Introduction

Rickettsiae are obligate intracellular Gram-negative bacteria that are associated with arthropod vectors and are responsible for mild to severe diseases in humans (Raoult and Roux 1997).

Rickettsia raoultii, Rickettsia sp. genotypes DnS14, DnS28 and RpA4, was first identified as new rickettsiae of the Rickettsia massiliae genogroup in 1999, by rrs (16S rDNA), gltA and ompA sequencing from Dermacentor nutallii ticks collected in Siberia and Rhipicephalus pumilio ticks collected in Astrakhan (Rydkina et al. 1999). R. raoultii strains were isolated from PCR-positive Dermacentor ticks, Dermacentor silvarum, D. nuttalli, Dermacentor reticulatus and Dermacentor marginatus, collected in Russia, Kazakhstan, and France using cell cultures (Rydkina et al. 1999; Mediannikov et al. 2008). Herein, we describe the first cultivation of R. raoultii in Slovakia from D. marginatus. Since 1999, R. raoultii have been detected in Dermacentor ticks throughout Europe, in the European part of Russia (Shpynov et al. 2001), Germany (Dautela et al. 2006), Spain (Márquez 2008), Portugal (Vitorino et al. 2007), Netherlands (Nijhof et al. 2007), Slovakia (Boldiš et al. 2008), France and Croatia (Mediannikov et al. 2008), Poland (Chmielewski et al. 2009), UK (Tijsse-Klasen et al. 2011) and in Haemaphysalis punctata collected in Spain (Márquez 2008). In 2002, Mediannikov et al. (2008) detected R. raoultii DNA in D. marginatus tick taken from the scalp of a patient in whom TIBOLA (tick-borne lymphadenopathy)/DEBONEL (Dermacentor-borne necrosis erythema and lymphadenopathy) developed in France. It seems to be less pathogenic than Rickettsia slovaca (Parola et al. 2009).

Rickettsia slovaca was first isolated in 1968 from the tick D. marginatus collected in central Slovakia (Brezina et al. 1969). R. slovaca had been considered as a non-pathogenic microorganism for many years. In 1997 it was described as a human pathogen and the etiological agent of TIBOLA/DEBONEL human disease, which is associated with a tick bite, an inoculation eschar on the scalp, and cervical lymphadenopathies (Raoult et al. 1997). R. slovaca and human diseases caused by it were recorded across Europe (Parola et al. 2009).

Ixodes ricinus, D. marginatus, D. reticulatus, Haemaphysalis concinna, Haemaphysalis punctata, and Haemaphysalis inermis (the family Ixodidae, the order Acari) are exophillic tick species occurring in Slovakia. Ixodes ricinus ticks are widely distributed throughout the country whereas D. reticulatus and D. marginatus ticks are limited to along the rivers and Krupinská planina, respectively (Řeháček et al. 1991).

The aim of this study was to isolate, identify and characterize rickettsial species occurring in Dermacentor ticks inhabiting Slovakia.

Materials and methods

Collection of ticks

D. marginatus and D. reticulatus ticks were collected by blanket-dragging over the vegetation in Bratislava, Horný Bar, Martinský les, Moravský sv. Ján localities in western Slovakia and Veľký Lom, Kalonda, Píla, Sása, Pinciná, Budiná localities in central Slovakia during 2004–2010 years. They were classified to species, sex and maintained alive at +4°C prior to the examination.

Hemocyte test (HT)

Ticks were microscopically tested by HT, hemocytes from one droplet of hemolymph from ticks were stained by the Gimenez method (Burgdorfer 1970). HT can indicate the presence of microorganisms with rickettsial morphology, such as Rickettsia sp., Coxiella burnetii, rickettsia-like microorganisms, but can not differentiate microorganism species. Ticks were still alive after the screening by the HT method, which is important for the isolation and cultivation of rickettsiae in cell lines.

Isolation and cultivation of rickettsiae in Vero and L929 cell lines

Isolation of rickettsiae was attempted on the hemolymph-positive Dermacentor tick. One droplet of hemolymph obtained from previously unbroken leg of tick, was inoculated into one cultivation well (shell vial) containing monolayer of confluent Vero or L929 cells. After inoculation, the shell vials were centrifuged for 45 min at 1,000g, 25°C. Then the monolayer was incubated in a CO2 incubator for 120 min at 33°C. Finally, the cells were incubated in CO2 incubator at 33°C for 7–10 days. For study of intracellular growth, R. raoultii was cultivated in Vero and L929 cells (inoculum ~105 R. raoultii per flask). Negative controls of Vero and L929 cells without rickettsiae were also done. In three parallel series of static cultivation the growth medium was not replaced over the 14 days. Each 24 h intervals of cultivation infected cells in growth medium were scrapped, frozen at −70°C and after defrost centrifuged (5,000g, 10 min). Pellet was processed to extraction of DNA for quantification of rickettsial DNA copies by qPCR.

Infection of guinea pigs with Rickettsia raoultii

Experimental infection of guinea pigs through infected ticks was done analogous to study of Niebylski et al. (1999). Rickettsia raoultii-positive adult ticks fed 7 days on 3 guinea pigs. Blood from guinea pigs was collected every seventh day during 4 weeks and serum, heart, marrow, liver, lungs, bones, spleen and brain were collected from guinea pigs after their death. Guinea pigs died spontaneously. All organs and blood have been subjected to DNA extraction and subsequently to qPCR.

DNA extraction

All infected cell line, tick, blood and organ samples were individually processed by PCR. The DNA from infected mammalian cells, blood and organs from guinea pigs was isolated according to Wilson (1995) by phenol–chloroform extraction. The DNA from ticks collected from the vegetation was extracted using alkaline hydrolysis. Each ticks were washed with 70% ethanol and sterile water, crushed with sterile forceps and treated with 0.7 M ammonium hydroxide (NH4OH) for 15 min at 100°C in sealed PCR tubes. Subsequently, NH4OH was evaporated for 25 min at 100°C. DNA from ticks acquired from animals was extracted using Dneasy Blood and Tissue Kit (Qiagen) according to manufacturing protocol. DNA extractions were stored at −20°C and later used as templates for the PCR amplification.

PCR amplification, PCR–RFLP and DNA sequencing

The PCR for eubacteria was used for amplification of 470 bp part of the 16S rRNA gene using primers GA1B and 16S8FE (Bekker et al. 2002). Detection of Rickettsia sp. was done with specific primers RpCS.877p–RpCS.1258n amplified 381 bp part of the gltA gene, RR190.70F–RR190.701R amplified 632 bp part of ompA gene and D767f–D1390r primers amplified a part of 623 bp of the sca4 gene (Regnery et al. 1991; Roux et al. 1996). Enzymatic digestion for the identification of R. slovaca was performed as described by Špitalská et al. (2008). PCR and PCR–RFLP products were analyzed by eletrophoresis in a 1% agarose gel, stained with GelRed (Biotium), and visualized with UV transilluminator. Amplicons were purified using a QIAquick Spin PCR Purification Kit (Qiagen) as described by the manufacturer. The sequencing was performed by Macrogen (South Korea; http://www.macrogen.com). DNA sequences were compared with available databases in GenBank using the Basic Local Alignment Search Tool (BLAST) on http://blast.ncbi.nlm.nih.gov/. Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2011).

qPCR assay

PCR amplification was conducted using DyNAmo HS SYBR Green qPCR kit reagents according to the manufacturer’s instructions (Finnzymes, Finland). The reaction mixture and conditions for amplification were described by Boldiš and Špitalská (2010). Negative controls contained all PCR reaction components, as template DNA were DNAs of uninfected mammalian cells and nuclease-free water. The 632-bp fragment of the ompA gene of R. slovaca strain B13 was amplified and cloned into pGEM-T easy plasmid (Promega), vector was transformed into Escherichia coli competent cells and used as a standard for absolute quantification. To quantify the copy numbers of rickettsial gene in samples, tenfold serial dilutions of a plasmid were used to generate a standard curve.

Results

A total of 540 adult ticks, 272 D. marginatus, and 268 D. reticulatus were collected from the vegetation. Totally 249 adult Dermacentor ticks, 96 D. marginatus, 153 D. reticulatus were collected from horses, sheep, goats and dogs, and only 5 I. ricinus from horses (Table 1).

Table 1 Infection rate with Rickettsia sp. in Dermacentor ticks
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The DNA of Rickettsia sp. was found in 229 ticks, 69 D. reticulatus and 88 D. marginatus ticks from the vegetation and 44 D. reticulatus and 28 D. marginatus ticks collected from animals using PCR. The infection rate was from 25.49 to 36.17% depending on the collection and from 16.67 to 44% depending on sex of ticks (Table 1). PCR–RFLP and/or sequencing for identification of Rickettsia species showed that D. reticulatus ticks were more infected with R. raoultii, 86.67 and 94.12% infection of tick from vegetation and animals, respectively. Seventy-five percent of D. marginatus ticks from vegetation were infected with R. slovaca and R. slovaca-infection was found in 70.59% of D. marginatus ticks from animals. Table 2 shows the prevalence of each rickettsia in each species of Dermacentor ticks.

Table 2 The prevalence of Rickettsia raoultii and R. slovaca in Dermacentor marginatus and D. reticulatus ticks in Slovakia
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Isolation of rickettsiae was attempted on D. marginatus ticks, the hemolymphs of which were positive in the HT. Rickettsiae were detected in eukaryotic cells by genus-specific PCR followed by sequencing. The obtained isolate showed infection with R. slovaca (Boldiš and Špitalská 2010) and R. raoultii. Partial sequencing of ompA gene of R. raoultii isolate (R. raoultii DMS, Acc. No. JN398480) confirmed 99.6% identity with R. raoultii strain Marne (Acc. No. DQ365799), (Fig. 1). Intracellular growth of R. raoultii (Fig. 2) was evaluated by quantitative PCR assay using L929 cells (triangle) and Vero cells (circles). Curves of bacterial growth were modeled with lag, exponential, stationary and death phases. R. raoultii achieved the highest point of multiplication on the 7th and 8th day postinfection in Vero and L929 cells, respectively. The rickettsial copy number in Vero and L929 cells per flask at this point was 1.6 and 1.4 times greater than rickettsial DNA copy number of inoculum, respectively.

Fig. 1
figure 1

Evolutionary relationship of Rickettsia spp. inferred from the comparison of a portion of the ompA gene using the Neighbor-Joining method. Bootstrap values are reported at the nodes. Sequences of R. raoultii DMS (highlighted by the black circle) were compared with sequences downloaded from the GenBank

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Fig. 2
figure 2

The growth kinetics of Rickettsia raoultii in Vero and L929 determined by the quantitative PCR assay. Eukaryotic cells approaching confluence were infected with ~105 copy number of bacteria per flask. Values are the average ± SE of three replicates

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Figure 3 shows the rickettsial copy number in blood and organs of guinea pigs, on which R. raoultii-positive adult ticks fed. The rickettsial infection was recorded in all organs, the highest infection was in spleen, liver and heart. The infection increased in time in blood samples.

Fig. 3
figure 3

Infection with Rickettsia raoultii in blood and organs of guinea pigs. Each point corresponds to the mean of three distinct experiments

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Discussion

For long time is known, that Dermacentor ticks are the main vectors of some species of Rickettsia. To this time only prevalence of Rickettsia spp. in Dermacentor ticks in Slovakia was determined, but no information exists regarding the prevalence of R. slovaca and R. raoultii in D. reticulatus and D. marginatus ticks. Herein we provide detailed data related to the infection with these rickettsiae in Dermacentor ticks. Infection rates of R. raoultii in D. reticulatus were 86.7 and 94.1% from vegetation and animals, respectively. Contrary, infection rates of R. slovaca in D. marginatus were 75 and 70.6% from vegetation and animals, respectively. Similar study was conducted by Milhano et al. (2010) in Portugal, where 58.5% of D. marginatus ticks were infected with R. raoultii and 41.5% were R. slovaca positive. Socolovschi et al. (2009) defined infection rate with R. slovaca in D. marginatus ticks, which is 7.2–40.6% and with R. raoultii in D. reticulatus is 5.6–23%, in D. marginatus 22.5–83.3%. Part of our findings is in accordance with Socolovschi’s et al. (2009) data. In our study, more D. marginatus ticks were infected by R. slovaca with prevalence 20.6 and 24.3% in ticks from animals and from vegetation, respectively. More D. reticulatus were infected by R. raoultii with 22.3% prevalence in ticks from vegetation and 27.0% in ticks from animals. Infection of D. marginatus with R. raoultii (8.0–8.6%) is lower in comparisson with Socolovschi’s et al. (2009) data (22.5–83.3%). In previously published studies, R. raoultii has been more frequently detected in D. marginatus in Spain (73%) and Portugal (65%), in 57% of D. reticulatus ticks in Poland (Márquez et al. 2006; Vitorino et al. 2007; Chmielewski et al. 2009). The most (13 from 17) D. reticulatus ticks from Wales and England showed 100% homology with R. raoultii (Tijsse-Klasen et al. 2011). Infection of ticks removed from animals was studied by Dautela et al. (2006), Nijhof et al. (2007) and Selmi et al. (2009). They found that 14–23% D. reticulatus ticks were infected with R. raoultii and recorded 1.8 and 32.1% infection prevalence of D. marginatus with R. raoultii and R. slovaca, respectively. Their results are similar to ours. Differences in infection rates of both rickettsia in both Dermacentor species could be explained by the sampling methods, size of the samples, the potential PCR inhibitors, PCR reaction mixture and different detection surveys. Herein, we also described successful the first cultivation of R. raoultii in Slovakia from D. marginatus ticks. According to our knowledge, the bacterial growth kinetics of R. raoultii in mammalian cells was not done up to date. In general, bacterial growth can be modeled with known four different phases. Similar phases of R. raoultii growth curves were seen in our study. Our findings added growth data that bacteria might be equally accustomed to growth in L929 and Vero cell lines at 33°C supplemented with RPMI 1,640 cell culture medium (PAA Laboratories, Austria) containing 5% fetal bovine serum (Gibco, BRL, USA). R. raoultii was transmitted during feeding of R. raoultii-positive ticks to guinea pigs. It is human pathogen causing a mild rickettsiosis in humans, but pathogenicity for guinea pigs is not known and requires more studies. Pathological assessment of R. raoultii was not the objective of the study, therefore it is not possible to identify whether R. raoultii caused any disease in guinea pigs.

Anyway, the high percentage of D. reticulatus and D. marginatus ticks infected with R. slovaca and R. raoultii strongly indicates increasing needs of medical attention for people in localities where these ticks occur. Data of this study indicate that clinicians should be aware that patient with tick-borne lymphadenopathy may be in Slovakia.