Co-occurrence of potential tick-borne pathogens of the order Rickettsiales and Borrelia burgdorferi s. l. and their link to season and area in Germany

Background: Ixodes ricinus, the most common hard tick species in central Europe, carries a variety of microorganisms, including tick-borne pathogens (TBPs), which are a potential human hazard. Recently it was shown that the relative abundances of potential human pathogenic members of the order Rickettsiales differed between Borrelia burgdorferi sensu lato-positive and -negative tick microbiomes. In this study, the co-occurrence of members of the order Rickettsiales, such as the genera Rickettsia spp., the species Anaplasma phagocytophilum, Wolbachia pipientis, and Neoehrlichia mikurensis as well as B. burgdorferi s.l. in the tick microbiome was addressed.Methods: This study used conventional PCRs to investigate the diversity and abundance of the before-mentioned bacteria in 760 nucleic acid extracts of I. ricinus ticks detached from humans, which were previously tested for B. burgdorferi s.l.. A 340 bp-sized gltA gene-based amplicon sequencing approach was performed to identify Rickettsia species. Results were correlated to the variables season, different areas of Germany, as well as B. burgdorferi s. l. occurrence in each tick extract via Fisher exact test and canonical correspondence analysis (CCA).Results: The prevalence of Rickettsia spp. (16.7%, n = 127) and W. pipientis (15.9%, n = 121) were similar, while A. phagocytophilum was found in 2.8% (n = 21) and N. mikurensis in 0.1% (n = 1) of all ticks. Co-occurrence of B. burgdorferi s. l. with Rickettsia spp. was most frequent. GltA gene sequencing results showed that Rickettsia helvetica predominantly occurred in tick microbiomes. Moreover, Rickettsia monacensis and Rickettsia raoultii were correlated with autumn and area south, respectively, and a negative B. burgdorferi s. l. finding. Further, R. aeschlimannii was correlated to a B. burgdorferi s. l. - positive microbiome.Conclusion: Almost every fifth tick carried DNA of at least two bacteria studied here that have human pathogenic effects. The lack of morphological determination of the tick in the diagnostic laboratory must be considered here. In future, this should be integrated into the analysis process because only based on these data exclusion criteria of (potential) TBPs can be created, which subsequently simplifies the evaluation of critical test results with very high probability.