Experimental and Applied Acarology

, Volume 68, Issue 1, pp 127–137 | Cite as

Prevalence of pathogenic bacteria in Ixodes ricinus ticks in Central Bohemia

  • Radek Klubal
  • Jan Kopecky
  • Marta Nesvorna
  • Olivier A. E. Sparagano
  • Jana Thomayerova
  • Jan HubertEmail author


Bacteria associated with the tick Ixodes ricinus were assessed in specimens unattached or attached to the skin of cats, dogs and humans, collected in the Czech Republic. The bacteria were detected by PCR in 97 of 142 pooled samples including 204 ticks, i.e. 1–7 ticks per sample, collected at the same time from one host. A fragment of the bacterial 16S rRNA gene was amplified, cloned and sequenced from 32 randomly selected samples. The most frequent sequences were those related to Candidatus Midichloria midichlori (71 % of cloned sequences), followed by Diplorickettsia (13 %), Spiroplasma (3 %), Rickettsia (3 %), Pasteurella (3 %), Morganella (3 %), Pseudomonas (2 %), Bacillus (1 %), Methylobacterium (1 %) and Phyllobacterium (1 %). The phylogenetic analysis of Spiroplasma 16S rRNA gene sequences showed two groups related to Spiroplasma eriocheiris and Spiroplasma melliferum, respectively. Using group-specific primers, the following potentially pathogenic bacteria were detected: Borellia (in 20 % of the 142 samples), Rickettsia (12 %), Spiroplasma (5 %), Diplorickettsia (5 %) and Anaplasma (2 %). In total, 68 % of I. ricinus samples (97/142) contained detectable bacteria and 13 % contained two or more putative pathogenic groups. The prevalence of tick-borne bacteria was similar to the observations in other European countries.


Tick Bacteria Central Bohemia Ixodes ricinus Pathogens 



The study was supported by project RO0415 of the Ministry of Agriculture of the Czech Republic. The authors thank to Martin Markovic for technical help.


  1. Alexeev D, Kostrjukova E, Aliper A, Popenko A, Bazaleev N, Tyakht A, Selezneva O, Akopian T, Prichodko E, Kondratov I, Chukin M, Demina I, Galyamina M, Kamashev D, Vanyushkina A, Ladygina V, Levitskii S, Lazarev V, Govorun V (2012) Application of Spiroplasma melliferum proteogenomic profiling for the discovery of virulence factors and pathogenicity mechanisms in host-associated spiroplasmas. J Proteome Res 11:224–236CrossRefPubMedGoogle Scholar
  2. Aureli S, Foley JE, Galuppi R, Rejmanek D, Bonoli C, Tampieri MP (2012) Anaplasma phagocytophilum in ticks from parks in the Emilia-Romagna region of northern Italy. Vet Ital 48:413–423PubMedGoogle Scholar
  3. Barbieri E, Paster BJ, Hughes D, Zurek L, Moser DP, Teske A, Sogin ML (2001) Phylogenetic characterization of epibiotic bacteria in the accessory nidamental gland and egg capsules of the squid Loligo pealei (Cephalopoda: Loliginidae). Environ Microbiol 3:151–167CrossRefPubMedGoogle Scholar
  4. Bazzocchi C, Mariconti M, Sassera D, Rinaldi L, Martin E, Cringoli G, Urbanelli S, Genchi C, Bandi C, Epis S (2013) Molecular and serological evidence for the circulation of the tick symbiont Midichloria (Rickettsiales: Midichloriaceae) in different mammalian species. Parasit Vectors 6:350. doi: 10.1186/1756-3305-6-350 PubMedCentralCrossRefPubMedGoogle Scholar
  5. Bi K, Huang H, Gu W, Wang J, Wang W (2008) Phylogenetic analysis of Spiroplasmas from three freshwater crustaceans (Eriocheir sinensis, Procambarus clarkia and Penaeus vannamei) in China. J Invertebr Pathol 99:57–65CrossRefPubMedGoogle Scholar
  6. Capelli G, Ravagnan S, Montarsi F, Ciocchetta S, Cazzin S, Porcellato E, Babiker AM, Cassini R, Salviato A, Cattoli G, Otranto D (2012) Occurrence and identification of risk areas of Ixodes ricinus-borne pathogens: a cost-effectiveness analysis in north-eastern Italy. Parasit Vectors 5:6. doi: 10.1186/1756-3305-5-61 CrossRefGoogle Scholar
  7. Carle P, Saillard C, Carrère N, Carrère S, Duret S, Eveillard S, Gaurivaud P, Gourgues G, Gouzy J, Salar P, Verdin E, Breton M, Blanchard A, Laigret F, Bové JM, Renaudin J, Foissac X (2010) Partial chromosome sequence of Spiroplasma citri reveals extensive viral invasion and important gene decay. Appl Environ Microbiol 76:3420–3426PubMedCentralCrossRefPubMedGoogle Scholar
  8. Claerebout E, Losson B, Cochez C, Casaert S, Dalemans A-C, De Cat A, Madder M, Saegerman C, Heyman P, Lempereur L (2013) Ticks and associated pathogens collected from dogs and cats in Belgium. Parasit Vectors 6:183. doi: 10.1186/1756-3305-6-183 PubMedCentralCrossRefPubMedGoogle Scholar
  9. Dally EL, Barros TS, Zhao Y, Lin S, Roe BA, Davis RE (2006) Physical and genetic map of the Spiroplasma kunkelii CR2-3x chromosome. Can J Microbiol 52:857–867CrossRefPubMedGoogle Scholar
  10. Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772PubMedCentralCrossRefPubMedGoogle Scholar
  11. Egyed L, Makrai L (2014) Cultivable internal bacterial flora of ticks isolated in Hungary. Exp Appl Acarol 63:107–122CrossRefPubMedGoogle Scholar
  12. Fenollar F, Raoult D (2004) Molecular genetic methods for the diagnosis of fastidious microorganisms. APMIS 112:785–807CrossRefPubMedGoogle Scholar
  13. Fukatsu T, Tsuchida T, Nikoh N, Koga R (2001) Spiroplasma symbiont of the pea aphid, Acyrthosiphon pisum (Insecta: Homoptera). Appl Environ Microbiol 67:1284–1291PubMedCentralCrossRefPubMedGoogle Scholar
  14. Gasparich GE, Whitcomb RF, Dodge D, French FE, Glass J, Williamson DL (2004) The genus Spiroplasma and its non-helical descendants: phylogenetic classification, correlation with phenotype and roots of the Mycoplasma mycoides clade. Int J Syst Evol Microbiol 54:893–918CrossRefPubMedGoogle Scholar
  15. Glatz M, Mullegger RR, Maurer F, Fingerle V, Achermann Y, Wilske B, Bloemberg GV (2014) Detection of Candidatus Neoehrlichia mikurensis, Borrelia burgdorferi sensu lato genospecies and Anaplasma phagocytophilum in a tick population from Austria. Ticks Tick Borne Dis 5:139–144CrossRefPubMedGoogle Scholar
  16. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704CrossRefPubMedGoogle Scholar
  17. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321CrossRefPubMedGoogle Scholar
  18. Hai VV, Almeras L, Socolovschi C, Raoult D, Parola P, Pages F (2014) Monitoring human tick-borne disease risk and tick bite exposure in Europe: available tools and promising future methods. Ticks Tick Borne Dis 5:607–619CrossRefPubMedGoogle Scholar
  19. Henning K, Greiner-Fischer S, Hotzel H, Ebsen M, Theegarten D (2006) Isolation of Spiroplasma sp. from an Ixodes tick. Int J Med Microbiol 296(Suppl 1):157–161CrossRefPubMedGoogle Scholar
  20. Hildebrandt A, Kramer A, Sachse S, Straube E (2010) Detection of Rickettsia spp. and Anaplasma phagocytophilum in Ixodes ricinus ticks in a region of Middle Germany (Thuringia). Ticks Tick Borne Dis 1:52–56CrossRefPubMedGoogle Scholar
  21. Hoy MA, Jeyaprakash A (2005) Microbial diversity in the predatory mite Metaseiulus occidentalis (Acari: Phytoseiidae) and its prey, Tetranychus urticae (Acari: Tetranychidae). Biol Control 32:427–441CrossRefGoogle Scholar
  22. Hurst GD, Graf von der Schulenburg JH, Majerus TM, Bertrand D, Zakharov IA, Baungaard J, Völkl W, Stouthamer R, Majerus ME (1999) Invasion of one insect species, Adalia bipunctata, by two different male-killing bacteria. Insect Mol Biol 8:133–139CrossRefPubMedGoogle Scholar
  23. Ishii Y, Matsuura Y, Kakizawa S, Nikoh N, Fukatsu T (2013) Diversity of bacterial endosymbionts associated with Macrosteles leafhoppers vectoring phytopathogenic phytoplasmas. Appl Environ Microbiol 79:5013–5022PubMedCentralCrossRefPubMedGoogle Scholar
  24. Jaffe JD, Stange-Thomann N, Smith C, DeCaprio D, Fisher S, Butler J, Calvo S, Elkins T, FitzGerald MG, Hafez N, Kodira CD, Major J, Wang S, Wilkinson J, Nicol R, Nusbaum C, Birren B, Berg HC, Church GM (2004) The complete genome and proteome of Mycoplasma mobile. Genome Res 14:1447–1461PubMedCentralCrossRefPubMedGoogle Scholar
  25. Jiggins FM, Hurst GD, Jiggins CD, Schulenburg JH, Majerus ME (2000) The butterfly Danaus chrysippus is infected by a male-killing Spiroplasma bacterium. Parasitology 120:439–446CrossRefPubMedGoogle Scholar
  26. Johnson G, Ayers M, McClure SCC, Richardson SE, Tellier R (2003) Detection and identification of Bartonella species pathogenic for humans by PCR amplification targeting the riboflavin synthase gene (ribC). J Clin Microbiol 41:1069–1072PubMedCentralCrossRefPubMedGoogle Scholar
  27. Kiewra D, Zalesny G, Czulowska A (2014) The prevalence of Anaplasma phagocytophilum in questing Ixodes ricinus ticks in SW Poland. Pol J Microbiol 63:89–93PubMedGoogle Scholar
  28. Kim E-J, Bauer C, Grevelding CG, Quack T (2013) Improved PCR/nested PCR approaches with increased sensitivity and specificity for the detection of pathogens in hard ticks. Ticks Tick Borne Dis 4:409–416CrossRefPubMedGoogle Scholar
  29. Konai M, Whitcomb RF, Tully JG, Rose DL, Carle P, Bové JM, Henegar RB, Hackett KJ, Clark TB, Williamson DL (1995) Spiroplasma velocicrescens sp. nov., from the vespid wasp Monobia quadridens. Int J Syst Bacteriol 45:203–206CrossRefPubMedGoogle Scholar
  30. Kopecky J, Nesvorna M, Hubert J (2014) Bartonella-like bacteria carried by domestic mite species. Exp Appl Acarol 64:21–32CrossRefPubMedGoogle Scholar
  31. Lartillot N, Lepage T, Blanquart S (2009) PhyloBayes 3: a Bayesian software package for phylogenetic reconstruction and molecular dating. Bioinformatics 25:2286–2288CrossRefPubMedGoogle Scholar
  32. Lazarev VN, Levitskii SA, Basovskii YI, Chukin MM, Akopian TA, Vereshchagin VV, Kostrjukova ES, Kovaleva GY, Kazanov MD, Malko DB, Vitreschak AG, Sernova NV, Gelfand MS, Demina IA, Serebryakova MV, Galyamina MA, Vtyurin NN, Rogov SI, Alexeev DG, Ladygina VG, Govorun VM (2011) Complete genome and proteome of Acholeplasma laidlawii. J Bacteriol 193(18):4943–4953PubMedCentralCrossRefPubMedGoogle Scholar
  33. Lichtensteiger CA, Steenbergen SM, Lee RM, Polson DD, Vimr ER (1996) Direct PCR analysis for toxigenic Pasteurella multocida. J Clin Microbiol 34:3035–3039PubMedCentralPubMedGoogle Scholar
  34. Lo W-S, Chen L-L, Chung W-C, Gasparich GE, Kuo C-H (2013) Comparative genome analysis of Spiroplasma melliferum IPMB4A, a honeybee-associated bacterium. BMC Genom 14:22. doi: 10.1186/1471-2164-14-22 CrossRefGoogle Scholar
  35. Majerus TM, Graf von der Schulenburg JH, Majerus ME, Hurst GD (1999) Molecular identification of a male-killing agent in the ladybird Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). Insect Mol Biol 8:551–555CrossRefPubMedGoogle Scholar
  36. Mariconti M, Epis S, Gaibani P, Dalla Valle C, Sassera D, Tomao P, Fabbi M, Castelli F, Marone P, Sambri V, Bazzocchi C, Bandi C (2012) Humans parasitized by the hard tick Ixodes ricinus are seropositive to Midichloria mitochondrii: is Midichloria a novel pathogen, or just a marker of tick bite? Pathog Glob Health 106:391–396PubMedCentralCrossRefPubMedGoogle Scholar
  37. May K, Strube C (2014) Prevalence of Rickettsiales (Anaplasma phagocytophilum and Rickettsia spp.) in hard ticks (Ixodes ricinus) in the city of Hamburg, Germany. Parasitol Res 113:2169–2175CrossRefPubMedGoogle Scholar
  38. Mediannikov O, Fenollar F (2014) Looking in ticks for human bacterial pathogens. Microb Pathog 77:142–148CrossRefPubMedGoogle Scholar
  39. Mediannikov O, Sekeyova Z, Birg M-L, Raoult D (2010) A novel obligate intracellular gamma-proteobacterium associated with ixodid ticks, Diplorickettsia massiliensis, gen. nov., sp. nov. PLoS ONE 5:e11478. doi: 10.1371/journal.pone.0011478 PubMedCentralCrossRefPubMedGoogle Scholar
  40. Meeus I, Vercruysse V, Smagghe G (2012) Molecular detection of Spiroplasma apis and Spiroplasma melliferum in bees. J Invertebr Pathol 109:172–174CrossRefPubMedGoogle Scholar
  41. Michelet L, Delannoy S, Devillers E, Umhang G, Aspan A, Juremalm M, Chirico J, van der Wal FJ, Sprong H, Boye Pihl TP, Klitgaard K, Bodker R, Fach P, Moutailler S (2014) High-throughput screening of tick-borne pathogens in Europe. Front Cell Infect Microbiol 4:103. doi: 10.3389/fcimb.2014.00103 PubMedCentralPubMedGoogle Scholar
  42. Movila A, Dubinina HV, Sitnicova N, Bespyatova L, Uspenskaia I, Efremova G, Toderas I, Alekseev AN (2014) Comparison of tick-borne microorganism communities in Ixodes spp. of the Ixodes ricinus species complex at distinct geographical regions. Exp Appl Acarol 63:65–76CrossRefPubMedGoogle Scholar
  43. Norman AF, Regnery R, Jameson P, Greene C, Krause DC (1995) Differentiation of Bartonella-like isolates at the species level by PCR-restriction fragment length polymorphism in the citrate synthase gene. J Clin Microbiol 33:1797–1803PubMedCentralPubMedGoogle Scholar
  44. Nunan LM, Lightner DV, Oduori MA, Gasparich GE (2005) Spiroplasma penaei sp. nov., associated with mortalities in Penaeus vannamei, Pacific white shrimp. Int J Syst Evol Microbiol 55:2317–2322CrossRefPubMedGoogle Scholar
  45. Pangracova L, Derdakova M, Pekarik L, Hviscova I, Vichova B, Stanko M, Hlavata H, Petko B (2013) Ixodes ricinus abundance and its infection with the tick-borne pathogens in urban and suburban areas of Eastern Slovakia. Parasit Vectors 6:238. doi: 10.1186/1756-3305-6-238 PubMedCentralCrossRefPubMedGoogle Scholar
  46. Papazisi L, Gorton TS, Kutish G, Markham PF, Browning GF, Nguyen DK, Swartzell S, Madan A, Mahairas G, Geary SJ (2003) The complete genome sequence of the avian pathogen Mycoplasma gallisepticum strain R(low). Microbiology 149:2307–2316CrossRefPubMedGoogle Scholar
  47. Pruesse E, Peplies J, Glockner FO (2012) SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics 28:1823–1829PubMedCentralCrossRefPubMedGoogle Scholar
  48. Qiu Y, Nakao R, Ohnuma A, Kawamori F, Sugimoto C (2014) Microbial population analysis of the salivary glands of ticks; a possible strategy for the surveillance of bacterial pathogens. PLoS ONE 9:e103961. doi: 10.1371/journal.pone.0103961 PubMedCentralCrossRefPubMedGoogle Scholar
  49. Rizzoli A, Silaghi C, Obiegala A, Rudolf I, Hubalek Z, Foldvari G, Plantard O, Vayssier-Taussat M, Bonnet S, Spitalska E, Kazimirova M (2014) Ixodes ricinus and its transmitted pathogens in urban and peri-urban areas in Europe: new hazards and relevance for public health. Front Public Health 2:251. doi: 10.3389/fpubh.2014.00251 PubMedCentralCrossRefPubMedGoogle Scholar
  50. Rudolf I, Mendel J, Sikutova S, Svec P, Masarikova J, Novakova D, Bunkova L, Sedlacek I, Hubalek Z (2009) 16S rRNA gene-based identification of cultured bacterial flora from host-seeking Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna ticks, vectors of vertebrate pathogens. Folia Microbiol (Praha) 54:419–428CrossRefGoogle Scholar
  51. Sasaki Y, Ishikawa J, Yamashita A, Oshima K, Kenri T, Furuya K, Yoshino C, Horino A, Shiba T, Sasaki T, Hattori M (2002) The complete genomic sequence of Mycoplasma penetrans, an intracellular bacterial pathogen in humans. Nucleic Acids Res 30:5293–5300PubMedCentralCrossRefPubMedGoogle Scholar
  52. Sparagano OAE, Allsopp MTEP, Mank RA, Rijpkema SGT, Figueroa JV, Jongejan F (1999) Molecular detection of pathogen DNA in ticks (Acari: Ixodidae): a review. Exp Appl Acarol 23:929–960CrossRefPubMedGoogle Scholar
  53. Stojek NM, Dutkiewicz J (2004) Studies on the occurrence of Gram-negative bacteria in ticks: Ixodes ricinus as a potential vector of Pasteurella. Ann Agric Environ Med 11:319–322PubMedGoogle Scholar
  54. Subramanian G, Mediannikov O, Angelakis E, Socolovschi C, Kaplanski G, Martzolff L, Raoult D (2012a) Diplorickettsia massiliensis as a human pathogen. Eur J Clin Microbiol Infect Dis 31:365–369CrossRefPubMedGoogle Scholar
  55. Subramanian G, Sekeyova Z, Raoult D, Mediannikov O (2012b) Multiple tick-associated bacteria in Ixodes ricinus from Slovakia. Ticks Tick Borne Dis 3:406–410CrossRefPubMedGoogle Scholar
  56. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefPubMedGoogle Scholar
  57. Uspensky I (2014) Tick pests and vectors (Acari: Ixodoidea) in European towns: introduction, persistence and management. Ticks Tick Borne Dis 5:41–47CrossRefPubMedGoogle Scholar
  58. Vayssier-Taussat M, Moutailler S, Michelet L, Devillers E, Bonnet S, Cheval J, Hebert C, Eloit M (2013) Next generation sequencing uncovers unexpected bacterial pathogens in ticks in western Europe. PLoS ONE 8:e81439. doi: 10.1371/journal.pone.0081439 PubMedCentralCrossRefPubMedGoogle Scholar
  59. Venclikova K, Betasova L, Sikutova S, Jedlickova P, Hubalek Z, Rudolf I (2014a) Human pathogenic borreliae in Ixodes ricinus ticks in natural and urban ecosystem (Czech Republic). Acta Parasitol 59:717–720CrossRefPubMedGoogle Scholar
  60. Venclikova K, Rudolf I, Mendel J, Betasova L, Hubalek Z (2014b) Rickettsiae in questing Ixodes ricinus ticks in the Czech Republic. Ticks Tick Borne Dis 5:135–138CrossRefPubMedGoogle Scholar
  61. Wang W, Gu W, Gasparich GE, Bi K, Ou J, Meng Q, Liang T, Feng Q, Zhang J, Zhang Y (2011) Spiroplasma eriocheiris sp. nov., associated with mortality in the Chinese mitten crab, Eriocheir sinensis. Int J Syst Evol Microbiol 61:703–708CrossRefPubMedGoogle Scholar
  62. Weisburg WG, Tully JG, Rose DL, Petzel JP, Oyaizu H, Yang D, Mandelco L, Sechrest J, Lawrence TG, Van Etten J, Maniloff J, Woese CR (1989) A phylogenetic analysis of the mycoplasmas: basis for their classification. J Bacteriol 171:6455–6467PubMedCentralPubMedGoogle Scholar
  63. Westberg J, Persson A, Holmberg A, Goesmann A, Lundeberg J, Johansson KE, Pettersson B, Uhlén M (2004) The genome sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP). Genome Res 14:221–227PubMedCentralCrossRefPubMedGoogle Scholar
  64. Zając V, Wójcik-Fatla A, Dutkiewicz J, Szymańska J (2015) Bartonella henselae in eastern Poland: the relationship between tick infection rates and the serological response of individuals occupationally exposed to tick bites. J Vector Ecol 40:75–82CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Radek Klubal
    • 1
  • Jan Kopecky
    • 2
  • Marta Nesvorna
    • 2
  • Olivier A. E. Sparagano
    • 3
  • Jana Thomayerova
    • 1
  • Jan Hubert
    • 1
    • 2
    Email author
  1. 1.Medical Centre PraguePragueCzech Republic
  2. 2.Crop Research InstitutePrague 6Czech Republic
  3. 3.Coventry UniversityCoventryUK

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