Optimization of polymerase chain reaction for monitoring of Borrelia burgdorferi infection by ixodid ticks

Determination of the infection rate of ixodid ticks with tick-borne borreliosis pathogens and determination of belonging to the pathogenic genotype by PCR is an important component for monitoring, risk assessment and control of the epizootic situation of Lyme borreliosis in different territories. The results of testing and optimization of the internal laboratory protocol of the classical polymerase chain reaction for the identification of Lyme disease pathogens are presented. Eight samples of extracted DNA from ixodid ticks collected from vegetation in the forest park tract "Golendernya", Bila Tserkva, Kyiv region, were examined by classical PCR. Samples were formed from pools of ten tick specimens: seven pools - ticks of the genus I. ricinus and one pool - ticks of the genus D. reticulatus. For detection of borrelia DNA, primer sets were used to detect DNA of Borrelia burgdorferi sensu lato complex; Borrelia burgdorferi and pathogenic borrelia: Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii. The protocol for nucleic acid extraction from ticks was modified using the commercial IndiSpin Pathogen Kit. Optimization of amplification temperature conditions was carried out by the annealing temperature gradient method for each primer pair. Based on the results of the study, internal laboratory protocols for classical PCR using specific oligonucleotide primers were developed. It was found that in each of the pools of I. ricinus and D. reticulatus there were infected tick specimens with the Borrelia burgdorferi sensu lato complex and Borrelia afzelii genus, and also identified the Borrelia burgdorferi sensu stricto genus in one of the pools of I. ricinus and D. reticulatus, DNA of the Borrelia garinii genus was not detected. The developed internal laboratory protocols of classical PCR will be further used to study the infection of ixodid ticks with tick-borne borreliosis pathogens: Borrelia burgdorferi sensu lato, Borrelia burgdorferi sensu stricto and Borrelia afzelii.

Key words: Lyme borreliosis, Ixodes ticks, polymerase chain reaction, Borrelia burgdorferi sensu lato, Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii.

PANTELEIENKO O.

https://orcid.org/0000-0002-4311-9680

TSARENKO T.

https://orcid.org/0000-0003-4373-5958

1. Cull, B., Hansford, K.M., McGinley, L., Gillingham, E. L., Vaux, A.G.C., Smith, R., Medlock, J.M. (2021). A nationwide study on Borrelia burgdorferi sl infection rates in questing Ixodes ricinus: a six‐year snapshot study in protected recreational areas in England and Wales. Medical and Veterinary Entomology. Vol. 35, no. 3, pp. 352–360. DOI:10.1111/mve.12503.

2. Zubriková, D., Wittmann, M., Hönig, V., Švec, P., Víchová, B., Essbauer, S., Dobler, G., Grubhoffer, L., Pfister, K. (2020). Prevalence of tick-borne encephalitis virus and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks in Lower Bavaria and Upper Palatinate, Germany. Ticks and tick-borne diseases. Vol. 11, no. 3, 101375 p. DOI:10.1016/j.ttbdis.2020.101375.

3. Hubálek, Z., Halouzka, J. (1998). Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe. Parasitol Res. Vol. 84, pp. 167–172. DOI:10.1007/s004360050378.

4. Nebogatkin, I.V., Shulhan, A.M. (2020). Epidemiolohichni ta epizootychni osoblyvosti khvoroby Laima v 2019 rotsi v Ukraini [Epidemiological and epizootic features of Lyme disease in 2019 in Ukraine]. Aktualna infektolohiia [Actual infectology]. Vol. 8, no. 5–6, pp. 44–48. (in Ukrainian) DOI:10.22141/2312- 413x.8.5-6.2020.217959.

5. Mysterud, A., Stigum, V.M., Jaarsma, R.I., Sprong, H. (2019). Genospecies of Borrelia burgdorferi sensu lato detected in 16 mammal species and questing ticks from northern Europe. Scientific reports. Vol. 9, no. 1, pp. 1–8. DOI:10.1038/s41598-019-41686-0.

6. Radolf, J.D., Caimano, M.J., Stevenson, B., Hu, L.T. (2012). Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nature reviews microbiology. Vol. 10, no. 2, pp. 87–99. DOI:10.1038/nrmicro2714.

7. Dulipati, V., Meri, S., Panelius, J. (2020). Complement evasion strategies of Borrelia burgdorferi sensu lato. FEBS letters. Vol. 594, no. 16, pp. 2645–2656. DOI:10.1002/1873-3468.13894.

8. Stanek, G., Reiter, M. (2011). The expanding Lyme Borrelia complex-clinical significance of genomic species? Clinical Microbiology and Infection. Vol. 17, no. 4, pp. 487–493. DOI:10.1111/j.1469- 0691.2011.03492.x.

9. Hubálek, Z., & Halouzka, J. (1998). Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe. Parasitology research. Vol. 84, no. 3, pp. 167–172. DOI:10.1007/ s004360050378.

10. Piesman, J., Schneider, B.S., & Zeidner, N.S. (2001). Use of quantitative PCR to measure density of Borrelia burgdorferi in the midgut and salivary glands of feeding tick vectors. Journal of clinical microbiology. Vol. 39, no. 11, pp. 4145–4148. DOI:10.1128/ JCM.39.11.4145-4148.2001.

11. Veinović, G., Ćakić, S., Mihaljica, D., Sukara, R., Tomanović, S. (2020). Comparison of growth and morphology of Borrelia burgdorferi sensu lato in BSK‐H and BSK‐II media stored for prolonged periods. Apmis. Vol. 128, no. 10, pp. 552–557. DOI:10.1111/ apm.13069.

12. Kučerová, H. L., Žákovská, A., Marková, J., & Bártová, E. (2019). Detection of antibodies to Borrelia burgdorferi sl in wild small mammals and sensitivity of PCR and cultivation. Veterinary microbiology. Vol. 230, pp. 241–243. DOI:10.1016/j. vetmic.2019.02.004.

13. Panteleienko, O., Tsarenko T. (2022). Vyvchennia ta porivniannia indeksu shchilnosti zaselennia iksodovymy klishchamy riznykh biotopiv Kyivskoi ta Cherkaskoi oblastei [Study and comparison of population density indices of Ixodes ticks of different biotopes of Kyiv and Cherkasy regions]. Naukovyi visnyk veterynarnoi medytsyny [Scientific Bulletin of Veterinary Medicine]. Vol. 1, pp. 63–71. (in Ukrainian) DOI:10.33245/2310-4902-2022-173-1-63-71.

14. Marconi, R.T., Garon, C.F. (1993). Development of Polymerase Chain Reaction Primer Sets for Diagnosis of Lyme Disease and for Species-Specific Identification of Lyme Disease Isolates by 16S rRNA Signature Nucleotide Analysis. Journal of Clinical Microbiology. Vol. 31, no. 4, 1026 p.

15. Levytska, V.A., Mushinsky, A.B., Zubrikova, D. (2021). Detection of pathogens in ixodid ticks collected from animals and vegetation in five regions of Ukraine. Ticks and Tick-borne Diseases. Vol. 12, no. 1. DOI:10.1016/j.ttbdis.2020. 101586.

16. Demaerschalck, I., Ben Messaoud, A., et all. (1995). Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients. Journal of clinical microbiology. Vol. 33, no. 3, pp. 602–608. DOI:10.1128/ jcm.33.3.602- 608.1995.

17. Guttman, D.S., Wang, P.W., Wang, I.N., Bosler, E.M., Luft, B.J., Dykhuizen, D.E. (1996). Multiple infections of Ixodes scapularis ticks by Borrelia burgdorferi as revealed by single-strand conformation polymorphism analysis. Journal of clinical microbiology. Vol. 34, no. 3, pp. 652–656. DOI:10.1128/ jcm.34.3.652-656.1996.

18. Doshi, R., Day, P.J., Carampin, P., Blanch, E., Stratford, I.J., Tirelli, N. (2010). Spectrophotomet ric analysis of nucleic acids: oxygenation-dependant hyperchromism of DNA. Analytical and bioanalytical chemistry. Vol. 396, no. 6, pp. 2331–2339. DOI:10.1007/s00216-010-3461-x.

19. Ishchenko, V.D., Voloshchuk, N.M., Sterlikova, O.M., Humenyuk, L.V., Sklyar, V.V., Kalakailo, L.I., Ishchenko, A.Ya., Ishchenko, L.M. (2019). Vnutrishnolaboratorna aprobatsiia praimeriv dlia molekuliarno-henetychnoi identyfikatsii hrybiv rodu Fusarium Link [Interlaboratory aprobation of primers for molecular genetic identification of Fusarium link fungus]. Naukovi dopovidi Natsionalnoho universytetu bioresursiv i pryrodokorystuvannia Ukrainy [Scientific Bulletin of the National University of Life and Environmental Sciences of Ukraine]. Vol. 6, no. 82. ( in Ukraine) DOI:10.31548/dopovidi2019.06.017.

20. Okeyo, M., Hartberger, C., Margos, G., Straubinger, R.K., Sing, A., Fingerle, V. (2019). Comparison of methods for economic and efficient tick and Borrelia DNA purification. Ticks and tick-borne diseases. Vol. 10, no. 5, pp. 1041–1045. DOI:10. 1016/j.ttbdis.2019.05.002.

21. Rogovskyy, A., Batool, M., Gillis, D.C. (2018). Diversity of Borrelia spirochetes and other zoonotic agents in ticks from Kyiv, Ukraine. Ticks and tick-borne diseases. Vol. 9, no. 2, pp. 404–409. DOI:10.1016/j.ttbdis.2017.12.006.

Attachment	Size
panteleienko_tsarenko_2_2022.pdf	864.33 KB

Scientific Journal of Veterinary Medicine 2'2022

You are here

Optimization of polymerase chain reaction for monitoring of Borrelia burgdorferi infection by ixodid ticks

Key words: Lyme borreliosis, Ixodes ticks, polymerase chain reaction, Borrelia burgdorferi sensu lato, Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii.

3. Hubálek, Z., Halouzka, J. (1998). Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe. Parasitol Res. Vol. 84, pp. 167–172. DOI:10.1007/s004360050378.

5. Mysterud, A., Stigum, V.M., Jaarsma, R.I., Sprong, H. (2019). Genospecies of Borrelia burgdorferi sensu lato detected in 16 mammal species and questing ticks from northern Europe. Scientific reports. Vol. 9, no. 1, pp. 1–8. DOI:10.1038/s41598-019-41686-0.

6. Radolf, J.D., Caimano, M.J., Stevenson, B., Hu, L.T. (2012). Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nature reviews microbiology. Vol. 10, no. 2, pp. 87–99. DOI:10.1038/nrmicro2714.

7. Dulipati, V., Meri, S., Panelius, J. (2020). Complement evasion strategies of Borrelia burgdorferi sensu lato. FEBS letters. Vol. 594, no. 16, pp. 2645–2656. DOI:10.1002/1873-3468.13894.

8. Stanek, G., Reiter, M. (2011). The expanding Lyme Borrelia complex-clinical significance of genomic species? Clinical Microbiology and Infection. Vol. 17, no. 4, pp. 487–493. DOI:10.1111/j.1469- 0691.2011.03492.x.

9. Hubálek, Z., & Halouzka, J. (1998). Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe. Parasitology research. Vol. 84, no. 3, pp. 167–172. DOI:10.1007/ s004360050378.

10. Piesman, J., Schneider, B.S., & Zeidner, N.S. (2001). Use of quantitative PCR to measure density of Borrelia burgdorferi in the midgut and salivary glands of feeding tick vectors. Journal of clinical microbiology. Vol. 39, no. 11, pp. 4145–4148. DOI:10.1128/ JCM.39.11.4145-4148.2001.

11. Veinović, G., Ćakić, S., Mihaljica, D., Sukara, R., Tomanović, S. (2020). Comparison of growth and morphology of Borrelia burgdorferi sensu lato in BSK‐H and BSK‐II media stored for prolonged periods. Apmis. Vol. 128, no. 10, pp. 552–557. DOI:10.1111/ apm.13069.

12. Kučerová, H. L., Žákovská, A., Marková, J., & Bártová, E. (2019). Detection of antibodies to Borrelia burgdorferi sl in wild small mammals and sensitivity of PCR and cultivation. Veterinary microbiology. Vol. 230, pp. 241–243. DOI:10.1016/j. vetmic.2019.02.004.

14. Marconi, R.T., Garon, C.F. (1993). Development of Polymerase Chain Reaction Primer Sets for Diagnosis of Lyme Disease and for Species-Specific Identification of Lyme Disease Isolates by 16S rRNA Signature Nucleotide Analysis. Journal of Clinical Microbiology. Vol. 31, no. 4, 1026 p.

15. Levytska, V.A., Mushinsky, A.B., Zubrikova, D. (2021). Detection of pathogens in ixodid ticks collected from animals and vegetation in five regions of Ukraine. Ticks and Tick-borne Diseases. Vol. 12, no. 1. DOI:10.1016/j.ttbdis.2020. 101586.

16. Demaerschalck, I., Ben Messaoud, A., et all. (1995). Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients. Journal of clinical microbiology. Vol. 33, no. 3, pp. 602–608. DOI:10.1128/ jcm.33.3.602- 608.1995.

18. Doshi, R., Day, P.J., Carampin, P., Blanch, E., Stratford, I.J., Tirelli, N. (2010). Spectrophotomet ric analysis of nucleic acids: oxygenation-dependant hyperchromism of DNA. Analytical and bioanalytical chemistry. Vol. 396, no. 6, pp. 2331–2339. DOI:10.1007/s00216-010-3461-x.

20. Okeyo, M., Hartberger, C., Margos, G., Straubinger, R.K., Sing, A., Fingerle, V. (2019). Comparison of methods for economic and efficient tick and Borrelia DNA purification. Ticks and tick-borne diseases. Vol. 10, no. 5, pp. 1041–1045. DOI:10. 1016/j.ttbdis.2019.05.002.

21. Rogovskyy, A., Batool, M., Gillis, D.C. (2018). Diversity of Borrelia spirochetes and other zoonotic agents in ticks from Kyiv, Ukraine. Ticks and tick-borne diseases. Vol. 9, no. 2, pp. 404–409. DOI:10.1016/j.ttbdis.2017.12.006.