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Journal of Clinical Microbiology, May 1999, p. 1361-1365, Vol. 37, No. 5
Ministry of Agriculture and Forestry,
Received 27 October 1998/Returned for modification 4 December
1998/Accepted 28 January 1999
Lyme borreliosis, an infection caused by the tick-borne spirochete
Borrelia burgdorferi, is a major health problem for
populations in areas of endemicity in the Northern Hemisphere. In the
present study we assessed the density of ticks and the prevalence of
B. burgdorferi sensu lato among ticks in popular urban
recreational areas of Helsinki, Finland. Altogether 1,688 Ixodes
ricinus ticks were collected from five areas located within 5 km
of the downtown section of Helsinki, and 726 of them (303 nymphs, 189 females, and 234 males) were randomly chosen for laboratory
analysis. The midguts of the ticks were divided into three pieces, one
for dark-field microscopy, one for cultivation in BSK-II medium,
and one for PCR analysis. Ticks were found in all the study
areas; their densities varied from 1 to 36 per 100 m along which a
cloth was dragged. The rate of tick infection with B. burgdorferi sensu lato varied from 19 to 55%, with the average
being 32%. Borellia afzelii was the most predominant
genospecies in all the areas, and no B. burgdorferi sensu
stricto isolates were detected. Only two ticks were concurrently infected with both B. afzelii and Borrelia
garinii. Dark-field microscopy gave more positive results
for B. burgdorferi than did cultivation or PCR analysis.
However, the agreement between all three methods was fairly good. We
conclude that Lyme borreliosis can be contracted even in urban
environments not populated with large mammals like deer or elk. The
disease should be taken into account in the
differential diagnosis of certain symptoms of patients from
these areas, and the use of measures to improve the awareness of the
general population and health care officials of the risk of contracting
the disease is warranted.
Lyme borreliosis is currently
the most important vector-borne disease in the developed countries of
the Northern Hemisphere. In Europe, the disease is most common in
central and eastern territories, where annual incidences of over 300 per 100,000 people have been reported (36). Since 1985, different clinical manifestations of Lyme borreliosis have been
reported in Finland, and the number of patients with late Lyme
borreliosis reported annually has varied between 300 and 450 (22). Epidemiological studies conducted in the southern
archipelago of Finland show an annual incidence of about 200 new cases
of Lyme borreliosis per 100,000 people (24).
The disease is caused by a spirochete, Borrelia burgdorferi.
B. burgdorferi sensu stricto is the dominating genospecies in North America, whereas several different genospecies of
B. burgdorferi sensu lato can be found in
Europe (2, 10, 34). In Finland, Borrelia
garinii and Borrelia afzelii appear to be the most
prevalent species (14).
Ticks of the Ixodes ricinus group are the most important
vectors of B. burgdorferi. These ticks have been found
in a wide variety of European habitats. In Finland they occur from the
south coast, especially in the southwestern archipelago, up to the
border of Lapland in the north (23). Ubiquitous small
rodents, Clethrionomys glareolus and Apodemus
flavicollis, are suspected of being the most important reservoir
animals for B. burgdorferi, while large or medium-sized
mammals are considered necessary for maintenance of the tick population
(12, 13). Most studies of the prevalence of infected ticks
have been conducted in rural or suburban settings known to harbor both
rodents and larger mammals. In Europe the reported mean rates of unfed
I. ricinus ticks infected with B. burgdorferi vary from 0 to 11% (mean, 1.9%) for larvae, from 2 to 43% (mean, 10.8%) for nymphs, and from 3 to 58% (mean,
17.4%) for adults (11). The risk of acquiring
borrelia infection in urban environments has generally been neglected,
and studies on the rates of infection of ticks in heavily populated
urban or suburban areas are limited in number (19, 20,
25-27). However, suitable habitats for ticks are known to exist
in cities (6, 9, 15-17, 33).
The main objectives of this study were to estimate the density of
I. ricinus ticks in popular recreational areas of Helsinki, Finland, and to estimate the prevalence of borreliae in these ticks. In
addition, the occurrence of different B. burgdorferi genospecies in the areas was studied, and three different methods of
detecting borreliae in ticks were evaluated.
Study areas.
Eighteen of the most popular
recreational areas in and around the city of Helsinki were screened in
a pilot study during the summer of 1995 by the same sampling method
used in the present study. Five areas representing the highest tick
densities (Table 1; Fig.
1) were chosen for study during the
summer of 1996. All the areas were located within a radius of 5 km from
the center of Helsinki, and the estimated number of annual visitors to
the areas is 1.4 million (5).
Estimation of tick density.
Ticks were collected during 20 visits to the areas between 17 May and 9 August 1996. They were caught
by dragging a 1-m2 cotton cloth through the vegetation. The
number of ticks attached to the cloth was counted every 5 m.
Dragging was performed along randomly chosen lines with a length of 100 to 200 m per each sampling day and area. The counts of larvae,
nymphs, females, and males were recorded, as was the length of the
drag. Tick density was expressed as the number of ticks caught
per a 100-m drag. Ticks were placed individually in sterile 1.5-ml
Eppendorf tubes containing a moist piece of paper and were stored at
4°C until they were prepared for further study.
Tick preparation.
Altogether, 1,688 I. ricinus
ticks were collected, and 726 of them (303 nymphs, 189 females, and 234 males) were randomly chosen for the laboratory analysis. No external
disinfectants were used during the preparation of the ticks. The ticks
were stored for a mean of 25 days (range, 7 to 54 days), and 154 of the
ticks died during storage. The midguts of the adults and nymphs were
removed under a stereomicroscope and were placed in a drop of BSK-II
medium with small sterile forceps, a disposable 28-gauge needle which
was used as a scalpel, and sterile insect needles. Each
midgut was divided into three equal parts. One portion was examined
immediately under a dark-field (DF) microscope, another portion was
inoculated in BSK-II medium, and the third was reserved for DNA
purification. Because the viability of ticks was suspected to affect
the results of all the methods, the vitality of each tick was recorded
for further statistical analyses.
DF microscopy.
One of the three parts of the tick midgut was
placed on a microscope glass in a drop of BSK-II medium and was covered
with a cover glass. The number of spirochetes in the midgut sample was
estimated by DF microscopy (Laborlux D; Leitz, Nürnberg, Germany)
by examining 100 fields at a magnification of ×400. Typical movement,
morphology, and size were used as the identification criteria for the borreliae.
Cultivation.
The samples were inoculated into tubes
containing BSK-II medium supplemented with rifampin (100 mg/ml) and
phosphomycin (50 mg/ml) and were incubated at 30°C for 8 weeks or
until growth was detected. The growth medium was examined by
DF microscopy every other week. If growth appeared, the cultures were
passaged into new tubes containing BSK-II medium without antibiotics.
DNA extraction.
DNA was extracted from the processed ticks
with the InstaGene DNA extraction matrix (Bio-Rad Laboratories,
Hercules, Calif.). A total of 200 µl of the matrix was added to the
sample, and the mixture was incubated at 56°C for 30 min. The
specimen was then mixed for 30 s, incubated at 100°C for 8 min,
mixed briefly, and centrifuged at 13,000 rpm for 3 min (Biofuge 13;
Heraeus Instruments GmbH, Haman, Germany). Five microliters of the
supernatant was used in the PCR analysis.
PCR amplification.
The nested PCR was carried out by the
method described by Schmidt et al. (29), which is based on
the flagellin gene. The 50-µl reaction mixture contained 50 mM KCl,
1.5 mM MgCl2, 10 mM Tris-HCl (pH 8.8), 0.1% Triton X-100,
each deoxynucleoside triphosphate (Pharmacia Biotech, Espoo, Finland)
at a concentration of 200 mM, 20 pmol of the outer or inner primers,
and 1 U of DynaZyme DNA polymerase (Finnzymes, Espoo, Finland). In the
first PCR, outer primers BBSCH31 and BBSCH42 were used. It consisted of
an initial denaturation at 95°C for 1 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C
for 30 s. The procedure was repeated 25 times, and the final
extension was done at 72°C for 10 min. Five microliters of the
amplicon from the first PCR was used as the template in the second PCR, for which primers FL59 and FL7 were used. The second PCR analysis was
carried out for 35 cycles by the same procedure used for the first PCR,
except that the annealing temperature was raised to 58°C. The final
277-bp PCR products were visualized by 1.5% agarose gel electrophoresis.
Identification of the positive cultures by PCR.
The
spirochetes grown from the ticks were identified to the species level
by the PCR method developed by Marconi and Garon (18) and
based on amplification of the 16S rRNA gene. DNA was extracted from the
cultures by the InstaGene procedure described above. The samples were
amplified with four sets of primers. The primer sets were specific for
B. burgdorferi sensu lato (LD primers), B. burgdorferi sensu stricto (BB primers), B. afzelii
(VS461 primers), and B. garinii (BG primers). The PCR
mixtures consisted of the components described above. For the LD
primers, 40 cycles of denaturation at 94°C for 1 min, annealing at
47°C for 30 s, and extension at 72°C for 1.5 min were carried
out. For the other primers, the PCR procedure was the same, except that
the annealing was performed at 42°C. The resulting PCR products were
visualized by agarose gel electrophoresis.
Confirmation of double infections by sequencing.
The samples
that gave ambiguous results in PCRs specific for B. afzelii and B. garinii were further analyzed by
PCR-based sequencing of the flagellin gene. A 277-bp product was
obtained by using primers FL7 (biotinylated) and FL59. The biotinylated PCR products were rendered single stranded with streptavidin-coated Dynabeads according to the instructions of the manufacturer (Dynabeads M-280 with streptavidin; Dynal AS, Oslo, Norway). Manual sequencing was
performed by Sanger's dideoxynucleotide chain termination method and with Sequenase 2.0 (United States Biochemical Corp., Cleveland, Ohio) as described previously (31). The sequences that were obtained were compared with the flagellin gene sequences of
the type strains B. afzelii Bo23 and B. garinii 387.
Statistical methods.
The student edition of Statistics,
version 4.0 (1992; Analytical Software, Torrance, Calif.) was used for
all the statistical analyses. A chi-square analysis was used to
test the association of borrelia prevalence and subspecies
distribution with the sampling area, developmental stage, and viability
of the ticks. After the preliminary association tests, log-linear
modeling was applied to control the interactions of sex, area, and
borrelia infection, as well as the possible confounding effect of
the viability of the ticks.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Prevalence of Borrelia burgdorferi in
Ixodes ricinus Ticks in Urban Recreational Areas of
Helsinki
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Annual number of visitors and tick densities of the
five recreational areas of Helsinki studied
View larger version (95K):
[in a new window]
FIG. 1.
The five areas in Helsinki studied, from north to south:
Meilahti, Seurasaari, Lehtisaari, Lauttasaari, and Pihlajasaari.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Tick densities in survey areas. Altogether, 1,688 I. ricinus ticks were collected from the study sites. The tick densities varied from 1 to 36 ticks/100 m (Table 1). Two of the areas (Seurasaari and Lehtisaari) had significantly higher densities than the others; Pihlajasaari had the lowest density. Nymphs and larvae were more abundant in Seurasaari and Lehtisaari, which partly explains the density differences.
Prevalence of ticks infected with B. burgdorferi.
Of the 1,688 ticks, 726 (303 nymphs, 189 females, and 234 males)
were randomly chosen for the laboratory analysis. The infection rate for the ticks varied from 19 to 55% (Table
2); the overall mean was 32% (234 of 726 ticks). The infection rate was significantly associated with area
(P < 0.001) but not with the developmental stage or
viability of the ticks (P = 0.06 and P = 0.19, respectively). The log-linear analysis supported the results
of the simple chi-square tests.
|
Comparison between methods used to detect borreliae.
From
the agreement (kappa statistics) between the results of DF
microscopy, cultivation, and PCR analysis (Table
3), the kappa values for all three
comparisons indicate fairly good agreement. The kappa values for
comparisons of cultivation and DF microscopy, cultivation and
PCR, and DF microscopy and PCR were 0.59, 0.53, and 0.57, respectively (P values were <0.001, 0.380, and <0.001, respectively, by McNemar's chi-square test). The discrepancies between
culture and PCR methods were numerous (N = 105), but
they were symmetrical and therefore not statistically significant
(Table 3). DF microscopy gave significantly more positive results than either culture or PCR analysis (P < 0.001 and
P < 0.001, respectively).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This study shows that a considerable risk of contracting a borrelia infection can be present in an urban environment, and therefore, a large human population can be at risk. Both the density of vector ticks and the prevalence of borreliae in these vectors were higher than expected in light of previously published studies (1, 3, 9, 21, 27, 30, 33). These observations indicate a need to take Lyme borreliosis into account as one possible differential diagnosis even for patients without a history of a visit to rural areas of endemicity. In addition, the predominance of B. afzelii and absence of B. burgdorferi sensu stricto may affect the clinical picture of Lyme borreliosis in this area.
The presence of large mammals has been considered a prerequisite for Lyme borreliosis in an area of endemicity (7, 8, 32). Our observations are in dispute with these presumptions and confirm the suspicions raised by previously published results (13). None of the study areas was known to be populated by deer or elk. The most numerous mammals are small rodents (Apodemus flavicollis, Apodemus sylvaticus, and Peromyscus leucopus) and insectivores (shrews and hedgehogs), with the largest ones being the hare (Lepus timidus). The areas where ticks were most abundant were also the most capable of supporting a permanent hare population.
Official statistics of the National Public Health Institute of Finland show that the incidence of late manifestations of Lyme borreliosis is twice as high in the district of Helsinki as in the surrounding, more rural areas (13 versus 6.6 cases per 100,000 inhabitants, respectively) (22). These incidence figures may be biased. However, if a bias existed, it should affect Helsinki and its surroundings in a similar manner and should therefore not explain the observed difference. This difference suggests that our findings also have relevance for the occurrence of Lyme borreliosis in the area.
The observed distribution of genospecies is in agreement with the results of earlier studies performed in Finland and Russia (14, 28). B. burgdorferi sensu stricto has been detected only in the southwestern parts of Finland, and the prevalence of B. garinii seems to increase toward the eastern border. The untypeable spirochetes could possibly represent new genospecies, but further studies are needed to confirm their identities.
If all reservoir animals were equally and nonexclusively favorable hosts for both B. afzelii and B. garinii, the expected number of mixed infections would be significantly higher than the number observed in this study. The low number of concurrent infections suggests that these two genospecies favor two distinct reservoir animal populations. Because these genospecies did, however, coexist in two adult ticks, significant competition between them in culture media or tick tissues is unlikely. The total lack of B. burgdorferi sensu stricto may indicate that there are no suitable reservoirs for this genospecies in the areas studied.
DF microscopy, culture, and PCR analysis have all been used in studies on the borrelia infestation rates of ticks (35). However, extensive comparisons between these methods have not been available. According to the present study, DF microscopy seems to be the method of choice in surveillance studies, with culture and PCR analysis being complementary in nature.
We conclude that dense populations of I. ricinus ticks heavily infested with B. burgdorferi can exist even in urban environments not populated with large mammals like deer or elk. Inhabitants and health care officials of cities should be made more aware of the risk of contracting Lyme borreliosis in parks or other recreational areas harboring infected ticks.
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ACKNOWLEDGMENTS |
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We thank Marja-Leena Helin, Ulla Toivonen, Leena Palmunen, and Risto Holma for excellent technical assistance. We also acknowledge funding for this work by the Clinical Research Institute of the University Central Hospital of Helsinki, the Orion Research Foundation, and the Academy of Finland.
The language of the manuscript was revised by Georgianna Oja.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Otolaryngology, Helsinki University Central Hospital, Haartmaninkatu 4 E, 00290 Helsinki, Finland. Phone: 358-9-471 5067. Fax: 358-9-288 359. E-mail: miikkap{at}nekku.pp.fi.
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Abstract
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Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, May 1999, p. 1361-1365, Vol. 37, No. 5
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[Abstract] [Full Text] -
Goodlad, J R, Davidson, M M, Gordon, P, Billington, R, Ho-Yen, D O
(2002). Morphoea and Borrelia burgdorferi: results from the Scottish Highlands in the context of the world literature. Mol. Pathol.
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PANELIUS, J., LAHDENNE, P., HEIKKILA, T., PELTOMAA, M., OKSI, J., SEPPALA, I.
(2002). Recombinant OspC from Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii in the serodiagnosis of Lyme borreliosis. J Med Microbiol
51: 731-739
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HEIKKILA, T., SEPPALA, I., SAXEN, H., PANELIUS, J., PELTOMAA, M., HUPPERTZ, H.-I., LAHDENNE, P.
(2002). Cloning of the gene encoding the decorin-binding protein B (DbpB) in Borrelia burgdorferi sensu lato and characterisation of the antibody responses to DbpB in Lyme borreliosis. J Med Microbiol
51: 641-648
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Heikkila, T., Seppala, I., Saxen, H., Panelius, J., Peltomaa, M., Julin, T., Carlsson, S.-A., Lahdenne, P.
(2002). Recombinant BBK32 Protein in Serodiagnosis of Early and Late Lyme Borreliosis. J. Clin. Microbiol.
40: 1174-1180
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Heikkila, T., Seppala, I., Saxen, H., Panelius, J., Yrjanainen, H., Lahdenne, P.
(2002). Species-Specific Serodiagnosis of Lyme Arthritis and Neuroborreliosis Due to Borrelia burgdorferi Sensu Stricto, B. afzelii, and B. garinii by Using Decorin Binding Protein A. J. Clin. Microbiol.
40: 453-460
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Rauter, C., Oehme, R., Diterich, I., Engele, M., Hartung, T.
(2002). Distribution of Clinically Relevant Borrelia Genospecies in Ticks Assessed by a Novel, Single-Run, Real-Time PCR. J. Clin. Microbiol.
40: 36-43
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Panelius, J., Lahdenne, P., Saxen, H., Heikkila, T., Seppala, I.
(2001). Recombinant Flagellin A Proteins from Borrelia burgdorferi Sensu Stricto, B. afzelii, and B. garinii in Serodiagnosis of Lyme Borreliosis. J. Clin. Microbiol.
39: 4013-4019
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Kurtenbach, K., De Michelis, S., Sewell, H.-S., Etti, S., Schafer, S. M., Hails, R., Collares-Pereira, M., Santos-Reis, M., Hanincova, K., Labuda, M., Bormane, A., Donaghy, M.
(2001). Distinct Combinations of Borrelia burgdorferi Sensu Lato Genospecies Found in Individual Questing Ticks from Europe. Appl. Environ. Microbiol.
67: 4926-4929
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Jenkins, A., Kristiansen, B.-E., Allum, A.-G., Aakre, R. K., Strand, L., Kleveland, E. J., van de Pol, I., Schouls, L.
(2001). Borrelia burgdorferi Sensu Lato and Ehrlichia spp. in Ixodes Ticks from Southern Norway. J. Clin. Microbiol.
39: 3666-3671
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Alekseev, A. N., Dubinina, H. V., Van De Pol, I., Schouls, L. M.
(2001). Identification of Ehrlichia spp. and Borrelia burgdorferi in Ixodes Ticks in the Baltic Regions of Russia. J. Clin. Microbiol.
39: 2237-2242
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Pietila, J., He, Q., Oksi, J., Viljanen, M. K.
(2000). Rapid Differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi Sensu Stricto by LightCycler Fluorescence Melting Curve Analysis of a PCR Product of the recA Gene. J. Clin. Microbiol.
38: 2756-2759
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De Michelis, S., Sewell, H.-S., Collares-Pereira, M., Santos-Reis, M., Schouls, L. M., Benes, V., Holmes, E. C., Kurtenbach, K.
(2000). Genetic Diversity of Borrelia burgdorferi Sensu Lato in Ticks from Mainland Portugal. J. Clin. Microbiol.
38: 2128-2133
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Ling, C L, Joss, A W L, Davidson, M M, Ho-Yen, D O
(2000). Identification of different Borrelia burgdorferi genomic groups from Scottish ticks. Mol. Pathol.
53: 94-98
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Hellwage, J., Meri, T., Heikkila, T., Alitalo, A., Panelius, J., Lahdenne, P., Seppala, I. J. T., Meri, S.
(2001). The Complement Regulator Factor H Binds to the Surface Protein OspE of Borrelia burgdorferi. J. Biol. Chem.
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[Abstract] [Full Text]
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