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Applied and Environmental Microbiology, May 2003, p. 2825-2830, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2825-2830.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Association of Borrelia garinii and B. valaisiana with Songbirds in Slovakia

Klára Hanincová,^1,2 Veronika Taragelová,² Juraj Koci,³ Stefanie M. Schäfer,^1,4,5 Rosie Hails,⁴ Amy J. Ullmann,⁶ Joseph Piesman,⁶ Milan Labuda,² and Klaus Kurtenbach^1*

Department of Infectious Disease Epidemiology, Imperial College of Science, Technology and Medicine, London W2 1PG,¹ NERC Centre of Ecology and Hydrology, Oxford OX 1 3SR,⁴ Department of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom,⁵ Institute of Zoology, Slovak Academy of Science, 81364 Bratislava,² Department of Biology, Microbiology and Immunology, University of Trnava, 918 43 Trnava, Slovakia,³ Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado 80522⁶

Received 13 September 2002/ Accepted 20 February 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lyme borreliosis is the most frequent arthropod-borne disease in humans living in moderate climates. Under the umbrella of the wider taxon Borrelia burgdorferi sensu lato, the bacteria constitute a group of 11 named genospecies, which were delineated based on DNA-DNA hybridization and sequence divergence of selected loci (2, 32, 34, 39, 40). In Europe, six genospecies are recorded as infecting Ixodes ricinus ticks (8, 15, 25), and the most prevalent genospecies are B. afzelii, B. garinii, and B. valaisiana (13, 25). In contrast to the case in northern America, B. burgdorferi sensu stricto and B. bissettii are relatively rare in Europe (8, 13, 15, 25). B. lusitaniae seems to be restricted to the western Mediterranean Basin, where its infection prevalence in ticks has been reported to be very high locally (3).

Since the delineation of genospecies in B. burgdorferi sensu lato, it has been discussed whether the species complex is differentiated ecologically (4, 17, 18, 26, 44). Our recent model of B. burgdorferi sensu lato transmission proposes that B. burgdorferi sensu lato comprises at least three major ecotypes which are associated with different sets of vertebrate host species (26, 28). Various testable predictions derived from this model have been validated in the field and in the laboratory. Field studies from Slovakia and Switzerland, for example, have shown that rodents preferentially transmit B. afzelii to ticks (13, 18). Furthermore, various studies indicate that some songbird species, seabirds, and pheasants are reservoir hosts of B. garinii and B. valaisiana (12, 17, 19, 27, 29). The transmission model also predicts that bird- and rodent-associated ecotypes of B. burgdorferi sensu lato segregate in individual questing ticks, a prediction that has also been validated (25).

The present work was carried out in a region of Europe where B. afzelii, B. garinii, and B. valaisiana are prevalent in questing ticks. In a previous study carried out in the same region, we have demonstrated that rodents are the reservoir hosts of B. afzelii but not of B. garinii and B. valaisiana (13). For this reason, hosts other than rodents must contribute to the prevalence of B. garinii and B. valaisiana in questing tick populations in this region. The major aim of this study, therefore, was to identify reservoir hosts of B. valaisiana and B. garinii, the agent of human neuroborreliosis, in Slovakia.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Field sites and collection of questing ticks.
The study was carried out in three localities within western Slovakia. Locality 1 (Podunajske Biskupice; 17°07'W, 48°08'N) is sylvatic and situated near the river Danube, 2 km southeast of Bratislava. The most prominent trees are Quercus robur (pedunculate oak), Populus alba (white poplar), and Ulmus minor (English elm). Humulus lupulus (hop), Clematis vitalba (traveler's joy), and Solidago gigantea (giant goldenrod) cover most parts of the ground. Locality 2 (Malacky; 17°08'W, 48°30'N) is a sylvatic park in the middle of the town Malacky, 35 km northwest or Bratislava. In this habitat Acer campestre (field maple), Carpinus betulus (hornbeam), Crateagus monogyna (hawthorn), P. alba, Prunus padus (European birdcherry), Tilia cordata (small-leaved lime), and Sambucus nigra (elder) predominate. Locality 3 (Zahorska Ves; 16°53'W, 48°22'N) is a mixed deciduous and coniferous woodland situated at alluvial sands close to the river Morava, 45 km northwest of Bratislava. The tree layer consists mainly of Q. robur, Pinus sylvestris (scotch pine), and Robinia pseudoacacia (false acacia). The undergrowth is dominated by S. nigra, Ligustrum vulgare (European privet), Calamagrostis epigeios (wood smallreed), C. monogyna, Dactylus glomerata (orchard grass), and R. pseudoacacia.

Within each locality, four sampling sites (each measuring 100 m²) were selected. Questing nymphal and adult ticks of I. ricinus were collected from the vegetation by blanket dragging at regular 2-week intervals in the spring and early summer of 1999. During 2000, field work was conducted only in locality 3 (Zahorska Ves) every 2 weeks from March until November. Field-derived ticks were preserved in 70% ethanol.

Capture of birds.
From March through June 2001, birds were mist-netted at a sylvatic locality near Jursky Sur (17°13'W, 48°15'N). The locality is situated 12 km northeast of Bratislava. The tree layer is formed by A. campestre, T. cordata, T. platyphylla (large-leaved lime), and Q. robur. The undergrowth is dense, consisting of S. nigra, Prunus spinosa (blackthorn), C. monogyna, Rubus caesius (dewberry), and L. vulgare. Netted birds were carefully removed, identified to species, weight, measured, identified as to sex, and examined for ticks. Feeding ticks were taken and preserved in 70% ethanol. Thereafter, all birds were released.

Detection of B. burgdorferi sensu lato in ticks.
I. ricinus ticks were examined for B. burgdorferi sensu lato genospecies by PCR, followed by the reverse line blot assay and, for a subsample, by nucleotide sequencing. Genomic DNA was extracted from ticks by alkaline hydrolysis (10). The nested PCR targeted the rrf (5S)-rrl (23S) intergenic spacer as described previously (13, 27, 45). To prevent DNA contamination, all stages of the PCR were carried out under strictly aseptic conditions with PCR hoods, and they were also separated temporally and spatially. In addition, negative controls at a ratio of 2:3 were included. As positive controls, serial dilutions of cultured B. andersonii, a genospecies confined to America, were used. The PCR products were hybridized to DNA probes specific for B. burgdorferi sensu lato, B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana as described previously (27, 45). In this study, a B. bissettii-specific probe was designed whose oligonucleotide sequence (5'-aminolink spacer-ATATAAAATTTAGAACTAAAATAAAATAC) is complementary to nucleotide positions 28 to 56 of the 5S-23S intergenic spacer region. Amplicons derived from cultured strains (B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, and B. bissettii) were used to confirm the specificities of the DNA probes.

DNA sequencing of PCR products.
Samples that could not be genotyped by the reverse line blot assay as well as randomly selected B. garinii samples were amplified at the ospA locus, and their nucleotide sequences were determined. Sequence analysis was carried out with the Lasergene99 system (DNASTAR, Inc.). Unique sequences have been deposited in GenBank.

Statistical analysis.
Differences in the infection prevalence of B. burgdorferi sensu lato genospecies between localities and developmental stages of questing ticks were analyzed by means of weighted logistical regression analysis (i.e., with binomial errors and weighted according to sample size).

Nucleotide sequence accession numbers.
The GenBank accession numbers for B. garinii alleles 4, 5, and 6 (see Tables 5 and 6) are AY226824, AY226822, and AY226823, respectively.

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TABLE 5. B. garinii ospA alleles in questing ticks

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TABLE 6. B. garinii ospA alleles in ticks engorged on songbirds

Top Abstract Introduction Materials and Methods Results Discussion References

 
Questing ticks.
A total of 1,294 questing I. ricinus ticks were tested for infection with B. burgdorferi sensu lato by PCR. Of these, 420 (33%) were infected with B. burgdorferi sensu lato (Table 1). Six genospecies of B. burgdorferi sensu lato were identified, with 34 ticks being infected with more than one genospecies. Considering the presence of multiple infections in individual ticks, the number of spirochete infections in the data set was calculated (n = 456). For example, an individual tick infected with three different genospecies was regarded as equivalent to three infections. The most frequent of all genospecies was B. afzelii (50.6%), followed by B. valasiana (26.5%) and B. garinii (19.1%). These three genospecies constituted the vast majority of spirochete infections (96.2%), whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae were very rare in the data set. The most frequent genospecies combination in individual ticks with mixed infections was B. garinii and B. valaisiana (27 of 34). In contrast, only few ticks harboring B. afzelii were concurrently infected with B. garinii (4 of 34) or B. valaisiana (3 of 34) (Table 1).

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TABLE 1. B. burgdorferi sensu lato infections in questing I. ricinus ticks

Significantly more adult ticks than nymphs were infected with B. burgdorferi sensu lato (² = 29.05; df = 1; P = 7.053 x 10^-8) (Table 1). B. afzelii contributed to 57% of the infections in nymphs and to 48% of those in adult ticks. Taken together, B. garinii and B. valaisiana contributed to 41% of the infections in nymphs and to 47% of those in adult ticks. The infection prevalences of B. burgdorferi sensu lato and of individual genospecies were compared between developmental tick stage and locality. First, B. burgdorferi sensu lato was most prevalent in locality 3 (² = 11.49; df = 1; P = 7.00 x 10^-4). Second, the major three genospecies in the data set, B. afzelii, B. garinii, and B. valaisiana, were found in all three localities. For B. afzelii, a higher infection prevalence was found in adult ticks than in nymphs (² = 5.812; df = 1; P = 0.0159), and it was most prevalent in locality 3 (² = 11.18; df = 1; P = 8.27 x 10^-4). The infection prevalence of B. garinii was also higher in adult ticks than in nymphs (² = 13.2; df = 1; P = 2.80 x 10^-4); however, no significant differences between localities were observed (² = 1.858; df = 2; P = 0.395). For B. valaisiana, the infection prevalence was higher in adult ticks than in nymphs (² = 6.42; df = 1; P = 0.0113), and it was more prevalent in localities 2 and 3 than in locality 1 (² = 9.82; df = 1; P = 0.00173).

In locality 3, questing ticks were also collected in March to November 2000, thus covering spring, summer, and autumn. Since both the structure of the host community and the activity of I. ricinus are known to vary with season, we were interested to see whether the infection prevalences of B. burgdorferi sensu lato and of the individual genospecies would also vary with season. However, no differences in the infection prevalence of any genospecies between the period of March to June and July to November 2000 was observed (² = 0.139; df = 1; P = 0.709). Furthermore, no difference in the overall infection prevalence of B. burgdorferi sensu lato between the years 1999 and 2000 was found (² = 0.2907; df = 1; P = 0.590). For B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae, no significant site-, stage-, or year-specific trend was determined, due to insufficient data.

Together, the data show that B. afzelii, B. garinii, and B. valaisiana are the most frequent genospecies in questing ticks in this region of Central Europe. About half of the infections in questing ticks were caused by B. garinii and B. valaisiana, and the other half were caused by B. afzelii.

Ticks feeding on birds.
Sixty-two birds of 17 bird species were captured in 2001. Of these, 14 species were either not infested with ticks or were infested with ticks that were found to be spirochete free (Table 2). Three species, the great tit (Parus major), the song thrush (Turdus philomelos), and the blackbird (T. merula), were infested with spirochete-infected ticks. Therefore, this paper focuses on these three songbird species.

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TABLE 2. List of birds not carrying ticks or B. burgdorferi sensu lato-infected ticks

Sixteen individual birds of these three songbird species were captured, and 69 engorged ticks were counted. The birds were more heavily infested with nymphs than with larvae. Of the 57 feeding ticks tested, 16 (28%) were infected with B. burgdorferi sensu lato. Genotyping revealed that feeding larvae and nymphs were infected with B. garinii (11 of 16) and B. valaisiana (6 of 16). Despite the high relative prevalence in questing nymphs, no B. afzelii infection in larvae and nymphs engorged on the birds was detected (Tables 3 and 4).

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TABLE 3. B. burgdorferi sensu lato infections in I. ricinus ticks feeding on songbirds

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TABLE 4. Infection prevalence of B. garinii, B. valaisiana, and B. afzelii in questing ticks and ticks feeding on birds

In order to fingerprint the B. garinii strains found in questing ticks and in ticks engorged on the birds, the ospA genes of randomly selected samples derived from both tick pools were amplified and sequenced. Among the 11 ospA sequences analyzed from questing ticks, five different alleles were identified. Three of these B. garinii alleles were identical to ospA sequences specific for previously characterized reference strains, whereas two alleles have so far not been described (Table 5). Among the eight ospA sequences obtained from ticks that had fed on songbirds, four alleles were found, three of which were also present in the questing tick pool. One allele was unique. None of the ospA alleles indicated infections with OspA serotype 4 or NT 29-like strains (Table 6).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study confirms that songbirds are reservoir hosts of B. garinii and B. valaisiana in Central Europe. The localities studied represent typical forest habitats of Central Europe. The overall infection prevalence of B. burgdorferi sensu lato of 33% in questing I. ricinus is consistent with previous findings (13, 25). Six genospecies were detected in questing ticks, the vast majority of which were identified as B. afzelii, B. valaisiana, and B. garinii. With some variation in prevalence, these three genospecies were present in all of the localities investigated in this study. They have also been found to be the most prevalent genospecies in other regions from Central Europe (8, 15, 18). The prevalences of B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae in questing ticks were very low, a finding that appears to be typical for Central Europe (15). In the western Mediterranean Basin, however, high prevalences of B. lusitaniae have been observed in some localities, suggesting that this genospecies has a narrow spectrum of reservoir hosts, restricting its geographical range (3).

In a recent study carried out in the same region as this study, we have shown that rodents, such as mice and voles, are reservoir hosts of B. afzelii but not of B. garinii or B. valaisiana (13). Thus, B. garinii and B. valaisiana must be maintained by tick hosts other than rodents. As emerging evidence from previous studies had pointed to ground-foraging birds as the prime candidates (12, 17, 27, 38), the present study focused on the analysis of songbirds. Three of the 17 captured songbird species were infested with spirochete-infected larvae and nymphs of I. ricinus. All infections were genotyped as B. garinii and B. valaisiana, showing that these songbirds are reservoir competent for these genospecies. Given the limited number of larvae engorged on the birds tested in this study, precise quantitative estimates of spirochete infectivity (35) of these birds cannot yet be provided. However, a combined infection prevalence of B. garinii and B. valaisiana of about 30% in larval ticks that had fed on these birds indicates that these three songbird species are as efficient in transmitting these two genospecies to ticks as European rodent populations are for B. afzelii (13, 18). Eight of the bird species captured in the present study were parasitized with spirochete-free I. ricinus ticks. The reasons for this finding are not known. Mathematical models of Lyme borreliosis will have to estimate the force of infection for each genospecies or even OspA serotype (42, 43).

B. garinii is highly polymorphic and comprises six OspA serotypes and different ribotypes (6, 14, 33). Recent evidence indicates that some Asian strains of B. garinii thrive in rodent hosts rather than in avian hosts (34, 36). Furthermore, recent studies suggest that OspA serotype 4 strains are associated with rodents rather than with birds (14, 16). Therefore, it was necessary to investigate whether the avian hosts analyzed in this study transmit the same B. garinii strains to feeding ticks as those prevalent in the questing tick pool. None of the sequenced B. garinii strains represented OspA serotype 4 strains or the NT 29-like strains. Three of the six ospA alleles found in the data set were shared by the questing tick pool and the tick pool engorged on the birds. Allele 3 is identical to the ospA sequence of reference strain ZQ1ox/Rio2 (6), and we have shown experimentally and epidemiologically that the pheasant is also reservoir competent for this strain of B. garinii (27, 29). Furthermore, this strain of B. garinii displays full resistance against avian complement but is readily lysed by complement of rodents and larger mammals, such as deer, cows, and sheep (30), a property also observed for the strains VSBP and VSBM (K. Kurtenbach, unpublished observation). Blackbirds have also been found to be reservoir hosts of B. garinii in Switzerland (7, 17). Therefore, it can be concluded that songbirds and other bird species, such as the pheasant (27), are important reservoir hosts of the vast majority of B. garinii and B. valaisiana strains in questing ticks in terrestrial habitats of Europe (19).

Of the questing nymphs collected in this study, 12% were infected with B. afzelii. Therefore, songbirds must encounter B. afzelii-infected ticks. The lack of B. afzelii amplification in ticks feeding on the birds indicates that songbirds are not transmission competent for this genospecies. A similar finding has recently been made experimentally with the pheasant as avian model of Lyme borreliosis (29). That study also showed that this genospecies was eliminated in preinfected ticks that had fed on the birds. Most interestingly, a reverse pattern of spirochete survival in engorged ticks was found for rodents captured in Slovakia (13). Not only did the rodents fail to infect tick larvae with B. garinii and B. valaisiana, but these two genospecies were also eliminated from nymphs feeding on the rodents.

As opposed to rodents (1), birds are highly mobile, and some songbird species are long-distance migrants (11, 19, 38, 41, 44). Migratory restlessness reactivates spirochete infection, which may enhance transmission (11). Therefore, it is likely that avian migration has a major impact on the population structure of B. burgdorferi sensu lato, as bird-associated spirochetes are likely to be spread over large distances within a time frame too short to allow for the accumulation of mutations. In fact, the ospA gene of the B. garinii strain, ZQ1ox, found in ticks engorged on the songbirds from Slovakia is identical to that of spirochetes detected in Spain (6), France (unpublished data), and the United Kingdom (6, 29).

The concept of host association of B. burgdorferi sensu lato is now firmly based on empirical evidence and can therefore be regarded as a scientific fact (26). The host association is determined by the interaction of spirochetes with host complement in an antibody-independent manner (23, 28, 30, 31, 37). B. burgdorferi sensu lato strains that are associated with a particular host species are always resistant to the alternative pathway of complement of this host. In contrast, sera from hosts that are reservoir incompetent for a B. burgdorferi sensu lato strain often lyse those strains (26, 28, 47). The patterns of differential survival of the B. burgdorferi sensu lato genospecies in ticks engorged on rodents and birds suggest that complement operates in the gut of the feeding tick (5, 13, 26, 29). Resistance to complement is now known to be mediated by the binding of host-derived complement control proteins, such as factor H (20-22). OspE and related proteins (Erps) which are expressed by spirochetes residing in the tick gut (9) have recently been identified as ligands of factor H (46). It has, therefore, been suggested that the particular erp gene repertoire of a B. burgdorferi sensu lato cell determines its ability to resist complement-mediated lysis, thereby defining its ecotype (26, 28, 46). The erp genes constitute a superfamily of plasmid-borne, multigene families. These represent phage genomes for which transduction as a mode of lateral gene transfer has been demonstrated (46). This mechanism of genetic recombination may in part explain adaptive radiation of B. burgdorferi sensu lato.

It is now apparent that the species complex of B. burgdorferi sensu lato comprises at least three ecotypes: (i) a rodent-associated ecotype, (ii) a bird-associated ecotype, and (iii) an ecotype that thrives in both rodent and avian hosts (24, 26, 28, 44). In Europe, bird-associated and rodent-associated B. burgdorferi sensu lato strains circulate in distinct transmission cycles involving mainly birds and rodents and one tick species, I. ricinus. In conclusion, complement-mediated selection of B. burgdorferi sensu lato appears to be a major factor in the evolution and ecology of Lyme borreliosis spirochetes.

 
We thank Ladislav Roller, Bratislava, Slovakia, for help with the field work.

This study was supported by The Wellcome Trust, London, United Kingdom (grants 050854/Z/97/Z and 054292/Z/98/Z); the Natural Environment Research Council, United Kingdom (studentship to S.M.S.); and the Slovak Academy of Sciences, Slovakia (grant PVT-51-004702).

 
* Corresponding author. Mailing address: Department of Infectious Disease Epidemiology, Imperial College of Science, Technology and Medicine, St. Mary's Campus, Norfolk Place, London W2 1PG, United Kingdom. Phone: 44-20-759-43787. Fax: 44-20-759-43693. E-mail: k.kurtenbach{at}ic.ac.uk.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, May 2003, p. 2825-2830, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2825-2830.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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