Tickborne Pathogen Detection, Western Siberia, Russia

Ixodes and Dermacentor ticks harbor Borrelia, Anaplasma/Ehrlichia, Bartonella, and Babesia species.

D. reticulatus is well known as the vector of a canine pathogen, Babesia canis canis (23); Rickettsia spp. (13), Francisella tularensis, and Coxiella burnetii were also found in this tick species (1). Borrelia spp. was also detected in different Dermacentor species, including D. reticulatus, by means of PCR (9) and an indirect immunofluorescence assay (24). Infection of D. reticulatus with Anaplasma/Ehrlichia and Bartonella species was previously unknown in spite of the detection of bacterial DNA in other Dermacentor species (17,25). Little or nothing was known of the genetic variability of the tickborne pathogens in ixodid ticks from Western Siberia (2); consequently, the aim of the present study was to study prevalence and genetic diversity of Borrelia, Anaplasma/ Ehrlichia, Bartonella, and Babesia among I. persulcatus and D. reticulatus ticks in Western Siberia, Russia.

Materials and Methods
Unfed adult I. persulcatus ticks were collected by flagging of lower vegetation in different suburban places of mixed aspen-birch and pine forests of Novosibirsk (55°N, 83°E) (115 ticks) and Tomsk regions (56°N, 85°E) (

PCR Assay
To prevent contamination, we performed DNA isolation, PCR master mix assembly, and amplifications in separate rooms. Aerosol-free pipette tips were also used at each stage. We included negative control reactions with bidistillated water in each experiment at both steps of nested PCR. All reactions were performed in 20 µL reaction mixture containing 67 mmol/L Tris-HCl (pH 8.9), 16.6 mmol/L (NH 4 ) 2 SO 4 , 2 mmol/L MgCl 2 , 0.01% Tween-20, 200 µmol/L each dNTP, 5% glycerol, 0.5 µmol/L specific primers, 2 U Taq DNA polymerase (Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences), and 2 µL tested DNA. Amplification was performed in a Tercic Thermal Cycler (DNA Technology, Moscow, Russia). For the inner reactions, 2 µL of the outer PCR products were added into the reaction mixture. PCR fragments were visualized under UV irradiation after electrophoresis in agarose gels containing ethidium bromide. To control DNA isolation from ticks, we performed PCR on an aliquot of purified DNA with the following universal primers targeted to 18S rRNA gene: forward 5′-AACCTGGTTGATCCTGCCAGTAGT-CAT-3′ and reverse 5′-GAATGATCCTTCCGCAGGTT-CACCTAC-3′ (26).
B. canis canis DNA isolated from blood samples of a dog with clinical signs of babesiosis confirmed by microscopic examination (GenBank accession no. AY527064) and B. microti DNA from Clethrionomys rutilus blood (AY943958) (V. Rar, unpub. data) were used as positive controls.

Sequencing of PCR Products
The PCR products were purified after gel electrophoresis in 1.5%-2% agarose gels with GFX Columns (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer's instructions. Nucleotide sequences of the PCR products were determined by using BigDye Terminator Cycle Sequencing Kit and the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) at the DNA Sequencing Centre of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia. For initial species identification, the nested PCR products were sequenced in 1 direction. Detailed confirmation for each genetic group was performed by sequencing with forward and reverse outer or inner primers as needed.
Nucleotide sequences of PCR products determined in this study were analyzed by BlastN and aligned with ClustalW (29). Phylogenetic analysis was performed with MEGA 3.0 software (30). We used the unweighted pairgroup method with arithmetic mean (UPGMA) and neighbor-joining algorithms with the Kimura 2-parameter model to generate the distance matrix as well as maximum parsimony and minimal evolution with a heuristic search. Bootstrap analysis was performed with 1,000 replications. GenBank accession numbers for the sequences used in the phylogenetic analysis are shown in Figures 2-5.

Results
Infection of I. persulcatus and D. reticulatus with 3 bacterial and 1 protozoan tickborne pathogens in Western Siberia, Russia were studied by nested PCR with genusspecific primers. To control DNA suitability for PCR analysis, we amplified the 18S rRNA gene in 125 of the 127 I. persulcatus samples tested and in 84 of the 87 D. reticulatus ticks studied. Therefore, the 5 samples in which we were unable to amplify tick DNA were excluded from further analysis. Both tick species contained Borrelia and Bartonella DNA, whereas Anaplasma/Ehrlichia DNA was detected only in I. persulcatus, and Babesia DNA was detected only in D. reticulatus ticks (Table).
In 37.6% ± 4.3% (standard deviation) of samples isolated from I. persulcatus and in 3.6% ± 2.0% of samples from D. reticulatus, DNA of B. burgdorferi sensu lato complex was found (Table). The nucleotide sequences of the 5S-23S intergenic spacer (216-237 bp) determined in this study were compared to those of other B. burgdorferi sensu lato sequences. The sequences from I. persulcatus ticks were placed in 2 clades of monophyletic origin, which corresponded to B. garinii and B. afzelii with excellent bootstrap support (99% and 100%, respectively), whereas samples from D. reticulatus were more closely related to B. garinii (Figure 2 (Table). For 2 PCRpositive samples from I. persulcatus, the hydrolysis of the PCR products with the Tru9I restriction endonuclease resulted in 6 fragments of 108, 68, 57, 50, 38, and 20 bp that corresponded to a mixture of patterns C and D (27) and, consequently, 2 species, B. garinii group NT29 and B. afzelii (Table). Among samples obtained from D. reticulatus ticks, 3 contained B. garinii group NT29 DNA, but no other variants were found.
Anaplasma/Ehrlichia DNA was found in 14 I. persulcatus ticks but not in D. reticulatus ticks from different areas of Novosibirsk region. PCR with primers specific to A. phagocytophilum 16S rRNA gene showed the human pathogen DNA in 3 samples, Ip-4, Ip-45, and Ip-68, collected from different areas of Novosibirsk region. The nucleotide sequences of 629 bp of all these samples were identical to each other (GenBank accession no. AY587607) and to the known A. phagocytophilum sequence (AF205140). Nucleotide sequences from 11 other DNA samples were identical to each other (GenBank accession  no. AY587608) and differed from E. muris DNA sequence (U15527) at the single position 91 (C→T). In a phylogenetic tree created by the UPGMA method, both A. phagocytophilum and E. muris sequences evidently formed the distinctive clusters (Figure 3).
Bartonella DNA was detected by using nested PCR with primers that corresponded to the groEL gene in 47 I. persulcatus and 18 D. reticulatus ticks (Table). Comparative analysis of the groEL gene fragment nucleotide sequences of 190 bp showed 2 species, B. henselae and B. quintana, in both tick species. Part of the data is shown in Figure 4. The evidently separated 2 clades, B. henselae and B. quintana, were monophyletic with good statistical support (99% and 90%, respectively).
Babesia DNA was found in 3 D. reticulatus ticks (Dr-2, Dr-4, Dr-5) by nested PCR and was not detected among I. persulcatus studied (Table). The nucleotide sequences of the Babesia 18S rRNA gene fragment of 1,203 bp determined in this study were similar to each other and to the single known full-length B. canis canis nucleotide sequence (GenBank accession no. AY072926). In the phylogenetic tree, nucleotide sequences from Dr-2 and Dr-5 as well as the B. canis canis sequence formed a distinctive cluster that was separated from other B. canis subspecies with excellent bootstrap support ( Figure 5). Direct sequencing of the PCR fragment from the tick Dr-4 showed a mixture of nucleotide sequences with 2 undetermined bases at positions 609 and 610. Diluting DNA 10 times allowed us to determine 2 nucleotide sequences. The first was identical to those from Dr-2 and the second to a B. canis canis sequence found in canine blood from Croatia (AY072926).
UPGMA analysis produced phylogenetic trees (Figures 2-5) that were almost identical to the neighbor-joining trees and results of phylogenetic analysis with maximal parsimony and minimal evolution approaches (trees not shown).

Discussion
I. persulcatus is believed to maintain spirochetes transtadially and to transmit Borrelia to animals (31). Previously, the spirochetelike cells were isolated from I. persulcatus in Barbour-Stoenner-Kelly-H cultural medium (2) and were observed by indirect immunofluorescence assay (24). The nested PCR with subsequent sequencing showed that I. persulcatus contained both B. afzelii and B. garinii DNA (Table) as was previously shown (2,10,32,33). B. garinii appeared to be the prevalent species in I. persulcatus in Western Siberia (33). The B. garinii NT29 group is widely spread not only in Western Siberia but in the Russian Far East (GenBank accession no. AY429014, AY429015), Japan (34,35), and China (36). The nested PCR with subsequent sequencing allowed us to detect DNA of B. garinii group NT29 in 3.6% ± 2.0% of D. reticulatus ticks. Although Borrelia-specific DNA was detected in samples from D. reticulatus, numerous previous attempts to cultivate the living spirochetes were unsuccessful (2). Therefore, the ability of D. reticulatus to transmit Borrelia spp. remains unknown.
E. muris was the prevalent species among Anaplasma/ Ehrlichia and was found in 8.8% ± 2.5% of I. persulcatus Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 11, No. 11, November 2005  ticks in Western Siberia. This finding coincided with the E. muris prevalence (3%-13%) described in Baltic regions of Russia (12) and Siberia (16). The infection rate of I. persulcatus ticks with the human pathogen A. phagocytophilum (2.4% ± 1.4%) was significantly lower than the rate of infection with E. muris. In other regions, the infection rate of I. persulcatus with A. phagocytophilum varied from 1% to 4% in both China and Russia (14)(15)(16)37). The comparison of the Anaplasma 16S rRNA gene fragment nucleotide sequences (Figure 3) showed several genovariants of A. phagocytophilum. In I. persulcatus ticks, 4 types of sequences were found: 3 in China (GenBank accession nos. AY079425, AF205140, AF227954) and 1 in Korea (AF470701). All 3 nucleotide sequences of A. phagocytophilum determined in this study coincided with 1 genovariant from China (AF205140) found only in I. persulcatus ( Figure 3) but not with the A. phagocytophilum isolated earlier in West Ural, Russia (GenBank accession no. AY094353) (38). No correlation was seen between genovariant and specific host or location.
Several tick species, such as deer ticks, I. persulcatus, I. ricinus, and I. pacificus, have been found to harbor Bartonella spp. (17)(18)(19)(20). Thus, PCR with primers specific to the 16S rRNA gene has shown Bartonella DNA in >70% of I. ricinus ticks in the Netherlands (18). Different Bartonella species, including B. henselae, have been detected in 19.2% of questing I. pacificus ticks in California by amplifying and sequencing the gltA gene fragment (17). More recently, B. henselae DNA has been found in 1.5% of I. ricinus ticks removed from humans in northwestern Italy (19) and in 38%-44% of I. persulcatus in Western Siberia (20). The high Bartonella infection rate of I. persulcatus in Western Siberia in 2003 and 2004 coin-cided with our observations from previous years (20). Moreover, both B. henselae and B. quintana were found not only in the 2 tick species studied (Figure 4) but also in Aedes mosquitos (O. Morozova, unpub. data). Only 2 human pathogens, B. henselae and B. quintana, were found in ixodid ticks in Siberia, despite sample collection for 4 years and phylogenetic analysis of all known Bartonella species (Figure 4).
We did not detect Babesia spp. in I. persulcatus. The only species of Babesia detected in D. reticulatus was B. canis canis, which causes babesiosis in dogs (7). D. reticulatus is the only known vector for B. canis canis (23,39). Comparison of the previously known Babesia canis canis 18S rRNA gene nucleotide sequences showed 3 genetic variants of B. canis canis in canine blood from Europe that differed at 2 variable positions 609 and 610 (26,40). Two of these variants were also seen in ticks in Novosibirsk. A new B. canis canis genetic variant that differed in a single nucleotide transition from those previously described was found. To our knowledge, this report is the first to identify nucleotide sequences of B. canis canis in ticks. B. microti was not found among tick samples studied, despite the presence of this human pathogen in small mammals in the same area (V. Rar, unpub. data).
When the 2 tick species were compared, I. persulcatus was more likely than D. reticulatus to be the host for tickborne bacterial infections examined in Western Siberia, Russia. The Borrelia, Anaplasma/Ehrlichia, and Bartonella infection rates for I. persulcatus exceeded those for D. reticulatus (Table). Moreover, Borrelia (10,33) and Bartonella (20) DNA from I. persulcatus could be easily detected in a single PCR, whereas nested PCR was required to detect DNA in samples from D. reticulatus. Neither Anaplasma nor Ehrlichia spp. were found in D. reticulatus. Conversely, Babesia spp. were detected only in D. reticulatus. The infection of unfed adult I. persulcatus and D. reticulatus ticks reflected transtadial transmission of tickborne infectious agents.
The experimentally observed and theoretically expected values of mixed infections of ticks with Borrelia, Ehrlichia, and Bartonella were statistically similar and consistent with independent distribution of these pathogens as previously reported (10). Thus, simultaneous coinfection with Borrelia, Anaplasma/Ehrlichia, and Bartonella found in 2.9% of I. persulcatus ticks slightly exceeded statistical probability of 1.8%. Further studies are required to establish the role of different tick species and biting arthropods as natural vectors of bacterial and protozoan agents. providing the B. burgdorferi sensu stricto strain B31, Edward I. Korenberg for providing the B. afzelii strain Ip-21, Dionysios Liveris for providing the A. phagocytophilum, Nikolay V. Rudakov for providing the A. marginale, and Michael Minnick for providing DNA isolated from B. henselae and B. quintana.
The study was supported in part by grant 02-01-113 from the Russian program "Vaccines of New Generation," grant N51 of the Program of the Integration in Basic Sciences of Siberian Branch of the Russian Academy of Sciences and grant "Fundamental Sciences to Medicine" of the Russian Academy of Sciences.
Dr Rar works in the Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences. Her research interests include molecular epidemiology, tickborne infections, and natural transmission cycles.