Identification of Borrelia burgdorferi sensu lato, Anaplasma and Ehrlichia Species, and Spotted Fever Group Rickettsiae in Ticks from Southeastern Europe

  • I. Christova
  • J. van de Pol
  • S. Yazar
  • E. Velo
  • L. SchoulsEmail author


Prevalence data for tick-borne pathogens are used to assess the risk for human health. In this study the presence and identity of Borrelia burgdorferi sensu lato, Ehrlichia, Anaplasma, and Rickettsia species in Bulgarian Ixodes ricinus ticks and in non-Ixodes ticks from Turkey and Albania was determined by polymerase chain reaction (PCR) and reverse line blot hybridization. In the adult Bulgarian ticks, the prevalence of Borrelia burgdorferi sensu lato infection was approximately 40%, while Borrelia afzelii was the predominant species, representing more than half of all Borrelia-positive ticks. Ehrlichia and Anaplasma species were detected in 35% of the adult Ixodes ricinus ticks and in 10% of the nymphs. Sequence analysis of PCR products reacting with the Anaplasma phagocytophila probe revealed a 16S rRNA gene identical to that of the Anaplasma phagocytophila prototype strain. Ehrlichia and Anaplasma species were found in approximately 7% of the non-Ixodes ticks. Sequence analysis of some of these samples revealed the presence of Anaplasma ovis, Ehrlichia canis, and a species closely resembling Ehrlichia chaffeensis. About half of all adult ticks examined and approximately 20% of all nymphs were infected with Rickettsia species. In Ixodes ricinus ticks, Rickettsia helvetica and a Rickettsia species designated as IRS3 were found in high prevalence. Rickettsia conorii was found in virtually all non-Ixodes tick species from Albania and Turkey. The results of this study show that many tick-borne diseases are most probably endemic in the Balkan area. Furthermore, the results suggest that there is a considerable chance for simultaneous transmission of tick-borne pathogens to human beings.


Borrelia Burgdorferi Lyme Borreliosis Adult Tick Spotted Fever Reverse Line Blot 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This study was supported in part by grant L909/2000 from the Ministry of Education and Science, Sofia, Bulgaria, and by the Institute of Public Health, Tirana, Albania. We thank Metin Atambay and Izzet Sahin, Turkey, and Et'hem Llabani and Bajram Kastrati, Albania, for the collection of ticks.


  1. 1.
    Baranton G, Postic D, Saint Girons I, Boerlin P, Piffaretti JC, Assous M, Grimont PA (1992) Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis. Int J Syst Bacteriol 42:378–383PubMedGoogle Scholar
  2. 2.
    Canica MM, Nato F, du Merle L, Mazie JC, Baranton G, Postic D (1993) Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis. Scand J Infect Dis 25:441–448PubMedGoogle Scholar
  3. 3.
    Johnson RC, Hyde FW, Schmid GP, Brenner DJ (1984) Borrelia burgdorferi sp. nov.: etiologic agent of Lyme disease. Int J Syst Bacteriol 34:496–497Google Scholar
  4. 4.
    Postic D, Assous MV, Grimont PA, Baranton G (1994) Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons. Int J Syst Bacteriol 44:743–752PubMedGoogle Scholar
  5. 5.
    Wang GQ, Vandam AP, Lefleche A, Postic D, Peter O, Baranton G, Deboer R, Spanjaard L, Dankert J (1997) Genetic and phenotypic analysis of Borrelia valaisiana sp. nov. (Borrelia genomic groups VS116 and M19). Int J Syst Bacteriol 47:926–932Google Scholar
  6. 6.
    Lefleche A, Postic D, Girardet K, Peter O, Baranton G (1997) Characterization of Borrelia lusitaniae sp. nov. by 16S ribosomal DNA sequence analysis. Int J Syst Bacteriol 47:921–925Google Scholar
  7. 7.
    Rijpkema SGT, Tazelaar DJ, Molkenboer MJCH, Noordhoek GT, Plantinga G, Schouls LM, Schellekens JFP (1997) Detection of Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii and group VS116 by PCR in skin biopsies of patients with erythema migrans and acrodermatitis chronica atrophicans. Clin Microbiol Infect 3:109–116PubMedGoogle Scholar
  8. 8.
    Smith M, Gettinby G, Granstrom M, Gray JS, Guy EC, Revie C, Robertson JN, Stanek G (1998) The European Union Concerted Action World Wide Web site for Lyme borreliosis. Zentralbl Bakteriol 287:266–269PubMedGoogle Scholar
  9. 9.
    Dumler JS, Walker DH (2001) Tick-borne ehrlichioses. Lancet Infect Dis 1:21–28CrossRefPubMedGoogle Scholar
  10. 10.
    Anderson BE, Dawson JE, Jones DC, Wilson KH (1991) Ehrlichia chaffeensis, a new species associated with human ehrlichiosis. J Clin Microbiol 29:2838–2842PubMedGoogle Scholar
  11. 11.
    Chen SM, Dumler JS, Bakken JS, Walker DH (1994) Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J Clin Microbiol 32:589–595PubMedGoogle Scholar
  12. 12.
    Karlsson U, Bjoersdorff A, Massung RF, Christensson B (2001) Human granulocytic ehrlichiosis—a clinical case in Scandinavia. Scand J Infect Dis 33:73–74CrossRefPubMedGoogle Scholar
  13. 13.
    Tylewska Wierzbanowska S, Chmielewski T, Kondrusik M, Hermanowska Szpakowicz T, Sawicki W, Sulek K (2001) First cases of acute human granulocytic ehrlichiosis in Poland. Eur J Clin Microbiol Infect Dis 20:196–198CrossRefPubMedGoogle Scholar
  14. 14.
    Arnez M, Petrovec M, Lotric Furlan S, Zupanc TA, Strle F (2001) First European pediatric case of human granulocytic ehrlichiosis. J Clin Microbiol 39:4591–4592CrossRefPubMedGoogle Scholar
  15. 15.
    Dumler JS, Barbet AF, Bekker CPJ, Dasch GA, Palmer GH, Ray SC, Rikihisa Y, Rurangirwa FR (2001) Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia, and Ehrlichia with Neorickettsia descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol 51:2145–2165PubMedGoogle Scholar
  16. 16.
    Eremeeva ME, Balayeva NM, Ignatovich VF, Raoult D (1995) Serologic response to rickettsial antigens in patients with Astrakhan fever. Eur J Epidemiol 11:383–387PubMedGoogle Scholar
  17. 17.
    Tissot-Dupont H, Brouqui P, Faugere B, Raoult D (1994) Prevalence of antibodies to Coxiella burnetii, Rickettsia conorii, and Rickettsia typhi in seven African countries. Clin Infect Dis 21:1126–1133Google Scholar
  18. 18.
    Raoult D, Roux V (1997) Rickettsioses as paradigms of new or emerging infectious diseases. Clin Microbiol Rev 10:694–719PubMedGoogle Scholar
  19. 19.
    Burgdorfer W, Aeschlimann A, Peter O, Hayes SF, Philip RN (1979) Ixodes ricinus: vector of a hitherto undescribed spotted fever group agent in Switzerland. Acta Trop 36:357–367PubMedGoogle Scholar
  20. 20.
    Nilsson K, Jaenson TGT, Uhnoo I, Lindquist O, Pettersson B, Uhlen M, Friman G, Pahlson C (1997) Characterization of a spotted fever group Rickettsia from Ixodes ricinus ticks in Sweden. J Clin Microbiol 35:243–247PubMedGoogle Scholar
  21. 21.
    Nilsson K, Lindquist O, Liu AJ, Jaenson TGT, Friman G, Pahlson C (1999) Rickettsia helvetica in Ixodes ricinus ticks in Sweden. J Clin Microbiol 37:400–403PubMedGoogle Scholar
  22. 22.
    Parola P, Beati L, Cambon M, Raoult D (1998) First isolation of Rickettsia helvetica from Ixodes ricinus ticks in France. Eur J Clin Microbiol Infect Dis 17:95–100PubMedGoogle Scholar
  23. 23.
    Nilsson K, Lindquist O, Pahlson C (1999) Association of Rickettsia helvetica with chronic perimyocarditis in sudden cardiac death. Lancet 354:1169–1173CrossRefPubMedGoogle Scholar
  24. 24.
    Sekeyova Z, Fournier PE, Rehacek J, Raoult D (2000) Characterization of a new spotted fever group Rickettsia detected in Ixodes ricinus (Acari: Ixodidae) collected in Slovakia. J Med Entomol 37:707–713PubMedGoogle Scholar
  25. 25.
    Marquez FJ, Muniain MA, Soriguer RC, Izquierdo G, Rodriguezbano J, Borobio MV (1998) Genotypic identification of an undescribed spotted fever group rickettsia in Ixodes ricinus from southwestern Spain. Am J Trop Med Hyg 58:570–577PubMedGoogle Scholar
  26. 26.
    Christova I, Schouls L, Van De Pol I, Park J, Panayotov S, Lefterova V, Kantardjiev T, Dumler JS (2001) High prevalence of granulocytic Ehrlichiae and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Bulgaria. J Clin Microbiol 39:4172–4174CrossRefPubMedGoogle Scholar
  27. 27.
    Rijpkema S, Golubic D, Molkenboer M, Verbeek, De Kruif N, Schellekens J (1996) Identification of four genomic groups of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in a Lyme borreliosis endemic region of northern Croatia. Exp Appl Acarol 20:23–30PubMedGoogle Scholar
  28. 28.
    Alekseev AN, Dubinina HV, Van De Pol I, Schouls LM (2001) Identification of Ehrlichia spp. and Borrelia burgdorferi in Ixodes ticks in the Baltic regions of Russia. J Clin Microbiol 39:2237–2242CrossRefPubMedGoogle Scholar
  29. 29.
    Schouls LM, Van De Pol I, Rijpkema SGT, Schot CS (1999) Detection and identification of Ehrlichia, Borrelia burgdorferi sensu lato, and Bartonella species in Dutch Ixodes ricinus ticks. J Clin Microbiol 37:2215–2222PubMedGoogle Scholar
  30. 30.
    Stuen S, Van De Pol, I Bergstrom K, Schouls LM (2002) Identification of Anaplasma phagocytophila (formerly Ehrlichia phagocytophila) variants in blood from sheep in Norway. J Clin Microbiol 3192–3197Google Scholar
  31. 31.
    Stoilov R, Stamenov B, Zarcheva V, Christova I, Manev C, Noeva K (1995) Clinical manifestations of Lyme borreliosis in Bulgaria. Reumatologica 23:26–31Google Scholar
  32. 32.
    Berglund J, Eitrem R, Ornstein K, Lindberg A, Ringer A, Elmrud H, Carlsson M, Runehagen A, Svanborg C, Norrby R (1995) An epidemiologic study of Lyme disease in southern Sweden. N Engl J Med 333:1319–1327Google Scholar
  33. 33.
    Lesnyak O, Laikovskaya E, Kufko I, Bruinink H, Baranova N, Rijpkema S (1998) Clinical features of Lyme borreliosis in the middle Urals, and distribution of Borrelia burgdorferi sensu lato species in local Ixodes persulcatus ticks. Zentralbl Bakteriol 288:111–119PubMedGoogle Scholar
  34. 34.
    Lipsker D, Hansmann Y, Limbach F, Clerc C, Tranchant C, Grunenberger F, Caro Sampara F, Attali P, Frey M, Kubina M, Piemont Y, Sibilia J, Jaulhac B (2001) Disease expression of Lyme borreliosis in northeastern France. Eur J Clin Microbiol Infect Dis 20:225–230CrossRefPubMedGoogle Scholar
  35. 35.
    O'Connell S (1995) Lyme disease in the United Kingdom. Br Med J 310:303–308Google Scholar
  36. 36.
    Junttila T, Peltomaa M, Soini H, Marjamaki M, Viljanen MK (1999) Prevalence of Borrelia burgdorferi in Ixodes ricinus ticks in urban recreational areas of Helsinki. J Clin Microbiol 37:1361–1365PubMedGoogle Scholar
  37. 37.
    Jenkins A, Kristiansen BE, Allum AG, Aakre RK, Strand L, Kleveland EJ, Van De Pol I, Schouls L (2001) Borrelia burgdorferi sensu lato and Ehrlichia spp. in Ixodes ticks from southern Norway. J Clin Microbiol 39:3666–3671CrossRefPubMedGoogle Scholar
  38. 38.
    Ciceroni L, Ciarrochi S, Ciervo A, Mondarini V, Guzzo F, Caruso G, Murgia R, Cinco M (2001) Isolation and characterization of Borrelia burgdorferi sensu lato strains in an area of Italy where Lyme borreliosis is endemic. J Clin Microbiol 39:2254–2260CrossRefPubMedGoogle Scholar
  39. 39.
    Ruzic Sabljic E, Strle F, Cimperman J, Maraspin V, Lotric Furlan S, Pleterski Rigler D (2000) Characterisation of Borrelia burgdorferi sensu lato strains isolated from patients with skin manifestations of Lyme borreliosis residing in Slovenia. J Med Microbiol 49:47–53PubMedGoogle Scholar
  40. 40.
    Ruzic Sabljic E, Arnez M, Lotric Furlan S, Maraspin V, Cimperman J, Strle F (2001) Genotypic and phenotypic characterisation of Borrelia burgdorferi sensu lato strains isolated from human blood. J Med Microbiol 50:896–901PubMedGoogle Scholar
  41. 41.
    Lawson ET, Mousseau TA, Klaper R, Hunter MD, Werren JH (2001) Rickettsia associated with male-killing in a buprestid beetle. Heredity 86:497–505CrossRefPubMedGoogle Scholar
  42. 42.
    Baumgarten BU, Rollinghoff M, Bogdan C (1999) Prevalence of Borrelia burgdorferi and granulocytic and monocytic Ehrlichiae in Ixodes ricinus ticks from southern Germany. J Clin Microbiol 37:3448–3451PubMedGoogle Scholar
  43. 43.
    Cinco M, Padovan D, Murgia R, Maroli M, Frusteri L, Heldtander M, Johansson KE, Engvall EO (1997) Coexistence of Ehrlichia phagocytophila and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Italy as determined by 16S rRNA gene sequencing. J Clin Microbiol 35:3365–3366PubMedGoogle Scholar
  44. 44.
    Leutenegger CM, Pusterla N, Mislin CN, Weber R, Lutz H (1999) Molecular evidence of coinfection of ticks with Borrelia burgdorferi sensu lato and the human granulocytic ehrlichiosis agent in Switzerland. J Clin Microbiol 37:3390–3391PubMedGoogle Scholar
  45. 45.
    Liz JS, Anderes L, Sumner JW, Massung RF, Gern L, Rutti B, Brossard M (2000) PCR detection of granulocytic ehrlichiae in Ixodes ricinus ticks and wild small mammals in western Switzerland. J Clin Microbiol 38:1002–1007PubMedGoogle Scholar
  46. 46.
    Parola P, Beati L, Cambon M, Brouqui P, Raoult D (1998) Ehrlichial DNA amplified from Ixodes ricinus (Acari: Ixodidae) in France. J Med Entomol 35:180–183PubMedGoogle Scholar
  47. 47.
    Schauber EM, Gertz SJ, Maple WT, Ostfeld RS (1998) Coinfection of blacklegged ticks (Acari: Ixodidae) in Dutchess County, New York with the agents of Lyme disease and human granulocytic ehrlichiosis. J Med Entomol 35:901–903PubMedGoogle Scholar
  48. 48.
    Stedingk LV von, Gurtelschmid M, Hanson HS, Gustafson R, Dotevall L, Engval EO, Granstrom M (1997) The human granulocytic ehrlichiosis (HGE) agent in Swedish ticks. Clin Microbiol Infect 3:573–574PubMedGoogle Scholar
  49. 49.
    Christova IS, Dumler JS (1999) Human granulocytic ehrlichiosis in Bulgaria. Am J Trop Med Hyg 60:58–61PubMedGoogle Scholar
  50. 50.
    Beninati T, Lo N, Noda H, Esposito F, Rizzoli A, Favia G, Genchi C (2002) First detection of spotted fever group rickettsiae in Ixodes ricinus from Italy. Emerg Infect Dis 8:983–986PubMedGoogle Scholar
  51. 51.
    Vural T, Ergin C, Sayin F (1998) Investigation of Rickettsia conorii antibodies in the Antalya area. Infection 26:170–172PubMedGoogle Scholar
  52. 52.
    Fournier PE, Grunnenberger F, Jaulhac B, Gastinger G, Raoult D (2000) Evidence of Rickettsia helvetica infection in humans in eastern France. Emerg Infect Dis 6:389–392PubMedGoogle Scholar
  53. 53.
    Nilsson K, Pahlson C, Lukinius A, Eriksson L, Nilsson L, Lindquist O (2002) Presence of Rickettsia helvetica in granulomatous tissue from patients with sarcoidosis. J Infect Dis 185:1128–1138CrossRefPubMedGoogle Scholar
  54. 54.
    Fingerle V, Munderloh UG, Liegl G, Wilske B (1999) Coexistence of Ehrlichiae of the phagocytophila group with Borrelia burgdorferi in Ixodes ricinus from southern Germany. Med Microbiol Immunol (Berl) 188:145–149CrossRefGoogle Scholar
  55. 55.
    Pichon B, Godfroid E, Hoyois B, Bollen A, Rodhain F, Perez, Eid C (1995) Simultaneous infection of Ixodes ricinus nymphs by two Borrelia burgdorferi sensu lato species: possible implications for clinical manifestations. Emerg Infect Dis 1:89–90PubMedGoogle Scholar
  56. 56.
    Nadelman RB, Horowitz HW, Hsieh TC, Wu JM, Aguero Rosenfeld ME, Schwartz I, Nowakowski J, Varde S, Wormser GP (1997) Simultaneous human granulocytic ehrlichiosis and Lyme borreliosis. N Engl J Med 337:27–30CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • I. Christova
    • 1
  • J. van de Pol
    • 2
  • S. Yazar
    • 3
  • E. Velo
    • 4
  • L. Schouls
    • 2
    Email author
  1. 1.Department of MicrobiologyNational Center of Infectious and Parasitic DiseasesSofiaBulgaria
  2. 2.Laboratory for Vaccine-Preventable DiseasesNational Institute of Public Health and the EnvironmentBilthovenThe Netherlands
  3. 3.Department of Parasitology, Medical FacultyErciyes UniversityMelikgazi, KayseriTurkey
  4. 4.Department of Microbiology, Laboratory of Vector Disease ControlInstitute of Public HealthTiranaAlbania

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