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Seasonal and habitat variation in the prevalence of Rickettsia helvetica in Ixodes ricinus ticks from Denmark
Ticks and Tick-borne Diseases 1 (2010) 101–103
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.de/ttbdis
Short communication
Seasonal and habitat variation in the prevalence of Rickettsia helvetica in Ixodes ricinus ticks from Denmark Bjørn Kantsø a,∗ , Claus Bo Svendsen a , Per Moestrup Jensen b , Jean Vennestrøm b , Karen A. Krogfelt a a b
Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, 5 Artillerivej, DK-2300 Copenhagen S, Denmark Department of Agriculture and Ecology, University of Copenhagen, Frederiksberg, Denmark
a r t i c l e
i n f o
Article history: Received 3 November 2009 Accepted 13 January 2010 Available online 4 February 2010 Keywords: Rickettsia Tick Polymerase chain reaction Ixodes ricinius
a b s t r a c t A total of 704 unfed ticks of the species Ixodes ricinus collected in Denmark were screened for Rickettsia DNA by a genus-specific real-time PCR. Of the nymphs, 4.7% (31/662) were positive for rickettsial DNA. Among the positive ticks, we observed a seasonal and habitat variation. The infection rate was highest in May as compared to July, August, and October. Ecotone (high tick density) showed an elevated prevalence as compared to spruce or beech forests. Sequencing revealed only DNA from R. helvetica. © 2010 Elsevier GmbH. All rights reserved.
Introduction
Materials and methods
The tick Ixodes ricinus is a well-known vector of zoonotic diseases such as tick-borne encephalitis, Lyme borreliosis, anaplasmosis, and rickettsiosis (Sonenshine, 1991). In Denmark, the main focus in tick-borne diseases has been on Lyme borreliosis and anaplasmosis, while only a limited number of studies have evaluated the potential risk of tick-borne Rickettsia infection. Previous studies have found varying prevalences of Rickettsia helvetica in Danish ticks: Nielsen et al. (2004) found an overall prevalence of 4.9% in adult ticks and 1.4% in nymphs, while Svendsen et al. (2009a) found rickettsial DNA in 13% of nymphs. Skarphedinsson et al. (2007) also found adult I. ricinus ticks positive for rickettsial DNA without evaluating the prevalence of Rickettsia. In 1996 and 1997, the prevalence of Borrelia burgdorferi sensu lato in ticks from the same location as in this study has been determined to 5–10% (Jensen and Frandsen, 2000), whereas the infection rate increased to 15.5% in 2005 (Vennestrøm et al., 2008). The aim of the present study was to examine possible causes underlying the observed variation in the prevalence of Rickettsia spp. in ticks, with special focus on prevalence between habitats with different tick densities.
We examined 704 unfed ticks (662 nymphs, 42 larvae) of the species I. ricinus from Grib Forest North Zealand (56◦ 0 0N, 12◦ 19 60E). The ticks had been collected by flagging as part of an earlier study on tick-borne diseases in Denmark (Skarphedinsson et al., 2005). We had information about the specific habitat of collection in 348 of the ticks. The ticks originated from 3 different habitats; ecotones with 60±32 (mean±SD) ticks per 100 m2 ; spruce forest with 14±7 ticks per 100 m2 , and beech forest with 29±12 ticks per 100 m2 , thus representing high, low, and middle tick density, respectively. The habitats have previously been described by Jensen and Frandsen (2000). Ticks collected without information about specific collection habitats were used as reference samples. For a decade preceding the study, the tick densities in the 3 habitats had been monitored and were found to be stable (P.M. Jensen, pers. comm.). In the present study, ticks were collected by daytime flagging in August 2003 and May, July, and October 2004. Each tick was mechanically homogenized using a Qiagen TissueLyser (Qiagen, USA) and treated with 0.7 M ammonium hydroxide (NH4 OH) for 15 min at 99 ◦ C in sealed PCR tubes. Subsequently, ammonium hydroxide was evaporated for 25 min at 99 ◦ C. Next, 50 l DNA buffer was added (TE buffer with addition of bovine thymus DNA, Statens Serum Institut, Copenhagen, Denmark). DNA extractions were stored at −20 ◦ C and later used as templates for the PCR amplification. Real-time PCR was used to amplify a 74-bp fragment of the citrate synthase (CS) gene (gltA) in Rickettsia spp. (Stenos et al., 2005). Details of the real-time PCR as applied in this study are described elsewhere (Svendsen et al., 2009a). DNA extracted from
∗ Corresponding author. Tel.: +45 32 68 36 88; fax: +45 32 68 31 47. E-mail address: [email protected] (B. Kantsø). 1877-959X/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ttbdis.2010.01.004
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B. Kantsø et al. / Ticks and Tick-borne Diseases 1 (2010) 101–103
R. rickettsii or R. australis in 10-fold dilutions were used as positive control; as no-template control (NTC), Gibco BRL PCR grade water (Gibco BRL, Roskilde, Denmark) was used. All samples were analyzed in duplicate. We used a conventional PCR based on the citrate synthase (gltA) gene as confirmatory PCR (Stenos et al., 1998), as previously described (Svendsen et al., 2009a). Direct sequencing was performed on the amplified fragments. The prevalence of rickettsiae in ticks was compared between different habitats and against the reference samples by chi square analysis. Hence, habitat differences were evaluated by three 2×2 tables, and seasonal differences by three 2×4 tables. The significance level was set at p=0.05. All statistical analyses were performed using SPSS statistical software (SPSS 17 for Windows, SPSS Inc. [Chicago, IL, USA], 1993–2007).
Results The real-time PCR found that 31 out of the 662 (4.7%) nymphal ticks were positive for Rickettsia spp. with a Ct (cycle threshold) level ranging from 21.7 to 31.2 (mean 24.8). The confirmatory conventional PCR was positive in 30 out of the 31 (96.7%) samples. Sequencing of 10 randomly chosen samples showed, when aligned, 99–100% homology with R. helvetica C9P9 citrate synthase (gltA) gene, partial cds (GenBank accession no. U59723.1). Six samples originated from ecotone, 2 from spruce, and 2 from beech forest. None of the 42 larvae carried any rickettsial DNA. With regard to the presence of rickettsial DNA depending on the collection site of the ticks (both nymphs and larvae), ecotones stood out: 7% of the ticks from these sites were positive for rickettsial DNA (14/208, Chi square=4.39, p=0.04). Spruce forest had 1.4% (1/72) positive ticks, and beech forest had 4.4% (3/68) positive ticks (Table 1). Spruce and beech forest were not significantly different from the reference samples (Chi square=1.01, p=0.32 and Chi square=0.08 p=0.78, respectively), the latter of which had 3.7% (13/352) positive ticks. Based on the collection date, May had the highest rate of positive ticks (7.1%, 12/168), August plus October had 4.0% (8/199), and July had 3.1% (11/358) positive ticks (Table 2). When investigating the seasonal variation for ecotone forest, a significant variation was found between collection dates, where May had an infection rate of 10.7%, July had 3.6%, and August/October 3.8% (Chi-square=11.00, p=0.01). There was a non-significant tendency in the data that rickettsial load (as determined by the Ct level) depended on the collection date (Kruskal-Wallis Test p=0.17); the rickettsial load/CT level also varied non-significantly by habitat type (Kruskal-Wallis Test p=0.55).
Table 1 Prevalence of Rickettsia spp. in I. ricinus ticks and tick density by habitat. Habitat
Tick density (mean±SD)
Prevalence (n)
Ecotone Spruce Beach Unknown (reference)
60∗32 14 ± 7 29 ± 12 Not evaluated
7.0% (208) 1.4% (72) 4.4% (68) 3.7% (352)
Table 2 Prevalence of Rickettsia spp. in unfed I. ricinus ticks by date of collection. Collection date
Prevalence (n)
May July August+October
7.1% (168) 3.1% (358) 4.0% (199)
Discussion The findings in this study correspond with previous findings in Denmark by Nielsen et al. (2004) who found R. helvetica in 4% of I. ricinus ticks collected from roe deer (n=235), dogs (n=155), humans (n=56), cats (n=12), or from vegetation (n=112). In contrast, the prevalence found in this series is lower than the prevalence found by Svendsen et al. (2009a) in Korsør Recreational Forest in south-western coastal Zealand. Based on different sampling strategies and large differences in tick densities, the stable prevalences reported here suggest that these variables are not the underlying cause for previously reported differences in Rickettsia prevalence. Rather, rickettsial prevalence may show annual variation comparable to that observed for Borrelia, due to annual differences in the transmission of rickettsiae. The background of this variation is not well understood. The difference in rickettsial prevalence between habitats with high versus low density of ticks could be explained by a spatial and/or temporal variation in the infection rate, because differing methodologies can be ruled out as a cause. The seasonal variation found in this study correlates with the findings of Jensen and Frandsen in studies of B. burgdorferi sensu lato prevalence, where the highest prevalence was observed in ticks collected in May and June (Jensen and Frandsen, 2000). Spatial differences in the infection rate of unfed nymphal I. ricinus ticks have previously been described for B. burgdorferi at the same locations, where the highest infection rate was found in ecotones (Jensen and Frandsen, 2000), corresponding well to the findings in this study. It therefore seems likely that the temporal and spatial risk parameters for tick-borne rickettsiosis parallel that of Lyme borreliosis. The rickettsial load in the tick samples was not statistically different with regard to habitat or collection date. This is probably related to the low number of positive samples in each group. In 2 studies, a human infection with R. helvetica was connected with sarcoidosis and chronic perimyocarditis (Nilsson et al., 1999, 2002). Recently, reasonable doubt has been cast on this association by 3 independent studies (Planck et al., 2004; Svendsen et al., 2009a, b). The findings in this study substantiate the previous findings of R. helvetica in I. ricinus ticks from Denmark as a low-prevalence country of rickettsiosis (Kantsø et al., 2009).Furthermore, this study emphasizes that rickettsiosis is a diagnosis to be considered along with that of Lyme borreliosis in patients presenting with fever after a tick bite in Denmark.
Disclosure statement None of the authors have any financial involvement in any organisation with a direct financial interest in the subject discussed in the submitted manuscript. None of the authors have conflicts of interest regarding this manuscript.
References Jensen, P.M., Frandsen, F., 2000. Temporal risk assessment for Lyme borreliosis in Denmark. Scand. J. Infect. Dis. 32, 539–544. Kantsø, B., Svendsen, C.B., Jørgensen, C.S., Krogfelt, K.A., 2009. Evaluation of serological tests for the diagnosis of rickettsiosis in Denmark. J. Microbiol. Methods 76, 285–288. Nielsen, H., Fournier, P.E., Pedersen, I.S., Krarup, H., Ejlertsen, T., Raoult, D., 2004. Serological and molecular evidence of Rickettsia helvetica in Denmark. Scand. J. Infect. Dis. 36, 559–563. Nilsson, K., Lindquist, O., Pahlson, C., 1999. Association of Rickettsia helvetica with chronic perimyocarditis in sudden cardiac death. Lancet 354, 1169–1173. Nilsson, K., Pahlson, C., Lukinius, A., Eriksson, L., Nilsson, L., Lindquist, O., 2002. Presence of Rickettsia helvetica in granulomatous tissue from patients with sarcoidosis. J. Infect. Dis. 185, 1128–1138.
B. Kantsø et al. / Ticks and Tick-borne Diseases 1 (2010) 101–103 Planck, A., Eklund, A., Grunewald, J., Vene, S., 2004. No serological evidence of Rickettsia helvetica infection in Scandinavian sarcoidosis patients. Eur. Respir. J. 24, 811–813. Skarphedinsson, S., Jensen, P.M., Kristiansen, K., 2005. Survey of tickborne infections in Denmark. Emerg. Infect. Dis. 11, 1055–1061. Skarphedinsson, S., Lyholm, B.F., Ljungberg, M., Sogaard, P., Kolmos, H.J., Nielsen, L.P., 2007. Detection and identification of Anaplasma phagocytophilum, Borrelia burgdorferi, and Rickettsia helvetica in Danish Ixodes ricinus ticks. APMIS 115, 225–230. Sonenshine, D.E., 1991. Biology of ticks. Oxford University Press, New York. Stenos, J., Graves, S.R., Unsworth, N.B., 2005. A highly sensitive and specific real-time PCR assay for the detection of spotted fever and typhus group rickettsiae. Am. J. Trop. Med. Hyg. 73, 1083–1085.
103
Stenos, J., Roux, V., Walker, D., Raoult, D., 1998. Rickettsia honei sp. nov., the aetiological agent of Flinders Island spotted fever in Australia. Int. J. Syst. Bacteriol. 48, 1399–1404. Svendsen, C.B., Krogfelt, K., Jensen, P.M., 2009a. Detection of Rickettsia spp. in Danish ticks (Acari: Ixodes ricinus) using real-time PCR. Scand. J. Infect. Dis. 41, 70–72. Svendsen, C.B., Milman, N., Nielsen, H.W., Krogfelt, K.A., Larsen, K.R., 2009b. A prospective study evaluating the presence of Rickettsia in Danish patients with sarcoidosis. Scand. J. Infect. Dis. 41, 745–752. Vennestrøm, J., Egholm, H., Jensen, P.M., 2008. Occurrence of multiple infections with different Borrelia burgdorferi genospecies in Danish Ixodes ricinus nymphs. Parasitol. Int. 57, 32–37.
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.de/ttbdis
Short communication
Seasonal and habitat variation in the prevalence of Rickettsia helvetica in Ixodes ricinus ticks from Denmark Bjørn Kantsø a,∗ , Claus Bo Svendsen a , Per Moestrup Jensen b , Jean Vennestrøm b , Karen A. Krogfelt a a b
Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, 5 Artillerivej, DK-2300 Copenhagen S, Denmark Department of Agriculture and Ecology, University of Copenhagen, Frederiksberg, Denmark
a r t i c l e
i n f o
Article history: Received 3 November 2009 Accepted 13 January 2010 Available online 4 February 2010 Keywords: Rickettsia Tick Polymerase chain reaction Ixodes ricinius
a b s t r a c t A total of 704 unfed ticks of the species Ixodes ricinus collected in Denmark were screened for Rickettsia DNA by a genus-specific real-time PCR. Of the nymphs, 4.7% (31/662) were positive for rickettsial DNA. Among the positive ticks, we observed a seasonal and habitat variation. The infection rate was highest in May as compared to July, August, and October. Ecotone (high tick density) showed an elevated prevalence as compared to spruce or beech forests. Sequencing revealed only DNA from R. helvetica. © 2010 Elsevier GmbH. All rights reserved.
Introduction
Materials and methods
The tick Ixodes ricinus is a well-known vector of zoonotic diseases such as tick-borne encephalitis, Lyme borreliosis, anaplasmosis, and rickettsiosis (Sonenshine, 1991). In Denmark, the main focus in tick-borne diseases has been on Lyme borreliosis and anaplasmosis, while only a limited number of studies have evaluated the potential risk of tick-borne Rickettsia infection. Previous studies have found varying prevalences of Rickettsia helvetica in Danish ticks: Nielsen et al. (2004) found an overall prevalence of 4.9% in adult ticks and 1.4% in nymphs, while Svendsen et al. (2009a) found rickettsial DNA in 13% of nymphs. Skarphedinsson et al. (2007) also found adult I. ricinus ticks positive for rickettsial DNA without evaluating the prevalence of Rickettsia. In 1996 and 1997, the prevalence of Borrelia burgdorferi sensu lato in ticks from the same location as in this study has been determined to 5–10% (Jensen and Frandsen, 2000), whereas the infection rate increased to 15.5% in 2005 (Vennestrøm et al., 2008). The aim of the present study was to examine possible causes underlying the observed variation in the prevalence of Rickettsia spp. in ticks, with special focus on prevalence between habitats with different tick densities.
We examined 704 unfed ticks (662 nymphs, 42 larvae) of the species I. ricinus from Grib Forest North Zealand (56◦ 0 0N, 12◦ 19 60E). The ticks had been collected by flagging as part of an earlier study on tick-borne diseases in Denmark (Skarphedinsson et al., 2005). We had information about the specific habitat of collection in 348 of the ticks. The ticks originated from 3 different habitats; ecotones with 60±32 (mean±SD) ticks per 100 m2 ; spruce forest with 14±7 ticks per 100 m2 , and beech forest with 29±12 ticks per 100 m2 , thus representing high, low, and middle tick density, respectively. The habitats have previously been described by Jensen and Frandsen (2000). Ticks collected without information about specific collection habitats were used as reference samples. For a decade preceding the study, the tick densities in the 3 habitats had been monitored and were found to be stable (P.M. Jensen, pers. comm.). In the present study, ticks were collected by daytime flagging in August 2003 and May, July, and October 2004. Each tick was mechanically homogenized using a Qiagen TissueLyser (Qiagen, USA) and treated with 0.7 M ammonium hydroxide (NH4 OH) for 15 min at 99 ◦ C in sealed PCR tubes. Subsequently, ammonium hydroxide was evaporated for 25 min at 99 ◦ C. Next, 50 l DNA buffer was added (TE buffer with addition of bovine thymus DNA, Statens Serum Institut, Copenhagen, Denmark). DNA extractions were stored at −20 ◦ C and later used as templates for the PCR amplification. Real-time PCR was used to amplify a 74-bp fragment of the citrate synthase (CS) gene (gltA) in Rickettsia spp. (Stenos et al., 2005). Details of the real-time PCR as applied in this study are described elsewhere (Svendsen et al., 2009a). DNA extracted from
∗ Corresponding author. Tel.: +45 32 68 36 88; fax: +45 32 68 31 47. E-mail address: [email protected] (B. Kantsø). 1877-959X/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ttbdis.2010.01.004
102
B. Kantsø et al. / Ticks and Tick-borne Diseases 1 (2010) 101–103
R. rickettsii or R. australis in 10-fold dilutions were used as positive control; as no-template control (NTC), Gibco BRL PCR grade water (Gibco BRL, Roskilde, Denmark) was used. All samples were analyzed in duplicate. We used a conventional PCR based on the citrate synthase (gltA) gene as confirmatory PCR (Stenos et al., 1998), as previously described (Svendsen et al., 2009a). Direct sequencing was performed on the amplified fragments. The prevalence of rickettsiae in ticks was compared between different habitats and against the reference samples by chi square analysis. Hence, habitat differences were evaluated by three 2×2 tables, and seasonal differences by three 2×4 tables. The significance level was set at p=0.05. All statistical analyses were performed using SPSS statistical software (SPSS 17 for Windows, SPSS Inc. [Chicago, IL, USA], 1993–2007).
Results The real-time PCR found that 31 out of the 662 (4.7%) nymphal ticks were positive for Rickettsia spp. with a Ct (cycle threshold) level ranging from 21.7 to 31.2 (mean 24.8). The confirmatory conventional PCR was positive in 30 out of the 31 (96.7%) samples. Sequencing of 10 randomly chosen samples showed, when aligned, 99–100% homology with R. helvetica C9P9 citrate synthase (gltA) gene, partial cds (GenBank accession no. U59723.1). Six samples originated from ecotone, 2 from spruce, and 2 from beech forest. None of the 42 larvae carried any rickettsial DNA. With regard to the presence of rickettsial DNA depending on the collection site of the ticks (both nymphs and larvae), ecotones stood out: 7% of the ticks from these sites were positive for rickettsial DNA (14/208, Chi square=4.39, p=0.04). Spruce forest had 1.4% (1/72) positive ticks, and beech forest had 4.4% (3/68) positive ticks (Table 1). Spruce and beech forest were not significantly different from the reference samples (Chi square=1.01, p=0.32 and Chi square=0.08 p=0.78, respectively), the latter of which had 3.7% (13/352) positive ticks. Based on the collection date, May had the highest rate of positive ticks (7.1%, 12/168), August plus October had 4.0% (8/199), and July had 3.1% (11/358) positive ticks (Table 2). When investigating the seasonal variation for ecotone forest, a significant variation was found between collection dates, where May had an infection rate of 10.7%, July had 3.6%, and August/October 3.8% (Chi-square=11.00, p=0.01). There was a non-significant tendency in the data that rickettsial load (as determined by the Ct level) depended on the collection date (Kruskal-Wallis Test p=0.17); the rickettsial load/CT level also varied non-significantly by habitat type (Kruskal-Wallis Test p=0.55).
Table 1 Prevalence of Rickettsia spp. in I. ricinus ticks and tick density by habitat. Habitat
Tick density (mean±SD)
Prevalence (n)
Ecotone Spruce Beach Unknown (reference)
60∗32 14 ± 7 29 ± 12 Not evaluated
7.0% (208) 1.4% (72) 4.4% (68) 3.7% (352)
Table 2 Prevalence of Rickettsia spp. in unfed I. ricinus ticks by date of collection. Collection date
Prevalence (n)
May July August+October
7.1% (168) 3.1% (358) 4.0% (199)
Discussion The findings in this study correspond with previous findings in Denmark by Nielsen et al. (2004) who found R. helvetica in 4% of I. ricinus ticks collected from roe deer (n=235), dogs (n=155), humans (n=56), cats (n=12), or from vegetation (n=112). In contrast, the prevalence found in this series is lower than the prevalence found by Svendsen et al. (2009a) in Korsør Recreational Forest in south-western coastal Zealand. Based on different sampling strategies and large differences in tick densities, the stable prevalences reported here suggest that these variables are not the underlying cause for previously reported differences in Rickettsia prevalence. Rather, rickettsial prevalence may show annual variation comparable to that observed for Borrelia, due to annual differences in the transmission of rickettsiae. The background of this variation is not well understood. The difference in rickettsial prevalence between habitats with high versus low density of ticks could be explained by a spatial and/or temporal variation in the infection rate, because differing methodologies can be ruled out as a cause. The seasonal variation found in this study correlates with the findings of Jensen and Frandsen in studies of B. burgdorferi sensu lato prevalence, where the highest prevalence was observed in ticks collected in May and June (Jensen and Frandsen, 2000). Spatial differences in the infection rate of unfed nymphal I. ricinus ticks have previously been described for B. burgdorferi at the same locations, where the highest infection rate was found in ecotones (Jensen and Frandsen, 2000), corresponding well to the findings in this study. It therefore seems likely that the temporal and spatial risk parameters for tick-borne rickettsiosis parallel that of Lyme borreliosis. The rickettsial load in the tick samples was not statistically different with regard to habitat or collection date. This is probably related to the low number of positive samples in each group. In 2 studies, a human infection with R. helvetica was connected with sarcoidosis and chronic perimyocarditis (Nilsson et al., 1999, 2002). Recently, reasonable doubt has been cast on this association by 3 independent studies (Planck et al., 2004; Svendsen et al., 2009a, b). The findings in this study substantiate the previous findings of R. helvetica in I. ricinus ticks from Denmark as a low-prevalence country of rickettsiosis (Kantsø et al., 2009).Furthermore, this study emphasizes that rickettsiosis is a diagnosis to be considered along with that of Lyme borreliosis in patients presenting with fever after a tick bite in Denmark.
Disclosure statement None of the authors have any financial involvement in any organisation with a direct financial interest in the subject discussed in the submitted manuscript. None of the authors have conflicts of interest regarding this manuscript.
References Jensen, P.M., Frandsen, F., 2000. Temporal risk assessment for Lyme borreliosis in Denmark. Scand. J. Infect. Dis. 32, 539–544. Kantsø, B., Svendsen, C.B., Jørgensen, C.S., Krogfelt, K.A., 2009. Evaluation of serological tests for the diagnosis of rickettsiosis in Denmark. J. Microbiol. Methods 76, 285–288. Nielsen, H., Fournier, P.E., Pedersen, I.S., Krarup, H., Ejlertsen, T., Raoult, D., 2004. Serological and molecular evidence of Rickettsia helvetica in Denmark. Scand. J. Infect. Dis. 36, 559–563. Nilsson, K., Lindquist, O., Pahlson, C., 1999. Association of Rickettsia helvetica with chronic perimyocarditis in sudden cardiac death. Lancet 354, 1169–1173. Nilsson, K., Pahlson, C., Lukinius, A., Eriksson, L., Nilsson, L., Lindquist, O., 2002. Presence of Rickettsia helvetica in granulomatous tissue from patients with sarcoidosis. J. Infect. Dis. 185, 1128–1138.
B. Kantsø et al. / Ticks and Tick-borne Diseases 1 (2010) 101–103 Planck, A., Eklund, A., Grunewald, J., Vene, S., 2004. No serological evidence of Rickettsia helvetica infection in Scandinavian sarcoidosis patients. Eur. Respir. J. 24, 811–813. Skarphedinsson, S., Jensen, P.M., Kristiansen, K., 2005. Survey of tickborne infections in Denmark. Emerg. Infect. Dis. 11, 1055–1061. Skarphedinsson, S., Lyholm, B.F., Ljungberg, M., Sogaard, P., Kolmos, H.J., Nielsen, L.P., 2007. Detection and identification of Anaplasma phagocytophilum, Borrelia burgdorferi, and Rickettsia helvetica in Danish Ixodes ricinus ticks. APMIS 115, 225–230. Sonenshine, D.E., 1991. Biology of ticks. Oxford University Press, New York. Stenos, J., Graves, S.R., Unsworth, N.B., 2005. A highly sensitive and specific real-time PCR assay for the detection of spotted fever and typhus group rickettsiae. Am. J. Trop. Med. Hyg. 73, 1083–1085.
103
Stenos, J., Roux, V., Walker, D., Raoult, D., 1998. Rickettsia honei sp. nov., the aetiological agent of Flinders Island spotted fever in Australia. Int. J. Syst. Bacteriol. 48, 1399–1404. Svendsen, C.B., Krogfelt, K., Jensen, P.M., 2009a. Detection of Rickettsia spp. in Danish ticks (Acari: Ixodes ricinus) using real-time PCR. Scand. J. Infect. Dis. 41, 70–72. Svendsen, C.B., Milman, N., Nielsen, H.W., Krogfelt, K.A., Larsen, K.R., 2009b. A prospective study evaluating the presence of Rickettsia in Danish patients with sarcoidosis. Scand. J. Infect. Dis. 41, 745–752. Vennestrøm, J., Egholm, H., Jensen, P.M., 2008. Occurrence of multiple infections with different Borrelia burgdorferi genospecies in Danish Ixodes ricinus nymphs. Parasitol. Int. 57, 32–37.