Ticks are key vectors of zoonotic pathogens, and their expanding distribution in Europe heightens public health concerns. In Bosnia and Herzegovina, while tick distribution is well documented, molecular data on tick-borne pathogens remain limited. This study aimed to illustrate the presence and diversity of these pathogens, focusing on areas with high human activity. Ticks (n = 556) were collected in April 2022 from eight diverse locations, including urban parks, private properties, and rural sites. PCR-based screening was employed to detect Anaplasmataceae, Borrelia, Francisella, Piroplasmida, Rickettsia, and tick-borne encephalitis virus (TBEV), with subsequent sequencing to confirm results. Further characterization of Borrelia burgdorferi sensu lato was achieved via reverse line blotting (RLB) hybridization and sequencing. Ixodes ricinus was the most prevalent species, followed by Dermacentor marginatus and D. reticulatus. Our analysis revealed an overall infection rate of 22.1% in questing ticks, with Rickettsia spp. and Borrelia spp. predominating. Notably, seven Borrelia species were identified in I. ricinus, alongside Anaplasma phagocytophilum, Rickettsia helvetica, and R. monacensis, with co-infections mainly observed in peri-urban areas. This study provides the first molecular evidence of multiple tick-borne pathogens in the region, underscoring the need for further surveillance and risk assessment of tick-borne diseases in the region.

Borrelia spp. are transmitted to humans by the bite of an infected tick. In Europe, Borrelia afzelii and Borrelia garinii are the main causative agents of Lyme borreliosis, one of the most prevalent tick-borne diseases in the northern hemisphere. In bacteria such as Borrelia spp., a restriction-modification system (RMS) protects against the harmful introduction of foreign DNA. The RMS comprises two activities: methyltransferase and endonuclease. This study is aimed to characterize the RMS of B. afzelii and B. garinii. First, we identified potential RMS genes. The predicted genes were cloned into a methylase-deficient Escherichia coli strain and digested with methylation-sensitive restriction enzymes to verify methyltransferase activity. Additionally, the RMS proteins were purified to evaluate endonuclease activity. Subsequently, methylated and unmethylated plasmids were used to investigate the effect of methylation on endonuclease activity and transformation efficiency. We identified four possible RMS genes in B. afzelii and four RMS genes in B. garinii. We analyzed the presence of these genes in patient isolates and observed a high degree of heterogeneity. The restriction pattern of DNA methylated by each of the four recombinantly expressed genes provided strong evidence that all encode adenine-specific methyltransferases. After 24 h of incubation with purified RMS proteins, we observed complete digestion of unmethylated plasmid DNA, demonstrating endonuclease activity. Finally, we proved that methylation protects against endonuclease activity and increases transformation efficiency.

Tick-borne pathogens (TBPs) are increasingly reported from migratory passerines, yet data from the South Caucasus remain limited. Warblers of the genus Phylloscopus differ in migratory strategies and elevational distributions, providing an opportunity to examine host-associated variation in the molecular occurrence of TBPs. We screened blood samples from three sympatric species (Phylloscopus collybita, Phylloscopus lorenzii, and Phylloscopus nitidus) breeding in Armenia for TBP DNA using genus-specific PCR and reverse line blot hybridization targeting Anaplasma/Ehrlichia/Neoehrlichia, Babesia/Theileria, Borrelia, and Rickettsia. After excluding incomplete samples, 66 individuals were included in the analyses. Differences in molecular prevalence, elevation effects, and co-detection patterns were assessed statistically. Overall TBP DNA positivity differed significantly among host species, with the highest value in P. collybita. Among pathogens, Candidatus Neoehrlichia mikurensis showed the strongest host-associated pattern, whereas Rickettsia helvetica and Rickettsia slovaca showed weaker differences. The probability of TBP DNA detection decreased with elevation in a univariable model, but this effect weakened after accounting for host species. Multiple detections were relatively uncommon and were dominated by Candidatus N. mikurensis, R. helvetica, and R. slovaca, with only weak evidence of non-random co-occurrence. Our findings demonstrate species- and elevation-associated variation in the molecular occurrence of TBPs in Phylloscopus warblers in Armenia and highlight the need for broader sampling and integrated bird-tick studies to clarify the drivers and epidemiological meaning of pathogen DNA detection.

• The Borrelia restriction-modification system (RMS) differs by species group. • The RMS affects the transformation efficiency of Borrelia. • Increase in transformation efficiency observed with an in vitro methylated vector. • Transformation efficiency increased in low-passage Borrelia strains. • In vitro methylation can be a facilitating tool for Borrelia genetic manipulation.

Background: Golden jackals ( Canis aureus ) are expanding their range across Europe and Western Asia, frequently inhabiting agricultural and peri-urban areas where they interface with humans and domestic animals. Purpose: The aim of this study was to assess their potential role as reservoirs for tick-borne pathogens in Armenia, Transcaucasia. Methods: Between August 2024 and March 2026, blood samples were collected from 77 golden jackals across six administrative regions of Armenia (Lori, Tavush, Armavir, Ararat, Vayots Dzor, and Syunik). PCR followed by reverse line blot (RLB) hybridization was employed to simultaneously screen for bacterial and protistan pathogens, including Anaplasma, Borrelia , Ehrlichia , and Rickettsia , and Babesia and Theileria species, respectively. Results: Six zoonotic pathogens were detected: Rickettsia helvetica (16.9%), Borrelia burgdorferi sensu stricto (3.9%), Babesia canis (3.9%), Rickettsia raoultii (2.6%), Candidatus Neoehrlichia mikurensis (2.6%), and Borrelia afzelii (1.3%). Co-infections were detected in 7.8% of all samples (35.3% of pathogen-positive samples), including one triple co-infection with R. helvetica , B. burgdorferi s.s., and Ca. N. mikurensis . Statistically significant associations were observed between R. helvetica and B. burgdorferi s.s. ( r _tet = 0.915) and between R. helvetica and R. raoultii ( r _tet = 0.878). Rickettsia helvetica prevalence was significantly higher in females than in males (27.8% vs 7.3%; p = 0.030). Age-related trends suggested higher overall infection rates in adults (32.3%) compared to juveniles (15.2%; p = 0.097). Conclusions: This study provides the first molecular evidence of tick-borne pathogens in golden jackals from Transcaucasia, highlighting them as potential reservoir hosts for zoonotic pathogens with One Health significance.