Rickettsia spp. and Coinfections With Other Pathogenic Microorganisms in Hard Ticks From Northern Germany

VECTOR-BORNE DISEASES, SURVEILLANCE, PREVENTION Rickettsia spp. and Coinfections With Other Pathogenic Microorganisms in Hard Ticks From Northern Germany SABINE SCHICHT, THOMAS SCHNIEDER, AND CHRISTINA STRUBE 1 Institute for Parasitology, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany J. Med. Entomol. 49(3): 766Ð771 (2012); DOI: http://dx.doi.org/10.1603/me11204 ABSTRACT Rickettsia species are the causative agent of different forms of spotted fever and thus, monitored in a number of prevalence studies. The current study examined the status of ticks from the city of Hanover, Northern Germany, regarding the presence of Rickettsia spp. and coinfections with Borrelia burgdorferi sensu lato (sl) and Anaplasma phagocytophilum. In total, 1,089 questing Ixodes ricinus L. ticks were analyzed using quantitative real time polymerase chain reaction. A duplex quantitative real time polymerase chain reaction for simultaneous detection of Rickettsia spp. and Ixodes spp.-dna as positive control for successful DNA-isolation was established. Rickettsia spp. were detected in 363 (33.3%) of the 1,089 investigated ticks. QuantiÞcation of Rickettsia showed that larvae contained up to 50,000 bacteria, nymphs up to 85 million and adults up to 200 million per tick. Species differentiation was possible in 178 out of 363 Rickettsia positive samples and resulted in a predominant occurrence of R. helvetica (98.9%, 176/178), whereas R. monacensis was rarely found (1.1%, 2/178). Besides detection of Rickettsia, positive ticks were compared with results from previous studies to examine coinfections with B. burgdorferi sl and A. phagocytophilum. The resulting coinfection rates were 9.1% (99/1,089) for B. burgdorferi sl and 2.8% (11/391) for A. phagocytophilum. Triple-infection with Rickettsia spp., B. burgdorferi sl, and A. phagocytophilum occurred in 5 (1.3%) out of 391 ticks. The current study is the Þrst presenting quantitative data concerning the load of Ixodes ticks with Rickettsia individuals. KEY WORDS Ixodes, Rickettsia, Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, quantitative real time PCR The hard tick Ixodes ricinus L., which is the most abundant tick species in Europe, is known to transmit protozoa, viruses, and several bacteria. In Central Europe I. ricinus is the main vector for Lyme borreliosis spirochetes (Gray et al. 2009). Besides Borrelia burgdorferi sensu lato (sl) it also transmits Anaplasma phagocytophilum and different species of the genus Rickettsia. The latter pathogen belongs to the order Rickettsiales and is an obligate intracellular gram-negative -proteobacterium. In Germany, at least six Rickettsia species were detected in ticks: R. helvetica, R. monacensis, R. massiliae, and R. felis, which were found in I. ricinus, as well as R. slovaca and R. raoultii, which were diagnosed in ticks of the genus Dermacentor (Simser et al. 2002, Wolfel et al. 2006, Silaghi et al. 2008, Dobler and Wolfel 2009). All mentioned Rickettsia species are known to cause rickettsiosis in humans (Mediannikov et al. 2008, Dobler and Wolfel 2009, Parola et al. 2009). Depending on the species, symptomes like an eschar, local lymphadenopathy, maculopapular rash and ßu-like symptoms, for example, fever, malaise, headache, or myalgia may develop with a mild to severe clinical phenotype (Fournier et al. 1 Corresponding author, e-mail: christina.strube@tiho-hannover.de. 2000, Richter et al. 2002, Baumann et al. 2003). Reported Rickettsia infection rates in I. ricinus in Germany vary from 5.3 to 12% in the federal state of Bavaria, 8.9% in the federal state of Baden-Wuerttemberg (both Southern Germany), 14.7% in the federal state of Thuringia (Middle Germany) to 14.2% in a district of the German capital Berlin, which is located in Eastern Germany (Simser et al. 2002, Hartelt et al. 2004, Pichon et al. 2006, Wolfel et al. 2006, Silaghi et al. 2008, Dobler and Wolfel 2009, Hildebrandt et al. 2010). R. helvetica was found to be the most frequent Rickettsia species (Hartelt et al. 2004, Wolfel et al. 2006, Silaghi et al. 2008, Hildebrandt et al. 2010). The aim of the current study was to investigate the presence of Rickettsia spp. in questing ticks from Northern Germany and to analyze the coinfection rate with B. burgdorferi sl. Additionally, a subset of the ticks were examined for coinfection with A. phagocytophilum. Moreover, the number of individual Rickettsia in infected ticks was quantiþed. Materials and Methods Tick Material. In 2005, 8,800 questing Ixodes ticks were collected in different recreation areas in the city 0022-2585/12/0766Ð0771$04.00/0 2012 Entomological Society of America

May 2012 SCHICHT ET AL.: Rickettsia SPP. AND COINFECTIONS IN GERMAN TICKS 767 of Hanover, the capital of the northern German federal state Lower Saxony, using the ßagging method. The tick species and developmental stage were identiþed by morphological characteristics using light microscopy (Montenegro 2008). From this cohort, a total of 1,089 I. ricinus ticks, consisting of 669 adults (341 males/328 females), 332 nymphs, and 88 larvae, were examined for Rickettsia DNA using duplex real time polymerase chain reaction (PCR). Isolation of genomic DNA was performed as described by Strube et al. (2010). Plasmid Standards for Quantitative Real Time PCR. Plasmid standards were used to calculate standard curves for the determination of target gene copy numbers. The Rickettsia plasmid standard comprised a 70 bp sequence fragment of the citrate synthase (glta) gene, which was ampliþed in subsequent quantitative real time PCRs (qpcrs). The insert was generated by overlapping primers based on three GenBank entries of R. helvetica (GenBank accession EU359285, RHU59723, and DQ131912). Primers were Rick_ Plasmid for (5 -ATA GGA CAA CCG TTT ATT TAT CCT GAT AAT TCG TTA GAT TTT ACC G-3 ) and Rick_Plasmid rev (5 -CAA ACA TCA TAT GCA GAA AAT TTT CGG TAA AAT CTA ACG AAT TAT C-3 ). PCR was set up as a 25 l reaction mixture containing 20.3 l double-distilled water, 1 l dntps (10 mm each), 0.5 l for/rev primer (10 M each), 2.5 l 10x buffer, and 0.2 l Advantage 2 Polymerase Mix (Clontech, Saint-Germain-en-Laye, France). Thermocycling included an initial denaturation of 95 C for 4 min, 40 cycles of 95 C for 20 s, 55 C for 20 s, 72 C for 30 s, and a Þnal extension at 72 C for 10 min. After gel electrophoresis on a 2.5% agarose gel the amplicon was ligated into the pcr4-topo vector and introduced into chemically competent One Shot Top10 Escherichia coli cells using the TOPO TA Cloning kit according to the manufacturerõs instructions (Invitrogen, Karlsruhe, Germany). Plasmid preparation was carried out using the NucleoSpin Plasmid kit (Marcherey-Nagel, Düren, Germany) and the inserted sequence was veriþed by sequencing. The Ixodes plasmid was prepared as described by Strube et al. (2010). Plasmids were linearized and dephosphorylated using the restriction enzyme PstI and the alkaline phosphatase FastAP (Fermentas, St. Leon-Rot, Germany). Plasmid DNA concentration and purity were quanti- Þed using the NanoDrop ND-1000 spectral photometer (Peqlab Biotechnologie GmbH, Erlangen, Germany) and the theoretical number of plasmid copies was calculated. Ten fold serial dilutions of plasmids ranging from 10 0 to 10 6 copies per 1 l were generated, aliquoted, and kept at 80 C. Each day, a new aliquot was used in qpcr runs. Duplex qpcr. I. ricinus DNA samples were analyzed using duplex qpcr for a simultaneous detection of DNA from Rickettsia spp. and Ixodes ticks, whereby the latter one served as positive control to verify successful DNA isolation. According to Stenos et al. (2005), the rickettsial glta gene was ampliþed by primers CS-F (5 -TCG CAA ATG TTC ACG GTA CTT T-3 ) and CS-R (5 -TCG TGC ATT TCT TTC CAT TGT G-3 ) and detected by the TaqMan-probe CS-P (5-6-FAM-TGC AAT AGC AAG AAC CGT AGG CTG GAT G-BHQ-1Ð3 ). For Ixodes spp.-dna detection, primers and the corresponding TaqMan-minor groove binder probe according to Strube et al. (2010) were used. The reaction contained 10.2 l doubledistilled H 2 O, 12.5 l Absolute Blue QPCR low Rox Mix (Thermo Fisher, Hamburg, Germany), 0.15 lof each forward and reverse primer (50 M), 0.06 l of each probe (10 M), and 2 l tick DNA-template resulting in a total reaction volume of 25 l. The plasmid standard reactions contained 1 l plasmid DNA of each Rickettsia and Ixodes plasmid standard. Thermocycling conditions were as follows: initial denaturation of 95 C for 15 min followed by 40 cycles of 95 C for 15 s and 60 C for 60 s. In each qpcr run the DNA sample, plasmid standards and a no-template control were included in duplicate. Experiments and data analyses were performed using the Mx3005 Multiplex Quantitative PCR System (Stratagene, Heidelberg, Germany). glta Gene-Copy Number in the Rickettsia Genome. To ensure correct extrapolation from the qpcr copy number to the number of individual Rickettsia bacteria per I. ricinus tick, the copy number of the glta gene in the rickettsial genome was checked. Because a complete genome sequence of R. helvetica was not available in the National Center for Biotechnology Information (NCBI) Gene databases, the genome data of the R. rickettsii strains Sheila Smith and Iowa (accession NC_009882 and CP000766.2) were used instead. The mean number of Rickettsia individuals as well as the standard deviation was calculated for each developmental stage. Rickettsia Species Differentiation. Rickettsia spp.- positive tick samples were ampliþed by conventional PCR using primers RpCS.877p (5 -GGG GGC CTG CTC ACG GCG G-3 ) and RpCS.1258n (5 -ATT GCA AAA AGT ACA GTG AAC A-3 ) according to Regnery et al. (1991). PCR resulted in a 381 bp amplicon that was subsequently sequenced (EuroÞns MWG GmbH, Ebersberg, Germany). To identify the Rickettsia species in the infected ticks, obtained nucleotide sequences were BLASTed against the NCBI nucleotide database. Coinfections With B. burgdorferi sl and A. phagocytophilum. All 1,089 ticks, which were investigated for Rickettsia spp. infection, were compared with data from a previous study (Montenegro 2008) to analyze the coinfection rate with B. burgdorferi sl. Additionally, a subsample of 391 out of the 1,089 ticks could be analyzed for coinfections with A. phagocytophilum by using data from Schicht et al. (2011). Statistical Analysis. Statistical analysis of Rickettsia spp. infection and coinfection with B. burgdorferi sl and A. phagocytophilum, respectively, in different tick developmental stages and sexes was carried out using the 2 test (SAS 9.1, SAS Institute Inc. 2004). The copy number distribution between different tick stages was statistically analyzed by KruskalÐWallis one way analysis of variance (ANOVA) on ranks and a subsequent

768 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 49, no. 3 Table 1. Rickettsia load in 363 I. ricinus DNA samples originating from different tick stages No. Rickettsia individuals Total ticks (%) Adults (%) Males (%) Females (%) Nymphs (%) Larvae (%) 100 113 (31.1) 70 (28.7) 29 (27.1) 41 (29.9) 34 (35.7) 9 (37.5) 1,000 37 (10.2) 34 (13.9) 7 (6.6) 27 (19.7) 3 (3.2) 0 (0) 10,000 33 (9.1) 21 (8.6) 4 (3.7) 17 (12.4) 10 (10.5) 2 (8.3) 100,000 57 (15.7) 19 (7.8) 14 (13.1) 5 (3.7) 25 (26.3) 13 (54.2) 1,000,000 87 (24.0) 67 (27.5) 41 (38.3) 26 (19.0) 20 (21.1) 0 (0) 10,000,000 32 (8.8) 32 (13.1) 11 (10.3) 21 (15.3) 0 (0) 0 (0) 100,000,000 3 (0.8) 0 (0) 0 (0) 0 (0) 3 (3.2) 0 (0) 1,000,000,000 1 (0.3) 1 (0.4) 1 (0.9) 0 (0) 0 (0) 0 (0) 363 (100) 244 (100) 107 (100) 137 (100) 95 (100) 24 (100) MannÐWhitney rank sum test (SigmaStat, SYSTAT 2004). A P value of 0.05 was considered statistically signiþcant. Results Standard Curves. The Rickettsia spp. plasmid standard containing 10 0 to 10 6 copies per reaction revealed a standard curve showing the following values: R 2 0.996, y-intercept 39.03 cycles and a slope 3.454. The resulting ampliþcation efþciency was 94.8%. The Ixodes spp. standard curve containing the same copy numbers resulted in a R 2 of 0.998, a y-intercept of 36.15 cycles, a slope of 3.271, and an ampliþcation efþciency of 102.2%. Therefore, the established duplex qpcr was considered suitable because standard curve values of both target gene sequences met required quality criteria (R 2 0.98, y-intercepts below 40 cycles and ampliþcation efþciencies between 90Ð110%.) Rickettsia spp. Infection in I. ricinus Ticks. The total Rickettsia spp. infection rate was 33.3% (363/1,089). Adult I. ricinus showed a signiþcantly higher Rickettsia spp. infection rate with 36.5% (244/669) compared with nymphs with 28.6% (95/332; P 0.0089). Female ticks were signiþcantly more infected (41.8%, 137/ 328) than males (31.4%, 107/341; P 0.0067). Rickettsia spp. infection in larvae was found in 27.3% (24/ 88). Number of Rickettsia Individuals per Tick. Gene copy number analysis of the glta gene in the rickettsial genome showed that it is a single copy gene. Therefore, each glta copy quantiþed by qpcr corresponds to one Rickettsia bacterium. For the calculation of the number of Rickettsia organisms in each individual tick, the qpcr copy number was multiplied by 50 because the used template amount (2 l) represented one- Þftieth of the elution volume (100 l) of the isolated genomic DNA. This calculation resulted in a mean burden of 14,946 16,408 Rickettsia individuals per larva, 1,359,399 9,086,586 Rickettsiae per nymph, and 1,136,885 11,791,366 Rickettsiae per adult tick. The KruskalÐWallis test revealed a statistically significant difference of the Rickettsia burden between different tick stages (P 0.022). Subsequent MannÐ Whitney test showed signiþcant differences only when comparing Rickettsia numbers of adults against larvae (P 0.033). No signiþcant differences were observed in Rickettsia numbers of adults versus nymphs (P 0.063) and nymphs versus larvae (P 0.055). The highest Rickettsia number detected in different tick stages was 49,300 in larvae, 84,800,000 in nymphs and 184,000,000 in adults. However, nearly one-third of all infected ticks (113/363) harbored 100 Rickettsia individuals. A detailed overview of distribution of Rickettsia numbers in different tick stages is given in Table 1. Rickettsia Species Differentiation. Conventional PCR for subsequent differentiation of the Rickettsia species via sequencing resulted in a visible agarose gel band in 180 (49.6%) out of the total of 363 Rickettsiapositive tick samples. From these, sequences were obtained from 178 samples. BLAST search revealed that 176 samples (98.9%) were identical to the nucleotides 329Ð636 of R. helvetica, whereas two samples (1.1%) showed 100% sequence identity to the nucleotides 829-1153 of R. monacensis. Coinfections With B. burgdorferi sl and A. phagocytophilum. The total coinfection rate of the 1,089 analyzed tick samples with B. burgdorferi sl was 9.1% (99/1,089). 12.3% (82/669) of the adults were coinfected, whereas nymphs showed a signiþcant lower coinfection rate of 5.1% (17/332; P 0.001). Females were signiþcantly higher coinfected (15.9%, 52/ 328) than males (8.8%, 30/341; P 0.008). More detailed data are given in Table 2. Furthermore, a subset of 391 of the 1,089 investigated tick samples could be analyzed for coinfections with B. burgdorferi sl and A. phagocytophilum. Of the 391 ticks, 34.5% were positive for Rickettsia spp. and coinfection rates with B. burgdorferi sl and A. phagocytophilum resulted in 10.7 and 2.8%, respectively. Adult ticks showed higher infection rates (Rickettsia Table 2. Rickettsia spp. infections and coinfections with B. burgdorferi sl in the total of 1,089 investigated I. ricinus ticks No. investigated ticks Rickettsia spp. infection B. burgdorferi sl coinfection No. (%) No. (%) Adults 669 244 (36.5) 82 (12.3) Males 341 107 (31.4) 30 (8.8) Females 328 137 (41.8) 52 (15.9) Nymphs 332 95 (28.6) 17 (5.1) Larvae 88 24 (27.3) 0 (0) All stages 1,089 363 (33.3) 99 (9.1)

May 2012 SCHICHT ET AL.: Rickettsia SPP. AND COINFECTIONS IN GERMAN TICKS 769 Table 3. ticks Rickettsia spp. infection and coinfections with B. burgdorferi sl and/or A. phagocytophilum in a subsample of 391 I. ricinus No. investigated ticks Rickettsia spp. infection Total coinfection B. burgdorferi sl coinfection A. phagocytophilum coinfection No. (%) No. (%) No. (%) No. (%) Males 104 33 (31.7) 16 (15.4) 12 (11.5) 4 (3.8) Females 115 44 (38.3) 21 (18.3) 17 (14.8) 4 (3.5) Adults 219 77 (35.2) 37 (16.9) 29 (13.2) 8 (3.7) Nymphs 172 58 (33.7) 16 (9.3) 13 (7.6) 3 (1.7) All stages 391 135 (34.5) 53 (13.6) 42 (10.7) 11 (2.8) spp.: 35.2%, B. burgdorferi sl: 13.2%, A. phagocytophilum: 3.7%) than nymphs (Rickettsia spp.: 33.7%, B. burgdorferi sl: 7.6%, A. phagocytophilum: 1.7%) irrespective of the pathogen. Female ticks were more frequently infected with Rickettsia spp. (38.3%) and coinfected with B. burgdorferi sl (14.8%) than males (31.7 and 11.5%, respectively), whereas coinfection rates with A. phagocytophilum were found to be approximately the same in both sexes (3.5% females, 3.8% males). Five out of the 391 samples (1.3%) were coinfected with all three pathogens. A detailed overview of infection rates of different tick stages and sexes is shown in Table 3. Discussion Ticks of the genus Ixodes are vectors for a number of human pathogenic microorganisms including the genus Rickettsia. Rickettsial species occurring in Ixodes ticks in Europe are the causative agent of different forms of spotted fever or feverish infections and thus monitored in a number of prevalence studies. However, so far published PCR detection methods focus on Rickettsia-DNA and, as no veriþcation of successful genomic DNA isolation was included, negative results could also have derived from unsuccessful DNA isolation. To overcome this issue, a duplex qpcr for simultaneous detection of both, Rickettsia- and Ixodes-DNA, were established to include a positive control for successful DNA isolation. With the established duplex qpcr, a total of 1,089 I. ricinus ticks was analyzed resulting in a Rickettsia spp. prevalence rate of 33.3% (363 ticks). Adult ticks were signiþcantly more infected (36.5%) than nymphs (28.6%). Prevalence variation between different tick stages was also shown in a study by Hildebrandt et al. (2010), in which adults were signiþcantly more infected (18.8%) than nymphs (9.3%), which may be explained by a further bloodmeal by adult ticks providing the possibility of super infections. Furthermore, females were signiþcantly more infected (41.8%) than males (31.4%). This may be explained by the fact that copulation of I. ricinus does not only occur on the host. Mating may also occur before the female has had a bloodmeal, namely shortly after the female molts to the adult stage (Balashov 1956, Graf 1978). Infected males may transmit R. helvetica to females through spermatids, spermatophores, and spermiophore ßuids (Burgdorfer et al. 1979, Hayes et al. 1980); this could provide enough time for massive infections of the female tick even before a subsequent bloodmeal. B. burgdorferi sl bacteria are transmitted transstadially during tick development, but rather inefþciently transovarially (Gern et al. 1997, Nefedova et al. 2004). In contrast, efþcient transovarial as well as transstadial transmission has been shown for R. helvetica. For these, the transovarial transmission rate in I. ricinus under laboratory conditions was 100% (Burgdorfer et al. 1979). In the current study, Rickettsia infection occurred in 27.3% of the larvae. Differentiation of the rickettsial species, which was possible for eleven larvae, revealed R. helvetica as expected because of the predominant occurrence of this species. These results are in accordance with Stanek (2009), who observed an infection rate of 21% in I. ricinus larvae in Austria, and support the Þndings concerning efþcient transovarial transmission of Rickettsia spp. under natural conditions. In comparison to studies from other German regions, the determined Rickettsia infection rate of 33.3% is much higher compared with 14.7% in Central Germany, 14.2% in Eastern Germany, and 5.3Ð13.3% in Southern Germany (Hartelt et al. 2004, Pichon et al. 2006, Wolfel et al. 2006, Silaghi et al. 2008, Hildebrandtet al. 2010). On one hand, this may result from a superior sensitivity of the established duplex qpcr, which was able to detect one gene copy in the reaction set-up, and however, from regional differences caused by ecological factors such as microclimate, host susceptibility, and tick density (Hildebrandt et al. 2003). Regional prevalence variations were also observed in other European countries. In The Netherlands, Rickettsia spp. infection rates in I. ricinus ranged from 6 to 66% (Sprong et al. 2009). A Rickettsia prevalence varying from 2.4 to 7.9% was detected in Luxembourg (Reye et al. 2010). Because real time PCR is more sensitive than conventional PCR, in only 180 out of 363 (49.6%) Rickettsia-qPCR positive ticks an amplicon was visible after agarose gel electrophoresis and thus, provided enough DNA for Rickettsia species differentiation by sequencing. From these 180 samples 178 sequences were obtained with a resulting predominant occurrence of R. helvetica (98.9%, 176/178) with R. monacensis as a rare species (1.1%, 2/178), comparable to other German studies (Silaghi et al. 2008, Hildebrandt et al. 2010). The infective dose for the highly pathogenic species R. prowazekii and R. typhi is considered to be 10 individuals (Azad and Radulovic 2003), whereas the

770 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 49, no. 3 infective doses for Rickettsia species occurring in Germany are unknown yet. The high Rickettsia prevalence found in the current study suggests that transmission of Rickettsia spp. to the tickõs host and thus to humans occur rather often. However, clinical cases of rickettsioses in Germany are rarely reported. This could mean that in most cases the number of bacteria from Rickettsia species occurring in Germany transferred with a tick bloodmeal is not causing clinical disease. However, infections could easily be misdiagnosed because of the ßu-like symptoms. The duplex qpcr detection limit of one glta gene copy per reaction set-up corresponds to a detection limit of 50 individual Rickettsiae per tick. By calculating individual bacterial loads for investigated ticks, larvae contained a mean burden of 15,000 Rickettsia per tick, nymphs of 1.36 million and adults 1.14 million. The existence of a maximum load of bacteria, which allows I. ricinus ticks to undergo ecdysis as larvae and nymphs and reproduction as adults, may be speculated because of the fact that Rickettsia load did not differ signiþcantly between the nymphal and adult stages. The 1,089 ticks investigated for Rickettsia infection were also analyzed for coinfections with B. burgdorferi sl, the agent of Lyme borreliosis. In addition, 9.1% (99/1,089) of the ticks exhibited coinfections whereby the infection rate of adult ticks compared with nymphs was twice as high (12.3 vs. 5.1%). Because questing larvae were collected and transovarial transmission of B. burgdorferi sl is rather inefþcient (Gern et al. 1997, Nefedova et al. 2004), no coinfection could be detected in larvae. The distribution of Borrelia coinfection reßects the prevalenceõs in different tick stages collected in the area of Hanover, which was 34.6% in adults, 18.1% in nymphs, and 1.7% in larvae (Strube et al. 2010). Besides coinfections with B. burgdorferi sl a subpopulation of 391 ticks could be evaluated for coinfections with A. phagocytophilum. As expected, the percentage of 34.5% Rickettsia spp. infected ticks and 10.7% B. burgdorferi sl coinfected ticks was comparable to the results of the total sample size of 1,089 ticks. Rickettsia and A. phagocytophilum coinfections were found in 2.8% of the analyzed ticks, which basically reßects the prevalence of 3.2% for the region of Hanover (Schicht et al. 2011). Again, adult ticks were about two times more coinfected (3.7%) than nymphs (1.7%) that is in accordance to data from Southern Germany, where a coinfection in 2.6% of I. ricinus ticks was found (Hartelt et al. 2004). All three pathogens were harbored by 1.3% (5/391) of the ticks investigated in the current study, corroborating the broad vector competence of I. ricinus for pathogenic microorganisms affecting humans as well as companion animals and livestock. 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