Ixodes ricinus ticks (Acari: Ixodidae): vectors for Lyme disease spirochetes in Romania.
ABSTRACT In this study 1,868 questing Ixodes ricinus ticks (nymphs and adults), collected in six 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% of the investigated ticks. The prevalence of infection did not differ significantly between nymphs (19.1%) and adults (15.4%). Three B. burgdorferi s.l. genospecies were detected: B. afzelii (61.1%), B. garinii (31.2%), and B. valaisiana (7.7%). No mixed infections were detected. The highest infection prevalence in nymphs was detected at Cristian (Sibiu County)--22.0%, whereas in adults it was at Comana (Giurgiu County)--19.8%. This preliminary study provides evidence that Lyme disease spirochetes are present in various regions of Romania, and at a relatively high prevalence in their vectors, thus posing a risk of infection to human subjects in the areas infested by ticks.
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ABSTRACT: The aims of our study were to determine (i) which tick species bite humans in Romania and (ii) the prevalence of Borrelia (B.) burgdorferi genospecies in these ticks. All ticks collected from patients who presented to the Clinic of Infectious Diseases Cluj Napoca in spring/summer 2010 were morphologically identified by an entomologist and tested for B. burgdorferi genospecies prevalence by a real-time PCR assay targeting the hbb gene and melting curve analysis. Out of 532 ticks, 518 were Ixodes ricinus, 10 Dermacentor marginatus, and 3 Haemaphysalis spp. ticks, and one unidentified tick due to destruction. Since evaluation of the hbb PCR revealed that it was not possible to differentiate between B. spielmanii/B. valaisiana and B. garinii/B. bavariensis, sequencing of an 800-bp fragment of the ospA gene was performed in these cases. Out of 389 investigated ticks, 43 were positive by hbb PCR for B. burgdorferi sensu lato. The positive samples were 42 Ixodes ricinus (11.1% B. burgdorferi sensu lato prevalence) and the one unidentified tick. Species identification revealed the presence of mainly B. afzelii, but also of B. garinii, B. burgdorferi sensu stricto, B. valaisiana, and B. lusitaniae. In 4 samples, differentiation between B. spielmanii/B. valaisiana was impossible. Our study shows that the most relevant human pathogenic B. burgdorferi genospecies - predominantly B. afzelii - are present in ticks collected from Romanian patients.Ticks and Tick-borne Diseases 06/2014; · 2.35 Impact Factor
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ABSTRACT: The diagnosis of Lyme borreliosis (LB) is very complex due to the diversity of clinical manifestations, very few being exclusive for Borrelia burgdorferi infection . In this context the diagnosis is based on clinical criteria (symptoms and clinical signs), exposure history and laboratory test results. Generally, the microbiological laboratory data are a major criterium for the clinical diagnosis of LB. The antibody detection methods are the main laboratory tests used to support a clinical diagnosis for most LB stages. In this study we investigated the detection specificity of antibodies against B. burgdorferi s.l by using three different Western blot (WB) tests to evaluate possible correlations between the clinical manifestations of LB and infecting B. burgdorferi genospecies in 26 Romanian patients with positive ELISA results for LB. Each of WB tests allows detection of specific antibodies against one of three B. burgdorferi s.l genospecies responsible for LB cases in Romanian patients, represented by B. afzelii, B. garinii and B. burgdorferi sensu stricto. The results of our study showed that both IgG and especially IgM antibodies cross-react mainly with antigens of B. garinii and B. afzelii, no specific correlation can be done between genospecies of B. burgdorferi s.l involved in infection and clinical manifestations of patients with clinical suspicion of LB. Therefore the WB tests used for the confirmation of ELISA results in the serum and cerebrospinal fluid (CSF) samples of patients suspected of LB must allow the simultaneous detection of the antibodies against antigens of all three B. burgdorferi s.l genospecies. It is also recommended the use of the latest generation of WB kits, which contain not only the antigens purified from three strains of B. burgdorferi genospecies, but also the antigens that are expressed during in vivo infection, obtained by recombinant DNA technology.Biointerface research in applied chemistry. 08/2013; 3(5):628-634.
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ABSTRACT: The paper reports the prevalence and geographical distribution of Borrelia burgdorferi sensu lato (s.l.) and its genospecies in 12,221 questing Ixodes ricinus ticks collected at 183 locations from all the 41 counties of Romania. The unfed ticks were examined for the presence of B. burgdorferi s.l. by PCR targeting the intergenic spacer 5S–23S. Reverse line blot hybridization (RLB) and restriction fragment length polymorphism (RFLP) analysis were performed for identification of B. burgdorferi genospecies. The overall prevalence of infection was 1.4%, with an average local prevalence between 0.75% and 18.8%. B. burgdorferi s.l. was found in ticks of 55 of the 183 localities. The overall prevalence B. burgdorferi s.l. in ticks in the infected localities was 3.8%. The total infection prevalence was higher in female ticks than in other developmental stages. Three Borrelia genospecies were detected. The most widely distributed genospecies was B. afzelii, followed by B. garinii and B. burgdorferi sensu stricto (s.s.). The study is the first countrywide study and the first report of B. burgdorferi s.s. in Romania. The distribution maps show that higher prevalences were recorded in hilly areas, but Lyme borreliosis spirochetes were also present in forested lowlands, albeit with a lower prevalence.Ticks and Tick-borne Diseases 07/2013; 4(5):403-408. · 2.35 Impact Factor
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.