Printer Friendly

Rickettsia helvetica in Dermacentor reticulatus ticks.

We report on the molecular evidence that Dermacentor reticulatus ticks in Croatia are infected with Rickettsia helvetica (10%) or Rickettsia slovaca (2%) or co-infected with both species (1%). These findings expand the knowledge of the geographic distribution of R. helvetica and D. reticulatus ticks.


Rickettsia helvetica organisms were first isolated from Ixodes ricinus ticks in Switzerland and were considered to be a new nonpathogenic species of the spotted fever group (SFG) rickettsiae (1,2). Recently, R. helvetica was linked to acute perimyocarditis, unexplained febrile illness, and sarcoidosis in humans in Europe (3-5). It is generally accepted that Dermacentor marginatus is the main vector of R. slovaca and that L ricinus is the main vector of R. helvetica (1,2,6).

Until now, only in the southern (Mediterranean) part of Croatia have R. conorii, R. slovaca, and R. aeschlimannii been detected in Rhipicephalus sanguineus, D. marginatus. and Hyalomma marginatum ticks, respectively (7). Human disease caused by R. conorii (Mediterranean spotted fever) has also been described in this region (8). No published reports of R. helvetica in Croatia are available. In a previous study, antibodies to SFG rickettsiae were found in dogs in the northwestern continental part of the country, where D. marginatus and I. ricinus ticks are common (9). Given the importance of this finding, we set up this study to provide the molecular evidence of the presence of R. helvetica and R. slovaca in Croatia. Prior to the study, D. reticulatus ticks had not been found in Croatia, although they were prevalent in neighboring Hungary (10). We used molecular methods to establish whether D. reticulatus ticks are also present in Croatia.

The Study

Using the cloth-dragging method, during March-May 2007 we collected 100 adult Dermacentor spp. ticks from meadows in 2 different locations near Cakovec, between the Drava and Mura rivers in the central part of Medjimurje County. This area is situated in the northwestern part of Croatia, at 46"38'N, 16"43'E, and has a continental climate with an average annual air temperature of 10.4[degrees]C at an altitude of 164 m.

To isolate DNA from ticks, we modified the method used by Nilsson et al. (11). Before DNA isolation, ticks were disinfected in 70% ethanol and dried. Each tick was mechanically crushed in a Dispomix 25 tube with lysis buffer by using the Dispomix (Medic Tools, Zug, Switzerland). Lysis of each of the crushed tick samples was carried out in a solution of 6.7% sucrose, 0.2% proteinase K, 20 mg/mL lysozyme, and 10 ng/ml RNase A for 16 h at 37[degrees]C; 0.5 molar EDTA, and 20% sodium dodecyl sulfate was added and further incubated for 1 h at 37[degrees]C. Extraction was performed twice with 80% phenol (1:1, vol:vol) and methylenchloride/isoamylalcohol (24:1, vol:vol). DNA was precipitated with isopropanol. The DNA-pellet was washed with 70% ethanol and centrifuged at 16.000 x g for 15 min. After the ethanol was removed, the pellet was dried at 50[degrees]C and dissolved in 50 [micro]L distilled water.

For the detection of Dermacentor spp., we used conventional PCR based on ribosomal internal transcribed spacer 2 (ITS2) sequences (12). In addition, we developed quantitative real-time PCR for the detection of R. helvetica based on the omp B gene and R. slovaca based on the ompA gene. Primer and probe sequences are shown in Table 1. Borrelia DNA was investigated with real-time PCR described by Schwaiger et al. (13).

All PCR-derived products generated from ticks (11 ompB, 3 ompA, and 13 ITS2) were sequenced. Sequencing reactions were performed by using a modified Sanger method and the BigDye Terminator Cycle Sequencing Kit version 1.2 (Applied Biosystems, Carlsbad, CA, USA) on an ABI 3730 capillary DNA Analyzer (Applied Biosystems), employing the same primer pairs as for amplification of the PCR products. Sequencing was performed by both Microsynth AG (Balgach, Switzerland) and our laboratory. All sequences were aligned with known sequences by using BLAST (

The sequences of the ITS2 spacer regions obtained from 13 Dermacentor spp. ticks infected with R. helvetica and R. slovaca were 99.8% identical to the corresponding D. reticulatus ITS2 sequence ($83080) and 85% identical to the corresponding D. marginatus sequence (S83081). The 646-bp ITS2 spacer fragment is sufficient to discriminate between the Dermacentor spp. The consensus sequence of the 13 D. reticulatus ITS2 spacer regions determined in this study was deposited at the European Molecular Biology Laboratory database under accession no. FM212280.

Results of the identification of R. helvetica and R. slovaca are shown in Table 2. The amplified ompB sequences of R. helvetica were 100% identical to the corresponding ompB gene of the R. helvetica strain C9P9 (AF123725), 92% identical to "Candidatus R. hoogstraalii" (EF629536), 89% identical to R. asiatica (DQ110870), 84% identical to R. rhipicephali (AF123719), and 83.3 % identical to R. raoultii (EU036984, DQ365798, DQ365797). The amplified ompA sequences were 100% identical to the corresponding ompA gene of R. slovaca and showed 2- to 12-bp differences to the corresponding ompA sequences of other Rickettsia spp.

In summary, R. helvetica DNA was detected in 10 of 100 D. reticulatus ticks, and the pathogen loads ranged from 380 to 1,700 copies per tick. R. slovaca DNA was found in 2 of 100 D. reticulatus ticks with copy numbers of 400 and 460. One D. reticulatus tick was co-infected with R. helvetica (410 copies) and R. slovaca (20,000 copies). No Borrelia burgdorferi DNA was found in D. reticulatus ticks.


Scientific literature supports the premise that D. marginatus ticks are the main vector of R. slovaca and that I. ricinus ticks are the main vector of R. helvetica (1,2,6). R. slovaca was also detected in D. reticulatus ticks (6,14). Additionally, the DNA of R. raoultii strain Marne, which is well separated from R. helvetica according to phylogenetic analyses of 16S rDNA sequences, was also detected in D. reticulatus ticks (14,15).

Previous studies showed that D. marginatus ticks are common in Croatia, and R. slovaca was identified in 36.8% of D. marginatus ticks collected in the southern part of the country (7). Further, R. helvetica as well as D. reticulatus ticks have never been detected in Croatia. In our study, 2% of D. reticulatus ticks were infected by R. slovaca, 10% were positive for R. helvetica, and 1% (1 tick) was coinfected by both pathogens. Our findings may explain the high seroprevalence (20.7%) of SFG antibodies in dogs detected in a previous study in the continental part of Croatia that is R. conorii free (9). This study suggests that these antibodies to SFG rickettsiae are presumably related to R. helvetica and R. slovaca infections, which can be transmitted by the same tick vector.

Because D. reticulatus is the second most common tick species occurring in all 16 counties of neighboring Hungary, we believe our findings point to an enlargement of its distribution area (10). Visual identification of Dermacentor spp. ticks has traditionally been confirmed on the basis of morphologic features. Because D. marginatus and D. reticulatus exhibit overlapping phenotypes, this means of identification can be very difficult (12). Therefore, we cannot exclude the possibility that D. reticulatus ticks were frequently misinterpreted as D. marginatus. Our study shows that the identification problem can be solved through use of molecular biology techniques.

We provide molecular evidence of the existence of D. reticulatus ticks in Croatia. Our results expand the knowledge of R. helvetica hosts. D. reticulatus ticks occur at far more sites than previously known and thus have probably expanded their habitats. Our data point out the need for further studies on the epidemiology of R. helvetica and other SFG rickettsiae in Croatia as well as their association with infections in humans and animals.


We are grateful to Mark Jaeggi, Priska Keller, Tobias Wermelinger, Andreas Steffen Stein, and Alexandra Schauerte for their help during this study.

This work was supported by grants from the Commission of Technology and Innovation of the Federal Office for Professional Education and Technology, Bern, Switzerland, and from the Ministry of Science, Education and Technology of the Republic of Croatia (No. 216-0481153-1148).


(1.) Burgdorfer W, Aeschlimann A, Peter O, Hayes SF, Philip RN. Ixodes ricinus: vector of a hitherto undescribed spotted fever group agent in Switzerland. Acta Trop. 1979;36:357-67.

(2.) Beati L, Peter O, Burgdorfer W, Aeschlimann A, Raoult D. Confirmation that Rickettsia helvetica sp. nov. is a distinct species of the spotted fever group of rickettsiae. Int J Syst Bacteriol. 1993;43:521-6.

(3.) Nilsson K, Lindquist O, Pahlson C. Association of Rickettsia helvetica with chronic perimyocarditis in sudden cardiac death. Lancet. 1999;354:1169-73. DOI: 10.1016/S0140-6736(99)04093-3

(4.) Fournier PE, Allombert C, Supputamongkol Y, Caruso G, Brouqui P, Raoult D. Aneruptive fever associated with antibodies to Rickettsia helvetica in Europe and Thailand. J Clin Microbiol. 2004;42:816-8. DOI: 10.1128/JCM.42.2.816-818.2004

(5.) Nilsson K, Pahlson C, Lukinius A, Eriksson L, Nilsson L, Lindquist O. Presence of Rickettsia helvetica in granulomatous tissue from patients with sarcoidosis. J Infect Dis. 2002;185:1128-38. DOI: 10.1086/339962

(6.) Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin Microbiol Rev. 2005;18:719-56. DOI: 10.1128/CMR. 18.4.719-756.2005

(7.) Punda-Polic V, Petrovec M, Trilar T, Duh D, Bradaric N, Klismanic Z, et al. Detection and identification of spotted fever group rickettsiae in ticks collected in southern Croatia. Exp Appl Acarol. 2002;28:169-76. DOI: 10.1023/A:1025334113190

(8.) Sardelic S, Fournier PE, Punda Polic V, Bradaric N, Grgic D, Ivic I, et al. First isolation of Rickettsia conorii from human blood in Croatia. Croat Med J. 2003;44:630-4.

(9.) Punda-Polic V, Bradaric N, Klismanic-Nuber Z, Mrljak V, Giljanovic M. Antibodies to spotted fever group rickettsiae in dogs in Croatia. Eur J Epidemiol. 1995;11:389-92. DOI: 10.1007/BF01721222

(10.) Sreter T, Szell Z, Varga I. Spatial distribution of Dermacentor reticulatus and Ixodes ricinus in Hungary: evidence for change? Vet Parasitol. 2005; 128:347-51. DOI: 10.1016/j.vetpar.2004.11.025

(11.) Nilsson K, Lindquist O, Liu AJ, Jaenson TG, Friman G, Pahlson C. Rickettsia helvetica in Ixodes ricinus ticks in Sweden. J Clin Microbiol. 1999;37:400-3.

(12.) Zahler M, Gothe R, Rinder H. Genetic evidence against a morphologically suggestive conspecificity of Dermacentor reticulatus and D. marginatus (Acari: Ixodidae). Int J Parasitol. 1995;25:1413-9. DOI: 10.1016/0020-7519(95)00081-X

(13.) Schwaiger M, Peter O, Cassinotti R Routine diagnosis of Borrelia burgdorferi (sensu lato) infections using a real-time PCR assay. Clin Microbiol Infect. 2001;7:461-9. DOI: 10.1046/j.l198-743-x.2001.00282.x

(14.) Dautel H, Dippel C, Oehme R, Hartelt K, Schettler E. Evidence for an increased geographical distribution of Dermacentor reticulatus in Germany and detection of Rickettsia sp. RpA4. Int J Med Microbiol. 2006;296(Suppl 40):149-56. DOI: 10.1016/j.ijmm.2006.01.013

(15.) Mediannikov O, Matsumoto K, Samoylenko I, Drancourt M, Roux V, Rydkina E, et al. Rickettsia raoultii sp. nov., a spotted fever group rickettsia associated with Dermacentor ticks in Europe and Russia. Int J Syst Evol Microbiol. 2008;58:1635-9. DOI: 10.1099/ ijs.0.64952-0

Author affiliations: Medizinische Laboratorien Dr F. Kaeppeli, Zurich, Switzerland (M. Dobec, F. Kaeppeli); University of Split School of Medicine, Split, Croatia (M. Dobec, V. Punda-Polic); County Hospital, Cakovec, Croatia (D. Golubic); Split University Hospital, Split (V. Punda-Polic); and Zurich University of Applied Sciences, Waedenswil, Switzerland (M. Sievers)

DOI: 10.3201/eid1501.080815

Address for correspondence: Marinko Dobec, medica, Medizinische Laboratorien Dr. F. Kaeppeli, Wolfbachstrasse 17, CH-8024 Zurich, Switzerland; email:

Dr Dobec is associate professor of medical microbiology at the University of Split, School of Medicine, Split, Croatia; scientific director of the Institute Virion, Ruschlikon, Switzerland; and head of the laboratory for infectious diseases and immunology at medica, Medizinische Laboratorien Dr F. Kaeppeli, Zurich, Switzerland. His research activities address tick-borne infections and zoonoses.
Table 1. Primers and probes designed for real-time PCR *

Species         Primers and probe       Sequence (5' [right arrow] 3')

Rickettsia        ompB-forward           GATTTCGACGGTAAAATTACC
helvetica         ompB_reverse            GCTACCGATATTACCTACAG
                   ompB_probe        ACTCTACTGCTACAAGTATGGTTGCTACAG

R. slovaca        ompA_forward         GTGATAATGTTGGCAATAATAATTGG
                  ompA_reverse           CTCTCGTTATAATCGAACCAAC
                   ompA_probe         CAGCAGGAGTACCATTAGCTACCCCTCC

Dermacentor        ITS forward          GTGCGTCCGTCGACTCGTTTTGA
spp.               ITS reverse           ACGGCGGACTACGACGGAATGC

* Amplified products: 162 by (R. helvetica); 228 by (R.
slovaca); 646 by (D. reticulatus); standard curve: slope;
Y-intercept and correlation coefficient: -3.634. 40.129. 0 9937
(R, helvetica); -0.23, 42.82, 0.9942 (R. slovaca); ITS,
internal transcribed spacer.

([dagger]) The TaqMan probes were labeled with the fluorescent
dyes FAM at the 5' end and TAMRA as quencher at the 3' end.

Table 2. Identification of Dermacentor, Rickettsia, and
Borrelia species by molecular methods

Species            Identification        Targeting        Confirmation
                       method             sequence

Dermacentorspp.          PCR           ITS2 (646 bp)       Sequencing

R. helvetica        Real-time PCR      ompB (162 bp)       Sequencing

R. slovaca          Real-time PCR      ompA (228 bp)       Sequencing

B. burgdorferi      Real-time PCR        flaB (p41)      Not done (PCR
Species                Results

Dermacentorspp.       13/13 (100%) positive; * 99.8% identical to
                         D. reticulatus ITS2 sequence (S83080)

R. helvetica       11/100 (11%) positive; ([dagger]) 100% identical
                        to R. helvetica strain C9P9 (AF 123725)

R. slovaca         3/100 (3%) positive; ([dagger]) 100% identical to
                         R. slovaca strains ([double dagger])

B. burgdorferi              0/100 positive; not detected in
                          Dermacentor spp. ticks ([section])

* Only infected ticks (13 ticks) were identified by molecular
methods (PCR and sequencing).

([dagger]) 100 ticks were analyzed; 10 were positive for R.
helvetica only, 2 for R. slovaca; 1 was co-infected with R.
helvetica and R. slovaca.

([double dagger]) GenBank accession nos. EU622810, DQ649052,
DQ649050, DQ649049, DQ649048, DQ649046, DQ649045, DQ649030,
DQ649029, Q649027, DQ649054, DQ649053, and U43808.

([section]) DNA of B. burgdorferi DSM 4680 and B. afzelii DSM
10508 were used as positive controls.
COPYRIGHT 2009 U.S. National Center for Infectious Diseases
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2009 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:DISPATCHES
Author:Dobec, Marinko; Golubic, Dragutin; Punda-Polic, Volga; Kaeppeli, Franz; Sievers, Martin
Publication:Emerging Infectious Diseases
Article Type:Clinical report
Geographic Code:4EXCR
Date:Jan 1, 2009
Previous Article:Rotavirus genotype distribution after vaccine introduction, Rio de Janeiro, Brazil.
Next Article:Variation in antimicrobial resistance in sporadic and outbreak-related Salmonella enterica serovar Typhimurium.

Terms of use | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters