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International Journal of Medical Microbiology 298 (2008) S1, 188–192 www.elsevier.de/ijmm
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Prevalence of Borrelia burgdorferi s.l. in ticks feeding on humans in Thuringia/Germany Jan Franke, Susanne Kipp, Charlotte Flu¨gel, Wolfram Dorn Department of Food and Environmental Hygiene, Institute of Nutrition, Friedrich-Schiller-University Jena, Dornburger Straße 29, D-07743 Jena, Germany Accepted 20 January 2008
Abstract In 2004, a total of 618 Ixodes ricinus ticks fed on humans were collected by physicians throughout Thuringia. The prevalence rates of Borrelia burgdorferi sensu lato (s.l.) genotypes were determined by nested PCR and restriction fragment length polymorphism, targeting a 0.8-kb fragment of the ospA gene. The total prevalence of B. burgdorferi s.l. was 6.1%. B. afzelii was found in 67%, B. valaisiana in 15% and B. garinii in 15% of the positive ticks (3% could not be determined). In one tick, a double infection with B. valaisiana type II and B. garinii OspA type V was detected. Female adult ticks had the highest infection rate (11.7%), followed by nymphs (4.5%) and larvae (3.4%). The overall prevalence increased from spring (4.0%) to autumn (10.0%). Nevertheless, the risk of infection was maximal in summer, because of a much higher infestation and consequently a higher absolute number of infected ticks. The predominance of B. afzelii probably results from its resistance against human serum. The unexpectedly low total prevalence is possibly caused by immune defence mechanisms (e.g. complement) effective against less resistant B. burgdorferi s.l. strains in the tick. r 2008 Elsevier GmbH. All rights reserved. Keywords: Borrelia burgdorferi sensu lato; Tick bite; Human-fed ticks; Prevalence; ospA
Introduction The Borrelia burgdorferi s.l. group includes the causative agents of Lyme borreliosis, the most frequent arthropod-borne human disease in the temperate northern hemisphere. Approximately 60,000 people contract Lyme borreliosis in Germany annually, but there is presumably a much higher number of unreported cases (Mehnert, 2005). The various B. burgdorferi s.l. genospecies are responsible for a multitude of different clinical manifestations of Lyme borreliosis (van Dam Corresponding author. Tel.: +49 3641 94 9694; fax: +49 3641 94 9602. E-mail address: [email protected] (J. Franke).
1438-4221/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2008.01.009
et al., 1993; Balmelli and Piffaretti, 1995; Lu¨nemann et al., 2001), and even the genetic diversity within those species leads to differences in the symptomatic occurrence of Lyme borreliosis (Baranton et al., 2001). Until now, seven species are known to be or at least considered pathogenic for humans: B. burgdorferi sensu stricto (s.s.), B. garinii, B. afzelii, B. bissettii, B. lusitaniae, B. valaisiana, and B. spielmanii (Strle et al., 1997; Ryffel et al., 1999; Wang et al., 1999a, b; Godfroid et al., 2003; Diza et al., 2004; Richter et al., 2004; da Franca et al., 2005). The federal state of Thuringia with its extensive areas of coniferous, deciduous, and mixed forests as well as moderate climatic conditions provides a large number of habitats for Ixodes ricinus ticks. The number of reported Lyme borreliosis cases in
ARTICLE IN PRESS J. Franke et al. / International Journal of Medical Microbiology 298 (2008) S1, 188–192
Thuringia/Germany in 2004 was 263 (incidence: 11.2 per 100,000), mainly manifested (92%) as erythema migrans (Thuringian Ministry of Social Affairs, Family and Health, unpublished). Recent studies in this region have shown prevalence rates of B. burgdorferi s.l. in questing ticks of up to 28.7%, with varying prevalences of different genospecies depending on the area of investigation (Dorn et al., 2000; Hildebrandt et al., 2003; Kipp et al., 2006). The aim of this study was to obtain information about the presence of B. burgdorferi s.l. genospecies in I. ricinus ticks, feeding on humans in contrast to host-seeking ticks. The results might be a contribution to the risk assessment of Lyme borreliosis, particularly in the region of Thuringia and might also be beneficial for further studies on vector–host transmission and immune evasion mechanisms of the different genospecies.
Materials and methods To assess the risk of human infection, 618 I. ricinus ticks feeding on humans from Thuringia were tested for the presence of B. burgdorferi s.l. The ticks were collected by physicians and stored at 80 1C until analysis. Spirochaete DNA was extracted directly from TM the ticks with the GenElute Bacterial Genomic DNA Kit (Sigma, St. Louis, USA). DNA amplification was realised with a nested polymerase chain reaction (PCR) targeting a 0.8-kb ospA fragment. The genospecies were identified by restriction fragment length polymorphism (RFLP), using HindIII, BglII, SspI, SfuI, and Kpn2I as restriction enzymes (Michel et al., 2004). After gel electrophoresis and ethidium bromide staining, the OspA species could then be determined under UV exposure by means of their typical band patterns according to Michel et al. (2004). Significance analysis was performed using the chi-square test.
Results and discussion The total prevalence of B. burgdorferi s.l. in human engorged ticks was 6.1%. In contrast, in one investigation in southern Germany, 37% of the ticks removed from humans were infected with B. burgdorferi s.l. (Fingerle et al., 1998). It should be mentioned that in that study a less exact detection method (immunofluorescence assay) was used. Former studies on questing ticks in southern and Central Germany, using PCR, have revealed prevalence rates of 10–36% (Fingerle et al., 1999; Rauter et al., 2002; Stanczak et al., 2002; Hildebrandt et al., 2003; Kipp et al., 2006). B. afzelii was found in 67%, B. valaisiana in 15%, and B. garinii also in 15% of the positive ticks (3% could not
B. valaisiana type I 10%
B. valaisiana type II 5%
189
unknown 3%
B. garinii OspA type IV 3%
B. afzelii OspA type II 67%
B. garinii OspA type V 8% B. garinii OspA type VI 5%
Fig. 1. OspA types of Borrelia burgdorferi s.l. found in 38 positive Ixodes ricinus ticks, feeding on humans from Thuringia/Germany in 2004. Percentages are rounded. Values refer to the number of infections (n ¼ 39), because one coinfection with B. valaisiana type II and B. garinii OspA type V was detected.
be determined). In one tick, a double infection with B. valaisiana type II and B. garinii OspA type V occurred. B. burgdorferi s.s. and other genospecies were not found (Fig. 1). Only five districts provided an adequate number of samples. Local prevalence rates between those districts were not significantly distinct. In every district, B. afzelii was the predominant species. Former studies on hostseeking ticks in Thuringia, using the same method, have shown that the prevalence of B. garinii was much higher than (Hildebrandt et al., 2003) or at least as high as that of B. afzelii (Kipp et al., 2006). Hildebrandt et al. (2003) and Kipp et al. (2006) also found B. burgdorferi s.s. in 32.4% and 9.8% of the positive ticks, respectively. The reason for the predominance of B. afzelii in ticks, feeding on humans, might be its better resistance against immune defence mechanisms (e.g. complement) as described in some studies (Breitner-Ruddock et al., 1997; Kraiczy et al., 2002; Bhide et al., 2005). The lack of resistance against human complement in some strains, leading to a rapid decline of the spirochaete count in the tick salivary glands, might also be a reason for the unexpectedly low total prevalence of B. burgdorferi s.l. Data provided by the Thuringian Ministry of Social Affairs, Family and Health show, that 92.0% (242/263) of the Thuringian Lyme borreliosis cases in 2004 were manifested exclusively as erythema migrans. That corresponds with the predominance of B. afzelii in engorged ticks, the genospecies mainly, but not exclusively associated with erythema migrans (Canica et al., 1993; van Dam et al., 1993; Anthonissen et al., 1994; Balmelli and Piffaretti, 1995).
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20 18
Prevalence (%)
16
Spring Summer Autumn
14 12 10 8 6 4 2 0 Total [n=618]
Nymphs [n=444]
Female Adults [n=145]
Fig. 2. Seasonal course of the prevalence of Borrelia burgdorferi s.l. in Ixodes ricinus ticks, feeding on humans in Thuringia/ Germany (2004). Larvae (n ¼ 29, one larva infected) are included in the total result.
Only a few patients (7.2%) had neurological symptoms, known to be mostly caused by B. garinii (Demaerschalck et al., 1995). Arthralgic symptoms, associated mainly with B. burgdorferi s.s. (Demaerschalck et al., 1995), appeared only in 1.9% of the cases, corresponding to the fact that no B. burgdorferi s.s.infected ticks have been found. However, some strains seem to cause a mixture of symptoms, and there is an overlap between clinical manifestations, caused by different genospecies, which was also evident in a number of the above-mentioned Lyme borreliosis cases (4.9%). Also coinfections with other tick-borne pathogens like Anaplasma phagocytophilum can lead to diverse clinical outcomes (Belongia, 2002; Swanson et al., 2006). The mean infection rate of female adult ticks was significantly higher (11.7%, po0.025) than in nymphs (4.5%) and in larvae (3.4%). The overall prevalence increased from 4.0% in spring to 6.5% in summer and reached its peak in autumn (10.0%, Fig. 2). Nymphs had the highest infection rate in summer (5.0%), whereas adult females were most frequently infected in autumn (17.6%). However, the total number of ticks collected in autumn was low (13 nymphs, 17 females) and only three out of these 30 ticks were infected. These data support findings from our former investigations on host-seeking ticks, showing the highest prevalence rates in autumn (Dorn and Su¨nder, 1997). Some studies also indicate that there is often a negative correlation between tick activity and prevalence rates as observed in this investigation (Su¨nder et al., 1997; Gray et al., 1999). Nevertheless, seasonal patterns in the prevalence of B. burgdorferi s.l. in ticks seem to be very heterogeneous even within one investigation area (Steil, 2002; Hildebrandt, 2003), probably mainly due to the humidity- and temperature-dependent host-seeking activity of I. ricinus and seasonal activity patterns of reservoir hosts (Gray 1998; Jensen, 2000). For example, diapause mechanisms, induced by disadvantageous climate conditions lead to lower infection rates, because
the possibility of ingesting spirochaetes from reservoir hosts declines. The infestation with nymphs (n ¼ 444) was more than three times higher than with female adults (n ¼ 145). Only a few subjects were infested with larvae (n ¼ 29). More persons were infested with ticks in summer (n ¼ 462) than in spring (n ¼ 126) and autumn (n ¼ 30). Although the mean prevalence of B. burgdorferi s.l. in I. ricinus reached its peak in autumn, the risk of infection was higher in June and July, due to a much higher absolute number of infected ticks in summer (n ¼ 30) than in autumn (n ¼ 3) and spring (n ¼ 5). The higher outdoor activity also enhances the risk of a tick bite and consequently of contracting Lyme borreliosis in summer. In conclusion, the results of this investigation show that the prevalence of B. burgdorferi s.l. in ticks feeding on humans is lower when compared to host-seeking ticks, analysed in former studies. Also the composition of genospecies is different. This supports studies indicating that some Borrelia strains are not able to develop effective immune evasion mechanisms against human complement within the tick body. Further studies should focus on the genetic and phenotypic fundamentals for these mechanisms.
Acknowledgements We would like to thank Dr. Gerrit Hesse from the Thuringian Ministry of Social Affairs, Family and Health for providing the data of Lyme borreliosis cases.
References Anthonissen, F.M., De Kesel, M., Hoet, P.P., Bigaignon, G.H., 1994. Evidence for the involvement of different
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genospecies of Borrelia in the clinical outcome of Lyme disease in Belgium. Res. Microbiol. 145, 327–331. Balmelli, T., Piffaretti, J.C., 1995. Association between different clinical manifestations of Lyme disease and different species of Borrelia burgdorferi sensu lato. Res. Microbiol. 146, 329–340. Baranton, G., Seinost, G., Theodore, G., Postic, D., Dykhuizen, D., 2001. Distinct levels of genetic diversity of Borrelia burgdorferi are associated with different aspects of pathogenicity. Res. Microbiol. 152, 149–156. Belongia, E.A., 2002. Epidemiology and impact of coinfections acquired from Ixodes ticks. Vector Borne Zoonotic Dis. 2, 265–273. Bhide, M.R., Travnicek, M., Levkutova, M., Curlik, J., Revajova, V., Levkut, M., 2005. Sensitivity of Borrelia genospecies to serum complement from different animals and human: a host–pathogen relationship. FEMS Immunol. Med. Microbiol. 43, 165–172. Breitner-Ruddock, S., Wurzner, R., Schulze, J., Brade, V., 1997. Heterogeneity in the complement-dependent bacteriolysis within the species of Borrelia burgdorferi. Med. Microbiol. Immunol. 185, 253–260. Canica, M.M., Nato, F., du Merle, L., Mazie, J.C., Baranton, G., Postic, D., 1993. Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis. Scand. J. Infect. Dis. 25, 441–448. da Franca, I., Santos, L., Mesquita, T., Collares-Pereira, M., Baptista, S., Vieira, L., Viana, I., Vale, E., Prates, C., 2005. Lyme borreliosis in Portugal caused by Borrelia lusitaniae? Clinical report on the first patient with a positive skin isolate. Wien. Klin. Wochenschr. 117, 429–432. Demaerschalck, I., Ben Messaoud, A., De Kesel, M., Hoyois, B., Lobet, Y., Hoet, P., Bigaignon, G., Bollen, A., Godfroid, E., 1995. Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients. J. Clin. Microbiol. 33, 602–608. Diza, E., Papa, A., Vezyri, E., Tsounis, S., Milonas, I., Antoniadis, A., 2004. Borrelia valaisiana in cerebrospinal fluid. Emerg. Infect. Dis. 10, 1692–1693. Dorn, W., Su¨nder, U., 1997. Lyme borreliosis in Thuringia (Germany): prevalence of the causative agent in fieldcollected ticks. In: Su¨ss, J., Kahl, O., (Eds.), Tick-borne Encephalitis and Lyme Borreliosis, Proceedings of the Fourth International Potsdam Symposium Tick-borne Disease, Berlin, 21–22 February 1997, Pabst, Lengerich, pp. 175–196. Dorn, W., Su¨nder, U., Steil, B., Flu¨gel, C., 2000. Zum Zusammenhang von saisonaler Dichte und Durchseuchungsrate mit Borrelia burgdorferi sensu lato bei freilandgefangenen Zecken der Art Ixodes ricinus L. 1758 (Acari: Ixodidae) – eine Fallstudie im Freistaat Thu¨ringen. Mitt. Dtsch. Ges. Allg. Angew. Ent. 12, 187–191. Fingerle, V., Liegl, G., Munderloh, U., Wilske, B., 1998. Expression of outer surface proteins A and C of Borrelia burgdorferi in Ixodes ricinus ticks removed from humans. Med. Microbiol. Immunol. 187, 121–126. Fingerle, V., Munderloh, U.G., Liegl, G., Wilske, B., 1999. Coexistence of ehrlichiae of the phagocytophila group with
191
Borrelia burgdorferi in Ixodes ricinus from Southern Germany. Med. Microbiol. Immunol. 188, 145–149. Godfroid, E., Min Hu, C., Humair, P.-F., Bollen, A., Gern, L., 2003. PCR-reverse line blot typing method underscores the genomic heterogeneity of Borrelia valaisiana species and suggests its potential involvement in Lyme disease. J. Clin. Microbiol. 41, 3690–3698. Gray, J.S., 1998. Review: the ecology of ticks transmitting Lyme borreliosis. Exp. Appl. Acarol. 22, 249–258. Gray, J.S., Kirstein, F., Robertson, J.N., Stein, J., Kahl, O., 1999. Borrelia burgdorferi sensu lato in Ixodes ricinus ticks and rodents in a recreational park in south-western Ireland. Exp. Appl. Acarol. 23, 717–729. Hildebrandt, A., 2003. Pra¨valenz von Ehrlichien der Ehrlichia phagocytophila Genogruppe und Borrelien des Borrelia burgdorferi sensu lato Komplexes in Ixodes ricinus Zecken aus dem Ilmtal. Ph.D. Thesis, Friedrich-Schiller-Universita¨t Jena, Germany. Hildebrandt, A., Schmidt, K.H., Wilske, B., Dorn, W., Straube, E., Fingerle, V., 2003. Prevalence of four species of Borrelia burgdorferi sensu lato and coinfection with Anaplasma phagocytophila in Ixodes ricinus ticks in central Germany. Eur. J. Clin. Microbiol. Infect. Dis. 22, 364–367. Jensen, P.M., 2000. Host-seeking activity of Ixodes ricinus ticks based on daily consecutive flagging samples. Exp. Appl. Acarol. 24, 695–708. Kipp, S., Dorn, W., Wilske, B., Fingerle, V., 2006. Heterogeneity in prevalence and genetic diversity of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in different localities in Thuringia, Germany. Int. J. Med. Microbiol. 296 (Suppl. 40), 119–121. Kraiczy, P., Skerka, C., Kirschfink, M., Zipfel, P.F., Brade, V., 2002. Immune evasion of Borrelia burgdorferi: insufficient killing of the pathogens by complement and antibody. Int. J. Med. Microbiol. 291 (Suppl. 33), 141–146. Lu¨nemann, J.D., Zarmas, S., Priem, S., Franz, J., Zschenderlein, R., Aberer, E., Klein, R., Schouls, L., Burmester, G.R., Krause, A., 2001. Rapid typing of Borrelia burgdorferi sensu lato species in specimens from patients with different manifestations of Lyme borreliosis. J. Clin. Microbiol. 39, 1130–1133. Mehnert, W.H., 2005. Zur Situation bei wichtigen Infektionskrankheiten in Deutschland: Neuerkrankungen an LymeBorreliose im Jahr 2004. Analyse auf der Basis der Meldedaten aus den sechs o¨stlichen Bundesla¨ndern. Epidemiol. Bull. 32, 285–288. Michel, H., Wilske, B., Hettche, G., Goettner, G., Heimerl, C., Reischl, U., Schulte-Spechtel, U., Fingerle, V., 2004. An ospA-polymerase chain reaction/restriction fragment length polymorphism-based method for sensitive detection and reliable differentiation of all European Borrelia burgdorferi sensu lato species and OspA types. Med. Microbiol. Immunol. 193, 219–226. Rauter, C., Oehme, R., Diterich, I., Engele, M., Hartung, T., 2002. Distribution of clinically relevant Borrelia genospecies in ticks assessed by a novel, single-run, real-time PCR. J. Clin. Microbiol. 40, 36–43. Richter, D., Schlee, D.B., Allgower, R., Matuschka, F.R., 2004. Relationships of a novel Lyme disease spirochete,
ARTICLE IN PRESS 192
J. Franke et al. / International Journal of Medical Microbiology 298 (2008) S1, 188–192
Borrelia spielmani sp. nov., with its hosts in Central Europe. Appl. Environ. Microbiol. 70, 6414–6419. Ryffel, K., Pe´ter, O., Rutti, B., Suard, A., Dayer, E., 1999. Scored antibody reactivity determined by immunoblotting shows an association between clinical manifestations and presence of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana in humans. J. Clin. Microbiol. 37, 4086–4092. Stanczak, J., Okroy-Rysop, G., Racewicz, M., KubicaBiernat, B., Kruminis-Lozowska, W., 2002. Prevalence of Borrelia burgdorferi sensu lato in the selected Ixodes ricinus (Acari: Ixodidae) population in Weilburg forests, Hesse, Germany. Int. J. Med. Microbiol. 291 (Suppl. 33), 206–209. Steil, B., 2002. Die genetische Heterogenita¨t von Borrelia burgdorferi sensu lato in ausgewa¨hlten Naturherden Deutschlands. Ph.D. Thesis, Friedrich-Schiller-Universita¨t, Jena, Germany. Strle, F., Picken, R.N., Cheng, Y., Cimperman, J., Maraspin, V., Lotric-Furlan, S., Ruzˇic´-Sabljic´, E., Picken, M.M., 1997. Clinical findings for patients with Lyme borreliosis caused by Borrelia burgdorferi sensu lato with genotypic
and phenotypic similarities to strain 25015. Clin. Infect. Dis. 25, 273–280. Su¨nder, U., Dorn, W., Flu¨gel, C., 1997. Untersuchungen zur saisonalen Aktivita¨t von Ixodes ricinus-Populationen (Acari: Ixodidae) in ausgewa¨hlten Habitaten Thu¨ringens. Mitt. Dtsch. Ges. Allg. Angew. Ent. 11, 629–632. Swanson, S.J., Neitzel, D., Reed, K.D., Belongia, E.A., 2006. Coinfections acquired from Ixodes ticks. Clin. Microbiol. Rev. 19, 708–727. van Dam, A.P., Kuiper, H., Vos, K., Widjojokusumo, A., de Jongh, B.M., Spanjaard, L., Ramselaar, A.S.P., Kramer, M.D., Dankert, J., 1993. Different genospecies of Borrelia burgdorferi are associated with clinical manifestations of Lyme borreliosis. Clin. Infect. Dis. 17, 708–717. Wang, G., van Dam, A.P., Dankert, J., 1999a. Phenotypic and genetic characterization of a novel Borrelia burgdorferi sensu lato isolate from a patient with Lyme borreliosis. J. Clin. Microbiol. 37, 3025–3028. Wang, G., van Dam, A.P., Schwartz, I., Dankert, J., 1999b. Molecular typing of Borrelia burgdorferi sensu lato: taxonomic, epidemiological, and clinical implications. Clin. Microbiol. Rev. 12, 633–653.
International Journal of Medical Microbiology 298 (2008) S1, 188–192 www.elsevier.de/ijmm
SHORT COMMUNICATION
Prevalence of Borrelia burgdorferi s.l. in ticks feeding on humans in Thuringia/Germany Jan Franke, Susanne Kipp, Charlotte Flu¨gel, Wolfram Dorn Department of Food and Environmental Hygiene, Institute of Nutrition, Friedrich-Schiller-University Jena, Dornburger Straße 29, D-07743 Jena, Germany Accepted 20 January 2008
Abstract In 2004, a total of 618 Ixodes ricinus ticks fed on humans were collected by physicians throughout Thuringia. The prevalence rates of Borrelia burgdorferi sensu lato (s.l.) genotypes were determined by nested PCR and restriction fragment length polymorphism, targeting a 0.8-kb fragment of the ospA gene. The total prevalence of B. burgdorferi s.l. was 6.1%. B. afzelii was found in 67%, B. valaisiana in 15% and B. garinii in 15% of the positive ticks (3% could not be determined). In one tick, a double infection with B. valaisiana type II and B. garinii OspA type V was detected. Female adult ticks had the highest infection rate (11.7%), followed by nymphs (4.5%) and larvae (3.4%). The overall prevalence increased from spring (4.0%) to autumn (10.0%). Nevertheless, the risk of infection was maximal in summer, because of a much higher infestation and consequently a higher absolute number of infected ticks. The predominance of B. afzelii probably results from its resistance against human serum. The unexpectedly low total prevalence is possibly caused by immune defence mechanisms (e.g. complement) effective against less resistant B. burgdorferi s.l. strains in the tick. r 2008 Elsevier GmbH. All rights reserved. Keywords: Borrelia burgdorferi sensu lato; Tick bite; Human-fed ticks; Prevalence; ospA
Introduction The Borrelia burgdorferi s.l. group includes the causative agents of Lyme borreliosis, the most frequent arthropod-borne human disease in the temperate northern hemisphere. Approximately 60,000 people contract Lyme borreliosis in Germany annually, but there is presumably a much higher number of unreported cases (Mehnert, 2005). The various B. burgdorferi s.l. genospecies are responsible for a multitude of different clinical manifestations of Lyme borreliosis (van Dam Corresponding author. Tel.: +49 3641 94 9694; fax: +49 3641 94 9602. E-mail address: [email protected] (J. Franke).
1438-4221/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2008.01.009
et al., 1993; Balmelli and Piffaretti, 1995; Lu¨nemann et al., 2001), and even the genetic diversity within those species leads to differences in the symptomatic occurrence of Lyme borreliosis (Baranton et al., 2001). Until now, seven species are known to be or at least considered pathogenic for humans: B. burgdorferi sensu stricto (s.s.), B. garinii, B. afzelii, B. bissettii, B. lusitaniae, B. valaisiana, and B. spielmanii (Strle et al., 1997; Ryffel et al., 1999; Wang et al., 1999a, b; Godfroid et al., 2003; Diza et al., 2004; Richter et al., 2004; da Franca et al., 2005). The federal state of Thuringia with its extensive areas of coniferous, deciduous, and mixed forests as well as moderate climatic conditions provides a large number of habitats for Ixodes ricinus ticks. The number of reported Lyme borreliosis cases in
ARTICLE IN PRESS J. Franke et al. / International Journal of Medical Microbiology 298 (2008) S1, 188–192
Thuringia/Germany in 2004 was 263 (incidence: 11.2 per 100,000), mainly manifested (92%) as erythema migrans (Thuringian Ministry of Social Affairs, Family and Health, unpublished). Recent studies in this region have shown prevalence rates of B. burgdorferi s.l. in questing ticks of up to 28.7%, with varying prevalences of different genospecies depending on the area of investigation (Dorn et al., 2000; Hildebrandt et al., 2003; Kipp et al., 2006). The aim of this study was to obtain information about the presence of B. burgdorferi s.l. genospecies in I. ricinus ticks, feeding on humans in contrast to host-seeking ticks. The results might be a contribution to the risk assessment of Lyme borreliosis, particularly in the region of Thuringia and might also be beneficial for further studies on vector–host transmission and immune evasion mechanisms of the different genospecies.
Materials and methods To assess the risk of human infection, 618 I. ricinus ticks feeding on humans from Thuringia were tested for the presence of B. burgdorferi s.l. The ticks were collected by physicians and stored at 80 1C until analysis. Spirochaete DNA was extracted directly from TM the ticks with the GenElute Bacterial Genomic DNA Kit (Sigma, St. Louis, USA). DNA amplification was realised with a nested polymerase chain reaction (PCR) targeting a 0.8-kb ospA fragment. The genospecies were identified by restriction fragment length polymorphism (RFLP), using HindIII, BglII, SspI, SfuI, and Kpn2I as restriction enzymes (Michel et al., 2004). After gel electrophoresis and ethidium bromide staining, the OspA species could then be determined under UV exposure by means of their typical band patterns according to Michel et al. (2004). Significance analysis was performed using the chi-square test.
Results and discussion The total prevalence of B. burgdorferi s.l. in human engorged ticks was 6.1%. In contrast, in one investigation in southern Germany, 37% of the ticks removed from humans were infected with B. burgdorferi s.l. (Fingerle et al., 1998). It should be mentioned that in that study a less exact detection method (immunofluorescence assay) was used. Former studies on questing ticks in southern and Central Germany, using PCR, have revealed prevalence rates of 10–36% (Fingerle et al., 1999; Rauter et al., 2002; Stanczak et al., 2002; Hildebrandt et al., 2003; Kipp et al., 2006). B. afzelii was found in 67%, B. valaisiana in 15%, and B. garinii also in 15% of the positive ticks (3% could not
B. valaisiana type I 10%
B. valaisiana type II 5%
189
unknown 3%
B. garinii OspA type IV 3%
B. afzelii OspA type II 67%
B. garinii OspA type V 8% B. garinii OspA type VI 5%
Fig. 1. OspA types of Borrelia burgdorferi s.l. found in 38 positive Ixodes ricinus ticks, feeding on humans from Thuringia/Germany in 2004. Percentages are rounded. Values refer to the number of infections (n ¼ 39), because one coinfection with B. valaisiana type II and B. garinii OspA type V was detected.
be determined). In one tick, a double infection with B. valaisiana type II and B. garinii OspA type V occurred. B. burgdorferi s.s. and other genospecies were not found (Fig. 1). Only five districts provided an adequate number of samples. Local prevalence rates between those districts were not significantly distinct. In every district, B. afzelii was the predominant species. Former studies on hostseeking ticks in Thuringia, using the same method, have shown that the prevalence of B. garinii was much higher than (Hildebrandt et al., 2003) or at least as high as that of B. afzelii (Kipp et al., 2006). Hildebrandt et al. (2003) and Kipp et al. (2006) also found B. burgdorferi s.s. in 32.4% and 9.8% of the positive ticks, respectively. The reason for the predominance of B. afzelii in ticks, feeding on humans, might be its better resistance against immune defence mechanisms (e.g. complement) as described in some studies (Breitner-Ruddock et al., 1997; Kraiczy et al., 2002; Bhide et al., 2005). The lack of resistance against human complement in some strains, leading to a rapid decline of the spirochaete count in the tick salivary glands, might also be a reason for the unexpectedly low total prevalence of B. burgdorferi s.l. Data provided by the Thuringian Ministry of Social Affairs, Family and Health show, that 92.0% (242/263) of the Thuringian Lyme borreliosis cases in 2004 were manifested exclusively as erythema migrans. That corresponds with the predominance of B. afzelii in engorged ticks, the genospecies mainly, but not exclusively associated with erythema migrans (Canica et al., 1993; van Dam et al., 1993; Anthonissen et al., 1994; Balmelli and Piffaretti, 1995).
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20 18
Prevalence (%)
16
Spring Summer Autumn
14 12 10 8 6 4 2 0 Total [n=618]
Nymphs [n=444]
Female Adults [n=145]
Fig. 2. Seasonal course of the prevalence of Borrelia burgdorferi s.l. in Ixodes ricinus ticks, feeding on humans in Thuringia/ Germany (2004). Larvae (n ¼ 29, one larva infected) are included in the total result.
Only a few patients (7.2%) had neurological symptoms, known to be mostly caused by B. garinii (Demaerschalck et al., 1995). Arthralgic symptoms, associated mainly with B. burgdorferi s.s. (Demaerschalck et al., 1995), appeared only in 1.9% of the cases, corresponding to the fact that no B. burgdorferi s.s.infected ticks have been found. However, some strains seem to cause a mixture of symptoms, and there is an overlap between clinical manifestations, caused by different genospecies, which was also evident in a number of the above-mentioned Lyme borreliosis cases (4.9%). Also coinfections with other tick-borne pathogens like Anaplasma phagocytophilum can lead to diverse clinical outcomes (Belongia, 2002; Swanson et al., 2006). The mean infection rate of female adult ticks was significantly higher (11.7%, po0.025) than in nymphs (4.5%) and in larvae (3.4%). The overall prevalence increased from 4.0% in spring to 6.5% in summer and reached its peak in autumn (10.0%, Fig. 2). Nymphs had the highest infection rate in summer (5.0%), whereas adult females were most frequently infected in autumn (17.6%). However, the total number of ticks collected in autumn was low (13 nymphs, 17 females) and only three out of these 30 ticks were infected. These data support findings from our former investigations on host-seeking ticks, showing the highest prevalence rates in autumn (Dorn and Su¨nder, 1997). Some studies also indicate that there is often a negative correlation between tick activity and prevalence rates as observed in this investigation (Su¨nder et al., 1997; Gray et al., 1999). Nevertheless, seasonal patterns in the prevalence of B. burgdorferi s.l. in ticks seem to be very heterogeneous even within one investigation area (Steil, 2002; Hildebrandt, 2003), probably mainly due to the humidity- and temperature-dependent host-seeking activity of I. ricinus and seasonal activity patterns of reservoir hosts (Gray 1998; Jensen, 2000). For example, diapause mechanisms, induced by disadvantageous climate conditions lead to lower infection rates, because
the possibility of ingesting spirochaetes from reservoir hosts declines. The infestation with nymphs (n ¼ 444) was more than three times higher than with female adults (n ¼ 145). Only a few subjects were infested with larvae (n ¼ 29). More persons were infested with ticks in summer (n ¼ 462) than in spring (n ¼ 126) and autumn (n ¼ 30). Although the mean prevalence of B. burgdorferi s.l. in I. ricinus reached its peak in autumn, the risk of infection was higher in June and July, due to a much higher absolute number of infected ticks in summer (n ¼ 30) than in autumn (n ¼ 3) and spring (n ¼ 5). The higher outdoor activity also enhances the risk of a tick bite and consequently of contracting Lyme borreliosis in summer. In conclusion, the results of this investigation show that the prevalence of B. burgdorferi s.l. in ticks feeding on humans is lower when compared to host-seeking ticks, analysed in former studies. Also the composition of genospecies is different. This supports studies indicating that some Borrelia strains are not able to develop effective immune evasion mechanisms against human complement within the tick body. Further studies should focus on the genetic and phenotypic fundamentals for these mechanisms.
Acknowledgements We would like to thank Dr. Gerrit Hesse from the Thuringian Ministry of Social Affairs, Family and Health for providing the data of Lyme borreliosis cases.
References Anthonissen, F.M., De Kesel, M., Hoet, P.P., Bigaignon, G.H., 1994. Evidence for the involvement of different
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genospecies of Borrelia in the clinical outcome of Lyme disease in Belgium. Res. Microbiol. 145, 327–331. Balmelli, T., Piffaretti, J.C., 1995. Association between different clinical manifestations of Lyme disease and different species of Borrelia burgdorferi sensu lato. Res. Microbiol. 146, 329–340. Baranton, G., Seinost, G., Theodore, G., Postic, D., Dykhuizen, D., 2001. Distinct levels of genetic diversity of Borrelia burgdorferi are associated with different aspects of pathogenicity. Res. Microbiol. 152, 149–156. Belongia, E.A., 2002. Epidemiology and impact of coinfections acquired from Ixodes ticks. Vector Borne Zoonotic Dis. 2, 265–273. Bhide, M.R., Travnicek, M., Levkutova, M., Curlik, J., Revajova, V., Levkut, M., 2005. Sensitivity of Borrelia genospecies to serum complement from different animals and human: a host–pathogen relationship. FEMS Immunol. Med. Microbiol. 43, 165–172. Breitner-Ruddock, S., Wurzner, R., Schulze, J., Brade, V., 1997. Heterogeneity in the complement-dependent bacteriolysis within the species of Borrelia burgdorferi. Med. Microbiol. Immunol. 185, 253–260. Canica, M.M., Nato, F., du Merle, L., Mazie, J.C., Baranton, G., Postic, D., 1993. Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis. Scand. J. Infect. Dis. 25, 441–448. da Franca, I., Santos, L., Mesquita, T., Collares-Pereira, M., Baptista, S., Vieira, L., Viana, I., Vale, E., Prates, C., 2005. Lyme borreliosis in Portugal caused by Borrelia lusitaniae? Clinical report on the first patient with a positive skin isolate. Wien. Klin. Wochenschr. 117, 429–432. Demaerschalck, I., Ben Messaoud, A., De Kesel, M., Hoyois, B., Lobet, Y., Hoet, P., Bigaignon, G., Bollen, A., Godfroid, E., 1995. Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients. J. Clin. Microbiol. 33, 602–608. Diza, E., Papa, A., Vezyri, E., Tsounis, S., Milonas, I., Antoniadis, A., 2004. Borrelia valaisiana in cerebrospinal fluid. Emerg. Infect. Dis. 10, 1692–1693. Dorn, W., Su¨nder, U., 1997. Lyme borreliosis in Thuringia (Germany): prevalence of the causative agent in fieldcollected ticks. In: Su¨ss, J., Kahl, O., (Eds.), Tick-borne Encephalitis and Lyme Borreliosis, Proceedings of the Fourth International Potsdam Symposium Tick-borne Disease, Berlin, 21–22 February 1997, Pabst, Lengerich, pp. 175–196. Dorn, W., Su¨nder, U., Steil, B., Flu¨gel, C., 2000. Zum Zusammenhang von saisonaler Dichte und Durchseuchungsrate mit Borrelia burgdorferi sensu lato bei freilandgefangenen Zecken der Art Ixodes ricinus L. 1758 (Acari: Ixodidae) – eine Fallstudie im Freistaat Thu¨ringen. Mitt. Dtsch. Ges. Allg. Angew. Ent. 12, 187–191. Fingerle, V., Liegl, G., Munderloh, U., Wilske, B., 1998. Expression of outer surface proteins A and C of Borrelia burgdorferi in Ixodes ricinus ticks removed from humans. Med. Microbiol. Immunol. 187, 121–126. Fingerle, V., Munderloh, U.G., Liegl, G., Wilske, B., 1999. Coexistence of ehrlichiae of the phagocytophila group with
191
Borrelia burgdorferi in Ixodes ricinus from Southern Germany. Med. Microbiol. Immunol. 188, 145–149. Godfroid, E., Min Hu, C., Humair, P.-F., Bollen, A., Gern, L., 2003. PCR-reverse line blot typing method underscores the genomic heterogeneity of Borrelia valaisiana species and suggests its potential involvement in Lyme disease. J. Clin. Microbiol. 41, 3690–3698. Gray, J.S., 1998. Review: the ecology of ticks transmitting Lyme borreliosis. Exp. Appl. Acarol. 22, 249–258. Gray, J.S., Kirstein, F., Robertson, J.N., Stein, J., Kahl, O., 1999. Borrelia burgdorferi sensu lato in Ixodes ricinus ticks and rodents in a recreational park in south-western Ireland. Exp. Appl. Acarol. 23, 717–729. Hildebrandt, A., 2003. Pra¨valenz von Ehrlichien der Ehrlichia phagocytophila Genogruppe und Borrelien des Borrelia burgdorferi sensu lato Komplexes in Ixodes ricinus Zecken aus dem Ilmtal. Ph.D. Thesis, Friedrich-Schiller-Universita¨t Jena, Germany. Hildebrandt, A., Schmidt, K.H., Wilske, B., Dorn, W., Straube, E., Fingerle, V., 2003. Prevalence of four species of Borrelia burgdorferi sensu lato and coinfection with Anaplasma phagocytophila in Ixodes ricinus ticks in central Germany. Eur. J. Clin. Microbiol. Infect. Dis. 22, 364–367. Jensen, P.M., 2000. Host-seeking activity of Ixodes ricinus ticks based on daily consecutive flagging samples. Exp. Appl. Acarol. 24, 695–708. Kipp, S., Dorn, W., Wilske, B., Fingerle, V., 2006. Heterogeneity in prevalence and genetic diversity of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in different localities in Thuringia, Germany. Int. J. Med. Microbiol. 296 (Suppl. 40), 119–121. Kraiczy, P., Skerka, C., Kirschfink, M., Zipfel, P.F., Brade, V., 2002. Immune evasion of Borrelia burgdorferi: insufficient killing of the pathogens by complement and antibody. Int. J. Med. Microbiol. 291 (Suppl. 33), 141–146. Lu¨nemann, J.D., Zarmas, S., Priem, S., Franz, J., Zschenderlein, R., Aberer, E., Klein, R., Schouls, L., Burmester, G.R., Krause, A., 2001. Rapid typing of Borrelia burgdorferi sensu lato species in specimens from patients with different manifestations of Lyme borreliosis. J. Clin. Microbiol. 39, 1130–1133. Mehnert, W.H., 2005. Zur Situation bei wichtigen Infektionskrankheiten in Deutschland: Neuerkrankungen an LymeBorreliose im Jahr 2004. Analyse auf der Basis der Meldedaten aus den sechs o¨stlichen Bundesla¨ndern. Epidemiol. Bull. 32, 285–288. Michel, H., Wilske, B., Hettche, G., Goettner, G., Heimerl, C., Reischl, U., Schulte-Spechtel, U., Fingerle, V., 2004. An ospA-polymerase chain reaction/restriction fragment length polymorphism-based method for sensitive detection and reliable differentiation of all European Borrelia burgdorferi sensu lato species and OspA types. Med. Microbiol. Immunol. 193, 219–226. Rauter, C., Oehme, R., Diterich, I., Engele, M., Hartung, T., 2002. Distribution of clinically relevant Borrelia genospecies in ticks assessed by a novel, single-run, real-time PCR. J. Clin. Microbiol. 40, 36–43. Richter, D., Schlee, D.B., Allgower, R., Matuschka, F.R., 2004. Relationships of a novel Lyme disease spirochete,
ARTICLE IN PRESS 192
J. Franke et al. / International Journal of Medical Microbiology 298 (2008) S1, 188–192
Borrelia spielmani sp. nov., with its hosts in Central Europe. Appl. Environ. Microbiol. 70, 6414–6419. Ryffel, K., Pe´ter, O., Rutti, B., Suard, A., Dayer, E., 1999. Scored antibody reactivity determined by immunoblotting shows an association between clinical manifestations and presence of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana in humans. J. Clin. Microbiol. 37, 4086–4092. Stanczak, J., Okroy-Rysop, G., Racewicz, M., KubicaBiernat, B., Kruminis-Lozowska, W., 2002. Prevalence of Borrelia burgdorferi sensu lato in the selected Ixodes ricinus (Acari: Ixodidae) population in Weilburg forests, Hesse, Germany. Int. J. Med. Microbiol. 291 (Suppl. 33), 206–209. Steil, B., 2002. Die genetische Heterogenita¨t von Borrelia burgdorferi sensu lato in ausgewa¨hlten Naturherden Deutschlands. Ph.D. Thesis, Friedrich-Schiller-Universita¨t, Jena, Germany. Strle, F., Picken, R.N., Cheng, Y., Cimperman, J., Maraspin, V., Lotric-Furlan, S., Ruzˇic´-Sabljic´, E., Picken, M.M., 1997. Clinical findings for patients with Lyme borreliosis caused by Borrelia burgdorferi sensu lato with genotypic
and phenotypic similarities to strain 25015. Clin. Infect. Dis. 25, 273–280. Su¨nder, U., Dorn, W., Flu¨gel, C., 1997. Untersuchungen zur saisonalen Aktivita¨t von Ixodes ricinus-Populationen (Acari: Ixodidae) in ausgewa¨hlten Habitaten Thu¨ringens. Mitt. Dtsch. Ges. Allg. Angew. Ent. 11, 629–632. Swanson, S.J., Neitzel, D., Reed, K.D., Belongia, E.A., 2006. Coinfections acquired from Ixodes ticks. Clin. Microbiol. Rev. 19, 708–727. van Dam, A.P., Kuiper, H., Vos, K., Widjojokusumo, A., de Jongh, B.M., Spanjaard, L., Ramselaar, A.S.P., Kramer, M.D., Dankert, J., 1993. Different genospecies of Borrelia burgdorferi are associated with clinical manifestations of Lyme borreliosis. Clin. Infect. Dis. 17, 708–717. Wang, G., van Dam, A.P., Dankert, J., 1999a. Phenotypic and genetic characterization of a novel Borrelia burgdorferi sensu lato isolate from a patient with Lyme borreliosis. J. Clin. Microbiol. 37, 3025–3028. Wang, G., van Dam, A.P., Schwartz, I., Dankert, J., 1999b. Molecular typing of Borrelia burgdorferi sensu lato: taxonomic, epidemiological, and clinical implications. Clin. Microbiol. Rev. 12, 633–653.