Borrelia burgdorferi genospecies detection by RLB hybridization in Ixodes cinus ticks from different sites of North-Eastern Poland
More details
Hide details
Department of Infectious Diseases and Neuroinfections, Medical University, Białystok, Poland
Institute of Biology, Laboratory of Eco-Epidemiology of Parasites, University of Neuchâtel, Neuchâtel, Switzerland
Ann Agric Environ Med. 2014;21(2):239–243
RLB (Reverse Line Blot Hybridization) is a molecular biology technique that might be used for Borrelia burgdorferi sensu lato (sl) DNA detection with genospecies specification. Among B. burgdorferi sl genospecies at least 7 are regarded as pathogenic in Europe.

The aim of the study was to evaluate the frequency of different Borrelia genospecies DNA detection in Ixodes ricinus ticks in the endemic area of North-Eastern Poland by using RLB.

Material and Methods:
Ixodes ricinus ticks were collected in May – June, from 6 different sites in North-Eastern Poland (Jakubin, Kolno, Grajewo, Suwałki, Siemiatycze, Białowieża) by flagging. Extracted DNA was amplified by polymerase chain reaction (PCR) targeting the intergenic spacer 5S 23S of B. burgdorferi sl. PCR products were hybridised to 15 different oligonucleotide probes for 9 different Borrelia genospecies (B. burgdorferi sl, B. burgdorferi ss, B. garinii, B. afzelii, B. valaisiana, B. lusitaniae, B. spielmanii, B. bissettii and B. relapsing fever-like spirochetes (B. myamotoi)) by RLB.

Borrelia genospecies DNA was detected in 205 Ixodes ricinus ticks. Among 14 infected with Borrelia ticks, 4 were identified as B. garinii and 10 as B. afzelii. Higher numbers of infected ticks were noticed in the eastern part of the research area, where large forest complexes dominate. Nymphs appeared to be the most frequently infected tick stage, which has an epidemiological meaning in the incidence of Lyme borreliosis.

The study demonstrated that RLB might be easily used in Borrelia DNA detection with genospecies-identification, and indicated the domination of B. afzelii and B. garinii in ticks from North-Eastern Poland.

Gern L, Douet V, López Z, Rais O, et al. Diversity of Borrelia genospecies in Ixodes ricinus ticks in a Lyme borreliosis endemic area in Switzerland identified by using new probes for reverse line blotting. Ticks Tick Borne Dis. 2010; 1: 23–29.
Hanincova K, Taragelova V, Koci J, Schäfer SM, Hails R, Ullmann AJ, Piesman J, Labuda M, Kurtenbach K. Association of B. garinii and B. valaisiana with songbirds in Slovakia. Appl Environ Microbiol. 2003; 69: 2825–2830.
Hulínská D, Votýpka J, Kríz B, Holínková N, Nováková J, Hulínský V. Phenotypic and genotypic analysis of Borrelia spp. isolated from Ixodes ricinus ticks by using electrophoretic chips and real-time polymerase chain reaction. Folia Microbiol. 2007; 52: 315–324.
Richter D, Postic D, Sertour N, Livey I, Matushka FR, Baranton G. Delineation of Borrelia burgdorferi sensu lato species by multilocus sequence analysis and confirmation of the delineation of Borrelia spielmanii sp. nov. Int J Syst Evol Microbiol. 2006; 56: 837–881.
Fraenkel CJ, Garpmo U, Berglund J. Determination of novel Borrelia genospecies in Swedish Ixodes ricinus ticks. J Clin Microbiol 2002; 40: 3308–3312.
Richter D, Schlee DB, Allgöwer R, Matuschka FR. Relationship of a novel Lyme disease spirochete Borrelia spielmani sp. nov. with its hosts in Central Europe. Appl Environ Microbiol. 2004; 70: 6414–6419.
Richter D, Schlee DB, Matushka FR. Relapsing-fever like spirochetes infecting European vector of Lyme disease agent. Emerg Infect Dis. 2003: 9: 697–701.
Wodecka B. Significance of red deer (Cervus elaphus) in the ecology of Borrelia burgdorferi sensu lato. Wiad Parazytol. 2007; 53: 231–237 (in Polish).
Barbour AG, Bunikis J, Travinsky B, Gatewood Hoen A, Diuk-Wasser M, Fish D, Tsao JI: Niche partitioning of Borrelia burgdorferi and Borrelia miyamotoi in the same tick vector and mammalian reservoir species. Am J Trop Med Hyg. 2009; 81(6): 1120–1131.
Cisak E, Chmielewska-Badora J, Zwoliński J, Wójcik-Fatla A, Polak J, Dutkiewicz J. Risk of tick-borne bacterial disease among workers of Roztocze National Park (South-Eastern Poland). Ann Agric Environ Med. 2005; 12: 127–132.
Stańczak J, Kubica-Biernat B, Racewicz M, Kruminis-Łozowska W, Dąbrowski J, Kur W. In: Detection of three genospecies of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from different regions of Poland. Int J Med Microbiol. 2000; 290: 559–566.
Godfroid E, Min Hu C, Humair PF, Bollen A, Gern L. PCR-Reverse Line Blot typing method underscores the genomic heterogeneity of Borrelia valaisiana species and suggests its potential involvment in Lyme disease. J Clin Microbiol. 2003; 41(8): 3690–3698.
Mitchel H, Wilske B, Hettche G, Göttner G, Heimerl C, Reischl U, Schulte-Spechtel U, Fingerle V. An OspA-polymerase chain reaction/restriction fragment length polymorphism-based method for sensitive detection and reliable differentiation of all European Borrelia burgdorferi sensu lato species and OspA types. Med Microbiol Immunol. 2003; 193: 219–226.
Alekseev ANH, Dubinina V, Van de Pol I, Schouls LM. Identification of Ehrlichia spp. and Borrelia burgdorferi in Ixodes ticks in the Baltic regions of Russia. J Clin Microbiol. 2001; 39: 2237–2242.
Burri C, Morán Cadenas F, Douet V, Moret J, Gern L. Ixodes ricinus density and infection prevalence of Borrelia burgdorferi sensu lato along a north-facing altitudinal gradient in Rhone Valley (Switzerland). Vector-Borne Zoontic Dis. 2007; 7:50–58.
Morán Cadenas F, Rais O, Jouda F, Douet V, Humair PF, Moret J, Gern L. Phenology of Ixodes ricinus and infection with Borrelia burgdorferi sensu lato along a north- and south – facing altitudinal gradient on Chaumont Mountain, Switzerland. J Med Entomol. 2007; 44: 683–693.
Morán Cadenas F, Schneider H, Lommano E, Burri C, Moret J, Gern L. A comparison of two DNA extraction approaches in the detection of Borrelia burgdorferi sensu lato from live Ixodes ricinus ticks by PCR and reverse line blotting. Vector-Borne Zoonotic Dis. 2007; 7: 555–561.
Poupon MA, Lommano E, Humair PF, Douet V, Rais O, Schaad M, Jenni L, Gern L. Prevalence of Borrelia burgdorferi sensu lato in ticks collected from migratory birds in Switzerland. Appl Environ Microbiol. 2006; 72: 976–979.
Rijpkema SG, Molkenboer MJ, Schouls LM, Jongejan F, Schellekens JF. Simultaneous detection and genotyping of tree genomic groups of Borrelia burgdorferi sensu lato in dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23 S rRNA genes. J Clin Microbiol. 1995; 33: 3091–3095.
Marconi RT, Garon CF. Development of polymerase chain reaction primers sets for diagnosis of Lyme disease and species-specific identification of Lyme disease isolates by 16S rRNA signature nucleotide analysis. J Clin Microbiol. 1992; 30(11): 2830–2834.
Missone MC, Van Impe G, Hoet PP. Genetic heterogeneity of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in Belgium. J Clin Microbiol. 1998; 36: 3352–3354.
Wang G, van Dam AP, Spanjaard L, Dankert J: Molecular typing of Borrelia burgdorferi sensu lato by randomly amplified polymorphic DNA fingerprinting analysis. J Clin Microbiol. 1998; 36: 768–776.
Casati S, Bernasconi MB, Gern L, Piffaretti JC. Diversity within Borrelia burgdorferi sensu lato genospecies in Switzerland by recA gene sequence. FEMS Microbiol Let. 2004; 238(1): 15–123.
Dutkiewicz J, Cisak E. Biologiczne czynniki zagrożenia w leśnictwie. Zdr Publ. 2008; 118(1): 85–90 (in Polish).
Reports of Polish National Institute of Public Health (access: 2014.05.19).
Gubbels MJ, de Vos S, van der Weide M, Viseras J, Schouls LM, de Vries E, Jongejan F. Simultaneous detection of bovine Theileria and Babesia species using reverse line blot hybridization. J Clin Microbiol. 1999; 37: 1782–1789.
Schouls LM, Van de Pol I, Rijpkema SG, Shot CS. Deletion and identification of Ehrlichia, Borrelia burgdorferi sensu lato and Bartonella species in Dutch Ixodes ricinus ticks. J Clin Microbiol. 1999; 37: 2215–2222.
Taoufik A, Nijhof A, Hamidjaja R, Jongejan F, Pillay V, Sonnvelt M. Reverse line blot hybridisation in the detection of tick-borne diseases. Biotech Magazine Online as published in BTi September 2004, (access: 2014.05.19).
Moter SE, Hofmann H, Wallich R, Simon MM, Kramer MD. Detection of Borrelia burgdorferi sensu lato in lesional skin of patients with erythema migrans and acrodermatitis chronic atropicans by OspA-specific PCR. J Clin Microbiol. 1994; 32: 2980–2988.
Chmielewska-Badora J, Cisak E, Wójcik-Fatla A, Zwoliński J, Buczek A, Dutkiewicz J. Correlation of tests for detection of Borrelia burgdorferi sensu lato infection in patients with diagnosed borreliosis. Ann Agric Environ Med. 2006; 13: 307–311.
Strzelczyk KJ, Wiczkowski A, Kwaśniewski M, Zalewska-Ziob M, Strzelczyk J, Gawron K, Adamek B, Spausta G. Prevalence of Borrelia burgdorferi genospecies in Ixodes ricinus ticks from recreational areas of Silesia. Adv Clin Exp Med. 2006; 15(6): 1003–1008.
Honegr K, Hulínská D, Beran J, Dostál V, Havlsasová J, Čermáková Z. Long term repeated electron microscopy and PCR detection of Borrelia burgdorferi sensu lato after an antibiotic treatment. Centr Eur J Publ Health. 2004; 12(1): 6–11.
Derdáková M, Beati L, Pet`ko B, Stanko M, Fish D. Genetic variability within Borrelia burgdorferi sensu lato genospecies established by PCR-single strand conformation polymorphism analysis of the rrfA-rrlB intergenic spacer in Ixodes ricinus ticks from Czech Republic. Appl Environ Microbiol. 2003; 69: 509–516.
Paulauskas A, Radzijevskaja J, Ambrasiene D, Rosef O. Detection of tick-borne pathogens by molecular methods. Biologija 2008; 54(3): 92–197.
Quessada T, Martial-Convert F, Arnaud S, Leudet de la Vallee H, Gilot B, Pichot J. Prevalence of Borrelia burgdorferi species and identification of Borrelia valaisiana in questing Ixodes ricinus in Lyon region of France as a determined by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism. Eur J Clin Microbiol Infect Dis. 2003; 22: 165–173.
Escudero R, Barral M, Perez A, Vitutia MM, Garcia-Perez AL, Jimenez S, Sellek RE, Anda P. Molecular and pathogenic characterization of Borrelia burgdorferi sensu lato isolates from Spain. J Clin Microbiol. 2000; 38: 4026–4033.
Jouda F, Perret JL, Gern L. Ixodes ricinus density, and distribution and prevalence of Borrelia burgdorferi sensu lato infection along an altitudinal gradient. J Med Entomol. 2004; 41: 162–169.
Jouda F, Perret JL, Gern L. Density of questing Ixodes ricinus nymphs and adults infected by Borrelia burgdorferi sensu lato in Switzerland: spatiotemporal pattern at a regional scale. Vector-Borne Zoonotic Dis. 2004; 4: 23–32.
Singh SK, Girschick HJ. Molecular survival of Lyme disease spirochete Borrelia burgdorferi. Lancet Infect Dis. 2004; 4: 575–583.
Kurtenbach K, Peacey M, Rijpkema AGT, Hoodless AN, Nutall PA, Randolph SE. Differential transmission of the genospecies of Borrelia burgdorferi sensu lato by game birds and small rodents in England. Appl Environ Microbiol. 1998; 64(4): 1169–1174.
Piesman J, Gern L. Lyme borreliosis in Europe and North America. Parasitology 2004; 129: 191–220.
EUCALB (access: 2014.05.19).