HARD TICKS (ACARI: IXODIDAE)-VECTORS
FOR LYME DISEASE SPIROCHETES IN ROMANIA
ELENA CLAUDIA COIPAN
In this study 1868 questing Ixodes ricinus ticks (nymphs and adults), collected in six
different sites from three counties (Giurgiu, Sibiu and Tulcea) in Romania, were
examined by polymerase chain reaction (PCR), followed by reverse line blot (RLB) for
detection of Borrelia burgdorferi sensu lato presence. The bacteria were found in
18.04 % of the investigated ticks. The prevalence of infection was higher in Ixodes
ricinus nymphs (19.1 %) than in adults (15.37 %). Three B. burgdorferi sensu lato
genospecies were detected: B. afzelii (61.13 %), B. garinii (31.16 %) and B. valaisiana
(7.72 %). No mixed infections were detected in the investigated ticks. The highest
infection prevalence in I. ricinus nymphs was detected at Cristian (Sibiu County) –
22.03 %, while in adults it was at Comana (Giurgiu County) – 19.77 %. This
preliminary study provides evidence that Lyme disease spirochetes are present in
different regions of Romania and at a relatively high prevalence in Ixodes ricinus
vector, thus posing a risk of infection to human subjects that undergo work or leisure
activities in the areas infested by ticks.
Key words: Borrelia burgdorferi sensu lato, Ixodes ricinus, ticks, Lyme disease, PCR-
Romanian fauna of hard ticks (Acari: Ixodidae) comprises 25 species (Feider,
1965; Bădescu, 1967; Horak et al., 2002; Kolonin, 2009). Among them, Ixodes
ricinus L. is the most common and widespread species in Romania (Feider, 1965).
Many of these tick species are important vectors for different pathogens of
both medical and veterinary importance. Ticks and the diseases they transmit have
a zoogeographical range restricted by host movement and, to some extent, climatic
factors. The increased mobility of pets has resulted in rapid extension of the
zoogeographical ranges for many species (Shaw et al., 2001). The zoogeographical
range is also increasing because tick species are finding niches in different climatic
conditions (Lindgren & Gustafson, 2001). Anthropogenic activities such as habitat
fragmentation and export of wild animals for different purposes could determine
the extension of ticks’ zoogeographical ranges and influence wildlife pathogen
outbreaks (Dobson & Foufopoulos, 2001). Thus, the dispersal of ticks could have
significant impact in terms of economic (livestock and wildlife losses) and health
aspects (modifications in the epidemiology of tick-borne diseases).
ROM. J. BIOL. – ZOOL., VOLUME 55, No 2, P. 177–184, BUCHAREST, 2010
Elena Claudia Coipan 2
Ixodes ricinus L. is the main vector of Borrelia burgdorferi sensu lato, the
etiological agents of Lyme borreliosis, in Europe (Eisen & Lane, 2002). Seven
Borrelia genospecies have been found associated with this tick species and the risk
of human infection with Borrelia depends on outdoor activity, on the density of
tick populations, and on the infection of the ticks with Borrelia. Therefore, data
describing the prevalence of Borrelia in ticks can be used to assess the risk of
Lyme borreliosis for public health (Rauter & Hartung, 2005).
The aim of this study was to investigate the presence of B. burgdorferi s.l. in
Ixodes ricinus ticks from different regions of the country.
MATERIAL AND METHODS
Study Areas and Tick Collection. The study was carried out in one site
from Giurgiu County – Comana (N 44° 09076′, E 26° 06589′), three sites from
Sibiu County: Brateiu (N 46° 10100′, E 024° 23825’), Cristian (N 45° 46783’, E
023° 57782’), and Sadu (N 45° 38696′, E 024° 08772’), and two sites from Tulcea
County: Ciucurova (N 44° 53352′, E 28° 29927′) and Măcin (N 45° 09599′, E 28°
18450′) (Fig. 1). Ixodes ricinus ticks were collected in wooded areas (mixed
forests) until 14.00 hours of the day. Host-seeking adult, nymphal, and larval ticks
were collected monthly from March to October in two consecutive years – 2007
and 2008 – by flagging the low vegetation with a 1 m2 white flag over a distance of
100 m. Collected ticks were kept in plastic tubes until they were identified at
species level by examination under stereomicroscope, following the keys described
by Feider (1965), Baker (1999) and Estrada-Peña et al. (2004). After that, they
were passed into cryotubes containing RNAlater® (Ambion®, Applied
Biosystems) solution and stored at –80 °C to the moment of examination for
Borrelia burgdorferi s.l. infection.
Detection of Borrelia burgdorferi sensu lato in ticks. A number of 1341
nymphs and 527 adults of Ixodes ricinus were examined for B. burgdorferi s.l.
genospecies by polymerase chain reaction (PCR), followed by the reverse line blot
DNA extraction was achieved by placing the tick in 100 µl 0.7 M
ammonium hydroxide and boiling it for 15 minutes at 100°C (Guy & Stanek 1991;
Rijpkema et al., 1995).
PCR. Primers used to amplify the variable spacer region between two
repeated genes encoding for ribosomal 23S and 5S were B5S-Bor and 23S-Bor
primers (Schouls et al., 1999; Alekseev et al., 2001) and DNA amplification was
performed in a 2720 Thermal Cycler (Applied Biosystems), following a touchdown
PCR program previously described by Morán-Cadenas et al. (2007).
Agarose gel electrophoresis. PCR products were stained with ethidium
bromide and visualized on a UV transilluminator (UVP), after agarose gel
3 Hard ticks-vectors for Lyme disease spirochetes in Romania 179
electrophoresis (2% agarose, 1xTAE, pH 8.0), at 70V, 45 min, and stored at 4°C
until RLB analysis. All samples that produced bands between 400 and 500 bp were
subjected to DNA-DNA hybridization by the reverse line blot method.
Reverse Line Blotting. Isolates of B. burgdorferi sensu stricto (B31), B.
garinii (NE11), and B. afzelii (NE632) (obtained by the amiability of Prof. Dr. Lise
Gern, Institut of Zoology, University of Neuchâtel, Switzerland) were used as
positive controls. Two negative controls were also used: one for DNA extraction
and the other for PCR. The RLB technique was performed as described by Schouls
et al. (1999) and Alekseev et al. (2001) using 5 different oligonucleotide probes
(75 pmol and 100 pmol): B. burgdorferi s.l. (SL), B. burgdorferi sensu stricto (SS),
B. afzelii (AF), B. garinii (GA), B. valaisiana (VS) (Rijpkema et al., 1995; Schouls
et al., 1999; Alekseev et al., 2001). All probes were blotted in lines on an EDAC
(N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, Sigma-Aldrich)
activated Biodyne C membrane (Pall Europe Ltd.) using a Miniblotter 45
(Immunetic). Hybridization was visualized by incubating the membrane with
enhanced chemiluminescence detection liquid (GE Healthcare Europe) and by
exposing the membrane to Amersham Hyperfilm ECL (GE Healthcare Europe).
Statistical analysis. Chi square and Fisher’s exact test were calculated by
using PASW® Statistics 17.0 (IBM SPSS®).
From the 1868 Ixodes ricinus ticks (1341 nymphs and 527 adults) analyzed
for B. burgdorferi infection the target region was successfully amplified for ~
18.04% of them. The number of infected nymphs was higher (19.1 %) than that of
infected adults (15.37%) but not significantly (χ2 test, p = 0.0697). Also, the
number of infected ticks was slightly higher in 2007 (18.47 %) than in 2008
Regarding the monthly distribution of B. burgdorferi s.l. infection in Ixodes
ricinus ticks a higher infection prevalence was noticed in May (27.21 % in 2007
and 27.33 % in 2008) and September (16.46 %) – October (19.45 %), in 2007 and
2008 respectively. Statistical analysis on the annual means of infection prevalence
both within (between nymphs and adults) and between the sites, did not reveal any
significant differences (χ2 and Fisher tests with p > 0.05).
The highest infection prevalence in 2007 was observed at Cristian, for both
stages (20 % in adults and 24 % in nymphs), while the lowest was observed at
Măcin (5.26 % in adults and 12.9 % in nymphs). In 2008 similar infection
prevalence patterns in nymphs were observed, meaning that the highest infection
prevalence was again recorded at Cristian (21.03%) and the lowest at Măcin
(8.33%). That was not the case for adults, where the highest infection prevalence
was noticed at Comana (20 %) and the lowest at Sadu (5.88 %). The highest mean
Elena Claudia Coipan 4
(between the two years) infection prevalence in nymphs was detected at Cristian
(Sibiu County) – 22.03 %, while in nymphs it was at Comana (Giurgiu County) –
The PCR products hybridized to the membrane on the rows corresponding to
the probes for Borrelia garinii, Borrelia afzelii and Borrelia valaisiana. So, three
Borrelia species were identified by PCR-RLB: B. afzelii was the most frequently
detected species (61.13 %), with B. garinii (31.16 %) less common and
B. valaisiana (7.72 %) quite rarely encountered and even missing from some of the
sites (Sadu) (Fig. 1). B. burgdorferi sensu stricto was not detected in any of the
sites. The ratio of spirochetes genospecies infecting I. ricinus ticks varied greatly
between the different instars and between the sites (χ2 p > 0.05). Thus, B. afzelii
was detected in 90.91 % of the infected ticks at Sadu and in 40.3 % of them at
Ciucurova; B. garinii varied from 47.76 % at Ciucurova to 9.09 % at Sadu, while
for B. valaisiana, the maximum infection prevalence was found at Ciucurova
(11.94 %) (Fig. 1).
The interannual and interstadial variation in the ratio of the genospecies
infecting ticks were very low, statistically insignifcant (interannual – χ2 = 0.23,
p = 0.89, interstadial – χ2 = 0.63, p = 0.73).
This study confirmed the presence of Borrelia burgdorferi s.l. in ≈18% of the
questing Ixodes ricinus ticks collected in different regions of Romania. This
prevalence is comparable to the overall prevalence average in Europe which ranges
from 0 to 47% (Gray et al., 1998; Jouda et al., 2004; Rauter & Hartung, 2005) and
is close to the the ones reported in countries like Slovenia (19% – Strle et al., 1995)
or Austria (23.2% – Hubalek et al., 2003) where human Lyme borreliosis incidence
reaches its utmost values for the Old World – 155 and 130, respectively, new cases
for every 100,000 inhabitants (Lindgren & Jaenson, 2006; EUCALB, 2010).
Both nymphal and adult Ixodes ricinus were proven to be infected with the
bacteria. The lower infection prevalence in adults than in nymphs indicates that a
large percentage of nymphs feed on host species non-competent to transmit B.
burgdorferi s.l., most likely deer (Jaenson & Tälleklint, 1992; Randolph & Craine,
1995; Gray et al., 1999). Furthermore, the results confirm the assertion that
B. afzelii is the dominant B. burgdorferi s.l. genospecies followed by B. garinii
(Rauter & Hartung, 2005). The fact that B. burgdorferi s.s., which across Europe is
almost equally prevalent with B. valaisiana, was not encountered may find an
explanation in that its frequency in Europe seems to decrease from west to east
(Saint Girons et al., 1998).
5 Hard ticks-vectors for Lyme disease spirochetes in Romania 181
Fig. 1 . Ratio of B. burgdorferi s.l. genospecies detected in I. ricinus in different sites.
Elena Claudia Coipan 6
The wide variations between sites in the proportion of infecting Borrelia
genospecies could be due to some differences in available vertebrate host
assemblages and particularly those with reservoir competence for the spirochetes.
It is well known the fact that B. burgdorferi s.l. genospecies show vertebrate host
specificity, independently of the extrinsic ecological factors (Kurtenbach et al.,
1998; Hu et al., 2001). Accordingly, the proportion of different B. burgdorferi s.l.
genospecies in a site is a function of the number and density of the reservoir-
competent species in that site. Thus, the high prevalence of B. afzelii (61.13 % of
the positive Ixodes ricinus ticks) comparing to B. garinii (31.16 %) may be related
to the enzootic cycles that are predominant in the investigated area. For example, in
Sibiu County there is an abundant rodent fauna, with Apodemus flavicollis and
A. agrarius as the dominant species (Benedek, 2008), and a high percentage of
Ixodes ricinus ticks infected with B. afzelii is consistent with the known host
specificity of ticks and spirochetes (Matuschka et al., 1991).
Most probably the interannual variation of genospecies ratio does not have
any biological significance and is the sheer result of hazard in collecting the ticks
that fed on different host species. The similar patterns of infecting genospecies in
both investigated tick stages may be proof of ticks’ equal chances of feeding on
reservoir-competent hosts during their life-cycle.
Ixodes ricinus (Acari: Ixodidae) is the most common and widespread tick
species in Romania. This is the first study to assess Borrelia burgdorferi s.l.
infection prevalence in Ixodes ricinus ticks from different regions of Romania (six
sites from three counties) using polymerase chain reaction (PCR), followed by the
reverse line blot (RLB) assay. In 1868 Ixodes ricinus ticks (1341 nymphs and 527
adults) analyzed for B. burgdorferi s.l. infection three genospecies were detected:
B. afzelii, B. garinii and B. valaisiana, at an overall infection prevalence of
18.04%, with maximum values detected at Cristian (Sibiu County) and Comana
(Giurgiu County) for Ixodes ricinus nymphs and adults respectively.
Interannual variation in the infection prevalence was assessed, indicating
comparable infection rates in the two consecutive years of study. Infection
prevalence and genospecies ratio varied greatly between sites, even from the same
region, indicating that different host and vegetation assemblages lead to different
combinations of circulating spirochetes.
The detection of only three Borrelia genospecies probably does not reflect
the reality from the field, and a larger number of investigated ticks, covering more
regions and habitats would be necessary for an ensemble view of B. burgdorferi s.l.
genospecies circulating in Romania.
This preliminary study provides evidence that Lyme disease spirochetes are
present in different regions of Romania, and at a relatively high prevalence in
7 Hard ticks-vectors for Lyme disease spirochetes in Romania 183
Ixodes ricinus vectors, thus posing a risk of infection to humans that undergo work
or leisure activities in the areas infested by ticks.
Acknowledgements. This publication was partially funded by EU grant GOCE-2003-010284 EDEN
and is catalogued by the EDEN Steering Committee as EDEN 0246 (www.eden-fp6project.net). The
contents of this publication are the sole responsibility of the author and do not necessarily reflect the
views of the European Commission.
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Received September 10, 2010
“Cantacuzino” National Institute of Research-Development
for Microbiology and Immunology
103 Splaiul Independenţei Street, 050096, Bucharest,