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Previous Article | Next Article 
Journal of Clinical Microbiology, November 2001, p. 4172-4174, Vol. 39, No. 11
Department of Microbiology, National Center
of Infectious and Parasitic Diseases, Sofia,
Bulgaria1; Research Laboratory for
Infectious Diseases, National Institute of Public Health and the
Environment, Bilthoven, The Netherlands2;
and Department of Pathology, Division of Medical
Microbiology, The Johns Hopkins University School of Medicine,
Baltimore, Maryland3
Received 26 April 2001/Returned for modification 10 June
2001/Accepted 2 September 2001
Bulgarian Ixodes ricinus ticks were examined for
Ehrlichia and Borrelia coinfection: 34 and 32%
of adult ticks and at least 2 and 10% of nymphs were positive for
these infections, respectively. Coinfections and dual or triple
Borrelia infections were frequent, although Ehrlichia
phagocytophila heterogeneity was minimal. Multiple tick-borne
bacteria coexist in I. ricinus ticks in southeastern Europe.
Lyme borreliosis and human
granulocytic ehrlichiosis (HGE) are emerging infections
sometimes cotransmitted by Ixodes species ticks, including
Ixodes ricinus in Europe (3, 4, 14, 16, 17, 19,
25). Lyme borreliosis is the most common vector-borne disease in
the Northern Hemisphere, but HGE is still poorly investigated and its
geographic range is unclear. Five different species of Borrelia
burgdorferi sensu lato are described in Europe Lyme borreliosis is endemic in Bulgaria (7). Despite the
lack of mandatory Lyme borreliosis reporting in Bulgaria, about 500 cases are reported annually (4 cases/100,000 persons). Similarly, 9%
of Bulgarian patients with tick bites have antibodies to the HGE agent
(8), suggesting that the HGE agent is probably frequent in
tick populations. In the present study, Bulgarian ticks were tested for
B. burgdorferi and the HGE agent, and the findings reveal a
higher prevalence of infection and coinfection than previously reported
in Europe.
In the summer of 2000, I. ricinus ticks were collected by
flagging vegetation in wooded areas near Sofia, Bulgaria. After identification, ticks were processed individually if adults and in
pools of five if nymphs. Each tick or pool was mechanically homogenized
in 10 mM Tris, 1 mM EDTA, 100 µg of proteinase K per ml, and 0.5%
sodium dodecyl sulfate lysing buffer, incubated at 60°C for 1 h,
boiled for 10 min, and treated with 5 M NaCl and hexadecyltrimethylammonium bromide at 65°C for 20 min, followed by
DNA extraction in phenol-chloroform. DNA was precipitated with isopropanol, washed with ethanol, dried, and dissolved in 30 µl of 10 mM Tris (pH 8).
For amplification of Ehrlichia DNA, the
ankA gene PCR, described by Walls et al. (27),
was performed. Each PCR run included a known positive control (E. equi-infected horse neutrophil DNA) and a negative control (water
blank). For detection of B. burgdorferi sensu lato DNA, a
similar PCR amplification method was used with the
fla gene primers BBSCH31 and BBSCH2 (23). To
confirm ankA PCR amplification, the 16S rRNA gene primers
ge9f and ge10r were used in a PCR conducted in a separate laboratory
(6). For specific detection of B. burgdorferi
sensu lato amplicons, the reverse line blotting technique was performed
as previously described (24).
Six 16S rRNA HGE agent PCR products were cloned into the pCR4-TOPO
(Invitrogen, Inc., San Diego, Calif.) vector and sequenced. Sequences
were compared with 16S rRNA sequences of the HGE agent from Wisconsin
(GenBank accession number U02521). Alignments of 16S rRNA gene
sequences in ticks with other Ehrlichia sequences were
conducted using ClustalX (version 3.5c) and were used to generate
distances and dendrograms.
Unfed I. ricinus ticks (202) were examined for the presence
of the E. phagocytophila genogroup and B. burgdorferi sensu lato DNA. The sex and stage distribution of
ticks by infection are given in Table 1.
Of 112 adult ticks, 38 (34%) and 36 (32%) contained the E. phagocytophila genogroup and B. burgdorferi sensu lato DNA, respectively, and 15 ticks were coinfected. Of 18 nymph
pools, 9 were infected with B. burgdorferi sensu
lato and 2 were coinfected. Of the 17 coinfected samples, 16 were
confirmed with reverse line blot assay (Table
2). The reverse line blotting also
detected Ehrlichia DNA in 5 of 20 B. burgdorferi-only PCR-positive samples, suggesting an even higher
proportion of coinfected ticks (Table 2). Overall, B. afzelii was detected in 19 (17%) of the 112 adult ticks (Table
2). Dual infections with Borrelia species were noted often.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.4172-4174.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
High Prevalence of Granulocytic Ehrlichiae and Borrelia
burgdorferi Sensu Lato in Ixodes ricinus Ticks
from Bulgaria
ABSTRACT
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B.
burgdorferi sensu stricto, Borrelia garinii, Borrelia
afzelii, Borrelia valaisiana, and Borrelia lusitaniae
(2, 5, 11-13, 20, 28). However, the causative agent of
HGE is a granulocytic ehrlichia conspecific with the veterinary
pathogens Ehrlichia phagocytophila and Ehrlichia equi.
TABLE 1.
Results of ankA and fla PCR
amplification for detection of the E. phagocytophila
genogroup and B. burgdorferi sensu lato DNA in I. ricinus ticks
TABLE 2.
Results of the reverse line blot assay of 17 DNA samples
positive by both the Ehrlichia (ankA) and
Borrelia (fla) PCR and 20 samples positive
by the Borrelia (fla) PCR but negative by the
Ehrlichia (ankA) PCR
Only 19 of 50 ankA-positive samples were also amplified with the 16S rRNA gene primers. A higher percentage of coinfected samples (73.3%, 11 of 15) was detected by reverse line blot assay than by the 16S rRNA gene PCR. However, all six sequenced PCR amplicons from ticks were identified as E. phagocytophila group (99.8 to 99.9% identity with the HGE agent [GenBank accession number U02521]) (1).
This study demonstrates coinfection of granulocytic ehrlichiae and B. burgdorferi sensu lato and heterogeneity of B. burgdorferi sensu lato in unfed I. ricinus ticks in Eastern Europe. Although E. phagocytophila group species have been frequently detected in I. ricinus ticks in Europe (3, 9, 14, 15, 18, 22, 24, 26), the 33.9% E. phagocytophila group tick infectivity rate in this study is high and potentially explains the rate of HGE seropositivity with undifferentiated febrile illnesses after tick bites in Bulgaria (8). Based on minimal 16S rRNA gene sequences, heterogeneity of the ehrlichiae appears minimal. The 13% prevalence of coinfection with B. burgdorferi sensu lato in adult ticks from Bulgaria is higher than reported previously in Europe and is similar to the 1.9 to 29.6% rates demonstrated in the United States (10, 11, 21, 22). The prevalence of coinfected ticks supports findings where 9.7% of Bulgarian patients with early Lyme borreliosis had serological evidence of HGE (8).
The adult ticks in this study demonstrated a prevalence of B. burgdorferi sensu lato similar to that of granulocytic ehrlichiae. Nymphs had a minimal infection rate of 10%, which is intermediate in prevalence compared with European rates that vary from 2 to 43% for nymphs and from 3 to 58% for adults. B. afzelii was the predominant (17%) genospecies detected in the Bulgarian adult ticks. A few ticks were infected with each of the other B. burgdorferi sensu lato, and dual Borrelia infections were found, including two cases of infection with B. valaisiana and B. afzelii and cases of infection with B. lusitaniae and B. garinii in one adult and two nymphal pools. One tick was infected with a B. afzelii-like species recently detected in ticks from St. Petersburg, Russia (1).
The results show that a high proportion of ticks infected with the E. phagocytophila genogroup and B. burgdorferi sensu lato are present in Bulgaria and southeastern Europe. These findings in Bulgarian ticks should alert southeastern Europe to the possibility of human infections. Moreover, since I. ricinus is frequently infected with both pathogens, simultaneous HGE and Lyme borreliosis is probably not uncommon. Thus, every case of Lyme borreliosis with atypical clinical manifestations (29) should be carefully examined for the possibility of concurrent HGE.
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ACKNOWLEDGMENTS |
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This work was supported in part by grant L 909/2000 from the Ministry of Education and Science, Sofia, Bulgaria; grant R01 AI44102-01A1 from the National Institutes of Health, Bethesda, Md.; and by the National Institute of Public Health and the Environment, Bilthoven, The Netherlands.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Medical Microbiology, Department of Pathology, The Johns Hopkins Medical Institutions, Meyer B1-193, 600 North Wolfe St., Baltimore, MD 21287. Phone: (410) 955-5077. Fax: (410) 614-8087. E-mail: sdumler{at}jhmi.edu.
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