Research Laboratory for Infectious Diseases,
National Institute of Public Health and the Environment, Bilthoven,
The Netherlands
Received 14 December 1998/Returned for modification 8 March
1999/Accepted 6 April 1999
A sensitive and specific PCR hybridization assay was developed for
the simultaneous detection and identification of Ehrlichia and Borrelia burgdorferi sensu lato. In separate assays the
16S rRNA gene of Ehrlichia species and the 23S-5S rRNA
spacer region of B. burgdorferi sensu lato were amplified
and labeled by PCR. These PCR products were used in a reverse line blot
hybridization assay in which oligonucleotide probes are covalently
linked to a membrane in parallel lines. Hybridization of the samples
with the oligonucleotide probes on this membrane enabled the
simultaneous detection and identification of Ehrlichia,
B. burgdorferi, and Bartonella species in 40 different samples. The application of the assay to DNA extracts from
121 Ixodes ricinus ticks collected from roe deer
demonstrated that 45% of these ticks carried Ehrlichia DNA. More than half of these positive ticks carried species with 16S
rRNA gene sequences closely related to those of E. phagocytophila and the human granulocytic ehrlichiosis agent. The
majority of the other positive ticks were infected with a newly
identified Ehrlichia-like species. In addition, 13% of the
ticks were infected with one or more B. burgdorferi
genospecies. In more than 70% of the ticks 16S rRNA gene sequences for
Bartonella species or other species closely related to
Bartonella were found. In five of the ticks both
Ehrlichia and B. burgdorferi species were detected.
 |
INTRODUCTION |
The zoonotic vector-borne diseases
form a large proportion of the emerging bacterial infectious diseases.
The most prominent of these diseases are Lyme disease, ehrlichiosis,
and bartonellosis. In The Netherlands 10 to 35% of the Ixodes
ricinus ticks are infected with Borrelia burgdorferi,
the causative agent of Lyme disease (25, 26). In 1994, general practitioners in The Netherlands reported seeing 33,000 patients who had sustained tick bites and approximately 6,500 patients
with erythema migrans (7). These findings not only underline
the importance of borreliosis but also suggest that other vector-borne
diseases may occur in The Netherlands.
Presently, two tick-transmitted Ehrlichia species have been
shown to cause human disease. The first is Ehrlichia
chaffeensis, which causes human monocytic ehrlichiosis and which
is transmitted by Amblyomma americanum, a tick species found
only in the United States. Until now, very few cases of E. chaffeensis infection in Europe have been described (5, 15,
21). The second Ehrlichia species pathogenic for
humans is the human granulocytic ehrlichiosis agent (HGE). The exact
nature of this organism is still unclear, but on the basis of its 16S
rRNA sequence it is shown to be closely related to Ehrlichia
phagocytophila and Ehrlichia equi. The HGE agent is
transmitted by Ixodes scapularis, but possibly also by other
vectors like I. ricinus. Again, the initial reports of
disease of human patients with HGE came from the United States.
Remarkably, there have been very few reports of cases of disease caused
by HGE in Europe. The major indication that ehrlichiosis may play a
role in Europe comes from serosurveys performed in several European countries including Sweden, Norway, Switzerland, and the United Kingdom
(5, 7, 28). However, von Stedingk et al. (28) have recently detected Ehrlichia species in Swedish I. ricinus ticks, and Ehrlichia was also detected in a
French I. ricinus tick (19). These findings
indicate that Ehrlichia species that are pathogenic for
humans may be present in Western Europe as well.
The clinical manifestations of HGE infection can vary from a flu-like
disease to severe life-threatening acute febrile disease with
thrombocytopenia, leukopenia, and elevated liver transaminase levels.
Because of the diffuse nonspecific symptoms of this disease, diagnosis
relies heavily on laboratory tests. Serology, particularly immunofluorescence, is commonly used, but serology often does not
detect antibodies in the acute phase of disease. Culture of HGE is
possible, but it is very labor intensive and has not been validated as
far as sensitivity is concerned. Microscopic examination of stained
blood smears can be used to detect characteristic enclosures in
infected leukocytes. However, this method is insensitive and requires
special expertise. The sensitivity and specificity of PCR for the
detection of the Ehrlichia probably exceed those of the
other methods. Several PCRs for detection of Ehrlichia
species have been described (1, 6, 9, 14); however, all of these assays enable the detection of just a single species. In this
report we describe a PCR-hybridization assay that enables the
simultaneous detection and species identification of a variety of
Ehrlichia, B. burgdorferi, and
Bartonella species in a single sample. This method
allowed us to screen a large number of Dutch tick samples for the
presence of these tick-borne pathogens.
| |
MATERIALS AND METHODS |
Ticks and bacterial strains.
I. ricinus ticks were
collected from infested roe deer (Capreolus capreolus) shot
in the Flevopolder in The Netherlands, an area where roe deer are
abundant (26). Immediately after collection, the ticks were
immersed in 70% ethanol and stored. The four genomic groups of
B. burgdorferi sensu lato were represented by B. burgdorferi sensu stricto HB4, Borrelia garinii AR-1,
Borrelia afzelii A39S, and Borrelia valaisiana
M19. Crude DNA extracts from the following Ehrlichia species
were used: E. phagocytophila (kindly provided by F. Jongejan) and HGE, Ehrlichia canis, and E. chaffeensis (kindly provided by S. Dumler). The two
Bartonella species used in this study were Bartonella
henselae ATCC 49882 and Bartonella quintana 90-268 (3).
Preparation of DNA extracts from ticks.
Ticks were processed
as described before (10, 23). Briefly, the ticks were taken
from the 70% ethanol solution, air dried, and boiled for 20 min in 100 µl of 0.7 M ammonium hydroxide to free the DNA. After cooling, the
vial with the lysate was left open for 10 min at 90°C to evaporate
the ammonia. The tick lysate either was used directly for PCR or was
stored at 
20°C until use.
PCR amplification.
PCR amplifications were performed in an
Omnigene thermal cycler (Hybaid Ltd., Teddington, United Kingdom). DNA
amplification was done in 50-µl reaction volumes. For the
amplification of Ehrlichia DNA, each reaction mixture
contained 10 pmol of primer 16S8FE and B-GA1B, 1.25 U of SuperTaq DNA
polymerase (HT Biotechnology Ltd., Cambridge, United Kingdom), 0.275 µg of the TaqStart antibody (Clontech Laboratories, Palo Alto,
Calif.), and standard amounts of amplification reagents (each
deoxynucleoside triphosphate at a concentration of 200 µM, 10 mM
Tris · HCl [pH 9.0], 50 mM KCl, 1.5 mM MgCl2,
0.01% gelatin, 0.1% Triton X-100). A 25-µl overlay of paraffin oil
was added to the tubes, followed by the addition of 5 µl of the tick
DNA extract. To minimize nonspecific amplification a touchdown PCR
program was used: 3 min at 94°C, two cycles of 20 s at 94°C,
30 s at 67°C, and 30 s at 72°C, and then two cycles with
conditions identical to the previous cycles but with an annealing temperature of 65°C. During subsequent two cycle sets the annealing temperature was lowered by 2°C until it reached 57°C. Then, an additional 40 cycles each consisting of 20 s at 94°C, 30 s
at 57°C, and 20 s at 72°C, followed the touchdown program,
were performed. The PCR was ended by an extra incubation for 7 min at
72°C. For the amplification of B. burgdorferi sensu lato
DNA, conditions similar to those described above were used, except that
40 pmol of the primers 23SN2 and 5SCB and double the amounts of
SuperTaq and TaqStart were used. In addition, the touchdown PCR
temperature ranged from 60 to 50°C. For the amplification of
Bartonella DNA, the previously described PCR protocol of
Bergmans et al. (4) was used.
To monitor for the occurrence of false-positive PCR results, negative
controls were included during extraction of the tick samples: one
control sample for each six tick samples, with a minimum of two
controls. In addition, each time that the PCR was performed, negative
and positive control samples were included. In order to minimize
contamination, the reagent setup, the extraction and sample addition,
and the PCR and sample analysis were performed in three separate rooms,
of which the first two rooms were kept at positive pressure and had airlocks.
Reverse line blot hybridization.
The reverse line blotting
technique has been described before (11, 12, 25). Briefly,
solutions with 5' amino-linked oligonucleotide probes ranging from 10 to 800 pmol were coupled covalently to an activated Biodyne C membrane
in a line pattern by using a miniblotter (Immunetics, Cambridge,
Mass.). After binding of the oligonucleotide probes the membrane was
taken from the miniblotter, washed in 2× SSPE (360 mM NaCl, 20 mM
Na2HPO4 · H2O, 2 mM EDTA)
with 0.1% sodium dodecyl sulfate (SDS) at 60°C, and again placed in
the miniblotter with the oligonucleotide lines perpendicular to the
slots. Ten microliters of the biotin-labeled PCR product was diluted in
150 µl of 2× SSPE-0.1% SDS, denatured for 10 min at 99°C, and
cooled rapidly on ice. The slots of the miniblotter were filled with
the denatured PCR product, and hybridization was performed for 1 h
at 42°C. The membrane was removed from the miniblotter and was washed
twice for 10 min each time in 2× SSPE-0.1% SDS at 51°C.
Subsequently, the membrane was incubated for 30 min at 42°C with
streptavidin-peroxidase (Boehringer Mannheim GmbH, Mannheim, Germany)
diluted 1:4,000 in 2× SSPE-0.5% SDS and was washed twice for 10 min
in 2× SSPE-0.5% SDS. Hybridization was visualized by incubating the
membrane with enhanced chemiluminescence detection liquid (Amersham
International plc, Den Bosch, The Netherlands) and exposing the
membrane to X-ray film (Hyperfilm; Amersham). For species
identification the biotinylated Ehrlichia PCR product was
hybridized with seven different oligonucleotide probes in the reverse
line blot assay. Similarly, the biotinylated spacer fragment of the
Borrelia PCR was hybridized with five B. burgdorferi genospecies-specific oligonucleotide probes. For
identification of Bartonella species a region between bases
964 and 1243 of the 16S rRNA gene was amplified and was used in a
reverse line blot assay. All primers and probes are described in Table
1.
DNA sequencing and data analysis.
The PCR products used for
DNA sequencing were purified with Qiaquick PCR purification kits
(Qiagen, Hilden, Germany). For DNA sequencing reactions, the
fluorescence-labeled dideoxynucleotide technology was used
(Perkin-Elmer, Applied Biosystems Division). The sequenced fragments
were separated, and data were collected on an ABI 377 automated DNA
sequencer (Perkin-Elmer, Applied Biosystems Division). The collected
sequences were assembled, edited, and analyzed with the DNAStar package
(DNAStar Inc., Madison, Wis.). The phylogenetic tree was constructed by
using the Clustal analysis in the Megalign module of the DNAStar package.
Nucleotide sequence accession number.
The 16S rRNA gene
sequence of the Ehrlichia-like organism found in this study
is available in the GenBank database under accession no. AF104680.
| |
RESULTS |
Sensitivity of the PCR for Ehrlichia and
Borrelia.
The sensitivity of the PCR for
Ehrlichia and Borrelia was assessed by spiking
the samples with known concentrations of previously produced PCR
products. Extracts from ticks that were negative by previous PCRs were
spiked with serial dilutions of E. phagocytophila or
B. burgdorferi PCR products. All experiments were performed in duplicate. Repeatedly, the detection limit for both assays in which
the PCR yielded a positive result was five copies of the target
sequence (data not shown). The detection limit of the PCR for
Bartonella has been determined in another study and was shown to correspond to one genome copy (4).
Specificity of the reverse line blot hybridization.
A reverse
line blot hybridization assay was designed to differentiate the various
Ehrlichia, B. burgdorferi, and
Bartonella species. Some of the Ehrlichia species
differ by only one nucleotide in the target sequence (Fig.
1). The oligonucleotide probes were designed in such a way that the melting temperature of all
oligonucleotides was approximately 55°C under the conditions used. As
a result the oligonucleotide probes differ in length. In order to
obtain specific and sensitive signals in the assay the optimal
oligonucleotide probe concentrations and hybridization conditions were
determined empirically. To assess the specificity of the assay, control
samples were amplified and used in the reverse line blot hybridization assay under stringent hybridization conditions. Figure
2 shows the reactivities of the control
samples for Ehrlichia, B. burgdorferi, and
Bartonella species in the optimized reverse line blot assay. No cross-hybridization between the various species occurred, indicating that the system distinguished target sequences that differed by only a
single base pair.
View larger version (8K):
[in this window]
[in a new window]
|
FIG. 1.
Multiple alignment of the variable part of the 16S rRNA
gene sequences of the E. phagocytophila group. The region
where differences were detected is shown for E. phagocytophila, E. equi, HGE, and the variants of
E. phagocytophila and HGE. The positions of the residues
that differ in the 16S rRNA gene are shown below the multiple
alignment. Residues identical to those of the E. phagocytophila sequence are indicated by a dot. The sequence of
E. equi was obtained from GenBank (accession no. M73223).
The 500 bp of the 5' end of the 16S rRNA gene sequences of E. phagocytophila and HGE were determined in this study and compared
with the sequences in the GenBank and EMBL database and were found to
be identical to published sequences (accession nos. M73320 and U02521,
respectively). The 16S rRNA sequences of the E. phagocytophila variant and the HGE variant were determined in this
study.
|
|
View larger version (47K):
[in this window]
[in a new window]
|
FIG. 2.
Reverse line blot hybridization assay analyses for the
detection and identification of Ehrlichia, B. burgdorferi, and Bartonella spp. in ticks. (A) Membrane
carrying Ehrlichia-specific oligonucleotide probes; (B)
membrane carrying B. burgdorferi genospecies-specific
probes; (C) combined membrane carrying probes for Ehrlichia,
B. burgdorferi, and Bartonella species. The
oligonucleotide probes are attached to the membrane in the horizontal
direction, and the PCR samples were applied perpendicularly in the
vertical direction. The numbered lanes represent tick-derived PCR
products, and the lanes marked with letters show the PCR products
obtained from the positive control samples. El,
Ehrlichia-like; Eca, E. canis; Ech, E. chaffeensis; Epv, E. phagocytophila variant; Hv, HGE
variant; Ep, E. phagocytophila; H, HGE; Bs, B. burgdorferi sensu stricto; Bg, B. garinii; Ba, B. afzelii; Bv, B. valaisiana; Bh, B. henselae;
Bq, B. quintana.
|
|
PCR detection of Ehrlichia, Borrelia, and
Bartonella DNAs in ticks.
The various PCRs were
applied to DNA extracts from 121 I. ricinus ticks collected
from 38 different roe deer. The majority of the ticks were adults,
mainly females; and some were nonengorged, some were semiengorged, and
some were fully engorged (Table 2). Regardless of whether these PCRs yielded a visible fragment on agarose
gels, all samples were analyzed by the reverse line blot assay.
Fifty-four of the 121 samples (45%) reacted with one or more of the
Ehrlichia-specific probes (Table 2). Of these, 3 reacted
with the HGE-specific probe, 3 reacted with the E. phagocytophila-specific probe, 11 reacted with the HGE
variant-specific probe, 9 reacted with the E. phagocytophila
variant-specific probe, and 7 reacted with both the HGE
variant-specific and the E. phagocytophila variant-specific probes. In addition, 19 of the samples reacted solely with the Ehrlichia genus-specific probe. Sixteen of the same 121 tick
samples (13%) reacted in the Borrelia PCR hybridization
assay with the B. burgdorferi species-specific probes (Table
2). Coinfection with two different B. burgdorferi
genospecies was detected in four tick samples. None of the ticks
analyzed was infected solely with B. garinii or with
B. burgdorferi sensu stricto. However, these genospecies
were found in ticks coinfected with different genospecies.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Results of reverse line blot assay analysis of PCR
products obtained from 121 ticks with Ehrlichia-specific and
Borrelia-specific oligonucleotide probes
|
|
The 121 ticks were collected from 38 roe deer, which implies that
several ticks originated from the same animal. The distribution of the
Ehrlichia and Borrelia species found in the ticks
and their origins are displayed in Table
3. These results indicate that ticks
collected from the same roe deer carried a variety of
Ehrlichia and Borrelia species. This suggests
that these bacterial species did not originate from the roe deer only
but must have been taken up by the ticks during previous feeds on other
animals.
Five of the tick samples carried various Ehrlichia and
B. burgdorferi species, indicating the occurrence of
coinfection with these two pathogens (Table
4). Hybridization of the
Bartonella PCR products yielded hybridization signals with
the Bartonella genus-specific probe in 73 of the 121 samples
(60%). However, none of these PCR products reacted with either the
B. henselae-specific or the B. quintana-specific
oligonucleotide probes.
DNA sequence analysis.
In order to confirm the results
obtained by the reverse line blot assay, 15 of the
Ehrlichia-positive samples were also analyzed by DNA
sequencing. Sequencing of the PCR product obtained by PCR for
Ehrlichia revealed that we had correctly identified the
various species by the reverse line blot hybridization assay.
Furthermore, sequence analysis of the samples that reacted with both
the HGE variant-specific and the E. phagocytophila
variant-specific probes revealed an ambiguous nucleotide at position
100 in the 16S rRNA gene. Cloning of these PCR products and subsequent
reverse line blot assay analysis and DNA sequencing of the clones
revealed the presence of two types of cloned 16S sequences, indicating that these tick samples indeed carried a mixture of two different Ehrlichia sequences.
Nineteen of the PCR products obtained by the PCR for
Ehrlichia reacted with the Ehrlichia
genus-specific probe only. To determine the phylogenetic positions of
these Ehrlichia-like organisms, the complete 16S rRNA gene
sequences of three of these samples were determined. For this purpose
two PCR fragments from each tick sample were generated and sequenced.
The first PCR fragment covered bases 8 through 476 and was amplified
with primer set 16S8FE and B-GA1B. The second fragment was generated by
PCR with oligonucleotides A-EhrAll and 16S1523R and covered the region from positions 203 through 1543 of the 16S rRNA gene. The 16S sequences
of these three samples were identical, and comparison with the DNA
sequences in the GenBank data bank revealed that this 16S rRNA gene
sequence differed markedly from all other published Ehrlichia sequences. The most closely related 16S rRNA gene
sequences were those of Cowdria ruminantium (96%
similarity) and those of members of the monocytic Ehrlichia
group (Fig. 3). For this reason we
designate this species Ehrlichia-like.
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 3.
Dendrogram showing the phylogenetic relationships of the
16S rRNA gene sequences of the newly identified
Ehrlichia-like and those of other rickettsiae. The tree was
constructed by comparing sequences of the segment of the 16S rRNA gene
ranging from bases 40 to 1434 (E. phagocytophila
coordinates). The scale beneath the tree measures the distance between
sequences expressed as the number of substitution events. The sequences
used for comparison were obtained from the GenBank and EMBL database.
|
|
On the basis of the sequence analysis, a new probe (A-Eschot) specific
for this Ehrlichia-like organism was designed for use in the
reverse line blot assay and was used to screen the products obtained
from the tick samples by PCR for Ehrlichia. Hybridization showed that of the 19 samples that initially reacted with the Ehrlichia genus-specific probe only, 8 reacted with the
newly designed probe. Only one of the other
Ehrlichia-positive samples reacted with this probe; this
comprised a tick sample which reacted with both the E. phagocytophila variant-specific probe and the newly designed
probe. As a result, the species infecting 11 of the samples that
reacted with the Ehrlichia genus-specific probe remained undetermined.
Sequencing of 11 of the products obtained by PCR for
Bartonella revealed that none represented B. henselae or B. quintana but closely resembled
Bartonella vinsonii. However, the region of the 16S rRNA
gene that was used for the PCR for Bartonella does not carry
enough variation to reliably distinguish B. vinsoni from
other closely related Bartonella and Rhizobium species.
| |
DISCUSSION |
We developed a PCR-based reverse line blot hybridization assay in
which Ehrlichia, B. burgdorferi, and
Bartonella species can be detected and differentiated. The
assay was specific enough to detect single-base-pair changes with
immobilized oligonucleotide probes and enabled us to differentiate
Ehrlichia variants. The reverse line blot technique is a
relatively easy and rapid method for the simultaneous detection and
identification of microorganisms in field samples such as ticks. In its
present form we can combine the hybridization of PCR products obtained
in separate PCRs. We are now developing a multiplex PCR that will
enable us to have an even more convenient method for the screening of
samples. These samples could be tick lysates but could also be other
material such as blood from patients suffering from a febrile disease
with an unknown origin.
In the study presented here we used this method to detect and identify
Ehrlichia and B. burgdorferi species in Dutch
I. ricinus ticks. Analysis of the ticks showed an unexpected
high rate of infection with Ehrlichia species (45%). The
high infection rate may be partly due to the fact that the ticks
originated from roe deer, which may serve as a reservoir for
Ehrlichia. However, there was no significant correlation
between sex and engorgement of the ticks and infection with
Ehrlichia species. In addition, ticks collected from the
same roe deer carried a variety of Ehrlichia and
Borrelia species. This suggests that the ticks may have been infected before feeding on the roe deer and that the
Ehrlichia spp. originated from other reservoirs. In order to
get a more accurate impression of the prevalence of
Ehrlichia infection in Dutch ticks, we are now analyzing a
large number of ticks collected from the vegetation. Whatever the
reservoir may be, the results obtained in this survey suggest that
Dutch ticks may pose a serious health threat to both humans and animals
and should be used to warn clinicians to be aware of the possible
presence of ehrlichiosis in The Netherlands.
The majority of the Ehrlichia species found in this study
belong to the E. phagocytophila group. As expected, neither
E. canis nor E. chaffeensis was found in any of
the ticks. Analysis of PCR products revealed that the 16S rRNA gene
sequences of the E. phagocytophila group showed slight
variations. In total, four types of E. phagocytophila-like
sequences were found: species with the E. phagocytophila or
the HGE 16S rRNA gene sequences and two variants of these sequences
that carried a substitution of a single base pair at position 92 of the
16S rRNA gene. This corroborates the findings of a Swedish group
(28) and a group from the United States (2) that
also found Ehrlichia species in which the A at position 92 of the 16S gene was substituted by a G. It remains to be determined
whether the 16S rRNA variants represent different Ehrlichia
species. It is possible that the HGE agent, E. phagocytophila, and the variants found in this study all belong to
the same species and should be designated E. phagocytophila subspecies. Furthermore, it is unclear whether these variants can cause
disease in humans or animals. It was remarkable that in none of the
samples of the E. phagocytophila group from which the 16S
rRNA gene sequences were determined was a C found at position 49 in the
16S rRNA gene. The presence of a C at this position may be
characteristic for E. equi. This would corroborate earlier observations that E. equi was not found in Europe.
More than 6% of the ticks were infected with an
Ehrlichia-like organism not described before. This organism
is closely related to but clearly distinct from the monocytic group of
Ehrlichia species and C. ruminantium. It is
unclear whether this organism can cause disease in mammals, but
experimental infection of animals may confirm its infectious nature.
The newly identified organism may represent an endosymbiont. Examples
of such endosymbionts in ticks are the Francisella and
Wolbachia species, which are found at high rates in
particular tick species (16-18). However, the relatively
low frequency of infection of the ticks would argue against this hypothesis.
Analysis of the 121 ticks showed that 13% of the ticks carried
B. burgdorferi species and confirmed earlier findings that 10 to 35% of the Dutch I. ricinus ticks are infected with
B. burgdorferi genospecies (24). Interestingly, 5 of the 121 ticks were coinfected with Ehrlichia and two
genospecies of B. burgdorferi. Due to its immunosuppressive
nature, coinfection with Ehrlichia and B. burgdorferi may increase the severity of Lyme borreliosis.
Transmission of Bartonella species by ticks is speculative.
However, at least one study reports on three patients with B. henselae bacteremia. These patients had no history of contact with
cats but sustained tick bites prior to the bacteremia (13). From the study presented here it is clear that a large proportion of
the ticks carry Bartonella species or species closely
related to Bartonella but not the human pathogens B. henselae and B. quintana. The Bartonella
species found might originate from small rodents on which the ticks may
have been feeding. This could indicate that transmission of
Bartonella species between rodents is, at least in some
part, tick mediated. Further studies with other arthropods such as body
lice and perhaps also blood from rodents such as rats may disclose the
reservoirs and vectors for B. quintana.
Until now there have been no reports of ehrlichiosis in Dutch patients.
Therefore, the high rate of infection of Dutch ticks with
Ehrlichia species raises the question of whether human
ehrlichiosis does occur in The Netherlands. It is known that
Ehrlichia species cause infections in cattle, sheep, and
dogs in Europe. However, until now there have been very few reports on
human ehrlichiosis in Europe (15, 20, 27). In fact, only
recently was the first case of granulocytic ehrlichiosis infection
reported, and that was in Slovenia (20). Although the
seroprevalence in several European serosurveys suggest that infections
with Ehrlichia do occur in Europe, there seems to be a
paucity of reported cases. There may be several explanations for this
phenomenon. First, it is possible that there really are very few cases
of human ehrlichiosis. Second, the majority of cases may go unnoted
because they are caused by less virulent variants of HGE that result in
a mild course of disease. Finally, cases of ehrlichiosis may remain
unnoted because clinicians do not recognize the disease. Relatively few clinicians know that the disease exists and therefore cannot make the
correct diagnosis. Furthermore, the tools used to diagnose ehrlichiosis
are usually lacking. Very few laboratories in The Netherlands are
equipped to perform serology studies for Ehrlichia, and PCR
is performed in none of these laboratories. Therefore, at least in The
Netherlands, ehrlichiosis may have been overlooked. Recently, a Swedish
group reported on three PCR-confirmed cases of HGE infection in humans
(PROMED file 980418193622). Two of the three patients were
seronegative, which forewarns us that serology may not suffice for the
diagnosis of ehrlichiosis. The patients showed a variety of clinical
symptoms, of which only fever and headache were seen in all three
patients. Remarkably, the initial diagnosis for one of the patients was
neuroborreliosis, and the patient was treated for this condition. These
findings indicate that HGE infections do occur in Europe and suggest
that there may indeed be an underdiagnosis of ehrlichiosis and that surveillance is required to determine the true extent of the problem.
| 1.
|
Anderson, B. E.,
J. E. Dawson,
D. C. Jones, and K. H. Wilson.
1991.
Ehrlichia chaffeensis, a new species associated with human ehrlichiosis.
J. Clin. Microbiol.
29:2838-2842[Abstract/Free Full Text].
|
| 2.
|
Belongia, E. A.,
K. D. Reed,
P. D. Mitchell,
C. P. Kolbert,
D. H. Persing,
J. S. Gill, and J. J. Kazmierczak.
1997.
Prevalence of granulocytic Ehrlichia infection among white-tailed deer in Wisconsin.
J. Clin. Microbiol.
35:1465-1468[Abstract].
|
| 3.
|
Bergmans, A. M. C.,
J. F. P. Schellekens,
J. D. A. Vanembden, and L. M. Schouls.
1996.
Predominance of two Bartonella henselae variants among cat-scratch disease patients in The Netherlands.
J. Clin. Microbiol.
34:254-260[Abstract].
|
| 4.
|
Bergmans, A. M.,
J. W. Groothedde,
J. F. Schellekens,
J. D. van Embden,
J. M. Ossewaarde, and L. M. Schouls.
1995.
Etiology of cat scratch disease: comparison of polymerase chain reaction detection of Bartonella (formerly Rochalimaea) and Afipia felis DNA with serology and skin tests.
J. Infect. Dis.
171:916-923[Medline].
|
| 5.
|
Brouqui, P.,
J. S. Dumler,
R. Lienhard,
M. Brossard, and D. Raoult.
1995.
Human granulocytic ehrlichiosis in Europe.
Lancet
346:782-783[Medline].
|
| 6.
|
Chen, S. M.,
J. S. Dumler,
J. S. Bakken, and D. H. Walker.
1994.
Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease.
J. Clin. Microbiol.
32:589-595[Abstract/Free Full Text].
|
| 7.
|
de Mik, E. L.,
W. van Pelt,
B. Docters van Leeuwen,
A. van der Veen,
J. F. P. Schellekens, and M. W. Borgdorff.
1997.
The geographic distribution of tick bites and erythema migrans in general practice in The Netherlands.
Int. J. Epidemiol.
26:451-457[Abstract/Free Full Text].
|
| 8.
|
Dumler, J. S.,
L. Dotevall,
R. Gustafson, and M. Granstrom.
1997.
A population-based seroepidemiologic study of human granulocytic ehrlichiosis and Lyme borreliosis on the west coast of Sweden.
J. Infect. Dis.
175:720-722[Medline].
|
| 9.
|
Everett, E. D.,
K. A. Evans,
R. B. Henry, and G. McDonald.
1994.
Human ehrlichiosis in adults after tick exposure. Diagnosis using polymerase chain reaction.
Ann. Intern. Med.
120:730-735[Abstract/Free Full Text].
|
| 10.
|
Guy, E. C., and G. Stanek.
1991.
Detection of Borrelia burgdorferi in patients with Lyme disease by the polymerase chain reaction.
J. Clin. Pathol.
44:610-611[Abstract/Free Full Text].
|
| 11.
|
Kamerbeek, J.,
L. Schouls,
A. Kolk,
M. Van Agterveld,
D. Van Soolingen,
S. Kuijper,
A. Bunschoten,
H. Molhuizen,
R. Shaw,
M. Goyal, and J. Van Embden.
1997.
Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.
J. Clin. Microbiol.
35:907-914[Abstract].
|
| 12.
|
Kaufhold, A.,
A. Podbielski,
G. Baumgarten,
M. Blokpoel,
J. Top, and L. Schouls.
1994.
Rapid typing of group A streptococci by the use of DNA amplification and non-radioactive allele-specific oligonucleotide probes.
FEMS Microbiol. Lett.
119:19-25[Medline].
|
| 13.
|
Lucey, D.,
M. J. Dolan,
C. W. Moss,
M. Garcia,
D. G. Hollis,
S. Wegner,
G. Morgan,
R. Almeida,
D. Leong,
K. S. Greisen,
D. F. Welch, and L. N. Slater.
1992.
Relapsing illness due to Rochalimaea henselae in immunocompetent hosts: implication for therapy and new epidemiological associations.
Clin. Infect. Dis.
14:683-688[Medline].
|
| 14.
|
Massung, R. F.,
K. Slater,
J. H. Owens,
W. L. Nicholson,
T. N. Mather,
V. B. Solberg, and J. G. Olson.
1998.
Nested PCR assay for detection of granulocytic ehrlichiae.
J. Clin. Microbiol.
36:1090-1095[Abstract/Free Full Text].
|
| 15.
|
Morais, J. D.,
J. E. Dawson,
C. Greene,
A. R. Filipe,
L. C. Galhardas, and F. Bacellar.
1991.
First European case of ehrlichiosis.
Lancet
338:633-634[Medline].
|
| 16.
|
Niebylski, M. L.,
M. G. Peacock,
E. R. Fischer,
S. F. Porcella, and T. G. Schwan.
1997.
Characterization of an endosymbiont infecting wood ticks, Dermacentor andersoni, as a member of the genus Francisella.
Appl. Environ. Microbiol.
63:3933-3940[Abstract].
|
| 17.
|
Niebylski, M. L.,
M. E. Schrumpf,
W. Burgdorfer,
E. R. Fischer,
K. L. Gage, and T. G. Schwan.
1997.
Rickettsia peacockii sp. nov., a new species infecting wood ticks, Dermacentor andersoni, in western Montana.
Int. J. Syst. Bacteriol.
47:446-452[Abstract].
|
| 18.
|
Noda, H.,
U. G. Munderloh, and T. J. Kurtti.
1997.
Endosymbionts of ticks and their relationship to Wolbachia spp. and tick-borne pathogens of humans and animals.
Appl. Environ. Microbiol.
63:3926-3932[Abstract].
|
| 19.
|
Parola, P.,
L. Beati,
M. Cambon,
P. Brouqui, and D. Raoult.
1998.
Ehrlichial DNA amplified from Ixodes ricinus (Acari: Ixodidae) in France.
J. Med. Entomol.
35:180-183[Medline].
|
| 20.
|
Petrovec, M.,
S. L. Furlan,
T. A. Zupanc,
F. Strle,
P. Brouqui,
V. Roux, and J. S. Dumler.
1997.
Human disease in Europe caused by a granulocytic Ehrlichia species.
J. Clin. Microbiol.
35:1556-1559[Abstract].
|
| 21.
|
Pierard, D.,
E. Levtchenko,
J. E. Dawson, and S. Lauwers.
1995.
Ehrlichiosis in Belgium.
Lancet
346:1233-1234[Medline].
|
| 22.
|
Relman, D. A.,
J. S. Loutit,
T. M. Schmidt,
S. Falkow, and L. S. Tompkins.
1990.
The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens.
N. Engl. J. Med.
323:1573-1580[Abstract].
|
| 23.
|
Rijpkema, S.,
D. Golubic,
M. Molkenboer,
N. Verbeek De Kruif, and J. Schellekens.
1996.
Identification of four genomic groups of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in a Lyme borreliosis endemic region of northern Croatia.
Exp. Appl. Acarol.
20:23-30[Medline].
|
| 24.
|
Rijpkema, S.,
J. Nieuwenhuijs,
F. F. Franssen, and F. Jongejan.
1994.
Infection rates of Borrelia burgdorferi in different instars of Ixodes ricinus ticks from the Dutch North Sea Island of Ameland.
Exp. Appl. Acarol.
18:531-542[Medline].
|
| 25.
|
Rijpkema, S. G.,
M. J. Molkenboer,
L. M. Schouls,
F. Jongejan, and J. F. 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 rRNA genes.
J. Clin. Microbiol.
33:3091-3095[Abstract].
|
| 26.
|
Rijpkema, S. G. T.,
R. G. Herbes,
N. Verbeekdekruif, and J. F. P. Schellekens.
1996.
Detection of four species of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from roe deer (Capreolus capreolus) in The Netherlands.
Epidemiol. Infect.
117:563-566[Medline].
|
| 27.
|
Sumption, K. J.,
D. J. Wright,
S. J. Cutler, and B. A. Dale.
1995.
Human ehrlichiosis in the UK.
Lancet
346:1487-1488[Medline].
|
| 28.
|
von Stedingk, L. V.,
M. Gurtelschmid,
H. S. Hanson,
R. Gustafson,
L. Dotevall,
E. O. Engval, and M. Granstrom.
1997.
The human granulocytic ehrlichiosis (HGE) agent in Swedish ticks.
Clin. Microbiol. Infect.
3:573-574.
[Medline] |
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
and
Top
Abstract
Introduction
Present address: Division of Bacteriology, National Institute of
Biological Standards and Control, Potters Bar, EN6 3QG United Kingdom.
