Previous Article | Next Article 
Applied and Environmental Microbiology, October 2001, p. 4926-4929, Vol. 67, No. 10
The Wellcome Trust Centre for the
Epidemiology of Infectious Disease, Department of
Zoology,1 and Department of Clinical
Neurology,7 University of Oxford, and
NERC Centre for Ecology and Hydrology,2
Oxford, United Kingdom; ULBL/CMDT, Institute of Hygiene and
Tropical Medicine, Universidade Nova de Lisboa,3
and CBA/Science Faculty, Lisbon
University,4 Lisbon, Portugal; Institute
of Zoology, Slovak Academy of Sciences, Bratislava,
Slovakia5; and Ministry of Welfare
of the Republic of Latvia, Riga, Latvia6
Received 27 December 2000/Accepted 8 July 2001
The genetic diversity of Borrelia burgdorferi sensu
lato was assessed in individual adult Ixodes ricinus
ticks from Europe by direct PCR amplification of spirochetal DNA
followed by genospecies-specific hybridization. Analysis of mixed
infections in the ticks showed that B.
garinii and B. valaisiana
segregate from B. afzelii. This and
previous findings suggest that host complement interacts with spirochetes in the tick, thereby playing an important role in the
ecology of Lyme borreliosis.
Borrelia burgdorferi
sensu lato is a bacterial species complex comprising 10 named
genospecies and several genomic groups. All known species and genotypes
of B. burgdorferi sensu lato are tick transmitted
and are maintained in nature by complex zoonotic transmission cycles,
involving more than 50 avian and mammalian wildlife species as
reservoir hosts for the spirochetes in Europe (3, 14).
Six genospecies, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and
B. burgdorferi sensu stricto, are known to be
prevalent in Europe (2, 4, 7, 28, 29). All these
genospecies may cocirculate in local tick populations, suggesting that
the local diversity of B. burgdorferi sensu lato can be as high as the regional or continental diversity (5, 7,
24, 26, 27, 29). Individual ticks may be infected with multiple
genospecies of B. burgdorferi sensu lato;
however, mixed infections seem to be much rarer than single infections (4, 7, 8, 11, 13, 16, 26, 27). Although information on the
pattern of mixed infections in individual ticks may reveal important
principles of the biology and ecology of B. burgdorferi sensu lato, only a few studies provide
information on this aspect (4, 5, 7, 10, 11, 13, 16, 26).
Adult questing Ixodes ricinus ticks were collected in
sylvatic habitats by blanket dragging (14) in the springs
of 1996 and 1999. The habitats were located around Riga (Latvia),
Bratislava (Slovakia), Bonn (Germany), and Lisbon (Portugal, two
distinct sites) and in the New Forest (United Kingdom). B. burgdorferi sensu lato infection status was determined by
PCR and the reverse line blot assay as described previously (2,
16, 23, 26).
The null hypothesis that the different genotypes were distributed
independently of each other in individual ticks was statistically tested. The frequency data from each country were considered
separately. The probability of a tick containing any one
genotype was calculated from the data as follows: A total of 1,483 questing adult ticks were analyzed, and 461 (31.0%)
were found to be infected with B. burgdorferi
sensu lato (Table 1). The most prevalent
genospecies was B. afzelii (39.3%), followed by
B. garinii (21.2%), B. valaisiana (12.8%), B. lusitaniae (5.8%), and B. burgdorferi sensu stricto
(1.5%). The three genospecies B. afzelii,
B. garinii, and B. valaisiana were present in all local tick populations
collected in Slovakia (Bratislava), Latvia (Riga), Germany (Bonn), and
Portugal (the Mafra site). These three genospecies accounted for 94.2%
of all infections (Table 1; Fig. 1). Of
the 410 typeable infections, 372 (90.7%) were single infections. Of
the ticks infected with B. garinii, 24% were
infected simultaneously with B. valaisiana, and
34% of the ticks harboring B. valaisiana were
concurrently infected with B. garinii (Fig. 1).
In contrast, only 1% of the B. afzelii
infections occurred together with B. garinii, and
no mixed infection of B. afzelii and
B. valaisiana was observed. B. afzelii was absent from the United Kingdom site (16) and the Grândola site in Portugal
(2). Therefore, these two sites were omitted in the
statistical analysis. Of the remaining four sets of frequency data
presented in the table, three have sufficient data to statistically
test the hypothesis that the different genotypes are distributed
independently of each other in individual ticks, namely, the data from
Slovakia, Latvia, and Germany. Fewer mixed infections of B. garinii-B. afzelii composition were detected in
individual ticks than expected in each of the three cases, and in two
out of three this difference was significant. Significantly more mixed
infections of B. garinii-B. valaisiana composition than expected under neutralist assumptions were observed in
the data set from Slovakia (Slovakia,
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4926-4929.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Distinct Combinations of Borrelia burgdorferi
Sensu Lato Genospecies Found in Individual Questing Ticks
from Europe
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
(all ticks
containing that genotype, whether as mixed or single infection)/(all
ticks tested). The probabilities of mixed and single infections were
calculated assuming independent distributions in order to generate a
set of expected frequencies.
22 = 96.1, P = 1.05 × 10
20; Latvia,
12 = 6.41, P = 0.0113; and Germany, 12 = 1.38, P = 0.240).
TABLE 1.
Infection of questing adult I. ricinus with
B. burgdorferi sensu latoa
View larger version (32K):
[in a new window]
FIG. 1.
Reverse line blot assay on PCR products of the 5S-23S
intergenic spacer of B. burgdorferi sensu
lato (B. bu. s.l.) derived from questing
adult I. ricinus ticks from Slovakia.
Representative samples are shown. Briefly, PCR products of
B. burgdorferi sensu lato were hybridized
to membrane-bound DNA probes specific for B.
burgdorferi sensu lato, B.
burgdorferi sensu stricto (s.s.), B.
garinii (B. ga.),
B. afzelii (B.
af.), and B.
valaisiana (B. va.). No
DNA probe was available for B.
bissettii. The PCR products per tick sample are ordered
from the left to the right.
The present study yielded two major findings: (i) the majority of infected ticks harbored one genospecies only, and (ii) B. garinii and B. valaisiana constituted the majority of multiple infections, whereas the combination of B. garinii and B. afzelii occurred significantly less frequently than expected.
It has previously been shown that the PCR targeting the 5S-23S intergenic spacer of B. burgdorferi sensu lato does not preferentially amplify Borrelia genotypes (2, 16, 26). In addition, genotyping of PCR products by the reverse line blot assay readily detects mixed infections at different ratios of DNA (16, 26), indicating that the present findings are likely to reflect the true genospecies diversity of B. burgdorferi sensu lato in the ticks analyzed. No DNA probe specific for B. bissettii was available. It is, therefore, possible that some of the untypeable samples fall into this genospecies.
It is now accepted that B. afzelii, B. garinii, and B. valaisiana are the most abundant genospecies in central Europe, often prevailing sympatrically in local I. ricinus populations (4, 7, 11, 16, 26, 27-29). The data from the present study are in line with these previous studies. As questing adult I. ricinus ticks have a history of two blood meals on diverse hosts (25), Borrelia infections may accumulate with the number of infectious blood meals (10, 12, 16). Cumulative acquisition of spirochetes upon consecutive infectious blood meals or superinfection of ticks already infected transovarially could explain the presence of dual infections in individual ticks. In addition, cotransmission of multiple strains from an individual host that contains a mixed infection may result in polyclonal infection of a single tick. Assuming equal transmission coefficients for the Borrelia genospecies throughout the transmission cycle (25), the frequency distribution of genotypes in individual ticks should match the frequency distribution at the population level. The pattern that emerges from the present study, however, indicates that this is not the case; B. garinii and B. valaisiana on the one hand and B. azfelii on the other hand seem to colonize individual ticks in a mutually exclusive way. This pattern cannot be attributed to geographic separation, since these three genospecies were found to circulate sympatrically in all of the central European sites analyzed. For this reason, it is likely that physiological or immunological factors shape the diversity of B. burgdorferi sensu lato in individual ticks.
In this paper an underlying mechanism is proposed for this observed pattern. It has recently been reported that the genospecies of B. burgdorferi sensu lato differ in their resistance to the alternative pathway of the hosts' complement system, depending on the genetic background of the spirochete and the source of serum (15). Rodent complement readily lyses particular genotypes of cultured B. garinii (type strains 20047, prevalent in western Europe) and B. valaisiana but not B. afzelii, whereas avian complement lyses the spirochetes in the reverse pattern. Interestingly, B. burgdorferi sensu stricto displays partial resistance to both avian and mammalian complement (15). The pattern of complement-mediated lysis matches the pattern of transmission competence of the major rodent and avian hosts for B. burgdorferi sensu lato (8-10, 16, 21), suggesting that resistance and/or sensitivity to complement is a key factor in Lyme disease ecology (15). The fact that B. burgdorferi sensu stricto exhibits only partial resistance to complement from mammalian and avian hosts from Europe may in part explain its low prevalence in the Old World.
Host complement has been demonstrated to be active in the midgut of feeding I. ricinus ticks (22). It is likely, therefore, that host complement selectively acts on spirochetes in the midgut of the tick with an effect similar to that observed in vitro, thereby effectively reducing the probability of avian- and rodent-associated Borrelia to coinfect individual ticks.
Although apparently rare, mixed infections of B. garinii and B. afzelii do occasionally occur in individual ticks (7, 8, 11, 13). This may be related to the presence of complement-deficient vertebrate hosts (1) or to the circulation of particular genotypes of B. burgdorferi sensu lato. In fact, diverse genotypes of B. garinii from localities in eastern Europe and Asia as well as OspA serotype 4 strains have been linked with rodent-tick transmission cycles (6, 18-20). In addition, it is possible that spirochetes evade complement-mediated selection in systemically infected ticks (17).
Altogether, the data corroborate earlier observations that the genotypes of B. burgdorferi sensu lato are not only differentially acquired from a host but are also differentially transmitted to the next developmental stage of the tick. In quantitative terms, host-to-tick and tick-to-tick transmission coefficients (25) seem to vary significantly with the genotype of Borrelia and source of blood meal. The findings of this study support the conclusion that B. burgdorferi sensu lato is maintained in nature through distinct transmission cycles, mainly involving small mammalian and avian hosts.
| |
ACKNOWLEDGMENTS |
|---|
We thank Brian Spratt, Patricia A. Nuttall, and Mike Harris for support.
This study was funded by the British Council, The Wellcome Trust (grants 050854/Z/97/Z and 054292/Z/98/Z), the Natural Environment Research Council (grant GT 04/98/TS/2290) (United Kingdom), and the German Academic Exchange Service (to S.E.) (Germany).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: The Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, Oxford, OX1 3PS, United Kingdom. Phone: 0044 (0) 1865-281547. E-mail: klaus.kurtenbach{at}ceid.ox.ac.uk.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. | Colten, H. R., and F. S. Rosen. 1992. Complement deficiencies. Annu. Rev. Immunol. 10:809-834[CrossRef][Medline]. |
| 2. |
De Michelis, S.,
H.-S. Sewell,
M. Collares-Pereira,
M. Santos-Reis,
L. M. Schouls,
V. Benes,
E. C. Holmes, and K. Kurtenbach.
2000.
Genetic diversity of Borrelia burgdorferi sensu lato in ticks from mainland Portugal.
J. Clin. Microbiol.
38:2128-2133 |
| 3. | Gern, L., A. Estrada-Pena, F. Frandsen, J. S. Gray, T. G. T. Jaenson, F. Jongejan, O. Kahl, E. Korenberg, R. Mehl, and P. A. Nuttall. 1998. European reservoir hosts of Borrelia burgdorferi sensu lato. Zentbl. Bakteriol. 287:196-204. |
| 4. | Gern, L., C. M. Hu, E. Kocianova, V. Vyrostekova, and J. Rehacek. 1999. Genetic diversity of Borrelia burgdorferi sensu lato isolates obtained from Ixodes ricinus ticks collected in Slovakia. Eur. J. Epidemiol. 15:665-669[CrossRef][Medline]. |
| 5. | Guttman, D. S., P. W. Wang, I. N. Wang, E. M. Bosler, B. J. Luft, and D. E. Dykhuizen. 1996. Multiple infections of Ixodes scapularis ticks by Borrelia burgdorferi as revealed by single-strand conformation polymorphism analysis. J. Clin. Microbiol. 34:652-656[Abstract]. |
| 6. |
Hu, C. M.,
B. Wilske,
V. Fingerle,
Y. Lobet, and L. Gern.
2001.
Transmission of Borrelia garinii OspA serotype 4 to BALB/c mice by Ixodes ricinus ticks collected in the field.
J. Clin. Microbiol.
39:1169-1171 |
| 7. | Hubalek, Z., and J. Halouzka. 1997. Distribution of Borrelia burgdorferi sensu lato genomic groups in Europe, a review. Eur. J. Epidemiol. 13:951-957[CrossRef][Medline]. |
| 8. | Humair, P. F., O. Peter, R. Wallich, and L. Gern. 1995. Strain variation of Lyme disease spirochetes isolated from Ixodes ricinus ticks and rodents collected in two endemic areas in Switzerland. J. Med. Entomol. 32:433-438[Medline]. |
| 9. | Humair, P. F., D. Postic, R. Wallich, and L. Gern. 1998. An avian reservoir (Turdus merula) of the Lyme borreliosis spirochetes. Zentbl. Bakteriol. 287:521-538. |
| 10. | Humair, P. F., O. Rais, and L. Gern. 1999. Transmission of Borrelia afzelii from Apodemus mice and Clethrionomys voles to Ixodes ricinus ticks: differential transmission pattern and overwintering maintenance. Parasitology 118:33-42. |
| 11. |
Junttila, T.,
M. Peltomaa,
H. Soini,
M. Marjamäki, and M. K. Viljanen.
1999.
Prevalence of Borrelia burgdorferi in Ixodes ricinus ticks in urban recreational areas of Helsinki.
J. Clin. Microbiol.
37:1361-1365 |
| 12. | Kahl, O., C. Janetzki, J. S. Gray, J. Stein, and R. J. Bauch. 1992. Tick infection rates with Borrelia: Ixodes ricinus versus Haemaphysalis concinna and Dermacentor reticulatus in two locations in eastern Germany. Med. Vet. Entomol. 6:363-366[Medline]. |
| 13. | Kirstein, F., S. Rijpkema, M. Molkenboer, and J. S. Gray. 1997. The distribution and prevalence of B. burgdorferi genomospecies in Ixodes ricinus ticks in Ireland. Eur. J. Epidemiol. 13:67-72[CrossRef][Medline]. |
| 14. | Kurtenbach, K., H. Kampen, A. Dizij, S. Arndt, H. M. Seitz, U. E. Schaible, and M. M. Simon. 1995. Infestation of rodents with larval Ixodes ricinus L. (Acari: Ixodidae) is an important factor in the transmission cycle of Borrelia burgdorferi s.l. in German woodlands. J. Med. Entomol. 32:807-817[Medline]. |
| 15. |
Kurtenbach, K.,
H.-S. Sewell,
N. H. Ogden,
S. E. Randolph, and P. A. Nuttall.
1998.
Serum complement sensitivity as a key factor in Lyme disease ecology.
Infect. Immun.
66:1248-1251 |
| 16. |
Kurtenbach, K.,
M. Peacey,
S. G. T. Rijpkema,
A. N. Hoodless,
P. A. Nuttall, and S. E. Randolph.
1998.
Differential transmission of the genospecies of Borrelia burgdorferi sensu lato by game birds and small rodents in England.
Appl. Environ. Microbiol.
64:1169-1174 |
| 17. | Lebet, N., and L. Gern. 1994. Histological examination of Borrelia burgdorferi infections in unfed Ixodes ricinus nymphs. Exp. Appl. Acarol. 18:177-183. |
| 18. | Masuzawa, T., A. Iwaki, Y. Sato, K. Miyamoto, E. I. Korenberg, and Y. Yanagihara. 1997. Genetic diversity of Borrelia burgdorferi sensu lato isolated in near eastern Russia. Microbiol. Immunol. 41:595-600[Medline]. |
| 19. | Nakao, M., K. Miyamoto, and M. Fukunaga. 1994. Lyme disease spirochetes in Japan: enzootic transmission cycles in birds, rodents, and Ixodes persulcatus ticks. J. Infect. Dis. 170:878-882[Medline]. |
| 20. | Nakao, M., K. Miyamoto, and M. Fukunaga. 1994. Borrelia japonica in nature: genotypic identification of spirochetes isolated from Japanese small mammals. Microbiol. Immunol. 38:805-808[Medline]. |
| 21. | Olsen, B., T. G. T. Jaenson, and S. A. Bergström. 1995. Prevalence of Borrelia burgdorferi sensu lato-infected ticks on migrating birds. Appl. Environ. Microbiol. 61:3082-3087[Abstract]. |
| 22. | Papatheodorou, V., and M. Brossard. 1987. C-3 levels in the sera of rabbits infested and reinfested with Ixodes ricinus L. and in midguts of fed ticks. Exp. Appl. Acarol. 3:53-59[CrossRef][Medline]. |
| 23. |
Postic, D.,
M. V. Assous,
P. A. D. Grimont, and G. Baranton.
1994.
Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons.
Int. J. Syst. Bacteriol.
44:743-752 |
| 24. | Qui, W.-G., E. Bosler, J. R. Campbell, G. D. Ugine, I.-N. Wang, B. J. Luft, and D. E. Dykhuizen. 1997. A population genetic study of Borrelia burgdorferi sensu stricto from eastern Long Island, New York, suggested frequency-dependent selection, gene flow and host adaptation. Hereditas 127:203-216[CrossRef][Medline]. |
| 25. | Randolph, S. E., and N. G. A. Craine. 1995. A general framework for comparative quantitative studies on the transmission of tick-borne diseases using Lyme borreliosis in Europe as an example. J. Med. Entomol. 32:765-777[Medline]. |
| 26. | Rijpkema, S. G. T., M. J. C. H. Molkenboer, L. M. Schouls, F. Jongejan, and J. F. P. Schellekens. 1995. Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S ribosomal RNA genes. J. Clin. Microbiol. 33:3091-3095[Abstract]. |
| 27. |
Schouls, L. M.,
I. van de Pol,
S. G. T. Rijpkema, and C. S. Schot.
1999.
Detection and identification of Ehrlichia, Borrelia burgdorferi sensu lato, and Bartonella species in Dutch Ixodes ricinus ticks.
J. Clin. Microbiol.
37:2215-2222 |
| 28. | Stanek, G., and F. Strle. 1998. Lyme borreliosis and emerging tick-borne diseases in Europe. Wien. Klin. Wochenschr. 110:847-849[Medline]. |
| 29. | Strle, F. 1999. Lyme borreliosis in Slovenia. Zentbl. Bakteriol. 289:643-652. |
Applied and Environmental Microbiology, October 2001, p. 4926-4929, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4926-4929.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
(2008). MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi. Proc. Natl. Acad. Sci. USA
105: 8730-8735
-
Rauter, C., Hartung, T.
(2005). Prevalence of Borrelia burgdorferi Sensu Lato Genospecies in Ixodes ricinus Ticks in Europe: a Metaanalysis. Appl. Environ. Microbiol.
71: 7203-7216
[Abstract] [Full Text] -
Ranka, R., Bormane, A., Salmina, K., Baumanis, V.
(2004). Identification of Three Clinically Relevant Borrelia burgdorferi Sensu Lato Genospecies by PCR-Restriction Fragment Length Polymorphism Analysis of 16S-23S Ribosomal DNA Spacer Amplicons. J. Clin. Microbiol.
42: 1444-1449
[Abstract] [Full Text] -
Kampen, H., Rotzel, D. C., Kurtenbach, K., Maier, W. A., Seitz, H. M.
(2004). Substantial Rise in the Prevalence of Lyme Borreliosis Spirochetes in a Region of Western Germany over a 10-Year Period. Appl. Environ. Microbiol.
70: 1576-1582
[Abstract] [Full Text] -
Collares-Pereira, M., Couceiro, S., Franca, I., Kurtenbach, K., Schafer, S. M., Vitorino, L., Goncalves, L., Baptista, S., Vieira, M. L., Cunha, C.
(2004). First Isolation of Borrelia lusitaniae from a Human Patient. J. Clin. Microbiol.
42: 1316-1318
[Abstract] [Full Text] -
Hanincova, K., Taragelova, V., Koci, J., Schafer, S. M., Hails, R., Ullmann, A. J., Piesman, J., Labuda, M., Kurtenbach, K.
(2003). Association of Borrelia garinii and B. valaisiana with Songbirds in Slovakia. Appl. Environ. Microbiol.
69: 2825-2830
[Abstract] [Full Text] -
Derdakova, M., Beati, L., Pet'ko, B., Stanko, M., Fish, D.
(2003). Genetic Variability within Borrelia burgdorferi Sensu Lato Genospecies Established by PCR-Single-Strand Conformation Polymorphism Analysis of the rrfA-rrlB Intergenic Spacer in Ixodes ricinus Ticks from the Czech Republic. Appl. Environ. Microbiol.
69: 509-516
[Abstract] [Full Text] -
Kurtenbach, K., Schafer, S. M., Sewell, H.-S., Peacey, M., Hoodless, A., Nuttall, P. A., Randolph, S. E.
(2002). Differential Survival of Lyme Borreliosis Spirochetes in Ticks That Feed on Birds. Infect. Immun.
70: 5893-5895
[Abstract] [Full Text] -
Alitalo, A., Meri, T., Lankinen, H., Seppala, I., Lahdenne, P., Hefty, P. S., Akins, D., Meri, S.
(2002). Complement Inhibitor Factor H Binding to Lyme Disease Spirochetes Is Mediated by Inducible Expression of Multiple Plasmid-Encoded Outer Surface Protein E Paralogs. J. Immunol.
169: 3847-3853
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»