Prevalence of granulocytic Ehrlichiae in Ixodes ricinus ticks inMiddle Germany (Thuringia) detected by PCR and sequencing

of a 16S ribosomal DNA fragment

Anke Hildebrandt a, Karl-Hermann Schmidt a;�, Volker Fingerle b, Bettina Wilske b,Eberhard Straube a

a Institute of Medical Microbiology, University Hospital, Friedrich-Schiller-University, Semmelweisstrasse 4, D-07743 Jena, Germanyb Max von Pettenkofer-Institut, Munich, Germany

Received 22 March 2002; accepted 18 April 2002

First published online 14 May 2002

Abstract

A total of 305 Ixodes ricinus ticks (243 nymphs and 62 adults) were collected from three different regions of Thuringia in MiddleGermany which are known to be endemic for Borrelia burgdorferi. Our aim was to investigate the carrier rate of ticks for granulocyticEhrlichia species. The presence of ehrlichial 16S ribosomal DNA was investigated by polymerase chain reaction. Using primers specific forthe Ehrlichia phagocytophila group PCR fragments of 151 bp and 943 bp, respectively, were produced in positive samples. Adult ticksshowed a significantly higher infection rate (4/62; 6.5%) compared to nymphs (3/243; 1.2%). Prevalence rates varied between 0 and 3.8%regarding the different areas under investigation. The nucleotide sequences showed high similarity (between 97.5% and 99% identity) tothe known sequences of the three E. phagocytophila group members HGE agent, E. phagocytophila and Ehrlichia equi. The sequence datadid not allow a final classification to a particular member of this group. = 2002 Published by Elsevier Science B.V. on behalf of theFederation of European Microbiological Societies.

Keywords: Ixodes ricinus tick; Phagocytophila group; Epidemiological survey; Ehrlichia

1. Introduction

Ehrlichiae are obligate intracellular microorganisms re-siding within cytoplasmic vacuoles of monocytes, granulo-cytes or platelets. Hosts are various mammalian species[8,18,28,38,33]. In contrast to Chlamydiae they are ableto synthesize their own ATP and they di¡er from Rick-ettsiae in multiplying within endosomal compartments ofthe host cells [33,41]. At present the genus Ehrlichia isdivided into three distinct groups according to the nucle-otide sequence homology of the 16S ribosomal RNA genes[33]. As general term the ¢rst characterized group memberis determining: Ehrlichia canis group (E. canis, Ehrlichiacha¡eensis, Ehrlichia muris, Ehrlichia ewingii), Ehrlichiaphagocytophila group (E. phagocytophila, Ehrlichia equi,

human granulocytic ehrlichiosis (HGE) agent, Ehrlichiaplatys), and Ehrlichia sennetsu group (E. sennetsu, Ehrli-chia risticii) [33]. In 1953, E. sennetsu was the ¢rst reportedehrlichial species found to be pathogenic for humanswhich causes the mononucleosis-like Sennetsu fever [24].While E. canis, described in 1935 [12], seems to be onlypathogenic for dogs, in 1991 [1] E. cha¡eensis was isolatedfrom a patient of Northern America and is now known tocause human monocytic ehrlichiosis (HME). The mainvector was found to be the tick species Amblyoma ameri-canum [2]. In 1994, a granulocytic Ehrlichia, named HGEagent, could be identi¢ed to be responsible for humangranulocytic ehrlichiosis [3,9]. The HGE agent is geneti-cally closely related if not identical to E. equi and E. phag-ocytophila, both members of the E. phagocytophila group.Granulocytic Ehrlichiae appear to be transmitted by ticksnot only in the USA [3,9^12,21^23,25,35,38] but also inEuropean areas where other diseases like Lyme borreliosisor tick-borne encephalitis are endemic [5^8,10,14,19,20,26,29^31,36]. The present study was performed to collect dataabout the prevalence of granulocytic Ehrlichia species in-

0378-1097 / 02 / $22.00 = 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 6 8 5 - 7

* Corresponding author. Tel. : +49 (3641) 93 42 23;Fax: +49 (3641) 93 34 74.E-mail address: karl-hermann.schmidt@med.uni-jena.de

(K.-H. Schmidt).

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www.fems-microbiology.org

fected ticks in three di¡erent places of Thuringia in MiddleGermany. The results of the present investigation contrib-ute to the knowledge of the carrier rate of ticks for Ehr-lichiae in Germany.

2. Materials and methods

2.1. Collection of ticks

A total of 28 males, 34 females and 243 nymphs of thespecies Ixodes ricinus were collected from three di¡erentplaces in the area of the Ilm Valley (Thuringia) in MiddleGermany: Belvedere near Weimar, Bad Berka (13 km up-river to the south), and Dienstedt (additionally 17 kmsouthwards). The ticks were collected from September1999 till October 2000. After £agging the ticks were storedin a 1.5-ml reaction tube at 380‡C.

2.2. Extraction of DNA from ticks

Ticks were washed with 70% ethanol and sterile aquadest. Dissected internal organs (adults) or whole ticks(nymphs) were crushed with sterile needles and suspendedin 200 Wl sterile phosphate bu¡ered saline (PBS). For eachpreparation new instruments had to be used to avoid con-taminations between the ticks. DNA extraction was car-ried out with the High Pure PCR Template PreparationKit (Roche Diagnostics GmbH, cat. No. 1796828). Thehomogenates were incubated at 55‡C overnight with200 Wl lysis bu¡er and 40 Wl (concentration 20 mg ml31)proteinase K.

The next day 200 Wl binding bu¡er were added and after10 min the mixture was completed with 100 Wl isopropa-nol. The sample was transferred to High Pure FilterTubes. Washing of bound DNA and elution was per-formed according to the instructions of the manufacturer.The isolated DNA was stored at 380‡C until further use.

2.3. DNA ampli¢cation by PCR

Ampli¢cation targeting the 16S rDNA of granulocyticEhrlichiae was performed with the following primers:GER3 (5P-TAG ATC CTT CTT AAC GGA AGGGCG-3P, forward) and GER4 (5P-AAG TGC CCG GCTTAA CCC GCT GGC-3P, reverse) for a 151-bp fragment[15,17], and e16Slarge.fw (CAA GCT TAA CAC ATGCAA GTC GAA C, forward) and e16Slarge.rv (CCCTTC CGT TAA GAA GGA TCT AAT C, reverse) fora 943-bp fragment. Primers e16Slarge.fw and e16Slarge.rvwere deduced from sequences of the NCBI sequence database described for species of the E. phagocytophila group(Fig. 1). DNA from a HGE patient isolate (courtesy fromU. Munderloh, University of Minnesota) served as posi-tive control, PBS and sterile aqua dest. served as negativecontrol.

Each PCR mixture contained 8 Wl dNTPs (1.25 mM),5 Wl 10Ubu¡er (with 50 mM KCl, 2 mM MgCl2), 0.5 Wlprimer forward, 0.5 Wl primer reverse, 0.25 Wl Taq poly-merase and 5 Wl isolated DNA as template. Finally, sterileaqua dest. was added to a reaction volume of 50 Wl.

Ampli¢cation was performed in a thermal cycler(Perkin-Elmer GeneAmp PCR-System 2400). DNA wasinitially denatured for 5 min at 94‡C followed by 40cycles of the three following steps: 1 min of denaturationat 94‡C, 1 min of annealing at 50‡C for the shortfragment, at 51‡C for the large fragment, and 1 min ofextension at 72‡C. The ¢nal extension was for 7 min at72‡C.

The 151-bp PCR products were separated by electro-phoresis in 2% agarose gel, the 943-bp PCR products in1.5% agarose gel, stained with SYBR-Green (Biozym Di-agnostic GmbH, Germany) and visualised under UV light.The bands were cut out under UV light control and ex-tracted with the QIAquick Gel Extraction Kit (QiagenGmbH, cat. No. 28706).

2.4. Cloning of the PCR products

The 151-bp PCR fragments were ligated into the plas-mid vector pGEM-T overnight at 4‡C using the pGEM-T0 Easy Vector System Kit (Promega Corp., Madison,WI, USA) and transformed in Escherichia coli TG1 com-petent cells. Some of the 943-bp PCR fragments were li-gated into the plasmid vector pGEM-T0 like the shortfragment and some others into the plasmid vector pDrive0

using the Qiagen PCR Cloning Kit (Qiagen GmbH, Ger-many cat. No. 231122) and transformed in E. coli TG1cells or EZ competent cells enclosed in the appropriatecloning kit.

The cells containing the plasmid with insert were se-lected by blue^white colour screening on Luria^Bertani(LB) agar added with ampicillin, isopropyl L-D-thiogalac-toside (C9H18O5S) and 5-brom-4-chlor-3-indoxyl L-D-gal-actoside (C14H15BrCINO6xF) according to standard pro-tocols. The E. coli cells were grown overnight at 37‡C.From each agar plate 10 white clones were picked andmultiplied in LB £uid medium containing 100 Wg ml31

ampicillin for 3^4 h at 37‡C. From each grown clone200 Wl were stored in glycerine at 320‡C for further ex-aminations. In the next step plasmid DNA was isolatedusing the Seq-Lab Plasmid-Miniprep-Kit (Sequence Labo-ratories Go«ttingen GmbH).

Plasmids were analysed by electrophoresis in 1.5% aga-rose gel, stained with SYBR-Green (Biozym DiagnosticGmbH, Germany) and visualised under UV light. Theinserts of both plasmids were ampli¢ed by PCR with theprimers T7 (5P-TCG CCC TAT AGT GAG TCG TATTA-3P, forward) and SP6 (5P-ATT TAG GTG ACACTA TAG AAT AC-3P, reverse) to detect clones carryingthe inserts, the 151-bp or 943-bp fragments. Both primerswere purchased from Promega.

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2.5. DNA sequencing and data analysis

Both strands of the inserted fragments were sequencedby the £uorescence-labelled didesoxynucleotide technology(Big Dye1 Terminator Cycle Sequencing Ready ReactionKit, PE Biosystems) in an ABI PRISM1 310 GeneticAnalyser (PE Biosystems) using primers T7 and SP6,and, to complete sequencing of the 943-bp fragment, twoadditional primers. The primers P5 (5P-GAT TTA TCGCTA TTA GAT GAG CCT ATG-3P, forward) and P6(5P-CAC AGA GAT AAA AAA TCC CCA CAT TCAG-3P, reverse) were constructed during the sequence ex-periments.

Obtained sequences were compared with known Ehrli-chiae 16S RNA sequences of the NCBI database (http://www.ncbi.nlm.nih.gov).

2.6. Statistics

For statistical analysis, Fisher’s exact test for dichoto-mous variables was performed. P6 0.05 was regarded assigni¢cant.

3. Results

3.1. Prevalence of the E. phagocytophila genogroup inI. ricinus ticks

A total of 305 I. ricinus ticks (28 males, 34 females and243 nymphs) were analysed by PCR for infection withgranulocytic Ehrlichia species. The primers were designedto detect fragments of the 16S ribosomal RNA genomic

Fig. 1. Primer positions outlined from the published sequence of HGE agent (accession No. U02521). Primer pairs ‘GER3/GER4’ and ‘e16Slarge.fw/e16Slarge.rv’ ampli¢ed a 151-bp and 943-bp fragment, respectively. Overlap of primers e16Slarge.rv and GER3 is indicated in bold numbers. P5 and P6were additionally included for sequencing.

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region. We found an average of 2.3% ticks infected withEhrlichia. Di¡erences were obtained in the distribution ofEhrlichia infected ticks comparing the three examinedareas: in Bad Berka a total of 2.8% (4 of 145), in Belve-dere 3.8% (3 of 80) and in Dienstedt 0% (0 of 80) ticks, i.e.altogether 2.3% (7 of 305) ticks were Ehrlichia positive.

A signi¢cantly higher prevalence rate was present inadult ticks (4/62; 6.5%) compared to nymphs (3/243;1.2%) (Table 1).

3.2. Sequence analysis

Sequencing was performed with the plasmids carryingthe short 151-bp and the large 943-bp PCR fragments. Theblast search of the sequenced 16S segments revealed thatthe ticks positive in PCR analysis were carriers of Ehrli-chiae belonging to the E. phagocytophila group.

With the short PCR fragment no clear assignment toone of the species E. equi, HGE agent, E. phagocytophilaor E. platys could be found. For this reason, the sequenc-ing of a larger fragment, the 943-bp fragment, was in-cluded. This fragment was produced with primers e16Slar-ge.fw and e16Slarge.rv. The reverse primer e16Slarge.rvnearly represents the complement strand of the forwardprimer GER3 of the short fragment. Thus, joining bothfragments to a ¢nal size up to 1074 bp, the classi¢cationwithin the phagocytophila group could be slightly im-proved.

Sequence alignment illustrated in the phylogenetic treerevealed that the isolates found in this study did not be-long to E. platys, but are closely related to E. equi, E.phagocytophila, and the HGE agent (Fig. 2). Thus, a de-tailed classi¢cation to a special member of this group alsofailed if a larger fragment of the 16S rRNA was analysed.Between the sequences of the isolates there also occurred

sequence di¡erences (Fig. 3). They were submitted to theGenBank under the following accession numbers: Jena 8(AJ313513), Jena 14 (AJ312941), Jena 16 (AJ313512), Jena22 (AJ312939), Jena 72 (AJ312940), Jena 84 (AJ312942),Jena 238 (AJ313511).

4. Discussion

The study was undertaken to get information about theprevalence of granulocytic Ehrlichia infections in I. ricinusticks collected in three di¡erent regions of Middle Ger-many (Thuringia) belonging to the Ilm Valley, which isknown to be endemic for Borrelia burgdorferi. The pres-ence of Ehrlichia was proved by PCR in a segment of the16S RNA gene and con¢rmed by sequencing of the PCRfragments. Until now, from Germany only little sequenceinformation of Ehrlichia isolates became available. Ehrli-chia species of the phagocytophila group cause humangranulocytic ehrlichiosis, which is associated in most caseswith chills, fever, myalgia, and headache [4,33,37]. Otherclinical symptoms are leucopenia (50%), thrombocytope-nia (92%), or elevated liver enzymes (91%) [4,27,33]. Espe-cially in old people and immunodepressed patients thisinfection may take a fatal course due to multi-organ fail-ure. Sequences of the 16S rDNA and the groESL heatshock operon indicate that the HGE agent is closely re-lated if not identical to granulocytotropic Ehrlichiae in theE. phagocytophila group, i.e., E. phagocytophila and E.equi [17,34].

Recent prevalence reports from several countries in Eu-rope described granulocytic Ehrlichia infected free-living I.ricinus ticks in Switzerland, 0.8% [31], in Slovenia, 3.2%[30], or at the east and west coasts of Sweden, 3.1% and

Table 1Distribution of free-living I. ricinus ticks, according to origin, sex, devel-opmental state, and to the number of Ehrlichia infected ticks

Collecting region E. phagocytophila genogroupPCR positive

number positive (%)

Bad Berka males 13 0 0females 18 1 5.6nymphs 114 3 2.6

145 4 2.8Belvedere males 7 1 14.3

females 7 2 28.6nymphs 66 0 0

80 3 3.8Dienstedt males 8 0 0

females 9 0 0nymphs 63 0 0

80 0 0Total males 28 1 3.6

females 34 3 8.8nymphs 243 3 1.2

305 7 2.3

Fig. 2. Phylogenetic relationships performed with the program DNASISv2.6 between the seven sequences of this study and four sequences ofcharacteristic members of the E. phagocytophila group: HGE agent (ac-cession No. U02521.1), E. phagocytophila (accession No. M73224.1), E.equi (accession No. M73223), and E. platys (accession No. AF286699.1).The sequences of this study appear in the NCBI database under the fol-lowing accession numbers: Jena 238 (AJ313511), Jena 84 (AJ312942),Jena 14 (AJ312941), Jena 22 (AJ312939), Jena 8 (AJ313513), Jena 72(AJ312940), and Jena 16 (AJ313512).

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9.2% [36], in a part of Italy, 24% [10], and 2.2% in twostudies from southern Germany [6,13,14].

Our data from Thuringia in Middle Germany with 2.3%infected ticks (7 of 305 adult and nymphal I. ricinus ticks)show a close correlation to the described infection rate of2.2% in Baden-Wu«rtemberg [6] and in Munich [14] insouthern Germany. In accordance to the results of otherinvestigators [14] Ehrlichiae seem to have a focal distribu-tion in Thuringia, because in Dienstedt, one of the threeexamined areas, no infected ticks were found. In contrastto the ¢ndings in Munich where no infection of a nymphwas detected [14], we found three Ehrlichia positivenymphs (Table 1).

Sequencing of the PCR products directly obtained fromtick extracted material did not produce clear results. Se-quencing was drastically improved after cloning the frag-ments into the plasmids pGEM-T0 or pDrive0.

The 16S rDNA segment which was analysed allowed theassignment of all seven ampli¢cates to the E. phagocyto-phila group, but not a clear subdivision in E. equi, HGEagent or E. phagocytophila. Higher sequence diversity wasonly found to E. platys (Fig. 2). 16S ribosomal RNA genesare relatively well conserved on an evolutionary scale,making them appropriate targets to study the relationshipof phylogenetically distinct bacterial species [40]. However,to separate closely related organisms, genes that evolve ata faster rate such as the groESL operon [34] or the citratesynthase gene [32] had to be taken into account. Alterna-tively other markers such as DNA^DNA hybridisation orphenotypic characteristics may be more suitable [16,39].This is especially true for very recently diverged species[16]. In the case of GE, additional genotypic and pheno-typic characteristics may need to be considered to clarify

the relationship and pathogenic potential of the di¡erentstrains from Europe and the USA that exhibit identical ornearly identical 16S rDNA sequences.

Acknowledgements

We want to thank Wolfram Dorn and Charlotte Flu«gel(Institute of Nutrition and Environment, Jena) for sup-porting the tick collection, Cecilia Hizo-Teufel (Max-von-Pettenkofer-Institute, Munich) for the possibility toteach A.H. the techniques necessary for analysing tickDNA, Ulrike Munderloh (College of Agriculture, Univer-sity of Minnesota, USA) for providing a sample of Ehrli-chia DNA, and Gisela Gaschler (Institute of Medical Mi-crobiology, Jena) for valuable advice. KHS was supportedby grant B307-01022 of the Thu«ringer Forschungsministe-rium TMWFK.

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