Rickettsia slovaca and Rickettsia raoultii in Dermacentor marginatus and Dermacentor reticulatus ticks from Slovak Republic

Abstract

Rickettsiae, obligate intracellular Gram-negative bacteria, responsible for mild to severe diseases in humans are associated with arthropod vectors. Dermacentor marginatus and Dermacentor reticulatus are known vectors of Rickettsia slovaca and Rickettsia raoultii distributed across Europe. A total of 794 D. marginatus, D. reticulatus and Ixodes ricinus adult ticks were collected from the vegetation, removed from horses, sheep, goats and dogs in Slovakia. The DNA of Rickettsia sp. was found in 229 ticks by PCR amplifying parts of gltA, ompA and sca4 genes. Next analyses of Rickettsia-positive samples by PCR-RFLP and/or sequencing showed D. reticulatus ticks were more infected with R. raoultii and D. marginatus were more infected with R. slovaca. The prevalence of R. raoultii was 8.1-8.6% and 22.3-27% in D. marginatus and D. reticulatus, respectively. The prevalence of R. slovaca was 20.6-24.3% in D. marginatus and 1.7-3.4% in D. reticulatus. Intracellular growth of R. raoultii isolate from D. marginatus tick was evaluated by rOmpA-based quantitative SybrGreen PCR assay. The highest point of multiplication was recorded on the 7th and 8th day postinfection in Vero and L929 cells, respectively. R. raoultii was transmitted during feeding of R. raoultii-positive ticks to guinea pigs and subsequently rickettsial infection was recorded in all organs, the highest infection was in spleen, liver and heart. Our study describes the detection and isolation of tick-borne pathogens R. raoultii and R. slovaca, show that they are spread in Slovakia and highlight their risk for humans.

Full text

Rickettsia slovaca and Rickettsia raoultii in Dermacentor
marginatus and Dermacentor reticulatus ticks
from Slovak Republic
Eva S
ˇpitalska
´•Katarı
´na S
ˇtefanidesova
´•Elena Kocianova
´•
Vojtech Boldis
ˇ
Received: 22 August 2011 / Accepted: 17 February 2012
ÓSpringer Science+Business Media B.V. 2012
Abstract Rickettsiae, obligate intracellular Gram-negative bacteria, responsible for mild
to severe diseases in humans are associated with arthropod vectors. Dermacentor mar-
ginatus and Dermacentor reticulatus are known vectors of Rickettsia slovaca and Rick-
ettsia raoultii distributed across Europe. A total of 794 D. marginatus,D. reticulatus and
Ixodes ricinus adult ticks were collected from the vegetation, removed from horses, sheep,
goats and dogs in Slovakia. The DNA of Rickettsia sp. was found in 229 ticks by PCR
amplifying parts of gltA,ompA and sca4 genes. Next analyses of Rickettsia-positive
samples by PCR–RFLP and/or sequencing showed D. reticulatus ticks were more infected
with R. raoultii and D. marginatus were more infected with R. slovaca. The prevalence of
R. raoultii was 8.1–8.6% and 22.3–27% in D. marginatus and D. reticulatus, respectively.
The prevalence of R. slovaca was 20.6–24.3% in D. marginatus and 1.7–3.4% in D.
reticulatus. Intracellular growth of R. raoultii isolate from D. marginatus tick was eval-
uated by rOmpA-based quantitative SybrGreen PCR assay. The highest point of multi-
plication was recorded on the 7th and 8th day postinfection in Vero and L929 cells,
respectively. R. raoultii was transmitted during feeding of R. raoultii-positive ticks to
guinea pigs and subsequently rickettsial infection was recorded in all organs, the highest
infection was in spleen, liver and heart. Our study describes the detection and isolation of
tick-borne pathogens R. raoultii and R. slovaca, show that they are spread in Slovakia and
highlight their risk for humans.
Keywords Rickettsia slovaca Rickettsia raoultii Dermacentor marginatus 
Dermacentor reticulatus Slovakia
E. S
ˇpitalska
´(&)K. S
ˇtefanidesova
´E. Kocianova
´
Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic
e-mail: eva.spitalska@savba.sk
V. Boldis
ˇ
HPL (Ltd) Medical Laboratories, Department of Parasitology, Bratislava, Slovak Republic
123
Exp Appl Acarol
DOI 10.1007/s10493-012-9539-8

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 sil-
varum,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 (Ma
´rquez 2008), Portugal (Vitorino et al. 2007), Netherlands (Nijhof et al.
2007), Slovakia (Boldis
ˇet 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 (Ma
´rquez 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 Krupinska
´planina, respectively (R
ˇeha
´c
ˇek 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 veg-
etation in Bratislava, Horny
´Bar, Martinsky
´les, Moravsky
´sv. Ja
´n localities in western
Slovakia and Vel
ˇky
´Lom, Kalonda, Pı
´la, Sa
´sa, Pincina
´, Budina
´localities in central Slo-
vakia during 2004–2010 years. They were classified to species, sex and maintained alive at
?4°C prior to the examination.
Exp Appl Acarol
123

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 pres-
ence 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 CO
2
incubator for 120 min at 33°C. Finally, the cells were
incubated in CO
2
incubator at 33°C for 7–10 days. For study of intracellular growth, R.
raoultii was cultivated in Vero and L929 cells (inoculum *10
5
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 col-
lected 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 (NH
4
OH) for 15 min at 100°C in sealed PCR tubes. Subsequently,
NH
4
OH 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 pro-
tocol. DNA extractions were stored at -20°C and later used as templates for the PCR
amplification.
Exp Appl Acarol
123

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
S
ˇpitalska
´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 Boldis
ˇand S
ˇpitalska
´(2010). Negative
controls contained all PCR reaction components, as template DNA were DNAs of unin-
fected 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).
The DNA of Rickettsia sp. was found in 229 ticks, 69 D. reticulatus and 88 D. mar-
ginatus ticks from the vegetation and 44 D. reticulatus and 28 D. marginatus ticks col-
lected 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 2shows the prevalence of each rickettsia in each species of
Dermacentor ticks.
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
Exp Appl Acarol
123

(Boldis
ˇand S
ˇpitalska
´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 (cir-
cles). 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.
Figure 3shows 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.
Table 1 Infection rate with
Rickettsia sp. in Dermacentor
ticks
No. of Rickettsia sp. positive
ticks/no. of tested ticks
Ticks from D. marginatus D. reticulatus
Vegetation 88/272 (32.35%) 69/268 (25.75%)
21/102 (20.59%) M 31/108 (28.70%) M
67/170 (39.41%) F 38/160 (23.75%) F
Horses 1/4 26/102 (25.49%)
0/1 M 8/48 (16.67%) M
1/3 F 18/54 (33.33%) F
Sheep 25/89 (31.46%)
7/41 (17.07%) M
18/48 (43.9%) F
Goats 2/3 M 1/4
0/1 M
1/3 F
Dogs 17/47 (36.17%)
11/25 (44.0%) M
6/22 (27.27%) F
Total from animals 26/93 (27.96%) 44/153 (28.76%)
9/45 (20.0%) M 19/74 (25.68%) M
19/51 (37.25%) F 25/79 (31.65%) F
Table 2 The prevalence of Rickettsia raoultii and R. slovaca in Dermacentor marginatus and D. reticulatus
ticks in Slovakia
In ticks from vegetation (%) In ticks from animals (%)
R. raoultii R. slovaca R. raoultii R. slovaca
D. marginatus 8.09 24.26 8.58 20.59
D. reticulatus 22.31 3.43 27.02 1.69
Exp Appl Acarol
123

Fig. 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
Fig. 2 The growth kinetics of Rickettsia raoultii in Vero and L929 determined by the quantitative PCR
assay. Eukaryotic cells approaching confluence were infected with *10
5
copy number of bacteria per flask.
Values are the average ±SE of three replicates
Exp Appl Acarol
123

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 (Ma
´rquez 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
Fig. 3 Infection with Rickettsia raoultii in blood and organs of guinea pigs. Each point corresponds to the
mean of three distinct experiments
Exp Appl Acarol
123

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.
Acknowledgments The study was financially supported by the grants VEGA Nos. 2/0065/09, 2/0142/10
and 2/0031/11 from the Scientific Grant Agency of Ministry of Education of Slovak Republic. We thank to
Marianna Pjechova
´and Veronika Bala
´z
ˇova
´for collection of samples and excellent technical assistance.
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