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Identifying  Borrelia burgdorferi  as the causative agent of Lyme disease in 1981 was a watershed moment in understanding the major impact that tick-borne zoonoses can have on public health worldwide, particularly in Europe and the USA. The medical importance of tick-borne diseases has long since been acknowledged, yet little is known regarding the occurrence of emerging tick-borne pathogens such as  Borrelia  spp.,  Anaplasma phagocytophilum, Rickettsia  spp.,  Bartonella  spp., “ Candidatus  Neoehrlichia mikurensis”, and tick-borne encephalitis virus in questing ticks in Romania, a gateway into Europe. The objective of our study was to identify the infection and co-infection rates of different  Borrelia  genospecies along with other tick-borne pathogens in questing ticks collected from three geographically distinct areas in eastern Romania. We collected 557 questing adult and nymph ticks of three different species (534  Ixodes ricinus , 19  Haemaphysalis punctata , and 4  Dermacentor reticulatus ) from three areas in Romania. We analyzed ticks individually for the presence of eight different  Borrelia  genospecies with high-throughput real-time PCR. Ticks with  Borrelia  were then tested for possible co-infections with  A. phagocytophilum, Rickettsia  spp.,  Bartonella  spp., “ Candidatus  Neoehrlichia mikurensis”, and tick-borne encephalitis virus.  Borrelia  spp. was detected in  I. ricinus  ticks from all sampling areas, with global prevalence rates of 25.8%. All eight  Borrelia  genospecies were detected in  I. ricinus  ticks:  Borrelia garinii  (14.8%),  B. afzelii  (8.8%),  B. valaisiana  (5.1%),  B. lusitaniae  (4.9%),  B. miyamotoi  (0.9%),  B. burgdorferi  s.s (0.4%), and  B. bissettii  (0.2%). Regarding pathogen co-infection 64.5% of infected  I. ricinus  were positive for more than one pathogen. Associations between different  Borrelia  genospecies were detected in 9.7% of ticks, and 6.9% of  I. ricinus  ticks tested positive for co-infection of  Borrelia  spp. with other tick-borne pathogens. The most common association was between  B. garinii  and  B. afzelii  (4.3%), followed by  B. garinii  and  B. lusitaniae  (3.0%). The most frequent dual co-infections were between  Borrelia  spp. and  Rickettsia  spp., (1.3%), and between  Borrelia  spp. and “ Candidatus  Neoehrlichia mikurensis” (1.3%). The diversity of tick-borne pathogens detected in this study and the frequency of co-infections should influence all infection risk evaluations following a tick bite.

Borrelia Diversity and Co-infection with Other Tick Borne Pathogens in Ticks