Appl Environ Microbiol, April 1998, p. 1169-1174, Vol. 64, No. 4
Department of Zoology,
Received 24 September 1997/Accepted 1 January 1998
The genetic diversity of Borrelia burgdorferi sensu
lato was assessed in a focus of Lyme borreliosis in southern Britain
dominated by game birds. Ticks, rodents, and pheasants were analyzed
for spirochete infections by PCR targeting the 23S-5S rRNA genes, followed by genotyping by the reverse line blot method. In questing Ixodes ricinus ticks, three genospecies of B. burgdorferi sensu lato were detected, with the highest
prevalences found for Borrelia garinii and Borrelia
valaisiana. B. burgdorferi sensu stricto was rare (<1%) in all
tick stages. Borrelia afzelii was not detected in any of
the samples. More than 50% of engorged nymphs collected from pheasants
were infected with borreliae, mainly B. garinii and/or
B. valaisiana. Although 19% of the rodents harbored
B. burgdorferi sensu stricto and/or B. garinii
in internal organs, only B. burgdorferi sensu stricto was
transmitted to xenodiagnostic tick larvae (it was transmitted to 1% of
the larvae). The data indicate that different genospecies of B. burgdorferi sensu lato can be maintained in nature by distinct
transmission cycles involving the same vector tick species but
different vertebrate host species. Wildlife management may have an
influence on the relative risk of different clinical forms of Lyme
borreliosis.
Lyme borreliosis is a tick-borne
disease of humans in temperate climates of the northern hemisphere,
whose causative agent, a spirochete belonging to the genus
Borrelia, was described and named Borrelia
burgdorferi in 1984 (13). On the basis of DNA-DNA relatedness and other molecular criteria, B. burgdorferi
sensu lato is now considered to comprise at least nine genospecies and genomic groups (1, 30, 33). Phylogenetic analyses of various genes have suggested that the population structure of B. burgdorferi sensu lato is clonal (6). Here we ask
whether the diverse spirochete strains have differential transmission
patterns.
In Eurasia, six genospecies of B. burgdorferi sensu lato
have been recorded; B. burgdorferi sensu stricto,
Borrelia garinii, and Borrelia afzelii are
causative agents of Lyme disease in humans (38), while the
pathogenic potentials of Borrelia japonica, Borrelia
valaisiana (formerly genomic group VS116 [39]),
and Borrelia lusitaniae (formerly genomic group PotiB2
[18]) have not yet been demonstrated. Culturing
Borrelia is commonly considered the "gold standard" for
detection of B. burgdorferi sensu lato. Approaches based on
the PCR (33), however, appear to be more accurate in
assessing the diversity and distribution of borreliae in nature, as
culturing may favor particular genotypes (27). Furthermore,
strains of B. burgdorferi sensu lato prevalent in the United
Kingdom (known to be variants of B. garinii, B. afzelii, B. burgdorferi sensu stricto, and B. valaisiana) have been found to be unusually difficult to isolate
and culture from ticks and hosts by standard techniques
(20). There is increasing evidence that the kinds of
borreliae in ticks and hosts vary considerably (9, 14, 28, 29, 32,
33). In The Netherlands, for example, B. afzelii
appears to be the most frequent genospecies in ticks, whereas in
Ireland B. garinii and B. valaisiana seem to
dominate (14, 32, 33). Surprisingly, B. burgdorferi sensu stricto, the most common genospecies in
northeastern North America, appears to be comparatively rare in Europe
and virtually absent in central and east Asia (7, 24, 26).
The reasons for this variation remain unknown but may be related to the
structure of the vertebrate host cenosis; it has been postulated that
genospecies are associated with particular groups of vertebrate hosts,
such as birds or rodents (24). This suggestion appears to
conflict with the observation that different genospecies of B. burgdorferi sensu lato may coexist in individual vertebrate hosts
(7, 26). Such concurrent infections, however, do not imply
that the transmissibilities of the genospecies or strains between hosts
and ticks are equal; any differential transmission of the genospecies
in the various natural tick-host systems would influence the prevalence
of the genospecies and the degree of ecological diversity observed.
While the transmission behavior of B. burgdorferi sensu
stricto has been studied in detail with both laboratory and natural
rodent hosts (5, 16, 22), the relative transmissibilities of
other genospecies of B. burgdorferi sensu lato in rodents
and other hosts have not been investigated previously.
Small mammals, particularly mice, have always been considered the
principal hosts of B. burgdorferi sensu lato (10, 16, 17, 22, 25), but a role for avian hosts as reservoirs of B. burgdorferi sensu lato is gradually gaining credence (11, 12,
15, 31, 36) despite early claims to the contrary (21, 23). The potential role of birds in the transmission dynamics of
B. burgdorferi sensu lato is substantial. In England,
approximately 20 million farm-reared pheasants (Phasianus
colchicus) are released into the woodlands each year to supplement
natural populations for recreational shooting. As a result, pheasants
constitute the vast majority of the land-based avifauna, especially in
woodlands of southern England (34), and are present
alongside high densities of mammals, such as woodmice, voles,
squirrels, and deer. All of these hosts feed considerable numbers of
Ixodes ricinus, the European vector of B. burgdorferi sensu lato (4, 15, 31).
In this paper, we present field data that reveal differential
transmission of B. burgdorferi sensu lato genospecies
through pheasant and rodent populations to different developmental
stages of I. ricinus ticks.
Study site.
Animals were caught in a Dorset woodland 10 miles west of Fordingbridge (1°56'W, 50°53'N) within a focus of
Lyme borreliosis in southern England. This site contains mainly oak
(Quercus spp.), ash (Fraxinus excelsior), and
patchy conifer plantations (mainly Pinus sylvestris), and
the undergrowth is dominated by bluebells (Hyacinthoides
non-scripta) and dog's mercury (Mercurialis perennis) in the spring and bracken fern (Pteridium aquilinum) from
late summer until winter.
Rodents.
In May 1996, 47 rodents belonging to the species
Apodemus sylvaticus (woodmouse) and Clethrionomys
glareolus (bank vole) were trapped alive with Longworth traps
(Penlon Ltd., Abingdon, United Kingdom) and taken to a laboratory. The
spirochetal infectivity of the rodents for ticks was assayed by larval
xenodiagnosis (16) commencing 1 week after trapping; 30 uninfected I. ricinus larvae (colony maintained at the
NERC Institute of Virology and Environmental Microbiology, Oxford,
United Kingdom) were introduced to each animal. After repletion, the
engorged ticks were kept for 14 days above a saturated solution of
MgSO4 at 18°C and then frozen at Birds.
Thirty adult male pheasants were shot in April 1996. All of the I. ricinus ticks infesting these birds were
recorded and allowed to drop off naturally. No fully engorged larvae or
adult ticks were recovered, but 150 fed nymphs from the 30 birds (five
ticks per bird) were collected and kept alive for 2 weeks before they were preserved in 70% ethanol at Questing ticks.
I. ricinus ticks were collected
fortnightly by blanket dragging from the same site as their vertebrate
hosts. Some of these ticks (larvae collected in the summer of 1995, nymphs collected in the spring, summer, and autumn of 1995 and spring
of 1996, and adults collected in the autumn of 1996) were analyzed to
determine whether they were infected with particular genospecies of
B. burgdorferi sensu lato by PCR and the reverse line
blot method.
PCR and reverse line blotting.
Genomic DNA was extracted
from ticks and animal biopsies by alkaline hydrolysis (8). A
PCR with a nested set of primers (primers 23SN1
[5'-ACCATAGACTCTTATTACTTTGAC], 23SC1
[5'-TAAGCTGACTAATACTAATTACCC], 23SN2
[5'-ACCATAGACTCTTATTACTTTGACCA], and 5SC2
[5'-biotin-GAGAGTAGGTTATTGCCAGGG]) was performed by
targeting the tandemly duplicated rrf (5S)-rrl (23S) rRNA gene clusters (19, 30, 33). All of the steps were
separated temporally and spatially (different laboratories) and were
performed under strictly aseptic conditions. As B. japonica is not present in Europe, a culture of this genospecies
was used as a positive control in order to avoid DNA contamination.
Negative controls at a ratio of approximately 2:3 were incorporated
into the PCR procedures at the DNA extraction level and at the first- and second-round amplification levels and into the electrophoresis of
PCR products. All amplicons were electrophoresed with 2% agarose gels,
stained with ethidium bromide, and visualized by UV transillumination. All samples that produced bands at approximately 380 and/or 225 to 270 bp were subjected to DNA-DNA hybridization by the reverse line blot
method, a modification of the reverse dot blot method performed with a
line blotter (Miniblotter 45; Immunetics, Cambridge, Mass.). Briefly,
biotin-labelled amplicons were hybridized with DNA probes which were
covalently bound to an activated membrane by the 5' aminolink (a)
group. The probes were specific for B. burgdorferi
sensu lato (5'-a-CTTTGACCATATTTTTATCTTCCA), B. burgdorferi sensu stricto (5'-a-AACACCAATATTTAAAAAACATAA),
B. afzelii (5'-a-AACATTTAAAAAATAAATTCAAGG), B. garinii (5'-a-AACATGAACATCTAAAAACATAAA),
and B. valaisiana (5'-a-CATTAAAAAAATATAAAAAATAAATTTAAGG). PCR products of DNA
templates derived from cloned cultures of B. burgdorferi sensu stricto, B. garinii,
B. afzelii, and B. japonica were
included as positive controls for the reverse line blot. After
incubation with streptavidin-peroxidase conjugate (Boehringer Mannheim
GmbH, Mannheim, Germany), hybrids were visualized with an enhanced
chemiluminescence system (type ECL; Amersham Life Sciences, Amersham,
United Kingdom).
A total of 780 questing I. ricinus ticks (100 larvae,
100 adults, and 580 nymphs) were analyzed for B. burgdorferi sensu lato infection by PCR. The levels of infection
with B. burgdorferi sensu lato in larvae and nymphs
were 1 and 2.6%, respectively (not significantly different), but the
level of infection in questing adult ticks was significantly higher
(16%) (
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Differential Transmission of the Genospecies of Borrelia
burgdorferi Sensu Lato by Game Birds and Small Rodents in
England
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
20°C. The rodents
were finally autopsied under aseptic conditions in a building in which
no Borrelia DNA was handled. Biopsies from internal organs
(heart, urinary bladder, kidney, brain) and earlobes were taken and
stored at 20°C until PCR analysis.
20°C. Of these, the 122 most fully engorged ticks were selected and analyzed for the presence of
spirochetal DNA.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
2 = 14.70, P < 0.001) (Table
1). All of the larvae and adults and 200 of the nymphs were subjected to genotyping, which revealed three
genospecies, B. burgdorferi sensu stricto,
B. garinii, and B. valaisiana (Table
1). The most abundant genospecies was B. garinii (found
in all developmental stages of I. ricinus), followed by
B. valaisiana, while B. burgdorferi
sensu stricto was rare (
1%) and there was no detectable difference
in the levels of B. burgdorferi sensu stricto infection
in nymphs and adult ticks. The levels of B. garinii and
B. valaisiana infection in questing larvae and nymphs
were low (3.0%), but the level of infection in questing adults was
significantly higher (13%) (2 = 25.85, P < 0.001). Three of the infected adult ticks were
infected with both B. garinii and B. valaisiana, and two B. burgdorferi sensu lato
infections in adult ticks could not be identified to genospecies.
B. afzelii was not detected in any tick.
TABLE 1.
Levels of infection for four genospecies of B. burgdorferi sensu lato in I. ricinus collected in
1995 and 1996 and in xenodiagnostic ticks
Spirochetal DNA was detected in 5 of 26 C. glareolus individuals (19.2%) and in 4 of 21 A. sylvaticus individuals (19.0%). Two genospecies were detected, B. burgdorferi sensu stricto and B. garinii. All nine infected rodents were positive for B. garinii (seven brain infections and two kidney infections) (Fig. 1), while two animals had mixed infections with B. garinii and B. burgdorferi sensu stricto in their brains (Table 2). None of the skin, urinary bladder, and heart samples proved to be infected.
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Spirochete-free I. ricinus larvae were introduced to the 47 rodents (30 larvae per animal). A total of 771 engorged larvae were analyzed by PCR. Ten of these were positive for B. burgdorferi sensu lato, giving an overall level of infection in xenodiagnostic ticks of 1.3% (Table 1). All positive samples were identified as containing B. burgdorferi sensu stricto (Fig. 1). The infected xenodiagnostic larvae came from four individual rodents, only one of which had tested positive for tissue infections with B. burgdorferi sensu lato (Table 2).
Of the 122 fed I. ricinus nymphs from pheasants that were analyzed, 69 (56.6%) were infected with B. burgdorferi sensu lato. The organisms in all but three of these samples could be identified to species; 33 ticks were infected with B. garinii, 20 ticks were infected with B. valaisiana, and 13 ticks were found to have mixed infections with both of these genospecies (Table 1). Of the 30 birds, 27 yielded at least one Borrelia-infected tick; 14 birds yielded at least one tick infected with B. garinii and another tick infected with B. valaisiana or ticks infected concurrently with both genospecies, while 13 birds yielded ticks infected with only one of the two genospecies.
None of the negative controls incorporated into the PCR procedures performed throughout the present study gave a positive signal.
In summary, the transmission of spirochetes to ticks from rodents and
the transmission of spirochetes to ticks from pheasants differed both
qualitatively and quantitatively. Not only were different genospecies
of B. burgdorferi sensu lato transmitted, but the level
of infection by any genospecies was significantly lower in
xenodiagnostic larvae that fed on rodents (1.3%) than in nymphs that
had fed on pheasants (56.6%) (2 = 398.6, df = 1, P 
0.001).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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This study showed that three different genospecies of the B. burgdorferi sensu lato species complex, B. burgdorferi sensu stricto, B. garinii, and B. valaisiana (formerly genomic group VS116), are circulating in an endemic focus of Lyme borreliosis in southern England. B. afzelii, one of the most abundant genospecies in continental Europe, was not detected. Although 19% of the rodents harbored B. garinii, only B. burgdorferi sensu stricto was transmitted by the rodents to ticks. The infectivity of the rodent population was surprisingly low; only 1.3% of the xenodiagnostic ticks were infected. In contrast, more than 50% of the nymphal ticks derived from pheasants were infected with B. garinii and B. valaisiana. This pattern and the matching genotypic composition of B. burgdorferi sensu lato in the questing ticks indicate that B. garinii and B. valaisiana are preferentially transmitted to ticks by pheasants, while B. burgdorferi sensu stricto appears to be maintained at a low level by a rodent-tick cycle.
The detection of spirochetes in the present study was based on successful amplification of spirochetal DNA, which cannot distinguish between viable and nonviable borreliae. However, as all of the ticks were allowed to engorge and digest their bloodmeal for 14 days postrepletion, it is unlikely that the PCR detected only naked DNA from spirochetes in the ticks' midguts, particularly because the target of this PCR is located on the chromosome rather than on a plasmid (19, 30). Similarly, the presence of naked chromosomal DNA in host tissues was unlikely as the animals were autopsied more than 2 weeks after trapping, their last possible contact with infected ticks.
The observed level of infection with B. burgdorferi sensu lato in questing larval I. ricinus, 1%, is within the range previously described for endemic foci of Lyme borreliosis in Europe (17, 32), while the level of infection in questing nymphs, 2.6%, appears to be rather low compared with the levels of infection in endemic foci in North America (37) and continental Europe (10, 17, 31). The overall level of infection in adults was fivefold higher than the level of infection in nymphs, indicating that hosts of I. ricinus nymphs were particularly infective. Within the questing tick population, B. garinii was the most frequent genospecies and was detected at all developmental stages, followed by B. valaisiana. The levels of infection for both of these genospecies were markedly higher in adults than in questing nymphs. In contrast, the level of infection with B. burgdorferi sensu stricto, the most abundant genospecies in northern North America, did not exceed 1% even in adult ticks. This pattern of infection indicates that the hosts that were feeding large numbers of nymphal ticks were selectively infective for B. garinii and B. valaisiana.
I. ricinus larvae feed in large numbers on mice and voles, in addition to squirrels and deer (4, 10, 17). The low level of infection with B. burgdorferi sensu lato in questing nymphs, as found in the present study, is consistent with the low infectivity of the trapped small rodents as assessed by larval xenodiagnosis. Compared with the data from the present study, the reservoir capacity of the rodent populations in foci in continental Europe (10, 17) and many parts of northern North America (22) is much higher. This may be related to the particular genospecies of B. burgdorferi sensu lato circulating in each location; B. afzelii, for which rodents are particularly transmission competent, is widely distributed in continental Europe (9, 32, 33), whereas in our study site B. afzelii was not found. In North America, rodents, particularly rodents belonging to the genus Peromyscus, are highly transmission competent for B. burgdorferi sensu stricto (5, 22), whereas European rodent species were found to have a lower degree of reservoir competence and reservoir capacity for this genospecies (16), perhaps explaining its rarity in the present study site and throughout Eurasia (7, 24, 26). B. garinii, on the other hand, has the potential to persist in rodents concurrently with other genospecies (7, 26), but there is emerging evidence that it is only rarely passed from rodents to ticks (9, 25, 26). This is consistent with the results of the present study, in which 19% of the small rodents were infected with B. garinii but none of the 771 xenodiagnostic I. ricinus ticks acquired B. garinii from these animals. Moreover, B. garinii infections were not detected in the skin of the rodents, but were confined to internal organs, particularly the brain. None of the rodents or the xenodiagnostic ticks fed on these rodents was found to be infected with B. valaisiana, despite the prevalence of this organism in questing I. ricinus ticks collected in the same study site. This finding, together with the fact that B. valaisiana has never been detected in rodent hosts, may indicate that this genospecies does not survive and persist in small mammals.
Ground-foraging birds, particularly pheasants, which may occur in the United Kingdom at densities up to 50 birds per hectare (34), constitute a major part of the tick host community in the woodland studied. Pheasants feed more than four times as many nymphs as larvae of I. ricinus (15). In the present study pheasants were highly infective to nymphs of I. ricinus. The vast majority of bird-derived infected nymphs carried B. garinii and B. valaisiana, but not B. burgdorferi sensu stricto. Most infraspecific variants of B. garinii, the most polymorphic genospecies of the B. burgdorferi sensu lato species complex (40), have previously been associated with ticks derived from birds (24, 28, 29). In the present study mixed infections in ticks were found only for B. garinii and B. valaisiana, suggesting that the transmission of these two genospecies is associated. This finding is consistent with the results of a recent study in Ireland on mixed infections of B. garinii and B. valaisiana in questing ticks (14), which suggested that birds are reservoirs of B. valaisiana. To our knowledge, the present study is the first study which provides direct evidence that there is an avian reservoir host for B. valaisiana.
The reason for the complete absence of B. afzelii in the present study is unclear, but the lack of B. afzelii may also be related to avian hosts; in the study site used, it is possible that a large and dense pheasant population substantially reduces the basic reproduction number of B. afzelii. Thus, it is possible that this genospecies is taken out of the ecosystem by means of a zooprophylactic role of such birds for B. afzelii, despite the presence of reservoir-competent rodents (mice, voles, and squirrels) in the study site. A lack of reservoir competence and a possible zooprophylactic role of ground-foraging birds in relation to B. burgdorferi sensu stricto and B. afzelii, respectively, would be consistent with results of previous studies on the reservoir incompetence of birds (21, 23).
Besides rodents and pheasants, other tick host species undoubtedly play a role in generating the observed pattern of species diversity of B. burgdorferi sensu lato. For example, the high level of infection (57%), mainly infection with B. garinii and/or B. valaisiana, in nymphs that fed on pheasants was reduced to 16% in questing adult ticks. This was probably the result of dilution by uninfected nymphs that fed on other hosts not competent to transmit these genospecies. Apart from deer, squirrels are likely candidates for this role, because they feed large numbers of nymphs (4) and are competent to transmit B. burgdorferi sensu lato but apparently are not competent to transmit B. garinii (3).
The overall pattern which emerged from the present study is one of differential transmission and maintenance of various genospecies of B. burgdorferi sensu lato depending on the variable interactions between the vertebrate host species and (i) each genospecies or (ii) each developmental stage of the tick. Many hosts may be exposed to multiple tick bites, followed by the possible establishment of mixed infections (7, 26). However, there is increasing evidence that the various host species do not transmit all B. burgdorferi sensu lato strains to ticks with equal efficiency (5, 16, 21-25, 29). Cautions about the transfer of parameter values (e.g., transmission coefficients) from system to system for use in models (31) are supported by these results. The mechanisms underlying the apparent differential transmission of the genospecies of B. burgdorferi sensu lato by the various groups of mammalian and avian hosts remain to be determined.
The genetic diversity of B. burgdorferi sensu lato and the strain-specific interaction with each host species add additional elements to the considerable ecological diversity and thus variation in risk factors for humans of this zoonotic tick-borne disease. For example, in a site dominated by pheasants, such as the site analyzed in the present study, there is potentially a greater risk of neuroborreliosis associated with increased prevalence of B. garinii (38). On the other hand, the finding that many ground-foraging birds (pheasants in this study) primarily feed nymphs which, later as questing adults, are not considered as great a risk to humans as the less easily detected and more numerous nymphs may counterbalance the inflated risk of B. garinii infection for humans.
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ACKNOWLEDGMENTS |
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This study was supported by grant GR3/09626 from the Natural Environment Research Council, United Kingdom, by grant 044488/Z/95/A from The Wellcome Trust, London, United Kingdom, and by grant EC Biomed BMHI-CT93-1183 from the Commission of the European Union.
We thank A. P. Van Dam (Amsterdam, The Netherlands) and M. M. Simon (Freiburg, Germany) for providing strains of B. burgdorferi sensu lato.
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FOOTNOTES |
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* Corresponding author. Mailing address: The Wellcome Trust Center for the Epidemiology of Infectious Diseases, Department of Zoology, University of Oxford, OX1 3PS, Oxford, United Kingdom. Phone: 0044-1865-281630. Fax: 0044-1865-281696. E-mail: kku{at}mail.nerc-oxford.ac.uk.
Present address: National Institute of Biological Standards and
Controls, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, United Kingdom.
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Abstract
Introduction
Materials & Methods
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Discussion
References
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