Tick-borne pathogen diversity and clinical impact of tick-infestation in Serbia and North Macedonia.

Ticks and tick-borne pathogens (TBPs) pose an expanding health threat across Europe, yet few studies link tick infection data directly to human clinical outcomes. We conducted a prospective, cross-border "One Health" study in Serbia and North Macedonia to assess the prevalence, diversity, and potential transmission of TBPs from ticks to humans. A total of 133 tick-infested patients were enrolled at two healthcare centers (112 in Skopje, 26 in Novi Sad), and 135 ticks were collected from these individuals, alongside paired blood and serum samples. Overall, TBP DNA was detected in 66.6 % of ticks, with Rickettsia helvetica (25.9 %) and Rickettsia monacensis (12.5 %) were most common. Mixed infections occurred in 15 % of ticks, with up to three pathogens co-occurring. Other detected TBPs included Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, Anaplasma phagocytophilum, Babesia microti, and Babesia divergens. Four patients (3 %) had detectable pathogen DNA in blood, primarily Rickettsia spp. and Anaplasma spp., yet remained asymptomatic. Most common clinical outcome during the observation period was local hypersensitivity reaction (21.1 %), while α-Gal sensitization was detected in two cases. Phylogenetic analysis revealed Balkan Rickettsia and Borrelia strains were mostly identical to Western and Central European lineages, with a notable exception: a Bo. garinii strain from North Macedonia clustered with strains from Russia and China, indicating possible long-distance introduction. The present study demonstrates that human exposure to multiple TBPs is common and while asymptomatic bloodstream infections do occur, local hypersensitivity reactions are much more common.