tested by isolation attempts on pork kidney cells (Danielova´ et al., 2002) and in suckling mice. The ticks were tested in pools (up to 20 nymphs) or individually according to locality, altitude, and date of collection. The ticks collected in 2003 were individually investigated for TBE virus by RT-PCR. All the collected ticks were tested individually for B. burgdorferi sensu stricto, B. afzelii, B. garinii, and B. valaisiana by PCR.

Polymerase chain reaction (PCR and RT-PCR) The tick samples were prepared with Chelexs 100 resin involving no RNA or DNA purification as it was described previously (Walsh et al., 1991; Danielova´ et al., 2004a; Rudenko et al., 2004, 2005). The final supernatant (up to 12.5 ml) was used as a template for RT-PCR or PCR. RT-PCR was carried out with SuperScriptTM II RNase H RT using SuperScriptTM One Step RT-PCR system (Invitrogen). The reaction volume consisted of 10 ml Reaction Mix (final concentration 1 ), 0.5 ml RT/Taq Mix, 1 ml (final concentration 1 mM) of each specific primer 1 and 2, and the chelex-buffered sample up to the total volume of 20 ml. The conditions used for viral RNA amplification are described by Rudenko et al. (2004). The specific oligonucleotide primers corresponding to the 50 -terminal noncoding region were successfully used to identify TBE virus sequences in ticks: TBE 2 (forward) 50 -GCGTTTGCT(C,T)CGGA-30 and TBE 1 (reverse) 50 -CTCTTTCGACACTCGTCGAGG-30 (Ramelow et al., 1993). The size of the TBE1/TBE2 PCR product was 175 nt. All primers for PCR and RT-PCR were synthesized by Generi-Biotech (Czech Republic). In case of spirochaete detection, the PCR primers based on B. burgdorferi s.l. ospA gene sequences were used to type all (Sensu Lato [SL] primers set) or each (GI, GII, and GIII primers sets) of the B. burgdorferi s.l. genospecies involved in Lyme borreliosis, as it was described (Marconi and Garon, 1992; Demaerschalck et al., 1995; Sparagano et al., 1999; Rudenko et al., 2005). For the detection of B. valaisiana, the primers designated by Liebisch et al. (1998) were used: P8+(50 -GCAAGTCAAACGGGATGTAGT-30 ) and P9-(50 GTATTTTATGCATAGACTTTATATG-30 ). The size of the PCR product was 549 bp. The PCR conditions were as follows: initial denaturation at 95 1C/5 min and 35 cycles at 94 1C/30 s, 55 1C/30 s, and 72 1C/60 s. In all runs, both negative and positive controls were included. The PCR products were resolved on 1% agarose electrophoresis gel and the bands were visualized under UV light using SYBR Green gel stain.


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Results I. ricinus ticks were collected between 760 and 1080 m a.s.l. in the Sˇumava Mts. and between 600 and 1270 m a.s.l. in the Krkonosˇ e Mts. The decrease of tick abundance with increasing altitude is evident (Table 1). In 2002, 372 I. ricinus ticks (246 nymphs, 61 females, 65 males) from 762 to 860 m a.s.l. in the Sˇumava Mts. and 294 I. ricinus nymphs from 600 to 1180 m a.s.l. in the Krkonosˇ e Mts. were examined for various tickborne arboviruses. The results were negative. In 2003, TBE virus was detected in two out of 491 nymphs tested (at 620 and 710–720 m a.s.l.) in Krkonosˇ e Mts. In contrast to the TBE virus, B. burgdorferi s.l. was detected in ticks collected at 41000 m a.s.l. In the Sˇumava Mts., B. afzelii was detected in three I. ricinus nymphs from 762 m a.s.l. (including two cases of co-infection with B. garinii) and in two nymphs at 1024–1039 m a.s.l. (Table 2). In the Krkonosˇ e Mts., B. burgdorferi s.l. was found in ticks up to the zone 960–1020 m a.s.l. (Table 3). The genospecies B. burgdorferi s.s., B. afzelii, B. garinii, and B. valaisiana were detected. Although the abundance of I. ricinus decreased with increasing altitude, the prevalence of B. burgdorferi s.l. in the ticks did not.

Seventy-seven out of 832 nymphs were infected, just as one out of 19 larvae. Another group of 25 pooled larvae which also was tested positive for B. burgdorferi s.l (strain not identified) is not included in the tables. Fourteen out of 77 infected nymphs carried multiple Borrelia infections (with 2–4 different genospecies). Surprisingly, co-infected ticks were detected more frequently at X700 m a.s.l. than beneath. The infection rate of the ticks tested in 2002 was 12.2% (n ¼ 221)–nymphs 13.6% (n ¼ 191), larvae 4.0% (a pool of 25 larvae), and five adult ticks were Borrelianegative. In 2003, the infection rate of the tested ticks was 7.8% (n ¼ 664)–nymphs 8.0% (n ¼ 641), larvae 5.3% (n ¼ 19), four adults were Borrelia-negative. Ticks infected with B. burgdorferi s.s., B. afzelii, or B. garinii were detected at a maximum altitude of approximately 1000 m a.s.l., ticks carrying B. valaisiana at 780–800 m a.s.l. (Table 4).

Discussion B. burgdorferi s.l. occurrence in I. ricinus ticks in both studied areas and two TBE human infections acquired at 900 m a.s.l. in the Sˇumava Mts. indicate the risk of

Table 1. Ixodes ricinus nymphs abundance at different altitudes in the central and eastern part of the Krkonosˇ e Mts., 2002 and 2003, and in the Sˇumava Mts., 2002 (Czech Republic) Altitude (m a.s.l.)

600–620 700–765 780–850 870–930 960–1020 1030–1100 1160–1270

Central Krkonosˇ e

Eastern Krkonosˇ e

Altitude (m a.s.l.)

No. of ticksa

Tick densityb

No. of ticksa

Tick densityb

171 96 36 59

24.4 15.2 5.8 9.6

358 163 130 33 27

55.8 25.7 23.3 8.1 1.5

15 2

1.6 0.2

Sˇumava No. of ticksa

Tick densityb

760–860

171

13.5

1024–1080

38

2.8

a.s.l.: above sea level. a Total number of ticks collected in the respective altitude. b Number of ticks collected per man-hour.

Table 2. 2002

Prevalence of Borrelia burgdorferi s.l. in Ixodes ricinus nymphs in different altitudes in the Sˇumava Mts. (Czech Republic),

Altitude (m a.s.l.)

762 927–1045 1024–1039 1047–1080 a.s.l.: above sea level.

Ticks

B. burgdorferi s.l. findings

B. afzelii

B. garinii

2

No. tested

No. infected

% infected

20 4 29 5

3 0 2 0

15

5

3

7

2

2


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Table 3. Prevalence of Borrelia burgdorferi s.l. in Ixodes ricinus ticks of all life stages in different altitudes in the Krkonosˇ e Mts. (Czech Republic), 2002 and 2003 Altitude (m a.s.l.)

B. burgdorferi s.l. findings

Ticks

600–620 700–765 780–850 870–930 960–1020 1030–1100 1160–1270

No. tested

No. infected

% infected

441 197 110 47 48 15 2

35 19 12 2 10 0 0

8 10 11 4 21

39 23 21 3 12 0 0

a.s.l.: above sea level.

Table 4. Borrelia burgdorferi s.l. findings in Ixodes ricinus ticks of all life stages at different altitudes in the Krkonosˇ e Mts. (Czech Republic), 2002 and 2003 Altitude (m a.s.l.)

B. burgdorferi s.s.

B. afzelii

B. garinii

B. valaisiana

Genospecies unknown

600–620 700–765 780–850 870–930 960–1020 1030–1100 1160–1270

4 2 4 0 1 0 0

15 10 5 2 6 0 0

16 6 7 1 4 0 0

2 4 4 0 0 0 0

2 1 1 0 1 0 0

a.s.l.: above sea level.

getting tick-borne infections in altitudes regarded free of that risk, as yet (Daniel et al., 2003). This is important information for the public health service, since many attractive destinations inviting for outdoor activities especially in the main tick activity season are situated at that altitude. Engorged I. ricinus ticks had certainly been introduced by hosts (birds and bisulcate animals) to altitudes exceeding the former vertical border also in former times, but they usually did not find adequate living conditions. Although they occasionally accomplished a certain part of the developmental cycle, a stable local population was never established (Daniel et al., 1988; Daniel, 1993). Climate modifications observed in the past two decades, primarily, higher temperature shown foremost in spring and autumn and thus providing longer season for the development of ticks in higher altitudes (Danielova´ et al., 2004b) has contributed to the creation of populations in mountains. Regular findings of all active host-seeking stages and also tick occurrence on home animals (dogs, cats) (Daniel et al., 2003; Materna et al., 2005) provide evidence of the existence of local populations (not only sporadic findings of surviving individuals occasionally brought in there by hosts). Verified repeated attacks by I. ricinus nymphs above 1000 m a.s.l. in the Krkonosˇ e Mts. (Materna et al., 2005) have made the increased human risk evident.

Negative results of TBE virus isolation from I. ricinus collected in the Sˇumava Mts. correspond to the relatively low number of tested ticks and our experience with low tick infection rates in active TBE foci (Danielova´ et al., 2002). Human infection demonstrably acquired at 900 m a.s.l. (Daniel et al., 2003) places this altitude more or less close to the present maximum altitude of TBE risk in the Sˇumava Mts. In the Krkonosˇ e Mts., the presence of TBE virus in I. ricinus was detected at maximum at 600 and 720 m a.s.l. Documented autochthonous human infections are lacking; the situation corresponds to that in adjacent submontane regions. Although TBE infections are being reported regularly from the Sˇumava foothills, they are extremely rare in north-eastern Bohemia. In both mountain ranges, however, B. burgdorferi s.l. was detected in I. ricinus in locations above 1000 m a.s.l. In the Krkonosˇ e Mts., the frequency of positive findings as well as the spectrum of demonstrated genospecies at 1000 m a.s.l. is rather high. It is possible that this finding reflects a high species diversity of reservoir hosts (small mammals and birds), ensuring the local existence of different B. burgdorferi genospecies. Although the fauna of the Krkonosˇ e Mts. has been well known (Ande˘ra et al., 1974; Flousek and Gramsz, 1999), the differences in host composition in localities under study were not analysed, as yet. A particular (and common) habitat in

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altitudes above 1000 m a.s.l. has been formed by solitary trees (or small groups of them) in grassy land with branches reaching the ground. These ‘canopy trees’ provide a specific favourable microclimate all the year round for ticks including more stable air temperature, air humidity, soil moisture and also hiding places for vertebrates–the source of blood for all I. ricinus life stages. B. burgdorferi s.l. generally reaches higher prevalences in I. ricinus than the TBE virus does. This finding was also confirmed in areas rather newly colonized by I. ricinus. While the circulation of borreliae among animal hosts and I. ricinus apparently rather quickly gains momentum when I. ricinus advances into a new area, zoonotic circulation of TBE virus in new conditions proceeds with much greater difficulty. The density of local populations of I. ricinus seems to be high enough for the circulation of borreliae but not for TBE virus, as yet. The practical conclusion for the public health service is to accept that there is a risk of human Borrelia infection at 1000 m a.s.l. through tick bite, but the risk is very limited thanks to the presently rather low population density of I. ricinus. In the case of TBE, we may assume that the risk of infection will gradually reach higher altitudes than presently as the tick population density increases due to the current trends in climate modification.

Acknowledgements It is a pleasure to thank to Dr. J. Noz˘icˇka, District Public Health Centre, Prachatice, and Ing. A. Jirsa, Administration of the Sˇumava National Park, Vimperk, for their aid in this investigation. This study was partly supported by WHO/EC project ‘Climate Change and Adaptation Strategies for Human Health in Europe’ (cCASHh), Contract no. EVK2–2000–0070. We are very grateful to Prof. D. Crossley, University of Georgia, Athens, USA, for linguistic review of the manuscript.

References Ande˘ra, M., Hana´k, V., Vohralı´ k, V., 1974. The mammals of the Krkonosˇ e Mountains. Opera Corcontica 11, 131–184 (in Czech). Cˇerny´, V., Rosicky´, B., Asˇ mera, J., Kadlcˇı´ k, K., Kobı´ k, V., Kratochvı´ lova´, E., Lauterer, P., Nova´k, D., Pauleova´, E., Sˇebek, Z., Sˇvec, J., Teˇmin, K., 1965. Results of investigations of Ixodes ricinus (L.) tick phenology in Czech lands in 1960–1962. Cˇesk. Parasitol. 2, 125–131 (in Czech). Chocholousˇ kova´, Z., Gutzerova´, N., 2003. Forests in Sˇumava Mts. In: Ande˘ra, M., Zavrˇ el, P. (Eds.), Sˇumava Mts., Nature, History, Life. Publ. House Baset, Prague, pp. 167–170 (in Czech).

Daniel, M., 1993. Influence of the microclimate on the vertical distribution of the tick Ixodes ricinus (L.) in Central Europe. Acarologia 34, 105–113. Daniel, M., Krˇ ı´ z˘, B., 2002. Tick-borne encephalitis in the Czech Republic: I. Predictive maps of Ixodes ricinus tick high-occurrence habitats and a tick-borne encephalitis risk assessment in Czech regions; II. Maps of tick-borne encephalitis incidence in the Czech Republic in 1971–2000. Project cCASHh EVK2—2000-0070, National Institute of Public Health, Prague, Czech Republic. Daniel, M., Cˇerny´, V., Albrecht, V., Honza´kova´, E., 1988. The microclimate at different altitudes of the Krkonosˇ e Mts. and its influence on the existence of the Ixodes ricinus (L.) tick. Opera Corcontica 25, 76–110 (in Czech). Daniel, M., Danielova´, V., Krˇ ı´ z˘, B., Jirsa, A., Noz˘icˇka, J., 2003. Shift of the tick Ixodes ricinus and tick-borne encephalitis to higher altitudes in Central Europe. Eur. J. Clin. Microbiol. Infect. Dis. 22, 327–328. Daniel, M., Danielova´, V., Krˇ ı´ z˘, B., Kott, I., 2004. An attempt to elucidate the increased incidence of tick-borne encephalitis and its spread to higher altitudes in the Czech Republic. Int. J. Med. Microbiol. 293 (Suppl. 37), 55–62. Danielova´, V., Holubova´, J., Daniel, M., 2002. Tick-borne encephalitis virus prevalence in Ixodes ricinus ticks collected in high risk habitats of the South-Bohemian region of the Czech Republic. Exp. Appl. Acarol. 26, 145–151. Danielova´, V., Daniel, M., Rudenko, N., Golovchenko, M., 2004a. Prevalence rate of Borrelia burgdorferi s.l. genospecies in host-seeking Ixodes ricinus ticks in certain SouthBohemian locations (Czech Republic). Cent. Eur. J. Publ. Health 12, 151–156. Danielova´, V., Krˇ ı´ z˘, B., Daniel, M., Benesˇ , Cˇ., Valter, J., Kott, I., 2004b. Effects of climate change on the incidence of tick-borne encephalitis in the Czech Republic in the past two decades. Epidemiol. Mikrobiol. Immunol. 53, 174–181 (in Czech). Demaerschalck, I., Messaoud, A.B., Kesel, M.D., Hoyois, B., Lobet, Y., Hoet, P., Bigaignon, G., Bollen, A., Godfroid, E., 1995. Simultaneous presence of different Borrelia burgdorferi sensu lato genospecies in biological fluids of Lyme disease patients. J. Clin. Microbiol. 33, 602–608. Filippova, N.A., 1977. Ixodid ticks (Ixodinae). Fauna of the USSR, vol. IV/4. Nauka Publ. House, Leningrad, 396pp. (in Russian). Flousek, J., Gramsz, B., 1999. Atlas of breeding birds in the Krkonosˇ e Mts. (Czech Republic/Poland) in 1991–1994. Krkonosˇ e National Park Administration, Vrchlabı´ 424pp. (in Czech and Polish). Liebisch, G., Sohns, B., Bautsch, W., 1998. Detection and typing of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks attached to human skin by PCR. J. Clin. Microbiol. 36, 3355–3358. Lindgren, E., Gustafson, R., 2001. Tick-borne encephalitis in Sweden and climate change. The Lancet 358, 16–18. Marconi, R.T., Garon, C.F., 1992. Development of polymerase chain reaction primer sets for diagnosis of Lyme disease and for species-specific identification of Lyme disease isolates by 16S rRNA signature nucleotide analysis. J. Clin. Microbiol. 30, 2830–2834.


Materna, J., Daniel, M., Danielova´, V., 2005. Altitudinal distribution limit of the tick Ixodes ricinus shifted considerably towards higher altitudes in Central Europe: results of three years monitoring in the Krkonosˇ e Mts. (Czech Republic). Centr. Eur. J. Publ. Health 13, 24–28. Ramelow, C., Su¨ss, J., Berndt, D., Roggendorf, M., Schreier, E., 1993. Detection of tick-borne encephalitis virus RNA in ticks (Ixodes ricinus) by the polymerase chain reaction. J. Virol. Methods 45, 115–119. Rudenko, N., Golovchenko, M., Cihla´rˇ ova´, V., Grubhoffer, L., 2004. Tick-borne encephalitis virus-specific RT-PCR – a rapid test for detection of the pathogen without viral RNA purification. Acta Virol. 48, 167–171. Rudenko, N., Golovchenko, M., Ne˘mec, J., Volkaert, J., Malla´tova´, N., Grubhoffer, L., 2005. Improved method of detection and molecular typing of Borrelia burgdorferi

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sensu lato in clinical samples by polymerase chain reaction without DNA purification. Folia Microbiol. 50, 31–39. Skarpaas, T., Ljøstad, U., Sundøy, A., 2004. First human cases of tickborne encephalitis, Norway. Emerg. Infect. Dis. 10, 2241–2243. Skarphe´dinsson, S., Jensen, P.M., Kristiansen, K., 2005. Survey of tickborne infections in Denmark. Emerg. Infect. Dis. 11, 1055–1061. Sparagano, O.A., Allsopp, M.T., Mank, R.A., Rijpkema, S.G., Figueroa, J.V., Jongejan, F., 1999. Molecular detection of pathogen DNA in ticks (Acari: Ixodidae): a review. Exp. Appl. Acarol. 23, 929–960. Walsh, P.S., Metzger, D.A., Higuchi, R., 1991. Chelex 100 as a medium for simple extraction of DNA for PCRbased typing from forensic material. Biotechniques 10, 506–513.