VECTOR-BORNE AND ZOONOTIC DISEASESVolume 4, Number 4, 2004© Mary Ann Liebert, Inc.

Research Paper

Semi-Nested PCR Detection of Bartonella henselae inIxodes persulcatus Ticks from Western Siberia, Russia

O.V. MOROZOVA,1 F.C. CABELLO,2 and A.K. DOBROTVORSKY3

ABSTRACT

Questing adult Ixodes persulcatus ticks from Western Siberia, Russia were tested for infection with Bartonellaspp. using seminested PCR assay with primers specific to the groEL gene. The proportion of ticks infected withBartonella spp. was 44% in 2002 (n � 50) and 38% in 2003 (n � 50). Nucleotide sequences of a portion of the PCRproducts corresponded to Bartonella henselae species. Key Words: Bartonella—PCR assay—Ixodes persulcatus.Vector-Borne Zoonotic Dis. 4, 306–309.

giomatosis, or endocarditis (Jacomo et al.,2002). In Russia, Bartonella infections associatedmostly with human body lice have recentlybeen reported (Rydkina et al., 1999; Ignatovichet al., 2001; Tarasevich et al., 2001).

As for many vector-borne disease agents, awide range of mammalian reservoir hosts in-cluding humans and various arthropod vectorssuch as sand flies, fleas, body lice and ticks areinvolved in Bartonella spp. natural cycles (Ja-como et al., 2002). Recently, several tick speciessuch as deer ticks, Ixodes ricinus and Ixodes paci-ficus have been found to harbor Bartonella spp.(Schouls et al., 1999; Eskow et al., 2001; Changet al., 2001; 2002; Sanogo et al., 2003). Thus, PCRwith primers specific to 16S rRNA gene has re-vealed infection with Bartonella spp. or otherclosely related organisms of more than 70% ofIxodes ricinus ticks collected from roe deerCapreolus capreolus in Netherlands (Schouls etal., 1999). Different Bartonella species including

INTRODUCTION

THE GENUS Bartonella contains numerous re-cently described species of intracellular

bacteria, many of which are new and emerginghuman pathogens. Bartonella are aerobic, gram-negative, oxidase-negative, slow-growing andpleomorphic organisms (Jacomo et al., 2002;Zeaiter et al., 2002). The diversity of the genusBartonella, including bacterial species andstrains, animal hosts and vectors, geographicdistribution, and pathogenic properties, ap-pears to be complex; consequently, phyloge-netic studies are needed (Houpikian andRaoult, 2001). Bartonella henselae is the maincausative agent of cat scratch disease (Zeaiteret al., 2002; Eskow et al., 2001). Bartonella quin-tana causes trench fever (Jacomo et al., 2002).Both closely related species, Bartonella henselaeand Bartonella quintana are distributed world-wide and cause fever, bacteremia, bacillary an-

1Institute of Chemical Biology and Fundamental Medicine of Siberian Branch of the Russian Academy of Sciences,Novosibirsk, Russia.

2Department of Microbiology and Immunology, New York Medical College, Valhalla, New York.3Institute of Systematics and Ecology of Animals of Siberian Branch of the Russian Academy of Sciences, Novosi-

birsk, Russia.

306

5387_04_p306-309 12/3/04 4:16 PM Page 306

B. henselae have been detected in 19.2% ofquesting Ixodes pacificus ticks in California byamplification and sequencing of a fragment ofthe gltA gene (Chang et al., 2001). More re-cently, B. henselae DNA has been found usingPCR in 1.5% of I. ricinus ticks removed fromhumans in northwestern Italy (Sanogo et al.,2003).

The taiga tick Ixodes persulcatus is the mostcommon tick species in northeastern Europeand northern Asia. I. persulcatus ticks often bitehumans, transmitting tick-borne encephalitisvirus, Borrelia burgorferi sensu lato (Korenberget al., 2002) and Anaplasma phagocytophilum(Morozova et al., 2002). However, the infectionof I. persulcatus ticks with Bartonella spp. has notbeen previously reported.

The present study was aimed at PCR-baseddetection and identification of Bartonella spp. inIxodes persulcatus ticks in Western Siberia, Russia.

MATERIALS AND METHODS

Unfed adult Ixodes persulcatus ticks were col-lected from vegetation by flagging in suburbanaspen-birch and pine forests near Novosibirsk(55° N, 83° E) in May 2002 and 2003 as previ-ously described (Livanova et al., 2003). Nucleicacids were isolated by lysis of 100 individualticks in 4M guanidine thiocyanate or using theIsoQuick Nucleic Acid Extraction Kit (ORCAResearch, Bothell, WA) according to manufac-turer’s instructions, followed by phenol-chlo-roform extraction and isopropanol precipita-tion overnight. The Bartonella-specific DNAwas detected by seminested PCR with primers(GenoSys Biothechnology, The Woodlands,TX) corresponding to groEL gene (Zeaiter et al.,2002). Reactions were performed in 20 �L con-taining 67 mM Tris-HCl (pH 8.9), 16.6 mM(NH4)2SO4, 2 mM MgCl2, 0.01% Tween-20, 200�M of each dNTP, 5% glycerol, cresol red, 0.5�M of each primers, 1 U Taq DNA polymerase(Institute of Chemical Biology and Fundamen-tal Medicine of Siberian Branch of the RussianAcademy of Sciences, Novosibirsk, Russia) and2 �l of tested nucleic acids isolated from a tick.DNA isolated from B. henselae and B. quintanawere kindly provided by Michael Minnick (Di-vision of Biological Sciences, University of

Montana) and used as positive control. Ampli-fication was performed in Tercic Thermal Cy-cler (DNA Technology, Moscow, Russia) in thefollowing regime: after initial denaturation at95°C for 3 min denaturation at 95°C for 30 sec,annealing at 58°C for 30 sec and extension at72°C for 45 sec. PCR fragments were visualizedunder UV irradiation after electrophoresis in2% agarose gels containing ethidium bromide.Part of the seminested PCR products were sub-cloned into pCRII-TOPO (Invitrogen Life Tech-nologies) according to the manufacturer’s in-structions.

Nucleotide sequences of the PCR productsand the recombinant plasmids with clonedPCR products (Invitrogen Life Technologies)were determined using Big Dye™ dideoxynu-cleotide Terminator kit and the ABI PRISM™310 Genetic Analyzer (Perkin Elmer AppliedBiosystems). Nucleotide sequences were com-pared and aligned using CLUSTALW method(Thompson et al., 1994).

RESULTS

Infection of individual I. persulcatus tickswith Bartonella spp. was detected by semi-nested PCR with primers specific to groEL gene(Fig. 1). Specific PCR product of 231 bp longwas observed in samples isolated from 22 outof 50 ticks (44%) collected in 2002, and in 19 outof 50 ticks (38%) studied in 2003. Totally 41from 100 studied ticks were infected with Bar-tonella spp. Quantities of the PCR products var-ied from minimal hardly detectable (Fig. 1, lane

INFECTION OF TICKS WITH BARTONELLA SPP. IN NOVOSIBIRSK REGION, RUSSIA 307

FIG. 1. Results of the semi-nested PCR with DNA iso-lated from individual Ixodes persulcatus ticks (lanes 1–9),water as negative control (�C) and Bartonella henselaeDNA as positive control (�C). Lane M, Hi-Lo™ DNAmarkers (Minnesota Molecular Inc.).

5387_04_p306-309 12/3/04 4:16 PM Page 307

4) to maximal (Fig. 1, lane 6) comparable withthe corresponding product with DNA of posi-tive control (Fig. 1, lane �C). Besides primerdimers at the bottom of the gel additionalbands didn’t be produced after the seminestedPCR (Fig. 1), consequently, the reaction withthe primers specific to groEL gene (Zeaiter etal., 2002) can be used for detection of BartonellaDNA in I. persulcatus ticks.

To identify Bartonella species the PCR prod-ucts were sequenced. Five from 27 determinednucleotide sequences were registered in Gen-Bank (GenBank accession numbers AY453166-AY453170). The homology level between 27 nu-cleotide sequences determined in this study

varied from 93% to 99% (Fig. 2). CLUSTALWanalysis indicated that all Bartonella isolates de-tected in I. persulcatus ticks belonged to B. hense-lae. One should note that intraspecies homol-ogy level was 88–100% for the B. henselae groELgene fragment from the United States andEurasia (Fig. 2).

DISCUSSION

Molecular evidence continues to accumulatethat Ixodes ticks are capable of harboring Bar-tonella spp. To our knowledge, this study is thefirst report of Bartonella infection detected in I.

MOROZOVA ET AL.308

FIG. 2. Alignment of nucleotide sequences of Bartonella henselae groEL gene fragment. *Conservative nucleotides aremarked in the last lane. Variable nucleotides are shaded. Nucleotide sequences of Western Siberian isolates are markedas GenBank accession numbers AY453167-AY453170. Nucleotide sequences of the fragment from other known Bartonellahenselae strains from the United States and France are marked as FIZZ, URLLY8, CAL-1, 90-615, BhUSA, SA-2.

5387_04_p306-309 12/3/04 4:16 PM Page 308

persulcatus ticks in Russia. The high rate of Bar-tonella infection among unfed adult I. persulca-tus suggested that immature stages of the tickswere naturally infected as result of effectivetransstadial transmission or feeding on infectedvertebrate hosts.

The sensitivity limit of the seminested PCRwas 10 molecules in reaction mixture (unpub-lished observations). Since 1/10 part from to-tal DNA isolated from an individual tick andadded to PCR mixture resulted in detectableproduct, then amounts of the bacterial cells ex-ceeded 100 in each PCR-positive tick. Conse-quently, Bartonella infection rate among indi-vidual I. persulcatus ticks in Western Siberia,Russia may even be higher than our first esti-mation since even with this low sensitivitylimit, Bartonella-specific DNA was detected inthe majority of the studied samples. A high in-fection rate of I. persulcatus ticks represents apotential risk of infection with Bartonella forpersons exposed to tick bites.

ACKNOWLEDGMENTS

The study was supported in part by grant 02-01-113 from the Russian program Vaccines ofNew Generation, grant N-51 of the Program forthe Integration in Basic Sciences of the SiberianBranch of the Russian Academy of Sciences,and grant RO1 AI43063 from the National In-stitute of Allergy and Infectious Diseases toF.C.C.

REFERENCES

1. Chang, CC, Chomel, BB, Kasten, RW, et al. Molecu-lar evidence of Bartonella spp. in questing adult Ixodespacificus ticks in California. J Clin Microbiol 2001;39:1121–1126.

2. Chang, CC, Hayashidani, H, Pusterla, N, et al. Inves-tigation of Bartonella infection in ixodid ticks fromCalifornia. Comp Immunol Microbiol Infect Dis 2002;25:229–236.

3. Eskow, E, Rao, RV, Mordechai, E. Concurrent infec-tion of the central nervous system by Borreliaburgdorferi and Bartonella henselae: evidence for anovel tick-borne disease complex. Arch Neurol 2001;58:1357–1363.

4. Ignatovich, VF, Lukin, EP, Umnova, NS, et al. Seroim-munologic monitoring of microorganisms of Rickettsia

and Bartonella species microorganisms in the Moscowregion [in Russian]. Zh Microbiol Epidemiol Immunol2001; 1:14–17.

5. Houpikian, P, Raoult, D. Molecular phylogeny of thegenus Bartonella: what is the current knowledge?FEMS Microbiol Lett 2001; 200:1–7.

6. Jacomo, V, Kelly, PJ, and Raoult, D. Natural historyof Bartonella infections (an exception to Koch’s postu-late). Clin Diagn Lab Immunol 2002; 9:8–18.

7. Korenberg, EI, Kovalevsky, YV, Gorelova, NB. Ecol-ogy of Borrelia burgorferi sensu lato in Russia. In: Gray,G, Kahl, O, Lane, RS, eds. Lyme Borreliosis Epidemiol-ogy and Control. Oxford: CAB International; 2002:175–200.

8. Livanova, NN, Morozova, OV, Morozov, IV, et al.Characterization of Borrelia burgdorferi sensu lato fromWest Siberia Based on Direct PCR. Eur J Epidemiol2003; 18:1155–1158.

9. Morozova, OV, Dobrotvorsky, AK, Livanova, NN, etal. PCR detection of Borrelia burgdorferi sensu lato,tick-borne encephalitis virus, and the human granu-locytic ehrlichiosis agent in Ixodes persulcatus ticksfrom Western Siberia, Russia. J Clin Microbiol 2002;40:3802–3804.

10. Rydkina, EB, Roux, V, Gagua, EM, et al. Bartonellaquintana in body lice collected from homeless personsin Russia. Emerg Infect Dis 1999; 5:176–178.

11. Sanogo, YO, Zeaiter, Z, Caruso, G, et al. Bartonellahenselae in Ixodes ricinus ticks (Acari: Ixodida) re-moved from humans, Belluno province, Italy. EmergInfect Dis 2003; 9:329–332.

12. Schouls, LM, Van De Pol, I, Rijpkema, SG, et al. De-tection and identification of Ehrlichia, Borrelia burgdor-feri sensu lato, and Bartonella species in Dutch Ixodesricinus ticks. J Clin Microbiol 1999; 37:2215–2222.

13. Tarasevich, IV. New and emerging rickettsial and bar-tonella infections [in Russian]. Vestn Ross Acad MedNauk 2001; 11:8–11.

14. Thompson, JD, Higgins, DG, Gibson, TJ. CLUSTALW: improving the sensitivity of progressive multiplesequence alignment through sequence weighting, po-sition-specific gap penalties and weight matrix choice.Nucl Acids Res 1994; 22:4673–4680.

15. Zeaiter, Z, Fournier, P-E, and Raoult D. Genomic vari-ation of Bartonella henselae strains detected in lymphnodes of patients with cat scratch disease. J Clin Mi-crobiol 2002; 40:1023–1030.

Address reprint requests to:Dr. Olga Morozova

Institute of Chemical Biology and Fundamental Medicine

Siberian Branch of the Russian Academy of Sciences

Lavrentyev’s Avenue 8630090 Novosibirsk, Russia

E-mail: mov@niboch.nsc.ru

INFECTION OF TICKS WITH BARTONELLA SPP. IN NOVOSIBIRSK REGION, RUSSIA 309

5387_04_p306-309 12/3/04 4:16 PM Page 309