DOI: 10.2478/s11686-008-0028-5 © 2008 W. Stefañski Institute of Parasitology, PAS Acta Parasitologica, 2008, 53(2), 186–192; ISSN 1230-2821 Identification and characterization of 31 isolates of burgdorferi (Spirochaetales, ) obtained from various hosts and vectors using PCR-RFLP and SDS-PAGE analysis

Alena Žákovská1*, Eva Janouškovcová1, Kater4ina Pejchalová1, Jir4í Halouzka2 and Miloš Dendis3 1Department of Animal Physiology and Immunology, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotláøská 2, 611 37 Brno; 2Institute of Vertebrate Biology AV CR Brno, Department of Medical Zoology, Klášterní 2, 691 42 Valtice; 3Genex CZ, Company Ltd., Køenová 50, 602 00 Brno;

Abstract sensu lato, the etiologic agent of Lyme borreliosis, circulates between ticks and vertebrate hosts. Two main genospecies typically occur in the Czech Republic Borrelia garinii and Borrelia afzelii, transmitted generally by Ixodes rici- nus (L., 1758) ticks. The aim of our study was to identify isolates focusing on Borrelia burgdorferi acquired from different sources: vectors (ticks), potential vectors (mosquitoes, small mites) and hosts (wild rodents). In the years 1996–2001 a total of 2398 ticks, 72 mites (from wild rodents), 2700 mosquito adults, 1798 mosquito larvae and organ parts (kidney and spleen) of 216 wild rodents were collected from seven localities in the Czech Republic. A total of 31 spirochaete strains were isolated: 13 strains from ticks, 1 strain from mite (Haemogamasus sp.), 15 strains from rodents, 1 strain from mosquito adults and 1 strain from mosquito larva. For the genospecies identification of these isolates PCR, PCR-RFLP was used and their char- acterization was also performed by SDS-PAGE. By nested PCR method all except one isolated strains were detected as Borrelia burgdorferi s.l. Following PCR-RFLP molecular analysis results, tick isolates were identified as B. garinii and B. afzelii, the strain isolated from the mite was identified as B. afzelii. This is the first isolated strain of B.b.s.l. from a different mite of infra- order Parasitiformes than tick. All of rodent isolates were identified as B. afzelii; mosquito adult isolate was identified as B. afzelii. Larval isolate from mosquito is spirochaete, but does not belong to Borrelia burgdorferi sensu lato group.

Keywords Borrelia, isolate, PCR-RFLP, SDS-PAGE

Introduction Seven genomic groups or genospecies of B. burgdorferi have been found in Europe until now: B. burgdorferi sensu Since the discovery of B. burgdorferi (Burgdorfer, 1982), stricto, B. garinii, B. afzelii (Baranton et al. 1992, Canica et al. many strains of this spirochaete have been isolated from ticks, 1993, Peter and Bretz 1992), B. valaisiana (Wang et al. 1997), reservoir hosts and humans. B. burgdorferi sensu lato is path- B. lusitaniae (Le Fleche et al. 1997), B. bissettii (Maraspin et ogenic spirochaete bacterium, at least 15 genospecies belong- al. 2002) and B. spielmani (Richter et al. 2004). Geographic ing to B. burgdorferi sensu lato complex (Postic et al. 2007) distribution of these genospecies includes 26 European coun- are known, but only five genospecies – B. burgdorferi sensu tries (Hubálek and Halouzka 1997). The most frequent Euro- stricto, B. garinii, B. afzelii, B. bissettii and B. spielmanii are pean genomic species, B. garinii and B. afzelii, occurs over the considered to be the etiological agent for human Lyme borre- whole continent inclusive of the Czech Republic. The main liosis (Richter et al. 2004). Lyme borreliosis is the most com- vector of this agent in Europe and also in the Czech Republic mon tick-borne disease in Eurasia (O’Connell et al. 1998) and is (L., 1758), which is the most common tick North America (Steere 1994). in Europe (Kmety et al. 1986; Hubálek et al. 1990, 1991).

*Corresponding author: [email protected] Borrelia burgdorferi strains from various hosts and vectors 187

Œl¹ski

The purpose of this study was to determine and to identi- of the centre of Brno city in about 350 m a.s.l., in the hill slope. fy isolated from various hosts and vectors and This area is used as a garden. potential transmitters of B. burgdorferi using PCR, PCR- Overwintering mosquitoes were collected with the aspira- RFLP and SDS-PAGE analysis. We also focused our attention tor in their typical shelters (several village buildings) from No- on the distribution and the occurrence of various genospecies vember to March. Summer mosquitoes were collected using in endemic areas in the Czech Republic. a clap-net. The samples were kept alive under cool (5°C) and humid (r.h. 90%) conditions until processing. Mosquito larvae were collected from a barrel with the rainwater or from a pond Materials and methods during summer season by a strainer and a pipette. Adults and larvae were dissected, the all abdomen content Ticks were triturated individually (or in a pool of five). The content A total of 2398 ticks, all belonging to the species Ixodes rici- of all individually triturated abdomens or five pooled ones nus, were collected during the years 1996–2000 in three areas were further examined for the presence of spirochaetes by of two different regions. The urban park Brno-Pisárky (land DFM and samples with more than 100 spirochaetes were inoc- register Brno) is a part of Brno conurbation 2 km far from the ulated into BSK-H complete medium for cultivation. city centre situated at 197–210 m above sea level and forms the bottom of the Pisárky valley. The hill side of this valley is Rodents and mites covered with mixed forest. The locality Bílý potok is one part Trapping was practiced in the year 1999 in the locality of Ba- of Natural Park Bílý potok (l.r. Velká Bíteš) situated in the z4antula (Studénka). The samples were trapped four times at northeast part of Bíteš Highlands about 250 m above sea level the turn of March/April, May/June, July/August and Septem- (a.s.l.) and is characterized by deciduous forest. The third ber/October. The snapping spring traps (200 pieces) were set locality is situated around a pond, near the town Vysoké Mýto in a network at a distance 10 m from each other on the area (l.r. Vysoké Mýto) 300 m a.s.l. in the western part of the dis- about 200 × 300 m and were baited with a piece of wick fried trict Ústí nad Orlicí. Vegetation here is typically deciduous. with flour and small amount of root vegetables. Trapping was Ixodes ricinus ticks were collected by the flagging method carried out on 2 consecutive days; the traps were checked after (dragging white flannel flags of the standardized size over low each 12 hours and set again. A total of 216 rodents of 5 species vegetation). All collected ticks were placed into tubes and were trapped: 3 species of family Microtidae: Clethrionomys stored alive at 5°C until examination for the presence of spi- glareolus (Schreber, 1780) (25 ex.), Microtus arvalis (Pallas, rochaetes. Midgut tissues from each tick were removed by dis- 1778) (96 ex.), M. agrestis (L., 1761) (6 ex.) and 2 species of section, suspended in a drop of saline and examined by dark- family Muridae: Apodemus flavicollis (Melchior, 1834) (64 field microscopy (DFM) at × 400 magnifications. Spirochaetes ex.), Apodemus agrarius (Pallas, 1771) (25 ex.). in positive samples were counted, and the highly infected tis- Trapped mammals were transferred to the laboratory for sues containing about more than 100 spirochaetes were trans- next investigation early in the morning. Necropsy was carried ferred into BSK-H medium (Sigma-Aldrich) supplemented out during 3 hours from morning to noon. The rodents were with 6% of rabbit serum and antibiotics (e.g. riphampicin 50 identified and sexed. The sections of kidney and spleen (3 mm µg/ml, phosphomycin 100 µg/ml) for isolation attempts. diameter) were transferred under aseptic conditions into BSK- H complete medium. Mosquitoes Entrapped rodents were examined using a pincette and a A total of 2700 adults (females) and 1798 mosquito larvae of total of 72 parasitic Acari mites (Mesostigmata) were collect- the genus Culiseta (Felt, 1904), Aedes (Meigen, 1818), Ano- ed from the coat. Every mite was dissected using a microscope pheles (Meigen, 1818), Theobaldia (Neveu-Lemaire, 1902) with a preparation needle. During dissecting process it was were collected from the 6 different localities in the Czech very important not to damage the parasite too much, because Republic (Bílý potok, Vysoké Mýto, Baz4antula, Bøeclav, Brno- preserving of some morphological signs is necessary for the Z4ebìtín, Brno-Obøany) during 1997–2001 and examined for right taxonomic determination. After removing gut the mites the presence of spirochaetes. were kept in 70% ethanol until later determination, but that The locality Baz4antula (l.r. Studénka) is situated near the was carried out only in samples which were positive for the town of Studénka about 2 km northeast in about 400 m a.s.l. presence of spirochaete by DFM method. For identification, The floodplain forest is the type of a damp elm-oak forest – each sample was suspended in a drop of a medium containing association Ficario-Ulmetum alnetosum. The locality Bøeclav chloralhydrate placed on a microscopic slide and covered with (l.r. Bøeclav) is situated in the town Bøeclav in South Moravia a sterile glass cover. The solutions with a content of this mat- about in 150 m a.s.l. The forest locality is known for annual ter are used for the determination of mites usually (Novák floods with mosquito population outbreaks during this period. 1969), in our case the Hoyer’s medium was used. The locality Brno-Z4ebìtín (l.r. Brno) is situated westwards Isolated strains were cultivated at least for 5 weeks until 10 km of the centre of Brno city in about 280 m a.s.l. in a the density of bacterial suspension reached 106–107 cells/ml recreational area and close to inhabited areas. The locality and further identified and characterized by using PCR-RFLP Brno-Obøany (l.r. Brno) is situated in the northeast part 4 km and SDS-PAGE techniques. 188 Zdzis³aw Alena Z4ákovská et al.

Stanis³a PCR-RFLP gellin gene was used. As an isolation-positive control a sus- Identification of isolated strains was based on genotyping by pension of Borrelia burgdorferi cells (6 × 102 cells/ml) was PCR-RFLP analysis. DNA of samples was isolated from ho- isolated with every group of samples and the same amount of mogenates using a DNA isolation kit (Malamite, Czech Re- isolate was added to the PCR. To prevent contamination by public). Volume of 5 µl this preparation was used for ampli- amplicons from previous reaction, dUTP and uracyl DNA gly- fication. PCR assay based on the specific 874 bp flagellin cosylase were used in each reaction. sequence amplification for detection of B. burgdorferi sensu lato was performed (Picken et al. 1996). Restriction analysis SDS-gradient PAGE of amplified PCR products was performed by AluI (Arthro- For protein characterisation of isolated strains we used a SDS- bacter luteus) endonuclease digestion (New England Bio- gradient PAGE test according to Laemmli (1970) of whole Labs). RFLP patterns obtained after AluI treatment of ampli- cells of Borrelia with slight modifications (Janouškovcová et fied specific B.b.s.l. flagellin sequence for B. burgdorferi al. 2004). Each of the protein samples was evaluated using sensu stricto 100, 198 and 327 bp fragments, for B. garinii videodensitometer and Molecular Analyst software (Bio-Rad) 102, 177 and 261 bp fragments and for B. afzelii 237 and 261 with Broad Range comparative standard (Bio-Rad) and com- bp fragments are typical. These restricted DNA fragments pared to standard samples – B. afzelii – Br-75 isolated from were analysed by agarose gel electrophoresis through 2% flea Ctenophthalmus agyrtes (female), locality Valtice, 1995 agarose gel, visualised by ethidium bromide staining, detect- (Hubálek et al. 1998); B. garinii – Br-97 isolated from Ixodes ed using UV transillumination (312 nm), and analysed by ricinus ticks (female), locality Valtice, 1995; B. burgdorferi ULTRA LUM gel detection and analysis system (Ultra-lum, sensu stricto – B31, isolated from Ixodes scapularis, locality Inc.). For identification of Borrelia strains only fragments Shelter Island (New York, USA), 1984, donor Dr. J.F. Ander- longer than 100 bp were taken into account. son. For the computer sequence analysis flagellin gene se- quences of B. burgdorferi sensu stricto (accession number X56334), B. afzelii (accession number X75202) and B. garinii Results (accession number AB017479) were gathered from GenBank database (NCBI). A search for primer target sequence homol- A total of 2398 ticks (from localities of Brno-Pisárky, Bílý ogy by the BLAST algorithm was made. The multiple align- potok and Vysoké Mýto), 2700 mosquito adults and 1798 ments by the CLUSTALW program were done (Thomson et al. mosquito larvae (localities Bílý potok, Vysoké Mýto, Baz4antu- 1994). The RFLP patterns were predicted using the WEB- la, Bøeclav, Brno-Z4ebìtín, Brno-Obøany), 216 rodents and 72 CUTTER 2.0 program. mites (from the locality Baz4antula), Czech Republic, were col- Sterile water was used as isolation-negative control. As a lected during 1996–2001 and examined by DFM for the pres- PCR negative control sterile TE buffer was added into the ence of spirochaetes. Detailed prevalence of spirochaetes has reaction. To avoid possible false-negative results due to the already been published (Janouškovcová et al. 2004). During presence of PCR inhibitors in the samples, the internal com- isolation attempts, 31 cultivable strains were obtained: 13 petitive standard was used. As a positive PCR control the from ticks, 1 spirochaete isolate from mite, 1 isolated strain amount of 102 copies of construct with a borrelia fla- from mosquito adult and 1 isolate from mosquito larva (Table

Table I. Strains of Borrelia burgdorferi sensu lato (B.b.s.l.) isolated from arthropods from the Czech Republic

Sample Source Locality PCR results RFLP results Gradient PAGE results BRZ 1 Ixodes ricinus Pisárky (Brno) B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2 Ixodes ricinus Pisárky (Brno) B.b.s.l. Borrelia garinii Borrelia garinii BRZ 3 Ixodes ricinus Bílý potok B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 4 Ixodes ricinus Pisárky (Brno) B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 7 Ixodes ricinus Pisárky (Brno) B.b.s.l. Borrelia garinii Borrelia garinii BRZ 9 Ixodes ricinus Pisárky (Brno) B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 10 Haemogamasus sp. Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 11 Ixodes ricinus V. Mýto B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 12 Ixodes ricinus V. Mýto B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 13 Culex (Culex) pipiens Obøany negative not done negative BRZ 14 Culex p. molestus V. Mýto B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 15 Ixodes ricinus Pisárky (Brno) B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 16 Ixodes ricinus Pisárky (Brno) B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 17 Ixodes ricinus Pisárky (Brno) B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 18 Ixodes ricinus Pisárky (Brno) B.b.s.l. Borrelia garinii Borrelia garinii BRZ 19 Ixodes ricinus Pisárky (Brno) B.b.s.l. Borrelia afzelii Borrelia afzelii Borrelia burgdorferi strains from various hosts and vectors 189

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Table II. Strains of Borrelia burgdorferi sensu lato (B.b.s.l.) isolated from rodents from the Czech Republic

Sample Source Locality PCR results RFLP results Gradient PAGE results BRZ 2242 Apodemus agrarius Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2243 Apodemus agrarius Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2244 Microtus arvalis Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2246 Apodemus agrarius Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2248 Apodemus agrarius Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2250 Apodemus agrarius Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2281 Apodemus flavicollis Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2292 Apodemus agrarius Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2304 Microtus agrestis Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2324 Microtus arvalis Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2456 Apodemus flavicollis Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2466 Clethrionomys glareolus Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2495 Microtus arvalis Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2505 Microtus arvalis Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii BRZ 2507 Microtus arvalis Studénka B.b.s.l. Borrelia afzelii Borrelia afzelii

I) and 15 isolates from rodents (Table II). According to PCR, strains B. burgdorferi sensu stricto, B. garinii and B. afzelii PCR-RFLP molecular analysis results (Fig. 1), the tick iso- using gradient SDS-PAGE (Fig. 2). Each of the protein sam- lates were identified as B. burgdorferi s.l. and followly by ples was evaluated using videodensitometer and Molecular PCR-RFLP as B. garinii and B. afzelii, all of rodent isolates Analyst software (Bio-Rad) with Broad Range comparative were identified as B. afzelii, mosquito adult isolate was iden- standard (Bio-Rad) and compared to standard stem of B. af- tified as B. afzelii and spirochaete strain isolated from mos- zelii, B. garinii and B.b.s.s. Typical protein areas for B. burg- quito larva do not belong to Borrelia burgdorferi group. Iden- dorferi s.l. were determined: e.g. OspA (31–34 kDa), OspB tification of isolates resulted in 3 strains of B. garinii (BRZ 2, (35–36 kDa), OspC (24–25 kDa), OspD (28–29 kDa), flagellin BRZ 7 and BRZ 18) and 28 strains of B. afzelii (Table I). The (39–41 kDa), but there are other protein bands visible in results protein characterization of yielded isolates was performed appear to be e.g. OspE, OspF or proteins 35, 56, 60 kDa, but also by comparison of the protein composition to referential this is difficult to determine without specific methods.

Fig. 1. PCR-RFLP genetic characterization visualised by agarose gel electrophoresis and stained by ethidium bromide. Resulting RFLP pat- terns obtained after AluI endonuclease treatment of amplified products: Lane 4 – BRZ 15 (B. afzelii), lane 5 – BRZ 16 (B. afzelii), lane 6 – BRZ 17 (B. afzelii), lane 7 – BRZ 18 (B. garinii), lane 8 – BRZ 19 (B. afzelii). Lanes 1 and 10 – 100 bp molecular weight standard (Malamité, ÈR), lane 2 – presents the isolate from Culex (Culex) pipiens (negative), lane 3 – the isolate from Culex pipiens molestus (B. afzelii), lane 9 – one isolate from Microtus arvalis (B. afzelii) 190 Alena Z4ákovská et al.

Fig. 2. SDS-gradient PAGE protein profiles. Lane 1 presents the isolate from Culex (Culex) pipiens (negative), lane 2 is the isolate from Culex pipiens molestus (B. afzelii), lane 3 – BRZ 15 (B. afzelii), lane 4 – BRZ 16 (B. afzelii), lane 5 – BRZ 17 (B. afzelii), lane 6 – BRZ 18 (B. garinii), lane 7 – BRZ 19 (B. afzelii), lane 8 – molecular weight standard Broad-Range (Bio-Rad), lanes 9 and 10 – B. afzelii (Br-75), lane 11 – B. garinii (Br-97) and lane 12 – B. burgdorferi sensu stricto (B 31). The typical proteins for B. afzelii are described in lane 5 – OspA (31–34 kDa), OspB (35–36 kDa), OspC (24–25 kDa), OspD (28–29 kDa), flagellin (41 kDa) and others. You can see differences between B. garinii and B. afzelii

Discussion Culex (L., 1758), which corresponds also with the findings of other authors (Hubálek et al. 1998, Halouzka et al. 1999, We were interested in the occurrence and infestation of I. rici- Sanogo et al. 2000). Positive spirochaete samples were detect- nus ticks by LB spirochaetes in the urban park surrounding the ed in the North-Eastern Poland as well (Stañczak 1995). First second largest city of the Czech Republic – Brno (Pisárky). isolations of Borrelia afzelii from mosquitoe species Aedes But ticks were not the only group investigated for borreliae. (Aedimorphus) vexans, Culex (Culex) pipiens molestus were We focused also on rodents (as main vector hosts) and further already announced (Sanogo et al. 2000, Halouzka et al. 1998). on other blood sucking groups of arthropods. The association These authors identified spirochaetes using PCR-RFLP between borreliae and insects belonging to the order Diptera method. In this work one of these positive samples was suc- or other parasitic mites than ticks was at the periphery of sci- cessfully cultivated under laboratory conditions and then entists’ attention for a long time and has just recently gener- detected also using PCR-RFLP method as B. afzelii. ated more interest. In ticks – a domination of B. afzelii con- While some reports about borreliae detection in mosquito firms the high occurrence of this genospecies, but there is also appeared, the presence of borrelian DNA in other groups of a moderate domination of B. garinii, e.g. in Prague region arthropods than ticks, especially small mites, is very rarely (Bašta et al. 1999). We did not find B. burgdorferi sensu stric- described (Netušil et al. 2005). It is necessary to say, that pure to, although this genospecies was reported in the Czech presence of borrelia in various arthropods does not mean that Republic (Štìpánová-Tresová et al. 2000a, Danielová et al. they surely transfer (Matuschka and Richter 2004), as well as in some localities in Southern Europe, e.g. 2002). In this study we focused our attention on the detection Northern Italy (Cinco et al. 1996), Austria (Stünzner et al. and possible isolation of borreliae in other ectoparasites of 1998), Eastern Slovakia (Štìpánová-Tresová et al. 2000b) and infraorder Parasitiformes than ticks, e.g. mites of Haemoga- other European countries (Hubálek and Halouzka 1997, Mae- masus sp. (Berlese, 1889). These arthropods represent very tzel et al. 2005, Michalik et al. 2005). However, our results frequent ectoparasites of rodents being found all over the prove the importance of investigation of the local distribution world. In addition, several other have been already of B. burgdorferi genospecies in endemic areas and reveal the confirmed in tissues of these parasites (Zuevskiy 1976, Lysý possibility that the human urban population in Brno may come et al. 1979, Koèíanová and Koz4uch 1988, Koèíanová 1989). in contact with the causative agent of Lyme borreliosis. The samples were collected from the hair of small rodents Generally the findings of spirochaetes in haematophagous which participate as hosts in a circulation of infectious Diptera appeared in some works from the beginning of the spirochaetes in the nature. Within the framework of the study 20th century (Sinton and Shute P.G. 1939, Masseguin and carried out in Eastern Slovakia the mite species Haemoga- Palinacci 1954) and also later (Burgdorfer et al. 1982). In our masus nidi (Michael, 1892) was the second most abundant study spirochaetes were detected using DFM method in species found in nests of observed species of rodents or on species Aedes, Ochlerotatus (Lynch Arribalzaga, 1891) and single rodents (Stanko and Miklišová 2000). Altogether six Borrelia burgdorferi strains from various hosts and vectors 191

mites were positive for the presence of B. burgdorferi s.l., – seeking Ixodes ricinus ticks in selected South Bohemian from which one was identified as Eugamasus sp. (Berlese, locations (Czech Republic). Central European Journal of 1892) (family Parasitidae) and two mites were determined as Public Health, 12, 151–156. Gray J.S., Schonberg A., Postic D., Belfaiza J., Saint-Girons I. 1996. the representatives of genus Haemogamasus (family Haemo- First isolation and characterisation of Borrelia garinii, agent gamasidae), in one of them the spirochaetal strain, success- of Lyme borreliosis, from Irish ticks. Irish Journal of Medical fully isolated, was further identified as B. afzelii. Science, 165, 24–26. Rodents generally belong to reservoir animals playing the Halouzka J., Postic D., Hubálek Z. 1998. Isolation of the spirochaete Borrelia afzelii from the mosquito Aedes vexans in the Czech principal role in maintaining and spreading the etiological Republic. Medical and Veterinary Entomology, 12, 103–105. agent of Lyme disease in natural conditions. Nevertheless, DOI: 10.1046/j.1365-2915.1998.00086.x. sparse information is available about vertebrate reservoirs of Halouzka J., Wilske B., Stunzner D., Sanogo Y.O., Hubálek Z. 1999. B. burgdorferi s.l. especially in the Czech Republic. Accord- Isolation of Borrelia afzelii from overwintering Culex pipiens ing to our investigation, species of rodents such as Clethrio- biotype molestus mosquitoes. Infection, 27, 275–277. Hanincová K., Taragelová V., Koèí J., Schafer S.M., Hails R., Ul- nomys glareolus, Microtus agrestis, M. arvalis, Apodemus ag- lmann A.J., Piesman J., Labuda M., Kurtenbach K. 2003. rarius and A. flavicollis were positive for the presence of liv- Association of Borrelia garinii and B. valaisiana with song- ing spirochaetes. This result proved that small rodents (and birds in Slovakia. 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(Accepted January 17, 2008)