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Journal of Clinical Microbiology, November 1998, p. 3355-3358, Vol. 36, No. 11
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection and Typing of Borrelia
burgdorferi Sensu Lato in Ixodes ricinus Ticks Attached
to Human Skin by PCR
Gabriele
Liebisch,1,2
Bettina
Sohns,1 and
Wilfried
Bautsch1,*
Institute of Medical Microbiology, Hannover
Medical School,1 and
Institute of
Parasitology, Veterinary School,2 Hannover,
Germany
Received 1 May 1998/Returned for modification 2 July 1998/Accepted 4 August 1998
 |
ABSTRACT |
Live Ixodes ricinus ticks attached to humans residing
in Germany were examined for borreliae by dark-field microscopy and PCR. Borrelia species were identified by 16S rRNA sequence
analysis, which showed the presence of several species, some not
yet defined, and a high prevalence of multiply infected ticks.
| |
TEXT |
Lyme disease is a multisystem
disorder involving the skin, joints, heart, and nervous system (1,
6, 20, 21). The etiological agent is a spirochete species
complex, Borrelia burgdorferi sensu lato, which is
transmitted via infected ticks of the Ixodes ricinus
species complex (5, 17). B. burgdorferi
sensu lato has been found in other ticks, as well, like Ixodes
hexagonus, Ixodes canisuga, and Dermacentor
reticulatus (9-12), whose epidemiological relevance to
human Lyme disease transmission is not entirely clear at the moment.
European B. burgdorferi sensu lato isolates have been
divided into several genospecies based on phenotypic analysis,
as well as sero- and genotyping, i.e., B. burgdorferi sensu stricto, the predominant species found in North
America, Borrelia afzelii, and Borrelia
garinii (3, 13, 16, 19, 25, 26). There is some evidence
that this division is of relevance to the clinical presentation of Lyme
disease in Europe: several studies have revealed an association between
the presence of B. burgdorferi sensu stricto and arthritis
and between B. afzelii and acrodermatitis chronica atrophicans (23). This picture, however, is still not
complete, since other B. burgdorferi sensu lato isolates,
VS116 and PotiB2, that cannot easily be attributed to the three known
pathogenic species and whose pathological relevance is currently
completely unclear have recently been described (18, 22).
Accordingly, the prevalence of the different genospecies of B. burgdorferi sensu lato in infected ticks should be the prime
determinant for the risk of acquiring Lyme disease and its clinical
presentation.
We therefore analyzed live ticks (nymphal, larval, and adult stages),
which had been found attached to human skin and submitted by patients
during the period 1995 to 1996 from all parts of Lower Saxony, Germany,
for the presence of four genospecies of B. burgdorferi sensu
lato, i.e. B. burgdorferi sensu stricto, B. afzelii, B. garinii, and isolate VS116. (B. afzelii NE 632 [7] was kindly donated by L. Gern,
Neuchatel, Switzerland, B. garinii N 34 was donated
[2] by G. Baranton, Paris, France, B. burdorferi sensu stricto B31 [4] was donated by
W. Burgdorfer, Hamilton, Mont., and VS116 [18] was
donated by O. Peter, Sion, Switzerland.) The ticks were identified to
the species level by standard morphological criteria. Altogether, 2,421 ticks, of which the overwhelming majority belonged to the species
I. ricinus, had been submitted for analysis (2,399 I. ricinus, 11 I. hexagonus,
6 Rhicicephalus sanguineus, 2 Dermacentor
marginatus, 1 D. reticulatus, 1 Amblyomma
cajannense, and 1 Argas reflexus tick), but only some
of these (1,654 ticks, all of the I. ricinus species,
confirming that this tick is the predominant vector for human Lyme
disease) were still alive on arrival and were included in this study.
To determine Borrelia infection of the ticks, the midgut of
each tick was removed under a stereomicroscope and homogenized in a
0.9% NaCl solution (ca. 100 µl for larvae, 150 µl for nymphs, and
300 µl for engorged adults). Ten microliters of this homogenate was
then quantitatively analyzed for the presence of borreliae by
dark-field microscopy. One hundred fifty-three tick homogenates (9.3%)
were found to be infected by Borrelia species, with numbers ranging from 1 to >3,000 borreliae per 10-µl aliquot. One hundred nineteen homogenates were further analyzed by PCR (see below); 34 homogenates were excluded from the analysis due to insufficient material (mainly larval stages of I. ricinus). The
final sample collection consisted of borrelia-positive midgut
homogenates from 61 adults, 57 nymphs, and 1 larval stage of
I. ricinus.
To identify the pathogenic species of the B. burgdorferi sensu lato complex within these homogenates by PCR, we
used the 16S rRNA PCR primer pairs originally described by Marconi
and Garon (14, 15). The homogenates were first
incubated with 200 µg of proteinase K per ml at 60°C
overnight to release the DNA, and the reaction was stopped by 10 min of
boiling. Five microliters of the supernatant was subjected to 40 cycles
of PCR amplification (94°C for 1.5 min, 42 to 64°C for 2 min,
72°C for 2 min) using Taq polymerase, 1 µM
oligonucleotide primers, and 1.5 mM MgCl2 under standard
conditions. PCR products were separated on a 1% (wt/vol) agarose gel
and visualized under UV light after ethidium bromide staining. To
analyze for the presence of inhibitory substances in PCR-negative
homogenates, DNA equivalent to 20 genomes of B. afzelii
(grown in BSK-H medium supplemented with 6% rabbit serum [Sigma] at
33°C) was added to one sample of the homogenate and amplified under
the conditions specified above for B. afzelii. In our
hands, the species-specific primer pairs for B. burgdorferi sensu stricto, B. afzelii, and
B. garinii were quite effective, with a
sensitivity of ca. 10 borreliae/PCR mixture (i.e., 50 µl). In
contrast, the genus-specific primer pair for the B. burgdorferi sensu lato complex was about fivefold less sensitive
in our hands, which prompted us to develop new genus-specific primers
which allowed amplification of all four genospecies of B. burgdorferi analyzed in this study (i.e., B. burgdorferi sensu stricto, B. afzelii,
B. garinii, and isolate VS116) with a similar
sensitivity of ca. 10 borreliae/PCR mixture and no amplification of
other bacteria, like Escherichia coli, Enterococcus
faecalis, and Staphylococcus aureus (data not shown).
Furthermore, this repertoire of primers did not allow detection of
strain VS116, a novel species within the B. burgdorferi
sensu lato complex which seems to be present in I. ricinus ticks in a remarkable proportion of cases (8). We therefore amplified a 1.5-kbp DNA fragment encompassing the almost
complete 16S rRNA locus of strain VS116 and sequenced both strands
(Fig. 1). Sequence analysis confirmed the
presence of several single base changes in VS116 in comparison to the
other genospecies of B. burgdorferi sensu lato, which
allowed for the construction of a VS116-specific primer pair.
Again, a VS116-specific amplification product was obtained with no
apparent cross-binding to the other three genospecies or other
bacterial genomes (as exemplified by PCRs with E. coli, E. faecalis, and S. aureus; data not
shown) and a sensitivity similar to that of the other primer pairs used
in this study. Oligonucleotide sequences and annealing temperatures are
listed in Table 1.
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FIG. 1.
Alignment of the 16S rRNA sequences of B. burgdorferi: sensu stricto (BB), B. afzelii (BA),
B. garinii (BG), strain VS116, and sample M94496. Only
parts of the 16S rRNA sequences (from position 39 to position 735) are
shown. Variant base positions are indicated by an asterisk and boldface
type; the positions of the genus-specific (P1 and P2) and
VS116-specific (P8 and P9) primer pairs are underlined.
|
|
Nucleotide sequence accession numbers.
16S rRNA
sequences for the following strains have been submmitted to GenBank:
VS116, under accession no. AJ 225165, identical to the recently
published sequence of Wang et al. (24), and M94496,
under accession no. AJ 229044.
Of 119 samples tested by PCR, only 50 gave a positive result and 69 samples were PCR negative despite the microscopy-determined presence of
borreliae. As shown in Table 2, about one
in three samples contained inhibitory factors preventing proper
PCR amplification, which accounted for 42% of the
borrelia-positive homogenates with negative PCR results. Another 40.5%
of these homogenates are probably accounted for by insufficient
sensitivity of the PCR, since a considerable number of the positive
homogenates contained only low numbers of borreliae (equivalent
to 1 to 10 borreliae/PCR mixture) which were below or at the
detection limits of this PCR procedure. However, two samples failed
to produce a PCR product despite the microscopy-determined presence of
high numbers of borreliae and the absence of inhibitory substances in
the PCR mixture. This would indicate the presence of still other
Borrelia species, either novel as-yet-undescribed species of
the B. burgdorferi complex or non-B.
burgdorferi species, which do not properly bind any of the
species-specific or genus-specific primers.
The PCR results of the noninhibited samples are shown in Fig.
2. A genospecies identification was
considered definite when
both PCR mixtures (that with the
genus-specific primers and that
with the species-specific primers) were
positive and yielded DNA
fragments of the correct size. Several
amplification products
(at least two of each group, i.e., fragments
specific for
B. afzelii,
B. garinii,
and isolate VS116, as well as those reacting only
with the
genus-specific primers) were also partially sequenced,
and this
confirmed the presence of
Borrelia 16S rRNA sequences
in all
cases tested. Samples reacting only with the genus-specific
primers (14 of 50 or 28%) are indicated in Fig.
2 as "genus spec."
DNA
sequence analysis could be performed for 7 of the 14 samples,
and this
revealed the presence of one additional
B. garinii
sequence
and two novel partial 16S rRNA sequences of as-yet-unknown
Borrelia species. Five of the six PCR fragments with novel
16S rRNA sequences
were almost identical to each other but were rather
distinct from
the other
B. burgdorferi sensu lato
sequences. Their sequences
are shown in Fig.
1 for sample
M94496.
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FIG. 2.
Typing results of the PCR-positive tick homogenates. (A)
Number of homogenates harboring the different genospecies; (B) combined
analysis of the PCR typing results. B. burgd. s.s., B. burgdorferi sensu stricto; genus spec., genus specific; Ba,
B. afzelii; Bg, B. garinii; Bb,
B. burgdorferi sensu stricto.
|
|
Our results show that
B. burgdorferi sensu stricto was
not identified once among 50 positive samples. This result confirms
that this species is probably of minor importance in Germany.
At least
in our series, a prevalence of <1/50 indicates that in
northern
Germany only a very small percentage of the infecting
ticks transmit
B. burgdorferi sensu stricto, although this species
is
not absent in Europe (
26). Notably, a recent report on the
distribution and prevalence of
B. burgdorferi sensu
lato genospecies
in field-collected
I. ricinus ticks
(
8) indicated a higher
incidence of
B. burgdorferi sensu stricto than of
B. afzelii (ca.
18% versus ca. 8%), which is in striking contrast to our own
observations
(0% versus 38%) (Fig.
2A). The reason for this
discrepancy is
unknown and may either reflect local variations in the
distribution
of the different genospecies of
B. burgdorferi sensu lato in Europe
or else be related to the fact
that in our series ticks that were
already attached to human skin were
being evaluated. Thus, our
data should provide the first reliable risk
assessment of acquiring
one or several of the different genospecies of
the
B. burgdorferi sensu lato complex by tick bites
rather than an estimation of
the distribution and prevalence of the
different genospecies in
I. ricinus ticks in Germany.
Strain VS116 was the most common
Borrelia species identified
in our infected tick collection, with
58% of all ticks harboring this
strain, which is in good agreement
with the aforementioned field study
(
8). Furthermore, there
are probably several additional
Borrelia species involved in tick
bites, as shown by
analyses of
M94496 (Fig.
1) and the two
tick homogenate samples that
remained PCR negative despite the
microscopic presence of many
borreliae and the lack of inhibition.
These results confirm the
remarkable heterogeneity among European
Lyme disease agents with
possible clinical significance for human
disease. And finally, despite
the fact that fewer than 10% of
all skin-attached ticks were infected
by
Borrelia species, a large
proportion of the infected
ticks were simultaneously positive
for several genospecies of
B. burgdorferi sensu lato (at least
20 of 50 or 40%;
Fig.
2B). Thus, the simultaneous presence of
multiple
B. burgdorferi sensu stricto species is probably the
rule rather than
the exception in European Lyme disease, a notion
of considerable
importance for all clinical studies. The reason
for this strain
clustering must reside in the ecology of the borrelia-tick-host
relationship. Further investigations are required to solve these
issues.
| |
ACKNOWLEDGMENTS |
We thank the heads of our departments, A. Liebisch and D. Bitter-Suermann, Hannover, Germany, for their continuous strong support
and M. Frosch, Würzburg, Germany, for his help in the organization of this project. We also acknowledge the donations of
strains by L. Gern, G. Baranton, W. Burgdorfer, and O. Peter.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Medical Microbiology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30623 Hannover, Germany. Phone: (49)-511-532-4342. Fax:
(49)-511-532-4366. E-mail:
Bautsch.Wilfried{at}MH-Hannover.de.
| |
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Journal of Clinical Microbiology, November 1998, p. 3355-3358, Vol. 36, No. 11
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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