Advertisement

Experimental and Applied Acarology

, Volume 50, Issue 4, pp 353–359 | Cite as

Dermacentor marginatus and Ixodes ricinus ticks versus L929 and Vero cell lines in Rickettsia slovaca life cycle evaluated by quantitative real time PCR

  • Vojtech Boldiš
  • Eva ŠpitalskáEmail author
Article

Abstract

Ticks transmit many different pathogens to animals, humans and their pets. Rickettsia slovaca, as a member of the spotted-fever-group rickettsiae is an agent of the human disease Tick-borne lymphadenopathy (TIBOLA), also called Dermacentor-borne necrosis erythema and lymphadenopathy (DEBONEL), which occurs from the Mediterranean to central Europe, transmitted by Dermacentor reticulatus and Dermacentor marginatus (Acari: Ixodidae). In this study, quantitative real time PCR was used to characterize the growth of R. slovaca, strain B in static (mammalian L929 and Vero cells without replacement of growth medium) and dynamic (D. marginatus and Ixodes ricinus ticks) cultivation systems. Curves of bacterial growth in static cultivations were modeled with exponential, stationary and death phases, whereas in dynamic systems the stationary phase was absent. The highest point of multiplication of R. slovaca was recorded on the 4th day post infection in both cell lines and the rickettsial DNA copy number in L929 and Vero cells at this point was 21 and 27 times greater than rickettsial DNA copy number of inoculum, respectively. In the dynamic system, the highest point of multiplication was on the 21th and 12th day after feeding of ticks and rickettsial DNA copy numbers were 7,482 and 865 times greater than the inoculum in D. marginatus and I. ricinus, respectively. Life cycle of R. slovaca in mammalian cell lines was shorter; supposedly, bacteria destroyed these cells and ticks, especially D. marginatus, were considered a more appropriate environment.

Keywords

Rickettsia slovaca Dermacentor marginatus Ixodes ricinus L929 Vero Quantitative real-time PCR Transmission electron microscopy 

Notes

Acknowledgment

The study was financially supported by the grant No. 2/0065 from the Scientific Grant Agency of Ministry of Education of Slovak Republic—VEGA. We thank to Jan Erhart, Institute of Parasitology, ASCR, České Budejovice, Czech Republic who provided I. ricinus adult ticks from a laboratory colony and Dr. Elena Kocianová, Institute of Virology, SAS, Bratislava, Slovak Republic for D. marginatus ticks.

References

  1. Balashov YS (1972) Bloodsucking ticks (Ixodoidea)—vectors of diseases of man and animals. Misc Pub Ent Soc Amer 8:5Google Scholar
  2. Beati L, Finidori JP, Raoult D (1993) First isolation of Rickettsia slovaca from Dermacentor marginatus in France. Am J Trop Med Hyg 48:257–268PubMedGoogle Scholar
  3. Boldiš V, Kocianová E, Štrus J, Tušek-Žnidarič M, Sparagano O, Štefanidesová K, Špitalská E (2008) Rickettsial agents in Slovakian ticks (Acarina, Ixodidae) and their ability to grow in Vero and L929 cell lines. Ann NY Acad Sci 1149:281–285CrossRefPubMedGoogle Scholar
  4. Boldiš V, Štrus J, Kocianová E, Tušek-Žnidarič M, Štefanidesová K, Schwarzová K, Kúdelová M, Sekeyová Z, Špitalská E (2009a) Life cycle of Rickettsia slovaca in L929 cell line studied by quantitave real-time PCR and transmission electron microscopy. FEMS Lett 293:102–106. doi: 10.1111/j.1574-6968.2009.01510.x CrossRefGoogle Scholar
  5. Boldiš V, Štrus J, Kocianová E, Tušek-Žnidarič M, Štefanidesová K, Špitalská E (2009b) Ultrastructural studies on life cycle of Rickettsia slovaca, wild and standard type, cultivated in L929 and Vero cell lines. Folia Microbiol 54:130–136CrossRefGoogle Scholar
  6. Eremeeva ME, Dasch GA (2000) Rickettsiae. In: Lederberg J (ed) Encyclopedia of microbiology, 2nd edn. Academic Press, New York, pp 140–180Google Scholar
  7. Eremeeva ME, Bosserman EA, Demma LJ, Zambrano ML, Blau DM, Dasch GA (2006) Isolation and identification of Rickettsia massiliae from Rhipicephalus sanguineus ticks collected in Arizona. Appl Environ Microbiol 72:5569–5577CrossRefPubMedGoogle Scholar
  8. Fenollar F, Maurin M, Raoult D (2003) Wolbachia pipientis growth kinetics and susceptibilities to 13 antibiotics determined by immunofluorescence staining and real-time PCR. Antimicrob Agents Chemother 47:1665–1671CrossRefPubMedGoogle Scholar
  9. Harden VA (1990) Rocky mountain spotted fever: history of a twentieth-century disease. The Johns Hopkins University Press, Baltimore, MarylandGoogle Scholar
  10. Harley JP, Prescott LM (2002) Laboratory exercises in microbiology, 5th edn. McGraw-Hill, New YorkGoogle Scholar
  11. Hayes SF, Burgdorfer W (1989) Interactions between rickettsial endocytobionts and their tick hosts. In: Selwemmler W, Gassner G (eds) Insect endocytobiosis: morphology, physiology, genetics, evolution. CRC Press, Boca Raton, pp 235–251Google Scholar
  12. Korshus JB, Munderloh UG, Bey RG, Kurtti TJ (2004) Experimental infection of dogs with Borrelia burgdorferi sensu stricto using Ixodes scapularis ticks artificially infected by capillary feeding. Med Microbiol Immunol 193:27–34CrossRefPubMedGoogle Scholar
  13. Macaluso KR, Sonenshine DE, Ceraul SM, Azad AF (2002) Rickettsial infection in Dermacentor variabilis (Acari: Ixodidae) inhibits transovarial transmission of a second rickettsia. J Med Entomol 39:808–813Google Scholar
  14. Munderloh UG, Kurtti TJ (1995) Cellular and molecular interrelationships between ticks and prokaryotic pathogens. Ann Rev Entomol 40:221–243CrossRefGoogle Scholar
  15. Regnery RL, Spruill CL, Plikaytis BD (1991) Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol 173:1576–1589PubMedGoogle Scholar
  16. Řeháček J (1984) Rickettsia slovaca, the organism and its ecology. Acta Sci Nat Acad Sci Bohemoslov Brno 18:1–50Google Scholar
  17. Roux V, Fournier PE, Raoult D (1996) Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the gene encoding the protein rOmpA. J Clin Microbiol 34:2058–2065PubMedGoogle Scholar
  18. Silverman DJ, CLJr Wisseman, Waddell AD, Jones M (1978) External layers of Rickettsia prowazekii and Rickettsia rickettsii: occurrence of a slime layer. Infect Immun 22:233–246PubMedGoogle Scholar
  19. Špitalská E, Kocianová E, Výrosteková V (2002) Natural focus of Coxiella burnetii and rickettsiae of SFG in southwestern Slovakia. Biologia 57:589–595Google Scholar
  20. Uchiyama T (2005) Growth of typhus group and spotted fever group rickettsiae in insect cells. Ann NY Acad Sci 1063:215–221CrossRefPubMedGoogle Scholar
  21. Wilson K (1995) Preparation of genomic DNA from bacteria. In: Ausubel MF, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology. John Wiley and Sons, New York, pp 2.4.1–2.4.5Google Scholar
  22. Zanetti AS, Pornwiroon W, Kearney MT, Macaluso KR (2008) Characterization of rickettsial infection in Amblyomma americanum (Acari: Ixodidae) by quantitative real-time polymerase chain reaction. J Med Entomol 45:267–275CrossRefGoogle Scholar
  23. Žnidaršič N, Štrus J, Drobne D (2003) Ultrastructural alterations of the hepatopancreas in Porcellio scaber under stress. Environ Toxicol Phar 13:161–174CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  1. 1.Institute of VirologySlovak Academy of SciencesBratislavaSlovakia

Personalised recommendations