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Keywords:

  • 16S rRNA gene;
  • Anaplasma phagocytophilum;
  • ankA gene;
  • groESL operon;
  • Ixodes persulcatus;
  • Ixodes ricinus;
  • prevalence;
  • ticks

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Clin Microbiol Infect 2012; 18: 40–46

Abstract

Anaplasma phagocytophilum is associated with diseases of goats, sheep, cattle, dogs and horses. In the beginning of the 1990s it was identified as a human pathogen, causing human granulocytic anaplasmosis (HGA) in the USA, Europe and the far east of Russia. A. phagocytophilum is maintained in nature in an enzootic cycle including ticks as the main vector and a wide range of mammalian species as reservoirs. Ixodes ricinus and I. persulcatus ticks were collected in Estonia, Belarus and the European part of Russia and screened for the presence of A. phagocytophilum by real-time PCR. Positive samples were found only among I. ricinus, in 13.4% in the European part of Russia, 4.2% in Belarus, 1.7% in mainland Estonia and 2.6% on Saaremaa Island. Positive samples were sequenced for partial 16S rRNA, groESL and ankA genes and phylogenetic analyses were performed. The results showed that A. phagocytophilum circulating in Eastern Europe belongs to different groESL lineages and 16S rRNA gene variants and also consists of variable numbers of repetitive elements within the ankA gene.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects leucocytes and multiplies in cytoplasmic vacuoles [1]. It has been identified as a human pathogen, causing human granulocytic anaplasmosis (HGA), since 1994, when the first human case was reported in the United States [2]. In Europe, the numbers of human cases are continuously increasing and HGA has been reported from Austria, Italy, the Netherlands, Spain, Poland, Slovenia, Sweden, Estonia and the far east of Russia [2]; however, to our knowledge no HGA cases in the European part of Russia and Belarus have been reported up to date. In spite of absent or low numbers of clinical cases in Europe, HGA-seroconversion was reported in 11.3% of the population in Belarus (N Mishaeva, unpublished data), in approximately 6% of the population in European countries [3] following tick bites, and in 8.3% of Slovenian forestry workers in areas endemic for tick-borne diseases [4].

A. phagocytophilum is maintained in nature in an enzootic cycle including ticks as the main vector and a wide range of mammalian species as reservoirs. I. ricinus ticks act as the main vector of A. phagocytophilum in Europe, while I. persulcatus is the vector in Asia, the Urals, Siberia, the Far East, and in the Russian Baltic region. In Europe, large wild mammals have been suggested to act as reservoir hosts, amongst them mainly roe deer and red deer [5], but also small mammals and rodents [6]. Different strains, variants and lineages of A. phagocytophilum circulate widely in natural foci and display different host and/or vector tropism and pathogenicity [5,7–9].

For genetic characterization of A. phagocytophilum, small subunits of rRNA (16S rRNA), a heat-shock protein (groESL) or the AnkA protein (ankA) genes have been utilized. Although the similarities between the 16S rRNA gene sequences are high, several 16S gene rRNA variants have been reported [9–11]. Sequences of the groESL and the ankA genes are more diverse and thereby more reliable for genetic classification [5,12].

The objectives of the present study were detection of A. phagocytophilum in ticks collected in Estonia, the European part of Russia and Belarus and genetic characterization by sequencing of the partial 16S rRNA gene, the groESL operon and the ankA gene.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Tick collection

In 2006–2008, I. ricinus and I. persulcatus ticks were collected by flagging vegetation at 14 sites on mainland Estonia, ten sites on Saaremaa island, four sites in the European part of Russia (St Petersburg surroundings, Curonian Spit, Borok and Tcherepovets) and at seven sites in Belarus (Minsk surroundings) (Fig. 1). Tick species were identified by morphological criteria independently by two entomologists. Ticks were homogenized in 300 μl of PBS by TissueLyzer (Retsch, Haan, Germany). Two hundred microlitres of suspensions were used for DNA extraction.

image

Figure 1.  Map of Estonia, the European part of Russia and Belarus where positive ticks were collected; percentages show the prevalence of A. phagocytophilum in the tick population. I. ric, Ixodes ricinus ticks; I. per, Ixodes persulcatus ticks.

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DNA extraction

DNA was extracted by the guanidinium thiocyanate-phenol-chloroform method using the TriPure isolation system (Roche Diagnostics, Lewes, UK) according to the manufacturer’s recommendations. Sterile water was included as a negative control for every DNA preparation set.

Real-time PCR

Samples were screened for the presence of A. phagocytophilum by real-time PCR using ApMSP2f and ApMSP2r primers, and an ApMS2-FAM probe as described by Courtney [13].

PCR amplification of the partial groESL, the 16S rRNA and the ankA genes

Twenty-one samples positive for A. phagocytophilum by real-time PCR were chosen for the partial groESL, the 16SrRNA and the ankA gene amplification.

Amplification of the partial groESL operon PCR was performed with primers HS1 and HS6 for the first PCR and HS43 and HSVR for the nested PCR as described by Sumner [14] and Lotric-Furlan [15].

The partial 16S rRNA gene was amplified with primers Ehr1 and Ehr6 for the first PCR and Ehr7 and Ehr8 for the nested PCR as described by Rar [16,17].

The 3′ portion of the ankA gene was amplified as described by von Loewenich and Massung [12,18]. Primers 1F and 4R1 were used for the first PCR and the product of this reaction was used for nested reactions with the following sets of primers: 1F1 and 4R1mod, 2F1 and 2R1, and 3F1 and 4R1mod.

To avoid contamination, the extraction of DNA, the preparation of the master mixes and the PCR were performed in separate rooms.

DNA sequencing

PCR products were purified by the GFX™ PCR DNA and Gel Band purification kit (GE Healthcare UK Limited, Little Chalfont, Buckinghamshire, UK). The BigDye® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Forest City, CA, USA) was used for the DNA sequencing reaction, according to the manufacturer’s recommendations, followed by sequencing on a 3100 DNA automated sequencer (Applied Biosystems). The obtained sequences were edited using the BioEdit program (http://www.mbio.ncsu.edu/BioEdit/bioedit.html), and aligned by the ClustalW multiple alignment option with the corresponding region of sequences retrieved from GenBank.

Phylogenetic analysis

The Maximum Likelihood model was used for phylogenetic tree reconstruction of the partial groESL operon (1160 bp) and partial ankA (1591–1672 bp) using the Tree Puzzle program and 10 000 puzzling steps were applied using the Hasegawa–Kishino–Yano (HKY) model of substitutions. The transition/transversion ratio and nucleotide frequencies were estimated from the dataset.

Identical sequences were excluded from the phylogenetic analyses and the sources and places of their origin are shown within parenthesis in the phylogenetic trees (Figs 2 and 3).

image

Figure 2.  Phylogenetic tree (Maximum Likelihood) based on the partial sequence of groESL operon (nt 423–1679). Only bootstrap values >70% (derived from 10 000 replicates of neighbour-joining trees estimated under the ML substitution model) are shown. Source and place of detection of identical sequences are shown in parentheses. Samples sequenced in the present study are indicated in bold and underlined.

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image

Figure 3.  Phylogenetic tree (Maximum Likelihood) based on the partial sequence of ankA. Only bootstrap values >70% (derived from 10 000 replicates of neighbour-joining trees estimated the under ML substitution model) are shown. Source and place of detection of identical sequences are shown in parentheses. Samples sequenced in the present study are indicated in bold and underlined.

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Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

A total of 3976 ticks were collected in Estonia, among them 2474 I. ricinus from mainland Estonia, 345 I. ricinus from Saaremaa island and 1157 I. persulcatus from south-eastern Estonia. In Russia, 477 ticks were collected, among them 82 I. ricinus from Curonian Spit and 395 I. persulcatus from the European part of Russia (St Petersburg, Borok, Tcherepovets). In Belarus, a total of 187 of I. ricinus were collected from the Minsk surroundings.

In our study A. phagocytophilum was found only in I. ricinus ticks even at regions where I. ricinus and I. persulcatus co-circulate. The prevalence of A. phagocytophilum in ticks was detected by real-time PCR and we found that 1.7% of I. ricinus (42/2 474) were infected in seven out of 14 geographical sites in mainland Estonia. On Saaremaa island A. phagocytophilum was detected in nine out 345 ticks, with an overall prevalence of 2.6%. However, positive ticks were found only at four sites (Kamri, Kotlandi, Rootsiküla, Üübide), where the prevalence varied from 6.3% to 12.5%. In Russia, 11 out of 82 (13.4%) I. ricinus collected on the Curonian Spit were found to be positive. In Belarus, A. phagocytophilum was detected in eight out of 187 ticks (4.2%) (Fig 1).

A total of 1262 bp of the groESL heat shock operon were analysed for the 21 samples found positive by the real-time PCR. The nucleotide identity of the sequenced samples was high, from 98.8% to 100%. By analysing the first 402 predicted amino acids of the groEL protein sequence we found amino acid substitutions at position 139 (Glu[RIGHTWARDS ARROW]Arg in two Estonian samples) and at position 398 (Glu[RIGHTWARDS ARROW]Gly in one of the Belarusian samples). According to the substitutions at amino acid position 242, all analysed sequences (21 from this study and 38 retrieved from GenBank) were divided into two groups, one with serine (lineage 1) and another with alanine (lineage 2) at this position. The splitting of A. phagocytophilum into two genetic lineages was confirmed by phylogenetic analysis (Fig 2). On the phylogenetic tree based on the partial groESL operon sequences (1160 bp), lineage 1 subdivided with high bootstrap support into the European and New World lineages, while lineage 2 included only A. phagocytophilum from Europe. According to the information available in GenBank, A. phagocytophilum pathogenic for humans or domestic animals (horses, dogs, sheep) have to date been reported only within lineage 1. The sequences analysed in the present study belonged to both A. phagocytophilum genetic lineages; moreover, lineages 1 and 2 co-circulated at the same sites in Estonia, Belarus and in the Russian part of the Curonian Spit.

The partial 16S rRNA gene (1168 bp) was sequenced for 15 samples out of 21 found positive for the groESL operon gene. We found that the sequences of A. phagocytophilum from Estonia, Belarus and Russia shared a high degree of similarity (98.9–100%). Ten sequences of the analysed samples were identical, and only five samples could be distinguished from each other. A variable region near the 5′end of the 16S rRNA gene at position 76–84 was used for distinguishing the A. phagocytophilum variants [9,11,19,20]. Four variants of A. phagocytophilum were revealed in our study (Table 1). Variant 1 was found in eight samples collected on mainland Estonia, the Russian part of the Curonian Spit and in Belarus; variant 2 was found in one sample from the Estonian island Saaremaa; variant 3 was found in five samples from mainland Estonia, Saaremaa island and the Curonian Spit (Russia); and variant 4 was found in only one sample from Belarus.

Table 1.   16S rRNA gene variants detected in I. ricinus ticks collected in Estonia, the European part of Russia and Belarus. Signature nucleotides for each 16S rRNA gene variant are enboldened
No. of samplesPlace of tick collection16S variantNucleotide positions
767778798081828384
Est3229, Est3238Mainland of Estonia1AAAGAATAG
Rus30-10, Rus30-13, Rus34-7Curonian Split, Russia
Bel 11-2-07, Bel 6-22-07, Bel Bmi11Minsk, Belarus
Est2589Estonia, Saaremaa2AAAGAATAA
Est1090Mainland of Estonia3GAAGAATAA
Est2477, Est2535, Est2540Estonia, Saaremaa
Rus 29-12Curonian Split, Russia
Bel Bmi37Minsk, Belarus4GAAGAATAG

In eight of 21 samples positive for the groESL gene, the partial ankA gene could be amplified. Sequencing of PCR products revealed different lengths of the 3′portion of the ankA gene: 1591 bp for the samples from Belarus (BelBmi11 and Bel6-22-07), 1642 bp for the samples from Estonia and Russia (Bel11-2-07, Rus34-7, Rus30-13, Est3238), 1672 bp for the Belarusian samples (BelBmi37), and 1717 bp for the Estonian sample (Est3239). Sequencing revealed four distinct models of ankA gene organization (Fig 4). In four samples (Bel11-2-07, Rus34-7, Rus30-13 and Est3238) 25-, 27- and 17-amino-acid repeats were found as single copies and 11-amino-acid repeats in two copies. A similar organization of the ankA gene has been described for samples from humans (Sweden and Slovenia) [18], horses (Germany and Switzerland), dogs (Germany and Denmark) and sheep (Germany) (F. D. von Loewenich, unpublished data, GenBank). Two samples (Bel6-22-07 and BelBmi11) showed one copy of 25- and 27-amino-acid repeat, no 17-amino-acid element and two copies of 11-amino-acid repeats. The same ankA gene sequence was found only in one sample from a dog in Austria (F. D. von Loewenich, unpublished data, GenBank accession number GU236826). The sequence for Estonian sample, Est3239, included two copies of a 25-amino-acid repeat, one copy of 27- and 17-amino-acid elements and two copies of an 11-amino-acid repeat. A similar organization has been detected in A. phagocytophilum from ticks in Germany [12], and in dogs from Germany and Switzerland (F. D. von Loewenich, unpublished data, GenBank). The ankA gene sequence of sample BelBmi37 consisted of one copy of 25-, 27-and 11-amino-acid elements, and a 33-amino-acid insertion located directly adjacent to and upstream from a 27-amino-acid repeat. A similar ankA gene organization has been described for A. phagocytophilum from I. ricinus collected in Germany [12] and samples from roe deers in Germany, Slovenia, Spain and Norway (F. D. von Loewenich, unpublished data, GenBank). A. phagocytophilum samples demonstrated 99.8–100% similarity on the nucleotide level within each ankA gene sequence type, while between different ankA sequence types the similarity was only 64.6–95.5%. We found that A. phagocytophilum with a different ankA gene organization may co-circulate in the same geographical region, for example in Estonia (Est3238 and Est3229) and in Belarus (Bel11-2-07, Bel6-2207 and BelBmi37) (Fig 4).

image

Figure 4.  Schematic presentation of the variable numbers of repetitive elements within the ankA gene detected in this study.

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On a phylogenetic tree based on the partial ankA gene, the sequences of A. phagocytophilum formed two lineages (Fig 3) and the distinction between lineages 1 and 2 corresponded well with the lineage splitting based on the groESL phylogenetic tree. Samples of A. phagocytophilum sequenced in both regions (ankA and groESL genes) had serine or alanine for lineage 1 and 2, respectively, at amino acid position 242 of the groEL protein. Similar to the phylogenetic tree based on the partial groESL operon, lineage 1 divided into European and New World sublineages and lineage 2 consisted of A. phagocytophilum sequences detected in Europe.

A. phagocytophilum within lineage 1 and sequenced for the groESL, ankA and the 16S rRNA genes were classified as 16S rRNA gene variants 1 and 2, while variants 3 and 4 were detected within lineage 2.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

In the present study positive samples were found only among I. ricinus, despite the fact that A. phagocytophilum has been detected in 1% of I. persulcatus in a neighbouring region (St Petersburg, Russia) [21]. In Siberia and the Far East, where I. persulcatus is the main vector for A. phagocytophilum, the prevalence varied from 1% in the Ural to 9.6% in Japan [16,22]. However, A. phagocytophilum has previously been reported simultaneously in I. persulcatus and I. ricinus at geographical sites where the two tick species are distributed. Moreover, sequences of the A. phagocytophilum p44/msp2 gene indicated that there are no differences between sequences derived from I. ricinus and I. persulcatus [23]. In Estonia, even at two sites in south-eastern Estonia, where both species of ticks co-circulate and I. ricinus constitutes the minority (10–20%) of the tick population, we found A. phagocytophilum only in I. ricinus; thus, the role of I. persulcatus for the circulation of A. phagocytophilum in Estonia is unclear and further tick collections are necessary.

In present study A. phagocytophilum was detected in 0.9–13.4% of I. ricinus ticks. The reported prevalence in Europe also varied significantly from 0.3% in western France [24] to 17.1% on Norwegian islands [25]. As described earlier, ticks collected at different time-points at the same locality had different rates of A. phagocytophilum infection [26], and differences in prevalence may reflect seasonal or annual variation of tick infection rates, or different sampling, storage and methodical approaches. Therefore comparisons of the prevalence between various countries or different areas should be performed with caution.

Direct comparison of the 16S rRNA genetic variants of A. phagocytophilum lineages based on the groESL and ankA genes is difficult due to the limited number of simultaneously available A. phagocytophilum sequences for all three genes. However, eight samples of A. phagocytophilum sequenced in all three regions (16S rRNA, groESL and ankA) in the present study and 25 sequences published previously [12] showed that A. phagocytophilum sequences formed similar lineages on the groESL and the ankA gene phylogenetic trees. Division into lineages corresponded also with the 16S rRNA genetic variants; variants 1 and 2 clustered within lineage 1, variants 3 and 4 within lineage 2. The A. phagocytophilum belonging to lineage 1 were widely distributed in Europe and the USA and have been detected in humans, ticks, sheep, goats, horses, dogs, red deer and roe deer. Sequences belonging to lineage 2 have been detected only in roe deer and I. ricinus and only in Europe. Previous studies have suggested that strains of A. phagocytophilum acquire different host tropism [5,27] and pathogenicity [8,28–30]. Up to date, A. phagocytophilum with a known pathogenicity for animals and humans have been reported within lineage 1; thus we suggest that the presence of serin at position 242 of groEL may predict a potential to cause disease in humans or domestic animals. The same observation has previously been reported by Petrovec [5].

In our study we found that A. phagocytophilum belonging to different groESL lineages, with different variants of 16S rRNA genes and with different organization of the ankA gene, co-circulated in the same geographical regions in Estonia, Russia and Belarus. We suggest that ticks may harbour all repertoires of A. phagocytophilum that are circulating in this region, while genetic variants of A. phagocytophilum are segregated in specific natural hosts. Thereby ticks may be an optimal source for studies on the diversity of A. phagocytophilum in various geographical regions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

The preliminary results of this work were presented at the EDEN International Conference, Montpellier, France, 10–12 May 2010. The sequences detected in the present study were deposited in the GenBank database under numbers from HQ629901 to HQ629932.

Transparency Declaration

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

This work was supported by the Estonian Science Foundation (Grant ETF 6938), VISBY project 01347/2007, and also by the EU Grant Goce-2003-010284 EDEN (catalogued EDEN0222).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References
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