Molecular identification, genotyping and phylogenetic analysis of and ticks and their associated spotted fever group species from a single location in northern Tunisia.

Introduction: Ticks and their associated spotted fever group (SFGR) represent an emerging zoonotic risk in Tunisia, where data on tick species distribution and pathogen prevalence remain limited. This study specifically aimed to investigate the diversity and phylogeny of and tick species and to identify and genetically characterize their associated SFGR species in northwestern Tunisia.

Methods: Tick sampling was conducted over a five-month period, from November 2022 to March 2023, in the Jouza district, Beja Governorate, northern Tunisia. A total of 236 ticks were collected both from vegetation using flag-dragging and manually from a red fox () carcass encountered opportunistically in the field. Tick species were morphologically identified and confirmed by Sanger sequencing of the mitochondrial 16S rRNA gene. detection was performed by nested PCR targeting the gene, followed by species-level identification through sequencing of and partial sequences. Phylogenetic analyses were conducted to assess genetic relationships. Additionally, chi-square tests were used to assess differences in infection rates between tick species, life stages, and collection sources.

Results: Ticks belonged to the complex ( and ), , and the sensu lato complex ( sensu strict and ) have been identified. Twelve and ten genotypes were recorded from 45 and 59 partial 16S rRNA mitochondrial sequences isolated from and , respectively. Additionally, one genotype was recorded from five specimens, and four genotypes were detected among 23 (temperate lineage) individuals. Overall, 52.1% of ticks tested positive for spp., with significantly higher infection rates in (75.6%, < 0.001) and (67.4%, < 0.01) compared to the group (37.2%) and , which tested negative. Three species were identified: and in the complex (including one co-infection), and exclusively in ticks. Moreover, phylogenetic analysis revealed that our tick isolates and associated spp. from questing ticks and the red fox clustered primarily with those from other North African and Southern European countries, suggesting trans-Mediterranean strain circulation and a potential link to wildlife reservoirs.

Conclusion: These findings enhance our understanding of tick and SFGR diversity in Tunisia and underscores the zoonotic risks from co-circulating species in shared environments. The high infection rates in and call for improved national tick surveillance. Public health implications include the need to consider tick-borne rickettsioses in unexplained febrile illness diagnoses. Future studies should assess infection status in hosts, explore seasonal tick dynamics, and evaluate environmental factors affecting transmission.