Coexistence of Borrelia spp. With different tick-borne pathogens in Ixodes ricinus ticks removed from humans in Poland

Abstract

Ixodes ricinus tick is a primary vector of Borrelia spirochetes and various tick-borne pathogens in Europe. Multi-species infections are common among ticks, however, the mechanism by which Borrelia spp. coexists with other pathogens within the tick vector is poorly understood. Furthermore, the extent to which Borrelia spp. interact with other pathogens or how multi-species infections influence pathogen loads in ticks and consequently, their transmission success and pathogenicity, remains unclear. The aim of this study was to evaluate the impact of co-infections on occurrence and loads of Borrelia spp. and other pathogens in I. ricinus. In the years 2021-2022, we collected 2073 I. ricinus ticks from tick-bitten individuals from around Poland and analyzed individually for the presence of Borrelia spp., Rickettsia spp., Neoehrlichia mikurensis, Anaplasma phagocytophilum, Babesia spp. and Bartonella spp. using molecular methods. Loads of pathogens were determined with droplet digital PCR technique. Of the 324 ticks positive for Borrelia spp., 76 were co-infected with at least one different pathogen. We observed higher prevalence of Babesia spp. and N. mikurensis among Borrelia spp. - positive ticks than in ticks uninfected with Borrelia spp. (3.4% vs. 1.3% and 10.2% vs. 4.2% respectively). A similar positive correlation was observed between Babesia spp. and N. mikurensis. Additionally, the loads of N. mikurensis were nearly twice as high in Babesia spp. - positive ticks compared to those not infected with this pathogen. This study is among the first to explore influence of co-infections on pathogen loads in multi-infected ticks feeding on humans. Understanding the relationships between pathogens coexisting in ticks may broaden our insight into epidemiology of tick-borne diseases.

Keywords: Borrelia; Ixodes ricinus; Co-infection; Droplet digital PCR; Pathogen load; Tick-borne pathogens.

Fig. 1

Prevalence of TBPs in individual Ixodes ricinus ticks removed from humans depending on the year of the study (2021–2022). N = 2073. For presence of Neoehrlichia mikurensis DNA only ticks collected in 2021 were tested (N = 832). N. mik Neoehrlichia mikurensis, A. phag Anaplasma phagocytophilum. Asterisk indicates P < 0.05 determined with maximum likelihood techniques based on loglinear analysis of contingency tables (HILOGLINEAR).

Fig. 2

Prevalence of Borrelia species in Ixodes ricinus ticks removed from humans. N = 262 out of 326 Borrelia spp. – positive ticks. B. afz Borrelia afzelii, B. gar Borrelia garinii, B. burg Borrelia burgdorferi s.s., B. lus Borrelia lusitaniae, B. val Borrelia valaisiana, B. spiel Borrelia spielmanii. B. miy Borrelia miyamotoi, B. fin Borrelia finlandensis, B. miy + B. bur co-infection of Borrelia miyamotoi and Borrelia burgdorferi s.s.

Fig. 3

Prevalence of TBPs in single-infected and co-infected ticks. (A) prevalence of Rickettsia spp. in Borrelia spp. – and Borrelia spp. + ticks, (B) prevalence of Neoehrlichia mikurensis in Borrelia spp. – and Borrelia spp. + ticks, (C) prevalence of Babesia spp. in Borrelia spp. – and Borrelia spp. + ticks, (D) prevalence of Anaplasma phagocytophilum in Borrelia spp. – and Borrelia spp. + ticks, (E) prevalence of Bartonella spp. in Borrelia spp. – and Borrelia spp. + ticks, (F) prevalence of Babesia spp. in Neoehrlichia mikurensis – and Neoehrlichia mikurensis + ticks. Asterisk indicates P < 0.05 determined with maximum likelihood techniques based on loglinear analysis of contingency tables (HILOGLINEAR).

Fig. 4

Loads of spirochetes in individual ticks infected with different Borrelia species determined by droplet digital PCR. N = 246 out of 326 Borrelia spp. – positive ticks. B. afz Borrelia afzelii, B. gar Borrelia garinii, B. burg Borrelia burgdorferi s.s., B. lus Borrelia lusitaniae, B. val Borrelia valaisiana, B. spiel Borrelia spielmanii, B. fin Borrelia finlandensis, B. miy Borrelia miyamotoi. The concentrations of target DNA copies in ticks are presented as copies of template per µL of the final 1 × ddPCR reaction. Ends of boxes represent the 25th and 75th percentiles with medians marked as horizontal lines and means as crosess. Vertical whiskers represent 10th and 90th percentiles. An asterisk indicates a P < 0.05 determined by Mann-Whitney U-test in comparision with the mean value obtained for B. burgdorferi s.l. complex.

Fig. 5

Loads of selected TBPs depending on presence/absence of other TBP in ticks determined by droplet digital PCR. B. burg Borrelia burgdorferi s.l., Rick Rickettsia spp., N. mik Neoehrlichia mikurensis, A. phag Anaplasma phagocytophilum, Bab Babesia spp. The concentrations of target DNA copies in ticks are presented as copies of template per µL of the final 1 × ddPCR reaction. Ends of boxes represent the 25th and 75th percentiles with medians marked as horizontal lines and means as crosess. Vertical whiskers represent 10th and 90th percentiless. An asterisk indicates a P < 0.05 determined by Mann-Whitney U-test.