Open Access
Translator Disclaimer
31 January 2020 First molecular detection of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from humans in the Sarajevo Canton (Bosnia and Herzegovina)
Lejla Lasić, Lejla Ušanović, Sanja Ćakić, Jasna Hanjalić, Belma Kalamujić Stroil
Author Affiliations +

Borrelia burgdorferi sensu lato is a complex of spirochetes which includes five pathogenic species that cause Lyme borreliosis (LB), the most common tick-borne disease in humans (Stanek & Strle 2018). The Sarajevo Canton, as a part of the central mountain and basin area of the Bosnian Dinarides, is influenced by the Central European continental climate from the north and the Mediterranean climate from the south (Drešković & Đug 2006).

In total, 1081 cases of LB were reported in Bosnia and Herzegovina in the period from 2002 to 2018 according to the statistics of authorized public health institutions ( https://www.zzjzfbih.ba/,https://www.phi.rs.ba). However, only one molecular study has been conducted in Bosnia and Herzegovina so far to test for the prevalence of B. burgdorferi sensu lato in free questing Ixodes ricinus (Linnaeus, 1758) (Hodžić et al. 2017). Although six samples showed to be positive for Rickettsia monacensis, none of 87 adult ticks gave positive findings for Borrelia. Spirochetes from B. burgdorferi sensu lato complex were detected and cultivated in Serbia and Croatia, the neighboring countries to Bosnia and Herzegovina (Ćakić et al. 2019; Rijpkema et al. 1996). According to this, positive findings of Borrelia are to be expected in ticks from our country as well.

This study aimed to utilize the nested polymerase chain reaction (nested PCR) to test for the presence of spirochetes from B. burgdorferi sensu lato complex in I. ricinus ticks removed from the patients in The Center for Emergency Medical Assistance of the Sarajevo Canton in period between March and June 2019. All samplings were done with the patients' consent.

Ticks were stored in separate tubes with 96% ethanol until further identification which was done following Estrada-Peña et al. (2004). Total genomic DNA was extracted using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Molecular identification of I. ricinus ticks was performed using primer pair dITS678 and rITS814 (Rumer et al. 2011). Nested PCR for detection of B. burgdorferi sensu lato targeting intergenic rrf (5S)–rrl (23S) region was performed using two primer pairs (Cerar et al. 2008; Postic et al. 1994). The first PCR was carried out according to Cerar et al. (2008) and the second PCR was performed according to Wang et al. (2014). In both PCRs, a positive AMPLIRUN Borrelia burgdorferi DNA control (Vircell, Spain) and a negative control were run in parallel with samples. PCR products were analyzed on 2% agarose gels. The sequencing of PCR products was done in Macrogene Europe B.V. (Amsterdam, the Netherlands). Sequences were identified and analyzed using nucleotide BLAST tool at the National Centre for Biotechnology Information ( https:/ /blast. ncbi.nlm.nih.gov/Blast.cgi) and FinchTV 1.4.0 (Geospiza, Inc.; Seattle, WA, USA;  http://ww w.ge os piza.com).

We received 50 tick samples removed from 50 patients (28 females and 22 males). Two samples did not have morphological characteristics of ticks and were excluded from further analysis. According to patients' statements, no ticks were subjected to any treatment before removal by medical staff. Based on age, patients were classified into four groups: (1) 0–18 (15 patients), (2) 19-35 (4 patients), (3) 36–65 (19 patients) and (4) +65 (12 patients). The number of ticks found per patient was one. Sixteen reported localities of possible tick infestation were registered in different parts of the Sarajevo Canton (Fig 1).

FIGURE 1.

Localities of the tick bite in the Sarajevo Canton reported by patients: a—Vlakovo, b—Ahatovići, c—hill Žuč, d—Donji Hotonj, e—Waterfall Skakavac, f—Buća Potok, g—Švrakino selo, h—Pofalići, i—Velešići, j—Wilson's Promenade, k—Ferhadija, l—Ilidža, m—Vojničko polje, n—Sedrenik, o—Bistrik and Hrid, p—Čobanija

img-z2-3_169.jpg

All 48 tested ticks were identified as I. ricinus using both morphological and molecular identification methods. Fourteen of them were determined as nymphs (29.20%) and 34 as females (70.80%). Ticks were classified according to the change of body volume after a blood meal as partially engorged midgut (diverticula filled with blood, volume changes of tick are not evident) (20, 41.60%), semi-engorged (volume changes of tick are evident) (14, 29.20%) and fully engorged (14, 29.20%).

Electrophoretic analysis of nested PCR products revealed that 32 of 48 tested ticks (66.70%) were positive on B. burgdorferi sensu lato, displaying the bands of 226–266 bp. Of all positively tested ticks, one was nymph (3.10%) and 31 were females (96.90%). From all positively tested ticks, eleven (34.40%) were fully engorged and 21 were semi-engorged (65.60%), indicating a higher risk of Borrelia infection for patients (Hofhuis et al. 2017).

Two sequences were obtained from PCR-positive I. ricinus. Sequences were deposited in the GenBank database under the accession numbers MN510850 and MN510851. Sequence under accession number MN510850 showed 100% similarity to rrs(5S)–rrf(23S) sequences of B. spielmanii deposited in GenBank. Sequence under accession number MN510851 showed 99% similarity to rrs(5S)–rrf(23S) sequences of B. lusitaniae deposited in GenBank. The results of this study represents the first molecular detection of pathogenic species of B. burgdorferi sensu lato complex in ticks inhabiting the area of the Sarajevo Canton, as well as those species with potential pathogenic risk such as B. lusitaniae. The results provided preliminary insight into the presence of Borrelia in the studied area and represent the starting point for further molecular detection and determination of B. burgdorferi sensu lato species across the whole territory of Bosnia and Herzegovina.

Funding

This work was supported by the Ministry of Education, Science and Youth of Sarajevo Canton (grant number 11-05-14-26465-1/18).

Acknowledgements

We are grateful to The Center for Emergency Medical Assistance of the Sarajevo Canton for their help in collecting ticks.

References

1.

Ćakić, S., Veinovic, G., Cerar, T., Mihaljica, D., Sukara, R., Ruzic-Sabljic, E. & Tomanović, S. (2019) Diversity of Lyme borreliosis spirochetes isolated from ticks in Serbia. Medical and Veterinary Entomology , 33(4), 512–520.  https://doi.org/10.1111/mve.12392  Google Scholar

2.

Cerar, T., Ružić-Sabljić, E., Glinsek, U., Zore, A. & Strle, F. (2008) Comparison of PCR methods and culture for the detection of Borrelia spp. in patients with erythema migrans. Clinical microbiology and infection: the official publication of the European. Society of Clinical Microbiology and Infectious Diseases , 14, 653–658.  https://doi.org/10.1111/j.1469-0691.2008.02013.x  Google Scholar

3.

Drešković, N. & Đug, S. (2006) Uspostava zaštićenih područja prirode u Kantonu Sarajevo i mogućnosti njihove ekoturističke valorizacije. Annales. Series historia et sociologia ,16, 233–246. (in Bosnian) Google Scholar

4.

Estrada-Pena, A., Bouattour, A., Camicas, J.L. & Walker, A.R. (2004) Ticks of domestic animals in the Mediterranean region: a guide to identification of species. University of Zaragoza, Zaragoza, Spain, 131 pp. Google Scholar

5.

Hodžić, A., Fuehrer, H. & Duscher, G.G. (2017) First Molecular Evidence of Zoonotic Bacteria in Ticks in Bosnia and Herzegovina. Transboundary and Emerging Diseases , 64, 1313–1316.  https://doi.org/10.1111/tbed.12473  Google Scholar

6.

Hofhuis, A., van de Kassteele, J., Sprong, H., van den Wijngaard, C.C., Harms, M.G., Fonville, M., van Leeuwen, A.D., Simões, M. & van Pelt, W. (2017) Predicting the risk of Lyme borreliosis after a tick bite, using a structural equation model. PLoS One , 2(7), e0181807.  https://doi.org/10.1371/journal.pone.0181807  Google Scholar

7.

Postic, D., Assous, M.V., Grimont, P.A. & Baranton, G. (1994) Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5S)–rrl (23S) intergenic spacer amplicons. International Journal of Systematic and Evolutionary Microbiology , 44, 743–752.  https://doi.org/10.1099/00207713-44-4-743  Google Scholar

8.

Rijpkema, S., Golubić, D., Molkenboer, M., Verbeek-De Kruif, N. & Schelleken, 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. Experimental & Applied Acarology , 20, 23–30.  https://doi.org/10.1007/BF00051474  Google Scholar

9.

Rumer, L., Sheshukova, O., Dautel, H., Donoso Mantke, O. & Niedrig, M. (2011) Differentiation of Medically Important Euro-Asian Tick Species Ixodes ricinus, Ixodes persulcatus, Ixodes hexagonus, and Dermacentor reticulates by Polymerase Chain Reaction. Vector-Borne & Zoonotic Disease , 11, 899–905.  https://doi.org/10.1089/vbz.2009.0191  Google Scholar

10.

Stanek, G. & Strle, F. (2018) Lyme borreliosis–from tick bite to diagnosis and treatment. FEMS Microbiology Reviews , 42, 233–258.  https://doi.org/10.1093/femsre/fux047  Google Scholar

11.

Wang, G., Liveris, D., Mukherjee, P., Jungnick, S., Margos, G. & Schwartz, I. (2014) Molecular Typing of Borrelia burgdorferi. Current Protocols in Microbiology , 34, 12C.5.1–12C.5.31.  https://doi.org/10.1002/9780471729259.mc12c05s34  Google Scholar

12.

The Institute of Public Health of the Federation of Bosnia and Herzegovina.  https://www.zzjzfbih.ba/ (Last date accessed: 15/09/2019). Google Scholar

13.

The Institute of Public Health of the Republic of Srpska.  https://www.phi.rs.ba/(Last date accessed: 15/09/ 2019). Google Scholar
© Systematic & Applied Acarology Society
Lejla Lasić, Lejla Ušanović, Sanja Ćakić, Jasna Hanjalić, and Belma Kalamujić Stroil "First molecular detection of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from humans in the Sarajevo Canton (Bosnia and Herzegovina)," Systematic and Applied Acarology 25(1), 169-172, (31 January 2020). https://doi.org/10.11158/saa.25.1.13
Received: 24 October 2019; Accepted: 16 December 2019; Published: 31 January 2020
JOURNAL ARTICLE
4 PAGES


Share
SHARE
RIGHTS & PERMISSIONS
Get copyright permission
Back to Top